WO2024006775A2 - Compositions et méthodes pour le traitement de dystrophies myotoniques - Google Patents

Compositions et méthodes pour le traitement de dystrophies myotoniques Download PDF

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WO2024006775A2
WO2024006775A2 PCT/US2023/069191 US2023069191W WO2024006775A2 WO 2024006775 A2 WO2024006775 A2 WO 2024006775A2 US 2023069191 W US2023069191 W US 2023069191W WO 2024006775 A2 WO2024006775 A2 WO 2024006775A2
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nucleic acid
acid sequence
seq
aav
vector
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WO2024006775A3 (fr
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Mikio Takeuchi
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Astellas Gene Therapies Inc
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Astellas Gene Therapies Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the invention relates to the field of therapeutic treatment of myotonic muscular dystrophies in patients, such as myotonic dystrophy type 1 in human patients.
  • Myotonic dystrophy type 1 (DM1 ) is an inherited autosomal dominant muscle degenerative disease caused by mutations in and subsequent improper splicing of the myotonic dystrophy protein kinase gene (DMPK), which results in the sequestration of various splicing factors, ultimately causing alternative splice events of a series of a group of developmentally regulated genes to revert to a fetal profile.
  • DM1 is one of the most common neuromuscular disorders in adults and results in progressive muscle wasting and weakness. Patients diagnosed with DM1 often experience prolonged muscle contractions (myotonia) and are not able to relax certain muscles after use (e.g., a person may experience difficulty in releasing their grip on a doorknob).
  • DM1 progression is characterized by abnormal heart rhythm (arrhythmia) or weakened heartbeat, respiratory muscle weakness, acute muscle pain, and endocrine disturbances leading to other conditions including thyroid problems and diabetes.
  • arrhythmia abnormal heart rhythm
  • DM1 a heartbeat
  • respiratory muscle weakness a muscle weakness
  • acute muscle pain a muscle pain
  • endocrine disturbances leading to other conditions including thyroid problems and diabetes.
  • Cellular and animal models of DM1 indicate that overexpression of one or more of these sequestered splicing factors improves the splicing outcome of DMPK.
  • DM1 is a pathology that is induced by mutations in, and subsequent improper splicing of, the gene DMPK, which results in the sequestration of splicing factors and subsequent reversion of the alternative splice profile of affected cells to their fetal stage splice profile.
  • compositions described herein that may be used to treat such disorders include nucleic acids encoding transgenes for splicing factors, such as muscleblind-like splicing regulator 1 (MBNL1 ), which are operably linked to a desmin (DES) promoter and promote the overexpression of the encoded transgene by way of various cellular processes.
  • the present disclosure additionally features vectors, such as viral vectors, encoding such transgenes operably linked to DES promoters.
  • Exemplary viral vectors described herein that encode transgenes operably linked to DES promoters for overexpressing splicing factors in target cells are adeno-associated viral (AAV) vectors, such as pseudotyped AAV2/8 and AAV2/9 vectors.
  • AAV adeno-associated viral
  • a patient diagnosed with or displaying one or more symptoms of a myotonic dystrophy can be administered a nucleic acid containing a transgene operably linked to a DES promoter, or a vector encoding the same, so as to increase the expression of splicing factor proteins that are sequestered by repeat- expanded RNA.
  • compositions and methods described herein can be used to treat patients having DM1 , wherein patients may be administered a nucleic acid construct including a MBNL1 transgene operably linked to a DES promoter or a viral vector, such as an AAV vector, encoding such a construct, thereby increasing the expression of MBNL1 .
  • MBNL1 -expressing cells in patients not diagnosed with a myotonic dystrophy, such as DM1 and/or not experiencing one or more symptoms of DM1 , such as myotonia, typically express a mature adult phenotype splicing profile.
  • MBNL1 -expressing cells in patients diagnosed with or displaying one or more symptoms of a myotonic dystrophy, such as DM1 contain sequestered splicing proteins, such as MBNL1 .
  • This sequestration of MBNL1 and other splicing factors causes the splicing profile of these cells to revert to the fetal profile and serves as an underlying cause of DM1 .
  • the compositions and methods described herein can be used to treat patients whose muscle and neuronal cells contain sequestered MBNL1 and other splicing proteins, thereby overexpressing MBNL1 to orchestrate the proper splicing of proteins associated with muscle function and treat this underlying cause of myotonic dystrophy.
  • compositions and methods described herein can be used to ameliorate symptoms of, and treat one or more underlying causes of, various other disorders associated with myotonic dystrophies and the sequestration of muscle-specific splicing factors such as MBNL1 .
  • the present disclosure is based, in part, on the surprising discovery that operably linking a transgene construct to a DES promoter can be used to effectively overexpress the transgene product in target cells in a rapid and robust manner in comparison to current methods.
  • the compositions and methods described herein can thus increase the expression of MBNL1 rapidly and to a greater extent than current therapies. This property provides an important clinical benefit.
  • the invention features an adeno-associated virus (AAV) containing a transgene that encodes human MBNL1 .
  • the transgene may be operably linked to a DES promoter.
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO:
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO:
  • the DES promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has the nucleic acid sequence of SEQ ID NO: 3.
  • the MBNL1 has an amino acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MBNL1 has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12.
  • the MBNL1 has an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MBNL1 has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the transgene has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 .
  • the transgene has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 .
  • the transgene has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has the nucleic acid sequence of any one of SEQ ID NOs: 4-11.
  • the transgene has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 4.
  • the transgene has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4.
  • the transgene has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4.
  • the transgene has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has the nucleic acid sequence of SEQ ID NO: 4.
  • the AAV includes capsid proteins from an AAV serotype selected from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhIO, and AAVrh74.
  • the AAV is a pseudotyped AAV.
  • the pseudotyped AAV is AAV2/8.
  • the pseudotyped AAV is AAV2/9.
  • the AAV further includes a polyadenylation site (pA), optionally wherein the pA is positioned 3’ to the transgene.
  • the pA site includes the simian virus 40 (SV40) late pA site or the SV40 early pA site.
  • the pA site is the SV40 late pA site.
  • the AAV further includes an intron, optionally wherein the intron is positioned 3’ to the promoter and 5’ to the transgene.
  • the intron is a betaglobin intron.
  • the invention features a pharmaceutical composition including the AAV of any of the foregoing aspects and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the invention features a nucleic acid molecule including, a DES promoter, a beta-globin intron, a transgene encoding MBNL1 , and a SV40 pA site, wherein the components are operably linked to each other in a 5’-to-3’ direction as DES-beta-globin intron-MBNL1 -SV40 pA.
  • the SV40 pA site includes the SV40 late pA site or the SV40 early pA site. In some embodiments, the pA site is the SV40 late pA site.
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region that has the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region that has the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 3.
  • 85% identical e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the DES promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has the nucleic acid sequence of SEQ ID NO: 3.
  • the MBNL1 has an amino acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MBNL1 has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12.
  • the MBNL1 has an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the MBNL1 has the amino acid sequence of SEQ ID NO: 12.
  • the transgene has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 .
  • the transgene has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 .
  • the transgene has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 .
  • the transgene has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 . In some embodiments, the transgene has the nucleic acid sequence of any one of SEQ ID NOs: 4-11.
  • the transgene has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 4.
  • the transgene has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4.
  • the transgene has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4.
  • the transgene has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the transgene has the nucleic acid sequence of SEQ ID NO: 4.
  • the invention features a vector including the nucleic acid molecule of the foregoing aspect, optionally wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector.
  • the vector is a viral vector.
  • the viral vector is selected from the group consisting of an AAV, an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, and a synthetic virus.
  • the viral vector is an AAV.
  • the AAV includes capsid proteins from an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhI O, and AAVrh74.
  • the AAV is a pseudotyped AAV.
  • the pseudotyped AAV is AAV2/8 or AAV2/9, optionally wherein the pseudotyped AAV is AAV2/8.
  • the AAV includes a recombinant capsid protein.
  • the AAV of the first aspect or the vector has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV of the first aspect or the vector has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV of the first aspect or the vector has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV of the first aspect or the vector has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV of the first aspect or the vector has the nucleic acid sequence of SEQ ID NO: 20.
  • the invention features a plasmid encoding the viral vector of the foregoing aspect.
  • the plasmid includes a promoter (e.g., a prokaryotic promoter) operably linked to a selectable marker gene.
  • the selectable marker gene is an antibiotic resistance gene.
  • the invention features a pharmaceutical composition including the nucleic acid molecule of any one of the foregoing aspects, the vector of any one of the foregoing aspects, or the plasmid of any one of the foregoing aspects, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the invention features a method of treating myotonic muscular dystrophy type 1 (DM1 ) in a human patient in need thereof, the method including administering to the patient a therapeutically effective amount of the nucleic acid molecule of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • DM1 myotonic muscular dystrophy type 1
  • the invention features a method of increasing MBNL1 expression in a human patient in need thereof, the method including administering to the patient a therapeutically effective amount of the nucleic acid molecule of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • the invention features a method of inducing corrective splicing of one or more RNA transcript substrates of MBNL1 in a human patient diagnosed as having DM1 , the method including administering to the patient a therapeutically effective amount of the nucleic acid molecule of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • the patient is at least 18 years of age. In some embodiments of any one of the foregoing method aspects, upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in whole blood MBNL1 level, optionally wherein the patient exhibits the increase in whole blood MBNL1 level by about 12 weeks after administration. In some embodiments of any one of the foregoing method aspects, upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in whole blood MBNL1 level, optionally wherein the patient exhibits the increase in whole blood MBNL1 level by about 12 weeks after administration.
  • the patient upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits a reduction in abnormal splicing of one or more genes selected from kinesin family member 13A (KIF13A); dystrophin (DMD) insulin receptor (/NSR); chloride voltage-gated channel 1 (CLCN1) troponin T2; cardiac type (TNNT2): troponin T3, fast skeletal type (TNNT3): titin (TTN); and bridging integrator 1 (BIN1), optionally wherein the patient exhibits the reduction in abnormal splicing by about 12 weeks after administration.
