TW202409290A - Compositions and methods for the treatment of myotonic dystrophies - Google Patents

Abstract

The present invention relates to adeno-associated virus (AAV) delivery of nucleic acids for treating myotonic dystrophy, e.g., myotonic dystrophy type 1 (DM1), in patients in need thereof, e.g., patients diagnosed with DM1 or displaying one or more symptoms of DM1, e.g., myotonia. The invention provides AAV products and methods of using the AAV in the treatment of myotonic dystrophy, e.g., DM1.

Description

Compositions and methods for treating myotonic dystrophy

The present invention is in the field of therapeutic treatment of myotonic dystrophy in patients, such as myotonic dystrophy type 1, in human patients.

Myotonic dystrophy type 1 (DM1) is an inherited somatic chromosomally dominant muscle degenerative disease caused by mutations in the myotonic dystrophy protein kinase gene ( DMPK ) and subsequent incorrect splicing, resulting in the sequestration of various splicing factors. , ultimately resulting from a series of alternative splicing events that return a set of developmentally regulated genes to the fetal profile. DM1 is one of the most common neuromuscular disorders in adults and causes progressive muscle atrophy and weakness. Patients diagnosed with DM1 often experience prolonged muscle contractions (myotonia) and an inability to relax certain muscles after use (for example, an individual may experience difficulty releasing their grip on a doorknob). Distal limb weakness and gastrointestinal problems such as constipation and diarrhea are common. DM1 progression is characterized by abnormal heart rhythms (arrhythmias) or weakened heartbeats, respiratory muscle weakness, acute muscle pain, and endocrine disruption leading to other conditions including thyroid problems and diabetes. Currently, there is no cure for DM1; therefore, treatment focuses on controlling the complications associated with the disease. Cellular and animal models of DM1 indicate that overexpression of one or more of these isolated splicing factors improves DMPK splicing outcomes. However, there is a need in the art to improve the efficacy of gene therapy methods for diagnosing human patients with or exhibiting one or more symptoms of DM1.

Described herein are compositions and methods suitable for ameliorating symptoms of myotonic dystrophy conditions such as myotonic dystrophy type 1 (DM1), such as myotonia, and treating myotonic dystrophy (eg, DM1). DM1 is a condition caused by mutations in the gene DMPK and subsequent incorrect splicing, leading to sequestration of splicing factors and subsequent return of the affected cells' alternative splicing profiles to their fetal-stage splicing profiles. Compositions described herein useful for treating such disorders include nucleic acids encoding transgenes for splicing factors such as blind muscle-like splicing regulator 1 (MBNL1) operably linked to the desmin (DES) promoter and promote Various cellular processes overexpress the encoded transgene. The present disclosure additionally provides vectors, such as viral vectors, encoding such transgenes operably linked to a DES promoter. Exemplary viral vectors described herein that encode transgenes operably linked to a DES promoter to overexpress splicing factors in target cells are adeno-associated virus (AAV) vectors, such as pseudotyped AAV2/8 and AAV2/9 carrier.

Using the compositions and methods described herein, a nucleic acid containing a transgene operably linked to a DES promoter, or a vector encoding the same, can be administered to a patient diagnosed with or showing one or more symptoms of myotonic dystrophy, particularly DM1, so as to increase the expression of a splicing factor protein sequestered by repeat-expanded RNA. For example, the compositions and methods described herein can be used to treat a patient with DM1, wherein a nucleic acid construct comprising a MBNL1 transgene operably linked to a DES promoter, or a viral vector encoding such a construct, such as an AAV vector, can be administered to the patient, thereby increasing the expression of MBNL1. MBNL1 expressing cells in patients who are not diagnosed with a myotonic dystrophy such as DM1, and/or who do not experience one or more symptoms of DM1 such as myotonia, typically express a mature adult phenotypic splicing profile. However, MBNL1 expressing cells in patients who are diagnosed with or who exhibit 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 return to a fetal profile and become a potential 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 causing MBNL1 to be overexpressed in order to coordinate the correct splicing of proteins associated with muscle function and treat this potential cause of myotonic dystrophy. Similarly, the compositions and methods described herein can be used to improve symptoms of various other disorders associated with myotonic dystrophy and sequestration of muscle-specific splicing factors such as MBNL1, and treat one or more potential causes of such disorders.

The present disclosure is based in part on the unexpected discovery that operably linking a transgene construct to a DES promoter can be used to efficiently overexpress transgene products in target cells in a rapid and robust manner compared to current methods. . Accordingly, the compositions and methods described herein can rapidly increase the expression of MBNL1 and to a greater extent than current therapies. This property provides important clinical benefits.

In a first aspect, the present invention provides an adeno-associated virus (AAV) containing a transgene encoding human MBNL1. The transgenic gene can be operably linked to the DES promoter. In some embodiments, the DES promoter includes a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, The 5' region of a nucleic acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical. 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. 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. In some embodiments, the DES promoter includes a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, The 3' region of a nucleic acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical. 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 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. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. 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 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.

In some embodiments, 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. In some embodiments, 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, 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. In some embodiments, 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, 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 transgene has a nucleic acid sequence that is at least 85% identical to any one of SEQ ID NOs: 4-11 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. In some embodiments, the transgenic gene 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 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 transgenic gene has the nucleic acid sequence of any of SEQ ID NOs: 4-11.

In some embodiments, the AAV comprises a capsid protein from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, and AAVrh74. In some embodiments, the AAV is a pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2/8. In some embodiments, the pseudotyped AAV is AAV2/9. In some embodiments, the AAV further comprises a polyadenylation site (pA), optionally wherein the pA is located 3' to the transgene. In some embodiments, the pA site comprises a Simian Virus 40 (SV40) late pA site or an SV40 early pA site. In some embodiments, the pA site is a SV40 late pA site.

In some embodiments, the AAV further includes an intron, optionally located 3' to the promoter and 5' to the transgene. In some embodiments, the intron is a beta-globin intron.

In a second aspect, the present invention provides a pharmaceutical composition comprising the AAV of any of the preceding aspects and a pharmaceutically acceptable carrier, diluent, or excipient.

In another aspect, the present invention provides a nucleic acid molecule comprising a DES promoter, a β-globin intron, a transgene encoding MBNL1, and an SV40 pA site, wherein the components are operably linked to each other in the 5' to 3' direction as DES-β-globin intron-MBNL1-SV40 pA. In some embodiments, the SV40 pA site comprises an SV40 late pA site or an SV40 early pA site. In some embodiments, the pA site is an SV40 late pA site.

In some embodiments, 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. 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. 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 a nucleic acid sequence of SEQ ID NO: 1. In some embodiments, 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. 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 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 a nucleic acid sequence of SEQ ID NO: 2. In some embodiments, 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. 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 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 a nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, 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. In some embodiments, 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, 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. In some embodiments, 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, 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 transgene 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: 4-11. In some embodiments, 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. 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 a nucleic acid sequence of any one of SEQ ID NOs: 4-11.

In another aspect, the present invention provides a vector comprising a nucleic acid molecule of the aforementioned aspect, wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: AAV, adenovirus, lentivirus, retrovirus, poxvirus, bacillus virus, herpes simplex virus, vaccinia virus, and synthetic virus. In some embodiments, the viral vector is AAV. In some embodiments, AAV includes a capsid protein from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, and AAVrh74. In some embodiments, AAV is a pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2/8 or AAV2/9, where appropriate wherein the pseudotyped AAV is AAV2/8. In some embodiments, the AAV comprises a recombinant capsid protein.

In some embodiments, the AAV or vector of the first aspect has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%) identical to SEQ ID NO: 20 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. In some embodiments, the AAV or vector of the first aspect 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 or vector of the first aspect 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 or vector of the first aspect 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 or vector of the first aspect has the nucleic acid sequence of SEQ ID NO: 20.

In another aspect, the invention provides plasmids encoding viral vectors of the foregoing aspects. In some embodiments, the plasmid includes a promoter operably linked to a selectable marker gene. In some embodiments, the selectable marker gene is an antibiotic resistance gene.

In another aspect, the invention provides a nucleic acid molecule comprising any of the foregoing aspects, a vector of any of the foregoing aspects, or a plasmid of any of the foregoing aspects, and a pharmaceutically acceptable pharmaceutical compositions containing carriers, diluents, or excipients.

In another aspect, the present invention provides a method for treating myotonic muscular dystrophy type 1 (DM1) in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid molecule of any of the foregoing aspects, an AAV of any of the foregoing aspects, a vector of any of the foregoing aspects, a plasmid of any of the foregoing aspects, or a pharmaceutical composition of any of the foregoing aspects.

In another aspect, the invention provides a method of increasing MBNL1 expression in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid molecule of any of the foregoing aspects, any of the foregoing aspects. The AAV of any of the foregoing aspects, the vector of any of the foregoing aspects, the plasmid of any of the foregoing aspects, or the pharmaceutical composition of any of the foregoing aspects.

In another aspect, the invention provides a method of inducing corrective splicing of one or more RNA transcript substrates of MBNL1 in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of the aforementioned state. A nucleic acid molecule of any of the foregoing aspects, an AAV of any of the foregoing aspects, a vector of any of the foregoing aspects, a plasmid of any of the foregoing aspects, or a medicine of any of the foregoing aspects composition.

In some embodiments of any of the foregoing method aspects, the patient is at least 18 years old. In some embodiments of any of the foregoing method aspects, after administration of a nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in whole blood MBNL1 levels, optionally wherein approximately At 12 weeks, patients showed an increase in whole blood MBNL1 levels. In some embodiments of any of the foregoing method aspects, after administration of a nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in whole blood MBNL1 levels, optionally wherein approximately At 12 weeks, patients showed an increase in whole blood MBNL1 levels. In some embodiments of any of the foregoing method aspects, after administration of a nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits a reduction in aberrant splicing of one or more genes selected from: driver Protein family member 13A ( KIF13A ); dystrophin ( DMD ); insulin receptor ( INSR ); voltage-gated chloride channel 1 ( CLCN1 ); troponin T2; cardiac type ( TNNT2 ); troponin T3, fast skeletal type ( TNNT3 ); titin ( TTN ); and bridging integron 1 ( BIN1 ), as appropriate, in which patients demonstrated a reduction in aberrant splicing approximately 12 weeks after administration. In some embodiments of any of the foregoing method aspects, the reduction in aberrant splicing of INSR after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient includes an increase in expression of the INSR-B isoform, Depending on the situation, patients show increased expression of INSR-B isoforms approximately 12 weeks after administration. In some embodiments of any of the foregoing method aspects, the reduction of aberrant splicing of CLCN1 after administration of a nucleic acid, vector, plasmid, or pharmaceutical composition to the patient includes excluding exon 7a of the CLCN1 transcript. Increase, optionally wherein approximately 12 weeks after administration, the patient exhibits an increase in CLCN1 transcript excluding exon 7a.

In another aspect, the present invention provides a method for treating DM1 in a human patient diagnosed with a splicing disorder of an endogenous ATP2A1 , CLCN1 , and/or LDB3 gene, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid of any of the foregoing aspects, an AAV of any of the foregoing aspects, a vector of any of the foregoing aspects, a plasmid of any of the foregoing aspects, or a pharmaceutical composition of any of the foregoing aspects.

In another aspect, the present invention provides a method for increasing the expression of functional sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 1 (SERCA1) protein in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid of any of the foregoing aspects, an AAV of any of the foregoing aspects, a vector of any of the foregoing aspects, a plasmid of any of the foregoing aspects, or a pharmaceutical composition of any of the foregoing aspects.

In another aspect, the present invention provides a method for increasing the expression of functional voltage-gated chloride channel 1 (CLCN1) protein in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid of any of the foregoing aspects, an AAV of any of the foregoing aspects, a vector of any of the foregoing aspects, a plasmid of any of the foregoing aspects, or a pharmaceutical composition of any of the foregoing aspects.

In another aspect, the present invention provides a method of increasing the expression of functional ZO-2-associated speckle protein (ZASP) in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of the aforementioned aspect. The nucleic acid of any of the foregoing aspects, the AAV of any of the foregoing aspects, the vector of any of the foregoing aspects, the plasmid of any of the foregoing aspects, or the pharmaceutical combination of any of the foregoing aspects things.

In some embodiments, expression of SERCA1 mRNA is increased 1.1-fold to 10-fold following administration of the vector or composition to the patient. In some embodiments, expression of CLCN1 mRNA is increased 1.1-fold to 10-fold following administration of the vector or composition to the patient. In some embodiments, the expression of ZASP mRNA is increased 1.1-fold to 10-fold following administration of the vector or composition to the patient.

In some embodiments, expression of CLCN1 mRNA is increased 1.1-fold to 10-fold following administration of a nucleic acid, vector, plasmid or pharmaceutical composition to a patient. In some embodiments, the patient exhibits improvement in myotonia after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, optionally wherein the patient exhibits improvement in myotonia about 12 weeks after the administration. In some embodiments, the patient exhibits a reduction in abnormal heart rhythms after administration of a nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, optionally wherein the patient exhibits a reduction in abnormal heart rhythms about 12 weeks after the administration. In some embodiments, after 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 a reduction in excessive daytime sleepiness about 12 weeks after the administration. . In some embodiments, after administration of the nucleic acid, AAV vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in hand muscle strength, optionally wherein the patient exhibits an increase in hand muscle strength approximately 12 weeks after administration. Increase in strength. In some embodiments, hand muscle strength is measured by a grip strength test. In some embodiments, the patient exhibits an improvement in sleep apnea symptoms after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, optionally wherein the patient exhibits sleep apnea symptoms about 12 weeks after the administration. improvement. In some embodiments, after administration of a nucleic acid, vector, plasmid, or pharmaceutical composition to a patient, the patient exhibits a protein encoding an insulin receptor, ryanodine receptor 1, cardiac troponin, and/or skeletal troponin. Increased corrective splicing of troponin RNA transcripts.

In some embodiments, prior to treatment, the patient was not diagnosed with DM1. In some embodiments, prior to treatment, the patient had one or more symptoms of DM1. In some embodiments, symptoms of DM1 include difficulty breathing, gasping, sleep apnea, intellectual disability, excessive daytime sleepiness, abnormal heart conditions, arrhythmias, cardiomyopathy, swallowing problems, muscle weakness, muscle atrophy, muscle stiffness, and/or muscle pain.

In some embodiments, the nucleic acid, vector, plasmid, or pharmaceutical composition is administered via intravenous, intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intradermal, transdermal, parenteral, intramuscular, intranasal , subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, or oral administration and/or administered to the patient by inhalation, perfusion, or lavage.

In another aspect, the present invention provides a kit comprising a nucleic acid of any of the foregoing aspects, an AAV of any of the foregoing aspects, a vector of any of the foregoing aspects, a plasmid of any of the foregoing aspects, or a pharmaceutical composition of any of the foregoing aspects, and instructions for use, wherein the instructions instruct a user of the kit to administer the nucleic acid, vector, plasmid, or pharmaceutical composition to a human patient diagnosed with DM1 or exhibiting one or more symptoms of DM1.

Definition

As used herein, the term "about" means a value that is within 10% above or below the stated value.

As used herein, "activity" refers to one or more forms of a nucleic acid or polypeptide that retain the biological activity of a native or naturally occurring nucleic acid or polypeptide, respectively, where "biological" activity refers to the biological function (e.g., splicing) caused by a native or naturally occurring nucleic acid or polypeptide, respectively.

As used herein, "administering" refers to providing or giving a therapeutic agent (e.g., an inhibitor) to a subject 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 can be systemic or local.

As used herein, "combination therapy" means administering to a subject two (or more) different agents or treatments as part of a treatment regimen defined for a particular disease or condition (eg, DM1). The treatment regimen defines the dosage and period of administration of each dose so that the effects of individual doses on subjects overlap. In some embodiments, delivery of two or more agents is simultaneous or simultaneous and the agents may be co-formulated. In other embodiments, two or more agents are not co-formulated and are administered in a sequential manner as part of a formulation regimen. In some embodiments, administration of a combination of two or more agents or treatments results in a reduction in symptoms or other parameters associated with the condition that is greater than using one agent or treatment delivered alone or in the absence of the other agent or treatment Observed situation. The effects of two treatments may be partially additive, fully additive, or greater than additive (eg, synergistic). Sequential or substantially simultaneous administration of each therapeutic agent may be accomplished by any appropriate route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination can be administered intravenously, and a second therapeutic agent of the combination can be administered orally.

As used herein, the term "conservative mutation,""conservativesubstitution," or "conservative amino acid substitution" refers to the substitution of one or more amino acids for one or more amino acids exhibiting similar physicochemical properties (such as polarity, electrostatic charge, and steric bulk). different amino acids. These properties for each of the twenty naturally occurring amino acids are summarized in Table 1 below. Table 1. Representative physicochemical properties of naturally occurring amino acids amino acids 3 letter code 1 letter code Side chain polarity Electrostatic characteristics at physiological pH (7.4) volume of space ^† alanine Ala A non-polar neutral Small Arginine Arg R polarity cation big asparagine Asn N polarity neutral medium aspartic acid Asp D polarity anion medium cysteine Cys C non-polar neutral medium glutamate Glu E polarity anion medium Glutamine gnc Q polarity neutral medium glycine Gly G non-polar neutral Small Histidine His H polarity Both neutral and cationic forms are in equilibrium at pH 7.4 big isoleucine Ile I non-polar neutral big Leucine Leu L non-polar neutral big lysine Lys K polarity cation big methionine Met M non-polar neutral big Phenylalanine Phe F non-polar neutral big proline Pro P non-polar neutral medium Serine Ser S polarity neutral Small threonine Thr T polarity neutral medium Tryptophan tp W non-polar neutral huge tyrosine Tyr Y polarity neutral big Valine Val V non-polar neutral medium ^† Based on the volume of A ³ : 50-100 series is small, 100-150 series is medium, 150-200 series are large, and >200 series are huge

It should be understood from this table that conserved amino acid families include, for example, (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. Thus, a conservative mutation or substitution is one in which an amino acid is substituted for a member of the same amino acid family (eg, Ser for Thr or Lys for Arg).

