COMPOSITIONS AND METHODS FOR INCREASING GENE EXPRESSION

专利摘要:
the present disclosure relates in general to nucleic acid molecules for use in the regulation of gene expression. what is disclosed in this document includes nucleic acid molecules containing one or more structural elements of the viral capsid enhancer operably linked to a coding sequence for a gene of interest. in some embodiments, the viral capsid enhancer comprises a downstream loop (dlp) of a viral capsid protein or a variant of dlp.
公开号:BR112019011661A2
申请号:R112019011661-3
申请日:2017-12-04
公开日:2020-01-07
发明作者:Iver Kamrud Kurt；Nyan Win Maung；Stephen Wang Nathaniel；DeHart Jason
申请人:Synthetic Genomics, Inc.；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: COMPOSITIONS AND METHODS FOR IMPROVING GENE EXPRESSION
Related Orders [001] This order claims the priority of Interim Order No. U.S. Series 62 / 430,250, filed December 5, 2016; US Serial Order No. 62 / 486,361, filed on April 17, 2017 and US Serial Order No. 62 / 587,954, filed on November 17, 2017. The content of the orders referenced above is incorporated herein in its entirety for reference.
Incorporation of the sequence listing [002] The material in the attached sequence listing is incorporated here for reference in this application. The attached string listing text file, named SGI012WO_SeqListing.txt, was created on December 4, 2017, and is 169 KB.
Field [003] The present disclosure relates to the field of molecular biology and genetic engineering, which includes nucleic acid molecules useful for regulating gene expression, and the use of nucleic acid molecules for, for example, production of desired products in suitable host cells in cell culture or in an individual, and to impart beneficial characteristics to the
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2/207 cells or host individuals.
Background [004] Advances in biotechnology and molecular biology have offered many opportunities to develop recombinant cells and organisms with commercially desirable characteristics or traits. In particular, modern genetic engineering techniques have greatly accelerated the introduction of genes and, consequently, new traits in recombinant cells and organisms. An appropriate level of expression of a desirable gene in, for example, a host cell or transgenic organism is useful to achieve this goal.
[005] However, despite the availability of many molecular tools, genetic modifications of host cells and organisms are often restricted by insufficient or uncontrolled expression of the gene of interest. Thus, there is still a need for regulatory elements capable of enhancing transgenic expression in host cells and organisms. The identification of innovative molecular tools that includes regulatory elements, expression vectors, and expression systems that work on various types of organisms can be useful in developing genetically enhanced cells and organisms.
summary
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3/207 [006] This section provides a general summary of this application, and does not cover its full scope or all of its features.
[007] The present disclosure refers, in general, to methods and compositions useful for regulating, for example, enhancing, gene expression in vitro, ex vivo or in vivo. The gene expression can be, for example, in animal cells and other eukaryotic cells. The gene can be, for example, a heterologous gene that encodes a protein of interest.
[008] In one aspect, some embodiments disclosed herein refer to a nucleic acid molecule, which includes (i) a first nucleic acid sequence that encodes one or more RNA stem-loop structures of a capsid enhancer viral or a variant thereof; and (ii) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence comprises a coding sequence for a gene of interest (GOI).
[009] Implants of nucleic acid molecule modalities according to the present disclosure may include one or more of the following resources. In some embodiments, the first nucleic acid sequence is operably linked upstream to the coding sequence for GOI. In some embodiments, the acid molecule
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The nucleic 4/207 further includes a promoter operably linked upstream to the first nucleic acid sequence. In some embodiments, the nucleic acid molecule further includes a 5 'UTR sequence operably linked upstream to the first nucleic acid sequence. In some embodiments, the 5 'UTR sequence is operably linked downstream to the promoter and upstream to the first nucleic acid sequence. In some embodiments, the nucleic acid molecule further includes a coding sequence for an auto-protease peptide operably linked upstream to the second nucleic acid sequence. In some embodiments, the coding sequence for the auto-protease peptide is operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the auto-protease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (EMDV) 2A (F2A), an Equine Rhinitis Virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof. In some embodiments, the nucleic acid molecule further includes a 3 'UTR sequence operably linked downstream to the second nucleic acid sequence.
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5/207 [010] In some embodiments, the viral capsid enhancer is derived from a capsid gene of a species of virus that belongs to the Togaviridae family. In some embodiments, the virus species belongs to the Alfavirus genus of the Togaviridae family. In some embodiments, the alphavirus species is eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus ( PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), virus Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), Alfavirus Salmonidae (SAV) or Buggy virus Creek. In some embodiments, the viral capsid enhancer comprises a downstream loop (DLP) motif of the virus species, and in which the DLP motif comprises at least one of one or more RNA rod-loop structures. In some embodiments, the viral capsid enhancer comprises a nucleic acid sequence that exhibits at least 80% sequence identity for at least one
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6/207 among SEQ ID NOs: 1 and 46 to 52. In some embodiments, the nucleic acid sequence exhibits at least 95% sequence identity for at least one of SEQ ID NOs: 1 and 4 6 to 52.
[011] In some embodiments, the coding sequence for GOI encodes a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide or a combination thereof. In some embodiments, the polypeptide is an antibody, an antigen, an immune modulator, a cytokine, an enzyme, or a combination thereof.
[012] In some embodiments, the nucleic acid molecule of the disclosure further includes a third nucleic acid sequence that encodes one or more RNA stem-loop structures of a second viral capsid enhancer or a variant thereof; and a fourth nucleic acid sequence operably linked to the third nucleic acid sequence, wherein the fourth nucleic acid sequence comprises a coding sequence for a second gene of interest (GOI). In some embodiments, the nucleic acid molecule further includes a coding sequence for a second auto-protease peptide operably linked downstream to the third acid sequence
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7/207 nucleic and upstream to the fourth nucleic acid sequence.
[013] In some embodiments, the nucleic acid molecule of the disclosure is either an mRNA molecule or an RNA replicon. In some embodiments, the nucleic acid molecule is either an expression vector or a transcription vector. In some embodiments, the expression vector or a transcription vector further includes one or more additional transcription regulatory sequences. In some embodiments, the expression vector or a transcription vector further includes one or more additional transcription regulatory sequences. In some embodiments, the expression vector or a transcription vector further includes one or more additional translation regulatory sequences. In some embodiments, the nucleic acid molecule is a plasmid, a bacteriophage vector, a cosmid, a phosmoid, a viral replicon, a carrier vector or a combination thereof. In some embodiments, the nucleic acid molecule is a prokaryotic or eukaryotic vector. In some embodiments, the nucleic acid molecule is produced by de novo synthesis.
[014] Also disclosed in some embodiments includes a method for producing a polypeptide of interest in a cell, which includes introducing a nucleic acid molecule according to the present disclosure into a cell
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8/207 thereby producing a polypeptide encoded by the GOI in the cell. In yet another related aspect, some embodiments disclosed in this document relating to a method for producing a polypeptide of interest in a cell, which includes introducing an RNA molecule into the cell, wherein the RNA molecule comprises one or more stem structures - RNA loop from a viral capsid enhancer or a variant thereof, and a coding sequence for the polypeptide of interest, thereby producing the polypeptide of interest in the cell.
[015] In some embodiments, the RNA molecule is a messenger RNA (mRNA) molecule or a replicon RNA molecule. In some embodiments, the RNA molecule is produced by de novo synthesis and / or in vitro transcription before being introduced into the cell. In some embodiments, the RNA molecule comprises a downstream loop motif (DLP) of a virus species, and the DLP motif comprises at least one of one or more RNA stem-loop structures of the enhancer. viral capsid. In some embodiments, the RNA molecule further comprises a coding sequence for an autoprotease peptide downstream for at least one of one or more RNA stem-loop structures and upstream to the coding sequence for the polypeptide of interest. In some embodiments, the auto-protease peptide comprises a
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9/207 peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof. In some embodiments, the polypeptide is a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide or a combination thereof. In some embodiments, the polypeptide is an antibody, an antigen, an immune modulator, a cytokine, an enzyme, or a combination thereof. In some embodiments, the cell is present in a tissue, an organ or an individual. In some modalities, the individual is a human being, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster or buffalo.
[016] Some modalities reveal a method for producing a messenger RNA (mRNA) in a cell. The method, in some embodiments, includes administering to the cell a nucleic acid molecule comprising a first nucleic acid sequence that encodes one or more RNA rod-loop structures of a viral capsid enhancer or a variant thereof, and a Monday
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10/207 nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence comprises a coding sequence for a gene of interest (GOI), thereby producing a GOI mRNA.
[017] In some embodiments, the first nucleic acid sequence is operably linked upstream to the coding sequence for GOI. In some embodiments, the nucleic acid molecule further includes a promoter operably linked upstream to the first nucleic acid sequence. In some embodiments, the nucleic acid molecule further includes a 5 'UTR sequence operably linked upstream to the first nucleic acid sequence. In some embodiments, the 5 'UTR sequence is operably linked downstream to the promoter and upstream to the first nucleic acid sequence. In some embodiments, the nucleic acid molecule further includes a coding sequence for an auto-protease peptide operably linked upstream to the second nucleic acid sequence. In some embodiments, the coding sequence for the auto-protease peptide is operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the auto-protease peptide comprises a sequence of peptides selected from the group that
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11/207 consists of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis Virus (ERAV) 2A (E2A), a Thosea asigna 2A (T2A) virus, a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof. In some embodiments, the nucleic acid molecule further includes a 3 'UTR sequence operably linked downstream to the second nucleic acid sequence.
[018] In some embodiments disclosed in this document, the viral capsid enhancer is derived from a capsid gene of a species of virus that belongs to the Togaviridae family. In some modalities, the virus species belongs to the Alfavirus genus of the Togaviridae family. In some embodiments, the alphavirus species is eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus ( PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), virus Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV),
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12/207 Fort Morgan virus (FMV), Ndumu (NDUV), alfavirus Salmonidae (SAV) or Buggy Creek virus. In some embodiments, the viral capsid enhancer comprises a downstream loop (DLP) motif of the virus species, and in which the DLP motif comprises at least one of one or more RNA rod-loop structures. In some embodiments, the viral capsid enhancer comprises a nucleic acid sequence that exhibits at least 80% sequence identity for at least one of SEQ ID NOs: 1 and 46 to 52. In some embodiments, the nucleic acid sequence exhibits at least 95% sequence identity for at least one of SEQ ID NOs: 1 and 46 to 52.
[019] In some embodiments disclosed herein, the coding sequence for GOI encodes a polypeptide. In some embodiments, the polypeptide is selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide and a combination thereof. In some embodiments, the polypeptide is an antibody, an antigen, an immune modulator, a cytokine, an enzyme, or a combination thereof. In some embodiments of the method for producing a messenger RNA (mRNA), according to the present disclosure, the nucleic acid molecule further includes a third sequence of acids
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13/207 nucleic acids encoding one or more RNA stem-loop structures of a second viral capsid enhancer or a variant thereof; and a fourth nucleic acid sequence operably linked to the third nucleic acid sequence, wherein the fourth nucleic acid sequence comprises a coding sequence for a second gene of interest (GOI). In some embodiments, the nucleic acid molecule further includes a coding sequence for a second auto-protease peptide operably linked downstream to the third nucleic acid sequence and upstream to the fourth nucleic acid sequence.
[020] The nucleic acid molecule of the present disclosure may, in some embodiments, be an RNA replicon. In some embodiments, the nucleic acid molecule is either an expression vector or a transcription vector. In some embodiments, the nucleic acid molecule further comprises one or more additional transcriptional regulatory sequences. In some embodiments, the nucleic acid molecule further comprises. In some embodiments, one or more additional translation regulatory sequences. In some embodiments, the nucleic acid molecule is an expression vector selected from the group consisting of a plasmid, a bacteriophage vector, a cosmid, a phosphid, a viral replicon, a carrier vector and a combination thereof. In some embodiments, the
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14/207 nucleic acid is a prokaryote expression vector or a eukaryotic expression vector. In some embodiments, the cell is present in a tissue, an organ or an individual. In some modalities, the individual is a human being, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster or buffalo. In some embodiments, the method for producing a messenger RNA (mRNA), according to the present disclosure, further includes producing a polypeptide encoded by the GOI mRNA in the cell. In some embodiments, the method also includes obtaining the mRNA produced from the GOI and introducing the mRNA obtained in a second cell to express a polypeptide encoded by the GOI mRNA in the second cell.
[021] On a aspect, some modalities gives revelation if refer to the molecule nucleic acid what comprises a acid sequence nucleic that encodes a viral RNA replicon modified, wherein replicon of RNA
modified viral comprises (i) a first nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and (ii) a second nucleic acid sequence that encodes at least one non-structural viral protein or a portion thereof, where
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15/207 the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence.
[022] In some modalities, at least one of the one or more elements structural of perfector in capsid viral comprises one or more stem-structures tie of RNA. In some modalities, O perfector in capsid viral is derived from a gene in capsid from an species of viruses that belongs to the family Togaviridae. In
in some modalities, the virus species belongs to the genus Alfavirus of the Togaviridae family. In some embodiments, the alphavirus species is eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus ( PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), virus Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) or Buggy Creek virus. In some embodiments, the viral capsid enhancer comprises a downstream loop (DLP) motif of the virus species, and in which the DLP motif comprises
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16/207 at least one of one or more RNA rod-loop structures. In some embodiments, the viral capsid enhancer comprises a nucleic acid sequence that exhibits at least 80% sequence identity for at least one of SEQ ID NOs: 1 and 46 to 52. In some embodiments, the nucleic acid sequence exhibits at least 95% sequence identity for at least one of SEQ ID NOs: 1 and 46 to 52.
[023] In some embodiments, the nucleic acid sequence encoding the modified viral RNA replicon further comprises a coding sequence for an auto-protease peptide operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the auto-protease peptide comprises a sequence of peptides selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (EMDV) 2A (F2A), an Equine Rhinitis Virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) or a combination thereof. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 1 to 1,000 nucleotides downstream of the 5 'terminal of the modified viral RNA replicon. The second sequence of
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17/207 nucleic acids comprise substantially the entire coding sequence for the viral non-structural proteins native to the corresponding unmodified viral RNA replicon.
[024] In some embodiments disclosed herein, the modified viral RNA replicon comprises a modified RNA replicon derived from a species of virus belonging to the Alfavirus genus of the Togaviridae family or to the Arterivirus genus of the Arteriviridae family.
[025] In some embodiments, the arterivirus virus species is equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevation virus (LDV) or Simian hemorrhagic fever virus (SHFV ). In some embodiments, the first nucleic acid sequence is operably positioned upstream of a second nucleic acid sequence that encodes a portion or all of the non-structural pplab protein of the modified artevirus RNA replicon. In some embodiments, the nucleic acid sequence encoding the modified artevirus RNA replicon further comprises one or more expression cassettes, wherein at least one of the one or more expression cassettes comprises a promoter operably linked to a sequence coding for a gene of interest (GOI). In some embodiments, the modified artevirus RNA replicon
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18/207 comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding a portion or all of the non-structural protein pplab of the modified artevirus RNA replicon. In some embodiments, at least one of the one or more expression cassettes is operably positioned downstream to a transcriptional regulatory sequence (TRS) of the modified artevirus RNA replicon, where the TRS is TRS1, TRS2, TRS3, TRS4 , TRS5, TRS6 or TRS7. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for the GOT.
[026] In some embodiments, the nucleic acid sequence encoding the modified artevirus RNA replicon further comprises a coding sequence for an auto-protease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for the GOT. In some embodiments, the coding sequence for the GOT encodes a therapeutic polypeptide, a prophylactic polypeptide, a
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19/207 diagnostic polypeptide, nutraceutical polypeptide, industrial enzyme, reporter polypeptide or any combination thereof. In some embodiments, the coding sequence for GOI encodes an antibody, antigen, immune modulator, cytokine, enzyme or any combination thereof.
[027] In some embodiments, the modified viral RNA replicon comprises a modified RNA replicon derived from a species of virus alphavirus selected from the group consisting of eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus ( VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), 0'Nyong-Nyong virus ( ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland virus J (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), alphavirus Salmonidae (SAV) and Buggy Creek virus. In some embodiments, the first nucleic acid sequence is operably positioned upstream of a second nucleic acid sequence that encodes
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20/207 one or more nspl-4 non-structural proteins or a portion thereof of the modified alphavirus RNA replicon. In some embodiments, the nucleic acid sequence encoding the modified alphavirus RNA replicon further comprises one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to a coding sequence for a gene of interest (GOI). In some embodiments, the modified alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of a nucleic acid sequence encoding one or more nspl-4 non-structural proteins or a portion thereof of the alphavirus RNA replicon modified. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream of the coding sequence for GOI. In some embodiments, the nucleic acid sequence encoding the modified alphavirus RNA replicon further comprises a coding sequence for an operably linked auto-protease peptide downstream of the third sequence of
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21/207 nucleic acids and upstream to the coding sequence for GOI. In some embodiments, the coding sequence for GOI encodes a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide or a combination thereof. In some embodiments, the coding sequence for GOI encodes an antibody, antigen, immune modulator, enzyme, cytokine or a combination thereof.
[028] In one aspect, some embodiments of the disclosure refer to a nucleic acid molecule comprising a nucleic acid sequence that encodes a modified non-alphavirus RNA replicon, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer or a variant thereof. In some embodiments, the nucleic acid sequence encoding the modified non-alphavirus RNA replicon further comprises a second nucleic acid sequence that encodes at least one non-structural viral protein or a portion thereof, wherein the first nucleic acid sequence it is operably linked upstream to the second nucleic acid sequence. In some embodiments, the nucleic acid sequence encoding the modified non-alphavirus RNA replicon
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22/207 further comprises a coding sequence for an auto-protease peptide operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the auto-protease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis Virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Virus Flacherie 2A (BmIFV2A) or a combination thereof. In some embodiments, the nucleic acid sequence encoding the modified non-alphavirus RNA replicon comprises a modified RNA replicon derived from a positive strand RNA virus. In some embodiments, the positive-stranded RNA virus is a species of virus that belongs to a family selected from the group consisting of Togaviridae family, Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family and Paramyxoviridae family. In some embodiments, the positive strand RNA virus is a species of virus that belongs to the genus Arterivirus of the family Arteriviridae.
[029] In some embodiments disclosed herein, the nucleic acid sequence encoding the modified non-alphavirus RNA replicon further comprises
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23/207 one or more expression cassettes, each expression cassette comprising a promoter operably linked to a coding sequence for a gene of interest (GOI). In some embodiments, the modified non-alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of the second nucleic acid sequence that encodes at least one non-structural viral protein or a portion thereof. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for GOI. In some embodiments, the nucleic acid sequence encoding the modified non-alphavirus RNA replicon further comprises a coding sequence for an operably linked auto-protease peptide downstream of the third nucleic acid sequence and upstream of the coding for GOI. In some embodiments, the nucleic acid molecule is produced by de novo synthesis.
[030] In one aspect, some modalities revealed
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24/207 herein refer to a recombinant cell that includes a nucleic acid molecule as disclosed herein. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes a modified RNA replicon, and in which expression of the modified replicon RNA confers resistance to the innate immune response in the recombinant cell. In a related aspect, some embodiments disclosed herein refer to a cell culture that includes at least one recombinant cell as disclosed herein.
[031] In some respects, some embodiments disclosed herein refer to a method for imparting resistance to the innate immune system in an individual which includes administering to the individual a nucleic acid molecule comprising a nucleic acid sequence encoding a replicon modified viral RNA, wherein the modified viral RNA replicon comprises (i) a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and (11)
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25/207 a second nucleic acid sequence that encodes at least one non-structural protein or a portion thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, and in which expression of the Modified replicon RNA encoded by the nucleic acid molecule confers resistance to the innate immune response in the individual. In some modalities, the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster and buffalo.
[032] In some aspect, some embodiments disclosed herein refer to a method for producing a polypeptide of interest in an individual which includes administering to the individual a nucleic acid molecule comprising a nucleic acid sequence that encodes an RNA replicon modified viral, wherein the modified viral RNA replicon comprises (i) a first nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to viral RNA replicon, and (ii) a second nucleic acid sequence that encodes at least one non-structural protein or a portion thereof, wherein the first nucleic acid sequence is linked
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26/207 operable upstream to the second nucleic acid sequence. In some modalities, the individual is a human being, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster or buffalo.
[033] In some aspect, some embodiments disclosed herein refer to a method for producing a polypeptide of interest, which includes cultivating a host cell that comprises a nucleic acid molecule that comprises a nucleic acid sequence that encodes a replicon of Modified viral RNA, wherein the modified viral RNA replicon comprises (i) a first nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and (ii) a second nucleic acid sequence that encodes at least one non-structural protein or a portion thereof, wherein the first nucleic acid sequence is operably linked upstream to the second acid sequence nucleic.
[034] In some embodiments of the method for producing a polypeptide of interest according to the present disclosure, the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle , swine, sheep,
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27/207 rabbit, cat, dog, bird, fish, goat, donkey, hamster and buffalo. In some embodiments, at least one of the one or more structural elements of the viral capsid enhancer comprises one or more RNA rod-loop structures. In some embodiments, the viral capsid enhancer is derived from a capsid gene of a species of virus that belongs to the Togaviridae family. In some modalities, the virus species belongs to the Alfavirus genus of the Togaviridae family. In some embodiments, the alphavirus species is eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus ( PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), virus Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), or Buggy Creek virus. In some embodiments, the viral capsid enhancer comprises a downstream loop motif (DLP) of the virus species, and the DLP motif comprises at least one of one or more stem-loop structures
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28/207 RNA. In some embodiments, the viral capsid enhancer comprises a nucleic acid sequence that exhibits at least 80% sequence identity for at least one of SEQ ID NOs: 1 and 46 to 52. In some embodiments, the nucleic acid sequence exhibits at least 95% sequence identity for at least one of SEQ ID NOs: 1 and 46 to 52.
[035] In some embodiments disclosed herein, the nucleic acid sequence encoding the modified viral RNA replicon further comprises a coding sequence for an operably linked auto-protease peptide downstream of the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the autoprotease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (EMDV) 2A (F2A), an Equine Rhinitis Virus ( ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) or a combination thereof.
In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 1 to 1,000 nucleotides downstream of the 5 'end of the modified viral RNA replicon. The second nucleic acid sequence comprises substantially all of the
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29/207 coding sequence for the viral non-structural proteins native to the corresponding unmodified viral RNA replicon.
[036] In some embodiments, the modified viral RNA replicon comprises a modified RNA replicon derived from a virus species belonging to the genus Alfavirus of the Togaviridae family or to the Arterivirus genus of the Arteriviridae family. In some embodiments, the arterivirus virus species is equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevation virus (LDV) or Simian hemorrhagic fever virus (SHFV).
[037] In some embodiments disclosed herein, the nucleic acid sequence encoding the modified artevirus RNA replicon further comprises one or more expression cassettes, and wherein at least one of the expression cassettes comprises a promoter linked in a manner operable to a coding sequence for a gene of interest (GOI). In some embodiments, the virus species is an arterivirus, and in which the first nucleic acid sequence is operably positioned upstream to a nucleic acid sequence that encodes a portion or all of the non-structural pplab protein of the RNA replicon of modified artevirus. In some embodiments, the modified artevirus RNA replicon further comprises at least two,
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30/207 three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding a portion or all of the non-structural protein pplab of the modified artevirus RNA replicon. In some embodiments, at least one of the one or more expression cassettes is operably positioned downstream to a transcriptional regulatory sequence (TRS) of the modified artevirus RNA replicon, where the TRS is TRS1, TRS2, TRS3, TRS4 , TRS5, TRS6 or TRS7. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for GOI. In some embodiments, the nucleic acid sequence encoding the modified artevirus RNA replicon further comprises a coding sequence for an operably linked auto-protease peptide downstream of the third nucleic acid sequence and upstream to the coding sequence for GOI. In some embodiments, the coding sequence for GOI encodes a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide,
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31/207 an industrial enzyme, a reporter polypeptide or any combination thereof. In some embodiments, the coding sequence for GOI encodes an antibody, antigen, immune modulator, cytokine, enzyme or any combination thereof.
[038] In some embodiments, the modified viral RNA replicon comprises a modified RNA replicon derived from a species of virus alphavirus selected from the group consisting of eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus ( VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), 0'Nyong-Nyong virus ( ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland virus J (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), alphavirus Salmonidae (SAV) and Buggy Creek virus. In some embodiments, the first nucleic acid sequence is operably positioned upstream of a nucleic acid sequence that encodes one or more nspl-4 non-structural proteins or a portion thereof
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32/207 of the modified alphavirus RNA replicon.
[039] In some embodiments, the nucleic acid sequence encoding the modified alphavirus RNA replicon further comprises one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to a coding sequence for a gene of interest (GOI). In some embodiments, the modified alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of a nucleic acid sequence encoding one or more nspl-4 non-structural proteins or a portion thereof of the alphavirus RNA replicon modified. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream of the coding sequence for GOI. In some embodiments, the modified alphavirus RNA replicon further comprises a coding sequence for an auto-protease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for GOI. In some
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33/207 modalities, the coding sequence for the GOI encodes a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide or any combination thereof. In some embodiments, the coding sequence for GOI encodes an antibody, antigen, immune modulator, cytokine, enzyme or any combination thereof.
[040] In another aspect, some embodiments disclosed herein refer to a method for imparting resistance to the innate immune system in an individual, which comprises administering to the individual a nucleic acid molecule that comprises a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence that encodes one or more structural elements of an alphavirus capsid enhancer and in which expression of the non-alphavirus RNA replicon modified by the nucleic acid molecule confers resistance to the innate immune response in the individual. In some modalities, the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster and
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34/207 buffalo.
[041] Also disclosed herein includes a method for producing a polypeptide of interest to an individual, wherein the method comprises administering to the individual a nucleic acid molecule comprising a nucleic acid sequence that encodes a non-alphavirus RNA replicon modified, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence that encodes one or more structural elements of an alphavirus capsid enhancer. In some modalities, the individual is a human being, horse, pig, primate, mouse, ferret, rat, sigmodon, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster or buffalo.
[042] Some embodiments disclosed herein refer to a method for producing a polypeptide of interest, wherein the method comprises culturing a host cell that comprises a nucleic acid molecule that comprises a nucleic acid sequence that encodes an RNA replicon non-alphavirus modified, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence that encodes one or more structural elements of an alphavirus capsid enhancer.
[043] In some modalities according to the
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35/207 aspects above the disclosure, the modified non-alphavirus RNA replicon further comprises a second nucleic acid sequence that encodes at least one non-structural viral protein or a portion thereof, wherein the first nucleic acid sequence is linked upstream operable to the second nucleic acid sequence. In some embodiments, the modified non-alphavirus RNA replicon further comprises a coding sequence for an auto-protease peptide operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments the auto-protease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (EMDV) 2A (F2A), an Equine Rhinitis Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof. In some embodiments, the modified non-alphavirus RNA replicon comprises a modified RNA replicon derived from a positive strand RNA virus. In some embodiments, the modified non-alphavirus RNA replicon comprises a modified RNA replicon derived from a virus species belonging to the Togaviridae family, Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family, or family
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36/207
Paramyxoviridae. In some embodiments, the modified non-alphavirus RNA replicon comprises a modified RNA replicon derived from a virus species that belongs to the Arterivirus genus of the Arteriviridae family. In some embodiments, the sequence encoding the modified non-alphavirus RNA replicon further comprises one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to a coding sequence for a gene of interest (GOI). In some embodiments, the modified non-alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding at least one non-structural viral protein or a portion thereof of the non-alphavirus RNA replicon modified. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of an alphavirus capsid enhancer, wherein the third nucleic acid sequence is linked operable upstream to the coding sequence for GOI. In some embodiments, the modified non-alphavirus RNA replicon further comprises a coding sequence for an auto-protease peptide
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37/207 operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for GOI.
[044] In some respects, some of the modalities disclosed herein refer to recombinant polypeptides produced by a method according to one or more of the modalities described in this document.
[045] Some embodiments disclosed herein refer to a composition that includes a recombinant polypeptide as described herein and a pharmaceutically acceptable carrier.
[046] Some embodiments disclosed herein refer to a composition that includes a nucleic acid molecule as disclosed herein and a pharmaceutically acceptable carrier.
[047] In some embodiments, one or more of the compositions and / or molecules of the present application, for example, nucleic acid molecules, RNA replicons, and polypeptides, is additionally formulated in a pharmaceutical formulation. In some embodiments, one or more of the compositions and / or molecules of the present application is formulated in a pharmaceutical formulation with covalent compounds, non-covalent compounds, physical compositions or pharmaceutically acceptable buffers.
[048] In some modalities revealed in the present
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38/207 document, one or more of the compositions and / or molecules of the present application, for example, nucleic acid molecules,
replicons in RNA, and polypeptides, is additionally formulated for use as a composition protective (for example, vaccine ) or therapeutic composition. In particular,
Protective compositions produced in accordance with the present disclosure have a variety of uses which include, but are not limited to, use as vaccines and other therapeutic agents, use as diagnostic agents and use as antigens in the production of polyclonal or monoclonal antibodies.
[049] The above summary is illustrative only and is not intended to be limiting in any way. In addition to the modalities and illustrative features described in this document, aspects, modalities, objectives and additional features of the order will become fully apparent from the drawings, detailed description and claims.
Brief description of the drawings [050] Figure 1 is a graphic illustration of an exemplary non-limiting RNA stem-loop structure of an alphavirus capsid enhancer.
[051] Figures 2A to 2D are graphic representations of four exemplary non-limiting nucleic acid molecules of the present disclosure, wherein each of the nucleic acid molecules comprises a sequence of
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39/207 coding for an alphavirus capsid enhancer (e.g., DLP motif) and a coding sequence for a gene of interest (GOI), for example, a red Firefly reporter gene (rFF). Figure 2A: rEx-DLP-rFF; Figure 2B: rEx-DLP-pplab-rFF; Figure 2C: rEx-DLP-2A-pplabrEE and Figure 2D: rEx-DLP-2A-pplab-DLP-rFF. DLP: Downstream Loop Sequence; 2A: auto-protease peptide; pplab: non-structural polypeptide sequence; and rFF: coding sequence for the Firefly red reporter gene.
[052] Figures 3A to 3D are graphic illustrations of four exemplary non-limiting nucleic acid molecules of the present disclosure, wherein each of the nucleic acid molecules comprises a coding sequence for an alphavirus capsid enhancer (for example, a DLP motif) and a coding sequence for a gene of interest (GOI), for example, a red Firefly reporter gene (rFF). Figure 3A: Alpha-R-rFF; Figure 3B: Alpha-R-DLP-rFF; Figure 3C: Alpha-R-DLP-2A-nsp-rFF and 3D Figure: Alpha-R-DLP-2A-nsp-DLP-rFF. DLP: Downstream Loop Sequence; 2A: auto-protease peptide; nspl-4: non-structural polypeptide sequence; and rFF: coding sequence for the Firefly red reporter gene.
[053] Figures 4A to 4B are graphic illustrations of two other exemplary non-limiting nucleic acid molecules of the present disclosure, each of which