  • KIF13A kinesin family member 13A
  • DMD dystrophin
  • TNNT2 chloride voltage-gated channel 1
  • TNNT2 cardiac type
  • TNNT3 fast skeletal type
  • TTN titin
  • BIN1 bridging integrator 1
  • the reduction in abnormal splicing of INSR upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, includes an increase in the expression of the INSR-B isoform, optionally wherein the patient exhibits the increase in the expression of the INSR-B isoform by about 12 weeks after administration.
  • the reduction in abnormal slicing of CLCN1 upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, includes the increase in CLCN1 transcripts including exclusion of the exon 7a, optionally wherein the patient exhibits the increase in CLCN1 transcripts that exclude exon 7a by about 12 weeks after administration.
  • the invention features a method of treating DM1 in a human patient diagnosed as having a spliceopathy of an endogenous ATP2A1, CLCN1, and/or LDB3 gene, the method including administering to the patient a therapeutically effective amount of the nucleic acid of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • the invention features a method of increasing expression of functional sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1 ) protein in a human patient diagnosed as having DM1 , the method including administering to the patient a therapeutically effective amount of the nucleic acid of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • SERCA1 functional sarcoplasmic/endoplasmic reticulum calcium ATPase 1
  • the invention features a method of increasing expression of functional chloride voltage-gated channel 1 (CLCN1 ) protein in a human patient diagnosed as having DM1 , the method including administering to the patient a therapeutically effective amount of the nucleic acid of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • CLCN1 functional chloride voltage-gated channel 1
  • the invention features a method of increasing expression of functional ZO- 2 associated speckle protein (ZASP) in a human patient diagnosed as having DM1 , the method including administering to the patient a therapeutically effective amount of the nucleic acid of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects.
  • ZASP functional ZO- 2 associated speckle protein
  • expression of SERCA1 mRNA increases by from 1 .1 -fold to 10-fold.
  • expression of CLCN1 mRNA increases by from 1 .1 -fold to 10-fold.
  • expression of ZASP mRNA increases by from 1 .1 -fold to 10-fold.
  • expression of CLCN1 mRNA increases by from 1 .1 -fold to 10-fold.
  • the patient upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an improvement in myotonia, optionally wherein the patient exhibits an improvement in myotonia by about 12 weeks after administration.
  • the patient upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits a reduction in abnormal heart rhythms, optionally wherein the patient exhibits the reduction in abnormal heart rhythms by about 12 weeks after administration.
  • the patient upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits a reduction in excessive daytime sleepiness, optionally wherein the patient exhibits the reduction in excessive daytime sleepiness by about 12 weeks after administration.
  • the patient upon administration of the nucleic acid, AAV vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits increased strength in the muscles in the hands, optionally wherein the patient exhibits increased strength in the muscles in the hands by about 12 weeks after administration. In some embodiments, the strength in the muscles in the hands is measured by a grip test.
  • the patient upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an improvement in sleep apnea symptoms, optionally wherein the patient exhibits the improvement in sleep apnea symptoms by about 12 weeks after administration.
  • the patient upon administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in corrective splicing of RNA transcripts encoding insulin receptor, ryanodine receptor 1 , cardiac muscle troponin, and/or skeletal muscle troponin.
  • the patient before treatment, is not diagnosed with DM1 . In some embodiments, before treatment, the patient has one or more symptoms of DM1 . In some embodiments, the symptoms of DM1 include breathing difficulties, aspiration, sleep apnea, intellectual impairment, excessive daytime sleepiness, heart condition abnormalities, arrhythmias, cardiomyopathy, swallowing issues, muscle weakness, muscle atrophy, myotonia, and/or muscle pain.
  • the nucleic acid, vector, plasmid, or pharmaceutical composition is administered to the patient by way of intravenous, intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, or oral administration and/or by inhalation, perfusion, or lavage.
  • the invention features a kit including the nucleic acid of any one of the foregoing aspects, the AAV of any one of the foregoing aspects, the vector of any one of the foregoing aspects, the plasmid of any one of the foregoing aspects, or the pharmaceutical composition of any one of the foregoing aspects, and a package insert, wherein the package insert instructs a user of the kit to administer the nucleic acid, vector, plasmid, or pharmaceutical composition to a human patient diagnosed as having DM1 or displaying one or more symptoms of DM1.
  • FIG. 1 is a schematic drawing of the RNA polypeptide design. From 5’ to 3’, the construct includes the human desmin (hDes) promoter sequence, an intron sequence, a human muscleblindlike splicing regulator 1 (MBNL1 ) cDNA sequence, and an SV40 polyadenylation (polyA) sequence.
  • hDes human desmin
  • MBNL1 human muscleblindlike splicing regulator 1
  • polyA polyadenylation
  • the term “about” refers to a value that is within 10% above or below the value being described.
  • “activity” refers to form(s) of a nucleic acid or polypeptide which retains a biological activity of the native or naturally occurring nucleic acid or polypeptide, respectively, wherein “biological” activity refers to a biological function (e.g., splicing) caused by a native or naturally occurring nucleic acid or polypeptide, respectively.
  • “administration” refers to providing or giving a subject a therapeutic agent (e.g., an inhibitory agent) by any effective route. Exemplary routes of administration are described herein and below (e.g., intracerebroventricular (ICV) injection, intrathecal (IT) injection, intraparenchymal (IP) injection, intravenous (IV) injection, and stereotactic injection). Administration may be systemic or local.
  • a “combination therapy” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition (e.g., DM1 ).
  • the treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap.
  • the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated.
  • the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen.
  • administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic).
  • Sequential or substantially simultaneous administration of each therapeutic agent can be done by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection while a second therapeutic agent of the combination may be administered orally.
  • the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1, below.
  • conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W.
  • a conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).
  • the terms “desmin promoter” and “DES promoter” refer to any of the nucleic acids set forth in SEQ ID NOs: 1 -3, as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the nucleic acid sequence of any one of SEQ ID NOs: 1 -3 and that promote the expression of a transgene in a cell (e.g., a eukaryotic cell, such as a mammalian cell, human cell, or human muscle cell) when the transgene is operably linked to the promoter.
  • a cell e.g., a eukaryotic cell, such as a mammalian cell, human cell, or human muscle cell
  • DMPK distrophia myotonica protein kinase
  • forms of the human DMPK gene that encode a DMPK protein having one or more (e.g., up to 25) conservative amino acid substitutions relative to a wild-type DMPK protein.
  • the expanded repeat region may contain, for example, 50 or more CUG trinucleotide repeats, such as from about 50 to about 4,000 CUG trinucleotide repeats (e.g., 50 trinucleotide repeats, 60 trinucleotide repeats, 70 trinucleotide repeats, 80 trinucleotide repeats, 90 trinucleotide repeats, 100 trinucleotide repeats, 110 trinucleotide repeats, 120 trinucleotide repeats, 130 trinucleotide repeats, 140 trinucleotide repeats, 150 trinucleotide repeats, 160 trinucleotide repeats, 170 trinucleotide repeats, 180 trinucleotide repeats, 190 trinucleotide repeats, 200 trinucleotide repeats, 210 trinucleotide repeats, 220 trinucleotide repeats,
  • an “effective amount” of any one of the compounds or a combination of any of the compounds or a pharmaceutically acceptable salt thereof is administered via any of the usual and acceptable methods known in the art, either singly or in combination.
  • the term “endogenous” describes a molecule (e.g., a metabolite, polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • a gene refers to a region of DNA that encodes a protein.
  • a gene may include regulatory regions and a protein-coding region.
  • a gene includes two or more introns and three or more exons, wherein each intron forms an intervening sequence between two exons.
  • IRES refers to an internal ribosome entry site.
  • an IRES sequence is a feature that allows eukaryotic ribosomes to bind an mRNA transcript and begin translation without binding to a 5' capped end.
  • An mRNA containing an IRES sequence produces two translation products, one initiating form the 5' end of the mRNA and the other from an internal translation mechanism mediated by the IRES.
  • the “length” of a nucleic acid refers to the linear size of the nucleic acid as assessed by measuring the quantity of nucleotides from the 5’ to the 3’ end of the nucleic acid. Exemplary molecular biology techniques that may be used to determine the length of a nucleic acid of interest are known in the art.
  • muscle dystrophy is meant the group of muscle diseases that weaken the musculoskeletal system and hamper locomotion. Muscular dystrophies are characterized by progressive deterioration of muscle function (e.g., weakness), defects in muscle proteins, and death of muscle cells and tissue. Examples of muscular dystrophies include but are not limited to congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.
  • the terms “muscleblind-like splicing regulator 1 ” and its abbreviation, “MBNL1 ,” refer to the RNA-binding splicing protein involved in the regulation of alternative splicing, for example, in human subjects.
  • the terms “muscleblind-like splicing regulator 1 ” and “MBNL1 ” are used interchangeably herein and refer not only to wild-type forms of MBNL1 resulting from processing in the cell, but also to any other naturally occurring variants of MBNL1 (e.g., splice variants or allelic variants as described in Table 2).
  • the nucleic acid sequence of MBNL1 variants are provided herein as SEQ ID NOs: 4-11 .
  • Accepted variants of MBNL1 have a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11 .
  • the amino acid sequence of MBNL1 isoforms are provided herein as SEQ ID NOs: 12-19.
  • Accepted isoforms of MBNL1 have an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • any amino acid sequence differing from SEQ ID NOs: 12-19 by one or more (e.g., two, three, four, or five) conservative substitutions are additionally considered to be acceptable isoforms of MBNL1 .
  • mutation refers to any change in the sequence of a gene, such that the sequence is not identical to that of the wild type gene.
  • a mutation may be selected from the group including a single nucleotide point mutation that results in a premature termination codon, a single nucleotide insertion, a single nucleotide deletion, the insertion of two or more contiguous nucleotides, the deletion of two or more contiguous nucleotides, the duplication of a contiguous region within a gene, or the deletion of a contiguous region within a gene.