As used herein, 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 that have at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-3 and that promote expression of a transgene in a cell (e.g., a eukaryotic cell, such as a mammalian cell, a human cell, or a human muscle cell) when the transgene is operably linked to the promoter.

As used herein, the terms "dystrophin kinase" and "DMPK" include, for example, forms of the human DMPK gene encoding a DMPK protein having one or more (e.g., up to 25) conservative amino acid substitutions relative to the wild-type DMPK protein. As used herein, the terms "dystrophin kinase" and "DMPK" further include DMPK RNA transcripts containing an expanded CUG trinucleotide repeat region relative to the length of the CUG trinucleotide repeat region of the wild-type DMPK mRNA transcript. The expanded repeat region can contain, for example, 50 or more CUG trinucleotide repeats, such as about 50 to about 4,000 CUG trinucleotide repeats (e.g., particularly 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, Acid repeats, 180 trinucleotide repeats, 190 trinucleotide repeats, 200 trinucleotide repeats, 210 trinucleotide repeats, 220 trinucleotide repeats, 230 trinucleotide repeats, 240 trinucleotide repeats, 250 trinucleotide repeats, 260 trinucleotide repeats, 270 trinucleotide repeats, 280 trinucleotide repeats, 290 trinucleotide repeats, 300 trinucleotide repeats, 310 trinucleotide repeats, 320 trinucleotide repeats, 330 trinucleotide repeats, 340 trinucleotide repeats 350 trinucleotide repeats, 360 trinucleotide repeats, 370 trinucleotide repeats, 380 trinucleotide repeats, 390 trinucleotide repeats, 400 trinucleotide repeats, 410 trinucleotide repeats, 420 trinucleotide repeats, 430 trinucleotide repeats, 440 trinucleotide repeats, 450 trinucleotide repeats, 460 trinucleotide repeats, 470 trinucleotide repeats, 480 trinucleotide repeats, 490 trinucleotide repeats, 500 trinucleotide repeats, 510 trinucleotide repeats Acid repeats, 520 trinucleotide repeats, 530 trinucleotide repeats, 540 trinucleotide repeats, 550 trinucleotide repeats, 560 trinucleotide repeats, 570 trinucleotide repeats, 580 trinucleotide repeats, 590 trinucleotide repeats, 600 trinucleotide repeats, 610 trinucleotide repeats, 620 trinucleotide repeats, 630 trinucleotide repeats, 640 trinucleotide repeats, 650 trinucleotide repeats, 660 trinucleotide repeats, 670 trinucleotide repeats, 680 trinucleotide repeats Repeats, 690 trinucleotide repeats, 700 trinucleotide repeats, 710 trinucleotide repeats, 720 trinucleotide repeats, 730 trinucleotide repeats, 740 trinucleotide repeats, 750 trinucleotide repeats, 760 trinucleotide repeats, 770 trinucleotide repeats, 780 trinucleotide repeats, 790 trinucleotide repeats, 800 trinucleotide repeats, 810 trinucleotide repeats, 820 trinucleotide repeats, 830 trinucleotide repeats, 840 trinucleotide repeats, 850 trinucleotide repeats repeats, 860 trinucleotide repeats, 870 trinucleotide repeats, 880 trinucleotide repeats, 890 trinucleotide repeats, 900 trinucleotide repeats, 910 trinucleotide repeats, 920 trinucleotide repeats, 930 trinucleotide repeats, 940 trinucleotide repeats, 950 trinucleotide repeats, 960 trinucleotide repeats, 970 trinucleotide repeats, 980 trinucleotide repeats, 990 trinucleotide repeats, 1,000 trinucleotide repeats, 1,100 trinucleotide repeats, 1,20 0 trinucleotide repeats, 1,300 trinucleotide repeats, 1,400 trinucleotide repeats, 1,500 trinucleotide repeats, 1,600 trinucleotide repeats, 1,700 trinucleotide repeats, 1,800 trinucleotide repeats, 1,900 trinucleotide repeats, 2,000 trinucleotide repeats, 2,100 trinucleotide repeats, 2,200 trinucleotide repeats, 2,300 trinucleotide repeats, 2,400 trinucleotide repeats, 2,500 trinucleotide repeats, 2,600 trinucleotide repeats 3,000 trinucleotide repeats, 3,100 trinucleotide repeats, 3,200 trinucleotide repeats, 3,300 trinucleotide repeats, 3,400 trinucleotide repeats, 3,500 trinucleotide repeats, 3,600 trinucleotide repeats, 3,700 trinucleotide repeats, 3,800 trinucleotide repeats, 3,900 trinucleotide repeats, or 4,000 trinucleotide repeats).

In practicing the methods of the present invention, an "effective amount" of any compound or a combination of any compound or a pharmaceutically acceptable salt thereof is administered alone or in combination by any common and acceptable method known in the art.

As used herein, the term "endogenous" describes molecules (e.g., metabolites, polypeptides, nucleic acids) that are naturally present in a specific organism (e.g., a human) or at a specific location within an organism (e.g., an organ, tissue, or cell, such as a human cell). or cofactor).

As used herein, the term "gene" refers to a region of DNA that codes for a protein. Genes may include regulatory regions and protein coding regions. In some embodiments, a gene includes two or more introns and three or more exons, where each intron forms an intervening sequence between two exons.

As used herein, the term "IRES" refers to an internal ribosome entry site. In general, an IRES sequence is a feature that allows eukaryotic ribosomes to bind to an mRNA transcript and begin translation without binding to the 5' capped end. mRNA containing an IRES sequence produces two translation products, one initiated from the 5' end of the mRNA and the other initiated by the IRES-mediated internal translation mechanism.

As used herein, "length" of a nucleic acid refers to the linear size of a nucleic acid as assessed by measuring the amount of nucleotides from the 5' end to the 3' end of the nucleic acid. Exemplary molecular biology techniques that can be used to determine the length of a target nucleic acid are known in the art.

"Muscular dystrophy" refers to a group of muscle diseases that weaken the musculoskeletal system and prevent movement. Muscular dystrophy is characterized by progressive deterioration of muscle function (eg, 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.

As used herein, the term "blind muscle-like splicing regulatory factor 1" and its abbreviation "MBNL1" refer to an RNA-binding splicing protein involved in regulating alternative splicing, e.g., in human subjects. The terms "blind muscle-like splicing regulatory factor 1" and "MBNL1" are used interchangeably herein and refer not only to the wild-type form of MBNL1 produced by processing in cells, but also to any other naturally occurring variants of MBNL1 (e.g., splicing variants or allelic variants as described in Table 2 ). Nucleic acid sequences of MBNL1 variants are provided herein as SEQ ID NOs: 4-11. Recognized 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. Amino acid sequences of MBNL1 isoforms are provided herein as SEQ ID NOs: 12-19. Recognized 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. Those skilled in the art will recognize that any amino acid sequence that differs from SEQ ID NOs: 12-19 by one or more (eg, two, three, four, or five) conservative substitutions is otherwise considered an accepted isoform of MBNL1.

As used herein, the term "mutation" refers to any change in the sequence of a gene such that the sequence differs from that of the wild-type gene. The mutation may be selected from the group consisting of: a single nucleotide point mutation resulting in a premature stop codon, a single nucleotide insertion, a single nucleotide deletion, the insertion of two or more adjacent nucleotides, two or Deletion of more contiguous nucleotides, duplication of contiguous regions within the gene, or deletion of contiguous regions within the gene. A mutated gene may include a single mutation or multiple mutations. Mutations can occur in any region of the gene.

As used herein, the term "myotonic dystrophy" refers to a genetic muscular wasting disorder characterized by nuclear retention of RNA transcripts encoding DMPK and containing an expanded CUG trinucleotide repeat region (e.g., an expanded CUG trinucleotide repeat region having 50 to 4000 CUG repeats) in the 3' untranslated region (UTR) encoded by exon 15. In contrast, wild-type DMPK RNA transcripts typically contain 5 to 37 CUG repeats in the 3' UTR encoded by exon 15. In patients with myotonic dystrophy, the expanded CUG repeat region interacts with RNA-binding splicing factors such as myoblind-like splicing regulator 1 (MBNL1). This interaction results in retention of mutant transcripts in nuclear foci and sequestration of RNA binding proteins away from other pre-mRNA substrates, thereby promoting spliceopathic changes in proteins involved in regulating muscle structure and function. In myotonic dystrophy type 1 (DM1), skeletal muscle is usually the most severely affected tissue, but the disease also has toxic effects on heart and smooth muscle, the lens of the eye, and the brain. The skull, distal limbs, and diaphragm muscles are preferentially affected. Hand dexterity is impaired very early, leading to severe disability for decades. The median age of death in patients with myotonic dystrophy is 55 years, usually caused by respiratory failure (de Die-Smulders CE et al., Brain 121:1557-1563 (1998)).

As used herein, the term "neuron" is used to refer to a type of electrically excitable cell in the brain and nervous system that consists of a cell body or body, dendrites, and axons. The axon terminals of one neuron are separated from the dendrites of adjacent neurons by a space called the synaptic cleft, through which signals can be transmitted and intercellular communication can occur. Thus, multiple neurons connect to form neural circuits. Neurons are the main component of animal nervous tissue.

As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably herein and refer to a polymer of nucleotides of any length. Examples of polynucleotides are DNA polynucleotides and RNA polynucleotides. All nucleic acid sequences herein are written in 5' to 3' orientation and should be interpreted accordingly.

As used herein, the term "operably linked" in the context of a nucleic acid refers to a nucleic acid that is in a structural or functional relationship with another nucleic acid. For example, if one segment of DNA and another segment of DNA are positioned relative to each other on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer positioned relative to a coding region to promote transcription of the coding region , one segment of DNA is operably linked to another segment of DNA. In other examples, operably linked nucleic acids are not contiguous, but are positioned in a manner such that they are in a functional relationship with each other either as nucleic acids or as proteins expressed by them. For example, enhancers are not necessarily contiguous. Linking can be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adapters or linkers. In some embodiments, a nucleic acid molecule described herein is operably linked to a desmin (DES) promoter.

"Percent (%) sequence complementarity" relative to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to nucleic acids in a reference polynucleotide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve maximum percent sequence complementarity. A given nucleotide is considered to be "complementary" to a reference nucleotide as described herein if the two nucleotides form a canonical Watson-Crick base pair. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. Appropriate Watson-Crick base pairs are referred to in the context as "matches," while each unpaired nucleotide and each incorrectly paired nucleotide is referred to as a "mismatch." For the purpose of determining percent nucleic acid sequence complementarity, alignment can be accomplished in a variety of ways known to those skilled in the art, such as 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 algorithm required to achieve maximum complementarity over the full length of the compared sequences. As an example, the percent sequence complementarity of a given nucleic acid sequence A with a given nucleic acid sequence B (which may alternatively be phrased as a given nucleic acid sequence A having a certain percent complementarity with a given nucleic acid sequence B) is calculated as follows: 100 times (fraction X/Y) where X is the number of complementary base pairs in the program's alignment of A and B (e.g., as performed by computer software such as BLAST), and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A with B will not be equal to the percent sequence complementarity of B with A. As used herein, a query nucleic acid sequence is considered to be "fully complementary" to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity with the reference nucleic acid sequence.

As used herein, the term "sufficient complementarity for hybridization" refers to a nucleic acid sequence or portion thereof that need not be completely complementary (e.g., 100% complementary) to a target region or nucleic acid sequence or portion thereof, which has one or more nucleotide mismatches relative to the target region, but is still able to hybridize with the target region under specific conditions. For example, a nucleic acid can be, for example, 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, but still forms sufficient base pairs with the target to hybridize throughout its length.

"Percent (%) sequence identity" relative to a reference polynucleotide or polypeptide sequence is defined as the number of sequences in a candidate sequence that is identical to the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity. The percentage of nucleic acids or amino acids that are identical to nucleic acids or amino acids in a polypeptide sequence. For the purpose of determining percent nucleic acid or amino acid sequence identity, alignment can be accomplished in a variety of ways known to those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2 or Megalign software. One skilled in the art can determine the parameters suitable for comparing sequences, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A relative to, compared to, or compared to a given nucleic acid or amino acid sequence B (which may alternatively be phrased as relative to, compared to, or compared to a given Nucleic acid or amino acid sequence B A given nucleic acid or amino acid sequence A) with a certain percentage of sequence identity is calculated as follows: 100 times (fraction X/Y) Where It should be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the % sequence identity of A relative to B will not be equal to the % sequence identity of B relative to A.

As used herein, the term "pharmaceutical composition" refers to a composition containing a nucleic acid described herein, formulated together with a pharmaceutically acceptable excipient, and manufactured or sold with approval by a governmental regulatory agency as part of a therapeutic regimen for treating a disease in a subject.

As used herein, the term "pharmaceutically acceptable" means suitable for contact with tissue of a subject, such as a mammal (e.g., human), without undue toxicity, irritation, allergic reaction, and commensurate with a reasonable benefit/risk ratio other problems complications of these compounds, materials, compositions and/or dosage forms.

As used herein, the term "plastid" refers to an extrachromosomal circular double-stranded DNA molecule to which additional DNA segments can be linked. A plastid is a type of vector, which is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Certain plastids are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial plastids with bacterial replication origins and episomal mammalian plastids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome after introduction into the host cell, and thereby replicated along with the host genome. Certain plastids are capable of directing the expression of genes to which they are operably linked.

As used herein, the term "promoter" refers to a region within a gene regulatory region that is capable of initiating transcription of a gene into messenger RNA, wherein transcription is initiated by binding of RNA polymerase to or near the promoter. In some embodiments, the promoter is a DES promoter.

The term "reading frame" refers to how ribosomes define codons within a gene, as determined by the binding of tRNA to trinucleotide codons during translation of mRNA. When a reading frame is "restored," this indicates that the reading frame was first altered by a frameshift or nonsense mutation and then somehow returned to the original reading frame.

"Reference" means any useful reference for comparing the level of a protein or nucleic acid (e.g., mRNA) associated with DM1 (e.g., DMPK or MBNL1). A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, such as a predetermined negative control value, such as a "normal control" or a previous 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 who does not have DM1 or does not show symptoms of DM1 (e.g., DMPK or MBNL1); a sample from a subject diagnosed with DM1 (e.g., DMPK or MBNL1); a sample from a subject who has been treated for DM1 (e.g., DMPK or MBNL1); or a sample of a purified protein (e.g., MBNL1) of known normal concentration. "Reference standard or level" means a value or value derived from a reference sample. A "normal control value" is a predetermined value indicative of a non-disease state, such as the expected value for a healthy control subject. Typically, normal control values are expressed as a range ("between X and Y"), an upper threshold ("no higher than X"), or a lower threshold ("no lower than X"). Subjects whose measured values are within the normal control values for a particular biomarker are often referred to as being "within normal limits for that biomarker." A normal reference standard or level can be a value or numerical value derived from a subject who does not suffer from muscular dystrophy (e.g., MD). In a preferred embodiment, 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 general health status. A standard curve of purified protein levels (e.g., any of the proteins described herein) within the normal reference range can also be used as a reference.

As used herein, the term "repeat region" refers to a segment within a gene or its RNA transcript that contains nucleic acid repeats, such as a poly-CTG sequence in the 3'UTR of the human DMPK gene (or a poly-CUG sequence in the 3'UTR of its RNA transcript). If the number of nucleotide repeats in the repeat region exceeds the number of repeats normally found in the repeat region of the wild-type form of the gene or its RNA transcript, the repeat region is considered an "expanded repeat region", "repeat expansion" or the like. For example, the 3'UTR of the wild-type human DMPK gene normally contains 5 to 37 CTG or CUG repeats. Thus, in the context of the DMPK gene or its RNA transcript, "expanded repeat region" and "repeat expansion" refer to a repeat region containing greater than 37 CTG or CUG repeats, such as about 50 to about 4,000 CUG trinucleotide repeats (e.g., particularly 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, 230 trinucleotide repeats, 240 trinucleotide repeats, 250 trinucleotide repeats, 260 trinucleotide repeats, 270 trinucleotide repeats, 280 trinucleotide repeats, 290 trinucleotide repeats, 300 trinucleotide repeats, 310 trinucleotide repeats, 320 trinucleotide repeats, 330 trinucleotide repeats, 340 trinucleotide repeats, 350 trinucleotide repeats, 360 trinucleotide repeats, 370 trinucleotide repeats, 380 trinucleotide repeats, 390 trinucleotide repeats, 400 trinucleotide repeats, 410 trinucleotide repeats, 420 trinucleotide repeats, 430 trinucleotide repeats, 440 trinucleotide repeats, 450 trinucleotide repeats, 460 trinucleotide repeats, 470 trinucleotide repeats, 480 trinucleotide repeats, 490 trinucleotide repeats Repeats, 500 trinucleotide repeats, 510 trinucleotide repeats, 520 trinucleotide repeats, 530 trinucleotide repeats, 540 trinucleotide repeats, 550 trinucleotide repeats, 560 trinucleotide repeats, 570 trinucleotide repeats, 580 trinucleotide repeats, 590 trinucleotide repeats, 600 trinucleotide repeats, 610 trinucleotide repeats, 620 trinucleotide repeats, 630 trinucleotide repeats, 640 trinucleotide repeats, 650 trinucleotide repeats, 660 trinucleotide repeats, 67 0 trinucleotide repeats, 680 trinucleotide repeats, 690 trinucleotide repeats, 700 trinucleotide repeats, 710 trinucleotide repeats, 720 trinucleotide repeats, 730 trinucleotide repeats, 740 trinucleotide repeats, 750 trinucleotide repeats, 760 trinucleotide repeats, 770 trinucleotide repeats, 780 trinucleotide repeats, 790 trinucleotide repeats, 800 trinucleotide repeats, 810 trinucleotide repeats, 820 trinucleotide repeats, 830 trinucleotide repeats, 840 trinucleotide repeats nucleotide repeats, 850 trinucleotide repeats, 860 trinucleotide repeats, 870 trinucleotide repeats, 880 trinucleotide repeats, 890 trinucleotide repeats, 900 trinucleotide repeats, 910 trinucleotide repeats, 920 trinucleotide repeats, 930 trinucleotide repeats, 940 trinucleotide repeats, 950 trinucleotide repeats, 960 trinucleotide repeats, 970 trinucleotide repeats, 980 trinucleotide repeats, 990 trinucleotide repeats, 1,000 trinucleotide repeats, 1,100 trinucleotide repeats 1,200 trinucleotide repeats, 1,300 trinucleotide repeats, 1,400 trinucleotide repeats, 1,500 trinucleotide repeats, 1,600 trinucleotide repeats, 1,700 trinucleotide repeats, 1,800 trinucleotide repeats, 1,900 trinucleotide repeats, 2,000 trinucleotide repeats, 2,100 trinucleotide repeats, 2,200 trinucleotide repeats, 2,300 trinucleotide repeats, 2,400 trinucleotide repeats, 2,500 trinucleotide repeats, 2,600 trinucleotide repeats, 2,700 trinucleotide repeats, 2,800 trinucleotide repeats, 2,900 trinucleotide repeats, 3,000 trinucleotide repeats, 3,100 trinucleotide repeats, 3,200 trinucleotide repeats, 3,300 trinucleotide repeats, 3,400 trinucleotide repeats, 3,500 trinucleotide repeats, 3,600 trinucleotide repeats, 3,700 trinucleotide repeats, 3,800 trinucleotide repeats, 3,900 trinucleotide repeats, or 4,000 trinucleotide repeats).