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40/207 nucleic acid molecules comprise a coding sequence for an alphavirus capsid enhancer (for example, a DLP motif) and a coding sequence for a gene of interest (GOI), for example, a red Firefly reporter gene ( rFF). Figure4A: Alpha-R-DLP2A-rFF and Figure 4B: Alpha-R-DLP-2A-nsp-DLP-2A-rFF. DLP: Downstream Loop Sequence; 2A: auto-protease peptide; nspl-4: non-structural polypeptide sequence; and rFF: coding sequence for the Firefly red reporter gene.
[054] Figures 5A to 5B graphically summarize the results of flow cytometry analysis and essential luciferase analyzes performed to demonstrate that incorporating a DLP motif upstream of a nucleic acid sequence encoding non-structural EAV protein genes or a gene of interest positioned in the subgenomic RNA, that is, rFF reporter gene, did not negatively impact genomic RNA replication. In these experiments, FACS analysis (Figure 5A) and essential cell luciferase assays (Figure 5B) were performed on electroporated cells.
[055] Figures 6A to B graphically summarize the results of another exemplary flow cytometry analysis and essential luciferase analysis performed to demonstrate that modified arterivirus replication RNAs with a DLP motif embedded upstream from the sequence that
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41/207 encodes non-structural protein genes can replicate and express themselves efficiently in host cells that have been treated with IFN to induce the innate cellular immune system. In these experiments, FACS analysis (Figure 6A) and essential cell luciferase assays (Figure 6B) were performed on electroporated cells. IFN was added to cell culture media five hours post-electroporation. Samples were collected in triplicate eighteen hours after electroporation for analysis.
[056] Figures 7A to C graphically summarize the results of another exemplary essential luciferase analysis performed to demonstrate that alphavirus replication RNAs modified with a DLP motif embedded upstream of the sequence encoding non-structural protein genes can replicate and express itself efficiently in host cells that have been treated with IFN to induce the innate cellular immune system. In these experiments, essential cell luciferase assays were performed on electroporated cells. IFN was added to cell culture media immediately after electroporation or three hours after electroporation. Samples were collected in triplicate eighteen hours after electroporation for analysis. Figure 7A: α-rFF versus alpha-R-rFF construct; Figure 7B: α-rFF versus a-DLP-2A-nsprFF and Figure 7C: α-rFF versus alpha-R-DLP-2A-nsp construct
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42/207 rFF.
[057] Figure 8 graphically summarizes the results of exemplary in vivo experiments performed to demonstrate that alphavirus replication RNAs modified with a DLR motif embedded upstream of the sequence encoding non-structural protein genes can replicate and express themselves efficient in Balb / c mice. In these experiments, full-body imaging of animals that were injected with a modified alphavirus replication RNA was conducted. Each animal received 7.5 pg of replicon RNA injected intramuscularly. Individual animals were imaged on day 1, day 3 and day 7. Original: mice injected with the alpha-R-rFF construct; DLP: mice injected with the alpha-R-DLP-2Ansp-rFF construct.
[058] Figure 9 schematically depicts an exemplary genomic structure and expression of non-limiting alphavirus genome (adapted from Strauss et al., Microbiological Reviews, pages 491 to 562, September 1994). Genome organization of a Sindbis virus (SINV) is shown. The names of non-structural genes and structural protein genes are given. References to the nomenclature of genes and proteins can be found in Strauss et al. , supra, 1994. The 49S genomic RNA is illustrated schematically in the center, with its translated ORF shown
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43/207 as an open box. Small black boxes are conserved sequence elements; the open diamond denotes the ebbing opal termination codon. Non-structural polyproteins and their processed products are shown above. The termination at the opal codon produces P123, whose main function in replication is believed to be as a proteinase that acts in trans to process polyproteins in active RNA replicases; this proteinase domain is found in the nsP2 region. Reading through the opal terminating codon produces P1234, which can form an active replicase. The 26S subgenomic mRNA is expanded below to show the structural ORF and its translation products. Polypeptides present in virion are shaded. VcRNA is the negative strand complement of genomic RNA.
[059] Figure 10 schematically depicts genomic structure and EAV genome expression strategy. The names of the replicase gene and structural protein genes are given (references to the nomenclature of genes and proteins can be found in Snijder et al., 2005). Below the genome organization, the genome structure relationships and sg mRNAs are depicted. The sequence and leading TRSs found at the 5 'end of the EAV mRNAs are indicated as blue and orange boxes, respectively. The ribosomal frame displacement element (RFS) found in the length of the mRNAl genome is indicated and the
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44/207 translated region of each mRNA is highlighted by a green line, while translationally silent regions are indicated by a red line. Only translated open reading frames are indicated for each mRNA. The panels on the right show a typical pattern of EAV mRNAs isolated from infected cells, visualized by hybridization to a probe complementary to the 3 'end of the genome and, therefore, recognizing all viral mRNA species.
[060] Figures 11A to B schematically show the predicted RNA rod-loop structure of the 5 'CDS region of 26S alpavirus mRNA with a peak and valley topology. Two-dimensional (2D) models of RNA structure as the basis for the first 70 to 140 nucleotides of the CDS from seven representative Alfavirus mRNAs (SINV, SFV, RRV, SAGV, GETV, MIDV, UNAV, BEBV, MAYV and AURAV). The sequences were numbered from the initiation codon (AUGi), where A is the +1 position. The predicted structures are built based on SHAPE data (selective 2'-hydroxyl initiator acylation and extension) (Toribio et al., 2016).
[061] Figures 12A to C graphically summarize the results of exemplary in vivo experiments performed to demonstrate that alphavirus replicon RNAs modified with a DLP motif affect immunogenicity in Balb / c mice. In this experiment, 6 to 8 week old BALB / c animals were prepared
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45/207 on Days 0 and 42 with the use of varying doses of replicon RNA. Spleens and serum were collected on Day 56, and (a) flow cytometry for specific HA T cell memory (CD8 + CD44 + CD62L ^Lo KLRG-l ^Lo IL-7Ra ^Hi CXCR3 ^Hi ) using Dextramers for detection ( H-2 Kd [IYSTVASSL; SEQ ID NO: 44]) and (b, c) ΙΕΝ-γ ELISpot to quantify CD8 + and CD4 + T cell effector responses. Statistics were made using multiple comparisons between matched doses using a common one-way analysis of variance (ANOVA). Figural2A: A significant increase in memory precursor effector cells (MPEGs) was observed in constructs containing the DLP motif compared to each comparable dose of unmodified replicon. Figural2B: Effector T cell responses were measured by the number of specific antigen HA cells that were secreting IFN-γ following stimulation with a CD8 + T cell peptide. Figure 12C: Effector T cell responses were measured by the number of specific antigen HA cells that were secreting ΙΕΝ-γ following stimulation with a CD4 + T cell peptide.
[062] Figure 13 graphically summarizes the results of exemplary in vivo experiments performed to demonstrate that alphavirus replication RNAs modified with a DLP motif embedded upstream of the
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46/207 sequence encoding non-structural protein genes effectively prevented the suppression of immune response through pretreatment with agents that simulate viral infection in Balb / c mice. BALB / c animals 6 to 8 weeks of age were pre-treated with 20 pg of Poly (I: C) or saline administered by hydrodynamic injection into the caudal vein 24 hours before vaccination to simulate an ongoing viral infection. The mice were then prepared on Day 0 and boosted on Day 28 using a 1.5 pg dose of RNA replicon encoding HA. Serum was collected on Day 42, and specific HA antibodies were measured in the serum. Serum antibody concentrations were calculated by dilution versus optical density interpolation in a quadriparametric logistic regression and using the specific HA monoclonal antibody 8D2 as a standard. Statistics between individual groups were conducted using a Mann-Whitney test (non-parametric).
[063] Figures 14A to 14C graphically summarize the results of in vivo experiments performed to demonstrate that the replicons containing DLP, according to the present disclosure, are compatible with LNP (cationic lipid nanoparticles) formulations. In this experiment, BALB / c animals 6 to 8 weeks old were prepared on Days 0 and reinforced on Day 28 with the use of varying doses
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47/207 of an RNA replicon encoding HA. Spleens and serum were collected on Day 42. Figure 14A: Specific HA antibodies were measured in the serum. Serum antibody titer is the inverse of EC 20% and was calculated by dilution versus optical density interpellation in a quadriparametric logistic regression. Figure 14Β: ΙΕΝ-γ ELISpot used to quantify CD8 + cell effector responses. For detection of antigen-specific CD8 + T cells, splenocytes were incubated with the Kd H-2 peptide (IYSTVASSL; SEQ ID NO: 44). Figure 14Ο: ΙΕΝ-γ ELISpot used to quantify CD4 + T cell effector responses. For detection of antigen-specific CD4 + T cells, splenocytes were incubated with CD4 T cell restricted with H2-D epitope KSSFFRNWWLIKKN (SEQ ID
NO: 45). Statistics in between groups individual were conducted with the use of one test of Mann-Whitney (no parametric ). [064] The figure 15 illustrates graphically an
exemplary non-limiting configuration of DLP-containing mRNA, in which a DLP element from Sindbis virus is placed upstream of a coding sequence for a gene of interest (GOI; dsGFP), and a 5 'UTR sequence is placed immediately at downstream of a T7 promoter and upstream of the DLP sequence of Sindbis virus. The coding sequence for dsGFP is linked to the DLP element by means of a P2A signal, which is a self-cleaving peptide
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48/207 autocatalytic (e.g., auto-protease peptide) derived from porcine teschovirus-1. Also shown in the lower portion of the Figure is another exemplary non-limiting configuration of DLP-containing mRNA, in which a coding sequence for a destabilized form of the EGFP reporter gene (dsGFP) used as a GOI is operably linked to the PEST degradation signal. proteolytic derived from a mouse ornithine decarboxylase (MODC) gene.
[065] Figures 16A to D graphically summarize the results of experiments carried out to demonstrate that modified mRNAs containing DLP can confer resistance to interferon. Figure 16A: inclusion of DLP in mRNA results in a statistically significant increase in the frequency of positive GFP cells in the presence of IFN. Average with 95% confidence intervals in Kruskai-Wallist test (nonparametric). Figure 16B: unmodified mRNA is sensitive to IFN treatment (mean with 95% confidence intervals in 2-way ANOVA. Interaction: p = 0.0083. Row: p = <0.0001. Column: p = 0, 0273. Sidak's multiple comparison test with * p = 0.0217 and # p = <0.0241). Figure 16C: DLP-modified mRNA produces a statistically significant 30% increase in protein production per cell compared to unmodified mRNA in the presence of IFN (mean with 95% confidence intervals in 2-way ANOVA: p = <0, 0001. Sidak's multiple comparison test with ***
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49/207 p = <0.0002 and **** p = <0.0001). Figure 16D: DLR-modified mRNA in the presence of IFN produces an equivalent amount of protein compared to unmodified mRNA in the absence of IFN treatment (mean with 95% confidence intervals in 2-way ANOVA. Interaction: p = <0, 0001. Row: p = <0.0001 Column: p = 0.0023 Sidak multiple comparison test with **** p = <0.0001 and ** p = <0.0023).
[066] The above and other resources of the present disclosure will become more fully apparent from the following description and attached claims, taken in conjunction with the attached drawings. It is understood that these drawings portray only some modalities according to the disclosure and should not be considered as limiting its scope; the disclosure will be described with specificity and additional details using the accompanying drawings.
Detailed description of the disclosure [067] The present disclosure, in general, relates to compositions and methods for use in regulating gene expression in cells. Some embodiments of the disclosure refer to expression systems, such as viral based expression systems, with superior expression potential which are suitable for expressing heterologous molecules, such as, for example, vaccines and therapeutic polypeptides, in recombinant cells. For example, some modalities of the disclosure refer to nucleic acid molecules containing
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50/207 one or more structural elements of a viral capsid enhancer or a variant thereof. In some embodiments, at least one of the one or more structural elements comprises an RNA rod-loop. In some embodiments, at least one of the one or more structural elements is operably linked to a coding sequence for a gene of interest. Some embodiments of the disclosure refer to nucleic acid molecules such as constructs and transcription and / or expression vectors, containing a nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer. Transcription and expression vectors, such as viral-based vectors, which comprise a coding sequence for a gene of interest are also disclosed in some embodiments. In some embodiments, the nucleic acid molecules of the present disclosure, for example, messenger (mRNA) and RNA replicon, are generated by means of de novo synthesis and / or in vitro transcription. Recombinant cells that are genetically modified to include one or more of the nucleic acid molecules disclosed herein, as well as biomaterials and recombinant products derived from such cells are also within the scope of the application. Compositions and kits that include one or more of the nucleic acid molecules and / or
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51/207 recombinant cells disclosed herein, as well as methods for conferring resistance to the innate immune system in a host cell.
[068] In the following detailed description, reference is made to the accompanying drawings, which form a part of this document. In the drawings, similar symbols typically identify similar components, unless the context dictates otherwise. The illustrative alternatives described in the detailed description, drawings and claims are not intended to be limiting. Other alternatives can be used, and other changes can be produced, without departing from the spirit or scope of the matter presented here. It will be readily understood that aspects, as generally described in this document, and illustrated in the Figures, can be arranged, replaced, combined and designed in a wide variety of different configurations, all of which are explicitly contemplated and are part of the present request.
[069] Unless otherwise defined, all terms of the technique, notations and other scientific terms or terminology used in this document are intended to have the meanings commonly understood by persons versed in the technique to which this application belongs. In some cases, terms with commonly understood meanings are defined in this document for clarity and / or prompt
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52/207 reference, and the inclusion of such definitions in this document should not necessarily be interpreted as representing a substantial difference in what is, in general, understood in the art. Many of the techniques and procedures described or referenced in this document are well understood and commonly used with the use of conventional methodology by people skilled in the art.
Some definitions [070] The singular forms one, one, o and a include plural references unless the context clearly determines otherwise. For example, the term a cell includes one or more cells, which comprise mixtures of them.
[071] The term about, as used herein, has its common meaning of approximately. If the degree of approximation is not clear otherwise from the context, it means within about 10% of the value provided, or rounded up to the nearest significant figure, in all cases including the value provided. Where ranges are provided, they are inclusive of limit values.
[072] The terms, cells, cell cultures, cell line, recombinant host cells, recipient cells and host cells as used in
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53/207 present document, include the primary individual cells and any progeny of them, without considering the number of transfers. In some situations, a progeny is not exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in the environment); however, such an altered progeny is included in these terms, provided that the progeny retains functionality that is the same or substantially similar to that of the originally transformed cell.
[073] As used herein, the term construct is intended to mean any recombinant nucleic acid molecule such as an expression cassette, plasmid, cosmid, phosphid, viral replicon, carrier vector, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular, single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule in which nucleic acid sequences are connected in a functionally operational manner, for example, operably connected.
[074] The term derived from used in this document refers to an origin or source, and may include unpurified or purified, recombinant, naturally occurring molecules. The molecules of the present disclosure
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54/207 can be derived from viral or non-viral molecules. A protein or polypeptide derived from an original protein or polypeptide can include the original protein or polypeptide, in part or in whole, and can be a fragment or variant of the original protein or polypeptide.
[075] The term gene is used widely to refer to any segment of a nucleic acid molecule that encodes a protein or that can be transcribed into a functional RNA. Genes can include sequences that are transcribed, but are not part of a final, mature and / or functional RNA transcription, and genes that encode proteins can further comprise sequences that are transcribed, but not translated, for example, 5 'untranslated regions , 3 'untranslated regions, introns, etc. In addition, genes can optionally further comprise regulatory sequences required for their expression, and such sequences can be, for example, sequences that are not transcribed or translated. Genes can be obtained from a variety of sources, which include cloning from a source of interest or synthesizing from known or predicted sequence information, and can include sequences designed to have desired parameters.
[076] The native term is used in this document to refer to nucleic acid sequences or amino acid sequences as they occur naturally in the
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55/207 host. The term non-native is used herein to refer to nucleic acid sequences or amino acid sequences that do not occur naturally in the host, or are not configured as they are naturally configured in the host. A nucleic acid sequence or sequence of amino acids that has been removed from a host cell, subjected to laboratory manipulation, and introduced or reintroduced into a host cell is considered to be non-native. Synthetic genes or partially synthetic genes introduced into a host cell or organism are non-native. Non-native genes further include genes endogenous to the host cell operably linked to one or more heterologous regulatory sequences that have been recombined in the host genome, or genes endogenous to the host cell or organism that are at a different locus in the genome than the same genome. occur naturally.
[077] The terms naturally occurring and wild type, as used in this document, refer to a form found in nature. For example, a naturally occurring or wild-type nucleic acid molecule, nucleic acid sequence or protein can be present in and isolated from a natural source, and is not intentionally modified by human manipulation. As described in detail below, the nucleic acid molecules according to
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56/207 with some embodiments of the present disclosure are naturally occurring nucleic acid molecules.
[078] The term heterologous when used in reference to a polynucleotide, gene or nucleic acid molecule refers to a polynucleotide, gene or nucleic acid molecule that is not derived from the host species. For example, heterologous gene or heterologous nucleic acid sequence as used herein, refers to a gene or nucleic acid sequence of a species other than the species of the host organism into which it is introduced. When referring to a gene regulatory sequence such as, for example, an enhancer sequence, or an auxiliary nucleic acid sequence used to manipulate the expression of a gene sequence (for example, a 5 'untranslated region, non-translated region translated 3 ', poly A addition sequence, etc.) or to a nucleic acid sequence encoding a protein domain or protein localization sequence, heterologous means that the regulatory or helper sequence or sequence encoding a domain or sequence protein localization is from a source other than the gene with which the regulatory or auxiliary nucleic acid sequence or nucleic acid sequence encoding a protein localization domain or sequence is juxtaposed in a genome. Thus, a promoter operably linked to a gene to which
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57/207 it is not operably linked in its natural state (for example, in the genome of a non-genetically modified organism) is referred to herein as a heterologous promoter, even if the promoter may be derived from the same species (or , in some cases, the same organism) as the gene to which it is linked. For example, in some embodiments disclosed herein, a coding sequence for a heterologous gene of interest (GOI) is not linked to the recombinant RNA replication sequence in its natural state. In some embodiments, the coding GOI sequence is derived from another organism, such as another virus, bacteria, fungi, human cell (tumor Ag), parasite (malaria), etc.
[079] The terms nucleic acid molecule and polynucleotide are used interchangeably in this document, and refer to both RNA and DNA molecules, which include nucleic acid molecules that comprise cDNA, genomic DNA, synthetic DNA and molecules of DNA or RNA containing nucleic acid analogs. Nucleic acid molecules can have any three-dimensional structure. A nucleic acid molecule can be double-stranded or single-stranded (for example, a sense strand or an antisense strand). Non-limiting examples of nucleic acid molecules include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, RNA
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58/207 ribosomal, siRNA, micro RNA, tracrRNAs, crRNAs, guide RNAs, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers. A nucleic acid molecule can contain unconventional or modified nucleotides. The terms polynucleotide sequence and nucleic acid sequence as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set out in title 37, paragraph 1.822, of the CFR is used in this document. The nucleic acid molecules of the present disclosure can be synthesized ex vitro by any means known in the art, for example, with the use of one or more chemical or enzymatic techniques (for example, with the use of chemical nucleic acid synthesis, or with the use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (which includes, for example, methylation), or recombination (which includes homologous and specific site recombination) of molecules In some embodiments, the nucleic acid molecules of the present disclosure are generated from de-synthesis again. In some embodiments, nucleic acid molecules can be synthesized again in whole or in part, using known chemical methods , enzymatic techniques
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59/207 known, or any combination thereof. For example, the component nucleic acid sequences can be synthesized by solid phase techniques, removed from the resin, and purified by preparative high performance liquid chromatography followed by chemical and / or enzymatic bonding to form a chimeric nucleic acid molecule. The composition of the synthetic nucleic acid molecules can be confirmed by nucleic acid analysis or sequencing. In some embodiments, the nucleic acid molecules of the present disclosure can be assembled enzymatically from chemically synthesized oligonucleotides using techniques known in the art.
[080] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, for example, between about 0.5 Kb and about 1,000 Kb, between about 0.5 Kb and about 500 Kb, between about 1 Kb to about 100 Kb, between about 2 Kb to about 50 Kb, or between about 5 Kb to about 20 Kb. In some embodiments, the nucleic acid molecule is, or is, about
in, 0.5 Kb, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb , 10 Kb, 15 Kb, 20 Kb, 25 Kb, 30 Kb, 4 0 Kb , 50 Kb , 100 Kb, 200 Kb, 500 Kb , 1 Mb, or more , or an banner in between
any two of those values.
[081] The polynucleotides of the present disclosure can be biologically active in relation to any one
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60/207 structural attribute, such as the ability of a nucleic acid to hybridize to another nucleic acid, or the ability of a polynucleotide sequence to be recognized and linked by one or more of a transcription factor, a ribosome and an acid polymerase nucleic.
[082] The term recombinant or modified nucleic acid molecule as used herein, refers to a nucleic acid molecule that has been altered through human intervention. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by polymerase reaction (or reactions) ex vitro, or to which ligands have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As non-limiting examples, a recombinant nucleic acid molecule: 1) was synthesized or modified ex vitro, for example, using chemical or enzymatic techniques (for example, using chemical nucleic acid synthesis, or using enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (which includes, for example, methylation), or recombination (which includes homologous and site specific recombination) of acid molecules 2) includes conjugated nucleotide sequences
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61/207 that are not conjugated in nature, 3) has been modified with the use of molecular cloning techniques so that it lacks one or more nucleotides in relation to the sequence of naturally occurring nucleic acid molecules, and / or 4 ) was manipulated using molecular cloning techniques so that it has one or more sequence changes or rearrangements in relation to the naturally occurring nucleic acid sequence. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by polymerase reaction (or reactions) ex vitro, or to which ligands have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. In some embodiments disclosed herein, the recombinant nucleic acid molecules of the present application are generated from de novo synthesis.
[083] The term protein variant used in this document refers to a polypeptide that has an amino acid sequence that is the same or essentially the same as that of the reference protein except that it has at least one modified amino acid, for example, deleted, inserted or replaced, respectively. The amino acid substitution can be a conservative amino acid substitution, preferably in a non-essential amino acid residue in the protein. An amino acid substitution
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A conservative 62/207 is one in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched beta side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A protein variant may have an amino acid sequence of at least about 80%, 90%, 95% or 99%, preferably at least about 90%, more preferably, at least about 95%, identical to the sequence of protein amino acids. Preferably, a variant is a functional variant of a protein that retains the same function as the protein. The terms variant, when used in reference to a nucleic acid sequence, refer to a nucleic acid sequence that differs by one or more nucleotides from another, nucleotide sequence usually
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63/207 related. As such, the term variant may refer to a change of one or more nucleotides from a reference nucleic acid that includes the insertion of one or more new nucleotides, deletion of one or more nucleotides and replacement of one or more existing nucleotides. A variation is a difference between two different nucleotide sequences; typically, a sequence is a reference sequence. Broadly, the term nucleotide variation as used in this document includes point mutation, multiple mutation, single nucleotide polymorphism (SNP), deletion, insertion and translocation. The term reference nucleic acid is used herein to describe a nucleotide sequence that has a known reference sequence of interest.
[084] As used herein, the terms, identical or percentage identity, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of residues of amino acids or nucleotides that are the same, when compared and aligned to the maximum match over a comparison window. Unless otherwise specified, the comparison window for a selected string, for example, SEQ ID NO: X is the entire length of SEQ ID NO: X, and, for example, the comparison window for 100
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64/207 bp of SEQ ID NO: X is the indicated 100 bp. The degree of amino acid or nucleic acid sequence identity can be determined by various computer programs to align the sequences to be compared based on designated program parameters. For example, sequences can be aligned and compared to using the Smith & Waterman Adv. Appl. Math.2: 482 to 89, 1981, the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol.48: 443 to 53, 1970, or the search for similarity method of Pearson & Lipman Proc. Nat'l. Acad. Know. USA 85: 2444 to 48, 1988, and can be aligned and compared based on visual inspection or can use computer programs for analysis (for example, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
[085] In addition to calculating percentage sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'1. Acad. Sei.USA 90: 5873 to 87 , 1993). The lowest probability of sum (P (N)), provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the least probability of a sum
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in a comparison of the acid nucleic of test to acid reference nucleus is smaller than what about in 0, 1, preferably, smaller than what about 0.01, and more preferably, less than about 0.001. [086] How used in gift document, O term
vector refers to a recombinant polynucleotide construct designed to transfer to a host cell, or between host cells, and which can be used for the purpose of transformation, for example, the introduction of heterologous DNA into a host cell. A vector can be, for example, a replicon, such as a plasmid, bacteriophage, or cosmid, into which another segment of DNA can be inserted in order to cause replication of the inserted segment. In general, a vector is capable of replication when associated with the appropriate control elements. The term vector includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. An expression vector is a vector that includes a regulatory region, thus, capable of expressing DNA sequences and fragments, for example, ex vitro, ex vivo and in vivo. In some embodiments, the vector is a plasmid, a bacteriophage vector, a cosmid, a phosphid, a viral replicon or a combination thereof. In some embodiments, the vector is a eukaryotic vector, a prokaryotic vector (for example, a bacterial plasmid) or a carrier vector.
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An expression system can be, for example, an expression vector or an expression cassette. In some embodiments, the vector is a transcription vector. The term transcription vector refers to a vector capable of being transcribed, but not translated. For example, transcription vectors can be used to amplify your insert.
[087] Virus-based replicon expression vectors can be used, for example, vaccines and therapeutic compositions. Replication vectors can be used in a variety of formats, including DNA, RNA and recombinant viral particles. A wide body of literature has now demonstrated the effectiveness of viral replicon vectors for applications such as vaccines. In addition, these terms can be collectively referred to as vectors, vector constructs or gene delivery vectors.
[088] As will be understood by a person of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all of the tracks disclosed in this document also cover any and all of the sub-bands and sub-band combinations possible of them. Any range listed can easily be recognized as describing and sufficiently allowing the same range to be broken down into at least halves, thirds, quarters, fifths, equal tenths, etc. As a non-limiting example, each track
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67/207 discussed in this document can be readily broken down into a lower third, central third and upper third, etc. As will also be understood by a person skilled in the art all languages such as up to, at least, greater than, less than, and the like include the number enumerated and refer to tracks that can be further decomposed into sub-tracks as discussed above. Finally, as will be understood by an expert on the subject, a banner comprises each individual member. Thus, for example, a group that has 1 to 3 articles refers to groups that have 1, 2 or 3 articles. Similarly, a group that has 1 to 5 articles refers to groups that have 1, 2, 3, 4 or 5 articles and so on.
Viral capsid enhancers [089] Some viruses have sequences capable of forming one or more stem-loop structures that regulate, for example, increase, capsid gene expression. The term viral capsid enhancer is used in this document to refer to a regulatory element that comprises sequences capable of forming such stem-loop structures. In some examples, stem-loop structures are formed by sequences within the coding sequence of a capsid protein and called the Downstream Loop (DLP) sequence. As disclosed in this document, these rod-loop structures or variants thereof
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68/207 be used to regulate, for example, increased level of expression of genes of interest. For example, these stem-loop structures or variants thereof can be used in a recombinant vector (for example, in a heterologous viral genome) to enhance transcription and / or translation of the operably linked coding sequence downstream. As an example, members of the Alfavirus genus can resist the activation of antiviral RNA-activated protein kinase (PKR) through a prominent RNA structure present within 26S viral transcripts, which allows an initiation of eIF2-independent translation of these mRNAs. This structure, called the downstream loop (DLP), is located downstream of the AUG in 26S SINV mRNA and other members of the genus Alfavirus. In the case of Sindbis virus, the DLP motif is found in the first ~ 150 nt of Sindbis subgenomic RNA. The hairpin is located downstream of the Sindbis capsid AUG initiation codon (AUG is collected in nt 50 of the Sindbis subgenomic RNA). Previous studies of sequence comparisons and structural RNA analysis revealed the evolutionary conservation of DLP in SINV and predicted the existence of DLP-equivalent structures in many members of the Alfavirus genus (see, for example, Ventoso, J. Virol. 9484 to 9494, Vol. 86, September 2012).
[090] PKR phosphorylates the initiation factor of
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69/207 eukaryotic translation 2α (eIF2 α). Phosphorylation of eIF2 α blocks initiation of mRNA translation and in doing so prevents viruses from completing a productive replication cycle. PKR is activated by interferon and double-stranded RNA. Alphavirus replication in host cells is known to induce double-stranded RNA-dependent protein kinase (PKR). For example, Sindbis virus infection of cells induces PKR which results in phosphorylation of eIF2 α yet the viral subgenomic mRNA is efficiently translated while the translation of all other cellular mRNAs is restricted. The Sindbis virus subgenomic mRNA has a stable RNA clamp-shaped loop located downstream of the wild-type AUG primer codon for the virus capsid protein (e.g., capsid enhancer). This clamp-shaped loop, also called the RNA rod-loop structure, is often referred to as the Downstream Loop structure (or DLP motif). It has been reported that the DLP structure can stop a ribosome in the wild type AUG and this supports translation of the subgenomic mRNA without the requirement for functional eIF2 α. Thus, subgenomic mRNAs from Sindbis virus (SINV) as well as from other alphaviruses are efficiently translated even in cells that have highly active PKR that results in complete phosphorylation of eIF2a.
Structure of Alfavirus DLPs
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70/207 [091] The structure of DLP was first characterized in Sindbis virus 26S mRNA (SINV) and also detected in Semliki Forest virus (SFV). DLP-like structures have been reported to be present in at least 14 other members of the Alfavirus genus that include members from the New World (for example, MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and the Old World (SV , SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV and ONNV). The predicted structures of these Alfavirus 26s mRNAs were constructed based on SHAPE data (selective 2'-hydroxyl primer extension and acylation) (Toribio et al., Nucleic Acids Res. May 19; 44 (9): 4368 to 80 , 2016), the content of which is incorporated into this document as a reference). Stable rod-loop structures were detected in all cases except for CHIKV and ONNV, while MAYV and EEEV showed lower stability DLPs (see Figures 11A to B and Toribio et al., 2016 supra). Higher DLP activities were reported for those Alfaviruses that contained the most stable DLP structures. In some cases, DLP activity depends on the distance between the DLP motif and the AUG initiation codon (AUGi). The AUG-DLP spacing in Alfavirus 26s mRNAs is adjusted to the topology of the ES6S region of the ribosomal 18S rRNA in a way that allows the positioning of the AUGi in the P site of the 40S subunit stopped by the DLP, which allows the incorporation of Met-tRNA without the participation of
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71/207 eIF2. Two main topologies were detected: a compact and stable structure in the SFV clade, and a more extended structure in the SINV group. In both cases, it was observed that DLP structures were preceded by a region of intense SHAPE reactivity, which suggests a single filament conformation for the AUG-DLP elongation. Consequently, this region showed a high content of and a low content of G which resulted in a low propensity to form secondary structures when compared to positions in equivalents in all mouse mRNA transcriptomes or in those alphavirus mRNAs lacking DLPs. These results reported by Toribio et al. (2016, supra) suggest that the occurrence of DLPs in Alfavirus is probably linked to a flattening of the preceding region, which results in a peak topology and is valid for that mRNA region.
[092] In the case of Sindbis virus, the DLP motif is found in the first ~ 150 nt of Sindbis subgenomic RNA. The clamp form is located downstream of the Sindbis capsid AUG initiation codon (AUG at nt 50 of the Sindbis subgenomic RNA) and results in stopping a ribosome so that the correct capsid gene AUG is used to initiate translation . This is due to the clamp shape causing ribsomes to pause eIF2a are not required to support translation initiation. Without being bound by any particular theory, it is believed that placing the
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72/207 DLP motif upstream of a coding sequence for any GOI typically results in an N-terminal capsid amino acid fusion protein that is encoded in the clip-shaped region for the GOI encoded protein due to initiation occurring in the Capsid AUG not in GOI AUG. In some embodiments disclosed herein, a porcine teschovirus-1 2A (P2A) peptide sequence was modified framed immediately after the DLP sequence and framed immediately upstream of all GOI. The incorporation of the P2A peptide in the modified viral RNA replicons of the present disclosure allows the release of an almost pure GOI protein from the capsid-GOI fusion; a single proline residue is added to all GOI proteins.
[093] Without being bound by any particular theory, DLP is believed to allow translation to occur in a manner independent of eIF2a, nucleic acid molecules and expression vectors (eg, RNA replicon vectors) modified to use the same to initiate translation of non-structural proteins has increased functionality in cells that are activated by the innate immune system. Therefore, it is contemplated that DLP-modified nucleic acid molecules and expression vectors (for example, RNA replication vectors) also work more uniformly in cells, individuals or populations
