• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention solves the problem of providing new therapies that are effective in the treatment of muscular dystrophies through the use of compositions comprising a compound capable of: To. Increase the expression of the pitx2 gene in muscle satellite stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells; i B. Reduce the expression of mirna-106b in muscle satellite stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2559106A1
    申请号:ES201530645
    申请日:2015-05-12
    公开日:2016-02-10
    发明作者:Amelia Eva Aránega Jiménez；Diego Franco Jaime；Francisco HERNÁNDEZ TORRES；Estefanía LOZANO VELASCO；Daniel VALLEJO PULIDO
    申请人:Universidad de Jaen；
    IPC主号:
    专利说明:

    Method of activating the expression of the Pitx2 gene to promote regenerationmuscular.
    Technical Field of the Invention The present invention relates to the field of biotechnology, in particular the use of Pitx2 gene expression for the treatment or prevention of muscular dystrophies.
    Background of the Invention The following discussion of the background of the invention is provided merely to help the reader understand the invention.
    Skeletal muscle has the ability to repair and regenerate due to the presence of resident stem cells, called muscle satellite cells. In mature muscle tissue, satellite cells constitute a small, dispersed population of mitotically and physiologically quiescent cells, marked by their expression of the transcription factor Pax7 (Figure 1). Satellite stem cells of the adult muscle are a lineage from the Pax3 / pax7 + embryonic myogenic progenitor cells that remain in the adult muscle in a quiescence state and after an injury they are activated, proliferate and enter the myogenic differentiation program due to the positive regulation of the myf5, MyoD and myogenin myogenic determination genes thus forming new myoblasts that eventually fuse together to generate new muscle tissue (Figure 1).
    Muscular dystrophies are a group of genetic conditions characterized by progressive degenerative muscle disorders. One of the most serious characteristics in these pathologies is the gradual loss of skeletal muscle tissue due to chronic degeneration accompanied by poor regeneration. Its most common forms in childhood are Duchenne (DMD) and Becker (BMD) muscular dystrophies and are characterized as inherited recessive disorders linked to the X chromosome caused by mutations in the dystrophin gene. Dystrophin plays an important structural role in muscle fiber serving as a connection between the extracellular matrix and the cytoskeleton. The N-terminal region of this protein binds to the actin cytoskeleton protein, while the e-terminal end is part of the dystrophin-associated glycoprotein complex (DGC) that connects with the muscle fiber membrane (sarcolemma). In the absence of dystrophin, mechanical stress leads to ruptures in the sarcolemma
     04-1 2-2015
    causing progressive muscle necrosis, loss of independent ambulation at the beginning of adolescence, cardiomyopathy, respiratory failure, and premature death in affected individuals.
    5 There is currently no cure for muscular dystrophies and existing therapies areineffective Although, gene therapies are likely to provide a cureFor these diseases there are important obstacles that limit their application. Thushave made potential approaches that have gone from gene augmentation strategiesusing viral vectors or plasmids intended for the restoration of the expression of
    10 dystrophin, up to the positive regulation of genes that could be used to overcome the lack of expression of the deserted gene. Although some of these approaches have proven to be partially effective, the results obtained so far have revealed their numerous limitations. In particular, the progressive loss of expression of the therapeutic gene observed after treatment has clearly indicated that the modification
    15 of the mature fiber alone is not enough to maintain the beneficial effects obtained by this therapeutic approach.
    Therefore, there is a need to provide new therapies that are effective in the treatment of muscular dystrophies, especially by identifying new
    20 approaches to improve muscle regeneration in these patients.
    Brief Description of the Invention
    The present invention solves the problem of providing new therapies that result
    25 effective in the treatment of muscular dystrophies through the use of compositions comprising a compound capable of:
    to. increase Pitx2 gene expression in muscle satellite stem cells
    of a human or animal subject with respect to that observed in the absence of the compound in said cells; yfo
    b. reduce miRNA-106b expression in muscle satellite stem cells
    of a human or animal subject with respect to that observed in the absence of the compound in said cells.
    35 3

    Brief description of the figures
    Figure 1. Embryonic origin of muscle satellite cells. (Buckingham and Vincent, Current Opinion in Genetics & Development, 2009)
    Figure 2. A and B) Overexpression of Pitx2 in mouse satellite cells by transfection with the bicistronic lentiviral vector LVX-Pitx2c-ZSGreen. C) The expression of the miRNAs: miR-15b, miR-106b, miR-23b and miR-503 (qRT-PCR) is decreased in satellite cells that overexpress Pitx2, both in cells in early stages of activation ( EPq) as in more advanced stages of activation (EPa) (qRT-PCR). D) Transfection with the LVX-Pitx2c-ZSGreen bicistronic lentiviral vector leads to a decrease in the expression of the cyclin D1 and cyclin D2 cell cycle control genes (q RT-PCR) indicating that, similar to what occurs in myoblasts, the Pitx2-miRNAs pathway controls proliferation in satellite cells. E) Quantification by immunohistochemistry of the number of proliferating cells (Ki67 +) in Pitx2 over-expressing satellite cell cultures.
