selective transgene expression for tissue

专利摘要:
here compositions and methods are provided for the selective expression of a transgene. compositions and methods for the selective expression of a transgene comprise one or more human regulatory elements, which, when operationally linked to a transgene, can facilitate the selective expression of a transgene (for example, selective expression for cell type) in a target cell compared to at least one or more non-target cells.
公开号:BR112019020777A2
申请号:R112019020777
申请日:2018-04-03
公开日:2020-04-28
发明作者:Szu-Ying Chen；David Oberkofler；Kartik Ramamoorthi；Stephanie Tagliatela；Andrew Young
申请人:Encoded Therapeutics Inc；
IPC主号:

专利说明:

EXPRESSION OF SELECTIVE TRANSGENE FOR FABRIC
CROSSED REFERENCE [0001] This order claims the benefit of US Provisional Order No. 62 / 480,998, filed on April 3, 2017, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING [0002] The present application contains a Sequence Listing that was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 29, 2018, is titled 46482-704_601_SL.txt and is 78,248 bytes in size.
BACKGROUND OF THE REPORT [0003] Gene therapy has long been recognized for its enormous potential in its approach and treatment of human diseases. Rather than relying on drugs or surgery, patients, especially those with underlying genetic factors, can be treated by direct orientation to the underlying cause. In addition, by targeting the underlying genetic cause, gene therapy has the potential to effectively cure patients or provide sustained treatment for a longer period of time. On the other hand, despite this, the clinical applications of gene therapy still require improvements in several aspects. One area of concern is that of effects outside the goal. An attractive approach to focusing off-purpose effects is to target gene therapy gene expression to the cell type (s) or tissue (s) of interest or the type (s) of target cell (s) or tissue (s). As such, there is a need to identify elements and methods to target gene therapy or gene expression to a type of tissue or cell of interest.
REPORT SUMMARY [0004] There is a considerable need for targeting gene therapy and gene / transgene expression to the type of tissue and / or cell desired in vivo, which can reduce off-target effects, increase effectiveness
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2/133 therapy in the type of tissue and / or target cell, and increase patient safety and tolerance by reducing the effective dose required to achieve efficacy.
[0005] Compositions and methods for the selective expression of a transgene in a type of target tissue or cell in one or more types of non-target tissue or cell are provided here. The compositions and methods for the selective expression of a transgene comprise one or more regulatory elements (ERs) which, when operationally linked to a transgene (for example, an ion channel subunit or a neurotransmission regulator, or a protein from syntaxin binding), can facilitate or result in the selective or preferential expression of the transgene in a type of target cell or tissue (eg, parvalbumin neurons (PV)) compared to one or more non-target cell types (eg, cells non-PV). In some cases, ERs are sequences of unnatural occurrence. In some cases, REs are human-derived regulatory elements. In some cases, REs comprise a sequence of a non-human species, such as a monkey or dog, or a rabbit or mouse. In some cases, the compositions described herein are administered to a cell in vivo, ex vivo, or in vitro using a viral vector and / or virus particles, such as an adeno-associated virus (AAV) or lentivirus. In some cases, the compositions described here are administered to a cell as gene therapy. Also contemplated here are methods and compositions for the treatment of a neurological condition or disorder associated with a genetic defect in the CNS. In some cases, the relevant type of cell or tissue affected by the genetic defect is a PV cell. In some cases, the neurological condition or disease is Dravet's syndrome, Alzheimer's disease, epilepsy, and / or seizures. In some cases, the neurological condition or disease is a psychiatric disorder (for example, schizophrenia, obsessive compulsive disorder, addiction, depression, anxiety, psychosis); an autism spectrum disorder (for example, fragile X syndrome, Rett syndrome); epilepsy (eg, chronic traumatic encephalopathy, generalized epilepsy with febrile plus seizures (GEFS +), epileptic encephalopathy, lobe epilepsy
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3/133 temporal, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological condition or disease is a seizure and / or epilepsy related to the condition or disease in which PV neurons are involved.
[0006] In one aspect, this report contemplates a nucleic acid cassette comprising one or more regulatory elements operatively linked to a transgene that results in selective expression in any type of target cell, for example, PV neurons in the CNS, in one or more types of non-target cells, or non-PV cells in the CNS. In some cases, each regulatory element comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity to (i) or (ii). In some cases, the sequence identity is determined using BLAST. In some cases, at least one of the regulatory elements is derived from human. In some cases, at least one of the regulatory elements is derived from a non-human mammal. In some cases, the regulatory elements are not naturally occurring. In some cases, regulatory elements result in selective transgene expression in PV neurons that is greater than the expression of the same transgene when operationally linked to a non-selective regulatory element, as measured by a co-location assay. In some cases, the non-selective regulatory element is a constitutive promoter. In some cases, the non-selective regulatory element is that of CAG, EFla, SV40, CMV, UBC, PGK, and CBA. In some cases, regulatory elements result in selective expression of the transgene in PV neurons at a level that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, or at least 20 times compared to the selective expression of the transgene in PV neurons when operationally linked to an element
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4/133 non-selective regulator, as measured by the co-location assay. In some cases, regulatory elements result in selective expression in PV neurons that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30 %, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, or at least 95% greater than the expression in PV neurons when the transgene is operationally linked to a non-selective regulatory element. In some cases, the regulatory elements result in selective expression in PV neurons that is about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times , 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times,
8.5 times, 9 times, 9.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, or 100 times greater than expected for the natural distribution of PV neurons in the CNS . In some cases, the co-localization assay is an immunohistochemical assay. In some cases, the immunohistochemical assay comprises an anti-PV antibody. In some cases, the co-location assay is performed as shown in Example 5 below. In some cases, the transgene encodes an ion channel subunit, a neurotransmitter regulator, a DNA-binding domain, a gene editing protein, or a functional variant or fragment thereof. In some cases, the ion channel subunit is an alpha subunit or a beta subunit of a sodium ion channel or a subunit of a potassium ion channel. In some cases, the transgene comprises any of (i) SEQ ID NOs: 37-43; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii). In some cases, the sequence identity is determined using BLAST. In some cases, the transgene comprises (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, or KV3,3; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii). In some cases, the transgene is a neurotransmitter regulator that comprises (i) STXBP1, (ii) a functional fragment thereof, or (iii) a sequence
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5/133 showing at least 80% sequence identity to (i) or (ii). In some cases, the transgene comprises a DNA-binding protein that modulates the expression of an endogenous gene. In some cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, or STXBP1. In some cases, the transgene comprises a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (e.g., a nuclease, a restriction enzyme, a recombinase, etc. .) where the DNA cleavage domain or nuclease domain has been disabled, for example, a deactivated Cas nuclease (dCas), a deactivated transcription activator-like nuclease, or a deactivated zinc finger protein for nuclease. In some cases, the transgene comprises a DNA binding domain linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the gene editing protein is a Cas protein. In some cases, the combined regulatory elements are less than 2.5 kb, less than 2 kb, less than
1.5 kb, less than 1 kb, or less than 500 bp in size. In some cases, non-PV cells comprise one or more of the non-PV cell types in the CNS. In some cases, non-PV cells comprise one or more of the excitatory neurons, dopaminergic neurons, astrocytes, microglia, and motor neurons. In some cases, the nucleic acid cassette is a linear construct. In some cases, the nucleic acid cassette is a vector. In some cases, the vector is a plasmid. In some cases, the vector is a viral vector. In some cases, the viral vector is an adeno-associated virus (AAV) vector. In some cases, the AAV vector is AAV1, AAV8, AAV9, scAAVl, scAAV8, or scAAV9. In some cases, the viral vector is a lentiviral vector.
[0007] In one aspect, the regulatory elements of any of the nucleic acid cassettes described here contain less than 600 bp of contiguous sequence within the 10 kb transcription start site of GAD2, GAD1, SYN1, NKX2,1, DLX1 , DLX5 / 6, SST, PV, and / or VIP.
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6/133 [0008] In one aspect, a method for treating a neurological disorder or condition in an individual in need of such treatment comprises administering a therapeutically effective amount of any of the nucleic acid cassettes described herein. In some cases, the neurological disorder or condition is a psychiatric disorder (for example, schizophrenia, obsessive compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (for example, chronic traumatic encephalopathy, generalized epilepsy with febrile seizures plus (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological disorder or condition is Dravet's syndrome or Alzheimer's disease. In some cases, the neurological condition or disease is any seizure and / or epilepsy related to the condition or disease in which the PV neurons are involved.
[0009] In one aspect, a method for increasing the selective expression of a transgene in PV neurons in the CNS comprises contacting a cell with a nucleic acid cassette described here.
[0010] In some respects, this report contemplates a method for directing the expression of any transgene to PV neurons in the CNS, the method comprising the operational binding of one or more of the selective regulatory elements of the PV neuron to a transgene. In some cases, each of the regulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence of at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % sequence identity to (i) or (ii). In some cases, the sequence identity is determined using BLAST. In some cases, regulatory elements result in the selective expression of the transgene in PV neurons that is greater than the expression of the same transgene when operationally linked to a non-selective regulatory element, such as
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7/133 measured by a co-location assay. In some cases, the immunohistochemical assay comprises an anti-PV antibody (for example, as described in Example 5 below). In some cases, the non-selective regulatory element is a constitutive promoter. In some cases, the non-selective regulatory element is any of CAG, EFla, SV40, CMV, UBC, PGK, and CBA. In some cases, regulatory elements result in the selective expression of the transgene in PV neurons at a level that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, or at least 20 times compared to a non-selective regulatory element when operationally linked to the transgene, such as measured by a co-location test. In some cases, regulatory elements result in selective expression in PV neurons that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30 %, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, or at least 95% greater than the expression in PV neurons when the transgene is operationally linked to a non-selective regulatory element. In some cases, the regulatory elements result in selective expression in PV neurons that is about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times , 5.5 times, 6 times,
6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, or 100 times greater than expected for the natural distribution of PV neurons in the CNS. In some cases, the transgene is any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STXBP1, a DNA binding protein, a gene editing protein, or a functional fragment thereof. In some cases, the regulatory elements and the transgene are in an AAV. In some cases, the AAV is AAV1, AAV8, AAV9, scAAVl, scAAV8, or scAAV9.
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8/133 [0011] In another aspect, this report contemplates a method for the treatment of a neurological condition or disorder in an individual in need of such treatment, the method comprising contacting a cell with a nucleic acid cassette comprising a or more regulatory elements operatively linked to a transgene that result in selective expression of the transgene in PV neurons in one or more non-PV cells in the CNS. In some cases, each of the regulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence of at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) or (ii). In some cases, the sequence identity is determined using BLAST. In some cases, the transgene is a voltage-driven ion channel subunit, or a variant or functional fragment thereof. In some cases, the subunit is a beta subunit of a sodium ion channel. In some cases, the subunit is an alpha subunit of a sodium ion channel. In some cases, the subunit is a potassium ion channel. In some cases, the transgene is any one of (i) SCN1A, SCN1B, SCN2B, KV3,1, or KV3,3; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii). In some cases, the transgene is a DNA-binding protein. In some cases, the DNA-binding protein modulates an endogenous gene. In some cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, or STXBP1. In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase etc.) where the DNA cleavage domain or nuclease domain has been deactivated, for example, a deactivated Cas nuclease (dCas), a deactivated transcription activator-like nuclease, or a deactivated zinc finger protein for nuclease. In some cases, the transgene comprises
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9/133 a DNA binding domain linked to a transcription modulator domain (e.g., a transcription activator or repressor domain). In some cases, ο transgene is a gene editing protein. In some cases, the gene editing protein is a Cas protein, for example, Cas9. In some cases, the neurological condition or disorder is associated with a haploinsufficiency or a mutation in any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, or STXBP1. In some cases, the neurological condition or disorder is epilepsy, neurodegeneration, taupathy, or neuronal hypoexcitability. In some cases, the neurological condition or disorder is Dravet's syndrome. In some cases, the neurological condition or disorder is Alzheimer's disease. In some cases, the neurological condition or disease is a psychiatric disorder (for example, schizophrenia, obsessive compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (for example, chronic traumatic encephalopathy, generalized epilepsy with febrile seizures plus (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological condition or disease is any seizure and / or epilepsy related to the condition or disease in which PV neurons are involved. In some cases, the regulatory elements in this report result in the selective expression of the transgene in PV neurons that is greater than the expression of the same transgene when operationally linked to a non-selective regulatory element, as measured by a co-location assay. In some cases, the non-selective regulatory element is a constitutive promoter. In some cases, the non-selective regulatory element is any of CAG, EFla, SV40, CMV, UBC, PGK, and CBA. In some cases, regulatory elements result in selective expression in PV neurons at a level that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, or at least 20 times compared to an element
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10/133 non-selective regulator when operationally linked to the transgene, as measured by a co-location assay. In some cases, regulatory elements result in selective expression in PV neurons that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30 %, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, or at least 95% greater than the expression in PV neurons when the transgene is operationally linked to a non-selective regulatory element. In some cases, the regulatory elements result in selective expression in PV neurons that is about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times , 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times,
9.5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, or 100 times greater than expected for the natural distribution of PV neurons in the CNS. In some cases, the nucleic acid cassette is in an AAV. In some cases, the AAV is AAV1, AAV8, AAV9, scAAVl, scAAV8, or scAAV9.
[0012] In one aspect, this report provides a method for the treatment of Dravet syndrome, comprising contact of a cell with an AAV comprising a transgene, where the transgene is any one of (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, or a DNA binding protein, (ii) a functional fragment thereof, or (iii) a sequence showing at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) or (ii) . In some cases, sequence identity is measured using BLAST. In some cases, the DNA-binding protein modulates an endogenous gene. In some cases, the DNA-binding protein is a transcription modulator. In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase
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11/133 etc.) where the DNA dividing domain or nuclease domain has been disabled, for example, a deactivated Cas nuclease (dCas), a deactivated transcription activator-like nuclease, or a deactivated zinc finger protein for nuclease . In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene comprises a gene editing protein, for example, a Cas, Cas9 protein. In some cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, or SCN2B. In some cases, the AAV additionally comprises one or more selective regulatory elements of the PV neuron or one or more regulatory elements described here operationally linked to the transgene. In some cases, each of the regulatory elements independently comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least minus 99% sequence identity to (i) or (ii).
[0013] In another aspect, this report provides a method for the treatment of Alzheimer's disease, comprising contact of a cell with an AAV comprising a transgene, where the transgene is any one of (i) SCN1A, SNC2A, SNC8A , SCN1B, SCN2B, KV3,1, KV3,3, STXBP1, or a DNA binding protein; (ii) a functional fragment thereof; or (iii) a sequence showing at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% sequence identity to (i) or (ii). In some cases, sequence identity is measured using BLAST. In some cases, the DNA-binding protein modulates an endogenous gene. In some cases, the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, or STXBP1. In some cases, the transgene is a DNA-binding protein comprising a modulator of
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12/133 transcript. In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA dividing protein (for example, a nuclease, a restriction enzyme, a recombinase etc.) where the DNA dividing domain or nuclease domain has been deactivated, for example, a deactivated Cas nuclease (dCas), a deactivated transcription activator-like nuclease, or a deactivated zinc finger protein for nuclease. In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene comprises a gene editing protein, for example, a Cas, Cas9 protein. In some cases, the AAV additionally comprises one or more selective regulatory elements of the PV neuron or one or more regulatory elements described here operationally linked to the transgene. In some cases, each of the regulatory elements independently comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least minus 99% sequence identity to (i) or (ii).
INCORPORATION BY REFERENCE [0014] All publications, patents and patent applications mentioned in this report are hereby incorporated by reference to the same extent that each individual publication, patent or patent application was specifically and individually indicated as being incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS [0015] The new features of the invention are presented with particularity in the attached claims. A better understanding of the characteristics and advantages of the present invention will be obtained with reference to the detailed description
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13/133 following that presents illustrative realizations, in which the principles of the invention are used, and in the attached drawings in which:
[0016] FIG. 1 illustrates the frequency of seizures (12 h interval seizures) in heterozygous SCN1A mice after treatment with a recombinant AAVDJ vector comprising either SCN1B or eGFP operably linked to a regulatory element comprising a sequence of SEQ ID NO: 32. The graph illustrates the mean values on each day of recording with the error bars representing the standard error of the means.
[0017] FIG. 2 illustrates the high gamma power (50-100 Hz) of different mice: wild type control (WT), untreated transgenic APP / PS1 mice (APP / PS1), or transgenic APP / PS1 mice treated with rAAV comprising SCN1B operatively linked to a regulatory element comprising a sequence of SEQ ID NO: 32 (APP / PS1 + SCN1B).
[0018] FIG. 3A illustrates the immunofluorescent assay for co-localization of puppies' CNS cells after neonatal systemic injections of AAV9 comprising the eGFP transgene operably linked to a regulatory element comprising a sequence of SEQ ID NO: 1 or SEQ ID NO: 8. AAV9 comprising the eGFP transgene operatively linked to CAG was used as a control. The images in the bottom row illustrate eGFP + cells. The images in the middle row illustrate PV + cells, which are labeled with an anti-PV antibody. The images in the top row (combination) illustrate a fluorescence overlap of PV +, eGFP + (with eGFP + and PV + cells that are shown as white or light gray cells indicated by arrowheads) and DAPI +.
[0019] FIG. 3B illustrates the quantification of the colocalization immunofluorescence studies illustrated in FIG. 3A, where selective expression in PV cells is expressed as the percentage of eGFP + cells that are also PV + compared to the CAG control, as measured by the immunofluorescent co-localization assay.
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14/133 [0020] FIG. 4A illustrates the immunofluorescent assay for CNS cell co-localization of adult mice after systemic injection of AAV9 comprising the eGFP transgene operably linked to a regulatory element comprising a sequence of SEQ ID NO: 1 or SEQ ID NO: 8. AAV9 comprising the eGFP transgene operationally linked to EFla was used as a control. The images in the bottom row illustrate eGFP + cells. The images in the middle row illustrate PV + cells, which are labeled with an antiPV antibody. The images in the top row (combination) illustrate an overlap of the fluorescence of PV + eGFP + (with eGFP + and representative PV + cells, or the white or light gray cells, indicated by arrowheads) and DAPI +.
[0021] FIG. 4B illustrates the quantification of the colocalization immunofluorescence studies illustrated in FIG. 4A, where selective expression in PV cells is expressed as the percentage of eGFP + cells that are also PV + compared to the EFla control, as measured by the immunofluorescent co-localization assay.
[0022] FIGS. 5A-5F illustrate the immunofluorescent assay for CNS cell co-localization of adult mice after direct injections into the CNS of AAVDJ comprising the eGFP transgene operably linked to a regulatory element comprising a sequence of SEQ ID NOs: 2-22. The images in the bottom row illustrate eGFP + cells. The images in the middle row illustrate PV cells that have been labeled with an anti-PV antibody. The images in the top row (combination) illustrate an overlap of the fluorescence of PV +, eGFP + (with eGFP + and representative PV + cells, or the white or light gray cells, indicated by the arrowheads) and DAPI +. FIG. 5A illustrates the immunofluorescent co-localization assay performed with AAVDJ comprising one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 operably linked to eGFP. FIG. 5B illustrates the immunofluorescent co-localization assay performed with AAVDJ comprising one of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9 operably linked to eGFP. FIG. 5C illustrates the immunofluorescent co-localization assay performed with AAVDJ
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15/133 comprising one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 operatively linked to eGFP. FIG. 5D illustrates the immunofluorescent co-localization assay performed with AAVDJ comprising one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 operationally linked to eGFP. FIG. 5E illustrates the immunofluorescent co-localization assay performed with AAVDJ comprising one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 operably linked to eGFP. FIG. 5F illustrates the immunofluorescent co-localization assay performed with AAVDJ comprising SEQ ID NO: 22 or SEQ ID NO: 34 operationally linked to eGFP, where SEQ ID NO: 34 is a previously characterized non-selective regulatory element and was used as a control for comparison.
[0023] FIG. 6 illustrates the quantification of the colocalization immunofluorescence studies illustrated in FIGs. 5A-5F, where selective expression in PV cells [and expressed as the percentage of eGFP + cells that were also PV + compared to SEQ ID NO: 34, as measured by the immunofluorescent co-localization assay.
[0024] FIG. 7 illustrates a schematic of an example of an expression cassette containing the REs in this report, for example, an amplifier, a promoter, and stability elements. REs can be located upstream and / or downstream of a transgene in an expression cassette, which can be a plasmid, vector, or a viral vector.
DETAILED DESCRIPTION OF THE DESCRIPTION [0025] This report addresses compositions and methods for using such compositions in gene therapy to treat a disease or condition associated with the central nervous system (CNS), for example, Dravet's syndrome, Alzheimer's disease, epilepsy and / or seizures.
[0026] Gene therapy can replace, modify, delete or add a specific gene or a specific nucleic acid sequence, such as an expression cassette, to confer a therapeutic effect on a cell. In some cases,
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16/133 gene therapy is used to apply an expression cassette to a cell that produces or results in a therapeutic effect. In some cases, a virus, such as AAV, comprising a viral vector that comprises an expression cassette can be used to apply a transgene to a cell. The expression cassette can contain a transgene that provides a therapeutic effect when expressed in a cell.
[0027] A challenge in gene therapy is to ensure that the transgene is expressed in an appropriate cell type of interest, or in the type of target cell, in order to effect or target gene expression. Traditional methods for targeting gene therapy have often relied on methods and / or vehicles of administration (for example, varying the viruses used or capsids from virus sequences). In addition to targeting, or selective expression, an expression cassette in the type of target cell in one or more types of non-target cells, another challenge in the field is to increase gene expression, especially when the gene is large, in a type of target cell or tissue in order to have a therapeutic effect.
[0028] This report provides a plurality of regulatory elements, which are non-coding nucleotide sequences, which can be operationally linked to any transgene to increase or improve the selectivity of transgenic expression in the CNS, for example, in PV neurons. By increasing the selectivity of gene expression using one or more of the regulatory elements described here, it is possible to increase the effectiveness of a gene therapy, reduce the effective dose necessary to result in a therapeutic effect, minimize adverse effects or effects outside the objective and / or increase patient safety and / or tolerance.
[0029] In one aspect, one or more regulatory elements can be operationally linked to any transgene in an expression cassette in order to modulate gene expression in a cell, such as guiding the expression of the transgene in a type of target cell or tissue (for example, PV cells) in one or more types of non-target cell or tissue (for example, types of
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17/133 non-PV cells in the CNS). In some cases, the orientation of transgene expression in a target cell or tissue type includes increased gene expression in the target cell or tissue type. One or more regulatory elements operatively linked to a transgene can be part of an expression cassette, which can be a linear or circular construct, a plasmid, a vector, a viral vector, for example, an adeno-associated virus vector (AAV). Such an expression cassette can be adapted for gene therapy or to be administered to an individual (for example, a human, a patient or a mammal). In some cases, the operational attachment of one or more regulatory elements to a gene results in the targeted expression of the gene in a type of tissue or target cell in the CNS, such as a parvalbumin neuron (PV). In some cases, one or more regulatory elements (for example, SEQ ID NOs: 1-32, or a functional fragment or a combination of these, or sequences showing at least 80%, at least 90%, at least 95%, or at least minus 99% sequence identity to these) increase the selectivity of gene expression in a type of tissue or target cell in the CNS, such as PV neurons. In some cases, gene therapy comprises one or more of the regulatory elements described here, where the regulatory elements are operationally linked to a transgene and guide the selective expression of the transgene in PV neurons.
[0030] In some cases, the selective expression of a gene in PV neurons is used to treat a disease or condition associated with haploinsufficiency and / or a genetic defect in an endogenous gene, where the genetic defect may be a mutation in the gene or deregulation of the gene. Such a genetic defect can result in a reduced level of the gene product and / or a gene product with impaired function and / or activity. In some cases, an expression cassette comprises a gene, a subunit, a variant or a functional fragment thereof, where the gene expression of the expression cassette is used to treat the disease or condition associated with the genetic defect, function and / or activity compromised, and / or deregulation of the endogenous gene. In some cases, the disease
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18/133 or condition is Dravet's syndrome, Alzheimer's disease, epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability and / or seizures.
[0031] In some cases, the transgene is an ion channel or a neurotransmission regulator, a DNA-binding protein, or a subunit, variant, or functional fragment thereof. In some cases, the transgene is an alpha subunit sodium ion channel, beta subunit sodium ion channel, or a functional variant or fragment thereof. In some cases, the transgene is from a potassium ion channel or a subunit thereof. In some cases, the transgene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, STXBP1, a DNA-binding protein (for example, a DNA-binding protein that modulates the expression of an endogenous gene), or a functional variant or fragment thereof. In some cases, the transgene comprises a sequence showing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity to any of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, a DNA-binding protein, or a functional variant or fragment thereof. In some cases, the transgene is a DNA-binding protein that modulates the expression of an endogenous gene, such as any of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, and STXBP1.
[0032] As used here, the forms in the singular “one”, “one” and “the / a” also include the forms in the plural, unless the context clearly indicates otherwise. In addition, to the extent that the terms "including", "includes", "presenting", "presenting", "with", or variants of these are used both in the detailed description and in the claims, these terms are intended to be inclusive in a similar way to the term "comprising".
[0033] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, ie , the limitations of the measurement system. For example, “about” can mean
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19/133 within one or more of a standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.
[0034] The terms "determination", "measurement", "evaluation", "trial", "analysis", and their grammatical equivalents can be used to refer to any form of measurement, and include determining whether an element is present or not (for example, detection). These terms can include both quantitative and qualitative determinations. The assessment can be relative or absolute.
[0035] The term "expression" refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from template DNA (such as in mRNA or other transcribed RNA) and / or the process by which a Transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcribed and encoded polypeptides can collectively be called a "gene product". If the polynucleotide is derived from genomic DNA, the expression can include the joining of mRNA in a eukaryotic cell. [0036] As used here, "operationally linked", "operational link" or grammatical equivalents of these refer to the juxtaposition of genetic elements, for example, a promoter, an amplifier, a polyadenylation sequence etc., where the elements are in a relationship that allows them to operate in an expected manner. For example, a regulatory element, which may comprise promoter and / or amplifier sequences, is operationally linked to a coding region if the regulatory element assists in initiating the transcription of the coding sequence. There may be intervening residues between the regulatory element and the coding region as long as this functional relationship is maintained.
[0037] A "vector" as used here refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and that can be used to modulate the application of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other vehicles of application. The vector generally comprises
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20/133 genetic elements, for example, regulatory elements, operationally linked to a gene in order to facilitate the expression of the gene in a target. The combination of regulatory elements and a gene or genes to which (which) they are operationally linked for expression is called an "expression cassette".
[0038] The term “AAV” is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or a derivative of it. The term covers all serotypes, subtypes, and both naturally occurring and recombinant forms, except where otherwise required. The abbreviation “rAAV” refers to the recombinant adeno-associated virus, also called the recombinant AAV vector (or “rAAV vector”). The term “AAV” includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, and hybrids thereof, Bird AAV, bovine AAV, canine AAV, canine AAV, AAV primate, non-primate AAV, and sheep AAV. The genomic sequences of various AAV serotypes, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such strings can be found in the literature or in public databases such as GenBank. A "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally two, inverted AAV terminal repeat sequences (ITRs). The term rAAV vector encompasses both particles of the rAAV vector and plasmids of the rAAV vector. An rAAV vector can be either single-stranded (ssAAV) or self-complementing (scAAV). An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsulated polynucleotide of the rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be applied to a
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21/133 mammal), is typically referred to as a “rAAV vector particle” or simply a “rAAV vector”. Thus, the production of the rAAV particle necessarily includes the production of the rAAV vector, insofar as such a vector is contained within an rAAV particle.