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73/207 of different individuals due to differences in the level of innate immune activation in each will naturally cause variability. In some embodiments, DLP can help to remove that variability due to the translation and replication of RNA replication vectors (as well as GOI expression) may be less impacted by pre-existing innate immune responses. One of the significant values of the compositions and methods disclosed in this document is that vaccine effectiveness can be increased in individuals who are in a chronic or acute state of immune activation. Causes of chronic or acute immune activation could be found in individuals suffering from a subclinical or clinical infection or individuals undergoing medical treatment for cancer or other illnesses (e.g., diabetes, malnutrition, high blood pressure, heart disease, Crohn's disease, muscular sclerosis, etc.).
[094] As described in this document, DLP-containing nucleic acid molecules (for example, transcription and expression vectors (for example, viral RNA replicons)) disclosed herein may be useful for conferring resistance to the innate immune system in an individual. Unmodified RNA replicates are sensitive to the state of the initial innate immune system of cells into which they are introduced. If the cells / individuals are in an immune system state
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74/207 highly active innate, RNA replication performance (eg, replication and expression of a GOI) can be negatively impacted. By modifying a DLP to control the initiation of protein translation, particularly non-structural proteins, the impact of the activation state of the pre-existing innate immune system to influence efficient RNA replication replication is removed or reduced. The result is more uniform and / or improved expression of a GOI that can impact vaccine effectiveness or therapeutic impact of a treatment.
Arterivirus [095] Arteriviruses (Family Arteriviridae, Genus Arterivirus) comprise an important group of positive-sense, single-stranded RNA viruses that infect domestic and wild animals. Arteriviruses share a genome organization and replication strategy similar to that of members of the Coronaviridae family (genera Coronavirus and Torovirus), but differ considerably in their genetic complexity, genome length, biophysical properties, size, architecture and structural protein composition of viral particles (e.g., virion). Currently, the Arterivirus genus is considered to include equine arteritis virus (VAS), porcine reproductive and respiratory syndrome virus (PRRSV), mouse lactate dehydrogenase elevation virus (LDV),
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75/207 Simian hemorrhagic fever virus (SHFV) and faltering skunk disease virus (WPDV).
[096] A typical arterivirus genome varies between 12.7 and 15.7 kb in length, but its genome organization is relatively consistent with some minor variations. The exemplary virion genome and architecture organization of an arterivirus is shown in Figure 10. The arterivirus genome is a polycistronic + RNA, with 5 'and 3' untranslated regions (NTRs) that border a matrix of 10 to 15 known ORFs . The large replicase ORFs la and 1b occupy the three proximal quarters of 5 of the genome, the size of ORFla being much more variable than that of ORFlb. ORFla translation produces polyprotein replicase (pp) la, while ORFlb is expressed by programmed ribosomal frame shift —1 (PRF), which extends C terminally from ppla to pplab. In addition, a short transquadro ORF has been reported to overlap the ORFla nsp2 coding region in frame +1 and is expressed by PRF —2. The 3 'proximal part of the genome has a compact organization and contains 8 to 12 relatively small genes, most of which overlap with surrounding genes. These ORFs encode structural proteins and are expressed from a nested set of coterminals in 3 'sg mRNAs. The organization of these ORFs is preserved, but downstream of ORFlb, SHFV and all
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76/207 Recently identified SHFV-like viruses contain three or four additional ORFs (~ 1.6 kb) that can be derived from an old duplication of ORFs 2 through 4. Along with the size variation in ORFla, this assumed duplication explains the genome size differences between arterivirus.
[097] Regarding equine arteritis virus (EAV), the wild type EAV genome is approximately 12.7 Kb in size. The three 5 'quarters of the genome code for two large replicase proteins la and lab; the amino acid sequences of the two proteins are N-terminally identical, but due to a shift in the ribosomal picture, the amino acid sequence of the C terminal region of lab is unique. The 3 'quarter of the EAV genome codes for the virus's structural protein genes, all of which are expressed from subgenomic RNAs. Subgenomic RNAs form a nested set of 3 'coterminal RNAs that are generated through a discontinuous transcriptional mechanism. Subgenomic RNAs are composed of sequences that are not contiguous with the genomic RNA. All EAV subgenomic RNAs share a common 5 'leader sequence (156 to 221 nt in length) that is identical to the 5' genomic sequence. The leader and body parts of subgenomic RNAs are connected by a conserved sequence called a transcriptional regulatory sequence (TRS). TRS is found at the 3 'end of the leader (TRS leader) as well as
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77/207 in the subgenomic promoter regions located upstream of each structural protein gene (body TRS). Subgenomic RNAs are generated as the intermediate negative strand replication RNA is transcribed. As transcription occurs, complex replication pauses as it arrives at each body TRS and then the nascent negative strand RNA becomes associated with the complementary positive strand leader TRS where the transcription of negative strand RNA continues. This discontinuous transcription mechanism results in subgenomic RNA with conserved EAV sequences of both 5 'and 3'. The negative strand subgenomic RNAs then become the model for producing subgenomic positive sense mRNA.
[098] Infectious cDNA clones, which represent the entire EAV genome, have been reported and have been used to study EAV RNA replication and transcription for nearly two decades. In addition, infectious clones have been generated that contain the chloramphenicol acetyltransferase (CAT) gene inserted in place of ORF2 and ORF7, and it has been shown that CAT protein is expressed in cells electroporated with those RNAs. Modifications of the infectious clone by means of local directed mutagenesis and deletion of the structural protein gene regions were used to determine the requirement for each structural gene in the support of RNA replication (Molenkamp 2000). The study reported by
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Molenkamp 2000 concluded that structural genes are not required to support RNA replication. Analysis of sequence homology requirements for TRS activity in subgenomic RNA production was conducted and used to better define how discontinuous transcription occurs mechanistically (van Marie 1999, Pasternak 2000, Pasternak 2001, Pasternak 2003, van den Born 2005) and interfering RNAs Defective cells were used to understand the minimum genomic sequences required for replication and packaging of RNA into virus particles (Molenkamp 2000a).
Alfavirus [099] Alfavirus is a genus of genetically, structurally and serologically related viruses in the group IV Togaviridae family that includes at least 30 members, each of which has positive polarity single-strand RNA genomes enclosed in a nucleocapsid surrounded by a envelope containing viral peak proteins. Currently, the alphavirus genus comprises among others the Sindbis virus (SIN), the Semliki Forest virus (SFV), the Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and eastern equine encephalitis virus ( EEEV), all of which are closely related and capable of infecting various vertebrates such as mammals, rodents, fish, avian species and larger mammals such as humans and horses as well as invertebrates such as
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79/207 like insects. Transmission between species and individuals occurs mainly through mosquitoes that make alphavirus a contributor to the collection of Arbovirus - or Arthropod-Transmitted Viruses. In particular, the Sindbis and Semliki Forest viruses have been extensively studied and, therefore, the life cycle, mode of replication, etc., of these viruses are well characterized. In particular, alphavirus has been shown to replicate very efficiently in animal cells which makes them valuable as vectors for the production of protein and nucleic acids in such cells.
[100] Alphavirus particles are involved, have a diameter of 70 nm, tend to be spherical (although slightly pleomorphic), and have an isometric nucleocapsid of approximately 40 nm. Figure 9 depicts a typical genomic structure and expression of alphavirus genomes. The Alfavirus genome is single-stranded RNA of positive polarity approximately 11 to 12 kb in length, comprising a 5 'cap, a 3' poly-A tail, and two open reading frames with a first frame encoding proteins non-structural with enzymatic function and a second frame encoding structural viral proteins (for example, C-capsid protein, E1 glycoprotein, E2 glycoprotein, E3 protein and 6K protein).
[101] The 5 'thirds of the alphavirus genome encode a number of necessary non-structural proteins
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80/207 for viral RNA transcription and replication. These proteins are translated directly from RNA and together with cellular proteins form the RNA-dependent RNA polymerase essential for replication and transcription of the subgenomic RNA viral genome. Four non-structural proteins (nsPl-4) are produced as a single polyprotein that makes up the virus's replication machinery. The processing of the polyprotein occurs in a highly regulated manner, with the dividing at the P2 / 3 junction influencing the use of the RNA model during genome replication. This location is located at the base of a narrow slit and is not readily accessible. Once cleaved, nsP3 creates a ring structure that surrounds nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce non-cytopathic viruses or a cluster of temperature-sensitive phenotypes in the P2 / P3 interface region. P3 mutations opposed to the location of nsP2 non-cytopathic mutations prevent efficient P2 / 3 dividing. This, in turn, can affect RNA infectivity by altering levels of viral RNA production.
[102] The 3 'third of the genome comprises subgenomic RNA that serves as a model for translating all the structural proteins required to form viral particles: the C-nucleocapsid protein, and the P62 and El envelope proteins that associate as one
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81/207 heterodimer. Surface glycoproteins anchored in a viral membrane are responsible for receptor recognition and entry into target cells through membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter present at the 3 'end of the RNA sequence encoding the nsp4 protein. The proteolytic maturation of P62 in E2 and E3 causes a change in the viral surface. The glycoprotein peaks E1, E2, and sometimes E3, together form an E1 / E2 dimer or an E1 / E2 / E3 trimer, where E2 extends from the center to the vertices, El fills the space between the vertices , and E3, if present, is at the distal end of the peak. Upon exposure of the virus to the acidity of the endosome, El dissociates from E2 to form an homotrimer of El, which is necessary for the fusion step to drive the cell and viral membranes together. The alphaviral glycoprotein El is a class II viral fusion protein, which is structurally different from the class I fusion proteins found in influenza and HIV viruses. The glycoprotein E2 works to interact with the nucleocapsid through its cytoplasmic domain, while its ectodomain is responsible for binding a cell receptor. Most alphaviruses lose peripheral E3 protein, while in Semliki viruses it remains associated with the viral surface.
[103] Alphavirus replication has been reported
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82/207 occurs on a membranous surface within the host cell.
In the first stage of the infectious cycle, the 5 'end of the genomic RNA is translated into a polyprotein (nsPl-4) with RNA polymerase activity that produces a complementary strand negative to the genomic RNA. In a second step, the negative strand is used as a model for the production of two RNAs, respectively: (1) a positive genomic RNA corresponding to the secondary virus genome that produces, by translation, other nsp proteins and acts as a genome for the virus; and (2) subgenomic RNA that encodes the structural proteins of the virus that form the infectious particles. The positive genomic RNA / subgenomic RNA ratio is regulated by proteolytic autocleavage of the polyprotein to nsp 1, nsp 2, nsp 3 and nsp 4. In practice, viral gene expression occurs in two phases. In a first phase, there is a main synthesis of positive and negative genomic strands. During the second phase, the synthesis of subgenomic RNA is virtually exclusive, thus resulting in the production of a large amount of structural protein.
Innate immunity [104] Since innate immune activation can occur due to many different stimuli, vaccine approaches that rely on self-amplifying RNA replicons to express antigen or therapeutic GOI can be negatively impacted by disabling global host protein
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83/207 associated with PKR phosphorylation of eIF2a. Modifying RNA replicons to function in a cellular environment where host protein translation is suppressed could provide those systems with a significant advantage over standard RNA replication systems.
[105] Consequently, RNA replicon systems that are negatively impacted by innate immune responses, such as systems derived from alphavirus and arterivirus, may be more effective at expressing their encoded GOI when modified to contain a DLP motif. The DLP motif confers efficient mRNA translation in cellular environments where cell mRNA translation is inhibited. When a DLP is linked with translation of non-structural protein gene replication vectors, the replicase and transcriptase proteins are capable of initiating functional replication in PKR-activated cellular environments. When a DLP is ligated with translation of subgenomic mRNAs, robust GOI expression is possible even when cellular mRNA is restricted due to innate immune activation. Consequently, modifying replicons that contain DLP structures to help trigger translation of both structural protein non-genes and subgenomic mRNAs provides yet another powerful way to overcome innate immune activation.
[106] Some disclosure modalities refer to DLP structures that have been modified to support
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84/207 translation of non-structural viral genes from replicon vectors derived from two different viruses, Venezuelan equine encephalitis virus (VEEV) and equine arteritis virus (EAV), thus carrying evasion of innate immune response to the systems. As described in more detail below, the incorporation of the DLP structures into the replicon vectors made both resistant to treatment with interferon (IFN) and unexpectedly also resulted in an overall increase in GOI expression potential. The combination of resistance to IFN and potential for higher protein expression transmitted by modifying a DLP for RNA replication systems has made them suitable for use in individuals or populations where innate immune activation is present in an acute or chronic form.
Revelation Nucleic Acid Molecules [107] Some aspects of the present disclosure refer to nucleic acid molecules, such as synthetic or recombinant nucleic acid molecules, which include one or more DLP motifs, a coding sequence for one or more motifs DLP or a combination thereof. In some embodiments, the nucleic acid molecules of the disclosure may include a coding sequence for a gene of interest (GOI) operably linked to the DLP motif (or motifs) and / or the coding sequence for the DLP motifs.
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85/207 [108] In one aspect, a nucleic acid molecule is disclosed herein which comprises (i) a first nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer or a variant thereof ; and (ii) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence comprises a coding sequence for a gene of interest (GOI). In some embodiments, at least one of the one or more structural elements of the viral capsid enhancer comprises one or more RNA rod-loop structures. In some embodiments, at least one of the one or more RNA rod-loop structures is comprised of a DLP motif present in the first nucleic acid sequence. In some embodiments, at least one of the one or more structural elements of the viral capsid enhancer does not comprise any RNA stem-loop.
[109] As described above, a viral capsid enhancer comprises sequences within the 5 'and / or 5' non-coding sequences (preferably, 5 'coding sequences) of that enhancement expression (for example, transcription and / or translation) of sequences operably linked to it. In some embodiments of the present disclosure, the one or more
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Structural elements of the viral capsid enhancer include one or two RNA rod-loop structures of the viral capsid enhancer. In some embodiments, the viral capsid enhancer of the present disclosure includes sequences containing the 26S subgenomic promoter. In some embodiments, the viral capsid enhancer of the disclosure contains the 5 'coding sequences in about 20 to 250 nucleotides, about 20 to 200 nucleotides, about 20 to 150 nucleotides, about 20 to 100 nucleotides, or about nucleotides 50 to 250, about nucleotides 100 to 250, about nucleotides 50 to 200, about nucleotides 75 to 250, about nucleotides 75 to 200, about nucleotides 75 to 150, about nucleotides 77 to 139, or about nucleotides 100 to 250, about nucleotides 150 to 250, about nucleotides 100 to 150, about nucleotides 100 to 200 of viral 26S RNA, which is capable of forming a clamp-like structure. In some embodiments, the first nucleic acid sequence encodes one or more structural elements of a viral capsid enhancer that are important for enhancing the expression of a heterologous sequence operably linked thereto. In some embodiments, the first nucleic acid sequence includes encoding a sequence for one or more RNA stem-loop structures of a viral capsid enhancer. In some modalities, the first sequence
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87/207 nucleic acids encode one or more structural elements of a viral capsid enhancer that are important for enhancing the translation of a heterologous sequence operably linked thereto. In some embodiments, the first nucleic acid sequence encodes one or more structural elements of a viral capsid enhancer which are important for enhancing the transcription of a heterologous sequence operably linked thereto.
[110] In some modalities, the first sequence
of acids nucleic gives molecule of acid nucleic includes at least about 50, fence in 75, fence of about 100 in 150, fence of 200, about 300 , or more nucleotides gives
5 'coding sequence for a viral capsid protein. In some embodiments, the first nucleic acid sequence of the nucleic acid molecule includes about 50, about 75, about 100, about 150, about 200, about 300, or more, or a range between any two of these values, nucleotides of the 5 'coding sequence for a viral capsid protein. In some embodiments, the viral capsid enhancer is derived from a capsid gene from an alphavirus species selected from the group consisting of eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus ( EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV),
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88/207 Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV) , Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) and Buggy Creek virus. In some embodiments, the viral capsid enhancer is derived from a capsid gene from a species of Sindbis virus or a species of Semliki Forest virus. In some particular embodiments, the viral capsid enhancer is derived from a capsid gene from a species of Sindbis virus. In addition, a person of ordinary skill in the art will assess what modifications can be produced in the 5 'coding sequences of the viral capsid protein without substantially reducing its enhancement activities. More information in this regard can be found in, for example, Frolov et al. , J. Virology 70: 1182, 1994; Frolov et al. , J. Virology 68: 8111, 1994. In some embodiments, it may be an advantage that such mutations substantially preserve the RNA clamp-shaped structure formed by the 5 'capsid coding sequences.
[111] In some modalities, the
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89/207 viral capsid disclosed herein does not contain one or more, or all, of the 5 'coding sequences of the capsid protein that are upstream of the clamp-like structure. In some embodiments, the viral capsid enhancer disclosed herein does not contain all of the 5 'coding sequences of the viral capsid protein that are upstream of the clamp-like structure. In some embodiments, the viral capsid enhancer sequence can encode all or part of the capsid protein. Consequently, in some embodiments disclosed herein, the capsid enhancer region will not encode the entire viral capsid protein. In some embodiments, the viral capsid enhancer sequence encodes an amino terminal fragment of the viral capsid protein. In those embodiments where a otherwise functional capsid protein is encoded by the capsid enhancer sequence, it may be desirable to excise the capsid auto-protease activity. Capsid mutations that reduce or excise the auto-protease activity of the capsid protein are known in the art (see, for example, WO199 6/37616). In addition to or alternatively, one or more of the amino acid residues in the capsid protein can be altered to reduce capsid protease activity.
[112] As noted above, previous studies of
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90/207 sequence comparisons and structural RNA analysis revealed the evolutionary conservation of DLP motifs in many members of the Alfavirus genus (see, for example, Ventoso, 2012 supra). Consequently, in some additional embodiments, the viral capsid enhancer sequence of the present disclosure can be any other variant sequence such as, for example, a synthetic sequence or a heterologous sequence, which can form a functional RNA clamp or structurally equivalent to one or more of the RNA stem-loop structures envisaged for a viral capsid enhancer and which can act to improve translation of operably linked RNA sequences downstream of it (for example, coding sequence for a gene of interest). Non-limiting examples of RNA-raising structures that can act as a transcriptional and / or translational enhancer include those shown in Figures 11A and B. 11A-B. In some embodiments, the nucleic acid molecule of the disclosure includes an alphavirus capsid enhancer as derived from Sindbis virus (SINV; NC 001547.1), Aura virus (AURAV; AF126284), Chikungunya virus (CHIKV; NC 004162), 0'Nyong-Nyong virus (ONNV; NC 001512), Eastern Equine Encephalitis virus (EEEV (SA); AF159559 and EEEV (NA); U01558), Mayaro virus (MAYV; DQ001069), Semliki Forest virus (SFV ; NC
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003215), Ross River virus (RRV; DQ226993 and Sagiyama virus (SAGV; AB032553), Getah virus (GETV; NG 006558), Middelburg virus (MIDV; EF536323), Una virus (UNAV; AF33948), or Bebaru virus (BEBV; AF339480) as described in Toribio et al., 2016 supra, the content of which is incorporated in its entirety into this document as a reference, or a variant thereof.
[113] Nucleic acid molecules that have a high degree of sequence identity (for example, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the coding sequence for a viral capsid enhancer disclosed herein can be identified and / or isolated using the sequence described herein (for example, SEQ ID NO : 1) or any other alphavirus capsid proteins as they are known in the art, for example, using the sequences of Sindbis virus (SINV; NC 001547.1), Aura virus (AURAV; AF126284), Chikungunya virus (CHIKV; NC 004162), O'Nyong-Nyong virus (ONNV; NC 001512), Eastern Equine Encephalitis virus (EEEV (SA); AF159559 and EEEV (NA); U01558), Mayaro virus (MAYV; DQ001069) , Semliki Forest virus (SFV; NC 003215), Ross River virus (RRV; DQ226993 and Sagiyama virus (SAGV; AB 032553), Getah virus (GETV; NC 006558),
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Middelburg (MIDV; EF536323), Una virus (UNAV; AF33948) and Bebaru virus (BEBV; AF339480), by genome sequence analysis, hybridization, and / or PCR with degenerate primers or specific gene primers from sequences identified in the respective alphavirus genome. For example, the viral capsid enhancer may comprise, or consist of, a DLP motif of a virus species belonging to the Togaviridae family, for example, an alphavirus species or a rubivirus species. In some embodiments, the nucleic acid molecule of the disclosure includes a viral capsid enhancer that has a nucleic acid sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99%, or 100% sequence identity for the 5 'CDS portion of an alphavirus capsid protein. In some embodiments, the 5 'CDS portion of an alphavirus capsid protein comprises at least the first 25, 50, 75, 80, 100, 150, or 200 nucleotides of the coding sequence for the alphavirus capsid protein. In some embodiments, the nucleic acid molecule of the disclosure includes a viral capsid enhancer that has a nucleic acid sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% or 100% sequence identity for the
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93/207 nucleic acid sequence of any one of SEQ ID NOs: 1 and 46 to 52. In some embodiments, the nucleic acid molecule comprises a viral capsid enhancer that has a nucleic acid sequence that exhibits 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of these values, from sequence identity to nucleic acid sequence of any of SEQ ID NOs: 1 and 46 to 52. In some embodiments, the nucleic acid molecule of the disclosure includes a viral capsid enhancer that has a nucleic acid sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for the sequence of SEQ ID NO: 1 revealed in this document. In some embodiments, the nucleic acid molecule of the disclosure includes a viral capsid enhancer that has a nucleic acid sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% or 100% sequence identity for any of the sequences described in Figures 11A to B and / or in Figure IA in the publication by Toribio et al. (2016 above), the content of which is incorporated in its entirety into this document as a reference.
[114] Consequently, in some modalities, the
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94/207 nucleic acid molecule of the disclosure includes a
capsid enhancer viral that has a sequence of nucleic acids that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity for the sequence of any one of SEQ ID NOS: 46 to 52 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a viral capsid enhancer that has a nucleic acid sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 46 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a
capsid enhancer viral that has a sequence of nucleic acids that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 47 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a
capsid enhancer viral that has a sequence of nucleic acids that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
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97%, at least 98%, at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 48 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a
perfector of capsid viral what has a sequence of nucleic acids what displays fur any less 80%, fur minus 85%, at least 90%, fur any less 95%, fur any less 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 49 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a
perfector of capsid viral what has a sequence of nucleic acids what displays fur any less 80%, fur minus 85%, at least 90%, fur any less 95%, fur any less 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 50 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a
perfector of capsid viral what has a sequence of nucleic acids what displays fur any less 80%, fur minus 85%, at least 90%, fur any less 95%, fur any less 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 51 disclosed herein. In some embodiments, the nucleic acid molecule of the disclosure includes a
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96/207 viral capsid enhancer that has a nucleic acid sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or 100% sequence identity for the sequence set out in SEQ ID NO: 52 disclosed herein.
[115] In the nucleic acid molecule, according to some embodiments of the present disclosure, one or more RNA stem-loop structures are operably positioned upstream of the coding sequence for the GOI of the second nucleic acid sequence. In some embodiments, one or more RNA rod-loop structures are operably positioned from about 1 to about 50 nucleotides, from about 10 to about 75 nucleotides, from about 30 to about 100 nucleotides, about 40 to about 150 nucleotides, about 50 to about 200 nucleotides, about 60 to about 250 nucleotides, about 100 to about 300 nucleotides, or about 150 to about 500 nucleotides upstream of the coding sequence for GOI. In some embodiments, one or more RNA rod-loop structures are operably positioned about 1, about 2, about 5, about 10, about 15, about 20, about 25, about about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300,
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97/207 about 400, about 500, or a range between any two of those values, nucleotides upstream of the coding sequence for GOI. In some embodiments, one or more RNA rod-loop structures are operably positioned immediately upstream of the coding sequence for the GOI.
[116] In some embodiments, the nucleic acid molecule further includes a 5 'untranslated region (5' RTU) sequence positioned operably upstream of the first nucleic acid sequence. In some embodiments, the 5 'RTU sequence is positioned so
operable from about 1 to about . in 50, from fence in 10 The fence 75, about 30 to about in 100 of fence in 40 The fence 150, about 50 to about in 200, from fence in 60 The fence
from 250, from about 100 to about 300, or from about 150 to about 500 nucleotides upstream of the first nucleic acid sequence. In some embodiments, the 5 'RTU sequence is operably positioned at about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or 100 nucleotides upstream of the first nucleic acid sequence. In some embodiments, the 5 'UTR sequence is operably positioned immediately upstream of the first nucleic acid sequence.
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98/207 [117] In some embodiments, the 5 'RTU sequence is operably positioned downstream of the promoter. In some embodiments, the 5 'RTU sequence is operably positioned from about 1 to about 50, from about 10 to about 75, from about 30 to about 100, from about 40 to about 150 , from about 50 to about 200, from about 60 to about 250, from about 100 to about 300, or from about 150 to about 500 nucleotides downstream of the promoter sequence. In some embodiments, the 5 'RTU sequence is operably positioned at about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or 100 nucleotides downstream of the promoter sequence. In some embodiments, the 5 'UTR sequence is operably positioned immediately downstream of the promoter sequence. In some embodiments, the 5 'UTR sequence is operably positioned downstream of the promoter and upstream of the first nucleic acid sequence.
[118] In some embodiments, the nucleic acid molecule comprises a 3 '(3' UTR) untranslated region sequence positioned operably downstream of the second nucleic acid sequence. In some embodiments, the 3 'UTR sequence is operably positioned from about 1 to about 50 nucleotides, from about 10 to about
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99/207 nucleotides, from about 30 to about 100 nucleotides, from about 40 to about 150 nucleotides, from about 50 to about 200 nucleotides, from about 60 to about 250 nucleotides, from about 100 to about 300 nucleotides, or about 150 to about 500 nucleotides downstream of the second nucleic acid sequence. In some embodiments, the 3 'RTU sequence is operably positioned at about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, or a range between any two of those values , nucleotides downstream of the second nucleic acid sequence. In some embodiments, the 3 'UTR sequence is operably positioned immediately downstream of the second nucleic acid sequence.
[119] In some embodiments disclosed herein, the coding sequence for GOI is transcribed into a messenger RNA (mRNA) or part of an mRNA. As used herein, the term mRNA or messenger RNA refers to a single-stranded RNA molecule that is synthesized during transcription, is complementary to one of the double-stranded DNA strands, and serves to transmit the genetic information contained in DNA for ribosomes for protein synthesis. The mRNA can be joined, partially
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100/207 linked or not linked, and can be eukaryotic or prokaryotic mRNA. As discussed above, mRNA molecules, according to some disclosure modalities, can be produced by de novo synthesis. In some embodiments disclosed herein, the coding sequence for GOI encodes a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide, or any combination thereof. In some embodiments, the polypeptide is an antibody, an antigen, an immune modulator, a cytokine, an enzyme or any combination thereof.
[120] In some embodiments, the nucleic acid molecule of the disclosure further includes a coding sequence for an auto-protease peptide (e.g., autocatalytic autocleaving peptide), where the coding sequence for the auto-protease is, optionally, operably linked upstream to the second nucleic acid sequence. In general, any proteolytic dividing site known in the art can be incorporated into the nucleic acid molecules of the disclosure and can, for example, be proteolytic dividing sequences that are cleaved post-production by a protease. Additional suitable proteolytic dividing sites also include sequences
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101/207 of proteolytic divage that can be cleaved following the addition of an external protease. As used herein, the term auto-protease refers to an autocleaving peptide that has autoproteolytic activity and is capable of cleaving itself from a larger chemical portion of the polypeptide. First identified in the foot-and-mouth disease virus (EMDV), a member of the picornavirus group, several auto-proteases were subsequently identified such as, for example, peptides similar to 2A Equine Rhinitis Virus (E2A), porcine teschovirus-1 ( P2A) and Thosea asigna virus (T2A), and their activities in proteolytic divage have been shown in several eukaryotic systems ex vitro and in vivo. As such, the concept of autoproteases is available to a person skilled in the art with many naturally occurring auto-protease systems having been identified. Well-studied autoprotease systems are, for example, viral proteases, development proteins (for example, HetR, Hedgehog proteins), RumA auto-protease domain, UmuD, etc.). Non-limiting examples of auto-protease peptides suitable for the compositions and methods of the present disclosure include porcine teschovirus-1 2A (P2A) peptide sequences, a foot-and-mouth disease virus (EMDV) 2A (F2A), a Rhinitis Virus Equine A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A),
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102/207 a Virus Flacherie 2A (BmIFV2A) or a combination thereof.
[121] In some embodiments, the coding sequence for an auto-protease peptide is operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the autoprotease peptide comprises, or consists of, a sequence of peptides selected from the group consisting of porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), a Virus Equine Rhinitis A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof. In some embodiments, the auto-protease peptide includes a sequence of porcine teschovirus-1 2A (P2A) peptides.
[122] A person skilled in the art will appreciate that different configurations of the viral capsid enhancer sequence, the sequence encoding the autoprotease peptide and the sequence encoding the gene of interest can be employed as long as the capsid enhancer sequence enhances the expression of the heterologous nucleic acid sequence (s), for example, a coding sequence for a GOI, as compared to the level seen in the absence of the capsid enhancer sequence. These strings will typically be configured so that the
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The polypeptide encoded by the gene of interest can be released from the protease and any capsid protein sequence after dividing by the auto-protease.
[123] A non-limiting list of exemplary combinations of auto-protease peptides described in this document (such as P2A, F2A, E2A, T2A, BmCPV2A and BmIFV2A) with one or more viral capsid enhancer sequences described in this document is provided in Tables 1 and 2. Table 1 provides a short name for each viral capsid enhancer (for example, CE01) and a short name for each auto-protease peptide (for example, AP01). Each 'X' peptide numbered in Table 2 has a corresponding auto-protease peptide provided in Table 1. Likewise, each enhancer Ύ 'numbered in Table 2 has a corresponding viral capsid enhancer provided in Table 1. Therefore, each entry X: Y in Table 2 provides an example of a combination of a viral capsid enhancer and an autoprotease peptide that can be used in the molecules, compositions and methods of the present disclosure. For example, the combination designated as AP01: CE16 in Table 2 provides a combination of viral capsid enhancer derived from Sindbis virus (SINV) and an auto-protease peptide from porcine teschovirus-1 2A (P2A).
Table 1: Viral capsid and peptide enhancers
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104/207 auto-proteases exemplary of the disclosure
Viral Capsid Enhancer (Y) Auto-protease peptide (X) Encephalitis virus (Eastern equine CE01 (EEEV)) teschovirus-1 2A'l ΤΊ Ο 7 ' -Α.Γ U 1)porcine (P2A) Encephalitis virus (Venezuelan equine CE02 (VEEV) ^} foot-and-mouth disease virus (FMDV) 2A (AP02) (F2A) Virus Everglades (CE03(EVEV)) Rhinitis VirusEquine A (ERAV) (AP03) 2A (E2A) Mucambo virus (MUCV) Virus Thosea asigna 2A (T2A) ^{k 1} forest virus (CE04Semliki (SFV)) virus fromcytoplasmic polyhedrosis 2A (BmCPV2A) (CE 0 5 Virus Pixuna (PIXV) Virus Flacherie 2A (B _m IFV2A) ^(AP ° ⁶⁾ Middleburg virus (CE06(MIDV))Chikungunya virus (CE07 (CHIKV))0'Nyong-Nyong virus (CE08 (ONNV))Ross River Virus (CE09(RRV))forest virus (CEIOBarmah (BE))/ ζ-ί t — i η η Getah Virus (GET)Sagiyama virus (CE12(SAGV))/ -pi 2Bebaru virus (BEBV)(CE 14 Mayaro virus (MAYV)/ QP 1 C Una virus (UNAV)
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Viral Capsid Enhancer (Y) Auto-protease peptide (X) Sindbis virus (CE16(SINV))/ -pi yAura virus (AURAV)Whataroa virus (CE18(WHAV))Babanki virus (CE19(BABV))Kyzylagach virus (CE20 (KYZV))Encephalitis virus. _{,, Ί} Western equine CE21 (WEEV)! T Ο Ο virus Highland J (HJV)Fort Morgan virus (CE23(FMV))(CE2 4 Ndumu (NDUV)alfavirus Salmonidae (CE25 (SAV))ί _π „ί (CE26 virus from Buggy Creek
Table 2:
X: Y X: Y X: Y X: Y X: Y X: Y AP01: AP02: AP03: AP04: AP05: APO 6: CE01 CE01 CE01 CE01 CE01 CE01 AP01: AP02: AP03: AP04: AP05: APO 6: CE02 CE02 CE02 CE02 CE02 CE02 AP01: AP02: AP03: AP04: AP05: APO 6: CE03 CE03 CE03 CE03 CE03 CE03 AP01: AP02: AP03: AP04: AP05: APO 6: CE04 CE04 CE04 CE04 CE04 CE04 AP01: AP02: AP03: AP04: AP05: APO 6: CE05 CE05 CE05 CE05 CE05 CE05 AP01: AP02: AP03: AP04: AP05: APO 6: CE06 CE06 CE06 CE06 CE06 CE06
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AP01: AP02: AP03: AP04: AP05: AP0 6: CE07 CE07 CE07 CE07 CE07 CE07 AP01: AP02: AP03: AP04: AP05: AP0 6: CE08 CE08 CE08 CE08 CE08 CE08 AP01: AP02: AP03: AP04: AP05: AP0 6: CE09 CE09 CE09 CE09 CE09 CE09 AP01: AP02: AP03: AP04: AP05: AP0 6: SUPPORT SUPPORT SUPPORT SUPPORT SUPPORT SUPPORT AP01: AP02: AP03: AP04: AP05: AP0 6: CE11 CE11 CE11 CE11 CE11 CE11 AP01: AP02: AP03: AP04: AP05: AP0 6: CE12 CE12 CE12 CE12 CE12 CE12 AP01: AP02: AP03: AP04: AP05: AP0 6: CE13 CE13 CE13 CE13 CE13 CE13 AP01: AP02: AP03: AP04: AP05: AP0 6: CE14 CE14 CE14 CE14 CE14 CE14 AP01: AP02: AP03: AP04: AP05: AP0 6: CE15 CE15 CE15 CE15 CE15 CE15 AP01: AP02: AP03: AP04: AP05: AP0 6: CE16 CE16 CE16 CE16 CE16 CE16 AP01: AP02: AP03: AP04: AP05: AP0 6: CE17 CE17 CE17 CE17 CE17 CE17 AP01: AP02: AP03: AP04: AP05: AP0 6: CE18 CE18 CE18 CE18 CE18 CE18 AP01: AP02: AP03: AP04: AP05: AP0 6: CE19 CE19 CE19 CE19 CE19 CE19 AP01: AP02: AP03: AP04: AP05: AP0 6: CE20 CE20 CE20 CE20 CE20 CE20 AP01: AP02: AP03: AP04: AP05: AP0 6: CE21 CE21 CE21 CE21 CE21 CE21 AP01: AP02: AP03: AP04: AP05: AP0 6: CE22 CE22 CE22 CE22 CE22 CE22 AP01: AP02: AP03: AP04: AP05: AP0 6: CE23 CE23 CE23 CE23 CE23 CE23 AP01: AP02: AP03: AP04: AP05: AP0 6: CE24 CE24 CE24 CE24 CE24 CE24 AP01: AP02: AP03: AP04: AP05: AP0 6: CE25 CE25 CE25 CE25 CE25 CE25 AP01: AP02: AP03: AP04: AP05: AP0 6: CE2 6 CE2 6 CE2 6 CE2 6 CE2 6 CE2 6
[124] In one aspect, innovative nucleic acid molecules that include a nucleic acid sequence encoding a replicon are disclosed in this document.
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107/207 modified viral RNA, wherein the modified viral RNA replicon includes a first nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer (for example, a DLP motif) or a variant thereof , in which the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence encoding at least one non-structural viral protein or a portion thereof, wherein the first nucleic acid sequence is linked operable upstream to the second nucleic acid sequence.