    Figure 3. A) The expression of Myf5 (qRT-PCR) increases in satellite cells that overexpress Pitx2 and B) quantification by immunohistochemistry shows a significant increase in Myf5 + cells. C) Overexpression of miR-106b leads to a
    decreased expression of Myf5 validating it as the target of this miRNA. Or: Normalized luciferase activity of the 3'-UTR Myf5 luciferase reporter (WT Myf5 3'-UTR), with the empty plasmid (Vector) or co-transfecting with pre-miR-106b shows the loss of luciferase activity with the miR-106b. There is no loss of luciferase activity when miR-1 06b "seed sequence" was mutated.
    Figure 4. A) Analysis by qRT-PCR of overexpression of Pitx2c in the muscles of MDX mice injected with dystrophic satellite cells transfected with the lentiviral vector LVX-Pitx2c-ZS-Green vector compared to the muscles injected with cells transfected with the lentivirus empty (LVX-ZS-Green vector); percentage of fibers formed "de novo" (ZS-Green + cells) in the transplanted muscles after 15 days and representative image. B) The decrease in the expression of miR-31 in the transplanted muscles with cells overexpressing Pitx2 leads to an increase in the levels of dyslrofin expression as well as a significant increase in the fibers expressing dyslrofin. C) Treadmill test shows the functional improvement of DMDmdx mice injected with cells that overexpress Pitx2. O) Immunohistochemical analysis of proliferating cells (Ki67 +) after 15 days of cell transplantation. E) Expression analysis using qRT-PCR of

    the miRNAS modulated by Pitx2 in the muscle of the OMOmdx mice that underwent cell transplantation with cells that overexpress Pitx2. F) The expression levels of cyclins 01 and 02 as well as the Myf5 transcription factor were increased in the transplanted muscles with cells that overexpress Pitx2 indicating
    5 that the Pitx2-miRNAS molecular cascade is conserved in the "inalive".
    Detailed description of the invention
    10 Definitions As used in the specification and the appended claims, the term "Pitx2 gene" is a member of the family of homeodomain bicoid transcription factors that plays a relevant role in the phogenesis (Genbank polynucleotide sequence reference with number of access NM 000325 identifying Homo sapiens paired-like
    15 homeodomain 2 (PITX2))
    The term "increase" or "increase" refers to increases above the baseline level. For example, basal levels are normal at in vivo levels before, or in the absence of, the addition of an activator compound.
    The term "reduction" or "reduce" or "inhibit" or "inhibition ~ refers to reductions below the baseline level. For example, baseline levels are normal at in vivo levels before, or in the absence of, the addition of an inhibitor compound.
    25 In this specification and in the claims that follow, reference will be made to a number of terms that will be defined to have the following meanings:
    "Optional" or "optionally" means that the event or circumstance described below may or may not occur, and that the description includes cases in which said event or circumstance occurs and cases in which it does not.
    As used herein, the terms "prevent", "preventing" and "prevention" refer to methods to prevent or prevent the development of a disease or disorder or delay the recurrence or occurrence of one or more symptoms of a disorder in a subject resulting from
    35 administration of a prophylactic agent.
     04-1 2-2015
    The term "pharmaceutically acceptable vehicle" is intended to include the formulation used
    to stabilize, solubilize and be mixed in some way with active ingredients that are administered to living animals, including humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retardants, and the like, compatible with pharmaceutical administration. Except to the extent that any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.
    The term "disease", as used herein, is intended to be generally synonymous, and is used interchangeably with the terms "disorder" and "condition" (as in the medical condition), in which all reflect an abnormal condition of the body or of one of its parts that impairs normal functioning and is typically manifested by distinctive signs and symptoms.
    The term "combination therapy" means the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present description. Such administration encompasses the ca-administration of these therapeutic agents.
    in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple separate capsules for each active ingredient. In addition, such administration also encompasses the use of each type of therapeutic agent in a sequential manner. In any case, the treatment regimen will provide beneficial effects of the combination of compounds in the treatment of the conditions or disorders described herein.
    The phrase "therapeutically effective" is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder. This amount will be necessary to achieve the objective of reducing or eliminating said disease or disorder.
    The term "subject" refers to all mammals, including humans. Examples of subjects include, but are not limited to, humans, cows, dogs, cats, goats, sheep, pigs, and rabbits.
    Throughout this application, reference is made to various publications. The descriptions of these publications in their entirety are incorporated by reference in this application in order to more fully describe the state of the art to which it belongs. The
     04-1 2-2015
    References described are also individually and specifically incorporated herein as a reference for the material contained therein which is discussed in the phrase upon which said reference is based.
    Description of the invention The present invention addresses the problem of providing new therapies that are effective in the treatment of muscular dystrophies, increasing the ability to regenerate lost tissue, as a result of muscular dystrophy, of skeletal muscle stem cells.
    For this purpose, the authors of the present invention have evaluated the contribution of the Pitx2 gene in the regulation of tissue-specific transcription of different microRNAs during myogenesis. The analysis of gene expression profiles (microRNA-microarrays) in a myoblast cell line (So18 cell line) has led to identify the authors of the present invention a series of microRNAs (miRNAs) that are differentially regulated in Sol8 myoblasts that overexpress Pitx2 (see figure 2). The analysis of the effects of these microRNAs on the proliferation of myoblasts and the identification of their supposed targets demonstrate that Pitx2 regulates a subset of microRNAs that has a profound effect on the progression of the myoblast cell cycle (miR15b, miR-23b, miR106b Y miR-503). Additionally, the authors of the invention found that this Pitx2-miRNAs pathway also regulates cell proliferation in satellite cells isolated from mouse skeletal muscle (Figure 1). These results indicate that Pitx2 acts by amplifying the population of myoblasts derived from satellite cells during the differentiation processes of regenerative myogenesis (Figure 3).