  • a mutated gene may include a single mutation or multiple mutations. A mutation may occur in any region of the gene.
  • myotonic dystrophy refers to an inherited muscle wasting disorder characterized by the nuclear retention of RNA transcripts encoding DMPK and containing an expanded CUG trinucleotide repeat region in the 3’ untranslated region (UTR) encoded by exon 15, such as an expanded CUG trinucleotide repeat region having from 50 to 4,000 CUG repeats. Wildtype DMPK RNA transcripts, by comparison, typically contain from 5 to 37 CUG repeats in the 3' UTR, encoded by exon 15. In patients having myotonic dystrophy, the expanded CUG repeat region interacts with RNA-binding splicing factors, such as muscleblind-like splicing regulator 1 (MBNL1 ).
  • MBNL1 muscleblind-like splicing regulator 1
  • DM1 myotonic dystrophy
  • skeletal muscle is often the most severely affected tissue, but the disease also imparts toxic effects on cardiac and smooth muscle, the ocular lens, and the brain.
  • the cranial, distal limb, and diaphragm muscles are preferentially affected.
  • Manual dexterity is compromised early, which causes several decades of severe disability.
  • the median age at death of myotonic dystrophy patients is 55 years, which is usually caused by respiratory failure (de Die-Smulders C E, et al., Brain 121 :1557-1563 (1998)).
  • neuron is used to refer to a type of electrically excitable cell in the brain and nervous system which consists of a cell body, or soma, dendrites, and an axon.
  • the terminal end of the axon of one neuron is separated from a dendrite of a neighboring neuron by a space called the synaptic cleft, through which signals can be passed and intercellular communication can be achieved.
  • the synaptic cleft a space called the synaptic cleft
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides of any length. Examples of polynucleotides are DNA polynucleotides and RNA polynucleotides. All nucleic acid sequences herein are written in the 5’-to-3’ direction and are to be construed accordingly.
  • operably linked in the context of a nucleic acid refers to a nucleic acid that is placed into a structural or functional relationship with another nucleic acid.
  • one segment of DNA may be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding region so as to facilitate transcription of the coding region.
  • the operably linked nucleic acids are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.
  • a nucleic acid molecule described herein is operably linked to a desmin (DES) promoter.
  • DES desmin
  • Percent (%) sequence complementarity with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity.
  • a given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs.
  • Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine- guanine base pairs.
  • a proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.”
  • Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared.
  • the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, is calculated as follows:
  • a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.
  • nucleic acid sequence or a portion thereof that need not be fully complementary (e.g., 100% complementary) to a target region or a nucleic acid sequence or a portion thereof that has one or more nucleotide mismatches relative to the target region but that is still capable of hybridizing to the target region under specified conditions.
  • the nucleic acid may be, e.g., 95% complementary, 90%, complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, but still form sufficient base pairs with the target so as to hybridize across its length.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • composition represents a composition containing a nucleic acid described herein, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a subject.
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit/risk ratio.
  • plasmid refers to an extrachromosomal circular double stranded DNA molecule into which additional DNA segments may be ligated.
  • a plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids).
  • Other vectors e.g., non-episomal mammalian vectors
  • promoter refers to a region within the regulatory region of a gene that enables initiation of the transcription of a gene into a messenger RNA, wherein transcription is initiated with the binding of an RNA polymerase on or nearby the promoter.
  • the promoter is a DES promoter.
  • reading frame refers to how a ribosome defines a codon within a gene, as determined by binding of a tRNA to a three-nucleotide codon during translation of an mRNA.
  • a reading frame is “restored,” this indicates that the reading frame had first been first altered by a frameshift or nonsense mutation, and subsequently returned to the original reading frame by some means.
  • a “reference” is meant any useful reference used to compare protein or nucleic acid (e.g., mRNA) levels related to DM1 (e.g., DMPKor MBNL1).
  • the reference can be any sample, standard, standard curve, or level that is used for comparison purposes.
  • the reference can be a normal reference sample or a reference standard or level.
  • a “reference sample” can be, for example, a control, e.g., a predetermined negative control value such as a “normal control” or a prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having DM1 or not displaying symptoms of DM1 (e.g., DMPK or MBNL1 , a sample from a subject that is diagnosed with DM1 (e.g., DMPK or MBNL1); a sample from a subject that has been treated for DM1 (e.g., DMPK or MBNL1); or a sample of a purified protein (e.g., MBNL1 ) at a known normal concentration.
  • a control e.g., a predetermined negative control value such as a “normal control” or a prior sample taken from the same subject
  • a sample from a normal healthy subject such as
  • reference standard or level is meant a value or number derived from a reference sample.
  • a “normal control value” is a predetermined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (“between X and Y”), a high threshold (“no higher than X”), or a low threshold (“no lower than X”).
  • a subject having a measured value within the normal control value for a particular biomarker is typically referred to as “within normal limits” for that biomarker.
  • a normal reference standard or level can be a value or number derived from a normal subject not having a muscular dystrophy (e.g., MD).
  • the reference sample, standard, or level is matched to the subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health.
  • a standard curve of levels of a purified protein, e.g., any described herein, within the normal reference range can also be used as a reference.
  • repeat region refers to segments within a gene or an RNA transcript thereof containing nucleic acid repeats, such as the poly CTG sequence in the 3’ UTR of the human DMPK gene (or the poly CUG sequence in the 3’ UTR of the RNA transcript thereof).
  • a repeat region is considered to be an “expanded repeat region,” a “repeat expansion,” or the like, if the number of nucleotide repeats in the repeat region exceeds the quantity of repeats ordinarily found in the repeat region of a wild-type form of the gene or RNA transcript thereof.
  • the 3’ UTRs of wild-type human DMPK genes typically contain from 5 to 37 CTG or CUG repeats.
  • “Expanded repeat regions” and “repeat expansions” in the context of the DMPK gene or an RNA transcript thereof thus refer to repeat regions containing greater than 37 CTG or CUG repeats, such as from about 50 to about 4,000 CUG trinucleotide repeats (e.g., 50 trinucleotide repeats, 60 trinucleotide repeats, 70 trinucleotide repeats, 80 trinucleotide repeats, 90 trinucleotide repeats, 100 trinucleotide repeats, 110 trinucleotide repeats, 120 trinucleotide repeats, 130 trinucleotide repeats, 140 trinucleotide repeats, 150 trinucleotide repeats, 160 trinucleotide repeats, 170 trinucleotide repeats, 180 trinucleotide repeats, 190 trinucleotide repeats, 200 trinucleotide repeats,
  • skeletal muscle is meant the form of striated muscle tissue which is under the control of the somatic nervous system, i.e., it is voluntarily controlled.
  • the term muscle refers to multiple bundles of muscle fibers held together by connective tissue. Skeletal muscles may be attached to bones by tendons.
  • Non-limiting examples of skeletal muscles include, for example, the diaphragm, extensor digitorum longus, tibialis anterior, gastrocnemius, soleus, plantaris, biceps, triceps, deltoids, pectoralis major, pectoralis minor, rhomboids, trapezius, sartorius, knee flexors and extensors, elbow flexors and extensors, shoulder abductors, and abdominal muscles.
  • spliceopathy refers to a change in the splicing pattern of an mRNA transcript that leads to the expression of one or more alternative splice products relative to a wild-type form of the mRNA transcript of interest. Spliceopathy can lead to a toxic loss of function if, for example, the mRNA transcript is spliced in such a way that one or more exons necessary for the activity of the encoded protein are no longer present in the mRNA transcript upon translation. Additionally or alternatively, toxic loss of function may occur due to the aberrant inclusion of one or more introns, for example, in a manner that precludes the proper folding of the encoded protein.
  • the terms “subject” and “patient” are interchangeable and refer to an organism that receives treatment for a particular disease or condition as described herein.
  • the subject is a human.
  • transduction and “transduce” refer to a method of introducing a viral vector construct or a part thereof into a cell and subsequent expression of a transgene encoded by the vector construct or part thereof in the cell.
  • transcription regulatory element refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help to control gene transcription. Examples of transcription regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990). In some embodiments, a transcription regulatory element of a composition herein is a DES promoter.
  • transfection refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium-phosphate precipitation, diethylaminoethyl (DEAE)-dextran transfection, NUCLEOFECTIONTM, squeeze-poration, sonoporation, optical transfection, MAGNETOFECTIONTM, impalefection, and the like.
  • electroporation lipofection
  • calcium-phosphate precipitation calcium-phosphate precipitation
  • DEAE diethylaminoethyl
  • NUCLEOFECTIONTM squeeze-poration
  • sonoporation sonoporation
  • optical transfection MAGNETOFECTIONTM
  • impalefection and the like.
  • the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a heritable muscle-wasting disorder, for example, myotonic dystrophy, and particularly, DM1 .
  • beneficial or desired clinical results that are indicative of successful treatment include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment of a patient having a myotonic dystrophy may manifest in one or more detectable changes, such as an increase in MBNL1 expression (e.g., an increase in MBNL1 expression by about 1 % or more, such as an increase by about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%), relative to the expression of MBNL1 by the patient prior to administration of a therapeutic agent, such as a vector or nucleic acid described herein.
  • a therapeutic agent such as a vector or nucleic acid described herein.
  • RNA transcripts that is spliced in a manner that is dependent upon MBNL1 .
  • observations that signal successful treatment of a patient having myotonic dystrophy include a finding that the patient exhibits an increase in corrective splicing of one or more RNA transcript substrates of MBNL1 following administration of a therapeutic agent, such as a therapeutic agent described herein.
  • indicators that signal successful treatment of myotonic dystrophy include a determination that the patient exhibits an increase in expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1 ) mRNA containing exon 22, such as an increase of about 1 .1 -fold to about 10-fold, or more (e.g., an increase in expression of SERCA1 mRNA containing exon 22 by about 1 .1 -fold, 1 .2-fold, 1 .3- fold, 1 .4-fold, 1 .5-fold, 2-fold, 2.1 -fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more), as assessed, for example, using an RNA or protein detection assay described herein.