"Skeletal muscle" means a form of striated muscle tissue that is under the control of the body's nervous system, that is, it is controlled voluntarily. The term muscle refers to bundles of muscle fibers connected together by connective tissue. Skeletal muscle can be attached to bones by tendons. Non-limiting examples of skeletal muscle include, for example, the diaphragm, extensor digitorum longus, tibialis anterior, gastrocnemius, soleus, plantars, biceps, triceps, deltoids, pectoralis major, pectoralis minor, rhomboids, trapezius, sartorius, knee flexors and extensors, elbow flexors and extensors, shoulder abductors, and abdominal muscles.

As used herein, the term "splicing disease" refers to an alteration in the splicing pattern of an mRNA transcript that results in the expression of one or more alternative splicing products relative to the wild-type form of the target mRNA transcript. Splicing disorders can result in toxic loss of function if, for example, the mRNA transcript is spliced in such a manner that one or more exons required for the activity of the encoded protein are no longer present in the mRNA transcript after translation. Additionally or alternatively, toxic loss of function may occur due to aberrant inclusion of one or more introns, for example in a manner that prevents the correct folding of the encoded protein.

As used herein, the terms "subject" and "patient" are interchangeable and refer to an organism that is receiving treatment for a specific disease or condition as described herein. In a preferred embodiment, the subject is a human.

As used herein, the term "transduction" or "transduce" refers to a method of introducing a viral vector construct or a portion thereof into a cell and subsequently expressing a transgene encoded by the vector construct or a portion thereof in the cell.

As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcriptional regulatory elements may include promoters, enhancers, and other nucleic acids that control or help control gene transcription (eg, polyadenylation signals). Examples of transcriptional regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990). In some embodiments, the transcriptional regulatory element of the compositions herein is a DES promoter.

As used herein, the term "transfection" refers to any of a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, diethylamine DEAE-dextran transfection, NUCLEOFECTION™, extrusion transfection, sonoporation, optical transfection, MAGNETOFECTION™, punch transfection and the like.

As used herein, the term "treatment" refers to therapeutic treatment in which the goal is to prevent or slow down (mitigate) undesirable physiological changes or conditions, such as hereditary muscle wasting disorders, such as myotonic dystrophy And especially the progress of DM1. In the context of myotonic dystrophy treatment, beneficial or desirable clinical outcomes indicative of successful treatment include, but are not limited to, alleviation of symptoms, reduction in disease severity, stable (i.e., non-worsening) status of disease, delay or reduction in Chronic disease progression, improvement or alleviation of disease status and remission (partial or total), whether detectable or undetectable. Treatment of patients with myotonic dystrophy (e.g., DM1) may be manifested by one or more detectable changes, such as relative to the patient's MBNL1 prior to administration of a therapeutic agent, such as a vector or nucleic acid described herein. Performance, an increase in the performance of MBNL1 (for example, an increase in the performance of MBNL1 by approximately 1% or more, such as an increase of approximately 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%). Methods that can be used to assess the level of protein expression are known in the art and include immunoblotting assays and immunostaining assays described herein. Additional clinical indications for successful treatment of DM1 patients include, for example, alleviation of splicing disorders of RNA transcripts that are spliced in an MBNL1-dependent manner. For example, observations indicative of successful treatment of patients with myotonic dystrophy include the finding that patients exhibit receptors for one or more RNA transcripts of MBNL1 following administration of a therapeutic agent, such as those described herein. Qualitative corrective splicing increases. For example, markers indicative of successful treatment of myotonic dystrophy include determining that the patient exhibits increased expression of sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 1 (SERCA1) containing exon 22, such as, for example, using herein An increase in the described RNA or protein detection assay assessment of about 1.1-fold to about 10-fold, or more (e.g., SERCA1 mRNA containing exon 22 increases 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold , 2 times, 2.1 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more). Myotonic dystrophy treatment may also be manifested by a decrease in the expression of voltage-gated chloride channel 1 (CLCN1) containing exon 7a mRNA, such as a decrease of approximately 1 as assessed, for example, using the RNA or protein detection assays described herein. % to about 100% (e.g., expression of CLCN1 mRNA containing exon 7a is reduced by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). Additionally, successful treatment may be marked by determining that the patient exhibits a reduction in expression of ZO-2-associated speck protein (ZASP) containing exon 11, such as a reduction of about 1% to about 100% (e.g., expression of ZASP mRNA containing exon 11 is reduced by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90% or 100%). Successful treatment of myotonic dystrophy is also marked by the finding that, following therapy, patients exhibit RNA encoding the insulin receptor, ryanodine receptor 1 (RYR1), cardiac troponin, and/or skeletal muscle troponin. Increased corrective splicing of transcripts, such as, for example, an increase of about 1.1-fold to about 10-fold, or more, as assessed, for example, using the RNA or protein detection assays described herein (e.g., encoding insulin receptor, RYR1, cardiac troponin, and /or increase the expression of correctly spliced RNA transcripts of skeletal muscle troponin by approximately 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 times, 8 times, 9 times, 10 times, or more). Additional clinical indications for successful treatment of myotonic dystrophy include improvements in muscle function, such as improvements in the skull, distal limbs, and diaphragm muscles.

As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors, such as plasmids, RNA vectors, viruses or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994/011026; the document is incorporated herein by reference with respect to vectors suitable for expressing target genes. Expression vectors suitable for the compositions and methods described herein contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express nucleic acid molecules as described herein include plasmids containing regulatory sequences (such as promoter and enhancer regions) that direct gene transcription. Other vectors that can be used to express nucleic acid molecules contain polynucleotide sequences that enhance the translation rate of such genes or improve the stability or nuclear export of mRNA produced by gene transcription. Such sequence elements include, for example, 5' and 3' non-translated regions, IRES, and polyadenylation signal sites that guide efficient transcription of genes carried on the expression vector. Expression vectors suitable for the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, conmycin, nourseothricin, or zeocin.

The compositions and methods described herein can be used to reduce the occurrence of spliceopathies and for treating disorders associated with RNA dominance, such as myotonic dystrophy (e.g., myotonic dystrophy type 1 (DM1)). The compositions described herein include nucleic acid molecules including a transgene encoding human myosin-blind splicing regulator 1 (MBNL1), wherein the transgene is operably linked to a desmin (DES) promoter, and methods of delivering the transgene into cells so as to overexpress the resulting protein MBNL1. Overexpression of MBNL1 provides important physiological benefits, including returning the expression of a set of developmentally regulated genes from their incorrect fetal expression to their appropriate mature expression. Without being limited by mechanism, the compositions described herein can improve such a condition by increasing the expression of MBNL1 in a human patient diagnosed with or showing one or more symptoms of DM1. For example, the compositions and methods described herein can be used to treat a disorder, such as DM1, or a symptom of such a disorder, such as myotonia.

Nucleic acids described herein can be encoded by vectors, such as viral vectors. For example, described herein are adeno-associated virus (AAV) vectors, such as pseudotyped AAV vectors (eg, AAV2/8 vectors), containing a transgene encoding MBNL1 operably linked to a DES promoter.

The present invention is based, at least in part, on the discovery that operably linking the DES promoter to a transgene encoding MBNL1 and encoding the resulting nucleic acid into a viral vector, such as a pseudotyped AAV2/8 vector, results in highly efficient targeted delivery of the nucleic acid into cells and rapid, robust overexpression of MBNL1.

Using the compositions and methods described herein, expression of MBNL1 can be rapidly increased in patients diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia, thereby allowing expression of a set of developmentally regulated genes to be derived from its Improperly adopted fetal stage representations are returned to the desired mature stage representations. This switch in gene expression and subsequent shift in protein expression alleviates the symptoms of DM1 and may ameliorate the disease.

The following sections provide descriptions of exemplary nucleic acids that can be used in conjunction with the compositions and methods described herein, as well as vectors encoding such nucleic acids and methods that can be used to treat patients diagnosed with or displaying one or more symptoms of DM1. Methods of Treating DM1

Muscular dystrophies are a group of genetic disorders characterized by progressive muscle weakness. Myotonic dystrophy is a hereditary somatic chromosomally dominant muscular dystrophy. Myotonic dystrophy type 1 (DM1) is a myotonic dystrophy disorder caused by mutations in the myotonic dystrophy protein kinase gene ( DMPK ) and subsequent incorrect splicing, resulting in the splicing protein blind myoid splicing regulator 1 ( MBNL1) is caused by sequestration in ribonuclear foci. This ultimately results in a series of alternative splicing events of a set of developmentally regulated genes returned to the fetal profile. Causes many phenotypic changes, the most common of which is myotonia, a long-term state of muscle overexcitation that results in sustained discharge and delayed force relaxation. Over time, persistence of this fetal splicing profile can lead to shortened lifespan, acute and chronic muscle pain, and the development of gastrointestinal problems, vision problems, heart problems, and endocrine-disrupting conditions, including thyroid problems and diabetes.

Using the compositions and methods described herein, a patient suffering from myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia, may be administered a code that is operably linked to A nucleic acid molecule encoding a transgene of MBNL1 with a desmin (DES) promoter, or a composition encoding the same, in order to overexpress MBNL1. Independently of the mechanism, this overexpression provides the beneficial effect of restoring correct mature-stage MBNL1 expression, thereby returning a series of alternative splicing events of developmentally regulated genes from its inappropriate fetal profile to its mature adult profile. Subsequently, symptoms of DM1 including but not limited to myotonia improved.

In some embodiments, the patient is at least 18 years old.

In some embodiments, the patient is not yet ambulatory or ambulatory. Myotonic dystrophy type I

Myotonic dystrophy type I (DM1) is the most common form of myotonic dystrophy in adults and has an estimated frequency of 1 in 7500 people (Harper P S., Myotonic Dystrophy. London: W.B. Saunders Company; 2001). The disease is a somatic chromosomal dominant disorder caused by the expansion of non-coding CTG repeats in the human DMPK1 gene. DMPK1 is a gene encoding a cytoplasmic serine/threonine kinase (Brook et al., Cell. 68:799-808 (1992)). The expanded CTG repeat sequence is located in the 3' untranslated region (UTR) of DMPK1 and is encoded by exon 15. This mutation results in RNA dominance, in which expression of RNA containing expanded CUG repeats (CUGexp) induces cellular dysfunction (Osborne R J and Thornton C A., Human Molecular Genetics. 15:R162-R169 (2006)) .

Mutant forms of DMPK mRNA carrying larger CUG repeats are fully transcribed and polyadenylated but remain trapped in the nucleus (Davis et al., Proc. Natl. Acad. Sci. USA 94:7388-7393 (1997)) . The nuclear retention of these mutated mRNAs is one of the most important pathological features of DM1. DMPK genes typically have about 5 to about 37 CTG repeats in the 3' UTR. In DM1, this number is significantly expanded and may range, for example, from 50 to greater than 4,000 repeats. The CUG _exp channel in subsequent RNA transcripts interacts with RNA-binding splicing factor proteins, including blind muscle-like splicing regulator 1 (MBNL1). The enhanced affinity resulting from amplification of the CUG repeat region results in mutant transcripts retaining such splicing factor proteins in nuclear foci. The toxicity of this mutant RNA results from the sequestration of the RNA-binding splicing factor protein away from other pre-mRNA substrates, including those encoding proteins important in regulating muscle function.

In DM1, skeletal muscle is the most affected tissue, but the disease also has important effects on the heart and smooth muscle, the lens of the eye, and the brain. Among the musculature, the skull, distal limbs, and diaphragm muscles are often preferentially affected. Hand dexterity is compromised early on, resulting in severe disability for decades. The median age at death is 55 years, usually from respiratory failure (de Die-Smulders CE et al., Brain 121(Pt 8):1557-1563 (1998)). Symptoms of myotonic dystrophy include, but are not limited to, myotonia, muscle stiffness, distal weakness, facial and jaw muscle weakness, dysphagia, drooping eyelids (ptosis), neck muscle weakness, arm and leg muscle weakness, Persistent muscle pain, drowsiness, muscle atrophy, difficulty swallowing, respiratory insufficiency, arrhythmia, myocardial damage, sluggishness, insulin resistance and cataracts. In children, symptoms may also include developmental delays, learning problems, language and speech difficulties, and personality development challenges. Pathogenic DMPK transcript

Patients that can be treated using the compositions and methods described herein include patients such as human patients with myotonic dystrophy, such as patients diagnosed with or showing one or more symptoms of DM1, such as myotonia, including those expressing DMPK RNA transcripts with CUG repeat expansions. Exemplary DMPK RNA transcripts that can be expressed by patients undergoing treatment with the compositions and methods described herein are described in GenBank Accession Nos. NM_001081560.1, NT_011109.15 (nucleotides 18540696 to 18555106), NT_039413.7 (nucleotides 16666001 to 16681000), NM_032418.1, AI007148.1, AI304033.1, BC024150.1, BC056615.1, BC075715.1, BU519245.1, CB247909.1, CX208906.1, CX732022.1, 560315.1, 560316.1, NM_001081562.1, and NM_001100.3. Correction of splicing disorders

In some embodiments, the compositions and methods described herein may be used to correct patients, such as patients suffering from myotonic dystrophy (e.g., patients diagnosed with or exhibiting one or more symptoms of DM1 such as myotonia ) one or more splicing diseases. Without being limited by mechanism, the compositions and methods herein increase MBNL1 expression in cells. This in turn enables corrective splicing of one or more (eg, two, three, four, five, or more) RNA transcript substrates of MBNL1. For example, upon administration of a composition described herein to a patient suffering from myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1, the patient may, for example, develop a muscle in the tibialis anterior, gastrocnemius, and/or quadriceps, showing increased expression of sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 1 (SERCA1) mRNA containing exon 22. An increase in the expression of SERCA1 mRNA transcripts containing exon 22 can be, for example, an increase of about 1.1-fold to about 10-fold or more, as assessed, for example, using the RNA or protein detection assays described herein (e.g., expression of SERCA1 mRNA transcripts containing exon 22 The expression of SERCA1 mRNA in Zi22 increased by about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times , 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7 times, 7.1 times, 7.2 times, 7.3 times, 7.4 times , 7.5 times, 7.6 times, 7.7 times, 7.8 times, 7.9 times, 8 times, 8.1 times, 8.2 times, 8.3 times, 8.4 times, 8.5 times, 8.6 times, 8.7 times, 8.8 times, 8.9 times, 9 times, 9.1 times, 9.2 times, 9.3 times, 9.4 times, 9.5 times, 9.6 times, 9.7 times, 9.8 times, 9.9 times, 10 times, or more times).

In some embodiments, after administering a composition described herein to a patient with myotonic dystrophy, such as a patient diagnosed with or showing one or more symptoms of DM1, such as myotonia, the patient may exhibit, for example, reduced expression of voltage-gated chloride channel 1 (CLCN1) mRNA containing exon 7a in the tibialis anterior, gastrocnemius, and/or quadriceps muscles. The reduced expression of CLCN1 mRNA transcripts containing exon 7a can be, for example, a reduction of about 1% to about 100% (e.g., CLCN1 containing exon 7a) as assessed, for example, using an RNA or protein detection assay described herein. The expression of mRNA decreased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%,

Additionally or alternatively, after administering a composition described herein to a patient with myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia, the patient may exhibit reduced expression of ZO-2 associated plaque protein (ZASP) containing exon 11, for example, in the tibialis anterior, gastrocnemius, and/or quadriceps muscles. The reduced expression of ZASP mRNA transcripts containing exon 11 can be, for example, a reduction of about 1% to about 100% (e.g., a reduction of ZASP containing exon 11) as assessed, for example, using an RNA or protein detection assay described herein. The expression of mRNA decreased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%,

Additionally or alternatively, following administration of a composition described herein to a patient with myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia, the patient may exhibit an increase in the correct splicing of RNA transcripts encoding insulin receptors, ryanodine receptor 1 (RYR1), cardiac sarcoma, and/or skeletal muscle sarcoma, such as an increase of about 1.1-fold to about 10-fold, or more (e.g., an increase of about 1.1-fold, 1.2-fold, 1.3-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-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, 2.5-fold, 2.7-fold, 2.8-fold, 2.9-fold, 2.5-fold, 2.8-fold, 2.9-fold, 2.5-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 2.5 ... .3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4 times, 4.1 times, 4.2 times , 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7 times, 7.1 times, 7. 2 times, 7.3 times, 7.4 times, 7.5 times, 7.6 times, 7.7 times, 7.8 times, 7.9 times, 8 times, 8.1 times, 8.2 times, 8.3 times, 8.4 times, 8.5 times, 8.6 times, 8.7 times, 8.8 times, 8.9 times, 9 times, 9.1 times, 9.2 times, 9.3 times, 9.4 times, 9.5 times, 9.6 times, 9.7 times, 9.8 times, 9.9 times, 10 times or more). Improvement of muscle function

Beneficial therapeutic effects of the compositions and methods described herein, such as the ability of the nucleic acid molecules described herein and compositions encoding them to overexpress MBNL1 and subsequently restore the correct splicing of proteins involved in regulating muscle function may manifest clinically in a variety of ways Come out. For example, patients with myotonic dystrophy (e.g., patients diagnosed with or exhibiting one or more symptoms of DM1 such as myotonia) may demonstrate improvements in cranial, distal limb, and/or diaphragm muscle function. . Improvements in muscle function may be observed, for example, as increases in muscle mass, muscle contraction frequency, and/or muscle contraction amplitude. For example, using the compositions and methods described herein, a patient suffering from myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia, can exhibit the skull, distal limbs, and/or an increase in diaphragm muscle mass, muscle contraction frequency, and/or muscle contraction amplitude. The increase in muscle mass, muscle contraction frequency and/or muscle contraction amplitude may be, for example, an increase of 1% or more, such as an increase of 1% to 25%, 1% to 50%, 1% to 75%, 1% to 100%, 1% to 500%, 1% to 1,000% or more, such as approximately 1%, 5%, 10%, 15%, 20%, 25%, 50% increase in muscle mass, muscle contraction frequency and/or muscle contraction amplitude , 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 80%, 900%, 1,000% or more.