    Additionally, these results demonstrate that reducing the expression of miRNA-106b in the muscle satellite stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells, amplifies the population of myoblasts derived from satellite cells during the differentiation processes of regenerative myogenesis (see figures 3 and 4).
    Accordingly, a first aspect of the present invention relates to the use of a composition (hereinafter "composition of the present invention") comprising a compound capable of
    P201 530645
    increase the expression of the Pitx2 gene, activator compound, in the satellite muscle stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells; I
    reduce the expression of the miRNA-106b, inhibitor compound, in the muscle satellite stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells;
    for the development of a medicine to promote muscle regeneration.
    Alternatively, the first aspect of the invention relates to a composition comprising a compound capable of
    increase the expression of the Pitx2 gene, activator compound, in the satellite muscle stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells; and / or reducing the expression of the miRNA-106b, inhibitor compound, in the muscle satellite stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells;
    For use in promoting muscle regeneration.
    "Increase the expression of the Pitx2 gene" means the increase on the baseline, or in comparison with the control, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17> 18,
    19,20,21,22,23,24,25,26,27,28,29,30, 31,32,33,34, 35, 36, 37, 38, 39,40, 41,42, 43, 44, 45, 46, 47, 48, 49.50, 51, 52, 53, 54.55, 56.57, 58, 59.60, 61, 62, 63, 64.65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13,14,15,20, 25, 30 , 35, 40, 45, 50, or more times.
    Activating compounds can be identified by a screening method that allows the ability to activate Pitx2 to be identified in a compound, which comprises contacting a cell, preferably a muscle satellite stem cell, with a compound suspected of activating Pitx2; by testing the content of the cells to determine the amount and the biological activity of Pitx2, and compare the determined amount and the biological activity of Pitx2 to a predetermined level, at which a change of said amount and the biological activity of Pitx2 is indicative of a compound

    that activates Pitx2. In a preferred embodiment, the detection is performed by RT-PCR
    quantitative in real time, using specific primers for each isoform.
    In the context of the present invention, muscle satellite stem cells are understood to be
    5 stem cells, or pre-muscle cells, that serve to help the regeneration of
    adult skeletal muscle. Following proliferation (when satellite cells are activated) and
    subsequent differentiation (when they begin to express transcription factors that
    commit to a myogenic lineage (myoblasts), satellite cells fuse between
    yes or with adjacent damaged muscle fibers, which increases the number of
    1O Myionuclei in the fibers for growth and repair. The activation and proliferation of
    Satellite cells are necessary in order to meet the training needs of new
    muscle fibers to regenerate the muscle. Differentiation is necessary for the
    satellite cells can first become myoblasts and after the fusion process, in
    fibers
    fifteen
    A preferred embodiment of the first aspect of the invention relates to the use of the
    composition of the invention, wherein said medicament is used to promote the
    muscle regeneration in the treatment of dystrophinopathy or dystrophy. Preferably,
    where said dystrophinopathy or dystrophy is selected from the list consisting of dystrophy
    twenty Duchenne muscle and Becker muscular dystrophy.
    Another preferred embodiment of the first aspect of the invention or any of its
    preferred embodiments, refers to the use of the composition of the invention, wherein said
    compound, activator compound, is a DNA polynucleotide (hereafter
    25 polynucleotide of the invention) comprising a sequence selected from the list that
    consists in :
    Genbank polynucleotide sequence with access number NM_000325 or its
    complementary sequence;
    30 a sequence that selectively hybridizes with the sequence of (a); Y
    a polynucleotide sequence of DNA encoding a sequence
    identical amino acid at least 90%, 92%, 94%, 96%, 98% or 99% based
    in the identity of all the amino acids of said sequence, to the sequence
    Genbank amino acid with access number NP_000316.