[0039] As used herein, the terms "treat", "treatment", "therapy" and the like refer to obtaining a desired pharmacological and / or physiological effect, including, but not limited to, relief, delay or reducing progress, reducing effects or symptoms, preventing establishment, improving the establishment of a disease or disorder, obtaining a beneficial or desired outcome with respect to a disease, disorder, or medical condition, such as a therapeutic benefit and / or a prophylactic benefit. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease in an individual who may be predisposed to the disease or be at risk to acquire the disease, but it has not yet been diagnosed as such; (b) the inhibition of the disease, that is, the interruption of its development; and (c) relieving the disease, that is, causing the disease to regress. A therapeutic benefit includes eradicating or ameliorating the disorder in question being treated. Also, a therapeutic benefit is achieved by eradicating or improving one or more of the physiological symptoms associated with the disorder in question in such a way that an improvement is seen in the individual, regardless of whether the individual may still be afflicted by the disorder in question. In some cases, for prophylactic benefit, the compositions are administered to an individual at risk of developing a particular disease, or to an individual who reports one or more of the physiological symptoms of an illness, even though a diagnosis of this illness has not yet been made. been done. The methods in this report can be used on any mammal. In some cases, treatment may result in a reduction or cessation of symptoms (for example, a reduction in the frequency or duration of seizures). A prophylactic effect includes delaying or eliminating the onset of a disease or condition, delaying or eliminating the symptoms of a disease or
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22/133 condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof.
[0040] The term "effective amount" or "therapeutically effective amount" refers to the amount of a composition described here that is sufficient to effect the intended application, including, but not limited to, treatment of the disease, as defined below . The therapeutically effective amount may vary depending on the intended treatment (in vivo), or the individual and disease or condition being treated, for example, the individual's weight and age, the severity of the disease or condition, the manner of administration and the like, that can be easily determined by a person skilled in the art. The term also applies to a dose that will induce a particular response in a target cell. The specific dose will vary depending on the particular composition chosen, the dosage regimen to be followed, whether it is administered in combination with other compounds, the administration schedule, the tissue to which it is administered and the physical administration system in which it is carried .
[0041] A "fragment" of a sequence of nucleotides or peptides refers to a sequence that is smaller than what is believed to be the "full length" sequence.
[0042 | A "variant" of a molecule refers to allelic variations of such sequences, that is, a sequence substantially similar in structure and biological activity to both the entire molecule and a fragment thereof.
[0043] The term "functional fragment" includes the "fragments", "variants", "analogues", or "chemical derivatives" of a molecule.
[0044] A "functional fragment" of a DNA or protein sequence has at least one fragment of the biological activity of the sequence, which refers to a fragment that retains a biological (or functional or structural) activity that is substantially similar to an activity biological sequence of the full length DNA or protein. A biological activity of a DNA sequence may be its ability to influence expression in a manner known to be attributed to the full-length sequence. For example, a fragment
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23/133 functional function of a regulatory element will retain the ability to influence the transcription of the full-length ER.
[0045] The terms "individual" and "individual" are used interchangeably here to refer to a vertebrate, preferably a mammal, more preferably a human. "Individual" refers to an animal, such as a mammal, for example, a human. The methods described here can be useful in human therapy, veterinary applications and / or preclinical studies in animal models of a disease or condition. In some cases, the individual is a mammal, and in some cases, the individual is human.
[0046] The term "zn vzvo" refers to an event that occurs in an individual's body.
[0047] The term "in vitro" refers to an event that occurs outside an individual's body. For example, an in vitro assay encompasses any assay performed outside an individual. In vitro assays encompass cell-based assays in which living or dead cells are employed. In vitro assays also include a cellless assay in which no intact cells are employed.
[0048] Sequence comparisons, such as for the purpose of evaluating identities, mutations, or where one or more positions in a test sequence are relative to one or more specified positions in a reference sequence, can be performed by any algorithm of proper alignment, including, but not limited to, the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with the default settings ), the BLAST algorithm (see, for example, the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with the default settings), and the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner available at www.ebi.ac.uk/ Tools / psa / emboss_water /, optionally with the default settings). The optional alignment can be evaluated using any suitable parameters of a chosen algorithm, including standard parameters.
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24/133 [0049] In general, "sequence identity" or "sequence homology", which can be used interchangeably, refer to an exact nucleotide-to-nucleotide or amino acid-to-amino acid match of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded therein and comprising these sequences with a second sequence of nucleotides or amino acids. Two or more sequences (polynucleotide or amino acids) can be compared by determining their "percent identity", also called "percent homology". The percent identity with a reference sequence (for example, nucleic acid or amino acid sequences), which can be a sequence within a longer molecule (for example, polynucleotide or polypeptide), can be calculated as the number of exact combinations between two ideally aligned sequences divided by the length of the reference sequence and multiplied by 100. The percent identity can also be determined, for example, by comparing the sequence information using the advanced BLAST computer program, including version 2.2.9, made available by the National Institutes of Health. The BLAST program is based on the Karlin and Altschul, Proc. Natl. Acad. Know. USA 87: 2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215: 403410 (1990); Karlin and Altschul, Proc. Natl. Acad. Know. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). In summary, the BLAST program defines identity as the number of identical aligned symbols (that is, nucleotides or amino acids), divided by the total number of symbols in the shortest sequence of the two. The program can be used to determine the percent identity over the entire length of the strings being compared. Standard parameters are provided to optimize searches with short query strings, for example, with the blastp program. The program also allows the use of a SEG filter to remove segments from the query strings
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25/133 as determined by the Wootton & Federhen SEG program, Computers and Chemistry 17: 149-163 (1993). The ranges of the desired degrees of sequence identity are approximately 80% to 100% and integer values between them. Typically, the percent identities between a described sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. In general, an exact match indicates 100% identity by the length of the reference sequence. In some cases, the reference to the percent sequence identity refers to the sequence identity as measured using the BLAST (Basic Local Alignment Search Tool). In other cases, ClustalW can be used for multiple sequence alignment.
[0050] Unless otherwise indicated, all terms used here have the same meaning that a person skilled in the art recognizes and the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and recombinant DNA technology, that are within the knowledge of the technician in the subject.
Regulatory elements [0051] Regulatory elements are a sequence of nucleic acids or genetic elements that are able to influence (for example, increase or decrease) the expression of a gene and / or confer selective expression of a gene (for example, a reporter gene such as such as eGFP, a transgene, or a therapeutic gene) in a particular type of tissue or cell of interest. In some cases, a regulatory element can be a transgene, an intron, a promoter, an amplifier, RTU, isolator, a repressor, an inverted terminal repeat sequence (ITR), a long terminal repeat sequence (LTR), stability element, response element after translation, or a polyA sequence, or a combination of these. In some cases, the regulatory element is a promoter or an amplifier, or a combination of these. In some cases, the regulatory element is derived from a human sequence.
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26/133 [0052] In some cases, the cell type of interest is a PV neuron. The regulatory elements can function at the level of DNA and / or RNA. The regulatory elements can work to modulate the selectivity of gene expression in a cell type of interest. The regulatory elements can work to modulate gene expression in the transcription phase, post-transcription phase or in the gene expression translation phase. Regulatory elements include, but are not limited to, promoter, amplifier, repressor, silencer, and insulator sequences. At the RNA level, regulation can occur at the translation level (for example, stability elements that stabilize the mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination. In some cases, regulatory elements can recruit transcription factors for a coding region, which increases the selectivity of gene expression in a cell type of interest. In some cases, regulatory elements can increase the rate at which RNA transcripts are produced, increase the stability of the produced RNA and / or increase the rate of protein synthesis from RNA transcripts. In some cases, regulatory elements can prevent RNA degradation and / or increase its stability in order to facilitate protein synthesis. In some cases, regulatory elements suppress the transcription and / or translation processes in off-target cell types. In some cases, off-target cell types include, but are not limited to, excitatory neurons, non-CNS cell types, and non-neuronal CNS cell types.
[0053] Various assays including, but not limited to, DNAase hypersensitivity, ATAC-Seq, and ChIP-Seq can be used to identify putative non-coding regulatory elements (REs). The enzymatic reaction in each of these assays is preferably directed to open / accessible crimatin states, a state that is believed to be a predictor of regulatory elements. In order to discover the selective regulatory elements for the cell type, an open chromatin sequence assay can be performed for the target cell type of interest (for example, parvalbumin neurons) and
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27/133 compare this with open chromatin sequences for non-target cell types (eg, excitatory neurons). Additional filters can be applied to further refine the target selection, including proximity to a selective gene for the cell type, species conservation and / or sequence motifs, such as transcription factor binding sites. DNA sequences that are uniquely identified in the target cell type can be synthesized and cloned into an expression vector. The selectivity of a regulatory element can be determined using immunohistochemical methods to quantify the co-location for proteins selective for the cell type.
[0054] For example, a method of isolating a selective regulatory element for the cell type includes isolating the nuclei of a brain tissue or cell type of interest from an animal model, which can be achieved by using a method of affinity purification which isolates the tissue or cell type of interest (for example, using spheres coated with an anti-PV antibody for the isolation of PV neurons), using high-throughput natural initiation and DNA synthesis in a generating a set of sequences from the open chromatin regions in the nuclei, sequencing the set of sequences in order to identify putative sequences that trigger gene expression in the tissue or cell type of interest, and verification of selective expression in a system reporter in an in vitro cell line and / or an animal model.
[0055] Another method for identifying candidate regulatory elements that are selective in a tissue or cell type of interest includes the use of knockin mice R26-CAG-LSL-Sunl-sfGFP-Myc for the collection of tissue or type of cell of interest, isolation of GFP + / Myc + nuclei from the mouse neocortex of this strain using affinity purification, for example, using anti-GFP or anti-Myc antibodies and magnetic beads coated with G protein in order to isolate the nuclei of the neocortex. The nuclear RNA of the purified nuclei or complete neocortical nuclei can be converted to cDNA and amplified with the Nugen Ovation RNA-seq System V2 (Nugen 7102), followed by
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28/133 of the sequencing using the Illumina HiSeq 2500. The genomic DNA from the purified nuclei can be fragmented and used to produce MethylC-seq libraries, which can be sequenced using the Illumina HiSeq 2000. To generate an ATAC library -seq, the ball-bound nuclei are transposed using Tn5 transposase (Illumina FC-121-1030). After 9-12 cycles of PCR amplification, the libraries are sequenced using an Illumina HiSeq 2500. To generate a ChIP-seq library, excitatory neuron nuclei can be digested for mononucleosomes using micrococcal nuclease, followed by extraction of the chromatin with salt, and native ChIP and library construction, which can be sequenced on an Illumina HiSeq 2500. After sequencing these libraries, the sequences are mapped to identify, for example, the correlation of cell type-specific hypomethylation in regions GC-rich, histone modifications, transcription factor binding sites, and patterns associated with highly expressed transcription factors. The overlapping characteristics and correlations from multiple assays and / or the libraries described above provide evidence for the identification of candidate sequences within such genomic regions as potential regulatory elements associated with selective expression and / or high expression in isolated cells of the neocortex . For example, a genomic region characterized by a strong overlap between the hypomethylation detected in the methyl C-seq library, ChIP assay, and an enrichment in the transcription factor binding motifs in the same region provide converging data that indicate the genomic region that contains a sequence of a putative regulatory element selective for the isolated tissue or cell type. As another example, in order to identify selective candidate neuron PV regulatory elements, it is possible to isolate PV neurons and purify isolated PV cell nuclei in such a way that the genomic sequences that are identified as active in multiple sequencing assays described above have a high probability of being selective regulatory elements for PV cell, for example, a genomic region that is identified as active in an ATAC-seq assay (corresponding
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29/133 to open chromatin regions), active in the RNA-seq assay (indicative of active gene expression and low DNA methylation patterns in the region), and active in the methylC-seq assay (which generates methyloma resolution maps single basis of a cell type of interest).
[0056] Once candidate genomic regions have been identified as selectively active in a cell type of interest, sequences within the region can be generated using PCR methods and tested in additional in vitro and / or in vivo assays in order to validate the selectivity for tissue or cell type of the sequences. Such validation assays include immunohistochemical assay for colocalization, where an antibody or any detectable marker is used to label the cell type of interest and a second detectable marker, for example, a fluorescent transgene, is operably linked to putative regulatory elements. Expression cassettes comprising such elements are applied to cells in vitro and / or in vivo. The selective expression triggered by one or more putative regulatory elements can be validated by measuring the overlap between the type of cell of interest (as measured by the signal or detectable fluorescence of its labeled marker, for example, an antiPV antibody) and the second marker detectable corresponding to the expression of the transgene (for example, eGFP or RFP) operationally linked to the regulatory elements. An overlap in the signals from both detectable markers indicates selectivity for the cell type in the labeled cell type if the amount of overlap observed is greater than the overlap observed when the regulatory elements are replaced by a control, such as CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), a non-selective regulatory element, or a previously characterized non-selective regulatory element. Several strains of mice adapted to express a detectable marker in a cell type of interest allow validation of selectivity for the cell type of a regulatory element in vivo. For example, a number of mouse strains that express Cre in a particular cell type can be used, since the expression
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30/133 selective selection of Cre in the cell type can direct Cre-induced expression of a fluorescent protein, such as RFP, in a cell type of interest. Labeling such a cell type of interest in vivo allows the level of selective expression to be determined for the cell type that is associated with a putative regulatory element operatively linked to a fluorescent transgene or reporter in the same mouse. Similar to the co-location assay, an overlap of the signals from both markers that exceeds the overlap detected for CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-regulatory element Selective is indicative of selectivity for the cell type for the tested regulatory elements. In some cases, the mouse lineage used is the B6 PV-Cre mouse (Jackson Laboratory), which is a knock-in B6 PV-Cre mouse that expresses Cre recombinase in neurons that express parvalbumin (for example, interneurons in the brain and neurons afferent sensory proprioceptives in the dorsal root ganglion), without disrupting the expression of endogenous Pvalb.
[0057] By validating the selectivity for the cell type of a regulatory element for a particular cell type, sequences of such regulatory elements can be varied by using various methods of mutagenesis, for example, error-prone PCR methods (“ error-prone PCR ”), in order to increase its selectivity. In some cases, two or more regulatory elements showing cell selectivity can be combined. In some cases, the combined regulatory elements exhibit increased cell type selectivity in the orientation of gene expression in the cell type of interest. In some cases, such regulatory elements are truncated on one or more bases at a time to determine the minimum amount of the sequence that retains its selectivity for the cell type. Smaller regulatory elements that retain selectivity for the type of cell are useful for carrying out gene therapy comprising a large transgene, or where the ability to clone a vector or plasmid is limited in view of the size of the transgene that is to be applied using gene therapy.
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31/133 [0058] This report provides a plurality of nucleotide sequences that are regulatory elements. In some cases, any one or more of the regulatory elements described here results in increased selectivity in gene expression in a parvalbumin cell. In some cases, the regulatory elements described here are selective for the PV cell. In some cases, the selective regulatory elements for PV cells are associated with selective gene expression in PV cells rather than the expression in CNS non-PV cell types. In some cases, selective regulatory elements for PV cells are associated with reduced gene expression in non-PV cell types from the CNS. [0059] Non-limiting examples of regulatory elements include SEQ ID NOs: 1-32, as provided in TABLE 1 below.
TABLE 1: List of nucleic acid sequences described here.
SEQIDAT THE: Nucleic acid sequence Genomic Source / Location 1 GGAGGAAGCCATCAACTAAACTACAATGACTGTAAGATACAAAATTGGGAATGGTAACATATTTTGAAGTTCTGTTGACATAAAGAATCATGATATTAATGCCCATGGAAATGAAAGGGCGATCAACACTATGGTTTGAAAAGGGGGAAATTGTAGAGCACAGATGTGTTCGTGTGGCAGTGTGCTGTCTCTAGCAATACTCAGAGAAGAGAGAGAACAATGAAATTCTGATTGGCCCCAGTGTGAGCCCAGATGAGGTTCAGCTGCCAACTTTCTCTTTCACATCTTATGAAAGTCATTTAAGCACAACTAACTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTTGCCCAGGACAGAGTGCAGTAGTGACTCAATCTCGGCTCACTGCAGCCTCCACCTCCTAGGCTCAAACGGTCCTCCTGCATCAGCCTCCCAAGTAGCTGGAATTACAGGAGTGGCCCACCATGCCCAGCTAATTTTTGTATTTTTAATAGATACGGGGGTTTCACCATATCACCCAGGCTGGTCTCGAACTCCTGGCCTCAAGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTCAGCCACTATGCCCAACCCGACCAACCTTTTTTAAAATAAATATTTAAAAAATTGGTATTTCACATATATACTAGT Human; hgl9: chr2: 171621900171622580 2 AGTTTGGACAAGAACTATAGTTCTAGCTTTCTCTGGGTCTCCAC Mouse
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SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationCTTGCAGAGAATGCAGCTTTCATTATCTCATGAGCCAAACTCTCATCATCTCTTTCCATATATCTGTCGGTGCTCTTCCATGAGTACTCTAACACACACAGAAGGAGCACTTACACAGGCTGTTGTTTTTCTCTTATTATCATAGCTGTTGTTCAGACATGTGCATTCTGTTCTTGTTGCTTCAATGCTAAAGGAGTCTCAGGATATGAGAACTGTACCAGCCGAGGCATCAGGAAACATGGGTGGAAATTCCCACAGTACTATTTGTTCACTGTGTGACCTTGGGCCAGTCACATCCCTTTCCTGAGGCTTCGATTCCCCAAGCTATAAAAGAAGCATCTCTTAACCTTTTTTTAGGTCATGAGTCAGGCCCAGCACACTCTCAGGGAGACTCATGAGAGTACAGATCATTTCCCATAGAAAAACCATAGTTTTATATCCAGAGGCTTTTCTGTAAG 5mml0: chr2: 36053858-36054359 3 GGTTCCAGTTCAGAGGCAGAGCATTTGGGGTTCCCAGTCAGGAGCTTTCCTCTCTCCGCTCCTTAGTTTCCTCTCTTTAAAAAAAAATGGGTGATAGTATAGAAAGGAAGCTCTGGGCTCGGGGACCAGGGCCCTGGGATCCCCGCTCCCAGCCACTCGCTCCTGACCCTTCCAGGGACAAGCTCCCCCCCACCCCGTCCTTTCCAGGCTGCCACTAGAAGAGATGGGGACGCGTGGTCAGCCGCTTCTGTCGCCCCCCAGGGAACGGTCTCACGCTGGAGGGGGCAGTGCCCTCGGAACAGGACAGTCAGCCCAAGCCAGCCAAGCGCGCGCGGACGTCCTTCACCGCAGAGCAATTGCAGGTACCCCGGGCAAGCCCCGAAGCGTGTGGGCGGGGCTTCGGAGTGGGCGTGGTTGTTCGGGACTTGTGACTCCGCCCCTTGTGCGGGGACCCGCGTGAGGCCGCTCCAAGGATGAAGCTGCCTGGGGCGTGGCCTCGGACCCTGAGCCTCTGATTGGGCGGAGGTCTCAGGGCCCTTCTGCGCCCCACAGGTTATGCAGGCGCAGTTCGCGCAGGACAACAACCCGGACGCGCAGACGCTGCAGAAGCTGGCGGACATGACGGGCCTCAGTCGCAGGGTCATCCAGGTGGGGCTCCGGGGTCTCGGCCTTCAGGTCTAGGGTGAACCTTAGGGAAGCGCTGAAGCTCGTAGTGGTACGGATGGTCGCGCGTGCACGTGGCCGCCCCTCTCCAGTGTGGCCTAAGGACCCCAGTCGGCACGGGTTGACCCTTTTCCTTGATTACTGAGAGTGCAGAGGCTGT Mouse 5 chr2: 36,091, 14436,091,966 4 TGGTGGGAAGACATGTCCAGGGAAGAAATGGCCTCCAGAGGCC Mouse