[125] The terms RNA replicon and RNA replicon used interchangeably in this document refer to RNA that contains all the genetic information required to direct its own amplification or self-replication within a permissive cell. To direct its own replication, the RNA molecule 1) encodes polymerase, replicase, or other proteins that can interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and 2) contain cis-acting RNA sequences required for replication and transcription of the RNA encoded in subgenomic replicon. These sequences can be linked during the replication process to their autocodified proteins, or proteins derived
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108/207 non-autocoded cell, nucleic acids or ribonucleoproteins, or complexes between any of these components. In some embodiments of the present disclosure, a modified viral replicon RNA molecule typically contains the following ordered elements: 5 'or defective interfering RNA sequence (or sequences) required in replication cycles, encoding sequences for biologically nonstructural proteins active, promoter for subgenomic RNA, 3 'viral sequences required in cycles for replication, and a polyadenylate tract. Furthermore, the term replicon RNA generally refers to a molecule of positive polarity, or sense of message, and the replicon RNA may be of a different length than that of any known, naturally occurring RNA virus. In some embodiments of the present disclosure, the replicon RNA does not contain coding sequences for at least one of the structural viral proteins. In such cases, sequences encoding structural genes can be replaced by one or more heterologous sequences such as, for example, a coding sequence for a gene of interest (GOI). In those cases in which the replicon RNA must be encased in a recombinant alphavirus particle, it must contain one or more sequences, so called packaging signals, which serve to initiate interactions with structural alphavirus proteins that lead
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109/207 to particle formation.
[126] As used herein, subgenomic RNA refers to an RNA molecule of a length or size that is smaller than the genomic RNA from which it was derived. The subgenomic viral RNA must be transcribed from an internal promoter, dirty sequences reside within the genomic RNA or its complement. The transcription of a subgenomic RNA can be mediated by polymerase (or polymerases) encoded in viruses associated with proteins encoded in a host cell, ribonucleoprotein (or ribonucleoproteins), or a combination thereof. In some embodiments of the present disclosure, the subgenomic RNA is produced from a modified replicon RNA as disclosed herein and encodes or expresses one or more genes of interest (GOI). Instead of the native subgenomic promoter, the subgenomic RNA can be placed under control of the internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV), Bovine Viral Diarrhea Virus (BVDV), poliovirus, FMD virus (EMD ), enterovirus 71 or hepatitis C.
[127] In some embodiments, the second nucleic acid sequence of the modified viral RNA replicon includes the coding sequence for at least one, at least two, at least three, or at least four proteins
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110/207 non-structural viruses. In some embodiments, the second nucleic acid sequence of the modified viral RNA replicon includes the coding sequence for a portion of at least one non-structural viral protein. For example, the second nucleic acid sequence of the modified viral RNA replicon can include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100 %, or a range between any two of these values, of the coding sequence for at least one non-structural viral protein. In some embodiments, the second nucleic acid sequence of the modified viral RNA replicon may include the coding sequence for a substantial portion of at least one non-structural viral protein. As used herein, a substantial portion of a nucleic acid sequence encoding a non-structural viral protein comprises enough of the nucleic acid sequence encoding the non-structural viral protein to allow putative identification of that protein, by manual evaluation of the sequence by a person skilled in the art, or by comparison and automated computer sequence identification using algorithms such as BLAST (see, for example, in Basic Local Alignment Search Tool; Altschul SE et al., J. Mol. Biol. 215 : 403 to 410, 1993). In some embodiments, the second nucleic acid sequence of the modified viral RNA replicon may
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111/207 include the entire coding sequence for at least one non-structural protein. In some embodiments, the second nucleic acid sequence comprises substantially the entire coding sequence for native viral non-structural proteins.
[128] The molecular techniques and methods by which these new nucleic acid molecules were constructed and characterized are more fully described in the Examples in the present document of the present application. As non-limiting examples, in the Examples section, Venezuelan equine encephalitis virus (VEEV) and equine arteritis virus (EAV) were used to illustrate the compositions and methods disclosed in this document.
[129] In some embodiments, the nucleic acid molecules disclosed herein are recombinant nucleic acid molecules. As used herein, the term recombinant means any molecule (for example, DNA, RNA, etc.), which is, or results, however indirectly, from human manipulation of a polynucleotide. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by polymerase reaction (or reactions) ex vitro, or to which ligands have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As examples
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112/207 non-limiting, recombinant nucleic acid molecule:
1) was synthesized or modified ex vitro, for example, with the use of chemical or enzymatic techniques (for example, with the use of chemical synthesis of nucleic acid, or with the use of enzymes for replication, polymerization, exonucleolytic digestion, digestion endonucleolytic, ligation, reverse transcription, transcription, base modification (which includes, for example, methylation), or recombination (which includes homologous and site specific recombination) of nucleic acid molecules; 2) includes conjugated nucleotide sequences that are not conjugated in nature; 3) it has been modified using molecular cloning techniques so that it lacks one or more nucleotides in relation to the naturally occurring nucleic acid sequence, and / or 4) it has been manipulated using the molecular cloning techniques of so that it has one or more sequence changes or rearrangements in relation to the naturally occurring nucleic acid sequence.
[130] A nucleic acid molecule, which includes a naturally occurring variant of a nucleic acid sequence, can be produced using a number of methods known to those skilled in the art. The sequence of a nucleic acid molecule can be modified from a naturally occurring sequence from which it is derived using a variety
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113/207 techniques that include, but are not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as, but not limited to, site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme of a nucleic acid fragment, binding of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning and chemical synthesis, which includes chemical synthesis of oligonucleotide mixtures and ligation of mixing groups to build a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of nucleic acid molecules modified by screening for protein function or the replicon encoded by the nucleic acid molecule and / or by hybridization with a wild-type gene or fragment of the even, or by PCR using primers that have homology to a target or wild-type nucleic acid molecule or sequence.
[131] In various embodiments disclosed herein, the nucleic acid molecule disclosed herein may include one or more of the following resources.
[132] In some embodiments, the modified viral RNA replicon includes a modified RNA replicon derived from a virus species belonging to the genus
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Alfavirus of the Togaviridae family or the Arterivirus genus of the Arterívírídae family. Suitable arterivirus species include equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevation virus (LDV), Simian hemorrhagic fever virus (SHFV) and wobbly possum disease virus (WPDV). Virulent and avirulent arterivirus strains are both suitable. Non-limiting examples of preferred arterivirus strains include, but are not limited to, virulent Bucyrus strain of EAV (VBS), LDVPlagemann, LDV-C, PRRSV type 1, and PRRSV type 2. Exemplary preferred EAV strains include, however, without limitation , EAV VB53, EAV ATCC VR-796, EAV HK25, EAV HK116, EAV ARVAC MLV, EAV Bucyrus strain (Ohio), Modified EAV Bucyrus, CA95 avirulent strain, Red Mile (Kentucky), 84KY-A1 (Kentucky), Wroclaw-2 (Poland), Bibuna (Switzerland), and Vienna (Austria). Preferred non-limiting examples of PRRSV strains include PRRSV LV4.2.1, PRRSV 16244B, PRRSV HB-1 (sh) / 2002, PRRSV HB-2 (sh) / 2002, PRRSV HN1, PRRSV SD 0108, PRRSV SD0802, PRRSV SD0803, PRRSV and VR2332. Preferred non-limiting examples of SHFV strains and variants include variants of SHFV, SHFV-krtgla and -krtglb (SHFVkrtgla / b), SHFVkrtg2a / b (adhering to GenBank on JX473847 for JX473850), SHFV-LVR, the SHFV prototype variant 420 / M6941 (NC_003092); SHFV-krcl and SHFVkrc2 from Kibale
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115/207 red colobus (HQ845737 and HQ845738, respectively). Other non-limiting examples of preferred arterivirus include PRRSV-Lelystad, the European type strain (type 1) (M96262); PRRSWR2332, the North American type strain (type 2) (U87392); EAV-Bucyrus (NC_002532); EAV-s3685 (GQ903794); LDV-P, the Plagemann strain (U15146); and LDV-C, the type C neurovirulent strain (L13298).
[133] In some embodiments, the first nucleic acid sequence is positioned upstream of a nucleic acid sequence that encodes a portion or all of the non-structural pplab protein of the modified artevirus RNA replicon. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 1 to 1,000 nucleotides downstream of the 5 'terminal of the modified viral RNA replicon. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 1 to 25, about 1 to 40, about 10 to 25, 10 to 50, about 10 to 100, about 20 to 50, about 20 to 75, about 25 to 100, about 25 to 100 nucleotides downstream of the 5 'terminal of the modified viral RNA replicon. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, or more, or a range between any two
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116/207 of these values, nucleotides downstream of the 5 'terminal of the modified viral RNA replicon. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 1 to 100, about 1 to 500, about 25 to 800, about 50 to 900, about 50 to 300, about 25 to 200, about 25 to 100, about 50 to
400, fence in 100 The 500, fence in 100 The 300, fence in 100 The 200, fence in 200 The 500, fence in 200 The 600, fence in 200 The 400, fence in 150 The 700, fence in 150 The 400, or fence of 50 0
1,000 nucleotides downstream of the 5 'terminal of the replicon of
Modified viral RNA.
[134] Without being limited by any particular theory, it is believed that translational enhancement activity for a viral DLP motif may depend, in some modalities, on the distance between the viral DLP motif and the AUGi initiation codon (Toribio et al., 2016 supra). Consequently, in some embodiments, the first nucleic acid sequence is operably positioned in a region of about 10 to 100 nucleotides downstream of the AUGi initiation codon of the modified viral RNA replicon. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 10 to 75, about 10 to 50, about 10 to 25, 15 to 75, about 15 to 50, about 15 to 25, about 25 to 75, about 25 to 50, about 25 to 100 nucleotides downstream
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of the codon AUGi initiation of viral RNA replicon
modified. In some embodiments, the first nucleic acid sequence is operably positioned within a region of about 25, 28, 31, 34, 37, 37, 40, 43, 46, 49, 50, or a range between any two of these values, nucleotides downstream of the AUGi initiation codon of the modified viral RNA replicon.
[135] In some modalities, the sequence that encodes the modified viral RNA replicon further comprises one or more expression cassettes, where each of the cassettes
of expression comprises a promoter operably linked to a coding sequence for a gene of interest (GOI). As used in this document, the term
expression refers to a construct of genetic material that
contains enough coding sequences and regulatory information to direct appropriate transcription and / or translation of the coding sequences into a recipient cell, in vivo and / or ex vivo. The expression cassette can be inserted into a vector to target a desired host cell and / or an individual. In addition, the term expression cassette can be used interchangeably with the term expression construct. The term cassette of
expression as used in this document, refers to a
nucleic acid construct that encodes a functional protein or RNA operably linked to
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118/207 expression, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.
[136] The term operably linked, as used herein, denotes a functional link between two or more sequences. For example, an operable link between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is a functional link that allows expression of the polynucleotide of interest. In this sense, the term operably linked refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective in regulating transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term operably linked denotes a configuration in which a regulatory sequence is placed in a relative position appropriate to a sequence that encodes a functional polypeptide or RNA so that the control sequence directs or regulates the expression or cellular location of the mRNA encoding the polypeptide, polypeptide and / or functional RNA. Thus, a promoter is operably linked to a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. Operationally connected elements can be contiguous or non-contiguous.
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119/207 [137] Basic techniques for operably linking together or more DNA sequences are familiar to a person of ordinary skill in the art, and such methods have been described in many books for standard molecular biological manipulation (see, for example, Maniatis et al, Molecular Cloning:... a Laboratory Manual , 2nd ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Gibson et al, Nature Methods 6: 343-45, 2009).
[138] In some embodiments disclosed herein, the nucleic acid molecules disclosed herein may include more than one expression cassette. In principle, the nucleic acid molecules disclosed herein can, in general, include any number of expression cassettes. In some particular embodiments, the modified viral RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably positioned downstream of a regulatory sequence
transcriptional ( TRS) of replicon in RNA from artevirus modified, where the TRS Can be TRS1, TRS2, TRS3, TRS4, TRS5, TRS6, TRS7 or an combination From themselves. In some
particular embodiments, at least one of the one or more expression cassettes is operably positioned downstream of the TRS7 of the modified artevirus RNA replicon.
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120/207 [139] Nucleic acid molecules as provided herein may find use, for example, as an expression or transcription vector that, when operably linked to a heterologous nucleic acid sequence such as, for example, a coding sequence for a gene of interest (GOI), can affect the expression of GOI. In some embodiments, the GOI coding sequence is optimized for expression at a higher level than the expression level of a coding reference sequence. In some embodiments, the coding reference sequence is not codon optimized. In some embodiments, the GOI coding sequence comprises codon optimization. Regarding codon optimization of nucleic acid sequences, degeneration of the genetic code provides the possibility to replace at least one base of the protein coding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced to from the gene is changed. Consequently, the nucleic acid molecules of the present disclosure can also have one or more nucleotide substitutions according to degeneracy of the genetic code. References describing codon usage are readily and publicly available. In some additional disclosure embodiments, polynucleotide sequence variants can be produced for a variety of reasons, for
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121/207 example, to optimize codon expression for a particular host (for example, changing codons in the arterivirus mRNA to those preferred by other organisms such as humans, hamsters, mice or monkeys).
[140] In some embodiments disclosed herein, the GOI sequence encodes a polypeptide. The type of polypeptide may vary depending on specific requests. For example, the polypeptide can be a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide, or any combination thereof. In some embodiments, the polypeptide is an antibody, an antigen, an immune modulator, a cytokine, an enzyme, or a combination thereof.
[141] In some embodiments, the nucleic acid molecule as disclosed herein may further comprise a third nucleic acid sequence that encodes one or more structural elements of a second viral capsid enhancer (e.g., a DLP motif), wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for GOI. The second DLP motif can be the same or different from the first DLP motif positioned upstream of the coding sequence for non-structural proteins. Consequently, in some
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122/207 modalities, the second DLP motif is the same as the first DLP motif positioned upstream of the coding sequence for non-structural proteins. In some embodiments, the second DLP motif is different from the first DLP motif positioned upstream of the coding sequence for non-structural proteins.
[142] In some embodiments, the sequence encoding the modified viral RNA replicon further comprises a coding sequence for a proteolytic dividing site operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for the GOI. In general, any proteolytic dividing site known in the art can be incorporated into the nucleic acid molecules of the disclosure and can, for example, be proteolytic dividing sequences that are cleaved post-production by a protease. Additional suitable proteolytic dividing sites also include proteolytic dividing sequences that can be cleaved following the addition of an external protease. In some embodiments, the sequence encoding the modified viral RNA replicon further comprises a coding sequence for an auto-protease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for the GOI. In some embodiments, the auto-protease peptide includes a
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123/207 peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof. In some embodiments, the auto-protease peptide includes a sequence of porcine teschovirus-1 2A (P2A) peptides.
[143] A person skilled in the art will appreciate that different configurations of the viral capsid enhancer sequence, the coding sequence for non-structural proteins, the sequence encoding the auto-protease peptide, and the sequence encoding the gene of interest can be employed as long as the capsid enhancer sequence increases the expression of the heterologous nucleic acid sequence (or sequences) when compared to the level seen in the absence of the capsid enhancer sequence. These sequences will typically be configured so that the polypeptide encoded by the gene of interest can be released from the protease and any sequence of capsid protein after dividing by the auto-protease.
[144] In some embodiments, the sequence of the nucleic acid molecule as disclosed herein includes a modified RNA replicon of one species
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124/207 of alfavirus virus. In some embodiments, the modified alphavirus RNA replicon is from an alphavirus that belongs to the VEEV / EEEV group, or to the SE group, or to the SIN group. Non-limiting examples of the SE group alphavirus include Semliki Forest virus, O'Nyong-Nyong virus, Ross River virus, Middelburg virus, Chikungunya virus, Barmah forest virus, Getah virus, Mayaro virus, Sagiyama, Bebaru virus and Una virus. Non-limiting examples of alphaviruses of the SIN group include Sindbis virus, Girdwood S.A. virus, South African Arbovirus No. 86, Ockelbo virus, Aura virus, Babanki virus, Whataroa virus and Kyzylagach virus. Non-limiting examples of alphaviruses of the VEEV / EEEV group include eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus ( MIDV), Chikungunya virus (CHIKV), 0'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV) , Bebaru virus (BEBV), Mayaro virus (MAYV) and Una virus (UNAV).
[145] Non-limiting examples of alphavirus species include eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), virus Pixuna (PIXV), Middleburg virus (MIDV),
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125/207 Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV) , Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) and Buggy Creek virus. Virulent and avirulent alphavirus strains are both suitable. In some embodiments, the modified alphavirus RNA replication is from a Sindbis virus (SIN), a Semliki Forest virus (SFV), a Ross River virus (RRV), a Venezuelan equine encephalitis virus (VEEV) or a eastern equine encephalitis virus (EEEV). In some embodiments, the modified alphavirus RNA replicon is from a Venezuelan equine encephalitis virus (VEEV).
[146] In some cases where the nucleic acid molecule as disclosed herein includes a modified RNA replicon of an alphavirus virus species, the first nucleic acid sequence is positioned upstream of a nucleic acid sequence that encodes a or more nspl-4 non-structural proteins or a portion thereof of the modified alphavirus RNA replicon. Consequently, in some modalities, the first
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126/207 nucleic acid sequence is positioned upstream of a nucleic acid sequence encoding nspl, nspl-2, nspl-3, nspl-4, nsp2-4, nsp34, nsp2-3, nsp2, nsp3 , nsp4, or a portion thereof of the modified alphavirus RNA replicon. In some embodiments, the sequence encoding the modified alphavirus RNA replicon further includes one or more expression cassettes, wherein each expression cassette includes a promoter operably linked to a coding sequence for a gene of interest ( GOI). In some embodiments, the modified alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of a nucleic acid sequence encoding one or more nspl-4 non-structural proteins or a portion thereof of the alphavirus RNA replicon modified. Consequently, in some embodiments, at least one of the one or more expression cassettes is operably linked downstream of a nucleic acid sequence encoding nspl, nspl-2, nspl-3, nspl-4 nonstructural proteins, nsp2-4, nsp3-4, nsp2-3, nsp2, nsp3, nsp4, or a portion thereof, of the modified alphavirus RNA replicon.
[147] In some modalities, at least one of the one or more expression cassettes further comprises a
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127/207 third nucleic acid sequence that encodes one or more structural elements of a second viral capsid enhancer (for example, a DLP motif), wherein the third nucleic acid sequence is operably linked upstream to the sequence of coding for GOI. The second DLP motif may be the same or may be different from the first DLP motif positioned upstream of the coding sequence for at least the nspl-4 non-structural proteins or a portion thereof. Consequently, in some embodiments, the second DLP motif is the same as the first DLP motif positioned upstream of the coding sequence for non-structural proteins. In some embodiments, the second DLP motif is different from the first DLP motif positioned upstream of the coding sequence for non-structural proteins.
[148] In some embodiments, the nucleic acid sequence of the present disclosure further comprises a coding sequence for an auto-protease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for GOI . The auto-protease peptide can, in general, be any auto-protease peptide known in the art. Non-limiting examples of auto-protease peptides include porcine teschovirus-1 2A (P2A) peptide sequences, a foot-and-mouth disease virus (EMDV) 2A (F2A), a
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Equine Rhinitis A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Virus Flacherie 2A (BmIFV2A), and any combinations thereof.
[149] In an additional aspect, some embodiments disclosed herein refer to a nucleic acid molecule that includes a nucleic acid sequence that encodes a modified non-alphavirus RNA replicon, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence that encodes a viral capsid enhancer (e.g., a DLP motif). In some embodiments, the modified non-alphavirus RNA replicon further comprises a second nucleic acid sequence that encodes at least one non-structural viral protein or a portion thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence.
[150] In some embodiments, the modified non-alphavirus RNA replicon further comprises a coding sequence for an auto-protease peptide operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence. In some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon from a positive strand RNA virus. In some modalities, the
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129/207 modified non-alphavirus RNA replicon includes a modified RNA replicon from a negative strand RNA virus.
[151] Non-limiting examples of modified non-alphavirus RNA replicons include modified RNA replicons of the virus species belonging to the Togaviridae family, Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family or Paramyxoviridae family. Consequently, in some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon from a negative strand RNA virus. Species of suitable negative-stranded RNA viruses include, but are not limited to, viral species from the families Orthomyxoviridae, Rhabdoviridae and Paramyxoviridae. In some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon from a positive stranded virus species that belongs to the Togaviridae family or Flaviviridae family. In some embodiments, the modified non-alphavirus RNA replicon includes a modified RNA replicon from a positive stranded virus species that belongs to the Arterivirus genus of the Arteriviridae family. Suitable arterivirus species include, but are not limited to, equine arteritis virus (EAV) species, porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevation virus (LDV), fever virus
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130/207 Simian hemorrhagic (SHFV) and wobbly skunk disease virus (WPDV).
[152] In some embodiments, the sequence encoding the modified non-alphavirus RNA replicon further includes one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to a coding sequence for one gene of interest (GOI). In some embodiments, the modified non-alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes. In some embodiments, at least one of the one or more expression cassettes is operably linked downstream of the second nucleic acid sequence that encodes at least one non-structural viral protein or a portion thereof. In some embodiments, at least one of the one or more expression cassettes further comprises a third nucleic acid sequence that encodes one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for GOI. In some embodiments, the modified non-alphavirus RNA replicon further includes a coding sequence for an auto-protease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for the GOI.
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131/207 [153] Some embodiments of the disclosure refer to a nucleic acid molecule that includes a nucleic acid sequence that encodes a modified viral RNA replicon that includes in the 5'-> 3 'direction a first nucleic acid sequence that encodes a Sindbis virus capsid enhancer, a second nucleic acid sequence that encodes an auto-protease peptide, and a third nucleic acid sequence that encodes all viral non-structural proteins. Some embodiments of the disclosure refer to a nucleic acid molecule that includes a nucleic acid sequence that encodes a modified viral RNA replicon, wherein the modified viral RNA replicon comprises a viral capsid enhancer and in which the sequence of the replicon of Modified viral RNA exhibits at least 80% sequence identity for the sequence of at least one of SEQ ID NOs: 15 to 18 and 27 to 29.
[154] Variants of the polynucleotides provided in this document are contemplated within the scope of this disclosure. Such variants may be naturally occurring, which include homologous polynucleotides of the same or different species, or they may be unnatural variants, for example, polynucleotides synthesized using chemical synthesis methods, or generated using recombinant DNA techniques . Regarding the sequences of
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132/207 nucleic acid, genetic code degeneration provides the possibility to replace at least one base of the protein coding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Consequently, the nucleic acid molecules of the present disclosure can also have any base sequence that has been changed from any polynucleotide sequence disclosed in the present document by substitution according to genetic code degeneration. References describing codon usage are readily and publicly available. In additional embodiments, polynucleotide sequence variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (for example, changing codons in the viral mRNA to those preferred by other organisms such as mammalian species or fish).
[155] In some embodiments, the nucleic acid molecules of the present disclosure comprise in the 5 '> 3' direction a nucleic acid sequence that encodes a capsid enhancer from a Sindbis virus, a nucleic acid sequence that encodes a auto-protease peptide, and a nucleic acid sequence that encodes all viral non-structural proteins of a modified viral RNA replicon. In some modalities, the
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133/207 nucleic acid molecule comprises in the 5'-> 3 'direction a 5 UTR sequence, a first capsid enhancer from a Sindbis virus, an auto-protease peptide, a sequence that encodes all non-proteins viral structural features of a modified viral RNA replicon, one or more expression cassettes, and a 3 'UTR sequence, wherein at least one of the one or more expression cassettes comprises a second capsid enhancer from a virus Sindbis operably linked upstream of a coding sequence for a gene of interest (GOI).
[156] Consequently, in some embodiments, the nucleic acid molecule of the present disclosure includes a nucleic acid sequence that encodes a modified viral RNA replicon, where the sequence exhibits at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99% or 100% sequence identity for the sequence of at least one of SEQ ID NOs: 15 to 18 and 27 to 29.
[157] In some embodiments, the nucleic acid molecule of the disclosure is an expression vector. In some embodiments, the expression vector further includes one or more additional regulatory sequences, which can be a transcriptional regulatory element or a translational regulatory element. The terms regulatory element and regulatory region, as used interchangeably in this
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134/207 disclosure, refer to a sequence of nucleic acids that influences initiation of transcription or translation and rate, and stability and / or mobility of a transcription or translation product. Such regulatory elements need not be naturally occurring sequences. Regulatory sequences include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 'and 3' untranslated regions (RTUs), transcriptional start sites , termination sequences, polyadenylation sequences, introns and combinations thereof. In some embodiments, the expression vector of the disclosure further includes one or more of the following: an origin of replication, one or more sequences to promote integration of the expression cassette into the host genome, a terminator sequence.
[158] In some embodiments, the expression vector comprises at least one source of replication sequence (ORI) for replication in a cell. The vectors may additionally comprise, optionally, one or more selectable markers under the control of one or more eukaryotic promoters, one or more selectable markers under the control of one or more prokaryotic promoters, and / or one or more sequences that mediate recombination of a sequence of exogenous nucleic acids in the cell's genome
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135/207 target.
[159] An ORI is the sequence in a DNA molecule at which replication begins. ORI serves as a mounting base for complex pre-replication. Depending on the ORI, such replication can proceed unidirectionally or bidirectionally. An expression vector as presented herein may include an ORI for replicating the expression vector in a cloning host, such as E. coli or yeast, and / or may include an ORI for replicating the expression vector in a target cell , which can be, for example, a mammalian cell. The biological structure of ORIs is largely conserved among prokaryotes, eukaryotes and viruses. Most ORIs have simple tri-, tetra-, or higher nucleotide repeat patterns. Most are rich in AT and contain inverted repetitions. People skilled in the art will be familiar with the most common ORIs, such as P15A and pUC 'ORI.
[160] The expression vector can also, in some modalities, carry a selectable marker. For example, a vector that includes an expression cassette may include, as a selectable marker, a gene that confers resistance to a poisonous substance, such as an antibiotic, herbicide, or some other toxin, so that transformants can be selected exposing the cells to the poison and selecting those cells that survive
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136/207 to the meeting. In some embodiments, the selectable marker may be under the control of a promoter. In some embodiments, the promoter that regulates expression of the selectable marker may be conditional or inducible. In some embodiments, the promoter that regulates expression of the selectable marker may preferably be constitutive, and may be, for example, any promoter described in this document or another promoter.
[161] In some embodiments, the expression vector is a plasmid, a bacteriophage vector, a cosmid, a phosmid, a viral replicon, a carrier vector or a combination thereof. In some embodiments, the expression vector is an RNA replicon. In some embodiments, the expression vector is a prokaryote expression vector. In some embodiments, the expression vector is a eukaryotic expression vector. In some embodiments, the nucleic acid molecule of the disclosure is produced by de novo synthesis. In some embodiments of the disclosure, de novo synthesis can be used to generate a synthetic mRNA molecule.
Recombinant Cells [162] In one aspect, some of the modalities disclosed in this document refer to a method of transforming a cell that includes introducing into a host cell, such as an animal cell, a nucleic acid molecule
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137/207 as presented in this document, and select or screen for a transformed cell. The terms host cell and recombinant host cell are used interchangeably in this document. It is understood that such terms refer not only to the particular target cell, but also to the progeny or potential progeny of that cell. Due to the fact that certain modifications can occur in successive generations due to mutation or environmental influence, such a progeny may not, in fact, be identical to the parent cell, but still be included within the scope of the term as used herein. In some embodiments, the nucleic acid molecule is introduced into a host cell by an electroporation procedure or a biological procedure.
[163] In a related aspect, some embodiments refer to host recombinant cells, for example, animal recombinant cells that include a nucleic acid molecule described herein. The nucleic acid molecule can be stably integrated into the host genome, or it can be episomic replication, or present in the recombinant host cell as a minicircle expression vector for stable or transient expression. Consequently, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the host cell
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138/207 recombinant as an episomic unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be completed with the use of classical random genomic recombination techniques or with more accurate genome editing techniques such as the use of CRISPR / Cas9 guided by RNA guide, or NgAgo (Natronobacteríum gregoryi Argonaute) genome editing DNA-guided endonuclease, or TALEN genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule present in the recombinant host cell as an expression vector for stable or transient expression.
[164] In some embodiments, host cells can be genetically modified (for example, transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a vector for homologous recombination that includes sequences nucleic acid homologous to a portion of the host cell genome, or it can be an expression vector for the expression of any one or a combination of the genes of interest. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. In some modalities, a vector for expression of a
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139/207 polypeptide of interest can also be designed for integration into the host, for example, by homologous recombination. The vector containing a polynucleotide sequence as described herein, for example, a nucleic acid molecule comprising a modified alphavirus genome or replicon RNA, as well as, optionally, a selectable marker or reporter gene, can be employed to transform a appropriate host cell.
[165] The methods and compositions disclosed in this document can be deployed for genetic engineering of any species, which includes, without limitation, prokaryote and eukaryotic species. Host cells suitable for modification using the compositions and methods according to the present disclosure may, however, include, without limitation, algae cells, bacterial cells, heterocontes, fungal cells, chytrid cells, microfungi, microalgae and animal cells . In some embodiments, animal cells are cells of invertebrate animals. In some embodiments, the cells of vertebrate animals are mammalian cells. Host cells can be untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
[166] The methods and compositions disclosed in this document can be used, for example, with cells
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140/207 target species and / or hosts that are important or interesting for aquaculture, agriculture, animal husbandry, and / or for therapeutic and medical applications, which include production of polypeptides used in the manufacture of vaccines, pharmaceuticals, industrial products, chemicals and similar. In some embodiments, the compositions and methods disclosed in this document can be used with host cells of species that are natural hosts of alphavirus, such as rodents, mice, fish, birds and larger mammals such as humans, horses, pig, monkey and apes as well as invertebrates. Particularly preferred species, in some modalities of the application, are vertebrate animal species and invertebrate animal species. In principle, any animal species can generally be used and can be, for example, human, dog, bird, fish, horse, pig, primate, mouse, sigmodon, ferret, cattle, swine, sheep, rabbit, cat , goat, donkey, hamster or buffalo. Non-limiting examples of suitable bird species include chicken, duck, goose, turkey, ostrich, emu, swan, peacock, pheasant, partridge and guinea fowl. In some particular modalities, fish is any species in the Salmonidae family. Primary mammalian cells and continuous / immortalized cell types are also suitable. Non-limiting examples of suitable animal host cells
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141/207 include, but are not limited to, equine pulmonary artery endothelial cell, equine dermis cell, baby hamster kidney cell (BHK), rabbit kidney cell, mouse muscle cell, mouse connective tissue cell , human cervical cell, be human epidermoid laryngeal cells, Chinese hamster ovary (CHO) cell, human HEK-293 cell, mouse 3T3 cell, Vero cell, Madin-Darby canine kidney epithelial cell (MDCK ), primary chicken fibroblast cell, a HuT78 cell, lung cell A549, HeLa cell, PER.C6® cell, WI-38 cell, MRC-5 cell, FRhL-2, and CEM T cell. In some embodiments, the host cell is a baby hamster kidney cell. In some embodiments, the baby hamster kidney cell is a BHK-21 cell.
[167] Techniques for transforming a wide variety of the host cells and species mentioned above are known in the art and described in the technical and scientific literature. Consequently, cell cultures that include at least one recombinant cell as disclosed herein are also within the scope of that application. Suitable methods and systems for generating and maintaining cell cultures are known in the art.
Heterologous Nucleic Acid Sequences [168] According to some embodiments of the present disclosure, a wide variety of acid sequences