    35

    In the context of the present invention, the Genbank polynucleotide sequence with
    Accession number NM_000325 (SEQ ID NO 1) is identified as Homo sapiens paired-like
    homeodomain 2 (PITX2), transcript variant 3, mRNA. Said nucleotide sequence is set forth below:
    5 GTTAGGCCAACAGGGAAGCGCGGAGCCGCAGATCTGGTCCGTCGCTCGCCTGGGTGC CTGGAGCTGAGCTGCGGCAAGGCCCGGCTCCTGTTCGACCGCCCGAGGGGTGTGCGT GTGCGCGTTGCGGAGGGTGCGCTCAGAGGGCCGCGTCGTGGCTGCAGCGGCTGCTG CCGCCGCAGGGGATCTAATATCACCTACCTGTCCCTGTCACTCTTGACACTTCTCTGTC
    10 AGGGCTGCCGCGTGGGGGGGGGGCGGGCAGAGCGCGGTCGGCGTTAGCTTTCCTTAT TGGAGGGGTTCTTGGGGGAGGGAGGGAGAGAAGAAGGGGGTCTTTGCCCACTCTTGT TTCGCTTTGGAGCTTGGAAGCCTGCTCCCTAAAGACGCTCTGAGTGGTGCCCTTCTGCC CACATCCCATGTCTTCGTTTGCCCGCTGACTTTCCGTCTCCGGACI January 1 IICGCTTGAGC CTTCCGGAGGAGACGGGGGCAGCTTGGCTTGAGAACTCGGCGGGGGTTGCGTCCCCT
    15 GGCTCTCCCCGCAGCGGGGAAACTCCGCGCCTAGAGCGCGACCCGGAGCGGGCAGC GGCGGCTACGGGGGCTCGGCGGGGCAGTAGCCAAGGACTAGTAGAGCGTCGCGCTC CCTCGTCCATGAACTGCATGAAAGGCCCGCTTCACTTGGAGCACCGAGCAGCGGGGAC CAAGCTGTCGGCCGTCTCCTCATCTTCCTGTCACCATCCCCAGCCGTTAGCCATGGCTT CGGTTCTGGCTCCCGGTCAGCCCCGGTCGCTGGACTCCTCCAAGCACAGGCTGGAGG
    20 TGCACACCATCTCCGACACCTCCAGCCCGGAGGCCGCAGAGAAAGATAAAAGCCAGCA GGGGAAGAATGAGGACGTGGGCGCCGAGGACCCGTCTAAGAAGAAGCGGCAAAGGCG GCAGCGGACTCACTTTACCAGCCAGCAGCTCCAGGAGCTGGAGGCCACTTTCCAGAGG AACCGCTACCCGGACATGTCCACACGCGAAGAAATCGCTGTGTGGACCAACCTTACGG AAGCCCGAGTCCGGGTTTGGTTCAAGAATCGTCGGGCCAAATGGAGAAAGAGGGAGCG
    25 CAACCAGCAGGCCGAGCTATGCAAGAATGGCTTCGGGCCGCAGTTCAATGGGCTCATG CAGCCCTACGACGACATGTACCCAGGCTATTCCTACAACAACTGGGCCGCCAAGGGCC TTACATCCGCCTCCCTATCCACCAAGAGCTTCCCCTTCTTCAACTCTATGAACGTCAACC CCCTGTCATCACAGAGCATGTTTTCCCCACCCAACTCTATCTCGTCCATGAGCATGTCG TCCAGCATGGTGCCCTCAGCAGTGACAGGCGTCCCGGGCTCCAGTCTCAACAGCCTGA
    30 ATAACTTGAACAACCTGAGTAGCCCGTCGCTGAATTCCGCGGTGCCGACGCCTGCCTG TCCTTACGCGCCGCCGACTCCTCCGTATGTTTATAGGGACACGTGTAACTCGAGCCTGG CCAGCCTGAGACTGAAAGCAAAGCAGCACTCCAGCTTCGGCTACGCCAGCGTGCAGAA CCCGGCCTCCAACCTGAGTGCTTGCCAGTATGCAGTGGACCGGCCCGTGTGAGCCGC ACCCACAGCGCCGGGATCCTAGGACCTTGCCGGATGGGGCAACTCCGCCCTTGAAAGA
    35 CTGGGAATTATGCTAGAAGGTCGTGGGCACTAAAGAAAGGGAGAGAAAGAGAAGCTAT ATAGAGAAAAGGAAACCACTGAATCAAAGAGAGAGCTCCTTTGATTTCAAAGGGATGTC 10
    P201 530645
    CTCAGTGTCTGACATCTTTCACTACAAGTATTTCTAACAGTTGCAAGGACACATACACAAACAAATGTTTGACTGGATATGACATTTTAACATTACTATAAGCTTGTTAIIIIIIAAGTTTAGCATTGTTAACATTTAAATGACTGAAAGGATGTATATATATCGAAATGTCAAATTAATTTTATAAAAGCAGTTGTTAGTAATATCACAACAGTGIIIIIAAAGGTTAGGCTTTAAAATAAAG
    5 CATGTTATACAGAAGCGATTAGGAIIIIICGCTTGCGAGCAAGGGAGTGTATATACTAAATGCCACACTGTATGTTTCTAACATATTATTATTATTATAAAAAATGTGTGAATATCAGTTTTAGAATAGTTTCTCTGGTGGATGCAATGATGTTTCTGAAACTGCTATGTACAACCTACCCTGTGTATAACATTTCGTACAATATTATTGTTTTACTTTTCAGCAAATATGAAACAAATGTGTT
    TTATTTCATGGGAGTAAAATATACTGCATACAAAAAAAAAAAAVVVVVVVV ~ AA
    In the context of the present invention, the amino acid sequence with access number NP _000316 (SEa ID NO 2) is identified as. pituitary homeobox 2 isoform c [Hamo sapiens]. Said amino acid sequence is set forth below:
    15 MNCMKGPLHLEHRAAGTKLSAVSSSSCHHPQPLAMASVLAPGQPRSLDSSKHRLEVHTIS DTSSPEAAEKDKSQQGKNEDVGAEDPSKKKRQRRQRTHFTSQQLQELEATFQRNRYPDM STREEIAVWTNLTEARVRVWFKNRRAKWRKRERNQQAELCKNGFGPQFNGLMQPYDDMY PGYSYNNWAAKGLTSASLSTKSFPFFNSMNVNPLSSQSMFSPPNSISSMSMSSSMVPSAV TGVPGSSLNSLNNLNNLSSPSLNSAVPTPACPYAPPTPPYVYRDTCNSSLASLRLKAKQHS
    20 SFGYASVQNPASNLSACQYAVDRPV
    In the context of the present invention, the degree of identity of an amino acid sequence is based on the identity of all amino acids of said sequence.