Petition 870190126085, of 11/29/2019, p. 37/153
33/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationTGAGGTGGGGAAATGCTGGAGGTGGAGAGAGGAACAACTGACTGAAAATGAGCTTCCACTGTGGCTTAGTAGCCTATACCAAGTCTAGAGTATAGGGTAGGAGAAGATTAGGAAAGCGATGGGTCTGAGAATGATGTGGCCTGTTGACTTTTGTAAACCCAAAGCACCTTGGACTAAACCCTATGAACAGTGTGGTGCCACCAAAGACTATAATGAGCTCAGGGAACAGAATTCTGTGTGCATGGTGATTTTTTTTTTTTTTTTCTGCTAACTGCAGTCTGGGTGATGCATTGACAAACCAATCCTGGAAAGTAAGAGGCAAGGGCAGCTGGGACGGTGAGAGGAGCCTGATGGGAACCAGGCCAAGCAGGGCAGCAGAGGCGATGAAGAGGATGTGGTGCATCCAGAGACTCACTTCATTAGCTGGAGGCACTGCTGGATAGGGTCTGAAGGTTCTGGTATCTGAGTTGGCGGGCTGGGTGAGTGGTGGCTCTGCTTCCTGAACAGTGTGTGCAAGAGGAAACAGGGTTAAGGGCTAGGACAGTCACAGGTGAGTCAGCCTCACAAGAGCAACCTTCCCCTAGTGCAGA 5 chr2: 36,095, 39636,096,028 5 GGAGGTCTCCTTTTGCCCCGGTTCCAACAAGAGAATGCAAGGCTGTATCTCAATTTCCTTGAGCCTCTCTGTATTATAGAAGAAAAGTAGGGAAGCCATACGCCCCTTCTGAGCTTCAGTGTCTCTCTGTCTCTGCAAATGAGGCTGGGGAGGCTGGGGGCGGGCGTGAAAGAGGCCCGCGCCAAGCCGACCCCCACCTCTGCCCCCTCCCCAGGTCAACAACCTCATCTGGCACGTGCGGTGCCTCGAGTGCTCCGTGTGTCGCACATCGCTGAGGCAGCAGAATAGCTGCTACATCAAGAACAAGGAGATCTACTGCAAGATGGACTACTTCAGGTAGGCAGCGGCCATCCCGCCAGCAAGCGCTGGAGCATGAACGCCTTGCACACGCGTGCCTAGGCCACTTGTGTGGCCTGTGCTCTCCAATTCCTGAGCCCTGCTGTTCAGAGTGCACAACGCGGCTCAGCGCACTGGCCCGGCCCTCCTACTCAGCACGTCTTACACAGAAGGGAGCGCCAGTCTCAGCCTGAGTTCTGGCGGGGGATCTGCCTCGGGTTCCTCCGATCTGACAGGCGCTGGCCACGGGTCTGGTTCCATCTCTGGTCTTTTCTGGCCCCGAGCACCAGTGTGTTCTGTTGAGCTCTGATGTCCGAGGCTCTGGCCCGGATCA Mousemml0: chr2: 36102524-36103193
Petition 870190126085, of 11/29/2019, p. 38/153
34/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / Location 6 CTCTGGCTACCTCTTATCTTGGGCATTCACGACAATTTCTAATTGCAGGTAGTTTGTGTGTGTGCGCGTGTTTTTTTTCCCCCTCAGAGGCTTGGATTGCAAAGGAACTAAGCGATTACTTCAAGAGCCACGGGTTAAGTGCAGGGAGAGGGGGAGAGAGAGGGAAAAAAACCCAATCCAAATTCAAATTGCTTCATTAGAGAGACACCGCTTTTGTGGGGAAGGGCTTTAAATGCCCACTACAAAGTTAGGACTCATTGTTCAGCGCCGGTTTATATAACAGGCGAGGGGAGGCGCTGGGCTCTGACAGCTCCGAGCCAGTTCAGCAGCCGCCGTCGCCTGCATTCCCTCCCCCTCCCCCAGGTGATGGCCCAGCCAGGGTCCGGCTGCAAAGCGACCACCCGCTGTCTCGAAGGGACCGCTCCGCCTGCCATGGTGAGTCCTTTCGGTCCTGCTTTCGGCCCCGAGTCCCCCCAACAGCACAGGCCAGGGCTTCTGGCTCAGCCTTCCGGCTACCAACCTCTACCCCTGCGCTGGAAAACTGCCGATAGGAGCCGCCTCTCGTTGAGCCTTGGTTTTTCTGGCCTGGAATGTGAGCTTTGGCTGCTTCCTGCACCCAGGATGCGCTGTGTTAAAAGTTGGGGGCCGTCCCTTCTTCTCCAATAGGTCCTTTCATTCTTGTACTCCAGCCTAGGGCGCGACATCCCTGGCACATTTCGGTGTCAGTCGGTGCGCGAGGAAACCAGATTCAACTCTGAGTACTCGGCTAAGCGCTTCGCTGTTCCTCTCTCCCATTTCAGGCTCAGTCAGACGCAGAGGCCTTGGCAGGCGCTCTGGACAAGGACGAAGGTAGAGCCTCCCCATGTACGCCCAGCACACCGTCTGTCTGCTCGCCGCCCTCTGCTGCCTCTTCCGTGCCGTCTGCCGGCAAGAATATCTGCTCCAGTTGCGGTCTGGAGATCCTGGACCGGTATCTGCTCAAGGTGAGTCAGGGTAGGTGTGCCTGCTTGCCCACGGGTGTGGTTTGCAGCCCCAAGAGCTGT Mouse 5mml0: chr2: 36103286-36104328 7 CAAGACTTTTAAAAGTTTAGATAAATAAACAAACATTTGACGGCTTTCCATCACATCTAGACTATAATCCAAAGATCTATATGGTCCCAAACGACTTACACTTAACTACCGTCTCCCATATGGCTTCTTCCCCCATCAGTCATTGTCCTCAGCCATAGTGGCCTCCCTGTTCCTTTGGGTACAAGGGAACAACTCCCTGAGAGGTTCCATTAGCTGCTGTTGCCTGAGATGCTCTTGAGCCCACACCATCTGCTCATTTCTCTCCTCACGTGTCAGTGATTAAGAGGCTGTCCTTGGCCTCCCGTCAA Mousemml0: chr2: 36114311-36114817
Petition 870190126085, of 11/29/2019, p. 39/153
35/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationAATTACATCCCTGCCGCTTTCCACTTCTTGCCTTCTTATTTTCTAAATAGAACTAACTCACCACTACCCAACATTCTATATAATTGGATATCTGTCCTCTGTTTAAATATAATGTTGACTTCAAGAAAGAACGTTGTCACTGCCCTGTCACCAGACTTTTAAACAGTGCCTATCGTGTGGCACATGCTCAGTGAAATTG8 TCAACAGGGGGACACTTGGGAAAGAAGGATGGGGACAGAGCCGAGAGGACTGTTACACATTAGAGAAACATCAGTGACTGTGCCAGCTTTGGGGTAGACTGCACAAAAGCCCTGAGGCAGCACAGGCAGGATCCAGTCTGCTGGTCCCAGGAAGCTAACCGTCTCAGACAGAGCACAAAGCACCGAGACATGTGCCACAAGGCTTGTGTAGAGAGGTCAGAGGACAGCGTACAGGTCCCAGAGATCAAACTCAACCTCACCAGGCTTGGCAGCAAGCCTTTACCAACCCACCCCCACCCCACCCACCCTGCACGCGCCCCTCTCCCCTCCCCATGGTCTCCCATGGCTATCTCACTTGGCCCTAAAATGTTTAAGGATGACACTGGCTGCTGAGTGGAAATGAGACAGCAGAAGTCAACAGTAGATTTTAGGAAAGCCAGAGAAAAAGGCTTGTGCTGTTTTTAGAAAGCCAAGGGACAAGCTAAGATAGGGCCCAAGTAAT Mousemml0: chrl5:78179109-78179610 9 AAATAGAACTGTGAGATAGGGGGAGAGGGGGCAGGAAGGACAAGAGACCCCTGTCTCATTGTGATCCCCACCTGTCTGCTCTGTGGGAGGGTACCCATGAGGGCCAGCCCACAGCCCTTAGGTGGACATTGTCTGGTCCTGTCTCACTGTCCCTCCCAGCAGCCCCAGAGGCCAGGAGACAGGGGTCTCAGTCCTCACTGAGAGATGTGTAAACTGAGGCCCAGTGAATGTTGAGGGCCAGGGCATGCCCTTGGTGGGATGTGACCTGGGTCTCCTTCGCACGGGCTTCCTCCCCGAAGCCGAGCTGAGCATTTGGAGTTTGAAATGTTTCCGTACTTAGCAATCTGCTCCTCTATTCCCGGGCGGACTTCCGATAGCTCCGGCCTTATGCTGCACTAGATAAGATGGAGCAGGGAGAGGACACGGCACTACTTATGTAACCGGCCTCTTGAAAAATGGAGCAGCGGTCAGGGCGGAACAAGACGTCCTCTCTCTACGCATCCCTCTCCTTTCCCTGCTAAGGCTGCAGCTGGAGTCAGAGGCAGGGCTGTTCCAATCTGTCTTTGATCAGTAACGCAGCCAGCCTCCAGCCTCCGTCAGCCTCCTCAT Mousemml0: chrl5:78195347-78196134
Petition 870190126085, of 11/29/2019, p. 40/153
36/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationGGCTGAGACCCGGCCTCAGTTTCCCCCACTTACATCCCGAGGATCAGAGCCTGTGAGGATGAAATGGGATAAGGTAGCTGGAACCGTCTGGCAGAGAGCGAGTCCTCAGGACTGTTGATGCCTGTGGCTGCCTGGCTTGACCCCAAGTGACCCCGCCTCCTCATCCTGCAGCAGGAGAA10 TCTATAGAATGTGTCCCCAGCCTTGTTTTCCACACTTGATACGCAAGGAATGCATACCACAGAGAGGGATGAGGGTAGCATCCAGCCTGCTTCCTGTGTGTCGGGGCGCTACAGCCACATCTCCCCAGTCCATCTCAGACCGTCACAGAGCTTCGCCGAATGTATAGCTTTGTTCTCTGTGCAGACAGGGAGACAGAGCCTTGGGAAGCATAGGTGCTTGCTTCTTTGCCCACTGAGTCTTAGCTGGACTTGCACACCACATGCCTCACAGCCGGGCGCACTTGCATTTGTCACCCAGGCCCAGTGATGATGGCTCTGCTTGCTTTGTGCTTTGTGCCAACTACAGCTCCAGCACCTGTGCCCTGGGTTTTCACTCCTTTAGTTGAACACGTAGTTACTGGGGTTGTAGGGATGGAGCCTTTCTGCTTCCTTCTGGCAAAGTCCTTAGCGGCCTGCTGCGGGGGTGGGGGGTGTTCAGGGGAGTGGTGATGAAGTATGACAG Mousemml0: chrl5:78196305-78196806 11 TCTCCAGTTGGAGAAACAGATGCTGTAACTGGGGCCACAGTATAAAGAGAGCCCAGACATTGAACTGTCAACACAGAAGCCTGGCACACTGGAACTGGCAGTCCAGCTGGGAACAAGGGGTAGAGGCTGAGGCCACTAAGTCAACTGAGGCAGGAGACATAGGAGCTAAAGCAGCTGAAGGGTGCAGGACAGCTGGGGGGTCTGAAGTGGGCCTCATGCCCAGAGCTATGAAGTCAGGGGCTGTAGCCTAGGAGCCTTGGAAGCCAGCTGGCAAGCTGTGGCCCAAAGACGCTGACTCACCAGGAGGGGGCAGCTGGAGCCAGGCACTCCTAAGGTTTCCAGGAAGGGCAGCCTTCCAGGGCTCAGCTAGGGGAGACAGTGTTGACAGCAAGTTGTCAGGCAACTTGAGCTACTGGGCAGCTGGGAAGCTGTCCCTTGGTCCCCAGTATCATCATCACCCCAGACGCTGCCCACCTGCCTCAGGTCCCACACAGTGATCCTCCCATCTTTAACACAACACATGACCAGAGAGA Mousemml0: chrl5:78205234-78205766 12 GTCACCCTCCCCCCAAACAACCCCTTCTTCTCTGGTTCGAGAAA Mouse
Petition 870190126085, of 11/29/2019, p. 41/153
37/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationTTACAGGCATGAAAGATATAAATCGGGATGCTTGACTTGGGAATATAAATCACTAAAGCTTGGGGGCAGGGGTGGGCGACCTTTGTGACCGTCCTTGTGCGTGCCAGTAAATCCTGTGGTCCAGGGGAGAAGAAAAGGCTGTGTGGCTTCTGCTCACAAAGCTGCAGAAACCATTCTTTAAGCCCAAAAGCACTTCCAGAGAGAGCAGAGCATCCCCAGGCTGCTGGCTCAGCAAGTTCACTGTGCTCAATCTCAGGAAGTGAGGATAAGAGCAGTGCCTGGAGAGTGCCTGGTGCTGAGCTGAGGGTTTCTGAACACATTAAAGCGGGGAGCATGGACCGGGCCTCAGGAGGGGTGTTGAACATCCCTAGGCAGAGGAGTCTAGCTTCCTGGGAAAAGATATCAGGTTAAGCACACACATGTCCTCTGGAATAAGATAATCTTTCTGATCACACACTATACACACACAAAAGCCTGCTC 5mml0: chrl5:78224841-78225364 13 GCCCTCTAGGCCACCTGACCAGGTCCCCTCAGTCCCCCCCTTCCCACACTCCCACACTCAGCCCCCCTCCCCCCCCCCCGACCCCTGCAGGATTATCCTGTCTGTGTTCCTGACTCAGCCTGGGAGCCACCTGGGCAGCAGGGGCCAAGGGTGTCCTAGAAGGGACCTGGAGTCCACGCTGGGCCAAGCCTGCCCTTTCTCCCTCTGTCTTCCGTCCCTGCTTGCGGTTCTGCTGAATGTGGTTATTTCTCTGGCTCCTTTTACAGAGAATGCTGCTGCTAATTTTATGTGGAGCTCTGAGGCAGTGTAATTGGAAGCCAGACACCCTGTCAGCAGTGGGCTCCCGTCCTGAGCTGCCATGCTTCCTGCTCTCCTCCCGTCCCGGCTCCTCATTTCATGCAGCCACCTGTCCCAGGGAGAGAGGAGTCACCCAGGCCCCTCAGTCCGCCCCTTAAATAAGAAAGCCTCCGTTGCTCGGCACACATACCAAGCAGCCGCTGGTGCAATCT Mousemml0: chrl5:78241348-78241856 14 GTGTTCTTCCCTTCCCCTTTGGACCCCCGAGACAAGCCAATAAAATACTCGGCAGGGTGGCTTCTCTCCTTTTTTTGCCAGTAATAAACAGACTCAGAGCAAGTTAAGGGTCTGGTCCAAGGTCATGGCTGGGATCAGTGACAGAGCCCAGAAGAGAACCTGAGACTTCTTGCTGAGCCAAGCTGGAGAGGACAGAAAGGAATGCGTCTACTCCATGCATGACCCTCTGCCAGCTTTGCTCCTTCCTAAGGGACCATGAACGATATGTGCACACCGCTCATACGTATGTGCACACCTGCAAGAG Mousemml0: chr9: l07340928-107341325
Petition 870190126085, of 11/29/2019, p. 42/153
38/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationGAGGCATCCCATGTACACCTATGAGACGCACAGAGAAACATATATGTAGCCATAGGCTAGAAATTCTTTCTCTTTCTAGGTCTGCCCCTCTGCA15 GGACCACTCAGTGTACACGGAATGTAGAATTGAGTCTGCCATTGGTCTTCCCTCAAAGTCTTGGAGGCTTGGGACTGATATTGGGAGCATCTGGGCAGAGAAGGCCACAAAGACAGGGTGGTTTTTCTACACTGGGACATACTCGTGAGCATGCACAGAGGCGTGTCCCCAACTTCCCTGTCACCCCTGTCCTCTGCCGGCTAGAGGGGATGCGGGGGTGGACATATGCTGCTATTGGGCAGATATCACATGTTAAGAGGTGGGGGGGGGCTCAAGAGGCGGAGGGCTAGGAGCATCCCATGGGGAGAGGTTCTGGTTTTCTTGCTGCCTCTAGCTGCTATAAATACGTTAGCACTTGAGCAACTGGAAAGCTCTGAGTAATTTAGGATGCACAAAGCTGTAATTTAACTCCAGCATCTCAGTGTGCGAGAGCATTAAAGATGTAATTAAGATGTTTACACAAAGAGATTGGAGTCTGTGACACTTGGGGTGCAAAACCCCAGGAAGGGACACAATGGGTGAGGTGAGGATCTGTGGGAGGCCTGGGGACAGTCACTTGGATCCCAGCTATGAGATGGCAGGCCACCCAGCTGTTTCTCCTTGGAAATGTTTTGGCCTGGGGGTTGGGGGTGGGGCATCACACTTTGATATGGAGATGGGGCAACAAAGCCTGCAATATCTGGGGGTGGAGAGGTCAAGTGGATGGAGTCTTTTGAGATCATGTCAGGAAGAGGGCTCGATCCCCCAAAATCATGGTGACATATGGTGTCTCGGGGTTCACAGGAGCTATGTCTAAAATACAAAAGTAAA Mousemml0: chr9: l07349227-107350036 16 TCTGCAGAAGCCTGCCATTCCACCATTTAAACCTGTGACTCCAGGCCTTAAGCCTGTTGAAGGTCGAGTCCCAGAAGGGTCATATGTGCAACTGCCTAGGGAGAGTTCCCACTCGCAGGGCCAAGAGGAGTCCCCCGGTCTGAGGTGTGGGGGCGGGGACGTGCACTGGGCGCTGGGACCACGGCTGGGGCTCAGGACTCGC Mousemml0: chr9: l07399438-107399639 17 TGCCTCAGTTTCTTCGCCTAGAAAGCCGGGTCTAAGGGTACATGCCCTGATTCTTTTCTGGGGTGTCTCGAATTTTAAACAACACATACTGTTCTGGGCTGATGACAAGAGGAAGTACTGGTCGGTGGCTGATGGACATCCACCATGGTGGCAACTGGAGGGAGGGGGAACGG Mousemml0: chr9: l07443292-
Petition 870190126085, of 11/29/2019, p. 43/153
39/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationACGTTGAAACCCTGCCCTCCTGGAATCTGTCGCATGCACGCACGTTGACAATGCTTGGCACTGGGGACAGGCTGGGATGGATGGAGCGGAGCGTGAGGAGGAGTGGGCATGCAGGCCCGAGTGTCTGTTTTGCTGATTGCTCCTTTTGCTTTCAAGGAGATTAAACTATTTTTAGTCCATGCCTACTGCTGGTGAGACGCTGGAGGAAGCCTTTCCATCGTTGAGATTTTCTGGAAGCTGCCAAGTGTGGTCTTCAGCTCAATTCTGGGAGCCTCCCAGAGTGGGAGGGAGGAACATTTCCATCTGGGGGCTTCGGGGACAGGCTAAGATCTTCCCTGGGGTCCTTGCTGCGCTGGCCTCCTCAAACCACGCTGCCTCGGCCTGCATAAAGCAGTAATCTGATGTGCCCGATGTTTGTAACGCTGTGTTTAAAAAAAGTAATTTATTTTCTAATTATTCCTTGTCTTGCATAACCATGCATTGCCAAAGTGTCGCTATTTAAAATATTTATCTCTCCACGCCGCAGGAGCAGCTCTGGAGCGTGGAGGGGGAAGAAATAAAAGTCCGCGTGCCAGTCGCAGGCATATTACTTTGACTCGTCCTGGTGGCTTTGACGTCTCCCTGTAAATACATTTATTTTTCATTAGGACGTTTCTGAGCTTGTGGCCCCCGGAGAGCGGAGTGATTACGCTGTTCATCTGCAAGCGATGCAATAGAGGGGTACTCGCAGAATGACTTCCGCCCAGAGCATCCTGCGCCTGTCT 107444228 18 TAAAATACCTTATTTTTTTCCAGTCTCTAAACTGCTAATCTCCCA GGCTAAGGGATTCTGGGACAAAGGCAAGGCCTGGAAGTGGAA ATCTGTAAAATTAGCTTCAGCGGTATTAGTGTTTGCAGTTGAAG ATTGAAAAACTGCTTTCCCAGGGCCTGATTGGAGGCTCCACTCT CCTCCAGGAAGAGGCAAGGACTCTGGGCTGGCACTGAGGACAA ATCCTGGGAGGCTGCTATGGGGCCTGGGAGCCAGGCTGCCTTG TGCTAGAGGCCTAGAGAGTGTCTGTGTCCCAAGTCCCAAGCTA CCCCCAGCAGCTAACAGCTTTTCCAGTTCTCAGGCACAGCAGGT GCCAAGATCACGCTCTGGAGTCCAGCTGGGCCCCTTCCTCTTCT _{TTTTTTTTTTTTTTTTTTAAGACCTCCTGGACACTGTTCCTCTCCC} CCCCCCCGTGACCCCCCCCCTCAGTTCTCAAACACGTGAGGGTT GGGGGAGGGTTCCACAGCCAGAGAGAGGGGCCAGCTCTGGTGC CTGTGGGTACGCCCGCCCGTATGGCCCATCAGGCCTCTTGTGTG CTTGATTGCCTCTGATTGGCTGCAGCTGAATTCAGCAAAAGCTA Mousemml0: chr9: l07444825-107445746
Petition 870190126085, of 11/29/2019, p. 44/153
40/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationTTATTTGCCCTTGATGAGCCAATCAGATGGCCTCATTGGCCATTCAGAGCAGGCACCGGAACCTGAGGGTGGGGTGGGGGGTGGGGGATGGAGATGGGACTCAGTGAGGGGGTGGGAAGCTCTAAAACAGATGCAGGACCTGAGCCTGTCTGTGTCCACCACGACCTTCACACAGGTCACACCCCCTTCCCCTGACTTGTCACCCCAAACCAGGGCTTGTTGCCCAACCCCACCTCACAATTCCCTCACTCTGTAACACCTTTCCATATACCTCTGCATGTCTAAACCCAAGACTTGCTCTATGAAATC19 AGACCCTGCTTAGCACAGCTCTTAGCGGGTCCTTTAGGGGGTCTCCCAGCGGGCCCAGTGGGAATGAGATAAGGAAGGACACAGCTGTCCATTCTCCCGTGCCTGCTAAGGAGGAAATGGGGCCGCCTTACATAATTGGGGCAATTTGTTCCACTCTTGTCCTCCTGGTATCATGGCTATCACCCCCTCCTTGCTCAGGGAGTCCTTGATTGAGCGAGAAGCTCAGGCCTCCCTCTCTCCCTCCTGCTGGGGGTTGCTGAACAGAGGGTGTAGGAGCCATAGGCTCTGTCACTGCTGAGATCTGCCAGATGTCTAGGCCAGGAGAAAATGGAAAGGGCTAAGTCACAGCATATGTGGCCACTCAGGCCTATAGCCCCAAATCTGCCTGGTAACCCATTATGTCCCCAGAGAATTTGCATGGGCGGACACCCTCATGCCGGGTCTCAGTAAGGGAAGGGGTGGGAGGCAAAAATATCCCTCCCCACCCTGAATCTCCACCCCCTCCCCCCAGAAACTGACACTTGGCCTTGTCTAAGGATGGGTTTTCCCAAAATCCTTCTGAAAAAAACAGAATTTCAAGAGTCACTCCCTCCGGGTCTCAGCCTAGAACATATGCAGTATCCCCTGACGTCCATAGGG Mousemml0: chr9: l07452080-107452718 20 AAACTGGCACAGTAATGGCGGGCTGACAGACAAGGGAGTCTGTAGCACCCGCTGCCTCCGCCCACCCCTTCTCCGAGCAATTAAAAGGTGTTTATGTGGGGCTGGCAGTGGCTTCTGCCTCCCTTCCATTACGAACATTAAGAGATCTTGACCCTTCCACTTTCCCCGCTCTTGAAAGGAGCTGCAGACACGTGGAGCCAATTAGGCGCACGCGTGGGCGCCAAGGGCCTGAGCAGCTTTTTCTCCCTGATTGCGGCGTTTACAGCTGATTATTCTCCCCTCACCCAAACAGTGCTGCTTCCTGGCAAGGTGCCACCCAGAGGAGCCGGCTGGGGGCCCCTGGGGACA Mousemml0: chr9: l07470414-107471129
Petition 870190126085, of 11/29/2019, p. 45/153
41/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationGGGGAGGACTGGATTAGTAAATGGGCATCTATCGAATGGCTTTCATATGTGTGGCTGGAAGGGAGAAGGGTAGGGCCAGGAATGGTGGCAGCAAGGGCCCAGGTAGCAATGAGGGTTCTTCTAACCCACCATTTAGGGATAGCGATCAGAAAAGGGCCCTCGAGGAGGTGACCTAAATGTGTGTAGAAGCTGACGGCCACTACACACACACACACACACACACACACACACATACACAAGCATCCTTGTCCTTGGAGTCGGTCAGCATGAGCAAGAGAAAGATGTTCCCAGTGGCCATGAGAGTGGAGCCCTCCTCCCTACTTACATCCAGGTTGGATGGCCAGGAGATCCTGAGATCCTTCAAGACTCC21 AAGCCACATCCTGGGTGGAAATATATGGCTTCAATTCCCACTCTTCCGGATGACCTCTGTGGGGAGCCCTGGCTTCACCTTGGTCCAGCTTCATCCCTTAGCCTCGCTGCCAGGAAGGCAGTGAGGTCAGAGGCTGGTGCTGGCGTG Mousemml0: chr9: l07484887-107485033 22 CCTACCTGGTGCCCGCCAACATCTGGGGGCCATCCTGGCCAGCGCCAGCGTGGTGGTGAAGGCACTGTGCGCCGTGGTACTGTTTCTCTACCTGCTTTCCTTCGCTGTGGACACGGGCTGCCTGGCCGTCA→AGGCTACCTTTTCCCACCCAACTTCTGGATCTGGACCCTGGCCACCCACGGGCTCATGGAACAGCACGTGTGGGACGTGGCCATTAGCCTGGCCACAGTGGTTGTGGCCGGGCGATTACTGGAGCCCCTCTGGGGAGCCTTGGAGCTGCTCATCTTCTTCTC Mousemml0: chr9: l07534490-107534786 23 AAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGCGCATAAATAACCCCTGCGTGCTGCACCACCTGGGGAGAGGGGGAGGACCACGGTAAAT Human;hgl9:chr2: 171672063-171672163 24 GGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGA Human;hgl9:chr2: 171672697-171672797
Petition 870190126085, of 11/29/2019, p. 46/153
42/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / Location 25 GCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATATGGGAGGGGGAGG Human;hgl9:chr2: 171672918-171673018 26 TTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCC Human;hgl9:chr2: 171673ISO-171673696 27 CAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCGCGCGCGGGGCTA Human;hgl9:chr2: 171673900-171674000 28 GAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCACCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCC Human;hgl9:chr2: 171674400-171674600 29 GCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGGGGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTGCAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTT Human;hgl9: chr2: 171674
Petition 870190126085, of 11/29/2019, p. 47/153
43/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationGCAGCCTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAGAGGCCCCGGGACGACCGAGCTG 903-171675101 30 AAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGCGCATAAATAACCCCTGCGTGCTGCACCACCTGGGGAGAGGGGGAGGACCACGGTAAATGGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGAGCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATATGGGAGGGGGAGGTTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCCCAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCGCGCGCGGGGCTAGAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCACCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCCGCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGGGGAGCCTCCGTGCHotFGGCTGCTCAGTCCCTCCGGTGTGCAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTGCAGCCTGTTTCTGCGCC Human
Petition 870190126085, of 11/29/2019, p. 48/153
44/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationGGACCAGTCGAGGACTCTGGACAGTAGAGGCCCCGGGACGACCGAGCTG31 GGAGGAAGCCATCAACTAAACTACAATGACTGTAAGATACAAAATTGGGAATGGTAACATATTTTGAAGTTCTGTTGACATAAAGAATCATGATATTAATGCCCATGGAAATGAAAGGGCGATCAACACTATGGTTTGAAAAGGGGGAAATTGTAGAGCACAGATGTGTTCGTGTGGCAGTGTGCTGTCTCTAGCAATACTCAGAGAAGAGAGAGAACAATGAAATTCTGATTGGCCCCAGTGTGAGCCCAGATGAGGTTCAGCTGCCAACTTTCTCTTTCACATCTTATGAAAGTCATTTAAGCACAACTAACTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTTGCCCAGGACAGAGTGCAGTAGTGACTCAATCTCGGCTCACTGCAGCCTCCACCTCCTAGGCTCAAACGGTCCTCCTGCATCAGCCTCCCAAGTAGCTGGAATTACAGGAGTGGCCCACCATGCCCAGCTAATTTTTGTATTTTTAATAGATACGGGGGTTTCACCATATCACCCAGGCTGGTCTCGAACTCCTGGCCTCAAGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTCAGCCACTATGCCCAACCCGACCAACCTTTTTTAAAATAAATATTTAAAAAATTGGTATTTCACATATATACTAGTATTTACATTTATCCACACAAAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGCGCATAAATAACCCCTGCGTGCTGCACCACCTGGGGAGAGGGGGAGGACCACGGTAAATGGAGCGAGCGCATAGCAAAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAGCTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGAGCTCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGGGAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATATGGGAGGGGGAGGTTGAGTACGTTCTGGATTACTCATAAGACCTTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATAATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGGAGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGCCTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGCCCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTCTCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACGAGGAAGCAGC Human
Petition 870190126085, of 11/29/2019, p. 49/153
45/133
SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationTGGAGGTGACGCCGGGCAGATTACGCCTGTCAGGGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGGCGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGCTAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAGCCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAGCGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCCCAGCAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCAGGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCGCGCGCGGGGCTAGAGTGCAAGGTGACTGTGGTTCTTCTCTGGCCAAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCTCACCTTGTCTCACCAAAGTCCCTAGTCCCCGGAGCAGTTAGCCTCTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAGACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCCGCTCGCTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGGGGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTGCAGGACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTGCAGCCTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAGAGGCCCCGGGACGACCGAGCTG32 TCAACAGGGGGACACTTGGGAAAGAAGGATGGGGACAGAGCCGAGAGGACTGTTACACATTAGAGAAACATCAGTGACTGTGCCAGCTTTGGGGTAGACTGCACAAAAGCCCTGAGGCAGCACAGGCAGGATCCAGTCTGCTGGTCCCAGGAAGCTAACCGTCTCAGACAGAGCACAAAGCACCGAGACATGTGCCACAAGGCTTGTGTAGAGAGGTCAGAGGACAGCGTACAGGTCCCAGAGATCAAACTCAACCTCACCAGGCTTGGCAGCAAGCCTTTACCAACCCACCCCCACCCCACCCACCCTGCACGCGCCCCTCTCCCCTCCCCATGGTCTCCCATGGCTATCTCACTTGGCCCTAAAATGTTTAAGGATGACACTGGCTGCTGAGTGGAAATGAGACAGCAGAAGTCAACAGTAGATTTTAGGAAAGCCAGAGAAAAAGGCTTGTGCTGTTTTTAGAAAGCCAAGGGACAAGCTAAGATAGGGCCCAAGTAATGCTAGTATTTACATTTATCCACACAAAACGGACGGGCCTCCGCTGAACCAGTGAGGCCCCAGACGTGCGCATAAATAACCCCTGCGTGCTGCACCACCTGG Human and mouse
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SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationGGAGAGGGGGAGGACCACGGTAAATGGAGCGAGCGCATAGCA AAAGGGACGCGGGGTCCTTTTCTCTGCCGGTGGCACTGGGTAG CTGTGGCCAGGTGTGGTACTTTGATGGGGCCCAGGGCTGGAGC TCAAGGAAGCGTCGCAGGGTCACAGATCTGGGGGAACCCCGGG GAAAAGCACTGAGGCAAAACCGCCGCTCGTCTCCTACAATATA TGGGAGGGGGAGGTTGAGTACGTTCTGGATTACTCATAAGACC _{TTTTTTTTTTCCTTCCGGGCGCAAAACCGTGAGCTGGATTTATA} ATCGCCCTATAAAGCTCCAGAGGCGGTCAGGCACCTGCAGAGG AGCCCCGCCGCTCCGCCGACTAGCTGCCCCCGCGAGCAACGGC CTCGTGATTTCCCCGCCGATCCGGTCCCCGCCTCCCCACTCTGC CCCCGCCTACCCCGGAGCCGTGCAGCCGCCTCTCCGAATCTCTC TCTTCTCCTGGCGCTCGCGTGCGAGAGGGAACTAGCGAGAACG AGGAAGCAGCTGGAGGTGACGCCGGGCAGATTACGCCTGTCAG GGCCGAGCCGAGCGGATCGCTGGGCGCTGTGCAGAGGAAAGG CGGGAGTGCCCGGCTCGCTGTCGCAGAGCCGAGGTGGGTAAGC TAGCGACCACCTGGACTTCCCAGCGCCCAACCGTGGCTTTTCAG CCAGGTCCTCTCCTCCCGCGGCTTCTCAACCAACCCCATCCCAG CGCCGGCCACCCAACCTCCCGAAATGAGTGCTTCCTGCCCCAG CAGCCGAAGGCGCTACTAGGAACGGTAACCTGTTACTTTTCCA GGGGCCGTAGTCGACCCGCTGCCCGAGTTGCTGTGCGACTGCG CGCGCGGGGCTAGAGTGCAAGGTGACTGTGGTTCTTCTCTGGCC AAGTCCGAGGGAGAACGTAAAGATATGGGCCTTTTTCCCCCTCT CACCTTGTCTCACCAAAGTCCCTAGTC CCCGGAGCAGTTAGCCT CTTTCTTTCCAGGGAATTAGCCAGACACAACAACGGGAACCAG ACACCGAACCAGACATGCCCGCCCCGTGCGCCCTCCCCGCTCG CTGCCTTTCCTCCCTCTTGTCTCTCCAGAGCCGGATCTTCAAGG GGAGCCTCCGTGCCCCCGGCTGCTCAGTCCCTCCGGTGTGCAGG ACCCCGGAAGTCCTCCCCGCACAGCTCTCGCTTCTCTTTGCAGC CTGTTTCTGCGCCGGACCAGTCGAGGACTCTGGACAGTAGAGG CCCCGGGACGACCGAGCTG33 ATTTACATTTATCCACACA Human
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SEQIDAT THE: Nucleic acid sequence Genomic Source / Location 34 TGCCGCTGGACTCTCTTCCAAGGAACTAGGAGAACCAAGATCC _{GTTTTTCTGCCAAGGGCTGCCCCCCCCACGCCCCCAACCCCCTC} ACCCCGATCCCCACAGAAAGAAATCTTGAGGTAGCTGGAGCTT CTTCTGTGGGTGTGACAGGACTGCCATTCTCCTCTGTAGTCTGC AGAAGCCTGCCATTCCACCATTTAAACCTGTGACTCCAGGCCTT AAGCCTGTTGAAGGTCGAGTCCCAGAAGGGTCATATGTGCAAC TGCCTAGGGAGAGTTCCCACTCGCAGGGCCAAGAGGAGTCCCC CGGTCTGAGGTGTGGGGGCGGGGACGTGCACTGGGCGCTGGGA CCACGGCTGGGGCTCAGGACTCGCGAGCTTGGATTCGGATCGG TTTGCGCGAGCCAGTAGGGCAGGCTCCGGGGTGAACGGGGACG AGGGGCGCGCGGGCACAGGCGGGCGCGTGACCGCGGCGGGGG CGCGCGGAGGCGGGCCGGCCAAGGAGAGGGAGGGAGGGAATG AGGGAGGGAGCGACAGGGGAGGGCGGCGCCGGCAGGTTGGCG GCGGCCGCTATTTGAGCGCAGGTCCCGGGCCAGGCGCTCAAAG CGCTTGGAGCCAGCGCGGCGGGGAGATCGCTGCGCGCAGCCCG CAGAGGCGCTGCGGCCAGTGCAGCCCCGGAGGCCCCGCGCGGA GAAGGAGGTGGAGAAGAGGCCGGCTTTCCGCCCGCCGCCCGCG CCCCCCCACCTCCATCCCGCCGCCGCCGTCCCCCCTCCCTCCCC GCGGCGCCGCATCTTGAATGGAAAC Mouse5chr9: 107,399, 268,107,400,067 35 GAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAG
Petition 870190126085, of 11/29/2019, p. 52/153
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SEQIDAT THE: Nucleic acid sequence Genomic Source / LocationTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA
[0060] In one aspect, the regulatory elements described here are selective for the cell type. In some cases, the regulatory elements described here are selective for PV neurons. In some cases, the regulatory elements described here are selective for PV neurons in the CNS. In some cases, the selective regulatory elements for PV cells or any regulatory elements described here can result in selective gene expression in PV neurons in relation to at least one, two, three, four, five or more non-PV cell types in the CNS. [0061] In some cases, any of the one or more regulatory elements described here are operationally linked to a transgene in an expression cassette to result in selective expression in a target cell type, for example, a PV neuron. In some cases, a regulatory element of any of the embodiments herein comprises or consists of any of (i) SEQ ID
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NOs: 1-33; (ii) a variant, functional fragment, or a combination thereof; or (iii) a sequence showing at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity to either of (i) or (ii). In some cases, a regulatory element comprises any of SEQ ID NOs: 1-32. In some cases, the sequence identity is measured by BLAST.