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142/207 nucleic acid may be carried by the nucleic acid molecules of the present disclosure. In some embodiments, nucleic acid molecules as described herein do not contain any additional heterologous nucleic acid sequences. In some embodiments, the nucleic acid molecules of the present disclosure contain one or more additional heterologous or exogenous nucleic acid sequences. In some embodiments, the one or more additional heterologous or exogenous nucleic acid sequences include a coding sequence for a gene of interest (GOI). In some embodiments disclosed herein, the coding sequence for GOI encodes a polypeptide or functional RNA. In some embodiments, the coding sequence for GOI encodes a functional RNA selected from a ribosomal RNA, a tRNA, a ribozyme, a transactivating RNA from a CRISPR system, a crispr RNA from a CRISPR system , a chimeric guide RNA from a CRISPR system, a micro RNA, an interfering RNA molecule (RNAi), a short clamp-shaped RNA (sh), or an antisense RNA molecule. In some embodiments, the coding sequence for GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide, or any combination
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143/207 of them. In some embodiments, the coding sequence for GOI encodes a polypeptide that is selected from the group consisting of an antibody, an antigen, an immune modulator and a cytokine.
[169] In some embodiments, the heterologous nucleic acid sequence comprises a heterologous nucleic acid sequence of at least about 100 bases, 2 kb, 3.5 kb, 5 kb, 7 kb or 8 kb. The heterologous RNA or heterologous nucleic acid sequence can be chosen from a wide variety of sequences derived from viruses, prokaryotes or eukaryotes. Examples of categories of heterologous sequences include, but are not limited to, immunogens (which include native, modified or synthetic antigenic proteins, peptides, epitopes or immunogenic fragments), cytokines, toxins, therapeutic proteins, enzymes, antisense sequences and immune response modulators.
[170] A wide variety of GOI immune response can be included in the nucleic acid molecules of the present disclosure to express a GOI polypeptide, which includes, without limitation, cytokines, toxins, prodrugs, antigens that stimulate an immune response, ribozymes, and proteins that assist or inhibit an immune response, as well as antisense sequences (or sense sequences for antisense applications). As noted above, within
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144/207 various embodiments of the disclosure the modified RNA replicon shown herein may contain the coding region for (and express, in some embodiments) two or more polypeptides of interest.
1) Cytokines [171] In some embodiments disclosed in this document, GOI encodes a cytokine. In general, cytokines act to proliferate, activate and / or differentiate immune effector cells. Examples of cytokines include, but are not limited to, macrophages, B lymphocytes, T lymphocytes, endothelial cells, fibroblasts, lymphokines such as gamma interferon, tumor necrosis factor, interleukin, IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1, IL-12, IL-13, IL-14, IL-15, GM-CSF, CSF-1 and G-CSF.
[172] In some related modalities, GOI encodes an immunomodulatory cofactor. As used within the context of the present disclosure, immunomodulatory cofactor refers to factors that, when manufactured by one or more of the cells involved in an immune response, or when added exogenously to the cells, causes the immune response to be different in quality or power that would have occurred in the absence of the cofactor. The quality or potency of a response can be measured by a variety of assays known to a person skilled in the art that include, for example, ex vitro assays that
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145/207 measure cell proliferation (for example, 3 H thymidine absorption), and ex vitro cytotoxic assays (for example, which measure Cr 51 release) (see Warner et al., AIDS Res. And Human Retroviruses 7: 645 to 655, 1991).
[173] Examples of immunomodulatory cofactors include, but are not limited to, alpha interferon, gamma interferons, G-CSF, GM-CSF, TNFs, Interleukin-2 (IL-2), IL-4, IL6, IL-12, IL -15, ICAM-1, ICAM-2, LFA-1, LFA-3, MHC class I molecules, MHC class II molecules, 2-microglobulin, chaperones, CD3, B7 / BB 1, MHC-bound carrier proteins and analogs of themselves.
[174] The choice of which immunomodulatory cofactor to include within the nucleic acid molecules of the present disclosure can be based on known therapeutic effects of the cofactor, or determined experimentally. For example, in chronic hepatitis B infections, alpha interferon has been found to be effective in compensating for a patient's immune deficit and thereby assist in recovery from the disease. In some situations, a suitable immunomodulatory cofactor can be determined experimentally. Briefly, blood samples are obtained first from patients with liver disease. Peripheral blood lymphocytes (PBLs) are restimulated ex vitro with autologous or matched HLA cells (for example, transformed EBV cells), and
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146/207 transduced with modified arterivirus genome or replicon RNA of the present disclosure that directs the expression of an immunogenic portion of a hepatitis antigen and the immunomodulatory cofactor. Stimulated PBLs are used as effectors in a CTL assay with the BLA-transduced cells matched as targets. An increase in CTL response over that seen in the same assay performed with the use of a corresponding HLA stimulator and target cells transduced with a vector that encodes the antigen alone, indicates a useful immunomodulatory cofactor. In some embodiments, the interferon gamma immunomodulatory cofactor is particularly preferred.
[175] Another non-limiting example of an immunomodulatory cofactor is the co-stimulating factor B7 / BB1. The activation of the complete functional activity of T cells requires two signals. One signal is provided by interaction of the antigen-specific T cell receptor with peptides that are bound to complex major histocompatibility (MHC) molecules, and the second signal, called co-stimulation, is delivered to the T cell by cells that have antigen. The second signal is required for the production of interleukin-2 (IL-
2) by T cells and seems to involve interaction of the B7 / BB 1 molecule in cells that have antigen with CD28 and CTLA-4 receptors on T lymphocytes. In some modalities, B7 / BB 1 can be introduced into tumor cells in order to provoke
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147/207 co-stimulation of CD8 + T cells, so that the CD8 + T cells produce enough IL-2 to expand and become fully activated. These CD8 + T cells can kill tumor cells that are not expressing B7 because cosimulation is no longer required for additional CTL function. Vectors that express both the co-stimulating B7 / BB1 factor and, for example, an immunogenic HBV core protein, can be constructed using methods that are described in this document. The cells transduced with these vectors will become more effective cells that have antigen. The specific CTL response of the HBV nucleus will be increased from the fully activated CD8 + T cell through the co-stimulator ligand B7 / BB 1.
2) Toxins [176] In some embodiments disclosed in this document, GOI encodes a toxin. In some embodiments, toxins act to directly inhibit the growth of a cell. Examples of toxins include, but are not limited to, ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed, antiviral protein, tritine, Shigella toxin, Pseudomonas A exotoxin, herpes simplex virus thymidine kinase (HSVTK), and phosphoribosyl guanine transferase from E. coli ..
3) Prodrugs [177] In some modalities disclosed in the present
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148/207 document, the GOI encodes a prodrug. As used within the context of the present disclosure, prodrug refers to a gene product that activates a compound with little or no cytotoxicity in a toxic product. Representative examples of such gene products include HSVTK and VZVTK (as well as analogs and derivatives thereof), which selectively monophosphorylates certain purine arabinosides and substituted pyrimidine compounds, which convert them to cytotoxic or cytostatic metabolites. More specifically, exposure of the drugs ganciclovir, acyclovir or any of their analogs (for example, FIAU, FIAC and DHPG) to HSVTK phosphorylates the drug to its corresponding active nucleotide triphosphate form.
[178] Non-limiting examples of prodrugs that can be used within the context of the present disclosure include: E. coli phosphoribosyl guanine transferase. which converts thioxanthine to toxic thioxanthin monophosphate; alkaline phosphatase, which will convert inactive phosphorylated compounds, such as mitomycin phosphate and doxorubicin phosphate to toxic dephosphorylated compounds; fungal (eg, Fusarium oxysporum) and bacterial cytosine deaminase, which can convert 5-fluorocytosine to the toxic compound 5-fluorouracil; carboxypeptidase G2, which will cleave glutamic acid from to N-bis (2chloroethyl) aminobenzoyl glutamic acid thereby creating a
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149/207 toxic benzoic acid mustard; and Penicillin-V amidase, which will convert phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compounds.
4) Antisense sequence [179] In some embodiments disclosed in this document, the coding sequence for GOI is an antisense sequence. Antisense sequences are designed to bind to RNA transcripts, thereby preventing cellular synthesis of a particular protein or preventing the cell from using that RNA sequence. Non-limiting examples of such sequences include antisense thymidine kinase, antisense dihydrofolate reductase, antisense HER2, antisense ABL, antisense Myc, antisense ras, as well as antisense sequences that block any of the enzymes in the nucleotide biosynthetic pathway. In addition, according to some modalities disclosed in this document, antisense sequences for interferon and 2 microglobulin can be used in order to decrease the immune response.
[180] In some embodiments, antisense RNA can be used as an antitumor agent in order to induce a potent Class I restricted response. In addition to binding RNA and thereby preventing translation of a specific mRNA, high levels of specific antisense sequences are believed to induce increased expression of interferons (which include gamma-interferon) due to the formation of large
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150/207 amounts of double-stranded RNA. The increased expression of gamma interferon, in turn, reinforces the expression of MHC Class I antigens. Preferred antisense sequences for use in this regard include actin RNA, myosin RNA and histone RNA. Anti-sense RNA that forms an incompatibility with actin RNA is particularly preferred.
5) Ribozymes [181] In some embodiments disclosed herein, nucleic acid molecules are provided that comprise one or more RNA loop-stem structures that produce ribozymes upon infection of a host cell. Ribozymes are used to cleave specific RNAs and are designed so that they can affect only one specific RNA sequence. In general, the substrate binding sequence of a ribozyme is between 10 and 20 nucleotides in length. The length of this sequence is sufficient to allow hybridization with target RNA and disassociation of the ribozyme from the cleaved RNA. Representative examples for creating ribozymes include those described in U.S. Patents 5,116,742; 5,225,337 and 5,246,921.
6) Proteins and Other Cellular Constituents [182] In some embodiments disclosed in this document, a wide variety of proteins or other cellular constituents can be carried by the molecules
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151/207 nucleic acid of the present disclosure. Non-limiting examples of such proteins include native or altered cellular components, as well as exogenous cellular proteins or constituents, found, for example, in viruses, bacteria, parasites, fungi or animals such as mammals.
Methods for Producing Polypeptides [183] The host cells of the present disclosure, such as a prokaryotic or eukaryotic host cell, can be used to produce (for example, express) a molecule of interest such as, for example, a polypeptide, encoded in a open reading frame of a gene of interest (GOI) as disclosed in this document. Thus, the present application further provides methods for producing a molecule of interest, such as, for example, a polypeptide, using the host cells and / or nucleic acid molecules of the present disclosure. Host cells can be, for example, isolated cells, cells in cell culture, cells in a living body, or a combination thereof.
[184] Some modalities disclosed in this document provide methods for producing a polypeptide of interest. The method may include introducing a nucleic acid molecule according to any of the aspects and modalities of the present disclosure into a host cell, thereby producing a polypeptide encoded by GOI
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152/207 in the host cell. In some embodiments in which the introduced nucleic acid molecule is an RNA molecule, for example, an mRNA molecule or an RNA replicon. The RNA molecule can be generated by any method known in the art, for example, by de novo synthesis in whole or in part. For example, RNA molecules, which include, but are not limited to, mRNA molecules and RNA replicons, can be produced using chemical methods, enzymatic techniques, or any combination thereof, for example, by chemical synthesis using re-assembly (such as with oligonucleotides) or in vitro transcription reactions (using appropriate enzymes, buffers, nucleotides, etc.). In some cases where the introduced nucleic acid molecule is an mRNA, the mRNA can be delivered directly to cells in vivo to produce a polypeptide of interest (for example, drug, antigen, etc.) in cells. The cells can be isolated cells; cells in cell cultures; cells in a tissue, an organ and / or an individual; or any combination thereof. In some embodiments, no new copies of mRNA are produced in the cells. As disclosed in this document, incorporation of one or more RNA stem-loop structures from a viral capsid enhancer (e.g., DLP motifs) into chemically synthesized RNA can confer the desired gene expression enhancement
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153/207 once the DLP-containing mRNA is introduced into the cells.
[185] In some embodiments where the introduced nucleic acid molecule is a vector such as, for example, an RNA replicon, new copies of mRNA may be generated that include coding sequence for a gene of interest operably linked to one or more DLP motifs. The incorporation of one or more DLP motifs into the vector, for example, RNA replication, can then confer the desired gene expression enhancement once the DLP-containing vector or replicon is introduced into cells [186]. in this document provide methods for producing a polypeptide of interest in a host cell. Such a method includes culturing a recombinant host cell, which includes a nucleic acid molecule according to any of the aspects and modalities of the present disclosure. In some embodiments, the methods include culturing the host cell of the present disclosure (in which a recombinant expression vector encoding the molecule of interest has been introduced) in a suitable medium so that the molecule of interest is produced. In some embodiments, the methods also include isolating the molecule of interest from the medium or the host cell.
[187] Methods are also disclosed for producing a polypeptide of interest in an individual, which include
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154/207 administer to the individual a nucleic acid molecule, according to any of the aspects and modalities.
[188] Suitable host cells and / or individuals for use in the methods and compositions disclosed in this document include, without limitation, prokaryote and eukaryotic species. Suitable host cells to be modified using the compositions and methods, in accordance with the present disclosure, may, however, include, without limitation, algae cells, bacterial cells, heterocontes, fungal cells, chytridic cells, microfungi, microalgae and animal cells . In some embodiments, animal cells are cells of invertebrate animals. In some embodiments, the cells of vertebrate animals are mammalian cells. Host cells can be untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. Consequently, biological samples, biomass and progeny of a recombinant cell, according to any of the aspects and modalities are also within the scope of the present application. Thus, as discussed in more detail below, polypeptides produced by a method, in accordance with this aspect of the application, are also within the scope of that application.
[189] In some embodiments, the recombinant cell is an animal cell. Production of therapeutic protein in
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155/207 small and large scale is an important field of development in the pharmaceutical industry, due to the belief that proteins produced in animal cells, in general, have post-translational modification with appropriate processing and, therefore, have adequate activity for the treatment of physiological condition. In principle, any animal species can generally be used and can be, for example, human, dog, bird, fish, horse, pig, primate, mouse, sigmodon, ferret, cattle, swine, sheep, rabbit, cat , goat, donkey, hamster or buffalo. Non-limiting examples of suitable bird species include chicken, duck, goose, turkey, ostrich, emu, swan, peacock, pheasant, partridge and guinea fowl. In some particular modalities, fish is any species in the Salmonidae family. Primary mammalian cells and continuous / immortalized cell types are also suitable. Non-limiting examples of suitable animal host cells include, but are not limited to, equine pulmonary artery endothelial cell, equine dermis cell, baby hamster kidney cell (BHK), rabbit kidney cell, mouse muscle cell, mouse connective tissue cell, human cervical cell, human epidermoid laryngeal cells, Chinese hamster ovary (CHO) cell, human HEK-293 cell, mouse 3T3 cell, Vero cell, canine kidney epithelial cell in
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156/207
Madin-Darby (MDCK), primary chicken fibroblast cell, a HuT78 cell, lung A549 cell, HeLa cell, PER.C6® cell, WI-38 cell, MRC-5 cell, FRhL-2, and EMC T cell . In some embodiments, the host cell is a baby hamster kidney cell. In some embodiments, the baby hamster kidney cell is a BHK-21 cell.
Recombinant Polypeptides [190] Some modalities disclosed in this document refer to recombinant polypeptides produced by a method, according to one or more modalities described in this document. The recombinant polypeptides of the present application can, in general, be any recombinant polypeptides and can be, for example, one or more among therapeutic polypeptides, prophylactic polypeptides, diagnostic polypeptides, nutraceutical polypeptides, industrial enzymes and reporter polypeptides. In some embodiments, the recombinant polypeptides may be one or more among antibodies, antigens, immune modulators and cytokines. In some embodiments, the polypeptide of interest may have therapeutic or prophylactic activity.
Compositions and Formulations [191] Some embodiments disclosed herein refer to a composition comprising any of the recombinant polypeptides described herein
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157/207 document. The composition can be, for example, a nutraceutical composition, a prophylactic composition, a pharmaceutical composition comprising a pharmaceutically acceptable carrier or a mixture thereof. In some embodiments, the compositions of the present application can be used as a vaccine.
[192] Some embodiments disclosed in this document refer to a composition that includes any of the nucleic acid molecules (for example, expression vectors) described in this document. The composition can be, for example, a nutraceutical composition, a prophylactic composition, a pharmaceutical composition comprising a pharmaceutically acceptable carrier or a mixture thereof. In some embodiments, the compositions of the present application can be used as a vaccine.
[193] Some embodiments disclosed in this document refer to a composition that includes any of the recombinant cells described in this document. The composition can be, for example, a nutraceutical composition, a prophylactic composition, a pharmaceutical composition comprising a pharmaceutically acceptable carrier or a mixture thereof. In some embodiments, the compositions of the present application can be used as a vaccine.
[194] As used in this document, the term
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158/207 pharmaceutically acceptable carrier means a carrier that is useful for preparing a pharmaceutical composition or formulation that is, in general, safe, non-toxic and not biologically or otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. In some embodiments, a pharmaceutically acceptable carrier is as simple as water, but it can also include, for example, a physiological saline solution. In some embodiments, a pharmaceutically acceptable carrier may be, or may include, stabilizers, diluents and buffers. Suitable stabilizers are, for example, SPGA, carbohydrates (such as powdered milk, serum albumin or casein) or degradation products thereof. Suitable buffers are, for example, alkali metal phosphates. Diluents include water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol). For administration to animals or humans, the composition according to the present application can be given by any enteral or parenteral route, which includes, inter alia, intranasally, by spraying, intradermally, subcutaneously, orally, by aerosol, intramuscularly or any combination thereof.
[195] In some embodiments, nucleic acid molecules (for example, mRNAs and / or expression vectors),
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159/207 protein molecules and / or compositions of the disclosure are in suitable formulations, for example, pharmaceutical formulations. Pharmaceutical formulations containing one or more of the molecules and / or compositions disclosed herein are provided in a pharmaceutically acceptable carrier in the present document. Some disclosure modalities refer to pharmaceutical formulations that comprise one or more of the expression vectors disclosed in this document. Some embodiments of the disclosure refer to pharmaceutical formulations containing one or more of the nucleic acid molecules disclosed herein.
Some disclosure modalities refer to pharmaceutical formulations containing one or more of the polypeptides disclosed herein. Some embodiments of the disclosure refer to pharmaceutical formulations containing one or more of the recombinant cells disclosed herein.
[196] The molecules (for example, protein and nucleic acid molecules) and compositions disclosed herein can be in various formulations, for example, pharmaceutical formulations. For example, nucleic acid molecules (for example, replicons, mRNAs and expression vectors), protein molecules and / or compositions of the disclosure can be formulated, for example, in a pharmaceutical formulation, with one or more compounds
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160/207 covalent (for example, by means of direct bonding), non-covalent compounds (for example, through combinations of LNP-charged base or cationic nanoemulsions), physical compositions (for example, vault proteins, unloaded lipid encapsulations ), pharmaceutically acceptable buffers (for example, saline, Ringer's lactate), and any combinations thereof. Many suitable methods, reagents and systems for generating the aforementioned pharmaceutical formulations are known in the art.
[197] In some embodiments, molecules and / or compositions disclosed in this document are formulated in a saline or lipid formulation. The lipid formulation can be selected from, however, without limitation, liposomes, lipoplexes, copolymers such as PLGA, and lipid nanoparticles.
Particles and Nanoparticles [198] In some embodiments, one or more of the nucleic acid molecules, polypeptide molecules and / or compositions disclosed herein may be incorporated into particles or nanoparticles. Particles comprising one or more of the molecules and compositions disclosed in this document may be polymeric particles, lipid particles, solid lipid particles, self-assembled particles, composite nanoparticles
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161/207 of conjugated phospholipids, surfactants, proteins, polyamino acids, inorganic particles or combinations thereof (for example, polymeric particles stabilized by lipids). In some embodiments, the molecules and / or compositions disclosed herein are substantially encapsulated or partially encapsulated in the particles. In some embodiments, the molecules and / or compositions disclosed in this document are deposited and / or absorbed on the surface of the particles. In some embodiments, the molecules and / or compositions disclosed in this document are incorporated into the particles. In some embodiments, the molecules and / or compositions disclosed in this document are part of or a component of the particle. The molecules and / or compositions of the development can, in some modalities, be fixed to the surface of the
particles with connections covalent or interactions no covalent. In some modalities, at molecules and / or compositions of self-assembling revelation in an particle.[199] As used in the present document, the term encapsulate ' ' means for delimi tar, encircle or
to involve. As it relates to the formulation of the molecules and / or compositions of the present disclosure, encapsulation can be substantial, complete or partial. The term encapsulated substantially means that at least more than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%,
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97%, 98%, 99%, 99.9%, 99.99% or 99.999% of the molecules and / or compositions of the present disclosure can be delimited, surrounded or enveloped within the particle. Partial encapsulation means that less than 10%, 15%, 20%, 30%, 40%, 50% of the molecules and / or compositions of the present disclosure can be bounded, surrounded or enveloped within the particle. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 99.999% of the molecules and / or compositions of the present disclosure are encapsulated in the particle. Encapsulation can be determined by any known method.
[200] In some embodiments, the particles are polymeric particles or contain a polymeric matrix. The particles can, in general, contain any of the polymers known in the art. The particles, in general, will contain one or more biocompatible polymers. The polymers can be biodegradable polymers. The polymers can be hydrophobic polymers, hydrophilic polymers or amphiphilic polymers. In some embodiments, the particles contain one or more polymers that have an additional target chemical portion attached to them. In some embodiments, the particles are inorganic particles, such as, without limitation, gold nanoparticles and iron oxide nanoparticles.
[201] The particle size can be adjusted
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163/207 for the intended application. The particles can be nanoparticles or microparticles. The particle can have a diameter of about 10 nm to about 10 microns, about 10 nm to about 1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm or about 25 nm at about 250 nm. In some embodiments, the particle is a nanoparticle that has a diameter of about 25 nm to about 250 nm. In some embodiments, the particle is a nanoparticle that has a diameter of about 50 nm to about 150 nm. In some embodiments, the particle is a nanoparticle that has a diameter of about 70 nm to about 130 nm. In some embodiments, the particle is a nanoparticle that has a diameter of about 100 nm. It is understood by those skilled in the art that a plurality of particles will have a size range and it is understood that the diameter is the median diameter of the particle size distribution.
[202] In some embodiments, the molecules and / or compositions disclosed in this document can be incorporated into particles that are responsive to temperature, pH and ionic conditions. For example, the particles may comprise an ionizable network of homopolymeric ionizable monomers covalently crosslinked where the ionizable network is covalently attached to a single terminal region of an amphiphilic copolymer to form a
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164/207 plurality of suspended chains and wherein the suspended chains of amphiphilic copolymer form immobile intraret aggregates in aqueous solution, as disclosed in U.S. Patent No. 7,204,997.
Liposomes, Lipoplexes, and Lipid Nanoparticles (LNPs) [203] The molecules and / or compositions of the disclosure can be formulated using one or more liposomes, lipoplexes and / or lipid nanoparticles. In one embodiment, pharmaceutical formulations of the disclosure molecules and / or compositions include liposomes. Liposomes are artificially prepared vesicles that can primarily be composed of a lipid bilayer and can be used as a transport vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but without limitation, a multilamellar vesicle (MLV) that can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) that it can be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) that can be between 50 and 500 nm in diameter. Liposome design may include, however, without limitation, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but without limitation,
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165/207 endocytosis. Liposomes can contain a low or a high pH in order to improve the delivery of pharmaceutical formulations.
[204] The formation of liposomes may depend on physico-chemical characteristics such as, without limitation, the retained pharmaceutical formulation and liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the retained substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, polydispersity and the expiry date of the vesicles for the intended application, and the reproducibility between batches and the possibility of large production range of safe and efficient liposomal products.
[205] In some embodiments, the molecules and / or compositions of the disclosure may be formulated in a lipid vesicle that may have cross-links between functionalized lipid bilayers. In some embodiments, the disclosure molecules and / or compositions can be formulated in a lipid-polycation complex. The formation of the lipid-polycyclic complex can be achieved by methods known in the art. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, however, without limitation, polylysine, polyanitine and / or polyarginine and cationic peptides. In some
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166/207 embodiments, the nucleic acid molecules and / or compositions disclosed herein can be formulated into a lipid-polycation complex that may further include a neutral lipid such as, however, without limitation, cholesterol or dioleyl phosphatidylethanolamine (DOPE). The liposome formulation can be influenced, however, without limitation, by the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation ratio of all components and biophysical parameters such as size.
[206] In some embodiments, the PEG ratio in lipid nanoparticulate (LNP) formulations can be increased or decreased and / or the carbon chain length of the lipid PEG can be changed from C14 to C18 to change the pharmacokinetics and / or biodistribution of LNP formulations. As a non-limiting example, LNP formulations can contain 1 to 5% of the molar lipid ratio of PEG-c-DOMG when compared to cationic lipid, DSPC and cholesterol. In another embodiment, PEG-cDOMG can be replaced by a PEG lipid such as, but without limitation, PEG-DSG (1,2-Distearoil-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoil -snglycerol, methoxy polyethylene glycol). The cationic lipid can be selected from any lipid known in the art such as, but without limitation, DLin-MC3-DMA,
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DLin-DMA, C12-200 and DLin-KC2-DMA.
[207] In some embodiments, LNP formulations described in this document may comprise a polycationic composition. In some embodiments, LNP formulations that comprise a polycationic composition can be used for the delivery of the modified RNA described herein in vivo and / or ex vitro. In some embodiments, the LNP formulations described in this document may further comprise a permeability enhancing molecule. The nanoparticle formulations can be a carbohydrate nanoparticle comprising a carbohydrate carrier and a modified nucleic acid molecule (for example, mRNA). As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phyto-glycogen or glycogen-like material, octenyl succinate phyto-glycogen, betadextrin phyto-glycogen, and anhydride-modified beta-dextrin phyto-glycogen.
[208] Lipid nanoparticle formulations can be improved by replacing the cationic lipid with a biodegradable cationic lipid that is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, without limitation, DLinDMA, DLin-KC2-DMA and DLin-MC3-DMA, have
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168/207 shown that they accumulate in plasma and tissues over time and can be a potential source of toxicity. The rapid metabolism of lipids eliminated quickly can improve the tolerability and therapeutic index of lipid nanoparticles by an order of magnitude from a dose of 1 mg / kg to a dose of 10 mg / kg in rats. The inclusion of an enzymatically degraded ester bond can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester bond can be located internally within the lipid chain or it can be located terminally at the terminal end of the lipid chain. The internal ester bond can replace any carbon in the lipid chain.
[209] Additional information related to cationic lipids suitable for LNP formulations can be found, for example, in U.S. Publication No. 2017/0151339, which is incorporated in its entirety into this document for reference.
[210] The molecules and / or compositions of the disclosure can also be formulated as a nanoparticle using a combination of polymers, lipids and / or other biodegradable agents, such as, without limitation, calcium phosphate. Components can be combined in a core-shell, hybrid and / or layer-by-layer architecture, to allow
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169/207 fine adjustment of the nanoparticle so that the delivery of the molecules and / or compositions of the development can be improved.
[211] The pharmaceutical formulations of the present disclosure may furthermore comprise one or more pharmaceutically acceptable excipients, which, as used herein, include any and all of solvents, dispersion media, diluents or other liquid vehicles, dispersion aids or suspension, surfactants, isotonic agents, thickening and emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. More information about this can be found in Remington's The Science and Practice of Pharmacy, 21 Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) which discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Except to the extent that any conventional excipient medium is incompatible with a substance or its derivatives, such as due to producing any undesirable biological effect or otherwise interacting in a harmful manner with any other component (or components) of the pharmaceutical composition, its use is contemplated within the scope of that revelation.
Examples [212] Additional alternatives are revealed in
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170/207 additional details in the following examples, which are in no way intended to limit the scope of the claims.
Example 1
General Experimental Procedure
DNA Model Preparation [213] Plasmid DNA models were purified (Qiagen Catalog Number 12163) from 300 ml of saturated E. coli TransforMax Epi300 cultures (Epicenter Catalog Number EC300105) grown in LB broth (Teknova Catalog number L8000 06) supplemented with 50ng / ml carbamicillin (Teknova Catalog number NC9730116). Plasmid DNA was linearized by NotI digestion (New England Biolabs NEB number in catalog R3189S) for one hour at 37 ° C. The linearized model DNA was then repurified (Zymo Number in catalog D4003), and analyzed by 0.8% agarose gel (Life Technologies Catalog number G5018-08) against a commercial 2-log DNA ladder (New England Biolabs, NEB Catalog number N3200S). The presence of a single band was confirmed in each sample, corresponding to the expected fragment size of the linear DNA model, before proceeding with ex vitro transcription.
Ex vitro transcription [214] Ex vitro transcription (IVT) reactions were performed using 1 pg of DNA model prepared as
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171/207 described above, in a 20 μΐ reaction over a one hour incubation at 37 ° C (NEB number in catalog E2065S). 1 DNase I unit, supplied by the supplier, was then added directly to the IVT reaction, and incubated at 37 ° C for an additional 15 minutes. Reactions were then placed on ice, and purified using the method suggested by the manufacturers (Qiagen Number in catalog 74104). Purified RNA was then quantified using a NanoDrop 2000c UV-Vis Spectrometer. The RNA was visualized by electrophoresis through 0.8% Agarose gels (Life Technologies Number in the catalog G5018-08) and compared with Millennium RNA Marker (Ambion Number in the catalog AM7150), before proceeding with the electroporation.
Transfection and Analysis [215] In a typical cell transfection experiment, replicon RNA was introduced into BHK-21 cells by electroporation using the SF Cell Line Nucleofector ™ kit for the 4D-Nucleofector ™ System (Lonza). BHK-21 cells were harvested using 0.25% trypsin and washed once with cold PBS. The cells were resuspended in Tamponante SF at a cell density of 1 χ 10 ⁶ cells per 20 pL of electroporation reaction. Three micrograms of RNA were electroporated into cells in triplicate in a 16-well cuvette strip and incubated at room temperature for 10 minutes. Electroporated cells were covered in plates
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172/207 containing Dulbecco's Modified Eagle Medium containing 10% serum
fetal bovine, followed by incubation per 16 to 18 H in cell culture conditions pattern. [216] Intracellular analyzes in efficiency in replicon transfection and production in protein were
flow cytometry. In these assays, transfected BHK-21 cells were fixed and permeabilized using fix / perm concentrate and permeabilization buffer (eBioscience). The cells were then incubated with antibodies to produce double-stranded RNA (monoclonal antibody J2 anti-dsRNA IgG2A, English & Scientific Company) conjugated to R-Ficoeritrina (Innova Biosciences). Antigen production was assessed by further incubation with PECy5-conjugated antigen-specific antibodies (Innova Biosciences) (eg antibodies to Firefly red, Renilla green, HA or RSV-FO (Abeam)). The cells were then washed once and analyzed using a FACSAria ™ Fusion Cell Classifier (BD Biosciences) or FACSAria ™ Cell Classifier II (BD Biosciences). Transfected BHK-21 cells stained with unique colors for compensation controls were performed before sample collection. Data were collected using FACSDiva (BD Biosciences) and further analyzed using FlowJo software. Initial channeling was performed to exclude dead cells and impurities with the use of wefts
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173/207 dispersed front and side. Additional channeling was conducted to identify cell populations that were positive for expression of both dsRNA (R-PE-positive) and protein (PE-Cy5-positive or FITC-positive for GFP expression). Frequency and average fluorescence intensities were collected and used for comparison and construct optimization.
Example 2
Construction of EAV Replication Projects containing DLP [217] This Example describes the generation of a number of expression vectors based on arterivirus RNA replicon with a DLP motif operably positioned upstream of the nonprotein polyprotein / protein genes structural and / or a reporter gene. These arterivirus RNA replicon-based expression vectors were further characterized and analyzed in the flow cytometry analysis and essential luciferase analyzes described in Example 4.
A. Project [218] The respective design features of four EV-based DLP replicon constructs are described below.
(1) rEX-DLP-rFF [219] In this construct, a DLP motif was placed immediately upstream from rFF and downstream from TRS7 that directs rFF transcription.
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174/207 (2) rEX-DLP-pplab-rFF [220] In this construct, a DLP motif was placed immediately upstream of the pplab genes with few careful design modifications described below to maintain the stem-loop structure at 5 ' The replicon RTU is known to be essential for subgenomic mRNA replication and transcription.
[221] (i) The first 79 nucleotides of the non-structural viral gene la are duplicated with their initial codon mutated from ATG to TAG, denoted as the ATG displacement region (in bold in the sequence of SEQ ID NO: 2 below).
[222] (ü) The corresponding nucleotides, located upstream of the la gene, in base pairing with their initial codon ATG and forming the stem, have also been appropriately changed from CAT to CTA (underlined in the sequence of SEQ ID NO: 2 below ).
[223] (iü) DLP (in italics in the sequence below) was placed immediately downstream of the ATG displacement region and upstream of the polyprotein lab genes (initial ATG codon shown in the sequence of SEQ ID NO: 2 below).
SEQ IP NO: 2 (partial sequence)
CGAAGTGTGTATGGTGCCATATACGGCTCACCACCATATACACTGCAAGAATTACTATT CTTGTGGGCCCCTCTCGGTAAATCCTAGAGGGCTTTCCTCTCGTTATTGCGAGATTCGT CGTTAGATAACGGCAAGTTCTCTGTAGTGTGT
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GTCACTGCCTACGTCGTCGATCTCTATCAACTACCCTTGCGACTTAGGCAACCTTCTCC
GO TAC TGGAT T TGGAGGGAGT T T TGT TAGGGAC TGGTCCC TGGAC T TACCCGACGC T TG