    In this embodiment of the invention, synthetic or modified nucleotides may be included within the polynucleotides of the invention. A number of different types of polynucleotide modification are known in the art. These include methyl phosphate and phosphorothioate main chains, addition of acridine or polylysine chains at the 3 'and 5' ends of the molecule. For the purposes of the present invention, it should be understood
    The polynucleotides described herein can be modified by any method available in the art.
    The polynucleotides according to the invention can be produced recombinantly, synthetically or by any means available to those skilled in the art.
    35 They can also be cloned by standard techniques. polynucleotides are typically provided in isolated and / or purified form.
    "
    P201 530645
    Another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments relates to the use of the composition of the invention, wherein the compound is a DNA polynucleotide comprising a sequence selected from the list consisting of the polynucleotide sequence. with access number NM_000325 (SEO ID NO 1) or its complementary sequence or a polynucleotide sequence that codes for an identical amino acid sequence by at least 99%, based on the identity of all amino acids of said sequence, to the sequence amino acid with access number NP 000316.
    In a preferred aspect of the additional invention, the polynucleotides of the invention, such as those set forth above, can be transported, without degradation, by plasmid or viral vectors that include a nucleic acid expression promoter in the cells in which it is supplied
    Thus, in a further embodiment of the invention, the activator compounds of the invention may comprise any of the polynucleotides of the invention described above or a plasmid or a vector capable of transporting or delivering said polynucleotides, preferably by means of a viral vector.
    Viral vectors are, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including among them, viruses with the structure of HIV. Viral families that share the properties of these viruses that make them suitable for use as vectors are also preferred. Retroviruses are Maloney murine leukemia viruses, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are capable of carrying a larger genetic payload, that is, a marker gene transgene, than other viral vectors, and for this reason they are a commonly used vector. However, they are not as useful in nonproliferative cells. Adenovirus vectors are relatively stable and easy to work, have high titers, and can be sent in the aerosol formulation, and can transfect cells that do not divide. Smallpox viral vectors are large and have several sites for gene insertion, which are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector that has been designed to suppress the host organism's immune response, caused by viral antigens.
    P201 530645
    The activator compounds may comprise, in addition to the described polynucleotides of the invention, plasmids or vectors or the peptides of the invention, for example, lipids such as liposomes, such as cationic liposomes (eg, DOTMA, DOPE,
    OC-cholesterol) or anionic liposomes.
    The liposomes may further comprise proteins to facilitate the particular cell direction, if desired. The administration of a composition comprising a compound and a cationic liposome that can be administered to afferent blood to a target organ. In addition, the activator can be administered as a component of a microcapsule that can be targeted to specific cell types, such as cardiomyocytes, or where diffusion of the compound or administration of the microcapsule compound is designed for a specific type or dose. .
    Therefore, another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments refers to the use of the composition of the invention, wherein said compound is a vector or plasmid capable of transporting or delivering the sequence of polynucleotides, as defined in the context of the present invention, to muscle satellite stem cells. Preferably said vector is a viral vector that encodes the polynucleotide sequence as defined above. More preferably said viral vector selected from the list consisting of adenoviral, lentiviral, retroviral and adeno-associated vectors.
    Another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments relates to the use of the composition of the invention, wherein said medicament comprises satellite stem cells of muscle of a human or animal subject treated, transformed, transfected. or transduced with the compound defined in the first aspect of the invention or in any of its preferred embodiments. Preferably, said cells treated, transformed, transfected or transduced with the compound, are cells of autologous origin. More preferably, said transformed cells, transfected or transduced with the compound, are satellite muscle stem cells of a human subject suffering from dystrophinopathy or dystrophy.
    On the other hand, and as previously mentioned, Figures 3 and 4 of the present invention demonstrate that reducing miRNA-106b expression in the muscle stem cells of a human or animal subject with respect to that observed in the absence of the
    P201 530645
    The compound in said cells amplifies the population of myoblasts derived from satellite cells during the differentiation processes of regenerative myogenesis.
    Accordingly, a second aspect of the present invention relates to a composition comprising a compound, inhibitor compound, capable of reducing the expression of miRNA-106b in the muscle satellite stem cells of a human or animal subject with respect to that observed. in the absence of the compound in said cells, for the preparation of a medicament to promote muscle regeneration.
    In the context of the present invention, "inhibiting or reducing the expression of miRNA-106b" means reduction on the baseline, or in comparison with a control, by 1, 2, 3,
    4,5,6,7,8,9,10, 11,12,13, 14,15,16,17,18,19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31.32.33.34, 35.36.37, 38, 39, 40, 41, 42.43, 44.45.46.47.48, 49, 50, 51.52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82.83, 84, 85.86.87.88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, 02.3, 4, 5,6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20,25,30, 35, 40, 45,50, or more times.