[0062] In some cases, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of SEQ ID NOs: 1- 32, or a functional fragment or a combination of these, or sequences having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to them, are combined to form a major regulatory element or they are operationally linked to a gene in an expression cassette. In some cases, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of SEQ ID NOs: 1-32, or a functional fragment or a combination of these, or sequences showing at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to them, are combined using a 1-50 linker sequence nucleotides. In some cases, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of SEQ ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to them, are combined without a linker sequence. In some cases, a sequence of SEQ ID NO: 33 is used as a linker between any two regulatory elements. In some cases, a linker sequence between any two regulatory elements comprises SEQ ID NO: 33 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
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50/133 sequence identity to SEQ ID NO: 33. In some cases, sequence identity is measured by BLAST.
[0063] In some cases, when two or more regulatory elements are combined or used in an expression cassette, the regulatory elements need not be adjacent or linked in an expression cassette. For example, a regulatory element can be located upstream of a transgene, while a second regulatory element and / or additional regulatory elements can be located downstream of the transgene. In some cases, one or more regulatory elements can be located upstream of a transgene. In some cases, one or more regulatory elements can be located downstream of a transgene.
[0064] In some cases, any one or more of SEQ ID NOs: 1-22, or a functional fragment or a combination thereof, or sequences showing at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to these can be combined with any one or more of SEQ ID NO: 23-30, or a functional fragment or a combination of these, or sequences showing at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to them, in order to form a major regulatory element. For example, a regulatory element comprises SEQ ID NO: 1 and SEQ ID NO: 30. A regulatory element comprises SEQ ID NO: 8 and SEQ ID NO: 30. A regulatory element comprises SEQ ID NO: 1 and to SEQ ID NOs: 23-29. A regulatory element comprises SEQ ID NO: 8 and SEQ ID NOs: 23-29. In some cases, a regulatory element comprises SEQ ID NO: 30 or any one or more of SEQ ID NOs: 23-29, or a functional fragment or a combination of these, or sequences showing at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to them.
[0065] In some cases, one or more regulatory elements in this report result in selective gene expression in a PV cell. In some cases, the regulatory elements that show selective activity or function in a type of
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51/133 target cell also shows minimal activity or function in one or more cell types outside the target, for example, non-PV cell types in the CNS, non-inhibitory neurons or excitatory neurons, non-PV cells.
[0066] In some cases, one or more regulatory elements operatively linked to a gene modulate gene expression in a cell, including, but not limited to, selective expression in a target cell type over non-target cell types. Selective expression in a target cell or cell type can be referred to as selective expression for cell or selective expression for cell type.
[0067] Selective expression generally refers to expression in a high fraction of cells of the cell type of interest (or of the target cell type) compared to other cells (or non-target cell type). Selective expression can be seen as the preferred expression in a target cell or type of target cell in relation to one or more non-target cells or non-target cell types. In some cases, the selective expression of one or more regulatory elements in this report is compared with CAG, EF la, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element that is known to direct expression and a cell or cell type without selectivity. In some cases, the selective expression of one or more regulatory elements in this report is compared with CAG, EF la, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), a non-selective regulatory element, or a expression cassette without the regulating elements.
[0068] Non-target cell types can include a different subset, subtype or cell type compared to the target cell or target cell type, or all non-target cell types. In some cases, one or more regulatory elements operationally linked to a gene result in selective expression in one type of target cell in relation to at least one type of non-target cell, or at least two, at least three, at least four, at least minus five, or more than five non-target cell types. In some cases, non-target cell types refer to all other cell types not including the target cell type. In some
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52/133 cases, non-target cell types are all other cell types within a relevant tissue or organ not including the target cell type, for example, all non-target cell types in the CNS, all types of non-target cells in the hippocampus. In some cases, a non-target cell or non-target cell type encompasses a subset or subtype of cells that is not the target cell. For example, non-PV cell types in the CNS may include GABAergic cells that express calretinin and / or somatostatin instead of parvalbumin, or all GABAergic cells that do not express parvalbumin. In some cases, cell types are distinguished by the fact that they have a cell marker, morphology, phenotype, genotype, different function and / or any other means for the classification of cell types.
[0069] The selectivity of expression driven by a regulatory element in a cell or cell type of interest can be measured in a number of ways. The selectivity of gene expression in a target cell type over non-target cell types can be measured by comparing the number of target cells that express a detectable level of a gene transcript that is operationally linked to one or more regulatory elements with the total number of cells expressing the gene. Such measurement, detection, and quantification can be done either in vivo or in vitro.
[0070] In some cases, selectivity for PV neurons can be determined using a co-localization assay. In some cases, the colocalization assay is based on immunohistochemistry. In some cases, a detectable reporter gene is used as a transgene to allow the detection and / or measurement of gene expression in a cell. In some cases, a detectable marker, for example, a fluorescent marker or an antibody, which specifically labels the target cell is used to detect and / or measure the target cells. In some cases, a co-location assay employs imaging, for example, fluorescent imaging, to determine the overlap between different fluorescent labels, for example, the overlap between a fluorescent signal indicative of a target cell and another signal indicative fluorescent
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53/133 of gene expression. In some cases, the fluorescent labels used for a co-localization assay include a red fluorescent protein (RFP), such as a tdTomato reporter gene, and a fluorescent reporter protein, such as eGFP.
[0071] In some cases, a gene operationally linked to one or more regulatory elements is a fluorescent protein, for example, eGFP or RFP, where the expression of the transgene provides a detectable signal. In some cases, tissue is labeled for eGFP or eGFP fluorescence is detected directly using a fluorescence microscope. A second fluorescent marker or reporter gene having a different fluorescence or detectable signal can be used to indicate the target cells, such as an antibody that identifies the target cells. For example, an anti-PV antibody that specifically interacts with PV neurons, can be used to produce a detectable signal that is distinguishable from the fluorescence used to measure gene expression, such as a red fluorescence or a red label. Thus, in an example where eGFP is a transgene operationally linked to one or more regulatory elements that direct selective expression in PV neurons, and where PV neurons are labeled with an anti-PV antibody, the selectivity of gene expression in cells PV is measured as the percentage of eGFP + cells that are also PV +. In such an assay, PV + cells that are also eGFP + are indicated by the overlap of both fluorescence signals, that is, an overlap of red and green fluorescence. Such measurement, analysis and / or detection can be performed by eye inspection or by a computer.
[0072] In some cases, the proportion of a cell type of interest (or target cell type) that expresses a transgene can also be measured compared to the proportion of non-target cell types (or other cells) that express the transgene in order to assess the selectivity of one or more regulatory elements operationally linked to the transgene. Similarly, selectivity of expression can also be measured by comparing the number of target cells that express a transgene operationally linked to one or more elements
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54/133 regulators with the total number of all cells expressing the transgene. In both approaches, the greater the number of target cells that express the transgene, the more selective are the regulatory elements for the target cells. In some cases, the target cells are PV neurons.
[0073] In some cases, one or more regulatory elements described here result in increased selectivity of gene expression in a PV neuron. In some cases, one or more regulatory elements described here result in increased selectivity of gene expression in PV neurons compared to non-PV cell types in the CNS. In some cases, one or more regulatory elements described here result in increased selectivity of gene expression in PV neurons compared to non-PV GABAergic cells, where non-PV GABAergic cells can be any one or more of calabrinin (CR) expressing GABAergic cells , somatostatin (SOM), cholecystokinin (CCK), neuropeptide Y (NPY), intestinal vessel polypeptide (VIP), choline acetyltransferase (ChAT), or a combination of these. In some cases, one or more of the regulatory elements described here result in increased selectivity of gene expression in PV neurons compared to at least one, at least two, at least three, at least four, at least five, or more than five subtypes Non-PV GABAergics. In some cases, one or more of the regulatory elements described here result in increased selectivity of gene expression in PV neurons compared to all other non-PV GABAergic cells, or all other GABAergic cells that do not express PV, or all other cells in the CNS that do not express PV, or all other neurons that do not express PV. In some cases, one or more of the regulatory elements described here will result in increased selectivity of gene expression in PV neurons compared to all non-PV cells in the CNS or all non-PV neurons.
[0074] In some cases, one or more of the regulatory elements operationally linked to a transgene result in selective expression of the transgene in PV cells, where the percentage of PV cells that express the transgene is a percentage
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55/133 higher than that of gene expression in PV cells in which the transgene is operationally linked to CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element , such as SEQ ID NO: 34, or a functional fragment thereof, or a sequence having at least 80% sequence identity to them. In some cases, one or more of the regulatory elements result in selective expression in PV neurons at a level that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 15 times, at least 15 times, at least 20 times, at least 25 times, or at least 50 times compared to the expression a gene operatively linked to CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element such as SEQ ID NO: 34, or a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to it.
[0075] In some aspects, a regulatory element is derived from human or comprises a sequence that is derived from human. In some cases, a regulatory element is derived from mice or comprises a sequence that is derived from mice. In some cases, a regulatory element comprises an unnaturally occurring sequence. In some cases, a regulatory element is not naturally occurring. In some cases, one or more human-derived regulatory elements are combined with another regulatory element in order to generate an unnaturally occurring regulatory element. In some cases, a human-derived regulatory element is combined with a mouse-derived regulatory element.
[0076] The term "human derived", as used here, refers to sequences that are found in a human genome (or a constructed human genome), or sequences homologous to them. A homologous sequence can be a sequence that contains a region with at least 80%
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56/133 sequence (for example, as measured by BLAST) compared to a region of the human genome. For example, a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% homologous to a human sequence is considered to be derived from human. In some cases, a regulatory element contains a human-derived sequence and a non-human-derived sequence in such a way that the total regulatory element has low sequence identity with the human genome, although part of the regulatory element has 100% human identity. sequence (or local sequence identity) to a sequence in the human genome.
[0077] In some cases, a human-derived regulatory element is a sequence that is 100% identical to a human sequence. In some cases, the sequence of a cell type selective regulatory element is 100% human derived.
[0078] In other cases, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the sequence of the regulatory element is derived from human. For example, a regulatory element can have 50% of its sequence derived from human, and the remaining 50% be non-human (for example, derived from mouse or fully synthetic). As an additional example, a regulatory element that is considered to be 50% human-derived and comprises 300 bp may present a 45% global sequence identity to a sequence in the human genome, although ER 1150 base pairs may have 90% of identity (local sequence identity) with a region of similar size in the human genome.
[0079] In some cases, a sequence that is homologous to a human-derived regulatory sequence is at least 90% identical to a human sequence. In some cases, a regulatory element here comprises a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% by
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57/133 minus 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOs: 1, 23-31, and 33. In some cases, the sequence identity is measured by BLAST. When a regulatory element comprises a sequence that is homologous (for example, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity) to any one or more of SEQ ID NOs: 23-29, or a functional fragment or combination thereof, such a regulatory element results in a higher expression of an operably linked transgene (when a promoter is also present in the expression vector or cassette), compared to a vector similar without the regulating element. Such higher expression of a transgene can be seen, for example, in HEK293T or CHO cells.
[0080] In some cases, one or more regulatory elements comprise any one or more of SEQ ID NO: 23-29 combined with or used in combination with any one or more of SEQ ID NOs: 1-22 with or without a linker sequence such as SEQ ID NO: 33. In some cases, any two regulatory elements in this report are linked together using a polynucleotide linker comprising 1-50 nucleotides, such as SEQ ID NO: 33 or a variant thereof. In some cases, the linker sequence is a derivative of the human sequence. In some cases, the linker sequence is derived from mice or occurs unnaturally. In some cases, two regulatory elements are joined without a linker or without any intervening sequence. In some cases, a binder comprises 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, or 30 nucleotides. In some cases, the linker sequence is a result of a restriction, binding, PCR and / or cloning enzyme site.
[0081] In some cases, a human-derived regulatory element is combined with a mouse-derived regulatory element, such as SEQ ID NO: 32, which is a combination of SEQ ID NO: 8 (a mouse-derived sequence) with SEQ ID NOs: 23-29 (derived from human sequences). In some cases, the selective regulatory elements for the cell type are combined
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58/133 directly without an additional linker sequence. In other cases, the regulatory elements selective for the cell type are combined with one or more short ligand sequences that can be deliberate or cloning artifacts. In some cases, a linker sequence comprises 1-50 bases. For example, SEQ ID NO: 31 comprises the sequence of SEQ ID NOs: 1 and 23-29 together with an additional 19 bp of the genomic sequence (SEQ ID NO: 33) immediately following the sequence of SEQ ID NO: 1. A SEQ ID NO: 32 also includes these 19bp, but without the sequence of SEQ ID NO: 1. In other examples, the combined regulatory elements for the cell type may include short sequences, generally less than 50 bp, less than 20 bp , less than 15 bp or less than 10 bp, from a cloning plasmid or restriction enzyme recognition site.
[0082] In some cases, regulatory elements may be derived from non-coding DNA sequences. In some cases, regulatory elements derived from non-coding DNA are associated with genes, such as upstream sequences, introns, untranslated 3 'and 5' regions (RTU's) and / or downstream regions. In other cases, regulatory elements derived from non-coding DNA sequences are not associated with a gene. In some cases, the regulatory elements are derived from coding sequences. In some cases, the genomic region from which a regulatory element is derived is distinct from the genomic region from which an operably linked transgene is derived. In some cases, an ER is derived from a genomic region or location distal from the genomic region or location from which the transgene is derived (such as a naturally occurring version or an endogenous version of the transgene).
[0083] In one aspect, a regulatory element is any non-coding sequence that modulates gene expression, for example, the selectivity of expression in a target cell. In some cases, the target cell is a PV neuron. In some cases, a regulatory element is derived from a genomic sequence upstream of a transcription initiation site, a 5 'UTR sequence, an exonic sequence, an intronic sequence, or a 3' UTR sequence. In some
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In 59/133 cases, a human-derived regulatory element comprises an intronic derivative of human sequence. In some cases, a regulatory element comprises an amplifier, and its presence in an expression cassette together with a promoter increases the expression of a transgene operably linked in the target cell type (eg, PV neurons) compared to the expression of the same transgene by the promoter without the amplifier. In some cases, an amplifier increases the expression of an operably linked transgene by means of either a transcriptional mechanism, a post-transcriptional mechanism, or both. In some cases, a regulatory element comprises an amplifying sequence, a promoter sequence or a combination of an amplifying sequence and a promoter sequence. In some cases, a regulatory element comprises one or more of a human-derived amplifier sequence, a human-derived promoter sequence, an intronic human-derived sequence and / or a combination thereof.
[0084] In some cases, a regulatory element comprises one or more of SEQ ID NOs: 1-32, or a fragment or combination of these or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these. In some cases, a regulatory element comprises one or more of SEQ ID NOs: 1-22, or a fragment or combination of these or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, a regulatory element comprises one or more of SEQ ID NOs: 23-29, or a fragment or combination of these or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, a regulatory element comprises one or more of SEQ ID NOs: 30-32, or a fragment or combination of these or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, a regulatory element comprises a sequence of SEQ ID NOs: 30-32, or a
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60/133 fragment or a combination of these or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them.
[0085] In some cases, a regulatory element is derived from non-human DNA sequences or both human and non-human genomic sequences. In some cases, the regulatory elements selective for the cell type, or parts thereof, are homologous to a mammalian genomic sequence. In some cases, regulatory elements have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a mammalian genomic sequence. In some cases, a regulatory element is derived from a mouse genomic sequence. In some cases, a regulatory element, or fragments thereof, has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to a mouse genomic sequence or a non-human mammalian genomic sequence. In some cases, the sequence identity is measured by BLAST. In some cases, the regulatory elements may comprise any of SEQ ID NOs: 1-33, or a fragment or combination thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them.
[0086] In some cases, the regulatory elements selective for the cell type are short. In some cases, the size of the regulatory elements is compatible with the ability to clone a vector, for example, a viral or rAAV vector, just as the combined size of a transgene and one or more of the regulatory elements does not exceed the cloning capacity. of a vector. In some cases, the selective regulatory elements for the cell type have a length of up to about 2050 bp, 2000 bp, 1900 bp, 1800 bp, 1700 bp, 1600 bp, 1500 bp, 1400 bp, 1300 bp, 1200 bp, 1100 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, or 100 bp. In some cases,
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61/133 selective regulatory elements for the cell type have a total length of no more than about 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp , 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1010 bp, 1020 bp, 1030 bp, 1040 bp, 1050 bp, 1060 bp, 1070 bp, 1080 bp, 1090 bp, 1100 bp, 1200 bp, 1300 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, or 2000 bp. In some cases, the selective regulatory elements for the cell type have a length of about 100 bp - 1100 bp, 100 bp - 1000 bp, 100 bp - 900 bp, 200 bp - 900 bp, 200 bp - 800 bp, 300 bp - 600 bp, 400 bp - 800 bp, 500 bp - 600 bp, or 600 bp - 900 bp. In some cases, a regulatory element is between about 400 - 600 bp, 400 - 600 bp, 400 - 700 bp, 400 - 800 bp, 400 - 900 bp, 400 - 1000 bp, or 400 - 1500 bp. In some cases, a regulatory element is between about 500 - 600 bp, 500 - 700 bp, 500 - 800 bp, 500 - 900 bp, 500 - 1000 bp, or 500 - 1500 bp. In some cases, two or more regulatory elements are combined to form a selective regulatory element for the larger cell type of 1300 - 2500 bp, 1300 - 2060 bp, about 1350 bp, about 2050 bp, or about 1880 bp.
[0087] In some cases, two or more regulatory elements selective for the cell type may be combined. For example, two, three, four, five, six, seven, eight, nine, ten or more regulatory elements selective for the cell type can be combined. For example, SEQ ID NO: 30 comprises sequences of seven regulatory elements, i.e., SEQ ID NOs: 23-29, all of which are derived from the human genomic sequence. In some cases, selective regulatory elements for the cell type refer to selective regulatory elements for the PV neuron.
[0088] In some cases, a selective regulatory element for the cell type is repeated two or more times to obtain a combined regulatory element that is also selective for the cell type or has selective property for the cell type. In some cases, two or more regulatory elements with different selectivities for the cell type are combined. In some cases, a selective regulatory element for the cell type is combined with a regulatory element
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62/133 nonselective, for example, a non-selective amplifier element that drives high gene expression. For example, a regulatory promoter element with high selectivity for a target cell can be combined with a regulatory element with high expression efficiency. In some cases, one or more regulatory elements selective for the cell type are combined with one or more high efficiency regulatory elements. For example, any one or more of SEQ ID NOs: 1-32, or a fragment or combination thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these can be combined with a constitutive promoter, such as a GAD2 promoter, a human synapsin promoter, a minCMV promoter, a TATA box, a super core promoter, or an EFla promoter, or a combination of these .
[0089] In some respects, this report provides a list of regulatory elements that can be added to any gene therapy to result in selective gene expression in a target cell type, such as a PV neuron in relation to one or more types of non-target cells, such as CNS non-PV cells, including, but not limited to, excitatory cells and / or non-PV GABAergic cells.
[0090] In some cases, the regulatory elements selective for the cell type can be combined with other regulatory elements such as a high expression promoter or a sequence that increases mRNA stability. In some cases, one or more regulatory elements selective for the cell type are combined with a human, non-human, or non-mammalian sequence, for example, an hSynl promoter, CBA promoter, CMV promoter, EFla promoter, signal polyA (e.g., polyA SV40 signal), or a post transcriptional regulatory element such as the post-transcriptional regulatory element of the groundhog hepatitis virus (WPRE).
[0091] In some cases, the combined regulatory elements come from different species. The combined regulatory elements may come from different genomic regions within a species. In some cases,
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63/133 regulatory elements are derived from distal genomic sequences, for example, sequences that do not normally or naturally associate with each other or with a cell type of interest, are combined. In some cases, the individual regulatory elements used to obtain a combined regulatory element may come from different human chromosomes.
[0092] In one aspect, a regulatory element of the report comprises a functional fragment of any of SEQ ID NOs: 1-32, or a combination thereof, or sequences showing at least 80%, at least 85%, at least 90% at least 95%, or at least 99% sequence identity to them. Such a functional fragment can increase the expression of a transgene in a cassette or expression vector when compared to a cassette or similar expression vector without the regulatory element. Such a functional fragment can function as an amplifier to increase selective expression for cell type when the fragment is operationally linked to a transgene compared to a similar vector or cassette without the functional fragment. A fragment is preferably more than 30, 40, 50, or 60 bp in length.
[0093] In some cases, a selective regulatory element for PV cell or any regulatory element in this report comprises any of SEQ ID NOs: 1-32, (ii) a nucleic acid sequence having at least 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-32, (iii) a functional fragment from any sequence of (i) or (ii), or (iv) a combination of any sequence of (i) , (ii) and / or (iii). In some cases, the sequence identity is measured by BLAST. In some cases, two or more of SEQ ID NOs: 1-29, or a functional fragment or a combination of these, or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least minus 99% sequence identity to these, are used as a regulatory element to increase the selectivity of transgenic expression in PV cells compared to non-PV cells in the CNS, or with any type of target cell compared to a non-cell type target. In
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64/133 In some cases, a functional fragment is one that results in selective expression in one type of target cell in relation to one or more types of non-target cells.
[0094] In some cases, two or more copies of a regulatory element can be used to increase selective expression in a target cell, for example, two or more copies of any of SEQ ID NOs: 1-29, or a fragment or a combination of these, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them.
[0095] In other cases, one or more of SEQ ID NOs: 1-32, or a fragment or combination thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these, are operationally linked to another regulatory element, such as a promoter or amplifier, in order to further increase selective expression in a target cell. In some cases, it is possible to improve any gene therapy by adding one or more regulatory elements as described here in order to improve or increase expression from gene therapy in a target cell compared to non-target cells. In some cases, the target cell is a PV neuron or GABAergic cells that express parvalbumin.
[0996] In some respects, one or more regulatory elements (for example, any one or more of SEQ ID NOs: 1-32, or a fragment or combination thereof, or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of sequence identity to them) described here show selectivity for the cell type for a target cell type over at least one, at least two, at least at least three, at least four, at least five, or more than five non-target cell types. In some cases, a regulatory element directs selective expression or preferential expression in a target cell subtype in relation to at least one, at least two, at least three, at least four, at least five, or more than five non-target subtypes , or all other known cell subtypes. For example,
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65/133 GAB Aergic cells comprise different subtypes, including PV cells. In some cases, the target cell type is a PV cell. In some cases, one or more regulatory elements are selective for PV cells in relation to at least one, at least two, at least three, at least four, at least five, or more than five types of non-target cells. In some cases, one or more regulatory elements are selective for PV cells over all other known types of CNS cells.
[0097] In some cases, any one or more of SEQ ID NOs: 1-32, or a fragment or combination thereof, or sequences showing at least 80%, at least 85%, at least 90%, at least 95% , or at least 99% sequence identity to them, can be combined with any one or more of SEQ ID NOs: 1-32, or a fragment or combination thereof, or sequences having at least 80%, at least 85% at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, such combined regulatory elements are linked using a 1-50 nucleotide linker. In some cases, such combined regulatory elements are not linked.
[0098] In some cases, one or more of the regulatory elements described here, when operationally linked to any transgene (for example, a reporter or therapeutic transgene), triggers selective expression or preferential expression in at least one type of target cell at a level that is statistically significantly higher than the expression triggered by CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element (for example, SEQ ID NO : 34, or a fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these) when operationally linked to the same transgene , or by the same construct without the regulatory elements. In some cases, statistically significantly higher means that the regulatory elements drive selective expression in the target cell type at a level that is at least 1.1, 1.2,
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66/133
1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times the level of expression by CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or an element non-selective regulator when operationally linked to the same transgene, or by the same construct without the regulatory elements. In some cases, such cell type selective expression is assayed using a colocalization assay as described here. In some cases, the target cell type is a parvalbumin cell. In some cases, such a co-localization assay is conducted using an anti-PV antibody. In some cases, such a co-localization assay is conducted using a PV-Cre mouse as described here. In some cases, the non-selective regulatory element is SEQ ID NO: 34, or a fragment thereof, or the sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 % sequence identity to this.
Expression cassettes [00991 The terms "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to a polynucleotide molecule or nucleic acid sequence. In some cases, an expression cassette comprises one or more of the regulatory elements described herein operably linked to a transgene. In some cases, an expression cassette comprises one or more regulatory elements. In some cases, an expression cassette comprises one or more regulatory elements selective for the cell type described here. In some cases, an expression cassette comprises one or more of the selective regulatory elements for PV cell described here. In some cases, the expression cassette additionally comprises a promoter. In some cases, an expression cassette comprises one or more sequences of SEQ ID NOs: 1-32 and / or any combination thereof. In some cases, an expression cassette comprises one or more of SEQ ID NOs: 1-32, (ii) a nucleic acid sequence having
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67/133 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-32, (iii) a functional fragment of the sequence of (i) or (ii), or (iv) a combination of any sequence of (i), (ii) and / or (iii). In some cases, the sequence identity is measured by BLAST. In some cases, a regulatory element is located upstream of a transgene in an expression cassette. In some cases, a regulatory element is located downstream of a transgene in an expression cassette. In some cases, an expression cassette further comprises a promoter, for example, a hSynl promoter, CBA promoter, a CMV promoter, an EFla promoter, a polyA signal (e.g., polyA SV40 signal), or a post transcriptional regulatory element such as the post transcription regulatory element of the groundhog hepatitis virus (WPRE).
[0100] In some respects, one or more of the regulatory elements described here are operationally linked to a transgene in an expression cassette. In some cases, gene therapy comprises an expression cassette comprising a transgene operatively linked to one or more, two or more, three or more, four or more, or five or more regulatory elements in this report to result in selective expression of the transgene in a type of tissue or target cell, such as PV neurons. In some cases, an expression cassette comprises one or more regulatory elements selective for PV cell or one or more of the regulatory elements described herein operably linked to a transgene, for example, a reporter gene, eGFP, SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, STX BP1, a DNA binding protein, or a variant or fragment thereof, or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them.
[0101] In some cases, an expression cassette is adapted for application via gene therapy. In some cases, an expression cassette is either a linear construct or a circular construct. In some cases, an expression cassette is part of a plasmid, vector, viral vector, or rAAV.
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68/133 [0102] In some cases, gene therapy is administered directly to the CNS of an individual needing such treatment or systemically by injection and / or infusion. In some cases, such an individual has been diagnosed with a disease or condition associated with a haploinsufficiency or a genetic mutation, such as a haploinsufficiency or a mutation in any of the following genes: SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, or STX BP1. In some cases, the individual is at risk or has Dravet's syndrome, Alzheimer's disease, epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability and / or seizures. In some cases, such gene therapy is applied using a virus or viral vector, such as rAAV. In some cases, an AAV serotype with a tropism for CNS cells and / or the ability to cross the cerebral blood barrier, such as AAV9 or a variant thereof, is used.
[0103] In some cases, one or more regulatory elements (for example, one or more of SEQ ID NOs: 1-32, or a fragment or combination thereof, or sequences showing at least 80%, at least 85%, at least minus 90%, at least 95%, or at least 99% sequence identity to them) operably linked to a transgene in an expression cassette results in selective gene expression in PV cells compared to non-PV cells in the CNS, or in comparison with a control element, such as a CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element (for example, SEQ ID NO: 34, or its functional fragment, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to it). In some cases, the regulatory elements result in the fact that at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of all cells expressing the transgene are PV neurons. In some cases, the regulatory elements result in selective gene expression in PV neurons that is about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 7.5 times, 8 times , 9 times, or 10 times higher than expected for
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69/133 the natural distribution of PV neurons in the CNS. In some cases, a regulatory element directs selective expression in PV cells, where the percentage of PV cells that express the transgene is a percentage that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times greater than the expected distribution of PV cells in the CNS, or at least 1-5 %, 5% -10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50- 55%, 55-60%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, or 90-95% greater than the expression in PV cells when the transgene is operationally linked to CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element having a sequence of SEQ ID NO: 34, or a functional fragment thereof, or a sequence showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity sequence thereto, and as measured in an immunohistochemical co-location assay. In some cases, the regulatory element in an expression cassette, or such regulatory element is used in an expression cassette, resulting in selective gene expression in PV cells, or PV cells in the CNS, or PV neurons, where about 10% , 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells expressing the transgene are positive for PV.