  • SERCA1 sarcoplasmic/endoplasmic reticulum calcium ATPase 1
  • Treatment of myotonic dystrophy may also manifest as a decrease in expression of chloride voltagegated channel 1 (CLCN1 ) mRNA containing exon 7a, such as a decrease of about 1 % to about 100% (e.g., a decrease in expression of CLCN1 mRNA containing exon 7a by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), as assessed, for example, using an RNA or protein detection assay described herein.
  • CLCN1 chloride voltagegated channel 1
  • successful treatment may be signaled by a determination that the patient exhibits a decrease in expression of ZO-2 associated speckle protein (ZASP) containing exon 1 1 , such as a decrease of about 1 % to about 100% (e.g., a decrease in expression of ZASP mRNA containing exon 1 1 by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), as assessed, for example, using an RNA or protein detection assay described herein.
  • ZASP ZO-2 associated speckle protein
  • Successful treatment of myotonic dystrophy may also be signaled by a finding that, following the therapy, the patient exhibits an increase in corrective splicing of RNA transcripts encoding insulin receptor, ryanodine receptor 1 (RYR1 ), cardiac muscle troponin, and/or skeletal muscle troponin, such as an increase of about 1 .1 -fold to about 10-fold, or more (e.g., an increase in expression of correctly spliced RNA transcripts encoding insulin receptor, RYR1 , cardiac muscle troponin, and/or skeletal muscle troponin by about 1 .1 -fold, 1 .2-fold, 1 .3-fold, 1 .4-fold, 1 .5-fold, 2-fold, 2.1 -fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more), as assessed, for example, using an RNA or protein detection assay described herein.
  • vector includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus, or other suitable replicon (e.g., viral vector).
  • a DNA vector such as a plasmid, an RNA vector, virus, or other suitable replicon (e.g., viral vector).
  • a variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell.
  • expression vectors are disclosed in, e.g., WO 1994/011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a gene of interest.
  • Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of nucleic acid molecules as described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • nucleic acid molecules contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an IRES, and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.
  • compositions and methods described herein are useful for reducing the occurrence of spliceopathy and for treating disorders associated with ribonucleic acid (RNA) dominance, such as myotonic dystrophy (e.g., myotonic dystrophy type 1 (DM1 )).
  • RNA ribonucleic acid
  • the compositions described herein include nucleic acid molecules that include a transgene encoding human muscleblind-like splicing regulator 1 (MBNL1 ), wherein the transgene is operably linked to a desmin (DES) promoter, and methods for the delivery of this transgene into cells to overexpress the resulting protein MBNL1 .
  • MBNL1 human muscleblind-like splicing regulator 1
  • DES desmin
  • compositions described herein may ameliorate this pathology by increasing the expression of MBNL1 in human patients diagnosed with or displaying one or more symptoms of DM1 .
  • the compositions and methods described herein may be used to treat disorders, such as DM1 , or symptoms of such disorders, such as myotonia.
  • the nucleic acids described herein may be encoded by a vector, such as a viral vector.
  • a vector such as a viral vector.
  • AAV adeno-associated viral
  • pseudotyped AAV vectors e.g., an AAV2/8 vector
  • the present invention is based, at least in part, on the discovery that operably linking a DES promoter to a transgene encoding MBNL1 and encoding the resulting nucleic acids into a viral vector, such as a pseudotyped AAV2/8 vector, leads to a highly efficient targeted delivery of the nucleic acids into cells and rapid, robust overexpression of MBNL1 .
  • the expression of MBNL1 can be rapidly increased in patients diagnosed with or displaying one or more symptoms of DM1 , such as myotonia, which allows for a reversion of the expression of a group of developmentally regulated genes from their inappropriately adopted fetal-stage expression to the desired mature-stage expression.
  • This switch in gene expression and subsequent shift in protein expression mitigates symptoms of DM1 and may ameliorate the disease.
  • nucleic acids that may be used in conjunction with the compositions and methods described herein, as well as a description of vectors encoding such nucleic acids and methods that may be used to treat patients diagnosed with or displaying one or more symptoms of DM1 .
  • Muscular dystrophies are a group of inherited disorders characterized by progressive muscle weakness. Myotonic dystrophies are inherited autosomal dominant muscular dystrophy disorders. Myotonic dystrophy type 1 (DM1 ) is a myotonic dystrophy disorder caused by mutations in and subsequent improper splicing of the myotonic dystrophy protein kinase gene (DMPK), which results in the sequestration of the splicing protein muscleblind-like splicing regulator 1 (MBNL1 ) in ribonuclear foci. This ultimately causes alternative splice events of a series of a group of developmentally regulated genes to revert to a fetal profile.
  • DMPK myotonic dystrophy protein kinase gene
  • a patient having a myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia
  • a nucleic acid molecule that includes a transgene encoding MBNL1 operably linked to a desmin (DES) promoter, or a composition encoding the same, so as to overexpress MBNL1 .
  • DES desmin
  • this overexpression provides the beneficial effect of restoring proper mature-stage MBNL1 expression, which in turn reverts the alternative splice events of a series of developmentally regulated genes from their inappropriate fetal profile to their mature adult profile.
  • symptoms of DM1 including, but not limited to, myotonia, are ameliorated.
  • the patient is at least 18 years of age. In some embodiments, the patient is pre-ambulant or ambulant.
  • Myotonic dystrophy type I (DM1 ) is the most common form of myotonic dystrophy in adults and occurs with an estimated frequency of 1 in 7,500 (Harper P S., Myotonic Dystrophy. London: W.B. Saunders Company; 2001 ).
  • This disease is an autosomal dominant disorder caused by expansion of a non-coding CTG repeat in the human DMPK1 gene.
  • DMPK1 is a gene encoding a cytosolic serine/threonine kinase (Brook et al., Cell. 68:799-808 (1992)).
  • the expanded CTG repeat is located in the 3' untranslated region (UTR) of DMPK1 and is encoded by exon 15.
  • UTR 3' untranslated region
  • the mutant form of the DMPK mRNA, harboring large CUG repeats, are fully transcribed and polyadenylated, but remain trapped in the nucleus (Davis et al., Proc. Natl. Acad. Sci. U.S.A 94:7388- 7393 (1997)). These mutant, nuclear-retained mRNAs are one of the most important pathological features of DM1 .
  • the DMPK gene normally has from about 5 to about 37 CTG repeats in the 3' UTR. In DM1, this number is significantly expanded, and may be in the range, for example, of from 50 to greater than 4,000 repeats.
  • the CUGexp tract in the ensuing RNA transcript interacts with RNA- binding splicing factor proteins, including muscleblind-like splicing regulator 1 (MBNL1 ).
  • MBNL1 muscleblind-like splicing regulator 1
  • the enhanced avidity engendered by the expanded CUG repeat region causes the mutant transcript to retain such splicing factor proteins in nuclear foci.
  • the toxicity of this mutant RNA stems from the sequestration of RNA-binding splicing factor proteins away from other pre-mRNA substrates, including those that encode proteins that have important roles in regulating muscle function.
  • skeletal muscle is the most severely affected tissue, but the disease also has important effects on cardiac and smooth muscle, ocular lens, and the brain.
  • muscle tissue the cranial, distal limb, and diaphragm muscles are often preferentially affected.
  • Manual dexterity is compromised early, which causes several decades of severe disability.
  • the median age at death is 55 years, which is usually from respiratory failure (de Die-Smulders C E, et al., Brain 121 (Pt 8):1557- 1563 (1998)).
  • Symptoms of myotonic dystrophy include, without limitation, myotonia, muscle stiffness, disabling distal weakness, weakness in the face and jaw muscles, difficulty in swallowing, drooping of the eyelids (ptosis), weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataracts. In children, symptoms may also include developmental delays, learning problems, language and speech difficulties, and personality development challenges.
  • Pathogenic DMPK Transcripts include, without limitation, myotonia, muscle stiffness, disabling distal weakness, weakness in the face and jaw muscles, difficulty in swallowing, drooping of the eyelids (ptosis), weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataract
  • Patients that may be treated using the compositions and methods described herein include patients, such as human patients having myotonic dystrophy, such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia, including those that express a DMPK RN transcript harboring a CUG repeat expansion.
  • DMPK RN transcripts that may be expressed by a patient undergoing treatment with the compositions and methods described herein are set forth in GenBank Accession Nos.
  • compositions and methods described herein can be used to correct one or more spliceopathies in a patient, such as a patient having a myotonic dystrophy (e.g., a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia).
  • a patient having a myotonic dystrophy e.g., a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia
  • the compositions and methods herein increase MBNL1 expression in cells. This in turn effectuates corrective splicing of one or more (e.g., two, three, four, five, or more) RNA transcript substrates of MBNL1 .
  • the patient may exhibit an increase in expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1 ) mRNA containing exon 22, for example, in the tibialis anterior, gastrocnemius, and/or quadriceps muscles.
  • SERCA1 sarcoplasmic/endoplasmic reticulum calcium ATPase 1
  • the increase in expression of SERCA1 mRNA transcripts containing exon 22 may be an increase of, for example, about 1 .1 -fold to about 10-fold, or more (e.g., an increase in expression of SERCA1 mRNA containing exon 22 by about 1 .1 -fold, 1 .2-fold, 1 .3-fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1 .7-fold, 1 .8-fold, 1 .9-fold, 2- fold, 2.1 -fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1 -fold, 4.2-fold, 4.3- fold, 4.4-fold, 4.5-fold
  • a composition described herein upon administration of a composition described herein to a patient having myotonic dystrophy, such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia, the patient may exhibit a decrease in expression of chloride voltage-gated channel 1 (CLCN1 ) mRNA containing exon 7a, for example, in the tibialis anterior, gastrocnemius, and/or quadriceps muscles.