TGAGCATAGTCAGCATAGTACATTTCATCTGACTAATACTACAACACCACCACCATGAA TAGAGGATTCTTTAACATGCTCGGCCGCCGCCCCTTCCCGGCCCCCACTGCCATGTGGA GGCCGCGGAGAAGGAGGCAGGCGGGGGGGGG
[224] This construct was essentially identical to the second construct, in which DLP was placed following the same three design modifications, except that a 2A protease sequence (SEQ ID NO: 3) was added immediately at the 3 'end of DLP so that, when transferred, the polyproteins could be released from the peptide derived from DLP through a selective divease by the protease. A comparative analysis of replicon performances of Construct 2 (described above) and Construct 3 would provide information on the need for protease 2A for a functional replicon (see Example 4 below).
SEQ IP NO: 3 GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCC TGGACCT (4) rEX-DLP-2A-pplab-DLP-rFF [225] This construct was essentially identical to the third construct described above, except that another DLP was placed immediately upstream of the rPP gene the same way that a DLP motif was placed in the construct
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1). A comparative analysis of replicate performances of Construct 3 (described above) and Construct 4 would provide information on whether the placement of additional DLP upstream of the reporter gene has an added value to the expression of the reporter gene.
B. Construction [226] rEx-DLP-rFF was built according to a 3-piece Gibson Assembly® procedure described in Gibson et al. (Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343 to 345, 2009) with rEx-rFF (c4; SEQ ID NO: 34) digested with SphI and EcoRI as a vector and a g block containing DLP as an insert. The nucleic acid sequence of the g block used to construct rEx-DLP-rFF is set out in SEQ ID NO: 4 in the Sequence Listing.
[227] The following primers were designed to amplify the corresponding fragments required to construct the 3 new DLP replicon constructs based on EV described above.
Table 3
Start Initiators designed to build DLP- (2A) -pplab-rFF / DLP-rFF replicons RP114 ppla-DLP-F GCCATGTGGAGGCCGCGGAGAAGGAGGCAGGCGGCCCCGATGATGGCAACCTTCTCCGCTACTGGAT (SEQ ID NO: 5) RP115 pBR322-3'SrfI-R ACAATGTTGCCTCCCACATCTGCAA (SEQ ID NO: 6)
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RP116 pBR322-3'SrfI-F GGGTCACAAGGTAGTCGCCGTGGTT (SEQ ID AT THE: 7) RP117 pBR322-bla-R ACGTCAGGTGGCACTTTTCGGGGAA (SEQ ID AT THE: 8) RP118 ppla-DLP-2A-F AGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAG AACCCTGGACCTATGGCAACCTTCTCCGCTACTGG AT (SEQ ID NO: 9)
Construction of rEx-DLP-pplab-rFF [228] For the construction of the rEx-DLP-pplab-rFF vector, three nucleic acid fragments were generated using a 3-piece Gibson Assembly® procedure, as follows.
[229] Fragment 1 was generated with primers RP114 and RP115 and the heart of the rEx-rFF model.
[230] Fragment 2 was generated with primers RP116 and RP117 and the heart of the rEx-rFF model.
[231] Fragment 3 was a g block for rEx-DLPpplab-rFF with the nucleic acid sequence set out in SEQ ID NO: 10 in the Sequence Listing.
Construction of rEx-DLP-2A-pplab-rFF [232] For the construction of the rEx-DLP-2A-pplabrEE vector, three nucleic acid fragments were generated using a 3-piece Gibson Assembly® procedure, as follows.
[233] Fragment 4 was generated with primers RP118 and RP115 and the heart of the rEx-rFF model.
[234] Fragment 5 was generated with primers RP116 and RP117 and the heart of the rEx-rFF model.
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178/207 [235] Fragment 6 was a g block for rEx-DLP-2Applab-rFF with the nucleic acid sequence set out in SEQ ID NO: 11 in the Sequence Listing.
Construction of rEx-DLP-2A-pplab-DLP-rFF [236] For the construction of the rEx-DLP-2A-pplabDLP-rFF vector, three nucleic acid fragments were generated using a 3-piece Gibson Assembly® procedure , as follows.
[237] Fragment 7 was generated with primers RP118 and RP115 and the heart of the rEx-DLP-rFF model.
[238] Fragment 8 was generated with RP116 and RP117 primers and the heart of the rEx-DLP-rFF model.
[239] Fragment 9 was a g block for rEx-DLP-2Applab-DLP-rFF with the nucleic acid sequence set out in SEQ ID NO: 12 in the Sequence Listing.
[240] The construct assembly was performed according to a 3-piece Gibson Assembly® procedure described in Gibson et al. (2009, supra). In particular, the rEx-DLP-pplab-rFF construct was built using fragments 1, 2 and 3; the construct rEx-DLP-2A-pplab-rFF was built using fragments 4, 5 and 6; and the rEx-DLP-2A-pplab-DLP-rFF construct was built using fragments 7, 8 and 9. The assembled products were subsequently transformed into EPI300 cells from the Epicenter. A total of 144 colonies were examined with the
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179/207 use of primers RP126 (SEQ ID NO: 13) and RP127 (SEQ ID NO: 14) for each transformation, which results in 4 POR positive clones for rEx-DLP-pplab-rFF, 3 POR positive clones for rEx- DLP-2A-pplab-rFF and 2 positive POR clones for rExDLP-2A-pplab-DLP-rFF. Subsequent MiSeq results revealed that clone 4, clones 3 and 15, and clones 18 and 20 were completely correct in the sequence for rEx-DLPpplab-rFF, rEx-DLP-2A-pplab-rFF, and rEx-DLP-2A-pplab- DLPrEE, respectively.
Table 4
Initiator Initiators designed for colony screening of DLP- (2A) -pplab replicons RP126 DLP-pplabscreen-F CAGCATCTTTTACTTTCACCAGCGTTTCTG(SEQ ID NO: 13) RP127 DLP-pplabscreen-R GGAACTGGCGAAGCCAGTTTTAACA(SEQ ID NO: 14)
[241] The rEx-DLP-rFF, rEx-DLP-pplab-rFF, rEx-DLP-2A-pplab-rFF, and rEx-DLP-2A-pplab-DLP-rFF maps are also shown in Figures 2A to 2D .
[242] The sequences of the resulting replicons are revealed in the Sequence Listing with a T7 promoter and a 65A polyA tail, as follows: rEx-DLP-rFF (SEQ ID NO: 15), rEx-DLP-pplab-rFF ( SEQ ID NO: 16), rEx-DLP-2A-pplab-rFF (SEQ ID NO: 17), and rEx-DLP-2A-pplab-DLP-rFF (SEQ ID NO: 18).
Example 3
Construction of Alfavirus Replication Projects
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Containing DLP [243] This Example describes the generation of a number of expression vectors based on Alfavirus RNA replicon with a DLP motif positioned upstream of the polyprotein / non-structural protein genes and / or a reporter gene. These expression vectors based on Alfavirus RNA replicon were further characterized and analyzed in the flow cytometry analysis and essential luciferase analyzes described in Example 5.
A. Project [244] The respective design capabilities of three DLP replicon constructs based on Alfavirus are described below.
(1) Alpha-R-DLP-rFF [245] In this construct, DLP was placed immediately upstream of the initial codon of the rFF reporter gene.
(2) Alpha-R-DLP-2A-nsp-rFF [246] In this construct, the sequence encoding the DLP motif and the 2A peptide sequence (which was the same sequence used in the rEx-DLP-2A-pplab replicon -rFF described in Example 2 above) were placed within the 5 'end of the replicon with a few careful design modifications described below, to potentially maintain the sequence-frame requirement for mRNA replication and transcription
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181/207 subgenomic.
[247] (i) The first 195 nucleotides of the nspl gene were duplicated with their initial codon mutated from ATG to TAG (in bold following SEQ ID NO: 19 below).
[248] (ü) This sequence of 195 duplicated nucleotides was placed immediately following the 5 'RTU of the wild-type Alfavirus (underlined in the sequence of SEQ ID NO: 19 below) and is followed by the sequence DLP-2A (in italics in the sequence below ).
[249] (ill) The initial codon of the nspl gene following the DLP-2A sequence has been removed (pierced in the sequence of SEQ ID NO: 19 below).
SEQ IP NO: 19 (partial sequence)
GATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAATAGGAGAAAG T CA CGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGT TTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCG CATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACAT GGGAATAGTCAGCATAGTACATTTCATCTGACTAATACTACAACACCACCACCATGAAT AGAGGATTCTTTAACATGCTCGGCCGCCGCCCCTTCCCGGCCCCCACTGCCATGTGGAG GCCGCGGAGAAGGAGGCAGGCGGCCCCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGA AGCAGGCTGGAGACGTGGAGGAGAACCCTGGACC19 ^ GAGAAAGGT! CAG ...
(3) Alpha-R-DLP-2A-nsp-DLP-rFF [250] This construct is essentially identical to
Construct 2 following the same three design modifications, except that another DLP motif was placed immediately at
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182/207 amount of the reporter rFF gene (in the same way that a DLP motif was placed in Construct 1). A comparative analysis of replicate performances of Constructs 2 and 3 would provide information on whether the placement of additional DLP upstream of the reporter gene has an added value to the expression of the reporter gene (see Example 5 below).
B. Construction
Construction of Alpha-R-DLP-rFF [251] Alf aR-DLP-rFF was built using the Gibson Assembly® procedure, using Alfa-R-eGFP (c6; SEQ ID NO: 35) digested with EcoRI / SapI as a vector and
DLP-rFF as an insert amplified per PCR a leave of model rEx-DLP-rFF (c2, SEQ ID AT THE: : 15) with the use From RP112 primers (SEQ ID AT THE: 20) and RP113 (SEQ ID NO: 21)
to replace eGFP with DLP-rFF. Clones 2 and 3 had the sequence confirmed to be completely correct by sequencing MiSeq.
Table 5
Initiator Initiators used to clone DLP-rFF to Alfa-R-GFP (EcoRV / SapI) RP112 DLPrEE-E C C T GAAT G GAC TAC GACATAG TCTAGTCCGC CAAGAT AT C G CAC CATAG T CAG CATAG TACAT T T CAT C T GAC TAATACT (SEQ ID NO: 20) RP113 DLPrEE-R GCAGCTTGCCAATTGCTGCTGTATCGATCAATTAATCACATCTTGGCCACGGGTTTCTTC (SEQ ID NO: 21)
Construction of Alfa-R-DLP-2A-nsp-rFF and Alfa-R-DLP-2APetition 870190059497, of 06/26/2019, p. 185/247
183/207 nsp-DLP-rFF [252] Alpha-R-DLP-2A-nsp-rFF (Construct 2) and AlfaR-DLP-2A-nsp-DLP-rFF (Construct 3) were constructed using Gibson's procedure Assembly®, using the respective g blocks as inserts and the vectors that were amplified by PCR from the respective models, AlfaR-rFF (c6; SEQ ID NO: 35) and Alfa-R-DLP-rFF (c2; SEQ ID NO: 26), using the primers RP124 (SEQ ID NO: 22) and RP125 (SEQ ID NO: 22) and RP125 (SEQ ID NO: 23). Clones 1 and 3 of Alfa-R-DLP-2A-nsp-rFF and clones 8 and 32 of Alfa-R-DLP-2A-nspDLP-rFF had the sequence confirmed to be completely correct by means of MiSeq.
Table 6
Initiator Initiators used for the construction of Alfa-DLPnsp-rFF / DLP-rFF RP124 5'Alfa-P2A-F GAAGCAGGCTGGAGACGTGGAGGAGAACCCTG GAC C T GAGAAAG T T C AC G T T GAC AT C GAG GA AGAC (SEQ ID NO: 22) RP125 5'Scal-R CAC CAG T CACAGAAAAG CAT C T TAC G GAT G (SEQ ID NO: 23)
[253] The g-block sequence used for the construction of Alpha-R-DLP-2A-nsp-rFF is provided in the Sequence Listing as SEQ ID NO: 24. The g-block sequence used for the construction of Alpha-R -DLP-2A-nsp-DLP-rFF is also provided in the Sequence Listing as SEQ ID NO: 25.
[254] Alfa-R-rFF, Alfa-R-DLP-rFF, AlfaR-DLP-2A-nsp-rFF and Alfa-R-DLP-2A-nsp-DLP-rFF maps are shown
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184/207 in Figures 3A to 3D.
[255] The sequences of the resulting replicons are also provided in the Sequence Listing with a T7 promoter and a 40A polyA tail, as follows: AlfaR-rFF (SEQ ID NO: 26), Alfa-R-DLP-rFF (SEQ ID NO: 27), AlfaR-DLP-2A-nsp-rFF (SEQ ID NO: 28), and Alfa-R-DLP-2A-nsp-DLPrEE (SEQ ID NO: 29).
Construction of Alpha-R-DLP-2A-rFF and Alpha-R-DLP-2A-nspDLP-2A-rFF [256] Without being limited by any particular theory, it is believed that placing a DLP motif immediately upstream of the reporter gene rFF without the inclusion of protease 2A among them may negatively impact protein expression of GOI; this negative impact could be due to the fact that rFF now becomes a fusion protein, which results from the presence of the DLP sequence translated into a peptide at the 5 'end of rFF. Therefore, 2 new constructs were designed and built, including the 2A protease sequence between the DLP motif and the rFF gene for the two Alfavirus replicon constructs, AlfaR-DLP-rEE and Alfa-R-DLP-2A-nsp- DLP-rFF, to generate Alfa-RDLP-2A-rFF and Alfa-R-DLP-2A-nsp-DLP-2A-rFF, respectively. Inclusion of the 2A protease peptide sequence would allow the peptide to be encoded by the DLP sequence from rFF (see Example 5 below).
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185/207 [257] For this purpose, two fragments of g-block were synthesized (SEQ ID NOS: 30 and 31) and cloned into their respective vectors digested with EcoRV / Sbfl using Gibson Assembly. Alfa-R-DLP-2A-rFF clone 1 and Alfa-R-DLP-2A-nsp-DLP-2A-rFF clones 8 and 9 had the sequence confirmed as completely correct by Sanger sequencing with the use of RP123 (SEQ ID NO: 32) and RP96 (P89; SEQ ID NO: 96).
Table 7
I niciad or Initiators used to sequence constructsAlpha-R- (DLP-2A-nsp) -DLP-2A-rFF RP123 Alpha-3'nsp4-F GGCTGTTTAAGCTTGGCAAACCTCT (SEQ ID NO: 32) RP96 rFF-seql AGCGAGAACTGCGAGGAATTCTT (SEQ ID NO:33)
[258] Schematic maps of Alpha-R-DLP-2A-rFF and Alpha-R-DLP-2A-nsp-DLP-2A-rFF are provided in Figures 4A to 4B.
Example 4
Expression Analysis of Replicons Containing DLP Based on EAV [259] As shown in Examples 2 and 3 above, a number of replicons containing DLP based on EAV were constructed to determine the impact of modifying a DLP motif positioned upstream of the genes of non-structural replicon protein or the GOI gene in a subgenomic mRNA
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186/207 (Table 8).
Table 8: LISTING OF EAV REPLICATIONS CONTAINING DLP AND
VEW REPLICATIONS CONTAINING DLP.
DLP EAV replications rEx-DLP-rFF rEx-DLP-2A-rFF rEx-DLP-pplab-rFF rEx-DLP-2A-pplab-rFF rEx-DLP-2A-pplab-DLP-rFF rEx-DLP-2A-pplab-DLP-2A-rFFDLP VEEV replications alfa-R-DLP-rFF alfa-R-DLP-2A-rFF alfa-R-DLP-2A-nsp-rFF alfa-R-DLP-2A-nsp-DLP-rFF alfa-R-DLP-2A-nsp-DLP-2A-rFF
[260] Initial characterization of the DLP replicon constructs was performed ex vitro. RNA was produced and used for electroporated BHK cells as described in Example 1 above. After electroporation, cells were analyzed for protein expression by FACs, Western blot analysis or bulk luciferase assay.
[261] A graphical summary of the results of experiments carried out to measure the expression level of an exemplary gene of interest (GOI), reporter luciferase rFF, from EAV-based DLP replicons is shown in Figure 5. Data from both FACs analysis as
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187/207 of bulk luciferase are shown. In these experiments, four different DLP EAV replicons were analyzed as follows:
[262] 1) rEx-DLP-rFF: an EAV-based replicon with a DLP motif positioned upstream to the rFF subgenomic mRNA transcript);
[263] 2) rEx-DLP-pplab-rFF: an EAV-based replicon with DLP positioned upstream to non-structural pplab genes);
[264] 3) rEx-DLP-2A-pplab-rFF: an EAV-based replicon with a DLP motif positioned upstream of the non-structural proteins and a 2A protease peptide positioned between the DLP and the pplab region); and [265] 4) rEx-DLP-2A-pplab-DLP-rFF: an EAV-based replicon with a first DLP motif positioned upstream of the non-structural proteins and a 2A protease peptide positioned between the DLP and the region pplab as well as a second DLP motif positioned upstream to the rFF subgenomic mRNA transcript).
[266] The results shown in Figures 5A and 5B demonstrated that modifying a DLP motif upstream of the EAV non-structural protein genes (for example, rEx-DLP-pplab-rFF, rEx-DLP-2A-pplab-rFF or rEx-DLP-2Applab-DLP-rFF) or rFF reporter gene subgenomic RNA (for example, rEx-DLP-rFF and rEx-DLP-2A-pplab-DLP-rFF) no
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188/207 negatively impacted the genomic RNA replication since all four constructs demonstrated almost identical electroporation efficiencies (Figure 5A) .5A). Interestingly, analysis of bulk luciferase activity demonstrated that the rEx-DLP-pplab-rFF replicon expressed significantly less luciferase than the other three replicon designs (Figure 5B) .5B). As stated above, the incorporation of a DLP motif upstream of any GOI would result in an N-terminal fusion of Sindbis capsid amino acids encoded in the framed codons found in the DLP sequence. The fusion protein generated with the DLP encoding amino acids and the nsPl EAV protein is believed to impact the production of subgenomic RNAs efficiently by the complex EAV replication and result in the observed reduced levels of rFF GOI expression. One of the most notable results of this study was that EAV replicon constructs with a DLP that controls the translation of non-structural protein genes (rEx-DLP-pplab-rFF, rEx-DLP-2A-pplab-rFF and rEx-DLP- 2A-pplab-DLP-rFF) were efficiently translated as efficiently as the replicon RNA that did not have a DLP in that position (rEx-DLP-rFF). This result would not have been predicted based on work conducted by other researchers. It has been previously reported that the incorporation of 5 'Sindbis virus subgenomic RNA sequences (which includes the DLP region) has been efficiently translated
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189/207 only in cells infected with the virus. Put another way, it has been reported that mRNA containing a DLP motif associated with a reporter gene is insufficiently translated into cells that have not been infected with Sindbis virus. The absence of innate immune activation in these cells turned the DLP-modified mRNA into a distinct translation disadvantage compared to the translation of mRNAs that lack DLP modification (all cellular mRNAs). The innate immune system was not activated in these cells at the time that DLP-containing replicon vectors were introduced, so those DLP-containing mRNAs (capable of self-amplification) must be translated very inefficiently. Unexpectedly, this has not been proven in the experiments presented in this document.
[267] Subsequently, the rEx-DLP-2A-pplabrFF EAV replicon was examined in cells that were treated with IFN to induce the innate cellular immune system. IFN treatment of BHK cells will induce activation of PKR and phosphorylation of eIF2a which, in turn, results in disabling translation of global cellular mRNA. It has been previously reported that arteriviruses are sensitive to IFN treatment (Luo et al. Antiviral Res., August 91 (2): 99 to 101, 2011), therefore, IFN treatment of BHK cells, which are able to respond exposure to IFN and induce the innate immune system, would result in the deactivation of
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190/207 arterivirus replication. A representative example of the expression capacity of the DLP-modified EAV replicon in the presence of activation of the innate immune system is shown in Figure 6. The rEx-DLP-2A-pplab-rFF replicon demonstrated significant resistance to activation of the innate immune system when compared to an EAV replicon that has not been modified to contain the DLP motif, that is, rEx-rFF. Both replication (Figure 6A) and expression (Figure 6B) of the rEx-DLP-2A-pplab-rFF replicon were significantly superior in IFN-treated cells, compared to the control rEx-rFF replicon. These data demonstrate that DLP-modified EAV replicons are able to overcome deactivation of the innate immune system and that this replicon vector represents a significant advance in self-amplifying RNA technology.
Example 5
Expression Analysis VEEV Replications Containing DLP [268] As shown in Examples 2 and 3 above, a number of VEEV-based DLP-containing replicons were constructed to determine the impact of modifying a DLP motif positioned upstream of the non-protein genes. structural replication or the GOI gene in a subgenomic mRNA.
[269] VEEV alphavirus replicon vectors have been modified to contain one or more DLP motifs using a similar strategy for building vector vectors.
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191/207 EAV based replicon. Mainly, unlike other members of the Alfavirus genus (most of the Old World virus members), the VEEV genome does not contain a DLP motif associated with the translation of its subgenomic mRNA. Initial analysis of VEEV DLP replicons was performed on BHK-21 cells as described in Example 1 above. BHK-21 cells do not secrete IFN in response to RNA replication but these cells are able to respond to exogenous IFN to induce innate immune activation. In this experiment, four different alphavirus replicon constructs were tested. The experimental data presented in Figure 7 show replication of alphavirus replication containing DLP and expression of the rFF luciferase gene in BHK cells that were treated at the time of electroporation (Oh) or in 3 h post-electroporation with 1,000 U / ml of exogenous IFN. The replicon RNAs tested were:
[270] 1) Alpha-R-rFF: a replication based on control VEEV without DLP present;
[271] 2) Alpha-R-DLP-rFF: a VEEV-based replicon with a DLP motif positioned upstream of the transcribed rFF subgenomic mRNA;
[272] 3) Alpha-R-DLP-2A-nsp-rFF: a VEEV-based replicon with a DLP motif positioned upstream of non-structural proteins with a 2A protease between the DLP and the nsp region; and
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192/207 [273] 4) Alpha-R-DLP-2A-nsp-DLP-rFF: based on VEEV replicon with a first DLP motif positioned upstream of the non-structural proteins with a 2A protease between the DLP and the nsp region as well as with a second DLP motif positioned upstream of the transcribed rFF subgenomic mRNA.
[274] The results of luciferase expression normalized to the number of positive cells detected by FAC analysis are shown in Figure 7. It was observed that the presence of a DLP motif that controls the translation of non-structural VEEV protein genes resulted in superior reporter gene expression both in the absence and in the presence of post-electroporation IFN treatment (Figures 7A to 7C). Although the increase in rFF expression may have been considered statistically insignificant, the trend in all conditions was towards increased protein expression. As set out above in Example 4 in relation to DLP-containing EAV replicons, it could be expected that a DLP motif would have a negative impact on mRNA translation in cells that are not in an activated state of innate immune response. In direct contrast to that expectation, BHK cells that were not treated with IFN (Figure 7A) in these experiments represent the sample with the greatest benefit for incorporating a DLP motif.
[275] Subsequently, the two RNA replicons
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193/207 alfa-R-rFF and alfa-DLP-2A-nsp-rFF were tested in vivo in Balb / c mice. In this experiment, mice were tested in groups of 10 animals. In these experiments, equal doses of RNA were injected intramuscularly into mice and the whole body IVIS (In vivo Imaging System) analysis was performed over the course of a week. Whole body imaging was performed on day 1, day 3 and day 7 post-injection. The total flow measured at the injection site is shown in Figure 8. Although only modest increases in protein expression were observed ex vitro (Figure 8) from the DLP-modified VEEV replicon, statistically significantly higher protein expression was detected at all times at points measured from the modified VEEV replicon RNA with DLP (Figure 8). This observation represents a significant advantage, due to how unmodified VEEV replication vectors are capable of very high protein expression that can reach up to 20% of the total cellular protein (Pushko et al 1997). The DLP-modified VEEV replicon even exceeded this expression potential and demonstrated superior protein expression; for this reason, the DLP-modified alphavirus replication vector represents a significant advance over existing alphavirus replication RNA technology.
[276] There are at least three unexpected results that can be extracted from experimental data
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194/207 shown in the Examples above. First, the DLP motif has shown that it negatively impacts mRNA translation when a cell is not in an activated state of the innate immune system. The DLP-containing replicon RNAs disclosed in this document were found not to be negatively impacted on cells in a baseline state of innate activation. Secondly, it was found that expression levels, especially for VEEV replicons containing DLP, were even higher than unmodified replicons in vivo; this observation demonstrated that expression levels even from an alphavirus replicon can be increased from previously high historical expression levels. Third, all positive strand RNA viruses have considerable sequence conservation at the 5 'and 3' ends of their genomes. The fact that both the VEEV replicon and the EAV replicon are flexible enough to accept incorporation of a rod-loop structure (the DLP) at the 5 'end of their RNAs is unexpected.
Example 6
In vivo Immunogenicity Response Using DLP Replication Expression Systems [277] Alphavirus replication vectors have been modified to contain one or more DLP motifs, as described above. The RNA replicon, Alpha-R-gDLP-HA, containing
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195/207 the DLP sequence was further analyzed in vivo in Balb / c mice. In this experiment, 15 pg, 1.5 pg, or 0.15 pg of RNA encoding Hemagglutinin from Influenza A / Vietnam / 1203/2004 (H5N1) were injected into mice at 6-week intervals. Fourteen days after the final boost, spleens and serum were collected to analyze immune responses to AH. A summary of the results of these experiments is shown in Figures 12A to 12C. In Figure 12A, a significant increase in memory precursor effector cells (MPECs) was observed in constructs containing the DLP motif compared to each comparable dose of an unmodified replicon. Specific HA MPECs were detected using dextramers (H-2 Kd (IYSTVASSL; SEQ ID NO: 44)) along with other markers for specific population (CD8 + CD44 + CD62L ^L ° KLRG-1 ^L ° IL-7Ra ^Hi CXCR3 ^Hi ). It is worth noting that this benefit was also observed in low doses. In Figures 12B and 12C, effector T cell responses were measured by the number of specific antigen HA cells that were secreting ΙΕΝ-γ following stimulation with a CD4 ⁺ T cell or CD8 + T cell peptide. Animals immunized with replicons containing the DLP motif that had a significantly higher frequency of cytokine expression of CD4 ⁺ and CD8 + T cells are at doses of 15 µg and 1.5 µg. Taken together, these data indicate a significant increase in both effector and
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196/207 memory of T cell responses in response to immunization with antigen expressed by replicons containing the DLP motif when compared to the unmodified version.
[278] The DLP-containing replicons above were further analyzed in vivo in Balb / c mice for compatibility with LNP formulations. In this experiment, 2 pg or 0.2 pg of RNA encoding Hemagglutinin from Influenza A / Vietnam / 1203/2004 (H5N1) were injected into mice at 4-week intervals. Fourteen days after the final boost, spleens and serum were collected to analyze the immune response to AH. A summary of these experiments is shown in Figures 14A to 14C. In Figures 14A to 14C, an increase in T cell and B cell responses was observed with the use of constructs containing the DLP motif when combined with LNP (cationic lipid nanoparticles) formulations. In Figural4A, IgG titers of total specific HA were significantly higher in all dosing groups with the use of LNP formulations compared to the group with replicon administered in saline. Furthermore, in Figures 14B and 14C, it was observed that CD8 + and CD4 + T cells of specific HA were also significantly superior in all dosing groups with the use of LNP formulations compared to the group with replicon administered in saline. Taken together, these data demonstrate that constructs
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197/207 replicon containing the DLP motif are compatible with representative formulations.
Example 7
Prevention of Suppression of Immune Response Using DLP-containing Replicons [279] DLP-containing replicates constructed as described above were further evaluated in vivo for the ability to prevent suppression of immune response in Balb / c mice. In these experiments, 1.5 pg of mRNA, with or without a DLP motif, and carrying a coding sequence for Hemagglutinin derived from Influenza A / Vietnam / 1203/2004 (H5N1) are injected into mice at separate intervals for 4 weeks. Approximately 24 hours before injection, BALB / c mice aged 6 to 8 weeks old are pre-treated with 20 pg of Poli (I: C) or saline by hydrodynamic injection into the caudal vein to simulate a viral infection. Fourteen days after the final boost, serum from these mice is collected to analyze the immune response to Hemagglutinin (HA). A summary of these experiments is presented in Figural3. In Figure 13, a significant decrease is observed in the serum concentration of specific HA antibodies in mice that were pretreated with Poli (I: C) and received doses of unmodified replicates. The levels in the Poli (I: C) group were not significantly above the antecedents. On the contrary,
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198/207 animals pretreated with Poli (I: C) and dosed with a construct containing the DLP motif did not show significant reductions in serum total antigen-specific IgG concentration. Taken together, these data show that the DLP motif protects against suppression of serum antibody levels in response to vaccination following a simulated viral infection compared to the unmodified version.
Example 8
Construction of Expression Cassettes containing DLP [280] This Example describes the generation of a plasmid vector for ex vitro transcription of an mRNA containing a DLP element from Sindbis virus upstream of a gene of interest, for example, a reporter gene , according to some disclosure modalities. The 5 'and 3' untranslated regions used in these experiments (SEQ ID NO: 36 and SEQ ID NO: 41, respectively) were derived from the human beta globin gene. The 5 'UTR sequence was placed immediately downstream of a T7 promoter (SEQ ID NO: 37) and upstream of the Sindbis virus DLP sequence (SEQ ID NO: 38). In some experiments, the coding sequence for a gene of interest (GOI) was linked to DLP by means of a P2A signal, which is an autocatalytic autocleaving peptide (eg, autoprotease peptide) derived from porcine tescovirus-1. In some experiments, a coding sequence for a shape
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199/207 destabilized EGFP reporter gene (dsGFP) which, in this case used as a GOI, was operably linked to the proteolytic PEST degradation signal derived from a mouse ornithine decarboxylase (MODC) gene. In some other experiments, a coding sequence for the red Firefly luciferase reporter gene was used as the gene of interest (see also, Example 9 below). However, it is contemplated that coding sequences for any gene of interest could be implanted in this configuration. In addition, as illustrated in Figural5, a 3 'UTR sequence derived from human beta globin, a polyA tail consisting of adenine residues 120, and a T7 terminator were inserted downstream and adjacent to the dsGFP stop codon. The nucleic acid sequences for each of the components described above are as follows:
Table 9
DLP dsGFP mRNA components 5 'human beta globin RTU 5'- ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCGCCACC-3 '(SEQ ID NO 36) Promoter T7 5'-TAATACGACTCACTATAG-3 '(SEQ ID NO 37) Reason forDLP 5'-ATAGTCAGCATAGTACAT T T CAT C T GAC TAATAC TACAACAC CACCACCATGAATAGAGGATTCTTTAACATGCTCGGCCGCCGC CCCTTCCCGGCCCCCACTGCCATGTGGAGGCCGCGGAGAAGGA GGCAGGCGGCC P2A Peptide 5'-GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT-3 '(SEQ ID NO 39) DsGFP 5'- ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC
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GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG ATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGT GACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCT AC CCC GAC Cacat GAAG CAG CAC GAC TTCTT CAAG TCC GCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT CTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT GGGGCACAAGCTGGAGTACAACTACAACAGCCACAACG TC TATAT CAT GGCCGACAAGCAGAAGAACGGCAT CAAG GT GAAC TT CAAGAT CCGC CACAACAT C GAG GAC GG AGC CGTG CAG CTCGCC GAC CAC TAG CAG CAGAACAC CCC CA TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTAC CTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGA GAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCG CCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAA GAAGCTTAGCCATGGCTTCCCGCCGGAGGTGGAGGAG CAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGA GAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTG CTAGGATCAATGTGTAG -3 '(SEQ) 40 Human beta globin 3 'RTU 5'-GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTT GTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGG GCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTT TCATTGCAA -3 '(SEQ) TerminatorT7 5'- AACCCCTCTCTAAACGGAGGGGTTTTTTT-3 '(SEQ ID NO: 42) DLP dsGFP Mrna sequence T TAATACGAC 5'-CAC AAC TATAGACAT TTGCTTCT GACAC TGTGTT CAC TAG CT CAAACAGACAC CGCCGC CAAC T CAG CAC CATAG CATAG TAGAT CAT TT T CT GAC AAT TAG AAC AC AC CAC CAT CAC gaat AGAG GATT CTTTAACATGCTCGGCCGCCGCCCCTTCCCGGCCCC CACTGCCATGTGGAGGCCGCGGAGAAGGAGGCAGG CGGCCCCGGGAAGCGGAGCTACTAACTTCAGCCTG CTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGG ACCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCG GGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACG TAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAA GTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTG GCCCACCCTCGTGACCACCCTGACCTACGGCGTGCA GTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCA CGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAAC TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGAC ACCCTGGTGAACCGCATCGAGCTGGGCA
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GAG TACAAC TACAACAG C CACAAC GTCTATATCATGG CCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC TCGCC GAC CAC TAC AGC CAGAACAC CCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG CCC C CAG TCCGCCCT GAG CAAAGAC DC CAAC Gagaa GCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCC GCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGA AGCTTAGCCATGGCTTCCCGCCGGAGGTGGAGGAGCA GGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAG AGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTG CTAGGATCAATGTGTAGGCTCGCTTTCTTGCTGTCCAA TTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTA CTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTG GAT TCT GC C TAATAAAAAACAT T TAT TTT CAT T GCAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAACCCCTCTCTAAACGGAGGGGTTTT TTT - 3 '(SEQ ID NO: 43)
[281] In the above experiments, a DLP sequence from Sindbis virus was used. Additional experiments are carried out to incorporate DLP sequences from other Old World alphavirus members such as SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV and ONNV, into the nucleic acid molecules of the present disclosure. The binding of DLP to the gene of interest can be configured with or without an autocleaving peptide such as P2A. With no connection to any particular theory, it is believed that the requirement for a 2A sequence or other autocleaving peptide is dependent on the individual gene being inserted into the gene cassette and whether the additional amino acids added by the inclusion of DLP would affect the function of translated proteins. It is contemplated
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202/207 additionally that the 5 'and 3' RTU sequences used here can also be changed to any other set of functional RTUs regardless of origin.
Example 9
Ex vivo Evaluation of Gene Expression in Expression Cassettes containing DLP [282] mRNAs derived from expression cassettes containing DLP modified to contain one or more DLP motifs, as described above, were evaluated ex vivo for the ability to enhance gene expression of interest in BHK-21 cells. As a control, mRNA samples lacking the DLP sequence, but otherwise identical to the DLP-containing mRNAs described above were subjected to testing in parallel under the same conditions. In these experiments, BHK-21 cells were pretreated with 300, 600 or 1,000 U / ml of type I universal interferon or control vehicle for 2 hours. Following the pre-treatment, the cells were electroporated, in triplicate, with 2.5 pg of mRNA containing or lacking in DLP motifs. The cells were placed back in the media containing the same concentrations of interferon used during the pretreatment. The frequency of positive GFP cells and Mean Fluorescence Intensity (MFI) was tested at 2, 4 and 24 hours post-electroporation by flow cytometry. It was observed that mRNA containing DLP produces a frequency significantly
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203/207 higher GFP positive cells compared to non-DLP mRNA in the presence of interferon (Figure 16A).
[283] Furthermore, when the GFP MFI was normalized to the frequency of positive GFP cells and plotted against time, it was observed that the unmodified mRNA was sensitive to interferon treatment as exhibited by a statistically significant reduction of 30 % in global protein produced over the 24-hour time course (Figure 16B). In contrast, the modified DLP-containing mRNA demonstrated resistance to interferon treatment as exhibited by a statistically significant 30% increase in overall protein production over the unmodified control mRNA over the same 24-hour time course (Figure 16C). The treatment of interferon resistance conferred by the presence of the DLP motifs was further strengthened by the finding that cells treated with interferon and electroporated with a mRNA containing DLP produced as much protein as untreated cells electroporated with an unmodified mRNA (Figure 16C).
Example 10
In Vivo Evaluation of Gene Expression in Expression Cassettes Containing Dip [284] mRNAs derived from expression cassettes containing DLP modified to contain one or more DLP motifs, as described above, are further evaluated in
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204/207 alive for the ability to enhance expression of the gene of interest in Balb / c mice. In this experiment, 30 pg, 15 pg or 1.5 pg of mRNA containing DLP encoding Firefly red luciferase are injected into mice at 6-week intervals. The red Firefly luciferase expression is subsequently monitored by IVIS (In vivo Imaging System) analysis at 1, 3, 7, 10, 14, 21 and 28 days post-injection. A significant increase in luciferase expression is seen in mice that receive DLP-containing mRNAs when compared to control animals that receive mRNA lacking the DLP motif.
Example 11
Prevention of Suppression of Immune Response with the Use of DLP-Containing mRNAs [285] DLP-containing mRNAs as described above are further evaluated in vivo for the ability to enhance expression of the gene of interest in Balb / c mice. In this experiment, 30 pg, 15 pg or 1.5 pg of mRNA, with or without DLP motif, and carrying a coding sequence for Influenza A / Hemagglutinin derived / Vietnam / 1203/2004 (H5N1) are injected into mice at intervals separated by 4 weeks. Approximately 24 hours before the injection, mice are pre-treated with 20 pg of Poli (I: C) or saline by hydrodynamic injection into the caudal vein to simulate a viral infection. Fourteen days
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205/207 after the final boost, serum from these mice is collected to analyze the immune response to Hemagglutinin (HA). A significant decrease in the serum concentration of specific HA antibodies is expected to be observed in mice that are pretreated with Poli (I: C) and receive a dose of mRNA lacking the DLP sequence. On the contrary, it is expected that animals pretreated with Poli (I: C) and dosed with mRNA containing the DLP motif will not show significant reductions in total IgG concentration of specific antigen in serum.
[286] Although particular alternatives to the present disclosure have been revealed, it must be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the attached claims. There is, therefore, no intention of limitations on the exact summary and disclosure presented in this document.
[287] All references disclosed in this document, which include, without limitation, newspaper articles, textbooks, publications, patents and patent applications are incorporated in their entirety into this document for reference to the same extent if each reference was specifically and individually indicated to be incorporated as a reference.
[288] No admission is produced that any
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206/207 reference cited in this document constitutes prior art. The discussion of references expresses what their authors claim, and the inventors reserve the right to challenge the accuracy and relevance of the documents cited. It will be clearly understood that, although a number of sources of information, which include scientific journal articles, patent documents and textbooks, are referred to in this document; any discussion and comment on a specific source of information should in no way be taken as an admission that such comment has been widely accepted as the general opinion in the field.
[289] The discussion of the general compositions and methods presented in this document is for illustrative purposes only. It is not intended to be exhaustive or to limit disclosure. Individual aspects or features of a particular modality are generally not limited to that particular modality, but, where applicable, are interchangeable and can be used in a selected modality, even if not shown or described specifically. It is expressly contemplated that any aspect or feature of the present disclosure may be combined with any other aspect, feature or combination of aspect and feature disclosed in this document. Other compositions, methods, and alternative modalities will be apparent to people skilled in the art in the review
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207/207 of that disclosure, and should be included in the spirit and scope of that request.