    The inhibitor compounds can be identified by a screening method that allows identifying in a compound the ability to inhibit or reduce the intracellular expression of miRNA-106b, which comprises contacting a cell, preferably a muscle satellite stem cell, with a compound that it is suspected that it may inhibit or reduce the expression of miRNA-106b; testing the content of the cells to determine the amount and / or biological activity of miRNA-106b expression, and compare the determined amount and / or the biological activity of miRNA-106b at a predetermined level, at which a change in said The amount and / or biological activity of miRNA-106b expression is indicative of a compound with the ability to inhibit or reduce intracellular expression of miRNA-106b. In a preferred embodiment, the detection is performed by quantitative real-time RT-PCR.
    In a preferred embodiment of the second aspect of the invention, said medicament is used to promote muscle regeneration in the treatment of dystrophinopathy.
    In another preferred embodiment of the second aspect of the invention or any of its preferred embodiments, said dystrophy or dystronifopathy is selected from the list consisting of Duchenne muscular dystrophy and Becker muscular dystrophy.
     04-1 2-2015
    In yet another preferred embodiment of the second aspect of the invention or any of its preferred embodiments, said compound is an interference RNA (siRNA) of miRNA-106b such as an antisense RNA oligonucleotide of miRNA-106b or a polynucleotide that expresses said antisense oligonucleotide. Preferably, said compound is a synthetic antisense RNA oligonucleotide of miRNA-106b which optionally presents modifications to increase its resistance to nucleases. More preferably, said compound is a vector or plasmid capable of transporting or delivering said miRNA-106b interference RNA (siRNA) to muscle satellite stem cells. Preferably said vector is a viral vector and more preferably it is
    10 selects from any of those mentioned in the first aspect of the present invention.
    In another preferred embodiment of the second aspect of the invention or of any of its preferred embodiments, said medicament comprises satellite cells of muscle of a human or animal subject treated, transformed, transfected or transduced with the compound as defined herein. in the second aspect of the invention or in any of its preferred embodiments. Preferably, said cells treated, transformed, transfected or transduced with the compound, are cells of autologous origin. More preferably, said transformed cells, transfected or transduced with the compound, are satellite muscle stem cells of a human subject suffering from
    20 a dystrophinopathy (a dystrophy).
    Finally, a third aspect of the invention relates to a method of screening a compound capable of promoting muscle regeneration comprising:
    25 1. Select compounds from a library of compounds;
    2. Test whether any of these compounds is capable of:
    to. increase the expression of the Pitx2 gene in the muscle satellite stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells; I
    30 b. reduce the expression of miRNA-106b in the satellite muscle stem cells of a human or animal subject with respect to that observed in the absence of the compound in said cells; Y
    3. Select that or those compounds capable of carrying out the mentioned in any of the previous sections.

    In the context of the present invention, the DNA polynucleotides of the invention, such as those described above, which are supplied to the cells, can be integrated into the host gene of the host cell, usually through integration sequences. These sequences are often related to viral sequences, particularly in virus-based systems when used. These viral integration systems can also be incorporated into the nucleic acids to be delivered using a delivery based non-nucleic acid addition system, such as a liposome, so that the nucleic acid contained in the delivery system can be integrated in the host genome.
    Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems are typically based on the flanking sequence of the nucleic acid to be expressed that has sufficient homology with a target sequence in the genome of the host cell where recombination between the nucleic acid vector and the target nucleic acid takes place, causing The supplied nucleic acid is integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those skilled in the art.
    The activator or inhibitor compounds described herein can be administered in a pharmaceutically acceptable carrier and can be sent to the subject's cells in vivo and I or ex vivo by a variety of mechanisms well known in the art as discussed above.
    If ex vivo methods are used, cells or tissues can be removed and kept out of the body according to standard protocols well known in the art. Activator or inhibitor compounds can be introduced into cells through any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (for example, in a pharmaceutically acceptable carrier) or homotopically transplanted into the subject by standard methods for the type of cell or tissue. Standard methods are known for the transplantation or infusion of several cells in a subject.
    The activator or inhibitor compounds of the present invention can be used in conjunction with other treatment methods. 16
    P201 530645
    In addition, provided herein, a method is included to increase or improve the clinical condition and the perception of well-being of a subject with dystrophy or with dystrophinopathy, which comprises administering to an individual in need thereof an effective amount of a compound. inhibitor activator, which increases or improves the clinical status of the subject treated for a certain period of time.
    Current methods of treatment also include a method for increasing the efficacy of other agents proposed for the same disease, which comprises administering to an individual in need thereof an effective amount of an activator or inhibitor compound, and, optionally, a pharmaceutically acceptable carrier. , thus increasing the effectiveness of the other agent or agents.
    In any case, the compositions comprising the activator or inhibitor compound can be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, that is, the material can be administered to a subject, together with the nucleic acid or vector, without causing any undesirable biological effect or interacting in a harmful manner. with any of the other components of the pharmaceutical composition in which it is contained. The vehicle, of course, is selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to one skilled in the art.
    The effective dosages and administration schedules of the compositions comprising the activator or inhibitor compound described herein can be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration of the compositions are those large enough to produce the desired anti-hypertrophic effect on the disorder. The dosage should not be so large as to cause adverse side effects, such as unwanted cross reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, the route of administration, or if other drugs are included in the regimen, and can be determined by one skilled in the art. The dosage can be adjusted by the individual doctor in case of any contraindication. The dose may vary, and may be administered in one or more daily dose administrations, during
    P201 530645
    One or several days. Guidance can be found in the literature for appropriate dosages for the given classes of pharmaceutical products.
