Gene therapy for lafora disease

Abstract

Compositions and methods for treating LaFora disease are provided. The compositions and methods herein comprise AAV9 vector comprising a low expressing promoter driving the expression of a gene for e.g., EPM2A or EPM2B. Further provided are method of administering the composition for reducing one or more symptoms associated with or preventing LaFora disease.

Description

GENE THERAPY FOR LAFORA DISEASERELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 542,966 filed October 6, 2023, the contents of which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] This application contains references to amino acid sequences and / or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing xml file entitled “824996_sequencelisting.xml”, file size 40,960 bytes, created on October 4, 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(5).BACKGROUND

[0003] 1. Field

[0004] The present disclosure is directed to compositions and methods for inducing expression of laforin or malin polypeptides in patients in need thereof.

[0005] 2. Discussion of Related Art

[0006] Lafora Disease (LD) is a monogenic neurodegenerative disorder affecting adolescents and is associated with mutations in either EPM2A or EPM2B genes which encode for laforin and malin respectively. In the absence of either protein, there is an excessive accumulation of poorly branched glycogen which become aggregated and are called lafora bodies (LB). These lafora bodies are formed in neurons and glial cells and drive neurodegeneration and neuroinflammation.BRIEF SUMMARY

[0007] Various aspects of the present disclosure are directed to a recombinant adeno- associated virus (rAAV) vector comprising a nucleic acid sequence encoding a laforin polypeptide or a malin polypeptide operably linked to a weak promoter sequence. In some aspects, the nucleic acid sequence encoding a laforin polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 3-6. In further aspects, the nucleic acid sequence encoding a laforin polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 3. In some aspects, the nucleic acid sequenceencoding a malin polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 9-12. In certain aspects, the nucleic acid sequence encoding a malin polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 9.

[0008] In some aspects, the weak promoter sequence of the rAAV vector comprises a JeT promoter or a JeT-1 promoter. In some aspects, the JeT promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15. In some aspects, the JeT promoter comprises SEQ ID NO: 15.

[0009] In some aspects, the rAAV vector disclosed herein, further comprising at least one other mRNA stabilizing element. In some aspects, the mRNA stabilizing element comprises a polyA tail. In some aspects, the disclosed rAAV vector has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18 or 19.

[0010] In further aspects, the disclosure encompasses an rAAV viral vector comprising an AAV capsid protein; and a disclosed rAAV vector. In some aspects, the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein or an AAVrh.10 capsid protein. In certain aspects, the AAV capsid protein is an AAV9 capsid protein.

[0011] In some aspects, further provided herein is a pharmaceutical composition comprising the rAAV viral vector disclosed herein and at least one pharmaceutically appropriate carrier or excipient.

[0012] In some aspects, a method for treating a subject having a disease and / or disorder involving a mutation in a EPM2A gene and / or a NHLRC1 gene, the method comprising administering to the subject at least one therapeutically effective amount of the disclosed rAAV viral vector or the disclosed pharmaceutical composition. In some aspects, further disclosed is rAAV viral vector or the pharmaceutical composition for use in treating a disease and / or disorder involving an EPM2A gene and / or a NHLRC1 gene in a subject in need thereof.

[0013] In some aspects, the disease and / or disorder is Lafora Disease. In some aspects, the rAAV viral vector or the pharmaceutical composition is administered to the subject at adose ranging from about 1011to about 1018viral vector particles. In some aspects of the method, the dose is about 1x 1011to about 1x1012viral particles. In some aspects, the dose is about 1x1011or about 5x1011viral particles. In some aspects, the rAAV viral vector or the pharmaceutical composition is administered to the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intra-aurally, intra-ocularly, or peri-ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intranervally, intrapleurally, topically, intralymphatically, intracisternally or intranerve. In further aspects, the rAAV viral vector or pharmaceutical composition is administered intrathecally. In some aspects of the method the subject is human.BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1A-1 B show periodic acid schiff diastase (PASD) staining of LBs in Mouse Hippocampus in Lafora Disease Mouse Model and wild type mouse.

[0015] FIG 2A shows a schematic of the AAV9-CBh-hEPM2A for evaluating the toxicity in wild type LD moue model.

[0016] FIG. 2B shows a schematic of the experiments performed to evaluate the toxicity of CBh driven EPM2A (laforin) expression in wild type LD mouse model.

[0017] FIG. 2C shows histological assessment showing toxicity in tissues proximal to high dose of virus.

[0018] FIG. 3 shows representative images showing laforin distribution in dorsal root ganglion from virus treated and control wild type LD mice using IHC and PASD staining.

[0019] FIG. 4 shows representative images showing laforin distribution in spinal column from virus treated and control wild type LD mice using IHC and PASD staining.

[0020] FIG. 5A shows representative images showing laforin distribution in cerebellum from virus treated and control wild type LD mice using IHC and PASD staining.