  • CLCN1 chloride voltage-gated channel 1
  • the decrease in expression of CLCN1 mRNA transcripts containing exon 7a may be a decrease of, for example, about 1 % to about 100% (e.g., a decrease in expression of CLCN1 mRNA containing exon 7a by about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
  • a composition described herein upon administration of a composition described herein to a patient having myotonic dystrophy, such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia, the patient may exhibit a decrease in expression of ZO-2 associated speckle protein (ZASP) containing exon 1 1 , for example, in the tibialis anterior, gastrocnemius, and/or quadriceps muscles.
  • ZASP ZO-2 associated speckle protein
  • the decrease in expression of ZASP mRNA transcripts containing exon 1 1 may be a decrease of, for example, about 1 % to about 100% (e.g., a decrease in expression of ZASP mRNA containing exon 1 1 by about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%,
  • a composition described herein upon administration of a composition described herein to a patient having myotonic dystrophy, such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia, the patient may exhibit an increase in corrective splicing of RNA transcripts encoding insulin receptor, ryanodine receptor 1 (RYR1 ), cardiac muscle troponin, and/or skeletal muscle troponin, such as an increase of about 1 .1 -fold to about 10-fold, or more (e.g., an increase in expression of correctly spliced RNA transcripts encoding insulin receptor, RYR1 , cardiac muscle troponin, and/or skeletal muscle troponin by about 1 .1 -fold, 1 .2-fold, 1 .3-fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1 .7-fold, 1 .8-fold, 1 .9-fold, 2-fold, 2.1 -fold
  • beneficial treatment effects of the compositions and methods described herein such as the ability of the nucleic acid molecules described herein, and of the compositions encoding the same, to overexpress MBNL1 and subsequently restore correct splicing of proteins involved in regulating muscle function may manifest clinically in a variety of ways.
  • patients having myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia, may exhibit an improvement in cranial, distal limb, and/or diaphragmatic muscle function.
  • the improvement in muscle function may be observed, for example, as an increase in muscle mass, frequency of muscle contractions, and/or magnitude of muscle contractions.
  • a patient having myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia, may exhibit an increase in cranial, distal limb, and/or diaphragmatic muscle mass, frequency of muscle contractions, and/or magnitude of muscle contractions.
  • the increase in muscle mass, frequency of muscle contractions, and/or magnitude of muscle contractions may be, for example, an increase of 1 % or more, such as an increase of from 1 % to 25%, from 1 % to 50%, from 1 % to 75%, from 1 % to 100%, from 1 % to 500%, from 1 % to 1 ,000%, or more, such as an increase in muscle mass, frequency of muscle contractions, and/or magnitude of muscle contractions of about 1 %, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 80%, 900%, 1 ,000%, or more.
  • a patient having myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia
  • the beneficial therapeutic effects of the nucleic acid molecules described herein, and of the compositions encoding the same may manifest as a reduction in myotonia.
  • a patient having myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 , e.g., myotonia
  • compositions and methods described herein may be used to accelerate muscle relaxation by suppressing the onset of spontaneous action potentials caused by fluctuations in chloride ion concentration.
  • this beneficial activity may be caused by the restoration of correct splicing of CLCN1 mRNA, for example, such that the expression of CLCN1 mRNA containing exon 7a in the patient is reduced.
  • CLCN1 channel protein regulates chloride ion concentration
  • correcting the splicing pattern of CLCN1 mRNA transcripts may engender a reduction in the onset of spontaneous action potentials and an improvement in muscle relaxation speed, thereby ameliorating myotonia.
  • Suppression of myotonia can be evaluated using a variety of techniques known in the art, for example, by way of electromyography.
  • electromyography on the left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles, and/or lumbar paraspinals muscles can be performed to assess the effects of the compositions and methods described herein on myotonia in a patient, such as a human patient diagnosed with or displaying one or more symptoms of DM1 .
  • Electromyography protocols have been described, for example, in Kanadia et al., Science 302:1978-1980 (2003)).
  • electromyography may be performed using 30-gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle.
  • an average myotonia grade may be determined for a subject, such as a human patient or a model organism (e.g., a murine model of muscular dystrophy described herein). This grade can then be compared to the average myotonia grade of the patient or model organism determined prior to administration of a therapeutic agent described herein (e.g., a transgene encoding MBNL1 operably linked to a DES promoter or a vector encoding the same).
  • a therapeutic agent described herein e.g., a transgene encoding MBNL1 operably linked to a DES promoter or a vector encoding the same.
  • tests that can be used to assess myotonia grade of the patient or model organism include a grip strength test, in which the patient or model organism squeezes a hand-held dynamometer device.
  • the dynamometer may be squeezed three times with each hand. The measurement for each squeeze is recorded, and then an average score is calculated using the measurements from both hands.
  • Comparisons of tests performed before and after administration of a therapeutic agent described herein can serve as indications of the agent’s success.
  • an increase in the average score of the grip strength test after administration of the therapeutic agent can serve as an indication of successful amelioration of the symptom of myotonia and further, an indication of successful treatment of myotonic dystrophy.
  • a patient having myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 (e.g., myotonia), may be administered a composition described herein so as to attenuate or altogether eliminate one or more symptoms of DM1 .
  • symptoms of myotonic dystrophy include, without limitation, muscle stiffness, disabling distal weakness, weakness in the face and jaw muscles, difficulty in swallowing, ptosis, weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataracts.
  • compositions and methods described herein may be used to alleviate one or more (e.g., two, three, four, five, or more), or all, of the foregoing symptoms. Duration of Therapeutic Effects
  • compositions and methods described herein provide beneficial clinical effects that may last for extended periods of time.
  • a patient having myotonic dystrophy such as a patient diagnosed with or displaying one or more symptoms of DM1 (e.g., myotonia) may exhibit an improvement in muscle function (such as an improvement in muscle mass and/or muscle activity, e.g., in the cranial, distal limb, and diaphragm muscle) and/or alleviation of one or more symptoms of myotonic dystrophy, for a period of one or more days, weeks, months, or years.
  • the beneficial therapeutic effects described herein may be achieved for a period of at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 76 days, at least 77 days, at least 78 days, at least 79 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least
  • HSA human skeletal actin
  • LR long repeat
  • DM1 human skeletal actin
  • hACTAI human skeletal actin
  • mice carry a human skeletal actin (hACTAI ) transgene containing an expanded CTG region.
  • hACTAI transgene in HSA LR mice contains 220 CTG repeats inserted in the 3' UTR of the gene.
  • the hACTAI -CUGexp RNA transcript Upon transcription, the hACTAI -CUGexp RNA transcript accumulates in nuclear foci in skeletal muscles and results in myotonia similar to that observed in human DM1 , for example, due to the binding of CUG repeat expansions to splicing factors and the sequestration of these splicing factors from pre-mRNA transcripts encoding genes that play an important role in regulating muscle function (see, e.g., Mankodi et al., Mol. Cell 10:35 (2002), and Lin et al., Hum. Mol. Genet. 15:2087 (2006), the disclosures of each of which are incorporated herein by reference as they pertain to the HSA LR mouse).
  • HSA LR DM1 mice can be generated using methods known in the art, for example, by insertion of a hACTAI transgene with 250 CUG repeats in the 3' UTR of human skeletal actin into the genome of FVB/N mice. The transgene is subsequently expressed in the mice as a CUG repeat expansion in hACTAI RNA. This repeat-expanded RNA is retained in the nucleus, forming nuclear inclusions similar to those observed in human tissue samples of patients with DM1 .
  • RNA and protein detection methods known in the art and described herein.
  • total RNA may be purified from the HSA LR mouse from one or more, or all, of the tibialis anterior, gastrocnemius, and quadriceps muscle using the RNeasy Lipid Tissue Mini Kit (Qiagen®), according to the manufacturer's instructions.
  • RT-PCR may be performed with, for example, the SuperScript III One-Step RT-PCR System and Platinum Taq Polymerase (Invitrogen®), using gene-specific primers for cDNA synthesis and PCR amplification.
  • the forward and reverse primers for SERCA1 have been described, for example, in Bennett and Swayze, Annu Rev. Pharmacol. 50:259-293 (2010)).
  • PCR products may be separated on agarose gels, stained with SybrGreen I Nucleic Acid Gel Stain (Invitrogen®), and imaged using a Fujifilm LAS-3000 Intelligent Dark Box.
  • Restoration of correct splicing of the SERCA1 gene by an interfering RNA molecule, or vector encoding the same, for example, in the tibialis anterior, gastrocnemius, and/or quadriceps muscles of the HSA LR mouse, may be a predictor of the therapeutic efficacy of a composition described herein.
  • LC15 mice Line A, which are transgenic mice containing the entire human DMPK 3'UTR (developed by Wheeler et al, University of Rochester). These mice are the second generation of mice backcrossed to an FVB background. The DMPK transgene is expressed in these mice as a CUG repeat in the DMPK RNA transcript, which is retained in the nucleus, thereby forming nuclear inclusions similar to those observed in human tissue samples of patients with DM1 .
  • LC15 mice may express DMPK RNA transcripts containing from about 350 to about 400 CUG repeats. These mice display early signs of DM1 and do not display any myotonia in their muscle tissues.
  • DMSXL mice are generated by way of successive breeding of mice having a high level of CTG repeat instability, and, as a result, DMSXL mice express DMPK RNA transcripts containing >1 ,000 CUG trinucleotide repeats in the 3’ UTR.
  • DMSXL mice and methods for producing the same are described in detail, for example, in Gomes-Pereira et al., PLoS Genet. 3:e52 (2007), and Huguet et al., A, PLoS Genet. 8:e1003043 (2012), the disclosures of each of which are incorporated herein by reference in their entirety.
  • Nucleic acid molecules described herein include a transgene encoding MBNL1 operably linked to a desmin (DES) promoter. Operably linking an MBNL1 transgene to a DES promoter advantageously and robustly increases the transcription of MBNL1 gene products in target cells.
  • DES desmin
  • nucleic acids may be administered directly or may be encoded in a vector or other composition to optimize delivery into target cells.
  • Exemplary Nucleic Acids may be administered directly or may be encoded in a vector or other composition to optimize delivery into target cells.
  • MBNL1 has multiple isoforms.