Specifically, nucleic acid molecules described herein, and compositions encoding the same, are used in patients with myotonic dystrophy, such as patients diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia. A beneficial therapeutic effect may be manifested by a reduction in myotonia. Accordingly, using the compositions and methods described herein, a nucleic acid molecule or encoding the same may be administered to a patient suffering from myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1, such as myotonia. Compositions to promote and/or accelerate muscle relaxation. For example, the compositions and methods described herein can be used to accelerate muscle relaxation by inhibiting the occurrence of spontaneous action potentials caused by fluctuations in chloride ion concentration. Without being limited by mechanism, such beneficial activity may result from restoration of correct splicing of CLCN1 mRNA, for example, resulting in reduced expression of CLCN1 mRNA containing exon 7a in patients. Since the CLCN1 channel protein regulates chloride ion concentration, correcting the splicing pattern of CLCN1 mRNA transcripts may reduce the occurrence of spontaneous action potentials and increase the rate of muscle relaxation, thereby improving myotonia.

The suppression of myotonia can be evaluated using a variety of techniques known in the art, such as by electromyography. Specifically, electromyography can be performed on the left and right quadriceps, left and right gastrocnemius, left and right tibialis anterior, and/or lumbar paraspinal muscles to assess the effects of the compositions and methods described herein on myotonia in patients (e.g., human patients diagnosed with or showing one or more symptoms of DM1). Electromyography protocols have been described, for example, in Kanadia et al., Science 302: 1978-1980 (2003). For example, electromyography can be performed using a 30-gauge concentric needle electrode and at least 10 needle insertions per muscle. In this manner, the average myotonia level of a subject, such as a human patient or a model organism (e.g., a murine model of muscular dystrophy described herein) can be determined. This level can then be compared to the average myotonia level 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). The finding that the average myotonia level is reduced after administration of the therapeutic agent can serve as an indication of successful treatment of myotonic dystrophy and as an indicator of successful improvement of myotonic symptoms.

Other examples of tests that can be used to assess the level of myotonia in a patient or model organism include a grip strength test in which the patient or model organism squeezes a handheld dynamometer device. For example, the dynamometer can be squeezed three times with each hand. The measurements from each squeeze are recorded, and then the average score is calculated using the measurements from both hands. Comparison of tests performed before and after administration of a therapeutic agent described herein can serve as an indication of the success of the agent. For example, an increase in the average score on the grip strength test after administration of the therapeutic agent can serve as an indication of successful improvement of symptoms of myotonia and further as an indication of successful treatment of myotonic dystrophy. Improvement of Additional Symptoms of Myotonic Dystrophy

Using the compositions and methods described herein, a patient suffering from myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1 (e.g., myotonia), may be administered The composition is designed to attenuate or completely eliminate one or more symptoms of DM1. In addition to the myotonia mentioned above, symptoms of myotonic dystrophy include, but are not limited to, muscle stiffness, distal weakness, facial and jaw muscle weakness, dysphagia, ptosis, neck muscle weakness, arm and leg muscle weakness, Persistent muscle pain, drowsiness, muscle atrophy, difficulty swallowing, respiratory insufficiency, arrhythmia, myocardial damage, sluggishness, insulin resistance and cataracts. In children, symptoms may also include developmental delays, learning problems, language and speech difficulties, and personality development challenges. The compositions and methods described herein can be used to alleviate one or more (eg, two, three, four, five, or more) or all of the aforementioned symptoms. Duration of treatment effect

The compositions and methods described herein provide beneficial clinical effects that can last for long periods of time. For example, using one or more compositions described herein, patients with myotonic dystrophy, such as patients diagnosed with or showing one or more symptoms of DM1 (e.g., myotonia), can show improvements in muscle function (e.g., improvements in muscle mass and/or muscle activity in the skull, distal limbs, and diaphragm muscles) and/or a reduction in one or more symptoms of myotonic dystrophy for a period of one or more days, weeks, months, or years. For example, using the compositions and methods described herein, the beneficial therapeutic effects described herein can 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 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, or at least 1 year. Mouse Model of Myotonic Dystrophy

To examine the therapeutic effects of the compositions described herein, an appropriate mouse model can be utilized. For example, the human skeletal actin (HSA) ^LR (long repeat) mouse model is a model established for DM1 (see, e.g., Mankodi et al., Science 289:1769 (2000), which is incorporated herein by reference for its disclosure regarding HSA ^LR mice). These mice carry a human skeletal actin (hACTA1) transgene containing an expanded CTG region. Specifically, the hACTA1 transgene in the HSA ^LR mouse contains 220 CTG repeats inserted into the 3' UTR of the gene. Once transcribed, hACTA1-CUGexp RNA transcripts accumulate in nuclear foci of skeletal muscle and cause myotonia similar to that observed in human DM1, e.g., 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 important roles 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), each of which is incorporated herein by reference for its disclosure of HSA ^LR mice). Therefore, improving DM1 symptoms in HSA ^LR mice by suppressing the expression of hACTA1 RNA transcripts carrying CUG expanded repeats may be a predictor of improving similar symptoms in human patients by suppressing the expression of pathological DMPK RNA transcripts. HSA ^LR DM1 mice can be generated using methods known in the art, for example, by inserting an hACTA1 transgene with 250 CUG repeats in the 3' UTR of human skeletal actin into the genome of FVB/N mice. The transgene then manifests in mice as CUG repeat expansions in hACTA1 RNA. This repeat-expanded RNA is retained in the cell nucleus, forming nuclear inclusions similar to those observed in human tissue samples from patients with DM1.

As described above, in the HSA ^LR mouse model, accumulation of expanded CUG RNA in the nucleus leads to sequestration of poly(CUG)-binding splicing factor proteins such as MBNL1 (Miller et al., EMBO J. 19:4439 (2000)). Such splicing factors that control alternative splicing of the SERCA1 gene are thus sequestered in the expanded CUG foci in HSA ^LR mice. Such sequestration leads to dysregulation of alternative splicing of the SERCA1 gene. In order to assess the therapeutic efficacy of the compositions described herein, measurements of correctly spliced SERCA1 mRNA (and thus functional SERCA1 protein) may be obtained before and after administration of the compositions using RNA and protein detection methods known in the art and described herein. For example, to monitor increased expression of correctly spliced SERCA1 mRNA transcripts (e.g., SERCA1 mRNA transcripts containing exon 22) following administration of any of the compositions described herein, total RNA can be purified from one or more, or all, of the tibialis anterior, gastrocnemius, and quadriceps muscles of HSA ^LR mice using the RNeasy Lipid Tissue Mini Kit (Qiagen®) according to the manufacturer's instructions. RT-PCR can be performed using, 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. Forward and reverse primers for SERCA1 have been described, for example, in Bennett and Swayze, Annu Rev. Pharmacol. 50:259-293 (2010)). PCR products can 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 interfering with RNA molecules or vectors encoding them, for example, in the tibialis anterior, gastrocnemius, and/or quadriceps muscles of HSA ^LR mice may be a predictor of the therapeutic efficacy of the compositions described herein.

Additional mouse models of myotonic dystrophy include LC15 mice (strain A), which are transgenic mice containing an intact human DMPK 3′UTR (developed by Wheeler et al. at the University of Rochester) . These mice are second generation mice backcrossed to the FVB background. The DMPK transgene appears in these mice as CUG repeats in DMPK RNA transcripts that are retained in the nucleus, resulting in nuclear inclusions similar to those observed in human tissue samples from patients with DM1. LC15 mice express DMPK RNA transcripts containing about 350 to about 400 CUG repeats. These mice showed early signs of DM1 and did not show any myotonia in their muscle tissue.

Yet another murine model of myotonic dystrophy that can be used to assess the therapeutic efficacy of the nucleic acid molecules described herein, or vectors encoding the same, is the DMSXL model. The DMSXL mouse line was generated by serial breeding of mice with high levels of CTG repeat instability. As a result, DMSXL mice exhibit DMPK RNA transcripts containing >1,000 CUG trinucleotide repeats in the 3' UTR. DMSXL mice and methods for their production are described in detail in, for example, Gomes-Pereira et al., PLoS Genet. 3:e52 (2007) and Huguet et al., A, PLoS Genet. 8:e1003043 (2012), each of which The author's disclosures are incorporated into this article by full citation. Nucleic acid of this disclosure

Nucleic acid molecules described herein include a transgene encoding MBNL1 operably linked to the desmin (DES) promoter. Operably linking the MBNL1 transgene to the DES promoter advantageously and robustly increases transcription of the MBNL1 gene product in target cells. These nucleic acids can be administered directly or can be encoded in a vector or other composition to optimize delivery to target cells. Exemplary Nucleic Acids

MBNL1 has multiple isoforms. The nucleotide and amino acid sequences corresponding to this isoform are described in Table 2 below. Table 2. Exemplary MBNL1 sequences SEQ ID NO describe database identifier sequence 4 Exemplary MBNL1 transgenic gene -- ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGC GAACAGACAGACTTGAGGTATGTCGAGAGTACCAACGGTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAG CTGCCATGACTCAGTCGGCTGTCAAATCACTGAAGCGACCCCTCGAGGCAACCTTTGACCTGGGAATTCCTCAAGCTGTACTTCCCCCATTACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGGTCCAATATTGTGCATGACACCCGCTACAAGTGTTGTTCCCATGGTGCACGGTGC TACGCCAGCCACTGTGTCCGCAGCAACAACATCTGCCACAAGTGTTCCCTTCGCTGCAACAGCCACAGCCAACCAGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 5 MBNL1 variant 1 CCDS3163.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGC GAACAGACAGACTTGAGGTATGTCGAGAGTACCAACGGTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAG CTGCCATGGGAATTCCTCAAGCTGTACTTCCCCCATACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGGTCAATATTGTGCATGACACCCGCTACAAGTGTTGTTCCCATGGTGCACGGTGCTACGCCAGCCACTGTGTCCGCAGCAACAACATCTGCCACAAGTGTTCCCTTCGC TGCAACAGCCACAGCCAACCAGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 6 MBNL1 variant 2 CCDS3164.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGC GAACAGACAGACTTGAGGTATGTCGAGAGTACCAACGGTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAG CTGCCATGGGAATTCCTCAAGCTGTACTTCCCCCATACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGTTCCCATGGTGCACGGTGCTACGCCAGCCACTGTGTCCGCAGCAACAACATCTGCCACAAGTGTTCCCTTCGCTGCAACAGCCACAGCCAACCAGATACCCATATCTGCC GAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 7 MBNL1 variant 3 CCDS3165.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGC GAACAGACAGACTTGAGGTATGTCGAGAGTACCAACGGTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAG CTGCCATGACTCAGTCGGCTGTCAAATCACTGAAGCGACCCCTCGAGGCAACCTTTGACCTGGGAATTCCTCAAGCTGTACTTCCCCCATTACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGTTCCCATGGTGCACGGTGCTACGCCAGCCACTGTGTCCGCAGCAACATCTGCCA CAAGTGTTCCCTTCGCTGCAACAGCCACAGCCAACCAGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 8 MBNL1 variant 4 CCDS54656.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGGTATGTCGAGAGTACCAACGTTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACAATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAA GGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAGCTGCCATGGGAATTCCTCAAGCTGTACTTCCCCCATTACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTATTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGTTCCCATGGTGCACGGTGCTACGCCAGCCACTGTGTCCGCAGCAACATCT GCCACAAGTGTTCCCTTCGCTGCAACAGCCACAGCCAACCAGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 9 MBNL1 variant 5 CCDS3166.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGGTATGTCGAGAGTACCAACGTTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACAATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAA GGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAGCTGCCATGGGAATTCCTCAAGCTGTACTTCCCCCATTACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTATTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGGTCAATATTGTGCATGACACCCGCTACAAGTGTTGTTCCCATGGTGCACG GTGCTACGCCAGCCACTGTGTCCGCAGCAACAACATCTGCCACAAGTGTTCCCTTGCCTGCAACAGCCACAGCCAACCAGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 10 MBNL1 variant 6 CCDS3167.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGC GAACAGACAGACTTGAGGTATGTCGAGAGTACCAACGGTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAG CTGCCATGTTCCCATGGTGCACGGTGCTACGCCAGCCACTGTGTCCGCAGCAACAACATCTGCCACAAGTGTTCCCTTGCCTGCAACAGCCACAGCCAACCAGCCCCATCCTTGATGCTTCTACACTGTTGGGTGCAACATCCTGTCCTGCAGCAGCAGGAAAAATGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG 11 MBNL1 variant 7 CCDS3168.1 ATGGCTGTTAGTGTCACACCAATTCGGGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAAATGGACGCAATAACTTGATTCAGC AGAAGAACATGGCCATGTTGGCCCAGCAAATGCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGC GAACAGACAGACTTGAGGTATGTCGAGAGTACCAACGGTGGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAG CTGCCATGGGAATTCCTCAAGCTGTACTTCCCCCATACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGGTCAATATTGTGCATGACACCCGCTACAAGTGTTGATACCCATAATATCTGCCGAACATCTGACTAG 12 Exemplary MBNL1 protein -- MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIK GRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMTQSAVKSLKRPLEATFDLGIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPGSILCMTPATSVVPMVHGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVTQM 13 MBNL1 same function type 1 Q9NR56-1 MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIK GRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMTQSAVKSLKRPLEATFDLGIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPVPMVHGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVTQM 14 MBNL1 same function type 2 Q9NR56-2 MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIK GRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPVPMVHGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVTQM 15 MBNL1 same function type 3 Q9NR56-3 MAVSTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPA LEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPVPMVHGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVTQM 16 MBNL1 same function type 4 Q9NR56-4 MAVSTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPA LEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPGSILCMTPATSVVPMVHGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVTQM 17 MBNL1 same function type 5 Q9NR56-5 MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIK GRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPGSILCMTPATSVVPMVHGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVTQM 18 MBNL1 same function type 6 Q9NR56-6 MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIK GRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPGSILCMTPATSVDTHNICRTSD 19 MBNL1 same function type 7 Q9NR56-7 MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSCQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRNNLIQQKNMAMLAQQMQLANAMMPGAPLQPVPMFSVAPSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVPVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAHPADSTMIDTNDNTVTVCMDYIK GRCSREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAMFPWCTVLRQPLCPQQQHLPQVFPSLQQPQPTSPILDASTLLGATSCPAAAGKMIPIISAEHLTSHKYVTQM

In some embodiments, MBNL1 has an amino acid sequence that is at least 85% identical to any of SEQ ID NOs: 12-19 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical amino acid sequences. For example, in some embodiments, MBNL1 has an amino acid sequence that is at least 86% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 87% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 88% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 89% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 90% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 91% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 92% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 93% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 94% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 96% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 97% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 98% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has an amino acid sequence that is at least 99% identical to the amino acid sequence of any of SEQ ID NOs: 12-19. In some embodiments, MBNL1 has the amino acid sequence of any of SEQ ID NOs: 12-19. promoter

The DES promoter described herein may contain a DES promoter sequence or a functional portion thereof. For example, a DES promoter sequence may contain a DES promoter at nucleotides -984 to -644 of the human DES locus, relative to the DES transcription start site. The nucleic acid of this construct is set forth in SEQ ID NO: 1. A DES promoter sequence may contain a DES promoter at nucleotides -269 to +76 of the human DES locus, relative to the DES transcription start site. 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, and without intervening nucleic acids, to form a DES promoter comprising nucleotides -984 to -644 of the human DES locus and nucleotides -269 to +76 of the human DES locus relative to the DES transcription start site. The nucleic acid of this construct is set forth in SEQ ID NO: 3. Additional promoter sequences that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) relative to the above nucleic acid sequences.

Exemplary DES promoter sequences are described in Table 3 below. Table 3. Exemplary DES promoter sequences SEQ ID NO Description of DES promoter sequence nucleic acid sequence 1 Segment of the hDES promoter containing nucleotides -984 to -644 relative to the DES transcription start site TACCCCCTGCCCCCCTCTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAG CAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTCGAG 2 Segment of the hDES promoter containing nucleotides -269 to +76 relative to the DES transcription start site CGAGATAACCAGGGCTGAAAGAGGCCCGCCTGGGGGCTGGAGACATGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCACAGGACCCCCGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGCCGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATGTCAGGAGGGGATACAAATAGTGCCGACGGC TGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGGCCGCCTGTCCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCG 3 Exemplary desmin promoter containing SEQ ID NO: 1 fused to SEQ ID NO: 2 TACCCCCTGCCCCCCTCTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAG CAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTCGAGCGAGATAACCAGGGCTGAAAGAGGCCCGCCTGGGGGCTGGAGACATGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCACAGGACCCCCGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGCCGCCCTTTCCTGGCAGGACAGCGGGA TCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATGTCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGGCCGCCTGTCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCG

In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 5' region that is at least 85% (e.g., 85%, 86%) identical to the nucleic acid sequence of SEQ ID NO: 1 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. For example, in some embodiments, a transgene encoding MBNL1 is operably linked to a DES promoter having a 5' region having a nucleic acid that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 1 sequence. 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 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. 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 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. 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 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. 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 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. 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 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.