    The following example merely serves to illustrate the present invention.
    Examples
    Example 1. Pitx2 and muscle regeneration
    10 Based on the results shown in the figures, and taking into account that, interestingly, Pitx2 expression is significantly increased during mouse muscle regeneration and decreased in the murine model of Duchenne muscular dystrophy (DMDmdx mice), we lead to carried out an experimental approach of "in vivo" cell transplantation to test if Pitx2 could improve the regenerative capacity of
    15 satellite cells isolated from dystrophic muscles. It is important to note that the possibility of modifying the regenerative capacity of the dystrophic cells themselves is a significant advantage for their therapeutic application in humans (possibility of using dystrophic patient cells). The results obtained show that Pitx2 overexpression improves muscle regeneration carried out by satellite cells
    20 dystrophic increasing muscle regeneration in MDX mice. Thus, our results demonstrate that the transplantation of dystrophic satellite cells by overexpressing Pitx2 in DMDmdx mice leads to:
    An increase in the number of myofibers formed "de novo" (Figure 4A);
    25 Suppresses miR-31 expression producing a dystrophin restoration (Figure 48) Finally producing a very significant functional improvement of the muscle (Figure 4C).
    30 Therefore, these analyzes lead us to identify miR-31 as a PitR2-regulated miRNA during muscle regeneration In addition, we have also obtained additional evidence showing that the Pitx2-miRNAs pathway controlling cell proliferation is also present in our "in vivo" cell transplant model in DMDmdx mice (Figures 4D-F). Together, these results place Pitx2 as a regulatory molecule for
    35 different miRNAs that play a fundamental role in the molecular circuits that control the proliferation and / or differentiation of satellite cells. Revealing the important 18

    role of Pitx2 in the cellular biology of skeletal muscle satellite cells and identifying unknown functions of Pitx2 by modulating regenerative myogenesis in the dystrophic muscle.
    Example 2. Detailed statement of the results of the invention.
    First, as shown in Figure 2, sections A) and B), transfection with the bicistronic lentiviral vector LVX-Pitx2c-ZSGreen resulted in the overexpression of Pitx2 in mouse satellite cells. Additionally, this overexpression, see figure 2 section C), resulted in a decreased expression of the miRNAs: miR-15b, miR-106b, miR-23b and miR503 (qRT-PCR), both in cells in early stages of activation ( EPq) as in more advanced stages of activation (EPa) (qRT-PCR).
    On the other hand, as shown in Figure 2 O), transfection with the bicistronic lentiviral vector LVX-Pitx2c-ZSGreen leads to an increase in the expression of the cyclin 01 and cyclin 02 cell cycle control genes (qRT -PCR) indicating that, similar to what occurs in myoblasts, the Pitx2-miRNAs pathway controls proliferation in satellite cells.
    Second, as illustrated in Figure 3 A), the expression of Myf5 (qRT-PCR) increases in satellite cells that overexpress Pitx2, moreover, quantification by immunohistochemistry, see Figure 3 section B), shows a significant increase in Myf5 + cells. On the other hand, according to figure 3 section C), the overexpression of miR-106b leads to a decrease in the expression of Myf5 validating it as the target of this miRNA. Furthermore, several experiments were performed to calculate the normalized activity of the 3'-UTR mycilerase myf5 lucilerase reporter (WT Myf5 3'-UTR) by transfecting with the empty plasmid (Vector) or co-transfecting with pre-miR-106b. As shown in figure 3 section O, only co-transfecting with WT pre-miR106b produces a repression of the expression of Myf5 by miR-106b demonstrating, therefore, that Myf5 is a direct target for miR-106b In fact, there is no loss of luciferase activity when the miR-106b "seed sequence" was mutated, thus demonstrating the binding specificity of miR-106b to these "seed sequence" of the 3'UTR of Myf5 (see figure 3 section D))
    Third, as shown in Figure 4 section A), the qRT-PCR analysis shows the overexpression of Pitx2c in the muscles of MDX mice injected with dystrophic satellite cells transfected with the lentiviral vector LVX-Pitx2c-ZS- Green vector

    compared to the muscle injected with cells transfected with the empty ellentivirus(LVX-ZS-Green vector); percentage of "de novo" fused fibers (ZS-Green + celis) intransplanted muscles after 15 days and representative image. On the other hand, the figure4 section B), shows how the decrease of miR-31 expression in the muscles5 transplanted with cells that overexpress Pitx2 leads to increased levels ofdystrophin expression as well as a significant increase in the fibers that expressdystrophin Additionally, the authors of the present invention carried out a Treadmilltest, see figure 4 section C), which shows the functional improvement of DMDmdx miceinjected with cells that overexpress Pitx2. Finally, figure 4 section F shows
    10 as the expression levels of cyclins D1 and 02 as well as the Myf5 transcription factor were increased in the transplanted muscles with cells that overexpress Pitx2 indicating that the Pitx2-miRNAS molecular cascade is conserved in the live uin transplant system. "
     04-1 2-2015
    权利要求:
    Claims (7)
    [1]
    1. Use of a composition comprising an AON polynucleotide compound which in turn comprises a sequence consisting of the polynucleotide sequence
    5 SEO ID NO 1 or its complementary sequence, for the development of amedicine to promote muscle regeneration.
    [2]
    2. The use of the composition of claim 1, wherein said medicament is used to promote muscle regeneration in the treatment of dystrophinopathy.