  • the nucleotide and amino acid sequences corresponding to these isoforms are delineated in Table 2 below.
  • the MBNL1 has an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 86% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 87% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 88% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 89% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 91 % identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 93% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 94% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the MBNL1 has an amino acid sequence that is at least 96% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 97% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 98% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. In some embodiments, the MBNL1 has the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the DES promoter described herein may contain a DES promoter sequence or functional portion thereof.
  • the DES promoter sequence may contain a DES promoter that includes, with respect to the DES transcription start site, from nucleotide -984 to -644 of the human DES locus.
  • the nucleic acid of this construct is set forth in SEQ ID NO: 1 .
  • the DES promoter sequence may contain a DES promoter that includes, with respect to the DES transcription start site, from nucleotide -269 to +76 of the human DES locus.
  • the nucleic acid of this construct is set forth in SEQ ID NO: 2.
  • the DES promoter sequence may contain the nucleic acid of SEQ ID NO: 1 fused to the nucleic acid of SEQ ID NO: 2, with no intervening nucleic acids, to form a DES promoter that includes, with respect to the DES transcription start site, from nucleotide -984 to -644 of the human DES locus and from nucleotide -269 to +76 of the human DES locus.
  • the nucleic acid of this construct is set forth in SEQ ID NO: 3.
  • Additional promoter sequences useful in conjunction with the compositions and methods described herein include nucleic acid molecules that have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the above nucleic acid sequences.
  • DES promoter sequences are delineated in Table 3 below.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5’ region having the nucleic acid sequence of SEQ ID NO: 1 .
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3’ region having the nucleic acid sequence of SEQ ID NO: 2.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 85% (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the transgene encoding MBNL1 is operably linked to a DES promoter having a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having the nucleic acid sequence of SEQ ID NO: 3.
  • Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell (e.g., a muscle cell or a neuron). Viral genomes are particularly useful vectors for gene delivery as the nucleic acids contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
  • viral vectors are a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • retrovirus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example.
  • retroviruses are avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996)).
  • murine leukemia viruses murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.
  • vectors are described, for example, in McVey et al., (US 5,801 ,030), the teachings of which are incorporated herein by reference.
  • Nucleic acids of the compositions and methods described herein may be incorporated into a recombinant linear adeno-associated virus (rAAV) vector, a recombinant self-complementary AAV (scAAV) vector, and/or virions, in order to facilitate their introduction into a cell (e.g., a muscle cell or a neuron).
  • Adeno-associated virus (AAV) vectors can be used in the central nervous system, and appropriate promoters and serotypes are discussed in Pignataro et al., J Neural Transm., 125: 575 (2016), the disclosure of which is incorporated herein by reference as it pertains to promoters and AAV serotypes useful in CNS gene therapy.
  • the AAV is a single-stranded rAAV. In some embodiments, the AAV is a scAAV.
  • rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs (e.g., nucleic acids capable of expression in muscle cells or neurons) that include (1 ) a heterologous sequence to be expressed and (2) viral sequences that facilitate integration and expression of the heterologous genes.
  • the viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional inverted terminal repeat sequences (ITR)) of the DNA into a virion.
  • ITR functional inverted terminal repeat sequences
  • Such rAAV vectors may also contain marker or reporter genes.
  • Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences.
  • the AAV ITRs may be of any serotype suitable for a particular application.
  • the AAV ITR is an AAV2 ITR.
  • the nucleic acids and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell.
  • the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
  • the cap gene encodes three viral coat proteins, VP1 , VP2, and VP3, which are required for virion assembly.
  • the construction of rAAV virions has been described, for example, in US 5,173,414; US 5,139,941 ; US 5,863,541 ; US 5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol.
  • rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1 , 2, 3, 4, 5, 6, 7, 8, 9, rh10 and rh74.
  • AAV2 AAV9, and AAV10 may be particularly useful. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther.
  • Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.).
  • a representative pseudotyped vector is an AAV8 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 2.
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000).
  • Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001 ).
  • the nucleic acid molecule including a transgene encoding MBNL1 is operably linked to a DES promoter, optionally wherein the 5’ end of the portion of the nucleic acid molecule (e.g., including or encoding a transgene encoding MBNL1 ) is bound to the 3’ end of the DES promoter.
  • the transgene encoding MNBNL1 and DES promoter sequences are separated by a beta-globin intron sequence. It is anticipated that the beta-globin intron sequence will act as a linker sequence between the promoter and transgene sequences and will promote higher efficiency of transcription of the transgene.
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 5’ region having a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1 . In some embodiments, the DES promoter includes a 5’ region that has the nucleic acid sequence of SEQ ID NO: 1 .
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region having a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the DES promoter includes a 3’ region that has the nucleic acid sequence of SEQ ID NO: 2.
  • the DES promoter has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the DES promoter has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the DES promoter has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the DES promoter has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the DES promoter has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.
  • the DES promoter has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DES promoter has the nucleic acid sequence of SEQ ID NO: 3.
  • the AAV has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV has a nucleic acid sequence that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV has a nucleic acid sequence that is at least 87% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV has a nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 89% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 91% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV has a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 94% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 20.
  • the AAV has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the AAV has the nucleic acid sequence of SEQ ID NO: 20. SEQ ID NO: 20 is shown below:
  • exemplary AAV vector components include, for example, from 5’ to 3’, a DES promoter, a beta-globin intron, a transgene encoding MBNL1 , and an SV40 late polyadenylation (pA) sequence, as described herein.
  • transgene such as a transgene encoding MBNL1 , wherein the transgene is operably linked to a DES promoter
  • a target cell e.g., a mammalian cell such as a muscle cell or a neuron
  • electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest.
  • Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids (e.g., nucleic acids capable of expression in e.g., muscle cells or neurons).
  • NUCLEOFECTIONTM utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell.
  • NUCLEOFECTIONTM and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Exp. Dermatol. 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.
  • An additional technique useful for the transfection of target cells is the squeeze-poration methodology.
  • This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress.
  • This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., JoVE 81 :e50980 (2013), the disclosure of which is incorporated herein by reference.
  • Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US 7,442,386, the disclosure of which is incorporated herein by reference.
  • cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig, Top Curr Chem 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and DEAE-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Curr Protoc Mol Biol 40 :9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.
  • laserfection also called optical transfection
  • Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane.
  • the bioactivity of this technique is similar to, and in some cases found superior to, electroporation.
  • Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s).
  • An example of this technique is described in Shalek et al., PNAS 107:25 1870 (2010), the disclosure of which is incorporated herein by reference.
  • MAGNETOFECTIONTM can also be used to deliver nucleic acids to target cells.
  • the principle of MAGNETOFECTIONTM is to associate nucleic acids with cationic magnetic nanoparticles.
  • the magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications.
  • Their association with the gene vectors (DNA, asRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction.
  • the magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Ther. 9:102 (2002), the disclosure of which is incorporated herein by reference.
  • Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US2010/0227406, the disclosure of which is incorporated herein by reference.
  • sonoporation a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.
  • Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein.
  • microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence.
  • vesicles also referred to as Gesicles
  • Gesicles for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract].
  • Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.
  • RNA transcript expression can be ascertained, for example, by a variety of nucleic acid detection techniques. Additionally or alternatively, RNA transcript expression can be inferred by evaluating the concentration or relative abundance of an encoded protein produced by translation of the RNA transcript. Protein concentrations can also be assessed, for example, using functional assays. Using these techniques, an increase in the concentration of MBNL1 mRNA transcripts in response to the compositions and methods described herein can be observed, while monitoring the expression of the encoded protein.
  • the sections that follow describe exemplary techniques that can be used to measure the expression level of an RNA transcript and its downstream protein product.
  • RNA transcript expression can be evaluated by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescence in-situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis and/or PCR analysis of RNAs.
  • FISH fluorescence in-situ hybridization
  • FACS fluorescence activated cell sorting
  • Nucleic acid-based methods for detection of DNA or RNA transcript expression include imaging-based techniques (e.g., Northern blotting or Southern blotting) may be used in conjunction with cells obtained from a patient following administration of, for example, a vector encoding a transgene encoding MBNL1 operably linked to a DES promoter or a composition containing such a construct.
  • Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, NY 11803-2500.
  • RNA detection techniques that may be used in conjunction with the compositions and methods described herein to evaluate the efficacy of administration of any of the compositions described herein further include microarray sequencing experiments (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput sequencing or deep sequencing).
  • exemplary next generation sequencing technologies include, without limitation, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional methods of sequencing known in the art can also be used.
  • transgene expression at the mRNA level may be determined using RNA-Seq (e.g., as described in Mortazavi et al., Nat. Methods 5:621 -628 (2008), the disclosure of which is incorporated herein by reference in their entirety).
  • RNA-Seq is a robust technology for monitoring expression by direct sequencing the RNA molecules in a sample.
  • this methodology may involve fragmentation of RNA to an average length of 200 nucleotides, conversion to cDNA by random priming, and synthesis of double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology®). Then, the cDNA is converted into a molecular library for sequencing by addition of sequence adapters for each library (e.g., from lllumina®/Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome.
  • sequence adapters for each library e.g., from lllumina®/Solexa
  • RNA expression levels may be determined using microarray-based platforms (e.g., singlenucleotide polymorphism arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068 and Pollack et al., Nat. Genet. 23:41 -46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety.
  • nucleic acid microarrays mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then hybridize to one or more complementary nucleic acids arrayed and immobilized on a solid support.
  • the array can be configured, for example, such that the sequence and position of each member of the array is known.
  • Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • Expression level may be quantified according to the amount of signal detected from hybridized probe-sample complexes.
  • a typical microarray experiment involves the following steps: 1 ) preparation of fluorescently labeled target from RNA isolated from the sample, 2) hybridization of the labeled target to the microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image and 5) generation of gene expression profiles.
  • microarray processor is the Affymetrix GENECHIP® system, which is commercially available and includes arrays fabricated by direct synthesis of oligonucleotides on a glass surface. Other systems may be used as known to one skilled in the art.