In some embodiments, the transgene encoding MBNL1 is operably linked to a DES promoter having a 3' region that is at least 85% (e.g., 85%, 86%) identical to the nucleic acid sequence of SEQ ID NO: 2 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. For example, in some embodiments, a transgene encoding MBNL1 is operably linked to a DES promoter having a 3' region having a nucleic acid that is at least 86% identical to the nucleic acid sequence of SEQ ID NO: 2 sequence. 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 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. 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 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. 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 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. 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 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. 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 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.

In some embodiments, the transgene encoding MBNL1 is operably linked to a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, A DES promoter with a nucleic acid sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical. For example, in some embodiments, a 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Delivery I. Viral vectors for expression of therapeutic nucleic acid molecules

Viral genomes provide a rich source of vectors that can be used to efficiently deliver foreign genes into mammalian cells (eg, muscle cells or neurons). Viral gene systems are particularly useful as vectors for gene delivery because the nucleic acids contained within such genes are often incorporated into the nuclear genome of mammalian cells by general or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors are retroviruses (e.g., retroviridae vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative viruses RNA viruses (such as orthomyxoviruses (e.g., influenza virus)), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai virus), orthomyxoviruses (such as picornaviruses and alphaviruses) as well as double-stranded DNA viruses (including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus)) and poxviruses (e.g., , vaccinia, modified vaccinia Ankara (MVA), avian pox and canary pox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papillovesicular virus, hepadnavirus, human papilloma virus, human foamy virus and hepatitis virus. Examples of retroviruses are avian leukemia-sarcoma, avian C virus, mammalian C virus, B virus, D virus, oncogenic retrovirus, HTLV-BLV family, lentivirus, alpha retrovirus, gamma retrovirus Viruses, foamy viruses (Coffin, JM, Retroviridae: The viruses and their replication, Virology, 3rd ed. (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia virus, murine sarcoma virus, 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 leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentivirus. Other examples of vectors are described, for example, in McVey et al. (US 5,801,030), the teachings of which are incorporated herein by reference. II. Adeno-associated virus vector

The nucleic acids of the compositions and methods described herein can be incorporated into recombinant linear adeno-associated virus (rAAV) vectors, recombinant self-complementary AAV (scAAV) vectors, and/or virions to facilitate introduction of the nucleic acids into cells (e.g., muscle cells or neurons). 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 (2018), which is incorporated herein by reference for its disclosure of promoters and AAV serotypes that can be used in CNS gene therapy. In some embodiments, 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 promote integration and expression of the heterologous gene. The viral sequences may include those AAV sequences required for cis-replication and packaging of DNA into viral particles (e.g., functional inverted terminal repeat sequences (ITRs)). Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences. AAV ITRs may be of any serotype suitable for a particular application. In some embodiments, the AAV ITRs are AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate introduction of the nucleic acids or vectors into cells. The AAV capsid protein forms the outer portion (the non-nucleic acid part of the virion) and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virus particle 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; and Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), each of which is incorporated herein by reference for their disclosure of AAV vectors for gene delivery.

rAAV virions useful for use in conjunction with the compositions and methods described herein include those derived from multiple AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, and rh74. Virus particles. AAV2, AAV9 and AAV10 may be particularly useful for targeting or delivering to cells in the central nervous system. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000) ; Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al. , Hum. Molec. Genet. 10:3075 (2001), each of which is incorporated herein by reference for their disclosure of AAV vectors for gene delivery.

Pseudotyped rAAV vectors can also be used in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with capsid genes derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV8 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

AAV virions with mutations in the virion capsid can infect specific cell types more efficiently than unmutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations that promote AAV targeting to specific cell types. The construction and characterization of AAV capsid mutants (including insertion mutants, alanine selection mutants, and antigenic determinant tag mutants) are described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in the methods described herein include those capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000), and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

In some embodiments, a nucleic acid molecule comprising a transgene encoding MBNL1 is operably linked to a DES promoter, optionally the 5' end of a portion of the nucleic acid molecule (e.g., comprising or encoding a transgene encoding MBNL1) Binds to the 3' end of the DES promoter. In some embodiments, the transgene encoding MNBNL1 and the DES promoter sequence are separated by a β-globin intron sequence. It is expected that the β-globin intron sequence will serve as a linker sequence between the promoter and the transgene sequence and will promote more efficient transcription of the transgene.

In some embodiments, the DES promoter includes a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, The 5' region of a nucleic acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical. For example, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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% 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. In some embodiments, 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 having the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, 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. For example, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 having a nucleic acid sequence of SEQ ID NO: 2.

In some embodiments, 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. For example, in some embodiments, the DES promoter has a nucleic acid sequence that is at least 86% 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 87% 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 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. In some embodiments, 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. In some embodiments, 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 a nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, the AAV has a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. For example, in some embodiments, the AAV has a nucleic acid sequence that is at least 86% 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 87% 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 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. In some embodiments, 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. In some embodiments, 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: TACCCCCTGCCCCCCTCTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAG CAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTCGACAGTACCGGGGAGGAAGAGGGCCTGCACTAGTCCAGAGGGAAACTGAGGCTCAGGGCCAGCTCGCCCATAGACATACATGGCAGGCAGGCTTTGGCCAGGATCCCTCCGCCTGCCAGGCGTCTCCCTGCCCTCCCTTCCTGCCTAGAGACCCCCCTCAAGCCTGGCTGGTCTTTGCCTGAGACCCAATC TTCGACTTCAAGAGAATATTTAGGAACAAGGTGGTTTAGGGCCTTTCCTGGGAACAGGCCTTGACCCTTTAAGAAATGACCCAAAGTCTCCTTGACCAAAAAGGGGACCCTCAAACTAAAGGGAAGCCTCTCTTCTGCTGTCTCCCCTGACCCCACTCCCCCCCACCCCAGGACGAGGAGATAACCAGGGCTGAAAGAGGCCCGCCTGGGGGCTGCAGACATGCTTGCTGCCTGCCCTGGCGAAGGATTGGTAGGCTACCTGCCCGTCACAGGCCC GCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGCCGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATGTCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGGCCGCCTGCCCGCCGCCTCCTCCGTGCCGCCCGCCAGCCTCGCCCGGTGAGTCTATGGGACC CTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAAT ATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCCCACCATGGCTGTTAGTGTCACCAATTCG GGACACAAAATGGCTAACACTGGAAGTATGTAGAGAGTTCCAGAGGGGGACTTGCTCACGGCCAGACACGGAATGTAAATTTGCACATCCTTCGAAAAGCTGCCAAGTTGAAAATGGACGAGTAATCGCCTGCTTTGATTCATTGAAAGGCCGTTGCTCCAGGGAGAACTGCAAATATCTTCATCCACCCCCACATTTAAAAACGCAGTTGGAGATAAATGGACGCAATAACTTGATTCAGCAGAAGAACATGGCCATGTTGGCCCAGCAAAT GCAACTAGCCAATGCCATGATGCCTGGTGCCCCATTACAACCCGTGCCAATGTTTTTCAGTTGCACCAAGCTTAGCCACCAATGCATCAGCAGCCGCCTTTAATCCCTATCTGGGACCTGTTTCTCCAAGCCTGGTCCCGGCAGAGATCTTGCCGACTGCACCAATGTTGGTTACAGGGAATCCGGGTGTCCCTGTACCTGCAGCTGCTGCAGCTGCTGCACAGAAATTAATGCGAACAGACAGACTTGAGGTATGTCGAGA GTACCAACGTGCAATTGCAACCGAGGAGAAAATGATTGTCGGTTTGCTCATCCTGCTGACAGCACAATGATTGACACCAATGACAACACAGTCACTGTGTGTATGGATTACATCAAAGGGAGATGCTCTCGGGAAAAGTGCAAATACTTTCATCCCCCTGCACATTTGCAAGCCAAGATCAAGGCTGCCCAATACCAGGTCAACCAGGCTGCAGCTGCACAGGCTGCAGCCACCGCAGCTGCCATGACTCAGTCGGCTGTCAAATCACT GAAGCGACCCCTCGAGGCAACCTTTGACCTGGGAATTCCTCAAGCTGTACTTCCCCCATTACCAAAGAGGCCTGCTCTTGAAAAAACCAACGGTGCCACCGCAGTCTTTAACACTGGTATTTTCCAATACCAACAGGCTCTAGCCAACATGCAGTTACAACAGCATACAGCATTTCTCCCACCAGGCTCAATATTGTGCATGACACCCGCTACAAGTGTTGTTCCCATGGTGCACGGTGCTACGCCAGCCACTGTGTCCGCAGCAACAA CTGCCACAAGTGTTCCCTTCGCTGCAACAGCCACAGCCAACCAGATACCCATAATATCTGCCGAACATCTGACTAGCCACAAGTATGTTACCCAGATGTAG

As described herein, exemplary AAV vector components include from 5' to 3', for example, a DES promoter as described herein, a beta-globin intron, a transgene encoding MBNL1, and SV40 late polyadenylation (pA )sequence. III. Methods for delivering exogenous nucleic acids to target cells

Techniques that can be used to introduce a transgene, such as a transgene encoding MBNL1, wherein the transgene is operably linked to a DES promoter, into a target cell, such as a mammalian cell such as a muscle cell or a neuron, are well known in the art. For example, electroporation can be used to permeabilize a mammalian cell, such as a human target cell, by applying an electrostatic potential to the target cell. Mammalian cells, such as human cells, that are subjected to an external electric field in this manner are then susceptible to taking up exogenous nucleic acids, such as nucleic acids that can be expressed in, for example, muscle cells or neurons. Electroporation of mammalian cells is described in detail in, for example, Chu et al., Nucleic Acids Res 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, NUCLEOFECTION™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. NUCLEOFECTION™ and protocols that can be used to perform the technique are described in detail in, for example, Distler et al., Exp. Dermatol. 14:315 (2005), and US 2010/0317114, the disclosure of each of which is incorporated herein by reference.

An additional technique that can be used to transfect target cells is the squeeze-punch method. This technique induces rapid mechanical deformation of cells to stimulate the uptake of foreign DNA through membrane pores that form in response to applied stress. An advantage of this technology is that a vector is not necessary to deliver the nucleic acid into cells, such as human target cells. Extrusion-perforation is described in detail, for example, in Sharei et al., JoVE 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

Lipofection represents another technique that can be used to transfect target cells. This method involves loading nucleic acids into liposomes, which typically exhibit cationic functional groups, such as quaternary or protonated amines, toward the exterior of the liposome. This promotes electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, ultimately leading to the uptake of exogenous nucleic acids, for example, by guiding the fusion of liposomes with the cell membrane or through 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. A similar technique that utilizes ionic interactions with cell membranes to cause uptake of foreign nucleic acids is to contact cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that facilitates interaction with cell membranes are activated dendrimers (described, for example, in Dennig, Top Curr Chem 228:227 (2003), disclosed in (the contents of which are incorporated herein by reference), polyethylenimine and DEAE-dextran, the use of which as transfection agents is described in detail in, for example, Gulick et al., Curr Protoc Mol Biol 40:1:9.2:9.2.1 ( 1997), the disclosures of which are incorporated herein by reference.

Another tool that can be used to induce the uptake of exogenous nucleic acids by target cells is laser transfection, also known as phototransfection, which involves exposing cells to electromagnetic radiation of a specific wavelength to gently penetrate the cells and allow polynucleotides to pass through the cell membrane. The biological activity of this technique is similar to that of electroporation, and in some cases has been found to be superior to electroporation.

Piercing transfection 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 to be delivered into the cell is attached to the surface of the nanostructure. A chip with an array of these needles is then pressed against a cell or tissue. Cells pierced by the nanostructure can express the delivered gene. 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.

MAGNETOFECTION™ can also be used to deliver nucleic acids to target cells. The principle of MAGNETOFECTION™ is to associate nucleic acids with cationic magnetic nanoparticles. Magnetic nanoparticles are made from fully biodegradable iron oxide and coated with specific cationic proprietary molecules that vary depending on the application. Its association with gene carriers (DNA, asRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells by influencing the external magnetic field generated by the magnet. 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 gentle and efficient manner because this method uses an applied magnetic field to guide the uptake of nucleic acids. This technology is described in detail in, for example, US2010/0227406, the disclosure of which is incorporated herein by reference.

Another tool that can be used to induce uptake of exogenous nucleic acids by target cells is sonoporation, which is a technique that involves the use of sound (usually ultrasound frequencies) to modify the permeability of the plasma membrane of the cell to permeate the cell and allow polynucleotides to pass through the cell membrane. This technique is described in detail in, for example, Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.

Microvesicles represent another potential medium that can be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by co-expression of the glycoprotein VSV-G and, for example, genome modifying proteins (such as nucleases) can be used to efficiently deliver proteins into cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of the cell for covalent incorporation of target polynucleotides (such as genes or regulatory sequences). The use of such vesicles (also called gesicles) for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract], in Methylation changes in early embryonic genes in cancer [Abstract], and in Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122. Methods for detecting expression of RNA transcripts

The level of expression of an RNA transcript, such as the MBNL1 mRNA transcript, can be determined, for example, by various nucleic acid detection techniques. Additionally or alternatively, RNA transcript performance can be inferred by assessing the concentration or relative abundance of the encoded protein resulting from translation of the RNA transcript. Protein concentration can also be assessed, for example, using functional assays. Using these techniques, it is possible to observe an increase in MBNL1 mRNA transcript concentration in response to the compositions and methods described herein, while monitoring the expression of the encoded protein. The following section describes exemplary techniques that can be used to measure the performance levels of RNA transcripts and their downstream protein products. RNA transcript performance can be assessed by many methods known in the art, including but not limited to nucleic acid sequencing, microarray analysis, proteomics, in situ hybridization (e.g., fluorescent in situ hybridization (FISH)) , amplification-based assays, in situ hybridization, fluorescence-activated cell sorting (FACS), Northern analysis, and/or PCR analysis of RNA. Nucleic acid detection

Nucleic acid-based methods for detecting the expression of DNA or RNA transcripts include imaging-based techniques (e.g., Northern blotting or Southern blotting), which can be performed upon administration of, for example, a protein encoding a gene operably linked to a DES promoter. Vectors transgenic for MBNL1 or compositions containing such constructs are then used in combination with cells obtained from the patient. Northern blot analysis is a routine 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 in. Typical protocols for assessing the status of genes and gene products are found, for example, in Ausubel et al., 1995, Current Protocols in Molecular Biology, Unit 2 (Northern Blotting), Unit 4 (Southern Blotting), Unit 15 (Immunospotting) Method) and Unit 18 (PCR Analysis).

RNA detection technologies that can be used in conjunction with the compositions and methods described herein to assess the efficacy of administration of any composition 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, but are not limited to, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional sequencing methods known in the art may also be used. For example, expression of transgenic genes at the mRNA level can 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 by reference in its entirety). method incorporated into this article). RNA-Seq is a powerful technique for monitoring performance by directly sequencing RNA molecules in a sample. Briefly, the method can involve fragmenting 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®). Subsequently, the cDNA is converted into a molecular library for sequencing by adding sequence adapters (eg, from Illumina®/Solexa) to each library, and the resulting 50-100 nucleotide reads are mapped onto the gene body.

RNA performance levels can be determined using microarray-based platforms (eg, single nucleotide polymorphism arrays) because microarray technology provides high resolution. Detailed information on various microarray methods can be found in the literature. See, for example, U.S. Patent 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. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to generate cDNA. The probe can then hybridize to one or more complementary nucleic acids arranged and immobilized on a solid support. The array can be configured, for example, so that the sequence and location of each member of the array is known. Hybridization of a labeled probe to a specific array member indicates that the sample from which the probe is derived expresses that gene. The level of performance can be quantified based on the amount of signal detected from the hybridized probe-sample complex. A typical microarray experiment involves the following steps: 1) preparing fluorescently labeled targets from RNA isolated from the sample, 2) hybridizing the labeled targets to the microarray, 3) washing, staining, and scanning the array, 4) analyzing the scanned images and 5) Generate gene expression profiles. One example of a 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 those skilled in the art.

Amplification-based assays can also be used to measure the expression levels of specific RNA transcripts, such as the MBNL1 mRNA transcript. In such assays, the nucleic acid sequence of the transcript serves as a template in an amplification reaction (eg, PCR, such as qPCR or droplet digital PCR (ddPCR)). In a certain amount of amplification, the amount of amplification product is directly proportional to the amount of template in the original sample. In accordance with the principles described herein, comparison to appropriate controls provides a measure of the level of expression of the target transcript corresponding to the particular probe used. Real-time qPCR methods using TaqMan probes are well known in the art. Detailed protocols for real-time qPCR have been provided, for example, in Gibson et al., Genome Res. 6:995-1001 (1996) and Heid et al., Genome Res. 6:986-994 (1996), each of which The disclosure content of is incorporated into this article by full reference. For example, RNA transcript expression levels described herein can be determined by RT-PCR techniques. Probes used in PCR can be labeled with detectable labels such as radioisotopes, fluorescent compounds, biofluorescent compounds, chemofluorescent compounds, metal chelators or enzymes. protein detection

The expression of RNA constructs can also be inferred by analyzing the expression of proteins encoded by the constructs. Standard detection techniques known in the art can be used to assess protein levels. Protein expression assays suitable for use with the compositions and methods described herein include proteomic methods, immunohistochemistry and/or Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, AlphaLISA ^™ , enzyme-linked immunosorbent assay (ELIFA), mass spectrometry, mass spectrometry immunoassays, and biochemical enzyme activity assays. Specifically, proteomic methods can be used to generate large-scale multiplexed protein expression data sets. Proteomic methods can utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and/or peptide microarrays, using capture reagents (e.g., antibodies) specific for a set of target proteins to identify and measure the expression levels of proteins expressed in a sample (e.g., a single cell sample or a multicellular population).