    [3]
    3. The use according to claim 2, wherein said dystrophinopathy is selected from the list consisting of Ouchenne muscular dystrophy and Becker muscular dystrophy.
    [4]
    Four. The use according to any one of claims 1 to 3, wherein said compound is
    A vector or plasmid capable of transporting or delivering said polynucleotide sequence to the ex vivo muscle satellite stem cells.
    [5]
    5. The use according to claim 4, wherein said vector is a viral vector encoding the polynucleotide sequence defined in claim 1.
    [6]
    6. The use according to claim 5, wherein said vector is a viral vector selected from the list consisting of adenoviral, lentiviral, retroviral, and associated vectors.
    The use according to any of claims 1-6, wherein said medicament comprises satellite stem cells of muscle of a human or animal subject treated, transformed, transfected or transduced with the compound defined in claim 1.
    The use according to claim 7, wherein said cells treated, transformed, transfected or transduced with the compound defined in claim 1, are cells of autologous origin.

    [9]
    9. The use according to claim 7 or 8, wherein said cells transformed, transfected or transduced with the compound defined in claim 1, are satellite muscle stem cells of a human subject suffering from dystrophinopathy.
    类似技术:
    公开号 | 公开日 | 专利标题
    JP2020022513A|2020-02-13|C/EBPα SMALL MOLECULE-ACTIVATING RNA COMPOSITION
    Baba et al.2015|Treatment of neurological disorders by introducing mRNA in vivo using polyplex nanomicelles
    US20180258429A1|2018-09-13|Sarna compositions and methods of use
    Osman et al.2012|Bifunctional RNAs targeting the intronic splicing silencer N1 increase SMN levels and reduce disease severity in an animal model of spinal muscular atrophy
    ES2593836T3|2016-12-13|Oligonucleotide comprising an inosine to treat DMD
    ES2564563T3|2016-03-23|Means and methods to counteract muscle disorders
    ES2566553T3|2016-04-13|Modified U7 siRNA for the treatment of neuromuscular diseases
    Theis et al.2017|Lentiviral delivery of miR-133b improves functional recovery after spinal cord injury in mice
    JP2020508668A|2020-03-26|Compositions and methods relating to myomixer-enhanced muscle cell fusion
    US20140221464A1|2014-08-07|Compositions and Methods for Treating Skeletal Myopathy
    US20140213634A1|2014-07-31|Method for proliferation cardiomyocytes using micro-rna
    ES2559106B1|2016-11-25|Method of activating the expression of the Pitx2 gene to promote muscle regeneration
    US10870852B2|2020-12-22|Compositions and methods for treating diabetic retinopathy
    BR102019026837A2|2020-10-27|vlp for the treatment of a lysosomal storage disease
    KR20210009317A|2021-01-26|Gene therapy for diseases caused by unbalanced nucleotide pools, including mitochondrial DNA depletion syndrome
    US8685727B2|2014-04-01|Regulation of macrophage activation using miR-125b
    WO2014169087A2|2014-10-16|Treatment of proximal spinal muscular atrophy
    KR20220022126A|2022-02-24|Methods and compositions comprising TERT activation therapy
    US9458464B2|2016-10-04|Treatment of neuropathic pain
    US20210244828A1|2021-08-12|Miri26-5p for treating motor neuron diseases
    KR20210039495A|2021-04-09|Synthetic gene capable of feedback, target seed match cassette, and use thereof
    WO2021178668A1|2021-09-10|Compositions and methods for modulating rpgr expression
    US20150329857A1|2015-11-19|Rna interference to activate stem cells
    同族专利:
    公开号 | 公开日
    ES2559106B1|2016-11-25|
    EP3296399A1|2018-03-21|
    US10537591B2|2020-01-21|
    US20190111071A1|2019-04-18|
    WO2016181011A1|2016-11-17|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US10537591B2|2015-05-12|2020-01-21|Universidad De Jaen|Method for promoting muscle regeneration|ES2559106B1|2015-05-12|2016-11-25|Universidad De Jaén|Method of activating the expression of the Pitx2 gene to promote muscle regeneration|
    法律状态:
    2016-11-25| FG2A| Definitive protection|Ref document number: 2559106 Country of ref document: ES Kind code of ref document: B1 Effective date: 20161125 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201530645A|ES2559106B1|2015-05-12|2015-05-12|Method of activating the expression of the Pitx2 gene to promote muscle regeneration|ES201530645A| ES2559106B1|2015-05-12|2015-05-12|Method of activating the expression of the Pitx2 gene to promote muscle regeneration|
    PCT/ES2016/070362| WO2016181011A1|2015-05-12|2016-05-12|Method for promoting muscle regeneration|
    US15/573,417| US10537591B2|2015-05-12|2016-05-12|Method for promoting muscle regeneration|
    EP16735897.7A| EP3296399A1|2015-05-12|2016-05-12|Method for promoting muscle regeneration|
    [返回顶部]