  • Amplification-based assays also can be used to measure the expression level of a particular RNA transcript, such as an MBNL1 mRNA transcript.
  • the nucleic acid sequence of the transcript acts as a template in an amplification reaction (for example, PCR, such as qPCR or Droplet Digital PCR (ddPCR)).
  • PCR such as qPCR or Droplet Digital PCR (ddPCR)
  • ddPCR Droplet Digital PCR
  • the amount of amplification product is proportional to the amount of template in the original sample.
  • Comparison to appropriate controls provides a measure of the expression level of the transcript of interest, corresponding to the specific probe used, according to the principles described herein. Methods of real-time qPCR using TaqMan probes are well known in the art.
  • RNA transcript expression as described herein can be determined, for example, by RT-PCR technology.
  • Probes used for PCR may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
  • RNA construct may also be inferred by analyzing expression of the protein encoded by the construct. Protein levels can be assessed using standard detection techniques known in the art. Protein expression assays suitable for use with the compositions and methods described herein include proteomics approaches, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, AlphaLISATM, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays. In particular, proteomics methods can be used to generate large-scale protein expression datasets in multiplex.
  • Proteomics methods may utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and/or peptide microarrays utilizing capture reagents (e.g., antibodies) specific to a panel of target proteins to identify and measure expression levels of proteins expressed in a sample (e.g., a single cell sample or a multi-cell population).
  • polypeptides e.g., proteins
  • capture reagents e.g., antibodies
  • Exemplary peptide microarrays have a substrate-bound plurality of polypeptides, the binding of an oligonucleotide, a peptide, or a protein to each of the plurality of bound polypeptides being separately detectable.
  • the peptide microarray may include a plurality of binders, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins. Examples of peptide arrays may be found in U.S. Patent Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.
  • Mass spectrometry may be used in conjunction with the methods described herein to identify and characterize transgene expression in a cell from a patient (e.g., a human patient) following delivery of the transgene. Any method of MS known in the art may be used to determine, detect, and/or measure a protein or peptide fragment of interest, e.g., LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like.
  • Mass spectrometers generally contain an ion source and optics, mass analyzer, and data processing electronics.
  • Mass analyzers include scanning and ion-beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q), and trapping mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), may be used in the methods described herein. Details of various MS methods can be found in the literature. See, for example, Yates et al., Annu. Rev. Biomed. Eng. 11 :49-79, 2009, the disclosure of which is incorporated herein by reference in its entirety.
  • TOF time-of-flight
  • Q quadruple
  • trapping mass spectrometers such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR)
  • proteins in a sample obtained from the patient can be first digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion.
  • Complex peptide samples also benefit from the use of front-end separation techniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography.
  • the digested, and optionally separated, sample is then ionized using an ion source to create charged molecules for further analysis.
  • Ionization of the sample may be performed, e.g., by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)Zliqu id secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. Additional information relating to the choice of ionization method is known to those of skill in the art.
  • Tandem MS also known as MS/MS
  • Tandem MS may be particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring in between the stages, which may be accomplished with individual mass spectrometer elements separated in space or using a single mass spectrometer with the MS steps separated in time.
  • spatially separated tandem MS the elements are physically separated and distinct, with a physical connection between the elements to maintain high vacuum.
  • separation is accomplished with ions trapped in the same place, with multiple separation steps taking place over time.
  • Signature MS/MS spectra may then be compared against a peptide sequence database (e.g., SEQUEST).
  • Post-translational modifications to peptides may also be determined, for example, by searching spectra against a database while allowing for specific peptide modifications.
  • nucleic acid molecules described herein can be formulated into pharmaceutical compositions for administration to a patient, such as a human patient exhibiting or at risk of myotonic dystrophy, such as a patient diagnosed with or displaying one or more symptoms of DM1 (e.g., myotonia), in a biologically compatible form suitable for administration in vivo.
  • a pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and a nucleic acid.
  • the sterile saline is typically a pharmaceutical grade saline.
  • a pharmaceutical composition may include (e.g., consist of), e.g., sterile water and a nucleic acid.
  • the sterile water is typically a pharmaceutical grade water.
  • a pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and a nucleic acid.
  • PBS phosphate-buffered saline
  • pharmaceutical compositions include a plurality of the transgenes and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • nucleic acid molecules may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions including a transgene encoding a nucleic acid molecule encompass any pharmaceutically acceptable salts of the inhibitor, esters of the inhibitor, or salts of such esters.
  • pharmaceutical compositions including a transgene encoding a nucleic acid molecule upon administration to a subject (e.g., a human), are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • a subject e.g., a human
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • prodrugs include one or more conjugate group attached to a nucleic acid molecule, wherein the conjugate group is cleaved by endogenous nucleases within the body.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • compositions include a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types.
  • pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • compositions include a co-solvent system.
  • co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • compositions are prepared for oral administration.
  • pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intraocular, intravitreal, intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular etc.).
  • a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • Viral vectors such as AAV vectors and others described herein, containing an MBNL1 transgene operably linked to a DES promoter described herein may be administered to a patient (e.g., a human patient) by a variety of routes of administration.
  • the route of administration may vary, for example, with the onset and severity of disease, and may include, e.g., intravenous, intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, or oral administration and/or administration by inhalation, perfusion, or lavage.
  • Intravascular administration may include delivery into the vasculature of a patient.
  • the administration is into a vessel considered to be a vein (intravenous), and in some embodiments, the administration is into a vessel considered to be an artery (intraarterial).
  • Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein.
  • Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.
  • Treatment regimens may vary, and often depend on disease severity and the age, weight, and sex of the patient.
  • Treatment may include administration of vectors (e.g., viral vectors) or other agents described herein as useful for the introduction of a transgene into a target cell in various unit doses.
  • Each unit dose will ordinarily contain a predetermined-quantity of the therapeutic composition.
  • a nucleic acid molecule described herein can be administered in combination with one or more additional therapeutic agents for treatment of a patient having myotonic dystrophy, such as a patient diagnosed with or displaying one or more symptoms of DM1 (e.g., myotonia).
  • DM1 myotonia
  • the one or more additional therapeutic agents may include a corticosteroid (e.g., bethamethasone, prednisolone, triamcinolone, methylprednisolone, dexamethasone, hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone, or fludrocortisone) or an immunosuppressive drug (e.g., pomalidomide, methotrexate, azathioprine, lenalidomide, azathioprine, or thalidomide), or a combination thereof.
  • a corticosteroid e.g., bethamethasone, prednisolone, triamcinolone, methylprednisolone, dexamethasone, hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone
  • the compositions described herein can be provided in a kit for use in treating a patient diagnosed with or displaying one or more symptoms of DM1 (e.g., myotonia).
  • the kit may include one or more nucleic acid molecules as described herein.
  • the kit may include a viral vector as described herein.
  • the kit may include a pharmaceutical composition as described herein.
  • the kit can include a package insert that instructs a user of the kit, such as a physician, to perform any one of the methods described herein.
  • the kit may optionally include a syringe or other device for administering the composition.
  • the kit may include one or more additional therapeutic agents.
  • compositions useful for reducing the occurrence of spliceopathy and for treating disorders associated with ribonucleic acid (RNA) dominance a pathology that is induced by the expression and nuclear retention of messenger RNA (mRNA) transcripts containing expanded repeat regions that bind and sequester splicing factor proteins, thereby interfering with the proper splicing of various mRNA transcripts, such as myotonic dystrophy type 1 (DM1 ).
  • mRNA messenger RNA
  • DM1 myotonic dystrophy type 1
  • DM1 myotonic dystrophy type 1
  • DM1 is caused by expansion of a microsatellite repeat that leads to expression of a toxic expanded repeat mRNA.
  • This mRNA exhibits elevated avidity for splicing factor proteins, such as muscleblind-like splicing regulator 1 (MBNL1 ), and sequesters these proteins in ribonuclear foci, which subsequently promotes spliceopathy of MBNL1 RNA substrates, such as pre- mRNA molecules transcribed from ATP2A1, CLCN1, and LDB3, and therefore promotes a shift in the splicing profile of these cells to switch from the desired mature-stage splicing profile to a fetal-stage splicing profile.
  • splicing factor proteins such as muscleblind-like splicing regulator 1 (MBNL1 )
  • MBNL1 muscleblind-like splicing regulator 1
  • compositions described in this example were developed with the goal to manufacture AAV vectors encoding transgene constructs for MBNL1 , wherein the transgene is operably linked to a desmin (DES) promoter, so as to overexpress MBNL1 in target cells and reduce the occurrence of spliceopathy.
  • DES desmin
  • reduction of spliceopathy of MBNL1 RNA substrates promotes the development of the mature splicing profile over the fetal splicing profile, and results clinically in the amelioration of one or more symptoms of DM1 , such as myotonia.
  • the purpose of this example is to show the efficacy of the promoter at inducing robust overexpression of MBNL1 and therefore subsequently improving splicing outcomes in affected genes in a cellular model of DM1 .
  • the efficacy of nucleic acid constructs described herein were demonstrated to reduce the nuclear sequestration of MBNL1 and to improve the splicing of kinesin family member 13A (KIF13A) and dystrophin (DMD).
  • Immortalized non-DM1 human myoblast cells were used as control cells relative to immortalized DM1 patient-derived myoblast cells (DM1 cells) for all cellular experiments.
  • DM1 or control cells were seeded at 4,000 cells/well in collagen type l-coated 96 well plates (IWAKI # 4860- 010 for splicing assay and gene expression analysis, Thermo Fisher Scientific #152036 for RNA foci assay) in 160 pl growth medium [PromoCell Skeletal Muscle Cell Growth Medium Kit; part number: C- 23060 (note: media was supplemented with 20% FBS (Thermo Fisher Scientific #16000-044), rather than 5% as directed by kit, 50 pg/ml Gentamicin S (Thermo Fisher Scientific #15070-060))] and incubated at 37°C/5% CO2 for 5-6 hours.