权利要求:
Claims (174)
[1]
1. Nucleic Acid molecule, comprising:
a first nucleic acid sequence encoding one or more RNA stem-loops from a viral capsid enhancer or variant thereof; and a second nucleic acid sequence operably linked to the first nucleic acid sequence, characterized in that the second nucleic acid sequence comprises a coding sequence for a gene of interest (GOI).
[2]
Nucleic Acid Molecule according to Claim 1, characterized in that the first nucleic acid sequence is operationally linked upstream to the coding sequence for GOI.
[3]
Nucleic Acid Molecule according to Claim 1 or 2, characterized in that it further comprises a promoter operably linked upstream to the first nucleic acid sequence.
[4]
Nucleic Acid Molecule according to any one of Claims 1 to 3, characterized in that it further comprises a sequence of the 5 'UTR operably linked upstream to the first nucleic acid sequence.
[5]
Nucleic Acid Molecule according to Claim 4, characterized in that the 5 'RTU sequence is operably linked downstream to the promoter and upstream to the first nucleic acid sequence.
[6]
Nucleic Acid Molecule, according to any one of Claims 1 to 5, characterized in that it further comprises
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2/32 a coding sequence for an autoprotease peptide operably linked upstream to the second nucleic acid sequence.
[7]
Nucleic Acid Molecule according to Claim 6, characterized in that the coding sequence for the autoprotease peptide is operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence.
[8]
Nucleic Acid Molecule according to any one of Claims 6 to 7, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of swine teschovirus-1 2A (P2A), a foot-and-mouth virus ( FMDV) 2A (F2A), an equine rhinitis virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination of themselves.
[9]
Nucleic Acid Molecule according to any one of Claims 1 to 8, characterized in that it further comprises a 3 'UTR sequence operably linked downstream to the second nucleic acid sequence.
[10]
Nucleic Acid Molecule according to any one of Claims 1 to 9, characterized in that the viral capsid enhancer is derived from a capsid gene of a species of virus belonging to the Togaviridae family.
[11]
11. Nucleic Acid Molecule, according to Claim
10, characterized by the fact that the virus species belongs to the Alphavirus genus of the Togaviridae family.
[12]
12. Nucleic Acid Molecule, according to Claim
11, characterized in that the alphavirus species is selected from the group consisting of the Eastern Equine Encephalitis virus (EEEV), Venezuelan Equine Encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus ( SFC), Pixuna virus, Middleburg virus (MIDV), Chicungunha virus (CHIKV), 0'Nyong virus
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Nyong (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), West Equine Encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus ( FMV), Ndumu (NDUV), Salmonid alphavirus (SAV) and Buggy Creek virus.
[13]
13. Nucleic Acid molecule according to any one of Claims 10 to 12, characterized in that the viral capsid enhancer comprises a downstream loop motif (DLP) of virus species, and, in which, the DLP motif it comprises at least one of the one or more RNA stem loops.
[14]
Nucleic Acid Molecule according to any one of Claims 1 to 11, characterized in that the viral capsid enhancer comprises a nucleic acid sequence having at least 80% sequence identity with at least one of SEQ ID N ^os : 1 and 46-52.
[15]
15. Nucleic acid molecule according to Claim 14, characterized in that the nucleic acid sequence has at least 95% sequence identity with at least one of ^the SEQ ID NO: 1 and 46-52.
[16]
16. Nucleic Acid molecule according to any one of Claims 1 to 15, characterized in that the coding sequence for GOI encodes a polypeptide.
[17]
17. Nucleic Acid Molecule, according to any one of Claims 1 to 16, characterized in that the polypeptide is selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme , a reporter polypeptide and a combination thereof.
[18]
18. Nucleic Acid molecule according to any one of Claims 1 to 16, characterized in that the polypeptide is
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4/32 selected from the group consisting of an antibody, an antigen, an immune modulator, a cytokine, an enzyme and a combination thereof.
[19]
19. Nucleic Acid molecule according to any one of Claims 1 to 18, further comprising:
a third nucleic acid sequence encoding one or more RNA stem-loops from a second viral capsid enhancer or variant thereof; and a fourth nucleic acid sequence operably linked to the third nucleic acid sequence, characterized in that the fourth nucleic acid sequence comprises a coding sequence for a second gene of interest (GOI).
[20]
Nucleic Acid Molecule according to Claim 19, characterized in that it further comprises a coding sequence for a second autoprotease peptide operably linked downstream to the third nucleic acid sequence and upstream to the fourth nucleic acid sequence.
[21]
21. Nucleic Acid Molecule, according to any one of Claims 1 to 20, characterized in that the nucleic acid molecule is an mRNA molecule or an RNA replicon.
[22]
22. Nucleic Acid Molecule, according to any one of Claims 1 to 20, characterized in that the nucleic acid molecule is an expression vector or a transcription vector.
[23]
23. Nucleic Acid Molecule, according to Claim
22, characterized in that it further comprises one or more additional transcriptional regulatory sequences.
[24]
24. Nucleic Acid Molecule according to Claim 22 or 23, characterized in that it further comprises one or more
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5/32 regulatory translation sequences.
[25]
25. Nucleic Acid Molecule, according to any one of Claims 22 to 24, characterized in that the nucleic acid molecule is a plasmid, bacteriophage vector, cosmid, phosphid, viral replicon, shuttle vector or combination thereof.
[26]
26. Nucleic Acid Molecule, according to any one of Claims 22 to 25, characterized in that the nucleic acid molecule is a prokaryotic or eukaryotic vector.
[27]
27. Nucleic Acid Molecule, according to any one of Claims 1 to 26, characterized in that the nucleic acid molecule is produced by means of de novo synthesis.
[28]
28. Method for Producing Cell Polypeptide of Interest, characterized in that it comprises introducing a nucleic acid molecule, as defined in any one of Claims 16 to 27, into a cell, producing a polypeptide encoded by the GOI in the cell.
[29]
29. Method for Production of Polypeptide of Interest in Cell, comprising the introduction of an RNA molecule into the cell, characterized in that the RNA molecule comprises one or more RNA stem loops from a viral capsid enhancer or a variant of the same and a coding sequence for the polypeptide of interest, producing the polypeptide of interest in the cell.
[30]
30. Method for Producing Cell Polypeptide of Interest, according to Claim 29, characterized in that the RNA molecule is a messenger RNA (mRNA) molecule or a replicon RNA molecule.
[31]
31. Method for Producing Cell Polypeptide of Interest according to Claim 29 or 30, characterized in that the RNA molecule is produced by means of de novo synthesis and / or transcription in vitro, before being introduced into the cell .
[32]
32. Method for Production of Polypeptide of Interest in
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Cell according to any one of Claims 29 to 31, characterized in that the RNA molecule comprises a downstream loop (DLP) motif of a virus species and in which the DLP motif comprises at least one of the one or plus RNA stem-loops from the viral capsid enhancer.
[33]
33. Method for Producing Cell Polypeptide of Interest according to any one of Claims 29 to 32, characterized in that the RNA molecule further comprises a coding sequence for a downstream autoprotease peptide with respect to at least one of the one or more RNA stem-loops and upstream to the coding sequence for the polypeptide of interest.
[34]
34. Method for Producing Cell Polypeptide of Interest according to Claim 33, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of swine-1 2A (P2A) teschovirus, a foot-and-mouth disease virus ( FMDV) 2A (F2A), an equine rhinitis virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination of themselves.
[35]
35. Method for Producing Cell Polypeptide of Interest, according to any one of Claims 29 to 34, characterized in that the polypeptide is selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a polypeptide nutraceutical, an industrial enzyme, a reporter polypeptide and a combination thereof.
[36]
36. Method for Producing Cell Polypeptide of Interest, according to any one of Claims 29 to 34, characterized in that the polypeptide is selected from the group consisting of an antibody, an antigen, an immune modulator, a cytokine, an enzyme and a combination of them.
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[37]
37. Method for Producing Cell Polypeptide of Interest, according to any one of Claims 29 to 36, characterized in that the cell is present in a tissue, an organ or an individual.
[38]
38. Method for Producing Cell Polypeptide of Interest according to Claim 37, characterized in that the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster and buffalo.
[39]
39. Method for Production of Messenger RNA (mRNA) in Cell, comprising administering to the cell a nucleic acid molecule comprising a first nucleic acid sequence encoding one or more RNA stem loops from a viral capsid enhancer or a variant thereof and a second nucleic acid sequence operably linked to the first nucleic acid sequence, characterized in that the second nucleic acid sequence comprises a coding sequence for a gene of interest (GOI), thereby producing an mRNA of the GOI.
[40]
40. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 39, characterized in that the first nucleic acid sequence is operably linked upstream to the coding sequence for the GOI.
[41]
41. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 39 or 40, characterized in that it further comprises a promoter operably linked upstream to the first nucleic acid sequence.
[42]
42. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 39 to 41, characterized in that it further comprises a 5 'UTR sequence operably linked upstream to the first nucleic acid sequence .
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[43]
43. Method for Producing Cell Messenger RNA (mRNA) according to Claim 42, characterized in that the 5 'UTR sequence is operably linked downstream to the promoter and upstream to the first nucleic acid sequence.
[44]
44. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 39 to 43, characterized in that it further comprises a coding sequence for an autoprotease peptide operably linked upstream to the second sequence of nucleic acid.
[45]
45. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 44, characterized in that the coding sequence for the autoprotease peptide is operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence.
[46]
46. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 44 to 45, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of porcine-1 2A (P2A) ), a foot-and-mouth disease virus (FMDV) 2A (F2A), an equine rhinitis virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof.
[47]
47. Nucleic Acid Molecule according to any one of Claims 39 to 46, characterized in that it further comprises a 3 'UTR sequence operably linked downstream to the second nucleic acid sequence.
[48]
48. Method for the Production of Messenger RNA (mRNA) in Cell, according to any one of Claims 39 to 47, characterized in that the viral capsid enhancer is derived from a capsid gene of a virus species belonging to the family Togaviridae.
[49]
49. Method for the production of messenger RNA (mRNA) in
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Cell according to any one of Claims 39 to 48, characterized in that the virus species belongs to the Alphavirus genus of the Togaviridae family.
[50]
50. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 49, characterized in that the alphavirus species is selected from the group consisting of the Eastern Equine Encephalitis (EEEV) virus, Venezuelan Equine Encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFC), Pixuna virus, Middleburg virus (MIDV), Chicungunha virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), West Equine Encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) , Salmonid alphavirus (SAV) and Buggy Creek virus.
[51]
51. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 48 to 50, characterized in that the viral capsid enhancer comprises a downstream loop motif (DLP) of virus species, and, wherein, the DLP motif comprises at least one of the one or more RNA stem loops.
[52]
52. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 51, characterized in that the viral capsid enhancer comprises a nucleic acid sequence having at least 80% sequence identity with at least one of ^the SEQ ID NO: 1 and 46-52.
[53]
53. Nucleic acid molecule according to Claim 52, characterized in that the nucleic acid sequence has at least 95% sequence identity with at least one of ^the SEQ ID NO: 1 and 46-52.
[54]
54. Method for the production of messenger RNA (mRNA) in
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Cell according to any of Claims 39 to 53, characterized in that the coding sequence for GOI encodes a polypeptide.
[55]
55. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 54, characterized in that the polypeptide is selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide and a combination thereof.
[56]
56. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 54, characterized in that the polypeptide is selected from the group consisting of an antibody, an antigen, an immune modulator, a cytokine, an enzyme and a combination thereof.
[57]
57. Method for Producing Cellular Messenger RNA (mRNA) according to any one of Claims 39 to 56, further comprising:
a third nucleic acid sequence encoding one or more RNA stem-loops from a second viral capsid enhancer or variant thereof; and a fourth nucleic acid sequence operably linked to the third nucleic acid sequence, characterized in that the fourth nucleic acid sequence comprises a coding sequence for a second gene of interest (GOI).
[58]
58. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 57, characterized in that it further comprises a coding sequence for a second autoprotease peptide operably linked downstream to the third
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11/32 nucleic acid sequence and upstream to the fourth nucleic acid sequence.
[59]
59. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 39 to 58, characterized in that the nucleic acid molecule is an RNA replicon.
[60]
60. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 39 to 58, characterized in that the nucleic acid molecule is an expression vector or a transcription vector.
[61]
61. Method for the Production of Cell Messenger RNA (mRNA) according to Claim 59 or 60, characterized in that the nucleic acid molecule still comprises one or more additional transcriptional regulatory sequences.
[62]
62. Nucleic Acid Molecule according to Claim 60 or 61, characterized in that it further comprises one or more regulatory translation sequences.
[63]
63. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 60 to 62, characterized in that the nucleic acid molecule is an expression vector selected from the group consisting of a plasmid, a vector bacteriophage, a cosmid, a phosmid, a viral replicon, a shuttle vector, or a combination thereof.
[64]
64. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 60 to 62, characterized in that the nucleic acid molecule is a prokaryotic vector or a eukaryotic expression vector.
[65]
65. Method for the Production of Messenger RNA (mRNA) in Cell, according to any one of Claims 39 to 64, characterized in that the cell is present in a tissue, an organ or an individual.
[66]
66. Method for the production of messenger RNA (mRNA) in
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Cell according to Claim 65, characterized in that the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster and buffalo.
[67]
67. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 39 to 66, characterized in that it further comprises the production of a polypeptide encoded by the GOI mRNA in the cell.
[68]
68. Method for the Production of Cell Messenger RNA (mRNA) according to any one of Claims 39 to 66, characterized in that it further comprises:
obtain the mRNA produced from GOI;
introducing the mRNA obtained into a second cell for expression of a polypeptide encoded by the GOI mRNA in the second cell.
[69]
69. Nucleic Acid Molecule, comprising a nucleic acid sequence encoding a modified viral RNA replicon, characterized in that the modified viral RNA replicon comprises:
a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence encoding at at least one non-structural viral protein or a part thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence.
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[70]
70. Nucleic Acid Molecule, according to Claim
69, characterized in that at least one of one or more structural elements of the viral capsid enhancer comprises one or more RNA stem-loops.
[71]
71. Nucleic Acid Molecule according to Claim 69 or 70, characterized in that the viral capsid enhancer is derived from a capsid gene of a species of virus belonging to the Togaviridae family.
[72]
72. Nucleic Acid Molecule, according to Claim
71, characterized by the fact that the virus species belongs to the Alphavirus genus of the Togaviridae family.
[73]
73. Nucleic Acid Molecule, according to Claim
72, characterized in that the alphavirus species is selected from the group consisting of the Eastern Equine Encephalitis virus (EEEV), Venezuelan Equine Encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus ( SFC), Pixuna virus, Middleburg virus (MIDV), Chicungunha virus (CHIKV), 0'NyongNyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV ), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), West Equine Encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) and Buggy Creek virus.
[74]
74. Nucleic Acid Molecule according to any one of Claims 71 to 73, characterized in that the viral capsid enhancer comprises a downstream loop (DLP) motif of virus species, and, in which, the DLP motif it comprises at least one of the one or more RNA stem loops.
[75]
75. Nucleic acid molecule according to claim 69 or 70, characterized in that the viral capsid enhancer comprises a nucleic acid sequence having at least 80% sequence identity with at least one of SEQ ID ^Nos: 1 and
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46-52.
[76]
76. Nucleic Acid Molecule, according to Claim
75, characterized in that the nucleic acid sequence has at least 95% sequence identity with at least one of ^the SEQ ID NO: 1 and 46-52.
[77]
77. Nucleic Acid Molecule according to any one of Claims 69 to 76, characterized in that the nucleic acid sequence encoding the modified viral RNA replicon further comprising a coding sequence for an operably linked downstream autoprotease peptide to the first nucleic acid sequence and upstream to the second nucleic acid sequence.
[78]
78. Nucleic Acid Molecule, according to Claim
77, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an A virus of equine rhinitis (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.
[79]
79. Nucleic Acid Molecule according to any one of Claims 69 to 78, characterized in that the first nucleic acid sequence is operably positioned within a region of about 1 to 1,000 nucleotides downstream of the 5 'termination of the modified viral RNA replicon.
[80]
80. Nucleic Acid Molecule according to any one of Claims 69 to 79, characterized in that the second nucleic acid sequence comprises substantially the entire coding sequence for the corresponding non-modified viral RNA replicon native viral proteins .
[81]
81. Nucleic Acid Molecule according to any one of Claims 69 to 80, characterized in that the modified viral RNA replicon comprises a modified RNA replicon derived
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15/32 of a virus species belonging to the Alphavirus genus of the Togaviridae family or to the Arterivirus genus of the Arteriviridae family.
[82]
82. Nucleic Acid Molecule, according to Claim
81, characterized in that the species of arterivirus virus is selected from the group consisting of equine arteritis virus (EAV), swine reproductive and respiratory syndrome virus (PRRSV), lactic dehydrogenase elevator virus (LDV) and simian hemorrhagic fever virus (SHFV).
[83]
83. Nucleic Acid Molecule, according to Claim
82, characterized in that the first nucleic acid sequence is operably positioned upstream to a second nucleic acid sequence encoding a part or all of the non-structural pplab protein of the modified arterivirus RNA replicon.
[84]
84. Nucleic Acid Molecule, according to Claim 82 or 83, characterized in that the nucleic acid sequence encoding the modified arterivirus RNA replicon still comprising one or more expression cassettes, wherein at least one of the one or more expression cassettes comprise a promoter operably linked to a coding sequence for a gene of interest (GOI).
[85]
85. Nucleic Acid Molecule according to Claim 84, characterized in that the modified arterivirus RNA replicon comprises at least two, three, four, five or six expression cassettes.
[86]
86. The Nucleic Acid molecule of Claims 84 or 85, characterized in that at least one of one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding a part of or all of the non-structural pplab protein of the modified arterivirus RNA replicon.
[87]
87. Nucleic Acid molecule according to any one of Claims 84 to 86, characterized in that at least one
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16/32 of one or more expression cassettes is operationally positioned downstream to a transcriptional regulatory sequence (TRS) of the modified arterivirus RNA replicon, in which TRS is selected from the group consisting of TRS1, TRS2, TRS3, TRS4 , TRS5, TRS6 and TRS7.
[88]
88. Nucleic Acid Molecule according to any one of Claims 84 to 87, characterized in that at least one of one or more expression cassettes further comprises a third nucleic acid sequence encoding one or more structural elements of an enhancer viral capsid, in which the third nucleic acid sequence is operationally linked upstream to the coding sequence for GOI.
[89]
89. Nucleic Acid Molecule, according to Claim
88, characterized in that the nucleic acid sequence encoding the modified arterivirus RNA replicon still comprises a coding sequence for an autoprotease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for GOI .
[90]
90. Nucleic Acid Molecule, according to any one of Claims 84 to 89, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, a reporter polypeptide and any combination thereof.
[91]
91. Nucleic Acid Molecule, according to any of Claims 84 to 89, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immune modulator, a cytokine, an enzyme and any combination thereof.
[92]
92. Nucleic Acid Molecule, according to any one
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17/32 of Claims 69 to 91, characterized in that the modified viral RNA replicon comprises a modified RNA replicon derived from an alphavirus virus species selected from the group consisting of Eastern Equine Encephalitis (EEEV) virus, Encephalitis virus Venezuelan Equine (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFC), Pixuna virus, Middleburg virus (MIDV), Chicungunha virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), West Equine Encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu ( NDUV), Salmonid alphavirus (SAV) and Buggy Creek virus.
[93]
93. Nucleic Acid Molecule according to Claim 92, characterized in that the first nucleic acid sequence is operatively positioned upstream to a second nucleic acid sequence encoding one or more non-structural proteins nspl-4 or a part same as the modified alphavirus RNA replicon.
[94]
94. Nucleic Acid Molecule according to Claim 92 or 93, characterized in that the nucleic acid sequence encoding the modified alphavirus RNA replicon still comprises one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to a coding sequence for a gene of interest (GOI).
[95]
95. Nucleic Acid Molecule, according to Claim 94, characterized in that the modified alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes.
[96]
96. Nucleic Acid Molecule, according to Claim
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18/32
94 or 95, characterized in that at least one of one or more expression cassettes is operably linked downstream of a nucleic acid sequence encoding one or more nspl-4 non-structural proteins or a portion thereof of the RNA replicon of modified alphavirus.
[97]
97. Nucleic Acid Molecule according to any one of Claims 94 to 96, characterized in that at least one of one or more expression cassettes further comprises a third nucleic acid sequence encoding one or more structural elements of an enhancer viral capsid, wherein the third nucleic acid sequence is operably linked upstream of the coding sequence for GOI.
[98]
98. Nucleic Acid Molecule according to Claim 97, characterized in that the nucleic acid sequence encoding the modified alphavirus RNA replicon further comprises a coding sequence for an autoprotease peptide operably linked downstream to the third sequence of nucleic acid and upstream to the coding sequence for GOI.
[99]
99. Nucleic Acid Molecule according to any one of Claims 94 to 98, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme and a reporter polypeptide.
[100]
100. Nucleic Acid molecule according to any one of Claims 94 to 98, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immune modulator, an enzyme and a cytokine.
[101]
101. Nucleic Acid Molecule, comprising a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, characterized in that the non-alphavirus RNA replicon
Petition 870190052919, of 6/4/2019, p. 458/477
The modified 19/32 comprises a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or a variant thereof.
[102]
102. Nucleic Acid Molecule according to Claim 101, characterized in that the nucleic acid sequence encoding the modified non-alphavirus RNA replicon further comprises a second nucleic acid sequence encoding at least one non-structural viral protein or one part thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence.
[103]
103. Nucleic Acid Molecule, according to Claim 101 or 102, the nucleic acid sequence encoding the modified non-alphavirus RNA replicon, characterized in that it further comprises a coding sequence for an autoprotease peptide operably linked to downstream of the first nucleic acid sequence and upstream to the second nucleic acid sequence.
[104]
104. Nucleic Acid Molecule according to Claim 103, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A ), an equine rhinitis virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof.
[105]
105. Nucleic Acid molecule according to any one of Claims 101 to 104, characterized in that the nucleic acid sequence encoding the modified non-alphavirus RNA replicon comprises a modified RNA replicon derived from an RNA virus from positive filament.
[106]
106. Nucleic Acid Molecule, according to Claim 105, characterized in that the positive strand RNA virus is a species of virus belonging to a family selected from the group consisting of the Togaviridae family, Flaviviridae family, family
Petition 870190052919, of 6/4/2019, p. 459/477
20/32
Orthomyxoviridae, family Rhabdoviridae and family Paramyxoviridae.
[107]
107. Nucleic Acid Molecule according to Claim 106, characterized in that the virus species belongs to the genus Arterivirus of the family Arteriviridae.
[108]
108. Nucleic Acid Molecule according to any one of Claims 101 to 107, characterized in that the nucleic acid sequence encoding the modified non-alphavirus RNA replicon still comprising one or more expression cassettes, each of which of the expression cassettes comprises a promoter operably linked to a coding sequence for a gene of interest (GOI).
[109]
109. Nucleic Acid Molecule according to any one of Claims 101 to 108, characterized in that the nucleic acid sequence encoding the modified non-alphavirus RNA replicon comprises at least two, three, four, five, or six expression cassettes.
[110]
110. Nucleic Acid Molecule according to any one of Claims 101 to 109, characterized in that at least one of one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding at least one non-structural viral protein or a part of it.
[111]
111. Nucleic Acid Molecule according to any one of Claims 101 to 110, characterized in that at least one of one or more expression cassettes further comprises a third nucleic acid sequence encoding one or more structural elements of an enhancer viral capsid, in which the third nucleic acid sequence is operationally linked upstream to the coding sequence for GOI.
[112]
112. Nucleic Acid Molecule according to Claim 111, characterized in that the nucleic acid sequence encoding the modified non-alphavirus RNA replicon still comprising a coding sequence for an autoprotease-linked peptide of
Petition 870190052919, of 6/4/2019, p. 460/477
21/32 operational form downstream of the third nucleic acid sequence and upstream to the coding sequence for GOI.
[113]
113. Nucleic Acid Molecule, according to any one of Claims 1-26 and 69-112, characterized in that the nucleic acid molecule is produced by means of de novo synthesis.
[114]
114. Recombinant cell, characterized in that it comprises a nucleic acid molecule as defined in any one of Claims 1-27 and 69-113.
[115]
115. Recombinant cell according to Claim 114, characterized in that the recombinant cell is a prokaryotic cell or a eukaryotic cell.
[116]
116. Recombinant cell according to Claim 114, characterized in that the recombinant cell is an animal cell.
[117]
117. Recombinant cell according to any one of Claims 114 to 116, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding a modified RNA replicon and wherein the expression of the modified replicon RNA confers resistance the innate immune response in the recombinant cell.
[118]
118. Cell Culture, characterized in that it comprises a recombinant cell as defined in any one of Claims 114 to 117.
[119]
119. Method for Conferring Resistance to the Individual's Innate Immune System, comprising administering to the individual a nucleic acid molecule comprising a nucleic acid sequence, which encodes a modified viral RNA replicon, characterized in that the modified viral RNA replicon comprises :
a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and
Petition 870190052919, of 6/4/2019, p. 461/477
22/32 a second nucleic acid sequence encoding at least one non-structural protein or a part thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, and, wherein the expression of the modified replicon RNA encoded by the nucleic acid molecule confers resistance to the innate immune response in the individual.
[120]
120. Method for Producing Individual Interest Polypeptide, comprising administering to the individual a nucleic acid molecule comprising a nucleic acid sequence encoding a modified viral RNA replicon, characterized in that the modified viral RNA replicon comprises:
a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or a variant thereof, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence encoding at least one non-structural protein or a part thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence.
[121]
121. Method for Production of Polypeptide of Interest, comprising culturing a host cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a modified viral RNA replicon, characterized in that the modified viral RNA replicon comprises:
a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or
Petition 870190052919, of 6/4/2019, p. 462/477
23/32 a variant thereof, in which the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence encoding at least one non-structural protein or a part thereof, in which the first acid sequence nucleic is operably linked upstream to the second nucleic acid sequence.
[122]
122. Method according to Claim 119 or 120, characterized in that the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster and buffalo.
[123]
123. The method of any one of Claims 119 to 122, characterized in that at least one of one or more structural elements of the viral capsid enhancer comprises one or more RNA stem loops.
[124]
124. Method according to any of Claims 119 to 123, characterized in that the viral capsid enhancer is derived from a capsid gene of a virus species belonging to the Togaviridae family.
[125]
125. Method according to Claim 124, characterized in that the virus species belongs to the Alphavirus genus of the Togaviridae family.
[126]
126. Method according to Claim 125, characterized in that the alphavirus species is selected from the group consisting of the Eastern Equine Encephalitis virus (EEEV), Venezuelan Equine Encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFC), Pixuna virus, Middleburg virus (MIDV), Chicungunha virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BE) , Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV),
Petition 870190052919, of 6/4/2019, p. 463/477
24/32
Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), West Equine Encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV) and Buggy Creek virus.
[127]
127. Method according to any of Claims 124 to 126, characterized in that the viral capsid enhancer comprises a downstream loop motif (DLP) of the virus species and wherein the DLP motif comprises at least one of one or more RNA stemloops.
[128]
128. Method according to any one of claims 119-127, characterized in that the viral capsid enhancer comprises a nucleic acid sequence having at least 80% sequence identity with at least one of SEQ ID ^Nos: 1 and 46-52.
[129]
129. The method according to claim 128, wherein the nucleic acid sequence has at least 95% sequence identity with at least one of ^the SEQ ID NO: 1 and 46-52.
[130]
130. Method according to any of Claims 119 to 129, characterized in that the nucleic acid sequence encoding the modified viral RNA replicon further comprising a coding sequence for an autoprotease peptide operably linked downstream to the first sequence nucleic acid and upstream to the second nucleic acid sequence.
[131]
131. Method according to Claim 130, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), a equine rhinitis virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.
[132]
132. Method according to any of Claims 119 to 131, characterized in that the first acid sequence
Petition 870190052919, of 6/4/2019, p. 464/477
The nucleic 25/32 is operably positioned within a region of about 1 to 1000 nucleotides downstream of the 5 'end of the modified viral RNA replicon.
[133]
133. Method according to any of Claims 119 to 132, characterized in that the second nucleic acid sequence comprises substantially the entire coding sequence for the viral non-structural proteins native to the corresponding unmodified viral RNA replicon.
[134]
134. Method according to any one of Claims 119 to 133, characterized in that the modified viral RNA replicon comprises a modified RNA replicon derived from a virus species belonging to the Alphavirus genus of the Togaviridae family or to the Arterivirus genus of the family Arteriviridae.
[135]
135. Method according to any one of Claims 119 to 134, characterized in that the species of arterivirus virus is selected from the group consisting of Equine arteritis virus (EAV), swine reproductive and respiratory syndrome virus (PRRSV), lactic dehydrogenase elevator virus (LDV) and simian hemorrhagic fever virus (SHFV).
[136]
136. Method according to any of Claims 134 to 135, characterized in that the virus species is an arterivirus and in which the first nucleic acid sequence is operably positioned upstream to a second nucleic acid sequence encoding a part or all of the non-structural protein pplab from the modified arterivirus RNA replicon.
[137]
137. The method of Claim 136, wherein the nucleic acid sequence encoding the modified arterivirus RNA replicon still comprises one or more expression cassettes, wherein at least one of the expression cassettes comprises a linked promoter. operationally to a coding sequence for a gene of interest (GOI).
[138]
138. Method according to Claim 137, characterized
Petition 870190052919, of 6/4/2019, p. 465/477
26/32 why the modified arterivirus RNA replicon still comprises at least two, three, four, five or six expression cassettes.
[139]
139. Method according to Claim 137, characterized in that at least one of one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding a part or all of the replicon pplab nonstructural protein of modified arterivirus RNA.
[140]
140. Method according to any of Claims 137 to 139, characterized in that at least one of one or more expression cassettes is operably positioned downstream of a transcriptional regulatory sequence (TRS) of the arterivirus RNA replicon modified, in which TRS is selected from the group consisting of TRS1, TRS2, TRS3, TRS4, TRS5, TRS6 and TRS7.
[141]
141. Method according to any of Claims 137 to 140, characterized in that at least one of one or more expression cassettes still comprises a third nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for GOI.
[142]
142. Method according to Claim 141, characterized in that the nucleic acid sequence encoding the modified arterivirus RNA replicon further comprising a coding sequence for an autoprotease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for GOI.
[143]
143. Method according to any of Claims 137 to 142, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide , an industrial enzyme, a reporter polypeptide and any combination thereof.
Petition 870190052919, of 6/4/2019, p. 466/477
27/32
[144]
144. Method according to any of Claims 137 to 142, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immune modulator, a cytokine, an enzyme and any combination of them.
[145]
145. Method according to any of Claims 119 to 134, characterized in that the modified viral RNA replicon comprises a modified RNA replicon derived from an alphavirus virus species selected from the group consisting of Eastern Equine Encephalitis virus ( Venezuelan Equine Encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFC), Pixuna virus, Middleburg virus (MIDV), Chicungunha virus (CHIKV), O'Nyong virus -Nyong (ONNV), Ross River virus (RRV), Barmah Forest virus (BE), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), West Equine Encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), Salmonid alphavirus (SAV) and Buggy Creek virus.
[146]
146. Method according to Claim 145, characterized in that the first nucleic acid sequence is operatively positioned upstream to a nucleic acid sequence encoding one or more nspl-4 non-structural proteins or a part thereof of modified alphavirus RNA.
[147]
147. Method according to any of Claims 145 to 146, characterized in that the nucleic acid sequence encoding the modified alphavirus RNA replicon still comprises one or more expression cassettes, wherein each of the expression cassettes comprises a promoter operably linked to a coding sequence for a gene of interest (GOI).
[148]
148. Method according to Claim 147, characterized
Petition 870190052919, of 6/4/2019, p. 467/477
28/32 why the modified alphavirus RNA replicon comprises at least two, three, four, five or six expression cassettes.
[149]
149. Method according to any of Claims 147 to 148, characterized in that at least one of one or more expression cassettes is operably linked downstream of a nucleic acid sequence encoding one or more non-structural nspl proteins -4 or a portion thereof from the modified alphavirus RNA replicon.
[150]
150. Method according to any of Claims 147 to 149, characterized in that at least one of one or more expression cassettes still comprises a third nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer, wherein the third nucleic acid sequence is operably linked upstream of the coding sequence for GOI.
[151]
151. Method according to Claim 150, characterized in that the modified alphavirus RNA replicon further comprising a coding sequence for an autoprotease peptide operably linked downstream to the third nucleic acid sequence and upstream to the coding sequence for GOI.
[152]
152. Method according to any of Claims 147 to 151, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide , an industrial enzyme, a reporter polypeptide and any combination thereof.
[153]
153. Method according to any of Claims 147 to 151, characterized in that the coding sequence for GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immune modulator, a cytokine, an enzyme and any combination of them.
[154]
154. Method to Check Resistance to the Immune System
Petition 870190052919, of 6/4/2019, p. 468/477
29/32
Individual inborn, comprising administering to the individual a nucleic acid molecule comprising a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, characterized in that the modified non-alphavirus RNA replicon comprises a first acid sequence nucleic encoding one or more structural elements of an alphavirus capsid enhancer and wherein the expression of the modified non-alphavirus RNA replicon encoded by the nucleic acid molecule confers resistance to the innate immune response in the individual.
[155]
155. Method for Producing Individual Polypeptide of Interest, comprising administering to the individual a nucleic acid molecule comprising a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, characterized in that the RNA replicon does not Modified alphavirus comprises a first nucleic acid sequence encoding one or more structural elements of an alphavirus capsid enhancer.
[156]
156. Method for Producing Polypeptide of Interest, comprising culturing a host cell comprising a nucleic acid molecule, which comprises a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, characterized in that the RNA replicon does not Modified alphavirus comprises a first nucleic acid sequence encoding one or more structural elements of an alphavirus capsid enhancer.
[157]
157. Method according to Claim 154 or 155, characterized in that the individual is selected from the group consisting of human, horse, pig, primate, mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog, bird, fish, goat, donkey, hamster and buffalo.
[158]
158. Method according to any of Claims 154 to 157, characterized in that the non-alphavirus RNA replicon
Petition 870190052919, of 6/4/2019, p. 469/477
The modified 30/32 further comprising a second nucleic acid sequence encoding at least one non-structural viral protein or a part thereof, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence.
[159]
159. Method according to any of Claims 154 to 158, characterized in that the modified non-alphavirus RNA replicon further comprises a coding sequence for an autoprotease peptide operably linked downstream to the first nucleic acid sequence and upstream to the second nucleic acid sequence.
[160]
160. Method according to Claim 159, characterized in that the autoprotease peptide comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), a equine rhinitis virus A (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A) and a combination thereof.
[161]
161. Method according to any one of Claims 154 to 160, characterized in that the modified non-alphavirus RNA replicon comprises a modified RNA replicon derived from a positive strand RNA virus.
[162]
162. Method according to Claim 161, characterized in that the positive strand RNA virus is a species of virus belonging to a family selected from the group consisting of the Togaviridae family, Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family and Paramyxoviridae family.
[163]
163. Method according to Claim 161, characterized in that the positive strand RNA virus is a species of virus belonging to the genus Arterivirus of the family Arteriviridae.
[164]
164. Method according to any of Claims 154 to 163, characterized in that the sequence encoding the replicon of
Petition 870190052919, of 6/4/2019, p. 470/477
31/32
Modified non-alphavirus RNA further comprises one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to a coding sequence for a gene of interest (GOI).
[165]
165. Method according to any one of Claims 154 to 164, characterized in that the modified non-alphavirus RNA replicon comprises at least two, three, four, five, or six expression cassettes.
[166]
166. Method according to any of Claims 154 to 165, characterized in that at least one of one or more expression cassettes is operably linked downstream of the second nucleic acid sequence encoding at least one non-viral protein structural or part of it from the modified non-alphavirus RNA replicon.
[167]
167. Method according to any one of Claims 154 to 166, characterized in that at least one of one or more expression cassettes further comprises a third nucleic acid sequence encoding one or more structural elements of an alphavirus capsid enhancer , wherein the third nucleic acid sequence is operably linked upstream to the coding sequence for GOI.
[168]
168. Method according to Claim 167, characterized in that the modified non-alphavirus RNA replicon further comprises a coding sequence for an autoprotease peptide operably linked downstream to the third nucleic acid sequence and upstream to the sequence coding for GOI.
[169]
169. Recombinant Polypeptide, characterized in that it is produced by the method as defined in any of Claims 121-153 and 155-168.
[170]
170. Composition, characterized in that it comprises a recombinant polypeptide as defined in Claim 169 and a pharmaceutically acceptable carrier.
Petition 870190052919, of 6/4/2019, p. 471/477
32/32
[171]
171. Composition, characterized in that it comprises a nucleic acid molecule as defined in any one of Claims 1-27 and 69-113 and a pharmaceutically acceptable carrier.
[172]
172. Composition, characterized in that it comprises a recombinant cell as defined in any one of Claims 114 to 117 and a pharmaceutically acceptable carrier.
[173]
173. Composition according to any one of Claims 170 to 172, characterized in that the composition is formulated in a pharmaceutical formulation.
[174]
174. Composition according to any one of Claims 170 to 173, characterized in that the composition is formulated in a pharmaceutical formulation with covalent compounds, non-covalent compounds, physical compositions, or pharmaceutically acceptable buffers.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US5116742A|1986-12-03|1992-05-26|University Patents, Inc.|RNA ribozyme restriction endoribonucleases and methods|