Exemplary peptide microarrays have a plurality of polypeptides bound to a substrate, and the binding of an oligonucleotide, peptide or protein to each of the plurality of bound polypeptides is individually detectable. Alternatively, the peptide microarray may include a plurality of binders, including but not limited to monoclonal antibodies, polyclonal antibodies, phage-displayed binders, yeast dihybrid binders, aptamers, which can specifically detect the binding of a particular oligonucleotide, peptide or protein. Examples of peptide arrays can 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 (MS) can be used in conjunction with the methods described herein to identify and characterize transgene expression in cells from a patient (e.g., a human patient) following transgene delivery. Any MS method known in the art can be used to identify, detect, and/or measure target protein or peptide fragments, such as LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like. A mass spectrometer typically contains an ion source and optics, a mass analyzer, and data processing electronics. Mass analyzers include scanning and ion beam mass spectrometers, such as time of flight (TOF) and quadrupole (Q), and capture mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), which can 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.

Prior to MS analysis, proteins in samples obtained from patients can first be digested into smaller peptides by chemical (e.g., cleavage by cyanogen bromide) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from the use of front-end separation techniques such as 2D-PAGE, HPLC, RPLC, and affinity chromatography. The digested and optionally separated samples are then ionized using an ion source to generate charged molecules for further analysis. The sample can be ionized, for example, by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix-assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermal/plasma spray ionization, and particle beam ionization. Additional information regarding the choice of ionization method is known to those skilled in the art.

Once freed, the digested peptides can then be fragmented to generate characteristic MS/MS spectra. Tandem MS (also known as MS/MS) is particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring between stages, either by spatially separated individual mass spectrometer elements or by a single mass spectrometer using temporally separated MS steps. Finish. In a spatially separated tandem MS, the elements are physically separate and distinct, with physical connections between elements to maintain a high vacuum. In temporally separated tandem MS, separation is accomplished by trapping ions at the same location, with multiple separation steps occurring over time. The characteristic MS/MS spectrum can then be compared to a peptide sequence database (eg, SEQUEST). Post-translational modifications of peptides can also be determined, for example, by searching spectra against a database while allowing for specific peptide modifications. Pharmaceutical composition

The nucleic acid molecules described herein can be formulated into pharmaceutical compositions for administration to a patient, such as a human patient who exhibits myotonic dystrophy or is at risk for myotonic dystrophy, such as a patient diagnosed with or exhibiting one or more symptoms of DM1 (e.g., myotonia), in a biocompatible form suitable for in vivo administration. Pharmaceutical compositions containing, for example, nucleic acid molecules including a transgene encoding MBNL1, wherein the transgene is operably linked to a DES promoter as described herein, typically include a pharmaceutically acceptable diluent or carrier. The pharmaceutical composition may include, for example, a sterile saline solution and a nucleic acid (e.g., consisting thereof). The sterile saline is typically pharmaceutical grade saline. The pharmaceutical composition may include, for example, sterile water and a nucleic acid (e.g., consisting thereof). The sterile water is typically pharmaceutical grade water. The pharmaceutical composition may include, for example, phosphate buffered saline (PBS) and nucleic acid (e.g., consisting thereof). The sterile PBS is typically pharmaceutical grade PBS.

In certain embodiments, pharmaceutical compositions include a plurality of transgenic genes and one or more excipients. In certain embodiments, the excipient is selected from the group consisting of: water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, Hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, nucleic acid molecules can be mixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. The composition and the method for formulating the pharmaceutical composition depend on many criteria, including but not limited to the route of administration, the extent of the disease or the dose to be administered.

In certain embodiments, the pharmaceutical composition comprising a transgene encoding a nucleic acid molecule encompasses any pharmaceutically acceptable salt of an inhibitor, an ester of an inhibitor, or a salt of such an ester. In certain embodiments, the pharmaceutical composition comprising a transgene encoding a nucleic acid molecule is capable of providing (directly or indirectly) a biologically active metabolite or its residue after administration to a subject (e.g., a human). Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts of inhibitors, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In certain embodiments, the prodrug comprises one or more conjugate groups linked to a nucleic acid molecule, wherein the conjugate groups are cleaved by endogenous nucleases in vivo.

Lipid moieties have been used in nucleic acid therapy in a variety of ways. In some such methods, nucleic acids are introduced into preformed liposomes or lipoplexes made from a mixture of cationic lipids and neutral lipids. In some methods, DNA complexes with monocationic or polycationic lipids are formed in the absence of neutral lipids. In some embodiments, lipid moieties are selected to increase the distribution of pharmaceutical agents to specific cells or tissues. In some embodiments, lipid moieties are selected to increase the distribution of pharmaceutical agents to adipose tissue. In some embodiments, lipid moieties are selected to increase the distribution of pharmaceutical agents to muscle tissue.

In certain embodiments, pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including those containing hydrophobic compounds. In certain embodiments, certain organic solvents are used, such as dimethylsulfoxide.

In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the invention to a specific tissue or cell type. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with tissue-specific antibodies.

In certain embodiments, the pharmaceutical composition comprises a co-solvent system. Certain such co-solvent systems include, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is a VPD co-solvent system, which includes 3% w/v benzyl alcohol, 8% w/v non-polar surfactant polysorbate 80™, and 65% w/v polyethylene glycol 300 in anhydrous ethanol. The proportions of such a co-solvent system can vary considerably without significantly changing its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other surfactants may be used instead of polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, such as polyvinylpyrrolidone; and other sugars or polysaccharides may replace glucose.

In certain embodiments, the pharmaceutical composition is prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intraocular, intravitreal, intravenous, subcutaneous, intramuscular, intrathecal, intraventricular, etc.). In certain such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or saline buffer. In certain embodiments, other ingredients (e.g., ingredients that aid solubility or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, and the like. Certain injectable pharmaceutical compositions are presented in unit dosage form, for example, in ampoules or multidose containers. Certain injectable pharmaceutical compositions are suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulators such as suspending agents, stabilizers and/or dispersants. Certain solvents suitable for injectable pharmaceutical compositions 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. Administration and Routes of Administration

Viral vectors (such as AAV vectors and other vectors described herein) containing the MBNL1 transgene operably linked to the DES promoter described herein can 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, depending on the onset and severity of the disease and may include, for example, intravenous, intrathecal, intraventricular, intraparenchymal, intracisternal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular or oral administration and/or administration by inhalation, infusion or lavage. Intravascular administration may include delivery into the patient's vascular system. In some embodiments, administration is into a blood vessel considered a vein (intravenous), and in some embodiments, administration is into a blood vessel considered an artery (intraarterial). Veins include, but are not limited to, the internal cervical vein, peripheral vein, coronary vein, hepatic vein, portal vein, great sinus vein, pulmonary vein, superior vena cava, inferior vena cava, gastric vein, splenic vein, subenteric vein, supracervical vein, cranial vein, and/or femoral vein. Arteries include, but are not limited to, coronary arteries, pulmonary arteries, brachial arteries, internal jugular arteries, aortic arcs, femoral arteries, peripheral arteries, and/or ciliary arteries. It is contemplated that delivery may be through or to arterioles or capillaries.

Treatment regimens may vary and generally depend on the severity of the disease and the age, weight, and sex of the patient. Treatment may include administration of vectors (e.g., viral vectors) or other agents described herein that can be used to introduce transgenes into target cells in various unit doses. Each unit dose typically contains a predetermined amount of the therapeutic composition. Combination Therapy

Nucleic acid molecules described herein may be administered in combination with one or more additional therapeutic agents for the treatment of patients suffering from myotonic dystrophy, such as those diagnosed with or exhibiting one or more symptoms of DM1 (e.g., myotonia). One or more additional therapeutic agents may include corticosteroids (e.g., betamethasone, prednisolone, triamcinolone, methylprednisolone, dexamethasone ), hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisone, triamcinolone, dexamethasone, or fludrocortisone (fludrocortisone) or immunosuppressive drugs (eg, pomalidomide, methotrexate, azathioprine, lenalidomide, azathioprine, or thalidomide amine (thalidomide), or combinations thereof. set

The compositions described herein may be provided in the form of a kit for treating a patient diagnosed with or exhibiting one or more symptoms of DM1 (e.g., myotonia). The kit may include one or more nucleic acid molecules as described herein. In some embodiments, the kit may include a viral vector as described herein. In some embodiments, the kit may include a pharmaceutical composition as described herein. The kit may include a drug instruction sheet to instruct the user of the kit (e.g., a physician) to perform any of the methods described herein. The kit may optionally include a syringe or other device for administering the composition. In some embodiments, the kit may include one or more additional therapeutic agents. Examples

The following examples are presented to provide those skilled in the art with a description of how the compositions and methods described herein may be used and evaluated, and are intended merely to be illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention. Example 1 Target effectiveness of MBNL1 overexpression in rescuing muscle cell splicing profiles

The example describes the development of compositions that can be used to reduce the occurrence of spliceopathies and for treating disorders associated with RNA dominance, which are pathologies 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 proper splicing of various mRNA transcripts, such as myotonic dystrophy type 1 (DM1). For example, DM1 is caused by the expansion of microsatellite repeats, which results in the expression of toxic expanded repeat mRNAs. This mRNA exhibits a higher affinity for splicing factor proteins, such as muscleblind-like splicing regulator 1 (MBNL1), and sequesters these proteins at ribonuclear foci, subsequently promoting spliceopathic splicing of MBNL1 RNA substrates, such as pre-mRNA molecules transcribed from ATP2A1 , CLCN1 , and LDB3 , and thereby promoting a shift in the splicing profile of these cells from a desired mature-stage splicing profile to a fetal-stage splicing profile. The compositions described in this example were developed with the goal of producing an AAV vector encoding a transgene construct of 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 spliceopathic splicing. Ultimately, reduction of the spliceopathic nature of the MBNL1 RNA substrate promotes the development of a mature splicing profile over a fetal splicing profile and clinically results in improvement of one or more symptoms of DM1 such as myotonia.

The purpose of this example is to demonstrate the efficacy of the promoter in inducing robust overexpression of MBNL1 and thereby subsequently improving the splicing outcome of the affected gene in a cell model of DM1. In line with these goals, the efficacy of the nucleic acid constructs described herein to reduce nuclear sequestration of MBNL1 and improve splicing of kinesin family member 13A (KIF13A) and dystrophin (DMD) was demonstrated using the DM1 cell model. DM1 model

For all cell experiments, immortalized non-DM1 human myoblasts (control cells) were used as control cells relative to immortalized DM1 patient-derived myoblasts (DM1 cells). DM1 or control cells were plated at 4,000 cells/well in 160 μl of growth medium [PromoCell Skeletal Muscle Cell Growth Medium Kit; Part No.: C-23060 (Note: the medium was supplemented with 20% FBS (Thermo Fisher Scientific #16000-044) instead of 5%, 50 µg/ml gentamicin S (Thermo Fisher Scientific #15070-060) as indicated in the kit)] on type I collagen-coated 96-well plates (IWAKI #4860-010 for splicing assays and gene expression analysis, Thermo Fisher Scientific #152036 for RNA foci assays) and incubated at 37°C/5% _CO2 for 5-6 hours. DM1 cells were infected with 40 μl of AAV8 vector encoding human MBNL1 construct as described above. Control wells were treated with PBS including 0.001% Pluronic F68 and mixed. Plates were incubated for 2 days and the medium was replaced with 200 μl of fresh growth medium including 10 μM etoposide (Wako #051-08431) and incubated for another 4 days at 37°C/5% _CO2 . RNA extraction and cDNA preparation

Cells were harvested using the TaqMan Gene Expression Cells-to-CT Kit (Thermo Fisher Scientific #AM1728) and stored at -80°C according to the manufacturer's instructions. Total RNA was converted to cDNA using the RT enzyme mix included in the kit. Store cDNA at -20°C. splicing analysis

PCR was performed using PrimeSTAR® GXL DNA Polymerase (TaKaRa # R050A) according to the manufacturer's instructions. 2 μl of cDNA was used as template. The PCR primers used are as follows: Table 4. target Fw 5'→3' Re 5'→3' KIF13A exon 21 ACCTGTGCAGCATTCAGGGACAC (SEQ ID No：21) CTCGTCGTTTAATGAGTGCATCTG (SEQ ID No：22) DMD exon 78 TTAGAGGAGGTGATGGAGCA (SEQ ID No：23) GATACTAAGGACTCCATCGC (SEQ ID No：24)

PCR cycling conditions were as follows: 35 cycles of 98°C for 10 sec, 60°C for 15 sec, and 68°C for 30 sec, followed by 72°C for 7 min. PCR products were loaded onto Agilent DNA1000 Kit (Agilent # 5067-1504), electrophoresed, and analyzed using the Agilent 2100 BioAnalyzer system according to the manufacturer's instructions. The AUC of the peaks of normal and abnormal splicing products was measured, and the ratio of normal splicing products in each cell was calculated. Measuring human MBNL1 protein performance using AlphaLISA assay kit

Cells were harvested using the Human Blind Myelin 1 (MBNL1) AlphaLISA Immunoassay Kit (PerkinElmer, #AL3008) according to the manufacturer's instructions. Dilute 5X AlphaLISA Lysis Buffer (PerkinElmer, #AL003F) and 100x Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, #78441) in double distilled water. After washing twice with PBS, 100 μl/well of lysis buffer was added to each well. Shake the plate with a plate shaker for 10 minutes at room temperature. The plates were then incubated at 37°C for 10 min and then stored at -80°C. MBNL1 protein was measured in a 2 μl scale using Envision 2014-0020 (PerkinElmer) according to the manufacturer's protocol.

Total protein in lysates was also quantified using SPECTRA max PLUS 384 (Molecular Devices) using a 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 used as the amount of MBNL1 in 100% of DM1 cells to calculate the relative amount. result

DM1 cells were transduced with an AAV8 vector encoding the MBNL1 transgene operably linked to the DES promoter (AAV8-DES-MBNL1, as shown in Figure 1 ) or treated with PBS including 0.001% Pluronic F68. Compared to control immortalized non-DM1 human myoblasts, DM1 cells treated with the MBNL1 transgene showed increased expression of MBNL1, as measured by immunoblotting. DM1 cells treated with MBNL1 transgene also showed improvements in KIF13A and DMD splicing, as demonstrated by an increase in the percentage of KIF13A exon 21 inclusions and an increase in the percentage of DMD exon 78 inclusions, respectively ( Table 5-7 ).

Different doses of virus were tested and are indicated in Tables 5-7 : T contained a therapeutic concentration of 9x10 ¹¹ viral genomes (vg)/mL, H contained a therapeutic concentration of 3x10 ¹¹ viral genomes (vg)/mL, M contained a therapeutic concentration of 1x10 ¹¹ viral genomes (vg)/mL, L contained a therapeutic concentration of 3.3x10 ¹⁰ viral genomes (vg)/mL, and L2 contained a therapeutic concentration of 1.1x10 ¹⁰ viral genomes (vg)/mL. Table 5. AAV-Des-hMBNL1 Efficacy: In Vitro Data A Splicing (KIF13A) Splicing (DMD) Sample % KIF13A exon 21 inclusion bodies % KIF13A splicing improvement % DMD exon 78 inclusion bodies % DMD splicing improvement iDM_Des-hMBNL1_T_rep1 94.1% 105.3% 60.2% 94.1% iDM_Des-hMBNL1_T_rep2 95.6% 109.6% 58.6% 91.4% iDM_Des-hMBNL1_H_rep1 90.0% 93.7% 44.0% 66.2% iDM_Des-hMBNL1_H_rep2 95.3% 108.6% 38.4% 56.6% iDM_Des-hMBNL1_M_rep1 75.4% 52.9% 22.0% 28.3% iDM_Des-hMBNL1_M_rep2 89.4% 92.1% 31.3% 44.3% iDM_Des-hMBNL1_L_rep1 72.9% 45.6% 15.1% 16.5% iDM_Des-hMBNL1_L_rep2 73.7% 48.1% 17.4% 20.4% iDM_Des-hMBNL1_L2_rep1 59.7% 8.7% 9.3% 6.4% iDM_Des-hMBNL1_L2_rep2 52.5% -11.6% 8.8% 5.5% iDM_ctrl_rep1 63.8% 20.1% 6.8% 2.1% iDM_ctrl_rep2 59.1% 6.9% 4.0% -2.7% iDM_ctrl_rep3 52.3% -12.2% 5.9% 0.7% iDM_ctrl_rep4 51.3% -14.8% 5.5% -0.1% iCM_ctrl_rep1 91.1% 97.0% 65.8% 103.8% iCM_ctrl_rep2 93.3% 102.9% 67.6% 106.8% iCM_ctrl_rep3 90.3% 94.7% 60.8% 95.1% iCM_ctrl_rep4 94.1% 105.4% 60.3% 94.3% Table 6. AAV-Des-hMBNL1 efficacy: in vitro data B Splicing (KIF13A) Splicing (DMD) Sample % KIF13A exon 21 inclusion bodies % KIF13A splicing improvement % DMD exon 78 inclusion bodies % DMD splicing improvement iDM_Des-hMBNL1_T_rep1 94.7% 137.3% 60.3% 116.6% iDM_Des-hMBNL1_T_rep2 81.7% 87.3% 44.4% 81.8% iDM_Des-hMBNL1_H_rep1 86.2% 104.6% 33.1% 57.1% iDM_Des-hMBNL1_H_rep2 91.2% 124.1% 32.2% 55.1% iDM_Des-hMBNL1_M_rep1 80.1% 81.1% 25.1% 39.5% iDM_Des-hMBNL1_M_rep2 83.2% 93.3% 24.1% 37.4% iDM_Des-hMBNL1_L_rep1 71.5% 48.1% 12.8% 12.5% iDM_Des-hMBNL1_L_rep2 76.6% 67.8% 12.3% 11.4% iDM_Des-hMBNL1_L2_rep1 59.9% 3.4% 8.1% 2.3% iDM_Des-hMBNL1_L2_rep2 68.6% 37.1% 7.7% 1.3% iDM_ctrl_rep1 62.8% 14.8% 7.1% 0.0% iDM_ctrl_rep2 64.8% 22.5% 7.8% 1.6% iDM_ctrl_rep3 60.3% 5.1% 6.3% -1.8% iDM_ctrl_rep4 48.0% -42.3% 7.2% 0.2% iCM_ctrl_rep1 79.3% 78.2% 36.6% 64.7% iCM_ctrl_rep2 87.3% 108.9% 58.2% 112.1% iCM_ctrl_rep3 86.7% 106.8% 47.5% 88.5% iCM_ctrl_rep4 86.6% 106.2% 68.5% 134.6% Table 7. AAV-Des-hMBNL1 efficacy: in vitro data C Splicing (KIF13A) Splicing (DMD) Sample % KIF13A exon 21 inclusion bodies % KIF13A splicing improvement % DMD exon 78 inclusion bodies % DMD splicing improvement iDM_Des-hMBNL1_T_rep1 95.3% 139.7% 50.4% 91.3% iDM_Des-hMBNL1_T_rep2 87.6% 109.4% 48.3% 87.0% iDM_Des-hMBNL1_H_rep1 89.7% 117.7% 40.2% 70.3% iDM_Des-hMBNL1_H_rep2 87.3% 108.3% 40.9% 71.6% iDM_Des-hMBNL1_M_rep1 78.3% 73.0% 23.5% 35.5% iDM_Des-hMBNL1_M_rep2 85.1% 99.7% 19.9% 28.0% iDM_Des-hMBNL1_L_rep1 67.4% 29.7% 15.5% 18.8% iDM_Des-hMBNL1_L_rep2 63.7% 15.2% 10.0% 7.5% iDM_Des-hMBNL1_L2_rep1 63.0% 12.5% 7.3% 1.8% iDM_Des-hMBNL1_L2_rep2 62.2% 9.3% 8.2% 3.7% iDM_ctrl_rep1 61.8% 7.7% 5.9% -1.2% iDM_ctrl_rep2 60.4% 2.2% 7.5% 2.1% iDM_ctrl_rep3 65.0% 20.4% 7.8% 2.8% iDM_ctrl_rep4 52.1% -30.3% 4.6% -3.8% iCM_ctrl_rep1 86.0% 103.1% 67.5% 126.8% iCM_ctrl_rep2 94.3% 135.8% 53.5% 97.7% iCM_ctrl_rep3 83.7% 94.1% 49.7% 89.9% iCM_ctrl_rep4 76.8% 67.0% 47.6% 85.5%

To investigate the activity of other promoters, in separate experiments, DM1 cells were transduced with AAV8 vectors encoding the MBNL1 transgene operably linked to the CHAG promoter, CK8 promoter, PGK promoter, EF1α promoter, or eMCK promoter. As a control, cells treated with PBS containing 0.001% Pluronic F68 were included. As shown in Tables 8-9 , none of these alternative promoters achieved improvements in KIF13A splicing and DMD splicing comparable to those produced by the desmin promoter.