[0104] In some cases, an expression cassette or gene therapy comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more regulatory elements as described in TABLE 1, for example, SEQ ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these.
[0105] In some cases, one or more selective regulatory elements for PV cell or one or more of the regulatory elements described here are operationally
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70/133 attached to any transgene in an expression cassette. In some cases, the expression cassette is gene therapy. In some cases, the expression cassette is part of a vector or a plasmid, for example, a viral vector or an rAAV vector. In some cases, the expression cassette is part of AAV1, AAV8, AAV9, or AAVDJ or a variant or hybrid of these. In some cases, the expression cassette comprises one or more regulatory elements selective for PV cell or one or more of the regulatory elements described here operationally linked to a transgene, where the transgene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1 , KAV3,2, KV3,3, STX BP1, of the DNA binding protein (for example, transcription modulator of an endogenous gene), or a variant or functional fragment thereof, or a sequence having at least 80%, at least at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, such regulatory elements increase the selective expression of the transgene in PV neurons compared to non-PV cell types in the CNS. In some cases, such regulatory elements selectively increase the expression of the transgene in a type of target cell, such as a PV neuron. In some cases, the target cell type is a PV cell.
[0106] The techniques contemplated here for somatic cell gene therapy include application using a viral vector (eg retroviral, adenoviral, AAV, helper-dependent adenoviral systems, hybrid adenoviral systems, herpes simplex virus, smallpox virus , lentivirus, and Epstein-Barr virus), and non-viral systems, such as physical systems (naked DNA, DNA bombardment, electroporation, hydrodynamics, ultrasound, and magnetofection), and chemical system (cationic lipids, different cationic polymers, and lipid polymers).
[0107] The ability to clone vital expression vectors or vectors is a particular challenge for the expression of large transgenes. For example, AAV vectors typically have a packaging capacity of ~ 4.8 kb, lentiviruses typically have a capacity of --8 kb, adenoviruses typically have a capacity of ~ 7.5 kb and alphaviruses typically
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71/133 have a capacity of ~ 7.5 kb. Some viruses may have greater packaging capabilities, for example, herpesvirus may have a capacity of> 30 kb and vaccinia may have a capacity of ~ 25 kb. The advantages of using AAV for gene therapy include low pathogenicity, very low frequency of integration into the host genome, and the ability to infect cells in division and non-division.
[0108] In order to focus on restrictions on the size of certain viral vectors or to increase expression from viral vectors, this report contemplates the use of regulatory elements that are shorter than 2.5 kb, 2 kb,
1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, or 110 bp, but which are at least 10 bp, 50 bp or 100 bp in length. In some cases, the size of the combined regulatory element is about 2500 bp, 2000 bp, 1500 bp, 1400 bp, 1300 bp, 1200 bp, 1100 bp, or 1000 bp. In some cases, each combined regulatory element has a total length of about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp , 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, or 2500 bp. In some cases, the size of a combined RE has a total length of about 200 bp -3000 bp, 200 bp -2500 bp, 200 bp-2100 bp, 500 bp -2500 bp, 1000 bp2500 bp, 1500 bp -2500 bp , 1500b-2000 bp, or 2000 bp-2500 bp.
[0109] In some cases, a regulatory element of the report is preferably (i) one that selectively directs expression in a cell type of interest, such as PV cells; (ii) includes a human sequence derivative, and (iii) is less than 2.5 kb, 2 kb, 1.5 kb, or 1 kb.
[0110] Also included here are expression cassettes, which can be circular or linear nucleic acid molecules. In some cases, an expression cassette is applied to cells (for example, a plurality of different cells or cell types including target cells or cell types and / or non-target cell types) in a vector (for example, a vector expression). A vector can be an integral or non-integral vector, with respect to the vector's ability to
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72/133 integrate into the expression cassette and / or transgene in a cell genome. Both an integrating and a non-integrating vector can be used to apply an expression cassette containing a transgene operably linked to a regulatory element. Examples of vectors include, but are not limited to, (a) non-viral vectors such as nucleic acid vectors including linear oligonucleotides and circular plasmids; artificial chromosomes such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs or PACs); episomal vectors; transposons (for example, PiggyBac); and (b) viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, and AAV vectors. Viruses have several advantages for the application of nucleic acids, including high infectivity and / or tropism for certain target cells or tissues. In some cases, a virus is used to apply a nucleic acid molecule or expression cassette comprising one or more regulatory elements, as described herein, operably linked to a transgene.
[0111] The preferred characteristics of gene therapy with viral vectors or gene application vectors include the ability to be reproducible and propagated stably and purified for high titers; to mediate targeted application (for example, to apply the transgene specifically to a tissue or organ of interest without disseminating the vector to other parts or off-target application); and to mediate gene application and / or transgenic expression without inducing harmful side effects or off-target effects. In order to avoid harmful side effects, targeted expression or selective expression for tissue / cell type can be obtained by placing the transgene under the control of a selective regulatory element for the cell type, for example, one or more of the SEQs ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences having at least 80% ·, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity a these, an amplifier, promoter, stability element, RTU, or a combination of these. For example, viral particles containing a viral vector can be
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73/133 designed to infect many different cell types, but the transgene expression is increased and / or optimized in one cell type of interest (eg PV neurons), and the transgene expression is reduced and / or minimized in others non-target cell types (for example, CNS non-PV cells). Differential expression of the transgene in different cell types can be controlled, engineered or manipulated by using different transcription factors or regulatory elements that are selective for one or more cell types. In some cases, one or more regulatory elements, such as a promoter or amplifier, or a combination of these, are operationally linked to a transgene to direct the selective expression of the transgene in the tissue or cell. In some cases, one or more regulatory elements used in gene therapy or a vector selectively target gene expression in a cell type, that is, they confer selective gene expression in a target cell, cell type, or tissue and / or do not target gene expression in one or more (for example, at least one, two, three or four) non-target cells or non-target cell types. In some cases, one or more regulatory elements operatively linked to a transgene increase the selective expression of the transgene in a target cell, cell type, or tissue, while the one or more regulatory elements suppress transgenic expression in non-target cells, type of non-target cell, or non-target tissue, or confer significantly lower, minimal or statistically lower gene expression in one or more non-target cells, non-target cell types, or non-target tissue.
[0112] Several serotypes of AAV, a non-pathogenic parvo virus, have been engineered for the purpose of gene application, some of which are known to have tropism for certain tissues or cell types. Viruses used for various gene therapy applications can be engineered for poor replication or to have low toxicity and low pathogenicity in an individual or a host. Such virus-based vectors can be obtained by deleting all or some of the coding regions of the viral genome, and leaving those sequences intact (for example, repeat sequences)
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74/133 inverted terminal) that are necessary for functions such as packaging the vector genome into the virus capsid or integrating the nucleic acid vector (e.g., DNA) into the host chromatin. An expression cassette comprising a transgene, for example, can be cloned into a viral backbone such as a modified or engineered viral backbone that does not contain viral genes, and used in conjunction with additional vectors (for example, packaging vectors), which can, for example, when cotransfected, produce recombinant vector viral particles. In some cases, an AAV serotype that can cross the cerebral blood barrier or infect CNS cells is preferred. In some cases, AAV9 or a variant thereof is used to apply an expression cassette from this report, comprising one or more selective regulatory elements for PV operationally linked to a transgene. [0113] An advantage of applying the expression cassettes in this report using gene therapy, for example, rAAV, as described here, is that such therapies can provide more targeted and sustained therapeutic effects over time. In addition, the viral gene used in therapies can be engineered to display tropism for a cell or tissue type of interest in relation to non-target cell types or non-target tissues. For example, the one used in therapies can be engineered to infect and apply a payload or a therapeutic agent, for example, a transcription modulator or a transgene, in one or more regions, tissues, or cell types in the CNS (for example , PV cells), while having minimal effects on non-target tissue or cell types (eg non-CNS tissue or cell type, non-CNS non-PV cells). In some cases, the viral gene used in therapies may be engineered to apply a transgene across the brain blood barrier and / or target a specific region or tissue in the CNS (eg, hippocampus) or a cell type in the CNS, for example , PV cells.
[0114] In some cases, an AAV vector or an AAV viral particle, or virion, used to apply one or more regulatory elements and a transgene to a cell, cell type, or tissue, in vivo or in vitro, is preferably deficient in
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75/133 replication. In some cases, an AAV virus is engineered or genetically modified in such a way that it can be replicated and generate virions only in the presence of auxiliary factors.
[0115] In some cases, the expression cassette is designed for application by an AAV or a recombinant AAV (rAAV). In some cases, an expression cassette is applied using either a lentivirus or a lentiviral vector. In some cases, larger transgenes, that is, genes that exceed the AAV cloning capacity, are preferably applied using a lentivirus or a lentiviral vector.
[0116] The AAV used in the compositions and methods described here can be of any serotype (for example, AAV1, AAV2, AAV5, AAV8, AAV9, and AAVDJ), including hybrid or chimeric AAV serotypes. In some cases, AAV is used to apply and / or express a transgene operatively linked to one or more regulatory elements that are selective for PV neurons compared to non-PV cells in the CNS. In some cases, an AAV with a high tropism for CNS cells and / or that crosses the cerebral blood barrier is used. In some cases, AAV1, AAV8, AAV9, and / or AAVDJ are used to apply an expression cassette described here.
[0117] In some cases, an expression cassette comprises one or more regulatory elements selective for PV cell or one or more of the regulatory elements described here operationally linked to a transgene that is known to be insufficiently expressed in vivo, such as in a disease or condition associated with haploinsufficiency in the gene. In some cases, the transgene is a voltage-activated ion channel (for example, a sodium ion channel or a potassium ion channel), a neurotransmitter regulator, or a subunit or functional fragment thereof. In some respects, the transgene is a DNA-binding protein, an ion channel, a neurotransmission regulator, or an ion channel subunit or neurotransmission regulator. In some cases, the transgene is a DNA-binding protein that comprises one or more zinc fingers. In some cases, the protein of
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76/133 DNA binding comprises a Cas9 domain, a Cas family protein, deactivated Cas9 nuclease (or dCas9), a dCas family protein, or a transcription activator-like effector (TALE). In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase etc.) where the DNA cleavage domain or nuclease domain has been disabled, for example, a disabled Cas nuclease (dCas), an effected transcription activator-like nuclease, or a nuclease-deactivated zinc finger protein. In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene comprises a gene editing protein, for example, the Cas, Cas9 protein.
[0118] In some respects, the transgene is an ion channel driven by voltage or a subunit thereof, such as SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, or KV3.3, or a fragment functional or variant thereof. In some cases, the transgene is an alpha subunit of a sodium ion channel. In some cases, the transgene is a beta subunit of a sodium ion channel. In some respects, the neurotransmitter regulator is STXBP1 or a functional fragment or variant thereof.
[0119] In some ways, an expression cassette is applied as a viral vector, such as AAV. In some ways, AAV is AAV1, AAV8, AAV9, AAVDJ, scAAVl, scAAV8, or scAAV9. In some respects, gene therapy comprising an expression cassette from this report is administered to an individual in need of such treatment (for example, a human patient, a mammal, a transgenic animal, or an animal model). In some cases, the individual in need of such treatment has symptoms of, has been diagnosed with, or is at risk of developing Alzheimer's disease, Dravet's syndrome, epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability, and / or seizures. In some cases, the individual in need of such treatment presents
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77/133 insufficient gene expression or a mutation in any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, and STX BP1. [0120] In some cases, gene therapy, such as with rAAV9, is used to apply an expression cassette comprising one or more regulatory elements selective for PV cell or one or more of the regulatory elements described here operationally linked to a transgene, where the transgene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, STX BP1, a DNA binding protein, or a functional fragment thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, or 99% sequence identity to any of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, STX BP1, a DNA-binding protein, or fragment functional of these. In some cases, the transgene comprises a sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 37-43, or a functional fragment thereof, as provided in TABLE 2 below.
[0121] In some cases, the transgene is any one of SEQ ID NOs: 36-43, or a functional fragment thereof, or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, one or more of the regulatory elements described here are operationally linked to any of SEQ ID NOs: 3643 in an expression cassette, or a functional fragment thereof, or sequences showing at least 80%, at least 85%, at least at least 90%, at least 95%, or at least 99% sequence identity to them.
TABLE 2: List of amino acid sequences described here.
SEQIDAT THE. Gene Amino Acid Sequence 36 eGFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
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SEQIDAT THE. Gene Amino Acid Sequence ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL 37 SCN1B MGTLLALVVGAALVSSAWGGCVEVDSDTEAVYGMTFKILCISCKRRSETTAETFTEWTFRQKGTEEFVKILRYENEVLQLEEDERFEGRVVWNGSRGTKDLQDLSIFITNVTYNHSGDYECHVYRLLFFDNYEHNTSVVKKIHLEVVDKANRDMASIVSEIMMYVLIVVLTIWLVAEMVYCYKKIAAATEAAAQENASEYLAITSESKENCTGVQVAE 38 SCN2B MHRDAWLPRPAFSLTGLSLFFSLVPPGRSMEVTVPATLNVLNGSDARLPCTFNSCYTVNHKQFSLNWTYQECNNCSEEMFLQFRMKIINLKLERFQDRVEFSGNPSKYDVSVMLRNVQPEDEGIYNCYIMNPPDRHRGHGKIHLQVLMEEPPERDSTVAVIVGASVGGFLAVVILVLMVVKCVRRKKEQKLSTDDLKTEEEGKTDGEGNPDDGAK 39 SCN1A MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMIEQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEVIIDKPATDDNGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIA
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SEQIDAT THE. Gene Amino Acid Sequence MDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKL AKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYK DCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVA GQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEM NNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNK KDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSF INNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGS TVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQ WWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTML EYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSL TANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSI MNVLLVCLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNNHTD CLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIMYA AVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQK KKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFD FVTRQVFDISIMILICLNMVTMMVETDDQSEYVTTILSRINLVFIVLFTG ECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFR VIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILN SKPPDCDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVI LENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFMEFEKLSQFA AALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESG EMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRR HLLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTM STAACPPSYDRVTKPIVEKHEQEGKDEKAKGK 40 STXBP1 MAPIGLKAVVGEKIMHDVIKKVKKKGEWKVLVVDQLSMRMLSSCCKMTDIMTEGITIVEDINKRREPLPSLEAVYLITPSEKSVHSLISDFKDPPTAKYRAAHVFFTDSCPDALFNELVKSRAAKVIKTLTEINIAFLPYESQVYSLDSADSFQSFYSPHKAQMKNPILERLAEQIATLCATLKEYPAVRYRGEYKDNALLAQLIQDKLDAYKADDPTMGEGPDKARSQLLILDRGFDPSSPVLHELTFQAMSYDLLPIENDVYKYETSGIGEARVKEVLLDEDDDLWIALRHKHIAEVSQEVTRSLKDFSSSKRMNTGEKTTMRDLSQMLKK
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SEQIDAT THE. Gene Amino Acid Sequence MPQYQKELSKYSTHLHLAEDCMKHYQGTVDKLCRVEQDLAMGTDAEGEKIKDPMRAIVPILLDANVSTYDKIRIILLYIFLKNGITEENLNKLIQHAQIPPEDSEIITNMAHLGVPIVTDSTLRRRSKPERKERISEQTYQLSRWTPIIKDIMEDTIEDKLDTKHYPYISTRSSASFSTTAVSARYGHWHKNKAPGEYRSGPRLIIFILGGVSLNEMRCAYEVTQANGKWEVLIGSTHILTPTKFLMDLRHPDFRESSRVSFEDQAPTME 41 Kv3, l MGQGDESERIVINVGGTRHQTYRSTLRTLPGTRLAWLAEPDAHSHFDYDPRADEFFFDRHPGVFAHILNYYRTGKLHCPADVCGPLYEEELAFWGIDETDVEPCCWMTYRQHRDAEEALDSFGGAPLDNSADDADADGPGDSGDGEDELEMTKRLALSDSPDGRPGGFWRRWQPRIWALFEDPYSSRYARYVAFASLFFILVSITTFCLETHERFNPIVNKTEIENVRNGTQVRYYREAETEAFLTYIEGVCVVWFTFEFLMRVIFCPNKVEFIKNSLNIIDFVAILPFYLEVGLSGLSSKAAKDVLGFLRVVRFVRILRIFKLTRHFVGLRVLGHTLRASTNEFLLLIIFLALGVLIFATMIYYAERIGAQPNDPSASEHTHFKNIPIGFWWAVVTMTTLGYGDMYPQTWSGMLVGALCALAGVLTIAMPVPVIVNNFGMYYSLAMAKQKLPKKKKKHIPRPPQLGSPNYCKSVVNSPHHSTQSDTCPLAQEEILEINRAGRKPLRGMSI 42 Kv3.2 MGKIESNERVILNVGGTRHETYRSTLKTLPGTRLALLASSEPQGDCLT AAGDKLQPLPPPLSPPPRPPPLSPVPSGCFEGGAGNCSSHGGNGGNGG SDHPGGGREFFFDRHPGVFAYVLNYYRTGKLHCPADVCGPLFEEELA FWGIDETDVEPCCWMTYRQHRDAEEALDIFETPDLIGGDPGDDEDLA AKRLGIEDAAGLGGPDGKSGRWRKLQPRMWALFEDPYSSRAARFIAF ASLFFILVSITTFCLETHEAFNIVKNKTEPVINGTSPVLQYEIETDPALTY VEGVCVVWFTFEFLVRIVFSPNKLEFIKNLLNIIDFVAILPFYLEVGLSG LSSKAAKDVLGFLRVVRFVRILRIFKLTRHFVGLRVLGHTLRASTNEF LLLIIFLALGVLIFATMIYYAERVGAQPNDPSASEHTQFKNIPIGFWWA VVTMTTLGYGDMYPQTWSGMLVGALCALAGVLTIAMPVPVIVNNFGMYYSLAMAKQKLPRKRKKHIPPAPLASSPTFCKTELNMASNCTQSDT CLGKENRLLEHNRSVLSGDDSTGSEPPLSPPERLPIRRSSTRDKNRRGE TCFLLTTGDYTCASDGGIRKASTLEPMESTAQTKGDTRPEAHPTCAGG
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SEQIDAT THE. Gene Amino Acid Sequence 43 Kv3.3 MLSSVCVSSFRGRQGASKQQPAPPPQPPESPPPPPLPPQQQQPAQPGPA ASPAGPPAPRGPGDRRAEPCPGLPAAAMGRHGGGGGDSGKIVINVGG VRHETYRSTLRTLPGTRLAGLTEPEAAARFDYDPGADEFFFDRHPGVF AYVENYYRTGKEHCPADVCGPEFEEEEGFWGIDETDVEACCWMTYR QHRDAEEALDSFEAPDPAGAANAANAAGAHDGGLDDEAGAGGGGL DGAGGELKRLCFQDAGGGAGGPPGGAGGAGGTWWRRWQPRVWAL FEDPYSSRAARYVAFASLFFILISITTFCLETHEGFIHISNKTVTQASPIP GAPPENITNVEVETEPFLTYVEGVCVVWFTFEFLMRITFCPDKVEFLKS SLNIIDCVAILPFYLEVGLSGLSSKAAKDVLGFLRVVRFVRILRIFKLTR HFVGLRVLGHTLRASTNEFLLLIIFLALGVLIFATMIYYAERIGADPDDI LGSNHTYFKNIPIGFWWAVVTMTTLGYGDMYPKTWSGMLVGALCA LAGVLTIAMPVPVIVNNFGMYYSLAMAKQKLPKKKNKHIPRPPQPGSPNYCKPDPPPPPPPHPHHGSGGISPPPPITPPSMGVTVAGAYPAGPHTH PGLLRGGAGGLGIMGLPPLPAPGEPCPLAQEEVIEINRADPRPNGDPA AAALAHEDCPAIDQPAMSPEDKSPITPGSRGRYSRDRACFLLTDYAPSPPGP
[0122] In some cases, the one or more selective regulatory elements for PV cell comprise sequences of SEQ ID NOs: 1-32, a functional fragment or a combination thereof, or sequences comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or 100% sequence identity to them. In some cases, the sequence identity is measured by BLAST. In some cases, such gene therapy is used to treat epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability, Dravet's syndrome and / or Alzheimer's disease. In some cases, such gene therapy is used to treat epilepsy and / or seizures associated with Dravet's syndrome and / or Alzheimer's disease. In some cases, treatment using the gene therapy described here results in reduced seizure frequency and / or duration. In some cases, treatment using the gene therapy described here results in the increased formation of ion channels
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82/133 functional sodium, functional potassium ion channels or functional neurotransmission regulators in vivo.
[0123] In some cases, AAV serotypes 1, 8, and / or 9, or a hybrid thereof, can be used with the expression cassettes described here to target selective expression in PV cells. In some cases, an expression cassette designed for application by an AAV comprises a 5 'ITR, one or more regulatory elements selective for the cell type, an optional amplifier, an optional minimal promoter, a transgene, optionally one or more introns , an optional polyA signal, and a 3 'ITR. In some cases, an expression cassette may contain a 5 'ITR, two cell type-selective ERs, a basal promoter, a transgene, one or more post RNA regulatory elements, and a 3'ITR.
[0124] A typical AAV expression cassette is illustrated in FIG. 7. In some cases, the expression cassette contains a 5 'AAV ITR, an amplifier (for example, selective amplifier for PV cell or one or more regulatory elements combined), a promoter (for example, one or more selective promoters for cell PV or regulatory elements), a transgene (for example, SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, STX BP1, or a DNA binding protein), a post regulatory element transcription, and a 3 'AAV ITR. The promoter can be selective for PV cells, or be a constitutive promoter. In some cases, the transgene is a reporter gene, for example, a sequence that codes for eGFP, RFP, or a fluorescent marker. In other cases, the transgene is a DNA-binding protein that modulates gene expression.
[0125] In some cases, the transgene is a therapeutic transgene, for example, a sequence encoding SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, STX BP1, or a protein DNA binding, or a functional fragment or sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. The post transcriptional regulatory element can be any sequence that
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83/133 influence the expression of a protein from an mRNA or the stability of an RNA, for example, an intron, an internal ribosome entry site (IRES), or a post-transcriptional regulatory element of the groundhog hepatitis virus . In some cases, the post-transcriptional regulatory element is a combination of two or more post-transcriptional regulatory elements.
[0126] The expression cassette can be designed for application by a therapeutic optimized retroviral vector, for example, a lentiviral vector. The retroviral vector can be a lentiviral vector comprising a left (5 ') LTR; sequences that aid in the packaging and / or nuclear import of the virus, at least one selective regulatory element for the cell type, optionally a lentiviral reverse response element (RRE); optionally a promoter or an active part thereof; a transgene operationally linked to one or more regulatory elements; optionally an insulator; and a right (3 ') retroviral LTR.
[0127] In some cases, the expression cassette comprises one or more of the regulatory elements selective for the cell type described here. In some cases, the expression cassette comprises two or more combined regulatory elements. In some examples, the expression cassette comprises two or more regulatory elements that are not combined, for example, a promoter upstream of the transgene and an amplifier or stability element located downstream of the transgene.
[0128] In some cases, the expression cassette contains a selective regulatory element for the putative cell type that exhibits selective activity in a cell type of interest, for example, a selective regulatory element for a putative PV cell. The expression cassette containing the putative regulatory element can be packaged in a viral vector and transfected into an animal model in order to assess the activity of the selective regulatory element for the putative cell type. In some cases, a selective regulatory element for the putative cell type can be evaluated in vitro or ex vivo by applying a vector containing the selective regulatory element for the putative cell type to a plurality of cells or cell types that include a target cell or type of target cell, and
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84/133 then comparing the selective activity for the cell type of the putative regulatory element with a controlling regulatory element, such as a constitutive promoter or regulatory element, or a previously known regulatory element.
[0129] In some cases, selective expression is used to selectively express a therapeutic radical or a transgene in a cell type of interest (or tissue type of interest), such as PV neurons in the CNS. In some cases, a vector comprising a selective regulatory element for the cell type operatively linked to a transgene results in increased selective expression of the transgene in the cell type of interest compared to one or more (for example, at least two, three , four or five) other cells, cell types, tissues, or tissue types, or results in preferential expression of the transgene in the cell type of interest compared to one or more cells or cell types, for example, at least one, two, three, four or five non-target cell types.
[0130] Any known technique can be used to apply regulatory elements and a transgene, or compositions comprising regulatory elements and a transgene, to cells of interest (or a target cell or target cell type) in order to confer or induce expression in vitro, in vivo, or ex vivo of the transgene in a cell type selectively.
[0131] The expression cassettes containing regulatory elements selective for the type of cell in this report additionally comprise one or more transgenes. Transgenes can be genes that encode protein. In some cases, the expression cassette contains a transgene. The transgene can replace a missing or defective gene or compensate for defective expression of a protein within a cell. The transgene can be involved in a cell signaling pathway. In some cases, a transgene may encode a wild-type protein, a functional fragment thereof, a variant or mutant protein with improved therapeutic properties, for example, increased activity. In some cases, the transgene can encode a DNA-binding protein
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85/133 comprising either a zinc finger or a dCas9 domain, an ion channel, such as a potassium ion channel or a sodium ion channel, or a subunit thereof, a neurotransmission factor or a neurotransmission regulator . In some cases, a transgene may encode an ion channel subunit, a variant, or a mutant thereof. In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase etc.) where the DNA cleavage domain or nuclease domain has been disabled, for example, a disabled Cas nuclease (dCas), an effected transcription activator-like nuclease, or a nuclease-deactivated zinc finger protein. In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene comprises a gene editing protein, for example, a Cas, Cas9 protein.
[0132] The regulatory elements described here can be located in any position within an expression vector or cassette. For example, regulatory elements can be positioned upstream of an amplifier, downstream of an amplifier, but upstream of a promoter, within the 5 'RTU of a transgene, within an intron in the transgene, in the 3' RTU of the transgene , or downstream of the transgene. In some cases, one or more regulatory elements are positioned upstream or downstream of the operationally linked transgene.
[0133] In some instances, a regulatory element in this report results in the selective expression of an operationally bound transgene at a level that is at least 0.5, 1.0, 1.1, 1.2, 1.3, 1 , 4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3 lU / ml in a target cell type (for example, PV cells) such as as measured by ELISA. In some cases, the ability of a regulatory element to increase transgenic expression can be assessed in a mouse where the total amount of transgenic expression in the total mouse and / or the total number of cell types or tissue types showing transgenic expression is measured. .
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86/133 [0134] When evaluating the activity of an expression cassette or vector, the activity or expression can be represented as a level of activity or expression per unit dose, or normalized to a dose of the cassette or expression vector administered or applied to a cell, mouse, or an individual. In some cases, the expression or activity of a transgene is normalized to an amount of plasmid or DNA (eg, pg / kg per mouse), or viral particles (eg, normalized to an amount of genome copies / kg per mouse) or individual) used to allow comparison across different expression vectors or cassettes with or without a regulatory element. For example, when evaluating the activity of a regulatory element in a mouse, the selective expression or activity in tested PV cells can be normalized to a dose of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or above 10 or 720 pg of the expression vector, cassette or plasmid per mouse. In some cases, the level of expression or activity may be normalized to ΙΟ ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ gc / kg of viral particles containing an expression vector or cassette as described here by mouse.
[0135] In some respects, an expression cassette comprises one or more regulatory elements (for example, any one or more of SEQ ID NOs: 132, or a functional fragment or a combination of these, or sequences showing at least 80%, at least least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them) described herein operatively linked to a transgene to result in cell type selective expression, or preferential expression, of the transgene in one type of target cell relative to at least one, at least two, at least three, at least four, at least five, or more than five types of non-target cells. In some cases, an expression cassette comprises one or more regulatory elements operatively linked to a transgene to result in selective expression for cell type or preferential expression in a target cell subtype over at least one, at least two, at least at least three, at least four, at least
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87/133 five, or more than five non-target subtypes, or all other known subtypes of the cell. In some cases, the transgene is any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, and STX BP1, or a functional fragment thereof or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases the transgene is from a DNA-binding protein that modulates an endogenous gene (for example, an SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,2, KV3,3, or STX BP1 endogenous) . In some cases, the transgene is any of SEQ ID NOs: 36-43, or a functional fragment thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these. In some cases, the transgene is a transcription modulator. In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase etc.) where the DNA cleavage domain or nuclease domain has been disabled, for example, a disabled Cas nuclease (dCas), an effected transcription activator-like nuclease, or a nuclease-deactivated zinc finger protein. In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene is from a gene editing protein, such as a protein from the Cas family, Cas9, a zinc finger nuclease, or an effector nuclease similar to a transcription activator. In some cases, the transgene is a reporter gene or a fluorescent marker. In some cases, an expression cassette described here is in a viral vector. In some cases, an expression cassette described here is packaged in an rAAV, such as rAAV9 or rAAVDJ. In some cases, an expression cassette described here is applied to a cell as a gene therapy. In some cases, a gene therapy described here is applied to an individual, preferably a human or a mammal. In some cases, an expression cassette described here is used to treat an
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88/133 neurological condition or disease, such as epilepsy, a neurodegenerative disease, taupathy, neuronal hypoexcitability, Dravet syndrome or Alzheimer's disease.