  • DM1 cells were infected with 40 pl of AAV8 vector encoding a human MBNL1 construct, as described above. Control wells were treated with PBS including 0.001 % Pluronic F68 and mixed. The plates were cultured for 2 days and the medium was replaced with 200 pl of new growth medium including 10 pM of Etoposide (Wako #051 -08431 ) and incubated at 37°C/5% CO2 for 4 more days.
  • PCR was conducted using PrimeSTAR® GXL DNA Polymerase (TaKaRa # R050A) according to the manufacturer’s instructions. 2 pl of the cDNA was used as a template.
  • the PCR primers used were as follows:
  • the PCR cycle condition was as follows: 35 cycles at 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 30 seconds followed by 72°C for 7 minutes.
  • the PCR products were loaded on Agilent DNA1000 Kit (Agilent # 5067-1504), electrophoresed, and analyzed using Agilent 2100 BioAnalyzer system according to the manufacturer’s instructions. AUCs of the peaks of normally and abnormally spliced products were measured and the ratios of the normally spliced products in each cell were calculated.
  • MBNL1 Human Muscleblind-like Protein 1
  • MBNL1 AlphaLISA Immunoassay Kit
  • 5X AlphaLISA Lysis Buffer PerkinElmer, #AL003F
  • 100x Protease and Phosphatase Inhibitor Cocktail were diluted together with double distilled water.
  • 100 pl/well of lysis buffer was added to each well. Plates were shaken with a plate shaker for 10 minutes at room temperature. Then the plates were incubated for 10 minutes at 37°C before storage at -80°C.
  • MBNL1 protein was measured in 2 pl scale in accordance with the manufacturer’s protocol using Envision 2014-0020 (PerkinElmer).
  • Total protein of lysate was also quantified with SPECTRA max PLUS 384 (Molecular Devices) using DC Protein Assay kit (Bio-Rad, # 500-0112JA) according to the manufacturer’s protocol. The amount of MBNL1 protein was corrected by total protein, and then the relative amount was calculated with the amount of MBNL1 in DM1 cells as 100%. Results
  • DM1 cells were transduced with an AAV8 vector encoding an MBNL1 transgene operably linked to a DES promoter (AAV8-DES-MBNL1 , as shown in FIG. 1) or treated with PBS including 0.001 % Pluronic F68.
  • the MBNL1 transgene treated DM1 cells exhibited an increase in MBNL1 expression compared to control immortalized non-DM1 human myoblast cells, as measured by immunoblotting.
  • the MBNL1 transgene treated DM1 cells also displayed an improvement in KIF13A and DMD splicing, as shown by an increase in the percentage of KIF13A exon 21 inclusion and an increase in the percentage of DMD exon 78 inclusion respectively (Tables 5-7).
  • T contained a treatment concentration of 9x10 11 viral genomes (vg)/mL
  • H contained a treatment concentration of 3x10 11 viral genomes (vg)/mL
  • M contained a treatment concentration of 1 x10 11 viral genomes (vg)/mL
  • L contained a treatment concentration of 3.3x10 10 viral genomes (vg)/mL
  • L2 contained a treatment concentration of 1 .1 x10 10 viral genomes (vg)/mL.
  • Table 5 contained a treatment concentration of 1 .1 x10 10 viral genomes (vg)/mL.
  • DM1 cells were transduced with an AAV8 vector encoding an MBNL1 transgene operably linked to a CHAG promoter, a CK8 promoter, a PGK promoter, an EF1a promoter, or an eMCK promoter.
  • a control cells treated with PBS including 0.001% Pluronic F68 were included.
  • Pluronic F68 Pluronic F68
  • T contained a treatment concentration of 9x10 11 viral genomes (vg)/mL
  • H contained a treatment concentration of 3x10 11 viral genomes (vg)/mL
  • M contained a treatment concentration of 1 x10 11 viral genomes (vg)/mL
  • L contained a treatment concentration of 3.3x10 10 viral genomes (vg)/mL
  • L2 contained a treatment concentration of 1 .1 x10 10 viral genomes (vg)/mL.
  • DM1 cells were transduced with an AAV8 vector encoding an MBNL1 transgene operably linked to a desmin promoter or an alternative promoter (e.g., a CHAG promoter, a CK8 promoter, a PGK promoter, an EF1 a promoter, or an eMCK promoter).
  • a desmin promoter e.g., a CHAG promoter, a CK8 promoter, a PGK promoter, an EF1 a promoter, or an eMCK promoter.
  • PBS including 0.001% Pluronic F68
  • T contained a treatment concentration of 9x10 11 viral genomes (vg)/mL
  • H contained a treatment concentration of 3x10 11 viral genomes (vg)/mL
  • M contained a treatment concentration of 1 x10 11 viral genomes (vg)/mL
  • L contained a treatment concentration of 3.3x10 10 viral genomes (vg)/mL
  • L2 contained a treatment concentration of 1 .1 x10 10 viral genomes (vg)/mL.
  • Table 10-11 MBNL1 protein expression: in vitro data A
  • Example 2 Evaluation of effectiveness of adeno-associated viral vectors encoding MBNL1 transgene constructs operably linked to DES promoters for the treatment of disorders associated with RNA dominance
  • This example describes how an AAV vector encoding an MBNL1 transgene operably linked to a DES promoter could be designed and evaluated.
  • an AAV vector against murine HSA LR which is expressed in mouse models of myotonic dystrophy, could be designed for testing purposes.
  • an AAV vector against murine HSA LR which is expressed in mouse models of myotonic dystrophy, could be designed for testing purposes.
  • among the aims of this study would be translating the approach of reducing spliceopathy of MBNL1 RNA substrates to develop a paradigm for ameliorating symptoms of DM1 .
  • Example 1 Materials and methods for testing the therapeutic effects of AAV vectors encoding an MBNL1 transgene operably linked to a DES promoter are described in Example 1 .
  • Any suitable murine model of myotonic dystrophy can be used to develop AAV gene therapy for DM1 .
  • the gene therapy may be designed so that its efficacy could be tested in the HSA LR mouse model of DM1 .
  • the HSA LR mouse may be produced with insertion of an expanded CTG repeat in the 3’ UTR of the human skeletal actin gene (HSA) gene, a similar genetic context as the disease-causing repeat expansion in the DMPKgene in humans.
  • HSA human skeletal actin gene
  • the HSA LR mouse is known to display many of the genetic and phenotypic changes associated with DM1 in skeletal muscles, including myotonia, splicing changes in a variety of mRNAs, and sequestration of splicing factors in ribonuclear foci. In particular, mice may show characteristics of myotonic dystrophy resembling DM1 in humans.
  • the HSA LR transgene is derived from insertion of a (CTG)2so repeat in the 3’ UTR of the HSA gene.
  • transgene expression cassettes wherein the transgene encoding MBNL1 is operably linked to a DES promoter (DES-MBNL1 ), will be tested.
  • the AAV genomes may be packaged in a AAV2/8 capsid for targeting skeletal muscle cells and neuronal cells.
  • AAV2/8 DES-MBNL1 transgene constructs may be delivered by intravenous injection (IV) into the tail vein of HSA LR mice at 4 weeks of age.
  • HSA LR mice may be sacrificed and human placental alkaline phosphatase (AP) staining would indicate the presence of the viral genome with reporter gene expression. H&E staining of cryosections from treated mice may also be performed.
  • AP placental alkaline phosphatase
  • AAV transgene vectors encoding an MBNL1 transgene operably linked to a DES promoter may effectuate an increase in MBNL1 protein expression in HSA LR mice. Such a result could be evidenced by the restoration of correct splicing of SERCA1 mRNA.
  • AAV2/8 DES-MBNL1 transgene systemic injection may improve splicing of SERCA1 and CLCN1 in the tibialis anterior (TA) muscle.
  • a patient e.g., an adult patient of at least 18 years of age diagnosed with or displaying one or more symptoms of DM1 , such as a patient experiencing myotonia, may be administered a pseudotyped AAV2/8 vector including or encoding, from 5’ to 3’, a DES promoter, a beta-globin intron sequence, a transgene encoding MBNL1 , and an SV40 late polyadenylate site sequence.
  • a pseudotyped AAV2/8 vector including or encoding, from 5’ to 3’, a DES promoter, a beta-globin intron sequence, a transgene encoding MBNL1 , and an SV40 late polyadenylate site sequence.
  • the DES promoter has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NOs: 1 - 3.
  • the transgene encoding MBNL1 has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-1 1 .
  • the MBNL1 has an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.
  • the vector including the nucleic acid molecule may be delivered to a cell (e.g., the cell of a patient) harboring an expanded repeat region in exon 15 of the endogenous DMPK gene.
  • Increased expression of functional sarcoplasmic/endoplasmic reticulum calcium ATPasel (SERCA1 ), chloride voltage-gated channel 1 (CLCN1 ), and/or ZO-2 associated speckle protein (ZASP) can be evaluated by, e.g., immunoblotting of the respective proteins.
  • the patient may exhibit an increase in expression of MBNL1 , an increase in corrective splicing of RNA transcripts encoding insulin receptor, ryanodine receptor 1 , cardiac muscle troponin, and/or skeletal muscle troponin, and may experience amelioration of myotonia, as measured by a qualitative physical test, e.g., by a grip test to measure hand strength, within 12 weeks of treatment.
  • a qualitative physical test e.g., by a grip test to measure hand strength

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Abstract

La présente invention concerne l'administration de virus adéno-associés (AAV) d'acides nucléiques pour le traitement de la dystrophie myotonique, par exemple, la dystrophie myotonique de type 1 (DM1), chez des patients en ayant besoin, par exemple, des patients diagnostiqués avec DM1 ou affichant un ou plusieurs symptômes de DM1, par exemple, la myotonie. L'invention concerne des produits AAV et des procédés d'utilisation de l'AAV dans le traitement de la dystrophie myotonique, par exemple, DM1.
PCT/US2023/069191 2022-06-27 2023-06-27 Compositions et méthodes pour le traitement de dystrophies myotoniques Ceased WO2024006775A2 (fr)

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