To conduct the experiments, different doses of virus were tested and are indicated in Tables 8-9 : T contains a therapeutic concentration of 9x10 ¹¹ viral genomes (vg)/mL, H contains a treatment of 3x10 ¹¹ viral genomes (vg)/mL Concentrations, M contains a therapeutic concentration of ^1x10 viral genomes (vg)/mL, L contains a therapeutic concentration of ^3.3x10 viral genomes (vg)/mL, and L2 contains a therapeutic concentration of ^1.1x10 viral genomes (vg)/mL therapeutic concentration. Table 8. Alternative promoters: in vitro data A Splicing (KIF13A) Splicing(DMD) sample % KIF13A exon 21 inclusions % KIF13A splicing improvement % DMD exon 78 inclusions % DMD splicing improvement iDM_CHAG-hMBNL1_T_rep1 79.3% 78.2% 19.2% 26.6% iDM_CHAG-hMBNL1_T_rep2 81.8% 87.7% 17.3% 22.5% iDM_CHAG-hMBNL1_H_rep1 74.6% 60.1% 10.6% 7.8% iDM_CHAG-hMBNL1_H_rep2 76.5% 67.4% 16.5% 20.8% iDM_CHAG-hMBNL1_M_rep1 71.7% 48.9% 10.9% 8.4% iDM_CHAG-hMBNL1_M_rep2 64.7% 21.8% 14.7% 16.8% iDM_CHAG-hMBNL1_L_rep1 62.0% 11.7% 8.7% 3.7% iDM_CHAG-hMBNL1_L_rep2 61.7% 10.4% 8.1% 2.4% iDM_CHAG-hMBNL1_L2_rep1 55.8% -12.3% 5.7% -3.0% iDM_CHAG-hMBNL1_L2_rep2 52.6% -24.5% 9.0% 4.3% iDM_CK8-hMBNL1_T_rep1 88.9% 115.2% 17.5% 22.8% iDM_CK8-hMBNL1_T_rep2 86.2% 104.6% 28.6% 47.1% iDM_CK8-hMBNL1_H_rep1 87.1% 108.3% 21.9% 32.6% iDM_CK8-hMBNL1_H_rep2 88.8% 114.7% 22.3% 33.4% iDM_CK8-hMBNL1_M_rep1 72.1% 50.6% 14.7% 16.8% iDM_CK8-hMBNL1_M_rep2 79.7% 79.8% 21.6% 31.9% iDM_CK8-hMBNL1_L_rep1 56.8% -8.2% 9.6% 5.5% iDM_CK8-hMBNL1_L_rep2 65.7% 25.7% 12.4% 11.6% iDM_CK8-hMBNL1_L2_rep1 50.2% -33.9% 6.0% -2.2% iDM_CK8-hMBNL1_L2_rep2 61.6% 10.0% 9.0% 4.3% iDM_hPGK-hMBNL1_T_rep1 82.5% 90.5% 83.4% 167.4% iDM_hPGK-hMBNL1_T_rep2 70.4% 43.8% 70.8% 139.6% iDM_hPGK-hMBNL1_H_rep1 86.5% 105.7% 81.7% 163.5% iDM_hPGK-hMBNL1_H_rep2 88.8% 114.9% 56.4% 108.1% iDM_hPGK-hMBNL1_M_rep1 83.7% 95.1% 38.2% 68.3% iDM_hPGK-hMBNL1_M_rep2 88.2% 112.5% 56.8% 109.0% iDM_hPGK-hMBNL1_L_rep1 78.0% 73.0% 30.9% 52.2% iDM_hPGK-hMBNL1_L_rep2 70.5% 44.5% 18.8% 25.7% iDM_hPGK-hMBNL1_L2_rep1 71.5% 48.2% 14.1% 15.4% iDM_hPGK-hMBNL1_L2_rep2 60.8% 6.9% 14.4% 16.1% iDM_ctrl_rep1 62.8% 14.8% 7.1% 0.0% iDM_ctrl_rep2 64.8% 22.5% 7.8% 1.6% iDM_ctrl_rep3 60.3% 5.1% 6.3% -1.8% iDM_ctrl_rep4 48.0% -42.3% 7.2% 0.2% iCM_ctrl_rep1 79.3% 78.2% 36.6% 64.7% iCM_ctrl_rep2 87.3% 108.9% 58.2% 112.1% iCM_ctrl_rep3 86.7% 106.8% 47.5% 88.5% iCM_ctrl_rep4 86.6% 106.2% 68.5% 134.6% Table 9. Alternative promoters: in vitro data B Splicing (KIF13A) Splicing(DMD) sample % KIF13A exon 21 inclusions % KIF13A splicing improvement % DMD exon 78 inclusions % DMD splicing improvement iDM_Ef1a-hMBNL1_T_rep1 86.0% 103.3% 37.6% 64.7% iDM_Ef1a-hMBNL1_T_rep2 94.6% 137.1% 31.2% 51.5% iDM_Ef1a-hMBNL1_H_rep1 88.6% 113.5% 18.4% 24.9% iDM_Ef1a-hMBNL1_H_rep2 89.6% 117.5% 29.1% 47.1% iDM_Ef1a-hMBNL1_M_rep1 79.5% 77.7% 22.8% 34.1% iDM_Ef1a-hMBNL1_M_rep2 80.2% 80.5% 16.6% 21.2% iDM_Ef1a-hMBNL1_L_rep1 67.8% 31.6% 14.2% 16.1% iDM_Ef1a-hMBNL1_L_rep2 76.4% 65.4% 13.2% 14.0% iDM_Ef1a-hMBNL1_L2_rep1 62.8% 11.8% 5.8% -1.4% iDM_Ef1a-hMBNL1_L2_rep2 62.8% 12.0% 8.3% 3.9% iDM_eMCK-hMBNL1_T_rep1 83.8% 94.6% 13.1% 13.7% iDM_eMCK-hMBNL1_T_rep2 84.5% 97.3% 22.4% 33.2% iDM_eMCK-hMBNL1_H_rep1 85.4% 100.8% 15.4% 18.5% iDM_eMCK-hMBNL1_H_rep2 81.4% 85.1% 19.6% 27.3% iDM_eMCK-hMBNL1_M_rep1 78.4% 73.4% 9.9% 7.1% iDM_eMCK-hMBNL1_M_rep2 73.7% 54.7% 11.8% 11.1% iDM_eMCK-hMBNL1_L_rep1 62.1% 9.1% 11.9% 11.2% iDM_eMCK-hMBNL1_L_rep2 56.0% -15.0% 13.4% 14.5% iDM_eMCK-hMBNL1_L2_rep1 58.3% -5.8% 8.7% 4.7% iDM_eMCK-hMBNL1_L2_rep2 55.6% -16.5% 7.6% 2.5% iDM_ctrl_rep1 61.8% 7.7% 5.9% -1.2% iDM_ctrl_rep2 60.4% 2.2% 7.5% 2.1% iDM_ctrl_rep3 65.0% 20.4% 7.8% 2.8% iDM_ctrl_rep4 52.1% -30.3% 4.6% -3.8% iCM_ctrl_rep1 86.0% 103.1% 67.5% 126.8% iCM_ctrl_rep2 94.3% 135.8% 53.5% 97.7% iCM_ctrl_rep3 83.7% 94.1% 49.7% 89.9% iCM_ctrl_rep4 76.8% 67.0% 47.6% 85.5%

To evaluate the level of MBNL1 protein expression achieved by desmin and various other promoters, DM1 cells were transduced with AAV8 vectors encoding the MBNL1 transgene operably linked to the desmin promoter or alternative promoters (e.g., CHAG promoter, CK8 promoter, PGK promoter, EF1α promoter, or eMCK promoter). As a control, cells treated with PBS containing 0.001% Pluronic F68 were included. The results of these experiments are reported in Tables 10-11 . Surprisingly, although it was observed that some of these promoters were able to achieve MBNL1 protein expression levels equal to or exceeding that achieved by the desmin promoter, none of the alternative promoters achieved the same splicing improvements as those achieved by desmin.

For the experiments, different doses of virus were tested and indicated in Tables 10-11 : T contained a therapeutic concentration of 9x10 ¹¹ viral genomes (vg)/mL, H contained a therapeutic concentration of 3x10 ¹¹ viral genomes (vg)/mL, M contained a therapeutic concentration of 1x10 ¹¹ viral genomes (vg)/mL, L contained a therapeutic concentration of 3.3x10 ¹⁰ viral genomes (vg)/mL, and L2 contained a therapeutic concentration of 1.1x10 ¹⁰ viral genomes (vg)/mL. Table 10. MBNL1 protein expression: In vitro data A Sample Relative performance of MBNL1 iDM_CHAG-hMBNL1_T_rep1 1.54 iDM_CHAG-hMBNL1_T_rep2 1.65 iDM_CHAG-hMBNL1_H_rep1 1.19 iDM_CHAG-hMBNL1_H_rep2 1.44 iDM_CHAG-hMBNL1_M_rep1 1.06 iDM_CHAG-hMBNL1_M_rep2 0.95 iDM_CHAG-hMBNL1_L_rep1 0.93 iDM_CHAG-hMBNL1_L_rep2 1.07 iDM_CHAG-hMBNL1_L2_rep1 1.19 iDM_CHAG-hMBNL1_L2_rep2 1.09 iDM_Des-hMBNL1_T_rep1 1.90 iDM_Des-hMBNL1_T_rep2 1.86 iDM_Des-hMBNL1_H_rep1 1.26 iDM_Des-hMBNL1_H_rep2 1.18 iDM_Des-hMBNL1_M_rep1 1.31 iDM_Des-hMBNL1_M_rep2 1.38 iDM_Des-hMBNL1_L_rep1 1.45 iDM_Des-hMBNL1_L_rep2 0.91 iDM_Des-hMBNL1_L2_rep1 0.87 iDM_Des-hMBNL1_L2_rep2 1.03 iDM_CK8-hMBNL1_T_rep1 1.47 iDM_CK8-hMBNL1_T_rep2 1.33 iDM_CK8-hMBNL1_H_rep1 1.14 iDM_CK8-hMBNL1_H_rep2 1.49 iDM_CK8-hMBNL1_M_rep1 1.44 iDM_CK8-hMBNL1_M_rep2 1.10 iDM_CK8-hMBNL1_L_rep1 1.23 iDM_CK8-hMBNL1_L_rep2 1.17 iDM_CK8-hMBNL1_L2_rep1 0.94 iDM_CK8-hMBNL1_L2_rep2 1.05 iDM_hPGK-hMBNL1_T_rep1 12.89 iDM_hPGK-hMBNL1_T_rep2 14.04 iDM_hPGK-hMBNL1_H_rep1 6.82 iDM_hPGK-hMBNL1_H_rep2 6.86 iDM_hPGK-hMBNL1_M_rep1 2.70 iDM_hPGK-hMBNL1_M_rep2 3.18 iDM_hPGK-hMBNL1_L_rep1 1.81 iDM_hPGK-hMBNL1_L_rep2 1.65 iDM_hPGK-hMBNL1_L2_rep1 1.26 iDM_hPGK-hMBNL1_L2_rep2 1.43 iCM_ctrl_rep1 2.20 iCM_ctrl_rep2 2.16 iDM_ctrl_rep1 0.88 iDM_ctrl_rep2 1.10 iDM_ctrl_rep3 1.02 Table 11. MBNL1 protein expression: in vitro data B Sample Relative performance of MBNL1 iDM_Des-hMBNL1_T_rep1 4.03 iDM_Des-hMBNL1_T_rep2 2.04 iDM_Des-hMBNL1_H_rep1 1.77 iDM_Des-hMBNL1_H_rep2 1.66 iDM_Des-hMBNL1_M_rep1 1.35 iDM_Des-hMBNL1_M_rep2 1.25 iDM_Des-hMBNL1_L_rep1 1.19 iDM_Des-hMBNL1_L_rep2 1.30 iDM_Des-hMBNL1_L2_rep1 1.35 iDM_Des-hMBNL1_L2_rep2 1.19 iDM_Ef1a-hMBNL1_T_rep1 2.17 iDM_Ef1a-hMBNL1_T_rep2 1.82 iDM_Ef1a-hMBNL1_H_rep1 1.92 iDM_Ef1a-hMBNL1_H_rep2 1.73 iDM_Ef1a-hMBNL1_M_rep1 1.38 iDM_Ef1a-hMBNL1_M_rep2 1.53 iDM_Ef1a-hMBNL1_L_rep1 0.92 iDM_Ef1a-hMBNL1_L_rep2 0.97 iDM_Ef1a-hMBNL1_L2_rep1 1.15 iDM_Ef1a-hMBNL1_L2_rep2 1.10 iDM_eMCK-hMBNL1_T_rep1 1.47 iDM_eMCK-hMBNL1_T_rep2 1.69 iDM_eMCK-hMBNL1_H_rep1 1.35 iDM_eMCK-hMBNL1_H_rep2 1.08 iDM_eMCK-hMBNL1_M_rep1 1.43 iDM_eMCK-hMBNL1_M_rep2 1.66 iDM_eMCK-hMBNL1_L_rep1 1.32 iDM_eMCK-hMBNL1_L_rep2 1.05 iDM_eMCK-hMBNL1_L2_rep1 1.26 iDM_eMCK-hMBNL1_L2_rep2 1.03 iCM_ctrl_rep1 1.83 iCM_ctrl_rep2 1.69 iDM_ctrl_rep1 1.02 iDM_ctrl_rep2 0.99 iDM_ctrl_rep3 0.99 Example 2. Evaluation of the effectiveness of an adeno-associated viral vector encoding an MBNL1 transgenic construct operably linked to a DES promoter for the treatment of a disorder associated with RNAi dominance

This example describes how AAV vectors encoding the MBNL1 transgene operably linked to the DES promoter can be designed and evaluated. For example, AAV vectors targeting murine HSA ^LR expressed in a mouse myotonic dystrophy model can be designed for testing purposes. For example, one of the goals of this study is to translate methods to reduce splicing disorders of MBNL1 RNA substrates in order to develop paradigms that ameliorate the symptoms of DM1. Materials and methods

Materials and methods for testing the therapeutic efficacy of AAV vectors encoding the MBNL1 transgene operably linked to the DES promoter are described in Example 1.

For example, any suitable murine myotonic dystrophy model as described herein may be used to develop AAV gene therapy for DM1. Gene therapy can be designed so that its efficacy can be tested in the HSA ^LR mouse model of DM1. HSA ^LR mice can be generated by inserting an expanded CTG repeat in the 3'UTR of the human skeletal actin gene (HSA) gene, which is genetically similar to the disease-causing repeat expansion in the DMPK gene in humans. background. HSA ^LR mice are known to display a number of DM1-related genetic and phenotypic changes in skeletal muscle, including myotonia, splicing changes of various mRNAs, and sequestration of splicing factors in ribonucleosomes. Specifically, mice can exhibit features of myotonic dystrophy similar to DM1 in humans. The HSA ^LR transgene is derived from inserting (CTG) ₂₅₀ repeats into the 3'UTR of the HSA gene. When the transgene is expressed in mouse skeletal muscle, myotonic discharges are evident, splicing changes occur in various mRNAs, and nuclear foci containing amplified transgene mRNA and splicing factors are present.

A transgene expression cassette (DES-MBNL1) in which the transgene encoding MBNL1 is operably linked to the DES promoter will be tested. The AAV genome can be packaged in an AAV2/8 capsid for targeting skeletal muscle cells and neuronal cells. The AAV2/8 DES-MBNL1 transgene construct can be delivered by intravenous injection (IV) into the tail vein of 4-week-old HSA ^LR mice.