[0136] In some cases, an expression cassette (for example, gene therapy, viral vector, vector, or plasmid) comprises any one or more of SEQ ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, operably linked to a transgene. In some cases, such combined regulatory elements are linked using a 1-50 nucleotide linker. In some cases, such combined regulatory elements are not linked. In some cases, two or more regulatory elements are located upstream and / or downstream of the promoter. In some cases, two or more regulatory elements are located upstream and / or downstream of the transgene.
[0137] In some cases, an expression cassette comprises one or more of the regulatory elements described here, when operationally linked to any transgene (for example, a reporter transgene or a therapeutic transgene), and directs selective expression or preferential expression in at least at least one type of target cell at a level that is statistically significantly higher than expression directed by CAG, EFla, a constitutive promoter, or a non-selective regulatory element when operationally linked to the same transgene, or by the same construct without the regulatory elements. In some cases, “statistically significantly higher” means that the regulatory elements drive selective expression in the target cell type at a level that is at least 1.1, 1.2, 1.3, 1.4, 1, 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times the level of expression by CAG, EFla, a constitutive promoter, or a non-selective regulatory element when operationally linked to the same transgene in the target cell type, or by the same construct without the regulatory elements. In some cases, such an expression
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Cell type selective 89/133 is assayed using a co-localization assay as described here.
[0138] In other respects, an expression cassette comprising one or more of the regulatory elements described here operatively linked to any of the transgenes described here results in the selective expression or preferential expression of the transgene in a target cell type in at least one, at least two, at least three, at least four, at least five, or more than five non-target or subtype non-target cell types.
[0139] In some cases, the target cell type is a PV cell. In some cases, the non-target cell subtypes are at least one, at least two, at least three, or at least four of the non-PV GABAergic subtypes described here. In some cases, an expression cassette comprising a regulatory element described here is selective for PV cells over all non-PV GAB Aergic cells or all non-PV cells in the CNS. In some cases, selectivity for the cell type is measured according to a co-localization assay described here. In some cases, selectivity for cell type is measured using a mouse that expresses Cre in the target cell type.
Neurons of Parvalbumin (PV) [0140] GABAergic neurons produce gamma aminobutyric acid (GABA), the main inhibitory neurotransmitter in the CNS. GABA is important for reducing neural excitability throughout the nervous system. GABA acts on the inhibitory synapse by binding specific transmembrane receptors and causing the opening of ion channels which negatively change the polarization of the membrane. In general this results in the hyperpolarization of the cell and increases the signal required to trigger a potential action. Defects in GABAergic neurons can result in an imbalance between excitatory and inhibitory signals, and have been implicated in many neurological disorders, including Dravet's syndrome, epilepsy, neurodegeneration, tauopathies and Alzheimer's disease. Other neurological conditions or conditions involved include a psychiatric disorder (for example, schizophrenia, obsessive disorder
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Compulsive, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (for example, chronic traumatic encephalopathy, generalized epilepsy with febrile seizures plus (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); and / or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological condition or disease is any seizure and / or epilepsy related to the condition or disease in which the PV neurons are involved.
[0141] Parvalbumin is a calcium-binding protein, which is expressed in about 40% of the total GABAergic interneurons in the somatosensory cortex. Within the CNS, PV cells are generally considered to be GABAergic cells. Several studies have also identified that GABAergic cells include distinct cell subtypes, including cells that express PV, SOM, CR, CCK, NPY, VIP, or a combination of these.
[0142] PV neurons are particularly relevant for several neurological diseases or conditions, such as Dravet's syndrome, Alzheimer's disease, epilepsy, neurodegeneration, tauopathies and / or seizures. In some cases, a neurological condition or disease associated with a PV neuron is a psychiatric disorder (for example, schizophrenia, obsessive-compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (for example, chronic traumatic encephalopathy, generalized epilepsy with febrile seizures plus (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological condition or disease is any seizure and / or epilepsy related to a condition or disease in which the PV neurons are involved. In many ways, the target cell is a PV cell in the CNS, or GABAergic cells that express PV.
[0143] In several respects, interneurons that express PV are also called basket cells, which can be further subdivided by
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91/133 cell body size (for example, large basket cell, small basket cell and nest basket cell), and dendritic and axonal projection. Physiologically, basket cells that express PV are often fast-spiking (FS), characterized by a high frequency train of potential actions (APs) with little adaptation. It is widely accepted that PV basket neurons innervate proximal excitatory neurons and proximal neurons. Feedforward inhibition mediated by basket neurons that express FS PV, can be found in several cortical networks including thalamocortical, translaminar, and interareal circuits. The FS PV basket neurons strongly inhibit neighboring pyramidal excitatory neurons. It has been shown that basket neurons and pyramidal PV neurons that share common excitatory inputs tend to be connected reciprocally (feedback inhibition). These connections can serve to regulate the precise time window in which excitatory neurons can generate pulses in response to excitatory directions. In addition, thalamocortical and intracortical excitatory inputs in FS PV basket neurons are suppressed by high frequency stimulus, which mediates activity-dependent feedforward inhibition. Basket cells that express PV also innervate other interneurons including other basket cells, and are electrically coupled to each other through communicating junctions. It has been proposed that this feature can assist in the generation and maintenance of synchronization and oscillation of the cortical network.
[0144] In some cases, one or more of the regulatory elements described here result in increased selectivity of gene expression in PV neurons compared to at least one, at least two, at least three, at least four, or at least five neurons that do not express PV. In some cases, non-PV cells include all non-PV GABAergic cells. In some cases, non-PV GABAergic neurons include, but are not limited to, cells expressing calretinin (CR), somatostatin (SOM), cholecystokinin (CCK), CR + SOM, CR + neuropeptide Y (NPY), intestinal vessel polypeptide CR + (VIP), SOM + NPY, SOM + VIP, VIP + choline acetyltransferase (ChAT), CCK + NPY, CR + SOM + NPY, and CR + SOM + VIP.
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92/133 [0145] In some cases, either CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element that directs gene expression in a non selective for cell type can be used for comparison with selective regulatory elements for PV, or any selective regulatory elements for cell type described here. In some cases, a regulatory element that results in selective expression in PV cells at a level above expression of a gene operatively linked to CAG or EFla control is indicative of selectivity for PV cells. In some cases, a regulatory element described here shows selective gene expression in PV cells that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the level of PV expression from a transgene that is operationally linked to CAG, EFla, a constitutive promoter (for example, SV40 , CMV, UBC, PGK, and CBA), or a non-selective regulatory element (for example, SEQ ID NO: 34, or a functional fragment thereof or a sequence having at least 80%, at least 85%, at least 90% , at least 95%, or at least 99% sequence identity thereto), and as measured in a co-location assay. In some cases, a regulatory element described here shows selective gene expression in PV cells that is at least
1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the level of expression under CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element (for example, SEQ ID NO: 34, or a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto) and as measured in a co-location assay described herein.
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93/133 [0146] In preferred cases, one or more of the regulatory elements described here selectively directs the expression of a transgene in a GABAergic cell, such as a GABAergic cell that expresses parvalbumin in at least one other type of CNS cell (for example, example, at least two at least three, at least four, at least five non-PV cells, or two or more, three or more, or four or more non-PV cells and / or non-PV GABAergic neurons).
[0147] In some cases, a type of target cell is a GABAergic neuron that expresses parvalbumin, or PV cells.
[0148] One way to selectively express a transgene in a subpopulation of cells in the brain is to use a viral vector comprising a transgene operatively linked to a selective regulatory element for the cell type, or a regulatory element that is selective (or exhibits selective activity) ) in the subpopulation of cells in the brain, for example, PV cells. A viral vector can be selected to have high infectivity without selectivity for a particular cell type, while the regulatory element confers selectivity. For example, a selective regulatory element for the cell type can direct the expression of a transgene in PV neurons and not in other neurons.
[0149] In some cases, the present report involves the use of regulatory elements (ie, selective regulatory elements for PV cells) that selectively target expression in PV neurons.
[0150] GABAergic cells are inhibitory neurons that produce gamma-aminobutyric acid. GABAergic cells can be identified by the expression of glutamic acid decarboxylase 2 (GAD2). Other GABAergic cell markers include GAD1, NKX2,1, DLX1, DLX5, SST, PV and VIP.
[0151] In some cases, a CNS non-PV cell is an excitatory neuron, a dopaminergic neuron, an astrocyte, a microglia, a motor neuron or a vascular cell. In some cases, a non-GABAergic neuron is a cell that does not express one or more of GAD2, GAD1, NKX2,1, DLX1, DLX5, SST and VIP. In some cases, a non-PV neuron is a GABAergic neuron that does not express parvalbumin. In some cases, other CNS cells refer to types
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94/133 of CNS cells that never expressed any of PV, GAD2, GAD1, NKX2,1, DLX1, DLX5, SST and VIP.
[0152] In some cases, a regulatory element described here is selective for a cell that expresses PV in at least one, two, three, four, five, or more than five types of non-PV cells in the CNS. In some cases, non-PV cell types include non-PV GABAergic cells. In some cases, the cell type of interest is a PV cell. In some cases, selective ERs for PV cells are referred to as selective regulatory elements for PV cells.
[0153] In some cases, the selective regulatory elements for PV cell described here include the sequences of SEQ ID NOs: 1-32, or any combination thereof.
[0154] In some cases, one or more regulatory elements selective for PV cell or one or more of the regulatory elements described here are used to increase the expression of a transgene in cells that express PV by at least 2,5,10,15, 20,30,40,50,60,70,80,90,100, or more times compared to the expression without the regulatory element. In some cases, an ER in an expression cassette increases gene expression by at least 1.5%, 2%, 5%, 10%, 15%, 20%, or 50%, or more than 1.5%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, or 100% compared to the expression without the regulatory elements. In some cases, the compositions and methods of using these comprise expression cassettes containing one or more regulatory elements that result in a 10500% increase in transgenic expression, for example, the expression of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KCNC1 (also known as KV3,1), KCNC3 (also known as KV3,3), STX BP1, a DNA-binding protein, or a functional variant or fragment thereof, or a protein of these, compared to the level without the elements regulatory or in comparison with a non-selective regulatory element (eg CAG, EFla, a constitutive promoter, or SEQ ID NO: 34, or a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least least 90%, at least
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95/133 minus 95%, or at least 99% sequence identity thereto). In some cases, the increase in gene expression and / or the protein level of an SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KCNC1, KCNC3, and STX BP1 is 1.5-5%, 5% -10% , 10-15%, 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% , 100-150%, 150-200%, 250-300%, 300-350%, 350-400%, 400450%, 450-500%, or 1.5-20%, 20% -50%, 50% -100%, 100-200%, 200-300%, 300400%, or 400-500% compared to the level without the expression cassette or regulatory elements. In some cases, the expression of such a gene or protein is selective in PV cells compared to an expression cassette comprising a control (for example, CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), or a non-selective regulatory element) or a non-selective regulatory element for the cell type (for example, SEQ ID NO: 34, or a functional fragment thereof, or a sequence showing at least 80%, at least 85 %, at least 90%, at least 95%, or at least 99% sequence identity thereto).
[0155] In some cases, the selectivity of expression in PV cells can be calculated by dividing the number of cells that express both PV and eGFP (the transgene operatively linked to one or more regulatory elements) by the total number of cells that express eGFP, and multiplication by 100 to convert to a percentage. The selective regulatory elements for PV cells, as described here, can be highly selective for expression in PV cells. For example, the selective regulatory elements for PV cell or one or more of the regulatory elements described here can exhibit about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than about 99% selectivity for PV neurons.
[0156] In some cases, a PV cell selective regulatory element or any regulatory element described here confer selectivity in the expression of a transgene in PV neurons at a level that is statistically higher than that of a control regulatory element, for example, EFla or a previously known regulatory element. In some cases, the different statistic between a
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96/133 selective regulating element for PV cell and a control regulating element is at least 2 times, 5 times, 10 times, 20 times, or more than 2 times different, or more than 5 times, 10 times, or 20 times different as determined by any of the methods described here, such as a co-localization assay.
[0157] This report includes regulatory elements that are selective for PV cells. These PV cell selective REs or any cell type selective REs are preferably short, preferably less than about 1100 base pairs, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp , 300 bp, 200 bp, or less than about 110 bp. Selective REs for PV cell or any selective REs for cell type can be between 1050 bp and 100 bp, between 100 bp and 500 bp, or between 500 bp and 1050 bp. Some examples of selective regulatory elements for PV cells are provided by SEQ ID NOs 132, or a functional fragment or combination thereof. Other selective regulatory elements for PV cell contemplated in this report include sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of these sequences described here , or a part or fragment of one of the sequences described here.
[0158] In some cases, a selective regulatory element for PV cell or any regulatory element described here has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more than 99% identity to a sequence described here, or a fragment of a sequence described here. In some cases, a selective regulatory element for PV cell has at least about 80% identity to at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of a sequence described here, or a functional fragment thereof.
[0159] In some cases, a PV cell selective regulatory element comprises at least 80% identity to any one or more of SEQ ID NOs: 1-32, or a functional fragment or combination thereof, or sequences having at least 80 %, at least 85%, at least 90%, at least
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95%, or at least 99% sequence identity to these. In some cases, a PV cell selective regulatory element has 90% identity to 50% or more of a sequence of SEQ ID NOs: 1-22. In some cases, a selective regulatory element for PV is a functional fragment of any of SEQ ID NOs: 1-32 or a combination thereof. In some cases, the functional fragment is capable of selectively expressing a transgene in PV cells with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% selectivity of expression in PV cells.
[0160] In some cases, two or more regulatory elements selective for PV cell in this report or any two or more regulatory elements described here are combined to form a regulatory element combination. In some cases, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more selective regulatory elements for PV cell, or a plurality regulatory elements described here are combined. For example, SEQ ID NO: 31 is a combination of SEQ ID NOs 1 and 23-29. As another example, SEQ ID NO: 32 is a combination of SEQ ID NO: 8 and SEQ ID NO: 23-29. In some cases, fragments of two or more regulatory elements selective for PV can be combined to form a combination of regulatory elements. For example, 50% of SEQ ID NO: 1 can be combined with 30% of SEQ ID NO: 8 and 90% of SEQ ID NO: 30 to form a combination of regulatory elements.
[0161] In some cases, one or more regulatory elements selective for PV cell in this report or any one or more of the regulatory elements described here selectively express a transgene operably linked in PV neurons compared to one or more other types of CNS cells. This selective expression can be quantified by counting the number of PV neurons that express detectable levels of the bound transgene as a percentage of the total number of cells that express the transgene, including the number of non-PV neurons that express the transgene. In other words, the selectivity of a PV regulatory element in a particular cell type or target cell
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98/133 can be determined by measuring and / or comparing the number of PV neurons (or target cells) that express the transgene that is operationally linked to the regulatory element in relation to the number of non-target cell types that express the transgene (or the total number of cells expressing the transgene).
[0162] In some cases, the selective regulatory elements for PV cell can exhibit around 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or above about 99% selectivity for PV neurons, PV neurons in the CNS, or GABAergic neurons that also express PV. In some cases, one or more regulatory elements in this report may display around 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or above about 99% selectivity for PV neurons compared to CAG or EFla or a selective element for non-PV cell type, or in comparison with CNS non-PV cells, or compared with at least one, at least two, at least three, at least four, or at least five other non-PV GABAergic neuron subtypes in the CNS.
[0163] In some cases, a PV selective regulatory element confers selectivity in the expression of a transgene in PV neurons at a level that is statistically higher than that of a control regulatory element, for example, CAG, EFla, a constitutive promoter (for example, SV40, CMV, UBC, PGK, and CBA), a non-selective regulatory element, or a previously known regulatory element. In some cases, the different statistic between a PV cell selective ER and a control element is at least 2 times, 5 times, 10 times, 20 times, or more than 2 times different, or more than 5 times, 10 times , or 20 times different as determined by any of the methods described here. In some cases, selectivity for PV is measured using a co-localization assay as described here.
[0164] In some respects, the regulatory elements selective for the cell type described here are useful for modulating the selectivity of expression of a transgene in a CNS cell type compared to other cell types
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99/133 of the SNC. For example, the regulatory elements selective for the cell type described here may be useful for the selective modulation of the expression of a transgene in PV cells in relation to other cells of the CNS, including other types of neurons. For gene therapy, selective expression of a transgene in a target cell type and / or minimized expression of the transgene in relation to a non-target cell type may be desired. Expression of the transgene in an unwanted cell type (for example, non-target cell type) can result in an adverse effect for the individual. Expression of the transgene in an unwanted cell type can counteract the therapeutic effect of the transgene in the desired cell type. For example, a transgene for expression in PV cells can have a negative effect on the individual if expressed in glutamatergic neurons. The regulatory elements selective for the cell type described here can be used in expression cassettes to ensure appropriate expression of a transgene and / or to reduce the off-target effects of gene therapy.
[0165] The regulatory elements selective for the cell type here can be used in gene expression cassettes so they are operationally linked to one or more transgenes. Such gene expression cassettes are used to apply transgenes to cells for expression. The expression cassette can contain a cell type selective regulatory element as described here, a combination of cell type selective regulatory elements, or a fragment of a cell type selective regulatory element as described herein operatively. linked to a transgene.
[0166] Preferably, the expression cassettes here include one or more regulatory elements selective for the type of cell operationally linked to a transgene, so the two do not work together in their endogenous context in vivo. For example, a transgene for a sodium ion channel beta subunit, such as SCN1B, can be operationally linked to one or more regulatory elements that do not work in the same context in vivo, or do not detectably direct the expression of SCN1B endogenously .
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Similarly, a nucleic acid cassette can include a neurotransmitter regulator, such as STX BP1, operationally linked to a regulatory element that does not work in the same endogenous or in vivo context, or is not in the same open reading frame, or is not in same human chromosome, or does not detectably direct the expression of STX BP1 in vivo. In some cases, a selective regulatory element for the cell type is linked to a transgene, where the selective regulatory element for the cell type does not regulate the endogenous gene corresponding to the transgene in vivo.
[0167] In some respects, the regulatory elements selective for the cell type described here are derived from sequences isolated from a human chromosome locus other than a native gene locus corresponding to the transgene. Thus, in some cases, an expression cassette comprises selective regulatory element (s) for the cell type, presenting a sequence derived from a chromosome different from the chromosome corresponding to the transgene in the same cassette. In other cases, the regulatory element (s) of the report and a transgene that are operationally linked in an expression cassette are derived from sequences located more than 20 kb apart in the human genome, or at distal genomic sites. When two or more human-derived regulatory elements are used in an expression cassette, the two or more regulatory elements may have sequences located more than 5 kb apart, more than 10 kb apart, more than 15 kb apart or more than 20 kb apart in the human genome, or where the two or more regulatory elements do not naturally interact with each other in the genome.
[0168] In some cases, an expression cassette comprising selective regulatory elements for PV may exclude known sequences derived from hSynl or GAD2 promoter sequences. In some cases, the selective regulatory elements for PV do not comprise the complete promoter sequence of any of the promoters of GAD2, GAD1, SYN1, NKX2,1, DLX1, DLX5, SST and VIP. In some cases, the selective regulatory elements for PV do not comprise more than 500 contiguous base pairs of the sequence derived from the
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101/133 promoter or one or more of GAD2, GAD1, SYN1, NKX2,1, DLX1, DLX5, SST and VIP. In some cases, the selective regulatory elements for PV do not comprise sequences that are within 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, or 10 kb from the site of start of transcription of anyone from GAD2, SYN1, NKX2,1, DLX1, DLX5, SST, and VIP.
[0169] In some cases, a transgene is useful for treating a disease associated with a specific cell type of interest. In some cases, a cell type of interest is a neuron, an inhibitor neuron, a GABAergic neuron, or a PV neuron. In some cases, a transgene is any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, and STX BP1. In some cases, a transgene is a DNA-binding protein that modulates the expression of a gene (for example, a transcription activator or a transcriptional repressor that modulates the expression of an endogenous gene). In some cases, a transgene is a gene editing protein, such as a zinc finger nuclease, an effector nuclease similar to a transcription activator, a protein of the Cas family. In some cases, a transgene is a reporter gene or a detectable marker, such as eGFP, tdTomato, or RFP. In some cases, a transgene is a Cas protein, such as Cas9.
[0170] Transgenes useful for treating a condition associated with PV neuron cells can be incorporated into a vector, nucleic acid cassette, or method as described here. The transgenes used here generally do not contain introns, or do not contain more than one intron. A transgene can be obtained from a cDNA sequence instead of from a genomic sequence. In some cases, transgenes may contain some, or all, of their endogenous introns. In some examples, such a transgene encodes a DNA binding domain or an ion channel. Examples of DNA-binding domains that can be encoded in the expression cassettes in this report include zinc fingers, Cas9, a protein from the Cas family, dCas9, a protein from the dCas family, or a transcription activator-like effector (TALE). In some cases, the transgene is a DNA-binding protein that comprises a
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DNA from a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase, etc.) where the DNA cleavage domain or nuclease domain has been disabled, for example, a deactivated Cas nuclease (dCas), an effector nuclease similar to deactivated transcription activator, or a deactivated zinc finger protein for nuclease. In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene comprises a gene editing protein, for example, a Cas, Cas9 protein. In some cases, the transgene is a subunit or component of an ion channel or a membrane protein, or a gene associated with a neurological condition or disease described here. Examples of ion channel transgenes that can be used in the expression cassettes in this report include voltage-driven and ligand-driven ion channels. Voltage-driven ion channels include sodium channels, calcium channels, potassium channels and proton channels. In some cases, the transgene codes for a voltage driven sodium channel subunit. Examples of voltage driven sodium channel subunits include SCN1B (NM_001037,4), SCN1A (NM_001165963,1), and SCN2B, (NM_004588,4).
[0171] In some cases, the transgene codes for a subunit of a voltage-driven potassium channel. V Examples of voltage-driven potassium channel subunits include KCNC1 (NM_001112741.1), and KCNC3 (NM_004977.2). In some cases, a transgene is any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STX BP1, a variant and a functional fragment thereof. In some cases, a transgene is a sequence showing at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to any of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3 , 3, STX BP1, a variant or a
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103/133 functional fragment thereof. In some cases, such sequence identity is measured using BLAST.
[0172] In some cases, an expression cassette described here comprises one or more regulatory elements selective for PV or one or more regulatory elements of this report operationally linked to a transgene presenting at least 80%, at least 85%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99 , 5% sequence identity to a sequence encoding any of SEQ ID NOs: 37-43, or a functional fragment or variant thereof, or GenBank sequences that correspond to SEQ ID NOs: 37-43. In some cases, an expression cassette described here comprises a transgene having a sequence according to (i) a sequence of SEQ ID NOs: 37-43, or (ii) a functional fragment thereof, or (iii) a sequence presenting at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least at least 98%, at least 99%, or at least 99.5% sequence identity to (i) or (ii).
[0173] In some examples, the transgene is a neurotransmitter regulator, or a functional variant or fragment thereof. A neurotransmitter regulator may be involved in regulating the production or release of a neurotransmitter in the CNS. For example, a neurotransmitter regulator can assist in synaptic fusion to release neurotransmitters. An example of a neurotransmission regulator is STX BP1 (NM_001032221,3) or a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of sequence identity to this. The transgene can also be a subunit of a neurotransmitter regulator.
[0174] In some cases, an expression cassette in this report may contain an AAV2 5 'ITR, a selective amplifier for PV, a selective promoter for PV, or a combination of one or more promoters and selective amplifiers for PV,
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104/133 a SCN1B cDNA, a WPRE, a polyA hGH signal, a selective regulatory element for PV and an AAV2 3'ITR. In one example, the expression cassette comprises an AmpR promoter and an AmpR coding sequence, a bacterial origin of replication, an AAV2ITR, SEQ ID NO: 8, SEQ ID NOs: 2329, a transgene (coding sequence, a WPRE , a poly A signal from human growth hormone, an AAV2 ITR, and a fl origin.
[0175] As another example, an expression cassette in this report may contain an AAV2 5 'ITR, an amplifier, a promoter, an endogenous SCN1A gene transcription activator, a WPRE, a polyA hGH signal, a regulatory element, and an AAV2 3'ITR. In some cases, an expression cassette comprises AAV2 5 'ITR, a promoter, an intronic element, transcriptional modifier, synthetic polyA, and an AAV2 3' ITR. In some cases, an expression cassette in this report may contain an AAV2 5 'ITR, a selective amplifier for PV, a selective promoter for PV, a sequence encoding a transcription activator for SCN1A or SCN1B, a WPRE, a polyA hGH signal , a selective regulatory element for PV or any of the regulatory elements described here, and an AAV2 3'ITR.
[0176] An expression cassette comprising one or more regulatory elements of this report can be used to treat a medical condition. In some cases, an expression cassette containing a regulatory element in this report is used to treat a neurological condition or a neurodegenerative condition. The neurological condition can be caused by a known genetic event or it can have an unknown cause.
[0177] The neurological condition may be a disease associated with PV neurons. The neurological condition can be a disease associated with inhibitory neurons, such as PV neurons. Diseases or conditions associated with PV neurons can be treated by applying an expression cassette containing a transgene and one or more regulatory elements selective for PV cells or any of the regulatory elements as described herein to a cell in vivo. In some respects, expression cassettes comprising a transgene
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105/133 operatively linked to one or more regulatory elements selective for PV cell or any of the regulatory elements described here can be used to treat Dravet's syndrome, Alzheimer's disease, epilepsy, a neurodegenerative disorder, taupathy, neuronal hypoexcitability and / or convulsions. In some cases, an expression cassette from this report is used to treat a psychiatric disorder (for example, schizophrenia, obsessive-compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (eg, Dravet's syndrome, chronic traumatic encephalopathy, generalized epilepsy with febrile plus seizures (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); and / or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological condition or disease is any seizure and / or epilepsy related to a condition or disease in which PV neurons are involved.
[0178] Most cases of Dravet's syndrome are associated with mutations in the SCN1A and / or SCN2A genes. Mutations or abnormalities in SCN1A have also been associated with seizure disorders, epilepsy, autism, familial hemiplegic migraine type 3 (FHM3), genetic epilepsy with febrile plus seizures (GEFS +), and effectiveness of some anti-seizure medications. For example, the ICS5N + 5OA mutation in SCN1A is associated with the maximum safe amount (dose) of the anticonvulsant drugs phenytoin and carbamazepine.
[0179] In some Alzheimer's patients, the production of the amyloid β (Αβ) involving many peptides and proteases that can affect the excitability of neurons, causing seizures and the subregulation of the sodium channel Navl.l in PV neurons.
[0180] Diseases associated with dysfunctional PV neurons such as those due to loss of functional mutations in SCN1A or Navl.l include: Dravet's syndrome, Ohtahara's syndrome, epilepsy, early childhood epileptic encephalopathy 6 (EEEE6), familial febrile seizures 3A (FEB3A), childhood epilepsy not
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106/133 treatable with generalized tonic-clonic seizures (ICEGTC), migraine, familial hemiplegics 3 (FHM3), Panayiotopoulos syndrome, familial atrial fibrillation 13 (ATFB13), generalized epilepsy with febrile seizures plus type 1 (gefs + type 1), syndrome de Brugada, defect in non-specific cardiac conduction, generalized epilepsy with febrile seizures plus, benign familial infantile seizures, early childhood epileptic encephalopathy 11 (ISIS11), benign familial childhood epilepsy, neurodegeneration, tauopathies and Alzheimer's disease. In some cases, the neurological condition is Dravet's syndrome. Dravet's syndrome is associated with mutations in the SCN1A and / or SCN2A genes. In some cases, one or more regulatory elements in this report are used in gene therapy or an expression cassette to treat a neurological condition or disease associated with PV neurons, for example, a psychiatric disorder (eg, schizophrenia, obsessive compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (eg, Dravet's syndrome, chronic traumatic encephalopathy, generalized epilepsy with febrile plus seizures (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, one or more regulatory elements in this report (for example, selective PV neuron regulatory elements) are used to treat Dravet's syndrome and / or Alzheimer's disease (for example, in an expression cassette, a vector or gene therapy). In some cases, the neurological condition or disease is any seizure and / or epilepsy related to a condition or disease in which PV neurons are involved.