---

US5225337A|1989-09-25|1993-07-06|Innovir Laboratories, Inc.|Ribozyme compositions and methods for use|

---

US5246921A|1990-06-26|1993-09-21|The Wistar Institute Of Anatomy And Biology|Method for treating leukemias|

---

SE9401709D0|1994-05-18|1994-05-18|Mathilda Sjoeberg|Improved alphavirus vectors for expression of heterologous DNA|

---

US5792462A|1995-05-23|1998-08-11|University Of North Carolina At Chapel Hill|Alphavirus RNA replicon systems|

---

US7204997B2|2002-01-29|2007-04-17|Supratek Pharma Inc.|Responsive microgel and methods related thereto|

---

US6943015B2|2002-12-12|2005-09-13|Ilya Frolov|Large scale production of packaged alphavirus replicons|

---

EP1773403B1|2004-07-09|2018-04-25|The University of North Carolina At Chapel Hill|Alphavirus-based adjuvants|

---

US8961995B2|2012-09-20|2015-02-24|Uab Research Foundation|Methods and compositions for alphavirus replicons|

---

WO2016004043A1|2014-06-30|2016-01-07|Blend Therapeutics, Inc.|Targeted conjugates and particles and formulations thereof|EP3443107A1|2016-04-13|2019-02-20|Synthetic Genomics, Inc.|Recombinant arterivirus replicon systems and uses thereof|

---

EA202091513A1|2017-12-19|2020-09-09|Янссен Сайенсиз Айрлэнд Анлимитед Компани|VACCINES AGAINST HEPATITIS B VIRUSAND THEIR APPLICATION|

---

US11020476B2|2017-12-19|2021-06-01|Janssen Sciences Ireland Unlimited Company|Methods and compositions for inducing an immune response against Hepatitis B Virus |

---

KR20200111729A|2018-01-19|2020-09-29|얀센 파마슈티칼즈, 인코포레이티드|Induction and enhancement of immune response using recombinant replicon system|

---

BR112020016570A2|2018-02-17|2020-12-15|Flagship Pioneering Innovations V, Inc.|COMPOSITIONS AND METHODS FOR MEMBRANE PROTEIN RELEASE|

---

WO2020181058A1|2019-03-05|2020-09-10|Massachusetts Institute Of Technology|Dna launched rna replicon systemand uses thereof|

---

WO2020255055A1|2019-06-20|2020-12-24|Janssen Sciences Ireland Unlimited Company|Self-replicating rna molecules for hepatitis b virusvaccines and uses thereof|

---

US20210222133A1|2019-11-18|2021-07-22|Janssen Biotech, Inc.|Vaccines based on mutant calr and jak2 and their uses|

---

UY39079A|2020-02-14|2021-08-31|Janssen Biotech Inc|NEOANTIGES EXPRESSED IN MULTIPLE MYELOMA AND ITS USES|

---

UY39078A|2020-02-14|2021-08-31|Janssen Biotech Inc|NEW ANTIGES EXPRESSED IN OVARIAN CANCER AND ITS USES|

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WO2021209897A1|2020-04-13|2021-10-21|Janssen Biotech, Inc.|Psma and steap1 vaccines and their uses|

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US20210347828A1|2020-05-11|2021-11-11|Janssen Pharmaceuticals, Inc.|RNA Replicon Encoding a Stabilized Corona Virus Spike Protein|

---

WO2021229450A1|2020-05-11|2021-11-18|Janssen Pharmaceuticals, Inc.|Sars-cov-2 vaccines|

---

WO2022009049A1|2020-07-06|2022-01-13|Janssen Biotech, Inc.|Prostate neoantigens and their uses|

---

WO2022009052A2|2020-07-06|2022-01-13|Janssen Biotech, Inc.|Prostate neoantigens and their uses|

---

WO2022008613A1|2020-07-08|2022-01-13|Janssen Sciences Ireland Unlimited Company|Rna replicon vaccines against hbv|

---

CN112029781A|2020-08-14|2020-12-04|中山大学|Novel coronavirus SARS-CoV-2 safety replicon system and application thereof|

法律状态:
2021-10-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201662430250P| true| 2016-12-05|2016-12-05|

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US62/430,250|2016-12-05|

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US201762486361P| true| 2017-04-17|2017-04-17|

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US62/486,361|2017-04-17|

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US201762587954P| true| 2017-11-17|2017-11-17|

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US62/587,954|2017-11-17|

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PCT/US2017/064561|WO2018106615A2|2016-12-05|2017-12-04|Compositions and methods for enhancing gene expression|

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