After transduction with the AAV transgenes described herein, HSA ^LR mice can be sacrificed and human placental alkaline phosphatase (AP) staining indicates the presence of viral genomes with reporter gene expression. H&E staining of frozen sections from treated mice can also be performed.

AAV transgene vectors encoding the MBNL1 transgene operably linked to the DES promoter can achieve increased MBNL1 protein expression in HSA ^LR mice. This result can be demonstrated by restoring correct splicing of SERCA1 mRNA. Systemic injection of AAV2/8 DES-MBNL1 transgene can improve splicing of SERCA1 and CLCN1 in tibialis anterior (TA) muscle.

Taken together, such experiments indicate the utility of transgene therapy in treating DM1, where the transgene is operably linked to the DES promoter. Example 3. Treatment of myotonic dystrophy by AAV transgenic gene therapy

Using the compositions and methods of the present disclosure, pseudotyped AAV2/ 8 vector, which includes or encodes a DES promoter, a β-globin intron sequence, a transgene encoding MBNL1, and an SV40 late polyadenylation site sequence from 5' to 3'. In some embodiments, the DES promoter has a nucleic acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. In some embodiments, the transgene encoding MBNL1 has a nucleic acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%) identical to any one of SEQ ID NOs: 4-11 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequences. In some embodiments, MBNL1 has an amino acid sequence that is at least 85% identical to any of SEQ ID NOs: 12-19 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical amino acid sequences.

Vectors including nucleic acid molecules can be delivered to cells (eg, cells of a patient) that carry the expanded repeat region in exon 15 of the endogenous DMPK gene. Increased functional sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 1 (SERCA1), voltage-gated chloride channel 1 (CLCN1) and/or ZO-2-associated speckle protein (ZASP) proteins may be manifested by, for example, respective Western blot assessment of proteins.

After administration to patients of a viral vector including this transgene operably linked to the DES promoter, the patient exhibits increased expression of MBNL1, encoding the insulin receptor, lanodine receptor 1, cardiac troponin, and or an increase in corrective splicing of skeletal muscle troponin RNA transcripts, and may experience an improvement in myotonia within 12 weeks of treatment, as measured by qualitative physical testing, such as a grip strength test that measures hand strength. Test. Other embodiments

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. generally.

Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications and that this application is intended to cover any variations, uses or modifications of the invention (generally following the principles of the invention and including such departures from the invention) which are within known or customary practice in the art to which the invention pertains and which may be applied to the basic features described above and which are within the scope of the patent application.

Other embodiments are within the scope of the patent claims.

Figure 1 is a schematic diagram of RNA polypeptide design. From 5' to 3', the construct includes the human desmin (hDes) promoter sequence, intron sequence, human blind muscle-like splicing regulator 1 (MBNL1) cDNA sequence, and SV40 polyadenylation (polyA) sequence.

TW202409290A_112123915_SEQL.xml

Claims (107)

An adeno-associated virus (AAV) comprising a transgene encoding human blind muscle-like splicing regulator 1 (MBNL1), wherein the transgene is operably linked to a desmin (DES) promoter. The AAV of claim 1, wherein the DES promoter includes a 5' region having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 1. The AAV of claim 2, wherein 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 AAV of claim 3, wherein the DES promoter comprises a 5' region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1, and optionally wherein the DES promoter comprises 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. The AAV of claim 4, wherein the DES promoter comprises a 5' region having a nucleic acid sequence of SEQ ID NO: 1. The AAV of any one of claims 1 to 5, wherein the DES promoter includes a 3' region having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 2. The AAV of claim 6, wherein 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 AAV of claim 7, wherein the DES promoter comprises a 3' region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2, and optionally wherein the DES promoter comprises 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. The AAV of claim 8, wherein the DES promoter comprises a 3' region having a nucleic acid sequence of SEQ ID NO: 2. The AAV of any one of claims 1 to 9, wherein the DES promoter has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3. The AAV of claim 10, wherein the DES promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. The AAV of claim 11, wherein the DES promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3, optionally wherein the DES promoter has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 3 , 97%, 98%, or 99% identical nucleic acid sequences. The AAV of claim 12, wherein the DES promoter has the nucleic acid sequence of SEQ ID NO: 3. The AAV of any one of claims 1 to 13, wherein the MBNL1 has an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. The AAV of claim 14, wherein 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. The AAV of claim 15, wherein the MBNL1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 12-19, optionally wherein the MBNL1 has an amino acid sequence identical to SEQ ID NO: 12 - An amino acid sequence whose amino acid sequence is at least 96%, 97%, 98%, or 99% identical to any one of -19. The AAV of claim 16, wherein the MBNL1 has the amino acid sequence of any one of SEQ ID NOs: 12-19. The AAV of any one of claims 1 to 17, wherein the transgene has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11. The AAV of claim 18, wherein 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 AAV of claim 19, wherein 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 AAV of claim 20, wherein 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. The AAV of claim 21, wherein 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 AAV of claim 22, wherein 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. The AAV of claim 23, wherein 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. The AAV of claim 24, wherein the transgene has the nucleic acid sequence of any one of SEQ ID NOs: 4-11. The AAV of any one of claims 1 to 25, wherein the AAV comprises a capsid protein from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, and AAVrh74. Such as requesting the AAV of any one of items 1 to 26, wherein the AAV is a pseudo-type AAV. Such as the AAV of claim 27, wherein the pseudo-type AAV is AAV2/8. The AAV of claim 27, wherein the pseudotyped AAV is AAV2/9. The AAV of any one of claims 1 to 29, wherein the AAV further comprises a polyadenylation site (pA), optionally wherein the pA is located 3' to the transgene. The AAV of claim 30, wherein the pA site includes a simian virus 40 (SV40) late pA site or an SV40 early pA site. The AAV of claim 31, wherein the pA site comprises the SV40 late pA site. The AAV of any one of claims 1 to 32, wherein the AAV further includes an intron, optionally wherein the intron is located 3' of the promoter and 5' of the transgene. The AAV of claim 33, wherein the intron is a β-globin intron. A pharmaceutical composition comprising the AAV of any one of claims 1 to 34 and a pharmaceutically acceptable carrier, diluent, or excipient. A nucleic acid molecule comprising: (i) a DES promoter; (ii) a β-globin intron; (iii) a transgenic gene encoding MBNL1; and (iv) an SV40 pA site; wherein the components are operably linked to each other in the 5' to 3' direction as follows: DES-β-globin intron-MBNL1-SV40 pA. Such as the nucleic acid molecule of claim 36, wherein the SV40 pA site includes an SV40 late pA site or an SV40 early pA site. The nucleic acid molecule of claim 37, wherein the pA site includes an SV40 late pA site. The nucleic acid molecule of any one of claims 36 to 38, wherein the transgene encodes a protein having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. The nucleic acid molecule of claim 39, wherein the transgene encodes a protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. The nucleic acid molecule of claim 40, wherein the transgene encodes a protein having an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19, optionally wherein the transgene The gene encodes a protein having 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. The nucleic acid molecule of claim 41, wherein the transgene encodes a protein having an amino acid sequence of any one of SEQ ID NOs: 12-19. The nucleic acid molecule of any one of claims 36 to 42, wherein the transgene has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 4-11. The nucleic acid molecule of claim 43, wherein 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 nucleic acid molecule of claim 44, wherein 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 nucleic acid molecule of claim 45, wherein 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. The nucleic acid molecule of claim 46, wherein 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 nucleic acid molecule of claim 47, wherein the transgenic gene 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. The nucleic acid molecule of claim 48, wherein 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. The nucleic acid molecule of claim 49, wherein the transgenic gene has the nucleic acid sequence of any one of SEQ ID NOs: 4-11. A nucleic acid molecule as in any one of claims 36 to 50, wherein the DES promoter comprises a 5' region having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid molecule of claim 51, wherein 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. A nucleic acid molecule as in claim 52, wherein the DES promoter comprises a 5' region having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1, optionally wherein the DES promoter comprises 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. The nucleic acid molecule of claim 53, wherein the DES promoter comprises a 5' region having a nucleic acid sequence of SEQ ID NO: 1. A nucleic acid molecule as in any one of claims 36 to 54, wherein the DES promoter comprises a 3' region having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 2. The nucleic acid molecule of claim 55, wherein 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 nucleic acid molecule of claim 56, wherein 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, optionally wherein the DES promoter includes a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2. The 3' region of the nucleic acid sequence of 2 is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence. The nucleic acid molecule of claim 57, wherein the DES promoter comprises a 3' region having a nucleic acid sequence of SEQ ID NO: 2. The nucleic acid molecule of any one of claims 36 to 58, wherein the DES promoter has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 3. The nucleic acid molecule of claim 59, wherein the DES promoter has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. The nucleic acid molecule of claim 60, wherein the DES promoter has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3, optionally wherein the DES promoter has a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 3 %, 97%, 98%, or 99% identical nucleic acid sequences. The nucleic acid molecule of claim 61, wherein the DES promoter has the nucleic acid sequence of SEQ ID NO: 3. A vector comprising the nucleic acid molecule of any one of claims 36 to 62, optionally wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a virus vector. The vector of claim 63, wherein the vector is a viral vector. The vector of claim 64, wherein the viral vector is selected from the group consisting of AAV, adenovirus, lentivirus, retrovirus, poxvirus, bacillus virus, herpes simplex virus, vaccinia virus, and synthetic virus. Such as the vector of claim 65, wherein the viral vector is AAV. The vector of claim 66, wherein the AAV comprises a capsid protein from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, and AAVrh74. The vector of claim 66 or 67, wherein the AAV is a pseudotyped AAV. The vector of claim 68, wherein the pseudotyped AAV is AAV2/8 or AAV2/9, depending on the case, wherein the pseudotyped AAV is AAV2/8. The vector of any one of claims 66 to 69, wherein the AAV comprises a recombinant capsid protein. The AAV of any one of claims 1 to 34 and the vector of any one of claims 63 to 70, wherein the AAV or the vector has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 20. The AAV or the vector of claim 71, wherein the AAV 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 or vector of claim 72, wherein the AAV or the vector has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 20, optionally wherein the AAV or the vector has the nucleic acid sequence of SEQ ID NO: 20 A nucleic acid sequence whose sequence is at least 96%, 97%, 98%, or 99% identical. The AAV or the vector of claim 73, wherein the AAV or the vector has the nucleic acid sequence of SEQ ID NO: 20. A plasmid encoding a viral vector according to any one of claims 63 to 74. The plasmid of claim 63 or 75, wherein the plasmid comprises a promoter operably linked to a selectable marker gene. The plasmid of claim 76, wherein the selectable marker gene is an antibiotic resistance gene. A pharmaceutical composition comprising a nucleic acid molecule as described in any one of claims 36 to 62, a vector as described in any one of claims 63 to 74, or a plasmid as described in any one of claims 75 to 77, and a pharmaceutically acceptable carrier, diluent, or excipient. A method for treating myotonic muscular dystrophy type 1 (DM1) in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid as described in any one of claims 36 to 62, an AAV as described in any one of claims 1 to 34 and 71 to 74, a vector as described in any one of claims 63 to 74, a plasmid as described in any one of claims 63 and 75 to 77, or a pharmaceutical composition as described in claim 35 or 78. A method for increasing MBNL1 expression in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid as described in any one of claims 36 to 62, an AAV as described in any one of claims 1 to 34 and 71 to 74, a vector as described in any one of claims 63 to 74, a plasmid as described in any one of claims 63 and 75 to 77, or a pharmaceutical composition as described in claim 35 or 78. A method for inducing correct splicing of one or more RNA transcript substrates of MBNL1 in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid as described in any one of claims 36 to 62, an AAV as described in any one of claims 1 to 34 and 71 to 74, a vector as described in any one of claims 63 to 74, a plasmid as described in any one of claims 63 and 75 to 77, or a pharmaceutical composition as described in claim 35 or 78. The method of any one of claims 79 to 81, wherein the patient is at least 18 years old. The method of any one of claims 79 to 82, wherein the patient exhibits an increase in whole blood MBNL1 levels after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient. The method of claim 83, wherein the patient exhibits an increase in whole blood MBNL1 levels approximately 12 weeks after administration. The method of any of claims 79 to 84, wherein after administering the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits a reduction in aberrant splicing of one or more genes selected from: kinesin family member 13A ( KIF13A ); dystrophin ( DMD ); insulin receptor ( INSR ); voltage-gated chloride channel 1 ( CLCN1 ); calcification protein T2; cardiac type ( TNNT2 ); calcification protein T3, fast skeletal type ( TNNT3 ); titin ( TTN ); and bridging integrin 1 ( BIN1 ), optionally wherein the patient exhibits the reduction in aberrant splicing about 12 weeks after administration. The method of any one of claims 79 to 85, wherein the reduction in aberrant splicing of INSR after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient includes an increase in expression of the INSR-B isoform , where the patient exhibits increased expression of the INSR-B isoform approximately 12 weeks after administration. The method of any one of claims 79 to 86, wherein the reduction of aberrant splicing of CLCN1 after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient comprises excluding exon 7a of the CLCN1 transcript. of the increase, optionally wherein the patient exhibits an increase in CLCN1 transcript excluding exon 7a approximately 12 weeks after administration. A method of treating DM1 in a human patient diagnosed with a splicing disease of endogenous ATP2A1 , CLCN1 , and/or LDB3 genes, the method comprising administering to the patient a therapeutically effective amount of any of claims 36 to 62 The nucleic acid of any one of claims 1 to 34 and 71 to 74, the vector of any one of claims 63 to 74, the plasmid of any one of claims 63 and 75 to 77 , or the pharmaceutical composition of claim 35 or 78. A method of increasing expression of functional sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 1 (SERCA1) protein in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of claim 36 The nucleic acid of any one of claims 1 to 62, the AAV of any one of claims 1 to 34 and 71 to 74, the vector of any one of claims 63 to 74, the vector of any one of claims 63 to 75 to 77 The plastid of claim 35 or claim 78, or the pharmaceutical composition of claim 35 or 78. A method for increasing the expression of a functional voltage-gated chloride channel 1 (CLCN1) protein in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of a nucleic acid as described in any one of claims 36 to 62, an AAV as described in any one of claims 1 to 34 and 71 to 74, a vector as described in any one of claims 63 to 74, a plasmid as described in any one of claims 63 and 75 to 77, or a pharmaceutical composition as described in claim 35 or 78. A method of increasing the expression of functional ZO-2-associated speck protein (ZASP) in a human patient diagnosed with DM1, the method comprising administering to the patient a therapeutically effective amount of any one of claims 36 to 62 Nucleic acid, such as the AAV of any one of claims 1 to 34 and 71 to 74, the vector of any one of claims 63 to 74, the plasmid of any one of claims 63 and 75 to 77, or Such as the pharmaceutical composition of claim 35 or 78. The method of claim 89, wherein the expression of SERCA1 mRNA is increased 1.1-fold to 10-fold after administration of the vector or the composition to the patient. The method of claim 90, wherein the expression of CLCN1 mRNA is increased by 1.1-fold to 10-fold after administration of the vector or the composition to the patient. The method of claim 91, wherein after administering the vector or the composition to the patient, the expression of ZASP mRNA increases by 1.1 to 10 times. The method of any one of claims 85 to 94, wherein the expression of CLCN1 mRNA is increased by 1.1-fold to 10-fold after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient. The method of any one of claims 79 to 95, wherein after administration of the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an improvement in myotonia, wherein after administration, the patient exhibits an improvement in myotonia, as appropriate, wherein approximately At 12 weeks, the patient showed improvement in myotonia. The method of any one of claims 79 to 96, wherein after administering the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits a reduction in abnormal heart rhythm, optionally wherein the patient exhibits a reduction in abnormal heart rhythm about 12 weeks after administration. The method of any one of claims 79 to 97, wherein after administering 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 a reduction in excessive daytime sleepiness about 12 weeks after administration. The method of any of claims 79 to 98, wherein after administering the nucleic acid, AAV vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an increase in hand muscle strength, optionally wherein the patient exhibits an increase in hand muscle strength about 12 weeks after administration. The method of claim 99, wherein hand muscle strength is measured by a grip strength test. The method of any one of claims 79 to 100, wherein after administering the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits improvement in sleep apnea symptoms, optionally wherein the patient exhibits improvement in sleep apnea symptoms about 12 weeks after administration. The method of any one of claims 79 to 101, wherein prior to treatment, the patient has not been diagnosed with DM1. The method of any one of claims 79 to 102, wherein prior to treatment, the patient has one or more symptoms of DM1. Claim the method of any one of items 79 to 103, wherein the symptoms of DM1 include dyspnea, aspiration, sleep apnea, intellectual disability, excessive daytime sleepiness, abnormal heart conditions, cardiac arrhythmias, cardiomyopathy, swallowing problems, muscle weakness , muscle atrophy, myotonia and/or muscle pain. The method of any one of claims 79 to 104, wherein after administering the nucleic acid, vector, plasmid, or pharmaceutical composition to the patient, the patient exhibits an expression encoding an insulin receptor, a ryanodine receptor 1. Increase in corrective splicing of cardiac troponin and/or skeletal muscle troponin RNA transcripts. The method of any one of claims 79 to 105, wherein the nucleic acid, vector, plasmid, or pharmaceutical composition is administered via intravenous, intrathecal, intracerebroventricular, intraparenchymal, intracisternal, intracutaneous, transdermal, Administer parenterally, intramuscularly, intranasally, subcutaneously, transdermally, intratracheally, intraperitoneally, intraarterially, intravascularly, or orally and/or to the patient by inhalation, perfusion, or lavage. A vector comprising a nucleic acid according to any one of claims 36 to 62, an AAV according to any one of claims 1 to 34 and 71 to 74, a vector according to any one of claims 63 to 74, a vector according to claim 63 and A set of plasmids according to any one of claims 75 to 77, or a pharmaceutical composition as claimed in claim 35 or 78, and a set of drug instructions instructing a user of the set to report a diagnosis of DM1 or symptoms of DM1 Human patients with one or more symptoms are administered the nucleic acid, vector, plasmid, or pharmaceutical composition.