[0181] The methods and compositions in this report can be used to treat an individual who has been diagnosed with a disease, for example, a neurological or neurodegenerative disease. The individual may be a patient suffering from a form of epilepsy. In some cases, the individual is a patient with Dravet's syndrome. The individual may be a patient suffering from a neurodegenerative disease, for example, a patient with Alzheimer's disease. In
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107/133 some cases, epilepsy, encephalopathy and / or seizures are associated with a genetic mutation in SCN8A. In some cases, a genetic mutation in SCN8A can cause epileptic syndromes, for example, Dravet's syndrome. In some cases, a genetic mutation in STX BP1 is associated with encephalopathy with epilepsy, characterized by recurrent seizures.
[0182] In some cases, an individual treated with one or more of the compositions described here is one diagnosed with a genetic mutation or aberration in an ion channel or a neurotransmission regulator (for example, a syntaxin-binding protein). Examples of such mutations include mutations in SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KCNC1, KCNC3, and / or STX BP1, or a combination thereof. The expression cassette containing a cell type selective regulatory element as described herein can be applied to an individual to treat or prevent a disease with symptoms associated with a specific cell type. For example, an expression cassette comprising a transgene operatively linked to one or more regulatory elements selective for PV cell is applied to an individual who has symptoms, or is at risk of developing symptoms, associated with PV neurons.
[0183] In some cases, treatment may be given to an individual with, or at risk of developing, Dravet's syndrome. Symptoms associated with Dravet's syndrome include seizures, memory defects, developmental delay, low muscle tone and / or cognitive problems. Treatment with an expression cassette in this report may result in an improvement in one or more symptoms, such as a reduction in the number, duration and / or intensity of seizures. Administration of gene therapy as described here to an individual at risk of developing Dravet's syndrome may prevent the development or slow progression of one or more symptoms.
[0184] In another example, treatment can be administered to an individual suffering from Alzheimer's disease. Symptoms associated with Alzheimer's disease include short-term memory loss, cognitive difficulties, seizures and difficulties with language, executive functions, perception
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108/133 (agnosia), and execution of movements (apraxia). Treatment with an expression cassette in this report may result in an improvement in one or more symptoms of Alzheimer's disease, such as reduced progression of memory loss, or the prevention of one or more symptoms. In some cases, treatment may result in a correction of the high gamma power of brain activity. Treatment can result in a reduction in seizure frequency and / or seizure severity, or a reduction in high activity potency of 10%, 20%, 30%, 40%, 50%, 60%, or 70%. In some cases, treatment may result in an improvement in cognitive function. Learning and / or memory can be improved by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%.
[0185] The methods and compositions in this report can be used to treat an individual who is at risk of developing a disease. The individual may be known to be predisposed to a disease, for example, a neurological disease or a disease associated with epilepsy, seizures and / or encephalopathy. The individual may be predisposed to a disease due to a genetic event, or due to known risk factors. For example, an individual may have a mutation in SCN1A that is associated with Dravet's syndrome. In some cases, the individual may be predisposed to a disease such as Alzheimer's disease due to the individual's age.
[0186] Treatment may result in a reduction or elimination of symptoms. For example, treatment can improve learning, memory, cognitive function and / or motor function; reduce the frequency and / or duration of seizures; and / or reduce the sensitivity to temperature (or increase the temperature limit that triggers a seizure).
[0187] In some cases, the type of cell targeted by a gene therapy or expression cassette described here is a PV cell. In some cases, the non-target cell subtypes are at least one, at least two, at least three or at least four of the non-PV GABAergic subtypes described here. In some cases, an expression cassette comprising a regulatory element described here is
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109/133 selective for PV cells in relation to all non-PV cells of the CNS. In some cases, selectivity for the cell type is measured according to a co-localization assay described here. In some cases, selectivity for cell type is measured using a mouse that expresses Cre in the target cell type.
[0188] In some cases, treatment does not result in an adverse reaction for the individual. Treatment with a gene therapy containing a selective regulatory element for PV may cause fewer or less severe adverse reactions in an individual than treatment with a similar gene therapy containing the same transgene bound to a non-selective regulatory element.
[0189] In several respects, any expression cassette described here can be adapted for or used in gene therapy (for example, gene therapy with rAAV or rAAV9) to treat one or more between Dravet's syndrome, Alzheimer's disease, epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability and / or seizure. In some cases, a gene therapy comprises any one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of the SEQ ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to these, operationally linked to a transgene. In some cases, gene therapy comprises an expression cassette from this report. In some cases, a gene therapy comprises one or more of the regulatory elements described here operationally linked to any transgene (for example, a reporter or therapeutic transgene) in such a way that the regulatory elements direct selective expression or preferential expression in at least a type of target cell at a level that is statistically significantly greater than the expression directed by CAG or EFla or a non-selective regulatory element when operationally linked to the same transgene, or by the same construct without the regulatory elements. In some cases, “statistically significant” means that the regulatory elements drive selective expression in the type of
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110/133 target cell at a level that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 , 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times the level of expression by CAG, EFla, a constitutive promoter, or a non-selective regulatory element when operationally linked to the same transgene in the target cell type, or by the same construct without the regulatory elements. In some cases, such cell type selective expression is assayed using a co-localization assay as described here. In some cases, the transgene is any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STX BP1, and a functional fragment thereof. In some cases, the transgene is a DNA-binding protein that modulates the expression of an endogenous gene, such as a transcription modulator, a transcription activator, or a transcriptional repressor. In some cases, the transgene is a DNA-binding protein that comprises a DNA-binding domain of a DNA-binding protein or a DNA cleaving protein (for example, a nuclease, a restriction enzyme, a recombinase etc.) where the DNA cleavage domain or nuclease domain has been disabled, for example, a disabled Cas nuclease (dCas), an effected transcription activator-like nuclease, or a nuclease-deactivated zinc finger protein. In some cases, the DNA binding domain is linked to a transcription modulator domain (for example, a transcription activator or repressor domain). In some cases, the transgene is from a gene editing protein, such as a zinc finger nuclease or an effector nuclease similar to a transcription activator. In some cases, a transgene is a reporter gene or a detectable marker, such as eGFP, tdTomato, or RFP. In some cases, a transgene is a Cas protein, such as Cas9.
[0190J In some cases, a gene therapy comprising an expression cassette described here is used to treat a neurological condition or disease. In some cases, a gene therapy comprising an expression cassette described here is used to treat a neurological condition or disease, where the expression cassette comprises any one or more, two or more, three or
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111/133 more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of SEQ ID NOs: 1-32, or a functional fragment or a combination thereof , or sequences showing at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, operably linked to a transgene. In some cases, the transgene is any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STX BP1, a DNA-binding protein and a functional fragment thereof. In some cases, gene therapy comprising an expression cassette described here is used to treat Dravet's syndrome. In some respects, gene therapy comprising an expression cassette described here is used to treat Alzheimer's disease. In some cases, a gene therapy comprising an expression cassette described here is used to treat symptoms of epilepsy and / or seizures associated with Dravet's syndrome and / or Alzheimer's disease. In some cases, the treatment of anyone between Dravet's syndrome, Alzheimer's disease, epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability and / or seizures comprises the application or administration of gene therapy in this report to a cell of an individual in need of such treatment. In some cases, the individual in need of such treatment is at risk of or has any of Dravet's syndrome, Alzheimer's disease, epilepsy, and / or seizures. In some cases, the individual is a child or a minor. In some cases, gene therapy comprising an expression cassette described here is used to treat an infant, child or minor diagnosed with or at risk of developing Dravet's syndrome. In some cases, a gene therapy comprising an expression cassette described here is used to treat an individual comprising a mutation or a genetic defect in any one or more of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3 , and STX BP1.
[0191] In some respects, this report provides a method for treating anyone between Dravet's syndrome, Alzheimer's disease, epilepsy,
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112/133 neurodegeneration, taupathy, neuronal hypoexcitability and / or seizures, comprising administering a gene therapy to an individual's cell, where the gene therapy comprises an expression cassette described here. In some cases, such an expression cassette comprises any one or more of SEQ ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences having at least 80%, at least 85%, at least 90%, at least at least 95%, or at least 99% sequence identity to these, operationally linked to a transgene, where the transgene can be any of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STX BP1, and a functional fragment of these. In some cases, the transgene is a subunit of a sodium ion channel or a potassium ion channel. In some cases, the transgene is a syntaxin-binding protein. In some cases, the transgene is a transcription modulator, for example, a transcription activator or a transcriptional repressor. In some cases, the transgene is a transcription modulator that modulates the expression of an endogenous gene (for example, SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, or STX BP1). In some cases, a transgene is a gene editing protein, such as a zinc finger nuclease or an effector nuclease similar to a transcription activator. In some cases, the transgene is a Cas protein, such as Cas9.
[0192] In other respects, this report provides a method for modifying any gene therapy designed to treat Dravet's syndrome, Alzheimer's disease, epilepsy, neurodegeneration, taupathy, neuronal hypoexcitability and / or seizures by adding one or more of the regulatory elements described here in order to increase selectivity for the gene therapy cell type. In some cases, gene therapy is gene therapy with rAAV.
[0193] In some cases, treatment with an expression cassette described here reduces the duration and / or frequency of seizures, for example, seizures associated with Dravet's syndrome, by at least 1%, 2%, 3%, 4 %, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,
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23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to an untreated control or compared to the level prior to treatment.
[0194] In some cases, treatment with an expression cassette described here reduces the potency and high gamma activity (eg, high potency of gamma activity associated with Alzheimer's disease) by at least 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%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to a control untreated or compared to the pre-treatment level.
[0195] In one aspect, this report provides a nucleic acid cassette from this report that comprises one or more regulatory elements operatively linked to a transgene that result in selective expression in one target cell type over one or more cell types do not target, for example, selective expression in PV neurons in the CNS in relation to one or more types of non-PV cells in the CNS. In some cases, each of the regulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence with at least 80% sequence identity to (i) or (ii). In some cases, the percent sequence identity can be measured using BLAST. In some cases, at least one of the regulatory elements is derived from human. In some cases, at least one of the regulatory elements is derived from a non-human mammal. In some cases, the regulatory elements are not naturally occurring. In some cases, the regulatory elements result in an expression selectivity in PV cells that is greater than the expression of the transgene operatively linked to CAG or EFla or a non-selective regulatory element as measured by a co-location assay. In some cases, a transgene is any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, and
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STX BP1, or a functional fragment of these or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to them. In some cases, a transgene is a DNA-binding protein that modulates the expression of a gene (for example, an endogenous gene such as SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, and STX BP1 ), such as a transcription modulator, transcription activator or transcription repressor. In some cases, a transgene is a gene editing protein, such as a zinc finger nuclease or an effector nuclease similar to a transcription activator. In some cases, a transgene is a reporter gene or a detectable marker, such as eGFP, tdTomato, or RFP. In some cases, a transgene is a Cas protein, such as Cas9. In some cases, regulatory elements result in selective expression in PV cells at a level that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 11 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 25 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times compared to that of a CAG or EFla or a non-selective regulatory element, such as me measured by the co-location test. In some cases, a different one sometimes refers to a different one between the percentage of eGFP-ι-, PV + cells that results from one or more regulatory elements and that of a non-selective regulatory element. In some cases, the co-localization assay is an immunohistochemical assay, as described below in Example 5. In some cases, a co-localization assay is performed using a commercially available anti-PV antibody. In some cases, the transgene
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115/133 encodes an ion channel subunit, a neurotransmitter regulator, or a variant or functional fragment thereof. In some cases, the ion channel subunit is an alpha subunit or a beta subunit of a sodium ion channel or a subunit of a potassium ion channel. In some cases, the transgene is either (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, or a DNA binding protein; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii). In some cases, the neurotransmission regulator is (i) STX BP1, (ii) a functional fragment thereof, or (iii) a sequence having at least 80% sequence identity to (i) or (ii). In some cases, the regulatory elements and the operationally linked transgene are located on different chromosomes. In some cases, the combined regulatory elements are less than 2.5 kb, less than 1.5 kb, less than 1 kb, or less than 500 bp in size. In some cases, non-PV cells comprise any one or more of the types of non-PV cells in the CNS, including, but not limited to, excitatory neurons, dopaminergic neurons, astrocytes, microglia or motor neurons. In some cases, the nucleic acid cassette is a linear construct. In some cases, the nucleic acid cassette is a vector. In some cases, the nucleic acid cassette is a plasmid. In some cases, the vector is a viral vector. In some cases, the viral vector is an adeno-associated virus (AAV) vector. In some cases, the AAV vector is AAV1, AAV8, AAV9, scAAVl, scAAV8 or scAAV9. In some cases, the viral vector is a lentiviral vector. In some cases, the regulatory elements contain less than 600 bp of contiguous sequence within 10 kb of the GAD2, GAD1, SYN1, NKX2,1, DLX1, DLX5 / 6, SST, PV and / or VIP transcription start site. [0196] In several embodiments described here, a regulatory element is less than 2050 bp, 2000 bp, 1900 bp, 1800 bp, 1700 bp, 1600 bp, 1500 bp, 1400 bp, 1300 bp, 1200 bp, 1100 bp, 1000 bp , 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 20 bp, 10 bp, or 5 bp. In several embodiments described here, an expression cassette comprises a transgene that is larger than a typical-sized transgene in a
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116/133 conventional viral vector, for example, AAV. In some respects, an expression cassette of any of the embodiments described here comprises a transgene that has at least 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb,
4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb or 8 kb. In some respects, any embodiment described herein comprises an expression cassette (for example, AAV) that comprises a transgene that has more than 1, 1,2, 1,3, 1,4, 1,5, 1,6, 1 , 7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8, 3.9, or 4kb in size. In some aspects, any embodiment described herein may additionally comprise one or more nucleic acid sequences or heterologous elements.
[0197] In one aspect, a method for the treatment of a neurological disorder or condition in an individual in need of such treatment comprises applying a therapeutically effective amount of a nucleic acid cassette described herein. In another aspect, a method for increasing the selective expression of a transgene in PV neurons comprises contacting a cell with a nucleic acid cassette described here. In some cases, a method of any embodiment described here is used to treat a neurological condition or disease, for example, a psychiatric disorder (eg, schizophrenia, obsessive compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (eg, Dravet's syndrome, chronic traumatic encephalopathy, generalized epilepsy with febrile plus seizures (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, a method of any embodiment described here can be used to treat Dravet's syndrome. A method of any embodiment described here can be used to treat Alzheimer's disease. In some cases, the methods and / or compositions in this report may be used to treat any neurological condition or disease associated with seizure and / or epilepsy, and / or where PV neurons are involved.
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117/133 [0198] In one aspect, a neurological condition described here is treated with gene therapy, preferably one that results in preferential expression in one type of tissue or cell type over another, for example, a PV neuron as determined by means of a co-location assay. In some cases, gene therapy is an AAV.
[0199] In one aspect, a method for directing the expression of any transgene to PV neurons in the CNS comprises the operational attachment of one or more of the selective regulatory elements of the PV neuron to a transgene. In some cases, the regulatory elements comprise one or more sequences of SEQ ID NOs: 1-32, or sequences with at least 80% sequence identity to SEQ ID NOs: 1-32, or a functional fragment thereof. In some cases, regulatory elements result in selective expression in PV neurons at a level that is at least 2 times, at least 5 times, or at least 7 times, or at least 10 times compared to CAG or EFla or an element non-selective regulator operationally linked to the transgene, as measured by a co-localization assay. In some cases, the transgene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STX BP1, a DNA binding protein, or a functional fragment thereof. In some cases, the regulatory elements and the transgene are in an AAV. In some cases, the AAV is AAV9.
[0200] In one aspect, a method for treating a neurological condition or disorder in an individual in need of such treatment comprises contacting a cell with a nucleic acid cassette comprising one or more regulatory elements operatively linked to a transgene that results selective expression in PV neurons in relation to one or more non-PV cells in the CNS. In some cases, the regulatory elements comprise one or more of SEQ ID NOs: 1-32, or a functional fragment or a combination thereof, or sequences of at least 80%, at least 85%, at least 90%, at least 95 %, or at least 99% sequence identity to them. In some cases, the transgene is a voltage-driven ion channel subunit, or a variant or functional fragment thereof. In some cases, the subunit is
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118/133 a beta subunit of a sodium ion channel. In some cases, the subunit is an alpha subunit of a sodium ion channel. In some cases, the subunit is a potassium ion channel. In some cases, the transgene is any one of (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, a DNA binding protein or STX BP1; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii). In some cases, the neurological condition or disorder is associated with a haploinsufficiency or a mutation in either SCN1A, SCN1B, SCN2B, KV3,1, and KV3,3. In some cases, the neurological condition or disorder is Dravet's syndrome. In some cases, the neurological condition or disorder is Alzheimer's disease. In some cases, the neurological condition or disease is a psychiatric disorder (for example, schizophrenia, obsessive compulsive disorder, addiction, depression, anxiety, psychosis); a disorder of the autism spectrum (for example, fragile X syndrome, Rett syndrome); epilepsy (for example, chronic traumatic encephalopathy, generalized epilepsy with febrile seizures plus (GEFS +), epileptic encephalopathy, temporal lobe epilepsy, focal epilepsy, tuberous sclerosis); or neurodegeneration (for example, Alzheimer's disease, Parkinson's disease). In some cases, the neurological condition or disease is any seizure and / or epilepsy related to a condition or disease in which PV neurons are involved. In some cases, the nucleic acid cassette results in selective expression in PV neurons at a level that is at least 2 times, at least 5 times, or at least 7 times, or at least 10 times compared to CAG or EFla or a non-selective regulatory element operatively linked to the transgene, as measured by a co-location assay. In some cases, the nucleic acid cassette is in an AAV. In some cases, the AAV is AAV9.
[0201] In one aspect, a method for the treatment of Dravet's syndrome comprises contacting a cell with an AAV comprising a transgene which is any of (i) SCN1 A, SCNIB, SCN2B, a DNA-binding protein ( ii) a functional fragment of these, or (iii) a sequence showing at least
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80% sequence identity to (i) or (ii). In some cases, the AAV additionally comprises one or more selective regulatory elements for PV neuron or any of the regulatory elements described here operationally linked to the transgene. In some cases, each of the regulatory elements independently comprises a sequence comprising any of SEQ ID NOs: 1-32, or any functional fragment or combination thereof, or a sequence comprising at least 80% sequence identity to any of the SEQ IDs NOs: 1-32.
[0202] In another aspect, a method for the treatment of Alzheimer's disease comprises contacting a cell with an AAV comprising a transgene that is any of (i) SCN1A, SCN2B, KV3,1, KV3,3, and STX BP1, (ii) a functional fragment thereof and (iii) a sequence showing at least 80% sequence identity to (i) or (ii). In some cases, AAV additionally comprises one or more selective regulatory elements for PV neuron operatively linked to the transgene. In some cases, each of the regulatory elements independently comprises a sequence comprising any of SEQ ID NOs: 1-32, or any functional fragment or combination thereof, or a sequence comprising at least 80% sequence identity to any of the SEQs ID NOs: 1-32.
EXAMPLES [0203] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided here.
EXAMPLE 1
Identification of Selective Putative Regulatory Elements for PV [0204] In order to identify and select a putative regulatory element that is selective for PV cells, PV cells can be collected from a knockin mouse R26-CAG-LSL-Sunl-sfGFP-Myc using affinity purification, for example using anti-GFP antibodies or anti-Myc magnetic beads coated with G protein. PV cells can be enriched by using beads coated with anti-PV antibody or affinity purification matrix.
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The nuclei are then isolated from the PV cells. Nuclear RNA can be purified from the nuclei and converted to cDNA, and amplified with the Nugen Ovation RNA-seq System V2 (Nugen 7102), followed by sequencing using Illumina HiSeq 2500. Genomic DNA can be purified from the nuclei, fragmented and used to obtain methylC-seq libraries, which can be sequenced using Illumina HiSeq 2000. In order to generate an ATAC-seq library, the nuclei bound to the spheres are transposed using Tn5 transposase (Illumina FC- 121-1030). After 9-12 cycles of PCR amplification, the libraries are sequenced using an Illumina HiSeq 2500. In order to generate a ChIP-seq library, the PV cell nuclei are digested into mononucleosomes using micrococcal nuclease, followed by extraction chromatin saline and native ChIP and library construction, which can be sequenced on an Illumina HiSeq 2500. After sequencing these libraries, the sequences are mapped to identify the correlations and patterns in hypo-methylation in regions rich in CG, modifications of histone, transcription factor binding sites, and patterns associated with transcription factors highly expressed in PV cells. The overlapping and correlation characteristics of the multiple assays and / or libraries described above provide converging evidence for the identification of candidate sequences that are selective putative regulatory elements for PV. The putative selective regulatory elements for PV can be further tested using a co-localization assay as described in Example 5 below. The putative selective regulatory elements for PV can also be tested in B6 PVCre mouse (Jackson Laboratory), which is a B6 PV-Cre knock-in mouse that expresses Cre recombinase in cells that express parvalbumin, as described in Example 2 below. After validating the PV selectivity of the regulatory elements, the regulatory elements can be operationally linked to a transgene in order to target selective expression to PV cells in relation to at least one, two, three, four, five, or more than five non-PV cells.
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EXAMPLE 2 Selectivity for PV Neurons in Mouse PV-Cre [0205] Selectivity for PV neurons can be determined using fluorescent imaging. AAV9 vectors containing eGFP operably linked to (i) a control promoter (EFla); or (ii) a PV selective ER identified in Example 1 above; or (iii) a PV selective ER selected from SEQ ID NOs: 1-32; and AAV9 vectors containing a Cre-dependent tdTomato are injected together into a B6 PV-Cre mouse (Jackson Labs). PV-Cre is a knock-in mouse that expresses Cre recombinase in neurons that express parvalbumin (such as interneurons in the brain and proprioceptive afferent sensory neurons in the root ganglia), without disrupting the expression of endogenous Pvalb.
[0206] Mice are infused bilaterally with 1.5 pL of the AAV9 vector (5 ¹² to ¹³ gc / ml) in the dorsal and ventral hippocampus at a rate of 0.3 pL / min with a 4 min rest period after injection . The mice were anesthetized for injection. The animals were placed in a stereotaxic structure (Kopf instruments, USA), using the following coordinates for the dorsal hippocampus (AP -2.0 mm, lateral ± 1.5, DV -1.4 mm of dura) and for the ventral hippocampus (AP -3.1 mm, lateral ± 2.8, DV -3.8 mm of dura). A Hamilton syringe (model # 80308; 10 pL syringe with corresponding 30 gauge blunt needle) can be used with the stereotactic micro manipulator, to design and drill the holes. The drill is only used to penetrate the bone. After drilling, the infusion cannula is lowered into the brain to the depth of the desired injection site, for example, injection volume: 1.5 pL; injection rate: 0.3 pL / min. Before the infusion, the needle is left to equilibrate for 1 minute. Once the application is complete, the needle is left for 4 min and then removed for approximately 1 min. Once all infusions are complete, the skin incision is closed with sutures and an analgesic is administered after surgery. The treated mice are subjected to daily health checks for the remainder of the study and are weighed once a week to monitor body weight.
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122/133 [0207] For tissue collection, the mice were sacrificed using an overdose of isoflurane and perfused with 4% paraformaldehyde (PFA). A piece of brain tissue containing the hippocampus is extracted and placed in 4% PFA at 4 ° C for at least 12 hours. The brain tissue is then dehydrated in sucrose 30% (in phosphate buffered saline) at 4 ° C until the tissue sinks to the bottom of the tube. The brain tissue is incorporated in Tissue-Tek OCT for sectioning in a cryostat. The sectioned brain tissue is labeled for eGFP and tdTomato using standard immunohistochemical procedures rabbit polyclonal anti-RFP antibody (Rockland Antibodies and Essay) and chicken polyclonal anti-eGFP antibody (Birds Labs). Imaging using a fluorescence microscope is used to visualize the cells. eGFP, or green fluorescence, which corresponds to all gene expression. The red fluorescence of tdTomato corresponds to PV + cells. An overlap of the two fluorescent signals, which can be visualized as white yellow cells, represents the PV + cells that express the eGFP transgene. AAV9 vectors comprising a selective regulatory element for PV are expected to produce a greater number of cells that are eGFP-ι- and PV + compared to the control promoter (EFla). For example, fluorescent imaging of mouse cells injected with AAV9s comprising any of the PV-selective REs (for example, SEQ ID NOs: 1-32 or putative REs identified in Example 1) are expected to show a greater number of eGFP-ι- cells that are also PV +. Selectivity for PV cells can be quantified as the percentage of all eGFP-ι- cells that are also PV +.
EXAMPLE 3
Seizures reduction in Dravet Mouse Model [0208] Mice B6 (alkyl) -Scn7a ^{IM7 "7 £)} were obtained VJ of the European Federation of Dravet syndrome by Jackson Laboratories. These mice have Dravet's syndrome associated with a mutation in exon 24 of SCN1A (A to V at position 1783). The mice also have a floxed exon 24 with the wild type sequence. When not handled, this strain
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123/133 of mice expresses two copies of the WT allele of SCN1A. However, by applying an AAV expressing Cre recombinase, any cell targeted by the AAV will change to express a copy of the mutant allele. By expressing the mutant subunit of SCN1A, mice develop spontaneous seizures within 10 days.
[0209] B6 mice (alkyl) -5cn7a ^{i '7' £ 7)} C57B16 and control ÚJ were injected as in Example 2, AAVs expressing CRE recombinase under the control of promoter and an AAV EFLA comprising the regulatory element selective for cell PV, SEQ ID NO: 32 which directs the expression of either eGFP (SEQ ID NO: 36) or SCN1B (SEQ ID NO: 37). Once all four infusions were complete, implantation by telemetry was immediately performed (F20EET, Data Sciences International). The electrocorticogram data were monitored continuously for 14 days from 10 days after surgery. Data from the electrocorticogram data were analyzed and all seizure events were recorded, noted with date, start time, end time, duration of seizures, and severity score. FIG. 1 illustrates the frequency of seizures in a 12-hour window within 14 days after treatment. The mice treated with SCN1B showed a tendency towards a lower seizure frequency compared to control animals.
[0210] This observation was consistent with the notion that the sodium ion channel unit, for example, SCN1B, can contribute to the trafficking and assembly of the sodium ion channel and that the increase in the selective expression of the beta unit in PV neurons can result in increased trafficking and assembly of the Navl.l channel, thus leading to a trend towards lower seizure frequency and duration in mice treated with SCN1B gene therapy.
EXAMPLE 4
Treatment of Alzheimer's Disease in a Mouse Model [0211] Female APP / PS1 and WT mice bred at PsychoGenics were used in the study. APP / PS1 mice contain human transgenes for both Amyloid Precursor Protein Beta (APP) containing the Swedish mutation
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124/133 (670 G-T and 671 A-C) and Presenilin 1 (PSEN1) containing an L166P mutation, both under the control of the Thyl promoter. These mice develop symptoms of Alzheimer's disease, including amyloid plaques and memory defects. An additional description of these mice can be found in Radde et al, 2006 (Radde, Rebecca, et al. Ap42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO reports 7.9 (2006): 940-946).
[0212J APP / PS1 mice were used as a model to determine the effect of treatment with SCN1B under the control of an ER on the symptoms of Alzheimer's disease. The APP / PS1 mice and non-transgenic controls were injected either with a control vector expressing eGFP or a treatment vector expressing SCN1B, both under the control of SEQ ID NO: 32; and implanted with a TSE transmitter as in Example 3. Brain activity was assessed for 24 hours to 4 weeks after surgery. The electrocorticogram data were automatically analyzed and the power levels in different frequency bands were compared. FIG. 2 illustrates the high gamma power (50-100Hz) in non-transgenic controls (WT), APP / PS1, and APP / PS1 mice treated with SCN1B. An increased potency of high gamma activity is associated with seizures in patients with Alzheimer's and patients with epilepsy. APP / PS1 mice showed a higher level of high-activity potency than control mice. However, the increase was absent in the treated mice indicating the effectiveness of treatment with the vector.
EXAMPLE 5
Selectivity for PV Neurons in Mouse C57BL / 6J (WT) [0213] The selectivity of several REs described here was tested for selective gene expression in PV neurons using immunohistochemistry methods. The C57BL / 6J (WT) mouse strain was used for immunohistochemical assays. Expression cassettes comprising the transgene reporter
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125/133 eGFP operably linked to a regulatory element (SEQ ID NO: 1 or SEQ ID NO: 8) or a CAG promoter in an AAV9 construct.
[0214] Systemic infusions in puppies: C57BL / 6J mice with one day of birth were infused by injection into the facial vein with the AA9 vector (1 E ¹² to 3 E ¹² ) using a 300 U insulin syringe with a needle 31 G. For tissue collection, the mice were sacrificed 21 days after infusion with a sodium pentobarbital super (ip) and perfused with a heparinized saline solution (2.5 lU / ml) followed by perfusion with formaldehyde a 4%. The brains were removed and subsequently fixed by immersion in 4% formaldehyde for 24-48 hours at 4 degrees Celsius. The brain was then placed in PBS containing 30% sucrose and allowed to sink to 4 degrees Celsius (—2-3 days). By sinking the individual brain hemispheres were frozen in Tissue-Tek OCT with the midline facing downwards. The frozen brains were processed for sagittal sections in a cryostat and placed in free float in PBS. The sections were marked for eGFP and parvalbumin (PV) using standard immunohistochemical procedures with chicken anti-GFP (Aves Lab, GFP-1020) and mouse antiPV (Sigma, P3088).
[0215] Systemic infusions in adults: C57BL / 6 mice at 4 weeks of age were infused by injection into the tail vein with 60 pL of the AAV9 vector (4.9 ¹³ to ¹⁴ gc / ml) expressing eGFP. For tissue collection, the mice were sacrificed 21 days after the infusion by means of an isoflurane overdose and the total brains were extracted, washed with PBS and placed in separate 5 ml tubes containing 4% cold formaldehyde. The tissue was fixed at 4 degrees Celsius overnight. The next day, the brain was placed in PBS containing 30% sucrose and allowed to sink to 4 degrees Celsius. By sinking, the individual brain hemispheres were frozen in TissueTek OCT with the midline facing down. The frozen brains were processed for sagittal sections in a cryostat and placed in free float in PBS. The sections were marked for EGFP and parvalbumin (PV) using
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126/133 standard hyunohistochemistry procedures with chicken anti-GFP (Aves Lab, GFP-1020) and mouse anti-PV (Sigma, P3088).
[0216] Immunohistochemistry protocol: Immunohistochemistry was used to analyze the co-localization of the eGFP signal and the PV signal using the anti-PV antibody, where the overlapping of the signals displayed as white or light gray dots in the images of the upper panel (combination), where the representative overlap was indicated by the arrowheads. The overlapping of eGFP and PV fluorescences is indicative of expression in PV cells. Such experiments can be used to determine the selective expression of expression in PV cells. In order to perform immunohistochemical experiments, tissues obtained from each mouse were blocked with a Blocking Buffer Solution (comprising 3% BSA, 3% NGS, 0.3% Triton X-100, 0.2% Tween20 in IX PBS) for 1 hour at room temperature. The tissues were then incubated with primary antibodies in blocking buffer overnight at 4 ° C, washed three times with 1 ml of IX PBS, each at 5 minute intervals. Then the tissues were incubated with secondary antibodies in blocking buffer for 1 hour at room temperature, followed by washing three times, each time with Im L of IX PBS and 5 minutes apart. The tissues were incubated DAPI (1: 1000) in PBS buffer for 5 minutes and washed twice with ImL of IX PBS. The tissues were mounted on slides, formed in image and analyzed using a fluorescence microscope. The images were taken using a Vectra 3 image formation system (Perkin Elmer) and quantified for tagging co-labeling for eGFP and PV using the Inform-Tissue locator, an advanced image analysis software or manually classified. At least 80 GFP positive cells were counted in each panel before determining the percentage of co-localization.
[0217] FIGS. 3A-3C illustrate the results of immunohistochemical experiments performed on puppies after systemic injection of AAV9. FIGS. 4A-4C illustrate the results of similar immunohistochemical experiments performed on adult mice after AAV9 injections.
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127/133 [0218] FIG. 3A illustrates the overlap of immunohistochemical experiments performed on puppies after systemic AAV injections9. FIG. 3B illustrates the quantification of the co-location of immunohistochemical experiments, where selectivity for PV cells was measured as the percentage of GFP + cells that were also PV +, compared to the expression of eGFP under the control of the CAG promoter.
[0219] FIG. 4A illustrates the overlap of immunohistochemical experiments performed on adult mice after systemic AAV injections9. FIG. 4B illustrates the quantification of the co-location of immunohistochemical experiments, where selectivity for PV cells was measured as the percentage of GFP + cells that were also PV +, compared to the expression of eGFP under the control of EFla.
[0220] It is estimated that GABAergic neurons make up about 20% of the CNS, while PV cells make up about 40% of GABAergic neurons, meaning that PV cells represent approximately 8% of all neurons in the CNS. See Pelkey, KA et al., 2017; and Lee, S. et al., 2010. Thus, one could predict that about 8% of cells labeled by a non-selective regulatory element (for example, CAG, EFla, or a constitutive promoter) would be positive for PV, or within this range. Thus, expression in PV cells above 8% is indicative of increased selectivity in PV cells. Notably, injections of AAV9 comprising the regulatory element of SEQ ID NO: 8 resulted in about 60% of cells as positive for PV, which was 7.5 times greater than expected by the distribution of PV cells.
[0221] Immunohistochemical experiments similar to those described above were performed to determine the selective expression of additional regulatory elements, SEQ ID NOs: 2-7 and 9-22, compared to a non-selective regulatory element showing a sequence of SEQ ID NO: 34 , except that the viral vector AAVDJ was used to apply eGFP operatively linked to a regulatory element in C57BL / 6J (WT) mice. Such AAVDJ virus was injected directly into the CNS in the hippocampus of adult mice. At least
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128/133 GFP positive cells were counted in each experiment before calculating the percentage of co-localization, or selectivity, as a percentage of GFP positive cells that were also positive for PV. FIGS. 5A-5F illustrate the fluorescent imaging used to determine colocalization, or selectivity, measured as the percentage of eGFP positive cells that were also positive for PV and compared to the signal from the non-selective regulatory element, SEQ ID NO: 34. Cells that were positive for a marker appear as white / gray cells in the images. The combined images illustrate the overlap between the corresponding eGFP and anti-PV images. Cells that were positive for both eGFP and PV appear as white / light gray cells in the combined image. FIG. 6 illustrates quantification and co-localization analysis, measured as the percentage of eGFP + cells that were also PV +.
EXAMPLE 6
Treatment of Dravet's Syndrome in Different Mouse Strains [0222] The treatment of Dravet's syndrome and / or symptoms using the expression cassettes described here can be tested on several mouse strains, such as B6 (Cg) - ÒYn7 « ^íraAiD VJ as described above, and the mouse strains Scnla ^tmlKea and Scnla-R1470X. These mouse strains are established mouse models for Dravet's syndrome. The Sen 1% ^{! MlKea} and Scnla-R1470X strains of mice do not require CRE recombinase.
[0223] The Scnla ” ^{2A <M} mouse (made available by Jackson Laboratory; described in Hawkins etal., Scientific Reports, vol. 7: 15327 (2017)) presents a deletion of the first coding exon of SCN1 A. Homozygous mice for the knockout allele of SCN1A are characterized by tremors, ataxia, seizures and die after day 16 after parlo. Heterozygous mice on the C57BL / 6 base develop spontaneous seizures and die within weeks. Such a mouse strain can be used for safety and efficacy studies of
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129/133 treatment of epilepsy and Dravet's syndrome. See Miller et al., Genes Brain Behav. 2014 Feb; 13 (2): 163-72 for additional information.
[0224] The Scnla-R1470X mouse is a knock-in mouse presenting a premature termination codon, R1407X, in exon 21 of the SCN1A gene. The same mutation was identified as a pathogenic mutation in three unrelated SMEI patients. Scnla ^{RX / RX} puppies are characterized by recurrent spontaneous seizures in 12 days after giving birth, including tonic-clonic and clonic seizures in 12-16 days after giving birth, and sudden rhythmic movements and involuntary muscle contractions. See Ogiwara et al., Journal of Neuroscience, May 30, 2007, 27 (22) 5903-5914 for additional information.
[0225] In order to test the compositions described here, such as gene therapy with AAV and treatment using such gene therapy, Dravet mice from each of the mouse strains described above and control mice (for example, a wild type mouse or an untreated Dravet mouse for the strain) are injected (for example, administered by intraperitoneal injection) with AAVs expressing either eGFP or another reporter gene, or an expression cassette comprising one or more PV-selective ERs (for example, the SEQ ID NOs: 1-32) as described herein operably linked to a transgene described herein, such as SCN1A, SCN1B, or SCN2B, or any of SEQ ID NOs: 37-39, or a variant or functional fragment thereof. After AAV injections, mouse survival is monitored over time. All mice are monitored daily for general health (for example, weight, hydration, training and mobility) and deaths were recorded. Telemetry implantation can be performed immediately after AAV injections (F20-EET, Data Sciences International). Electrocorticogram data can be recorded and monitored continuously for at least 14 days from 10 days after surgery. All seizure events can be recorded for at least 14 days after treatment with AAV, noted with date, time of onset, time of thermal, duration and severity score. A reduction in frequency and / or
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The duration of seizures after treatment with an AAV as described above in comparison to the eGFP control or an untreated control is indicative of the effectiveness of gene therapy in reducing the symptoms and / or severity of Dravet's syndrome.
[0226] After treating the mice with AAV, the expression levels of the transgene (eg, SCN1A; SCN1B; SCN2B; a DNA-binding protein, such as a transcription activator, that modulates an SCN1 A, SCN1B, or Endogenous SCN2B; aa one of SEQ ID NOs: 37-39; or any variant or functional fragment thereof) can be monitored over time using various PCR and / or sequencing methods to demonstrate that treatment with AAV can result in a increased gene expression in PV cells. Northern blot analyzes and in situ hybridization can also be used to analyze transgenic expression in vivo. The level of the protein expressed from the protein can also be monitored after treatment to demonstrate that an increase in transgenic expression is correlated with an increase in the corresponding protein in vivo. Protein levels can be assayed using a variety of methods, including, but not limited to, Western blot analysis, immunohistochemical, histochemical immunofluorescence assays, and / or ELISA. The formation of ion channels activated by functional sodium voltage can also be tested using current clamp analysis.
[0227] Hyperthermia-induced seizures can be evaluated to compare wild type mice and / or Dravet mice not treated with Dravet mice treated with AAV gene therapy comprising an expression cassette described here (for example, an expression cassette) comprising one or more REs in this report operationally linked to a transgene in this report, such as SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, a functional fragment thereof, or a DNA binding protein that modulates a SCN1A, SNC2A, SNC8A, SCN1B, or endogenous SCN2B In such experiments, the temperature of the nuclear body is monitored with a RET-3 rectal temperature probe (Physitemp Instruments, Inc, New Jersey, USA) and controlled by a
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131/133 heating lamp connected to a rodent temperature regulator (TCAT-2DF, Physitemp) reconfigured with a Partlow 1160 + controller (West Control Solutions, Brighton, UK). Body temperature is increased by 0.5 ° C every two minutes until the first clonic seizure appears. In comparison to untreated Dravet mice, Dravet mice treated with AAV gene therapy are expected to have a higher threshold temperature before the appearance of the first clonic seizure and / or to have a higher proportion of mice that remain seizure-free. at the maximum temperature tested.
[0228] Different doses of AAV comprising an expression cassette can also be administered to mice to determine the safety and efficacy profile of each treatment with gene therapy. These preclinical studies may also inform the optimal dose (s) of the gene therapy to be used for the treatment of Dravet's syndrome.
EXAMPLE 7
Treatment of Alzheimer's Disease in Mice [0229] Female APP / PS1 and wild-type (WT) mice, which were bred at PsychoGenics and are an established mouse model for Alzheimer's disease, can be used for the study of safety and effectiveness of the compositions described herein in the treatment of Alzheimer's disease, comprising one or more selective Res for PV. APP / PS1 mice are described above in Example 4.
[0230] APP / PS1 mice and non-transgenic controls are injected either with a control AAV vector expressing eGFP or a treatment AAV vector comprising one or more PV-selective REs described here, for example, SEQ ID NOs: 1- 32, operationally linked to a transgene that is deficient or deficient in Alzheimer's disease, such as SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3,1, KV3,3, STX BP1, a DNA-binding protein that modulates a gene endogenous (e.g. SCN1A, SNC2A, SNC8A, SCN1B, SCN2B,
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KV3,1, KV3,3, or STX BP1), or any of SEQ ID NOs: 37-43 or a functional fragment thereof.
[0231] After AAV injections, mouse survival is monitored over time. All mice are monitored daily for general health (for example weight, hydration, training and mobility) and deaths were recorded. After injections of the AAVs, the mice are also implanted with an EET transmitter as described in Example 3 above. Brain activity can be recorded and monitored for 24 hours for at least 4 weeks after surgery. The electrocorticogram data can be analyzed automatically, and the power levels in the different frequency bands (50100Hz) can be compared by different groups: WT mice, untreated APP / PS1 mice and APP / PS1 mice treated with AAV, each treated with AAV gene therapy as described above. An increased potency of high gamma activity is associated with seizures in patients with Alzheimer's and patients with epilepsy. Thus, it is expected that untreated APP / PS1 mice will show a higher level of high-activity potency than control mice, while this increase is expected to be absent or reduced in treated mice, indicating effective treatment with a gene therapy by AAV.
[0232] After treating the mice with AAVs, the levels of expression of the transgene can be monitored over time using various methods of PCR and / or sequencing to demonstrate that treatment with AAV can result in an increase in endogenous expression of the transgene. Northern blot analysis and in situ hybridization can also be used to analyze gene expression in vivo. The level of the protein expressed from the transgene can also be monitored after treatment to demonstrate that an increase in gene expression is correlated with an increase in protein levels. The protein level can be assayed using a variety of methods, including, but not limited to, Western blot analysis, immunohistochemical and / or ELISA assays. The formation of voltage-activated sodium or potassium ion channels
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Functional 133/133 can also be tested using current clamp analysis.
[0233] Different doses of AAV comprising an expression cassette can also be administered to mice in order to determine the safety and efficacy profile of each treatment by gene therapy. These pre-clinical studies may also inform the optimal dose (s) of gene therapy to be used for the treatment of Alzheimer's disease,

权利要求:
Claims (80)
[1]
1. Nucleic acid cassette characterized by the fact that it comprises:
one or more regulatory elements operatively linked to a transgene which results in selective expression in neurons of parvalbumin (PV) in the CNS in one or more non-PV cells in the CNS.
[2]
2. Nucleic acid cassette according to claim 1, characterized in that each regulatory element comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence with at least 80% sequence identity to (i) or (ii).
[3]
Nucleic acid cassette according to any one of claims 1-2, characterized in that at least one of the regulatory elements is derived from human.
[4]
Nucleic acid cassette according to any one of claims 1-2, characterized in that at least one of the regulatory elements is derived from a non-human mammal.
[5]
Nucleic acid cassette according to any one of claims 1-2, characterized in that the regulatory elements are non-naturally occurring.
[6]
6. Nucleic acid cassette according to any one of claims 1-2, characterized in that the regulatory elements result in the selective expression of the transgene in PV neurons which is greater than the expression of the same transgene when operationally linked to a non-selective regulatory element , as measured by a co-location assay.
[7]
Nucleic acid cassette according to claim 6, characterized in that the non-selective regulatory element is a constitutive promoter.
[8]
Nucleic acid cassette according to claim 6, characterized in that the non-selective regulatory element is any one of CAG, EFla, SV40, CMV, UBC, PGK, and CBA.
[9]
Nucleic acid cassette according to claim 6, characterized
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2/10 because the regulatory elements result in the selective expression of the transgene in PV neurons at a level that is at least 2 times, at least 5 times, or at least 10 times compared to the selective expression of the transgene in PV neurons when operationally linked to a non-selective regulatory element, as measured by the co-location test.
[10]
10. Nucleic acid cassette according to claim 6, characterized in that the regulatory elements result in selective expression in PV neurons that is at least 2%, at least 5%, at least 10%, at least 15%, at least least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the expression in PV neurons when the transgene is operationally linked to a non-selective regulatory element.
[11]
Nucleic acid cassette according to claim 6, characterized in that the regulatory elements result in selective expression in PV neurons which is about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times,
7.5 times, 8 times, 9 times, or 10 times greater than expected for the natural distribution of PV neurons in the CNS.
[12]
Nucleic acid cassette according to claim 6, characterized in that the co-localization assay is an immunohistochemical assay.
[13]
Nucleic acid cassette according to claim 12, characterized in that the immunohistochemical assay comprises an anti-PV antibody.
[14]
Nucleic acid cassette according to any one of claims 1-2, characterized in that the transgene encodes an ion channel subunit, a neurotransmitter regulator, a DNA binding domain, a gene editing protein, or a variant or functional fragment thereof.
[15]
Nucleic acid cassette according to claim 14, characterized in that the ion channel subunit is an alpha subunit or a beta subunit of a sodium ion channel or a subunit of a potassium ion channel.
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3/10
[16]
16. Nucleic acid cassette according to claim 15, characterized in that the transgene comprises any one of (i) SEQ ID NOs: 37-43; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii).
[17]
Nucleic acid cassette according to claim 15, characterized in that the transgene comprises (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, or KV3.3; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii).
[18]
18. Nucleic acid cassette according to claim 14, characterized in that the transgene is a neurotransmitter regulator comprising (i) STXBP1, (ii) a functional fragment thereof, or (iii) a sequence showing at least 80% of sequence identity to (i) or (ii).
[19]
19. Nucleic acid cassette according to claim 14, characterized in that the transgene comprises a DNA binding protein that modulates the expression of an endogenous gene.
[20]
20. Nucleic acid cassette according to claim 19, characterized in that the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, or STXBP1.
[21]
21. Nucleic acid cassette according to claim 14, characterized in that the transgene comprises a gene editing protein.
[22]
22. Nucleic acid cassette according to claim 21, characterized in that the gene editing protein is a Cas protein.
[23]
23. Nucleic acid cassette according to any one of claims 1-2, characterized in that the combined regulatory elements are less than 2.5 kb, less than 1.5 kb, less than 1 kb, or less than 500 bp in size.
[24]
24. Nucleic acid cassette according to any one of claims 1-2, characterized in that the non-PV cells comprise one or more of the non-PV cell types in the CNS.
[25]
25. Nucleic acid cassette according to claim 24, characterized
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4/10 because non-PV cells comprise one or more of the excitatory neurons, dopaminergic neurons, astrocytes, microglia, and motor neurons.
[26]
26. Nucleic acid cassette according to any one of claims 1-2, characterized in that it is a linear construct.
[27]
27. Nucleic acid cassette according to any one of claims 1-2, characterized in that it is a vector.
[28]
28. Nucleic acid cassette according to claim 27, characterized in that the vector is a plasmid.
[29]
29. Nucleic acid cassette according to claim 28, characterized in that the vector is a viral vector.
[30]
30. Nucleic acid cassette according to claim 29, characterized in that the viral vector is an adeno-associated virus (AAV) vector.
[31]
31. The nucleic acid cassette according to claim 30, characterized in that the AAV vector is AAV1, AAV8, AAV9, scAAV1, scAAV8, or scAAV9.
[32]
32. Nucleic acid cassette according to claim 29, characterized in that the viral vector is a lentiviral vector.
[33]
33. Nucleic acid cassette according to any one of claims 1-2, characterized in that the regulatory elements contain less than 600 bp of contiguous sequence from within 10 kb of the GAD2, GAD1, SYN1 transcription start site , NKX2.1, DLX1, DLX5 / 6, SST, PV, or VIP.
[34]
34. Method for the treatment of a neurological disorder or condition in an individual in need of such treatment, characterized in that it comprises administering a therapeutically effective amount of the nucleic acid cassette as defined in any of claims 1-33.
[35]
35. Method for increasing the selective expression of a transgene in PV neurons in the CNS, characterized by the fact that it contacts a cell with the nucleic acid cassette as defined in any of claims 1-33.
[36]
36. Method to direct the expression of any transgene to PV neurons in the CNS, characterized by the fact that it operationally binds one or more of the
Petition 870190098818, of 10/02/2019, p. 14/134
5/10 regulatory elements of the PV neuron for a transgene.
[37]
37. Method according to claim 36, characterized in that each of the regulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence with at least 80% sequence identity to (i) or (ii).
[38]
38. Method according to any of claims 36-37, characterized in that the regulatory elements result in the selective expression of the transgene in PV neurons that is greater than the expression of the same transgene when operationally linked to a non-selective regulatory element, such as measured by a co-location test.
[39]
39. Method according to claim 38, characterized in that the non-selective regulatory element is a constitutive promoter.
[40]
40. Method according to claim 38, characterized in that the non-selective regulatory element is any one of CAG, EFla, SV40, CMV, UBC, PGK, and CBA.
[41]
41. Method according to claim 38, characterized in that the regulatory elements result in the selective expression of the transgene in PV neurons at a level that is at least 2 times, at least 5 times, or at least 7 times, or at least 10 times compared to a non-selective regulatory element when operationally linked to the transgene, as measured by a co-location assay.
[42]
42. Method according to claim 38, characterized in that the regulatory elements result in selective expression in PV neurons that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20% at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the expression in PV neurons when the transgene is operationally linked to a non-selective regulatory element.
[43]
43. Method according to claim 38, characterized by the fact that the
Petition 870190098818, of 10/02/2019, p. 15/134
6/10 regulatory elements result in selective expression in PV neurons which is about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 7.5 times, 8 times, 9 times , or 10 times greater than expected for the natural distribution of PV neurons in the CNS.
[44]
44. Method according to any one of claims 36-37, characterized in that the transgene is any one of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, STXBP1, a DNA binding protein, a gene editing protein, or a functional fragment thereof.
[45]
45. Method according to any of claims 36-37, characterized in that the regulatory elements and the transgene are in an AAV.
[46]
46. Method according to claim 45, characterized in that the AAV is AAV9.
[47]
47. Method for the treatment of a neurological condition or disorder in an individual in need of such treatment, characterized by the fact that it comprises the contact of a cell with a nucleic acid cassette comprising: one or more regulatory elements operatively linked to a transgene that results in the selective expression of the transgene in PV neurons in one or more non-PV cells in the CNS.
[48]
48. Method according to claim 47, characterized in that each of the regulatory elements comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence with at least 80% sequence identity to (i) or (ii).
[49]
49. Method according to any one of claims 47-48, characterized in that the transgene is a voltage-driven ion channel subunit, or a variant or functional fragment thereof.
[50]
50. Method according to claim 49, characterized in that the subunit is a beta subunit of a sodium ion channel.
[51]
51. Method according to claim 49, characterized in that the subunit is an alpha subunit of a sodium ion channel.
Petition 870190098818, of 10/02/2019, p. 16/134
7/10
[52]
52. The method of claim 49, characterized in that the subunit is a potassium ion channel.
[53]
53. Method according to any of claims 47-48, characterized in that the transgene is any one of (i) SCN1A, SCN1B, SCN2B, KV3.1, and KV3.3; (ii) a functional fragment thereof; or (iii) a sequence having at least 80% sequence identity to (i) or (ii).
[54]
54. Method according to any of claims 47-48, characterized in that the transgene is a DNA binding protein.
[55]
55. The method of claim 54, characterized in that the modular DNA-binding protein is an endogenous gene.
[56]
56. Method according to claim 55, characterized in that the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, or STXBP1.
[57]
57. Method according to any of claims 47-48, characterized in that the transgene is a gene editing protein.
[58]
58. Method according to claim 57, characterized in that the gene editing protein is a Cas protein.
[59]
59. Method according to any of claims 47-48, characterized in that the neurological condition or disorder is associated with a haploinsufficiency or a mutation in any of SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3. 3, and STXBP1.
[60]
60. Method according to any of claims 47-48, characterized in that the neurological condition or disorder is epilepsy, neurodegeneration, taupathy, or neuronal hypoexcitability.
[61]
61. Method according to any of claims 47-48, characterized in that the neurological condition or disorder is Dravet's syndrome.
[62]
62. Method according to any of claims 47-48, characterized in that the neurological condition or disorder is Alzheimer's disease.
Petition 870190098818, of 10/02/2019, p. 17/134
8/10
[63]
63. Method according to any of claims 47-48, characterized in that the regulatory elements result in the selective expression of the transgene in PV neurons that is greater than the expression of the same transgene when operationally linked to a non-selective regulatory element, such as measured by a co-location test.
[64]
64. Method according to claim 63, characterized in that the non-selective regulatory element is a constitutive promoter.
[65]
65. Method according to claim 64, characterized in that the non-selective regulatory element is any one of CAG, EFla, SV40, CMV, UBC, PGK, and CBA.
[66]
66. Method according to any of claims 64-65, characterized in that the regulatory elements result in selective expression in PV neurons at a level that is at least 2 times, at least 5 times, or at least 7 times, or at least 10 times compared to a non-selective regulatory element when operationally linked to the transgene, as measured by a co-location assay.
[67]
67. Method according to any of claims 64-65, characterized in that the regulatory elements result in selective expression in PV neurons that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the expression in PV neurons when the transgene is operationally linked to a non-selective regulatory element.
[68]
68. Method according to any of claims 64-65, characterized in that the regulatory elements result in selective expression in PV neurons which is about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 7.5 times, 8 times, 9 times, or 10 times greater than expected for the natural distribution of PV neurons in the CNS.
Petition 870190098818, of 10/02/2019, p. 18/134
9/10
[69]
69. Method according to any of claims 47-48, characterized in that the nucleic acid cassette is an AAV.
[70]
70. Method according to claim 69, characterized in that the AAV is AAV9.
[71]
71. Method for the treatment of Dravet's syndrome, characterized by the fact that it comprises the contact of a cell with an AAV comprising a transgene, where the transgene is any one of (i) SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, or a DNA binding protein, (ii) a functional fragment thereof, and (iii) a sequence having at least 80% sequence identity to (i) or (ii).
[72]
72. Method according to claim 71, characterized in that the modular DNA-binding protein is an endogenous gene.
[73]
73. Method according to claim 72, characterized in that the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, or SCN2B.
[74]
74. Method according to any of claims 71-73, characterized in that the AAV additionally comprises one or more selective regulatory elements of PV neuron operatively linked to the transgene.
[75]
75. Method according to claim 74, characterized in that each of the regulatory elements independently comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence with at least 80% sequence identity to (i) or (ii).
[76]
76. Method for the treatment of Alzheimer's disease, characterized by the fact of contacting a cell with an AAV comprising a transgene, where the transgene is any one of (i) SCN1 A, SNC2A, SNC8A, SCNIB, SCN2B, KV3. 1, KV3.3, STXBP1, or a DNA binding protein; (ii) a functional fragment thereof; and (iii) a sequence having at least 80% sequence identity to (i) or (ii).
[77]
77. Method according to claim 76, characterized in that the modular DNA-binding protein is an endogenous gene.
Petition 870190098818, of 10/02/2019, p. 19/134
10/10
[78]
78. The method of claim 77, characterized in that the endogenous gene is SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.3, or STXBP1.
[79]
79. Method according to any of claims 76-78, characterized in that the AAV additionally comprises one or more selective regulatory elements of the PV neuron operatively linked to the transgene.
[80]
80. Method according to claim 79, characterized in that each of the regulatory elements independently comprises (i) a sequence of SEQ ID NOs: 1-32, (ii) a functional fragment or a combination thereof, or (iii) a sequence with at least 80% sequence identity to (i) or (ii).

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法律状态:
2020-11-17| B25G| Requested change of headquarter approved|Owner name: ENCODED THERAPEUTICS, INC. (US) |

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2021-10-19| 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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US201762480998P| true| 2017-04-03|2017-04-03|

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PCT/US2018/025940|WO2018187363A1|2017-04-03|2018-04-03|Tissue selective transgene expression|

---