[0021] FIG. 5B shows representative images showing laforin distribution in dentate gyrus from virus treated and control wild type LD mice using IHC and PASD staining.

[0022] FIG. 5C shows representative images showing laforin distribution in skeletal muscle from virus treated and control wild type LD mice using IHC and PASD staining.

[0023] FIG. 6A shows a schematic of the AAV9-JeT-hEPM2A for evaluating toxicity in wild type LD mouse.

[0024] FIG. 6B shows a schematic of the experiments performed to evaluate the toxicity of JeT driven EPM2A (laforin) expression in wild type LD mouse model.

[0025] FIG. 7A shows representative images showing laforin distribution in dorsal root ganglion from high dose virus treated, low dose virus treated, and control wild type LD mice using IHC and PASD staining.

[0026] FIG. 7B shows representative images showing laforin distribution in spinal column from high dose virus treated, low dose virus treated, and control wild type LD mice using IHC and PASD staining.

[0027] FIG. 8A shows representative images showing laforin distribution in cerebellum from virus treated, and control wild type LD mice using IHC and PASD staining.

[0028] FIG. 8B shows representative images showing laforin distribution in dentate gyrus from virus treated, and control wild type LD mice using IHC and PASD staining.

[0029] FIG. 8C shows representative images showing laforin distribution in skeletal muscle from virus treated, and control wild type LD mice using IHC and PASD staining.

[0030] FIG. 9A shows a schematic of the AAV9-JeT-hEPM2A for evaluating the toxicity in LD knockout mouse.

[0031] FIG. 9B shows a schematic of the experiments performed to evaluate the toxicity of JeT driven EPM2A (laforin) expression in LD knockout mouse model.

[0032] FIG. 10A shows IHC Staining of human laforin across brain regions.

[0033] FIG. 10B shows insoluble glycogen measurement in control and knockout mice treated with low dose of AAV9-JeT-hEPM2A.

[0034] FIG. 10C shows insoluble glycogen measurement in control and knockout mice treated with high dose of AAV9-JeT-hEPM2A.

[0035] FIG. 10D shows quantification of PASD staining in cortex of knockout control and mice treated with low dose of AAV9-JeT-hEPM2A.

[0036] FIG. 10E shows quantification of PASD staining in cerebellum of knockout control and mice treated with low dose of AAV9-JeT-hEPM2A.

[0037] FIG. 10F shows quantification of PASD staining in hippocampus of knockout control and mice treated with low dose of AAV9-JeT-hEPM2A.

[0038] FIG. 10G shows quantification of PASD staining in cortex of knockout control and mice treated with high dose of AAV9-JeT-hEPM2A.

[0039] FIG. 10H shows quantification of PASD staining in cerebellum of knockout control and mice treated with high dose of AAV9-JeT-hEPM2A.

[0040] FIG. 101 shows quantification of PASD staining in hippocampus of knockout control and mice treated with high dose of AAV9-JeT-hEPM2A.

[0041] FIG. 11 A shows a schematic of the AAV9-JeT-hEPM2B for evaluating the toxicity in LD wild type mouse.

[0042] FIG. 11B shows a schematic of the experiments performed to evaluate the toxicity of JeT driven EPM2B (malin) expression in LD wild type mouse model.

[0043] FIG. 12 shows histological assessment showing absence of toxicity in tissues proximal to high dose of virus.

[0044] FIG. 13A shows a schematic of the AAV9-JeT-hEPM2B for evaluation of toxicity in LD knock out mice

[0045] FIG. 13B shows a schematic of the experiments performed to evaluate the toxicity of JeT driven EPM2B (malin) expression in LD knock out mice.

[0046] FIG. 13C shows quantification of relative expression of malin using qpCR in knockout and wild type mice treated with high or low dose of AAV9-JeT-hEPM2B.

[0047] FIG. 13D shows insoluble glycogen measurement in knockout control mice and knockout mice treated with low dose of AAV9-JeT-hEPM2B.

[0048] FIG. 13E shows insoluble glycogen measurement in knockout control mice and knockout mice treated with high dose of AAV9-JeT-hEPM2B.

[0049] FIG. 13F shows quantification of PASD staining in cortex of knockout control and mice treated with low dose of AAV9-JeT-hEPM2B.

[0050] FIG. 13G shows quantification of PASD staining in cerebellum of knockout control and mice treated with low dose of AAV9-JeT-hEPM2B.

[0051] FIG. 13H shows quantification of PASD staining in hippocampus of knockout control and mice treated with low dose of AAV9-JeT-hEPM2B.

[0052] FIG. 131 shows quantification of PASD staining in cortex of knockout control and mice treated with high dose of AAV9-JeT-hEPM2B.

[0053] FIG. 13J shows quantification of PASD staining in cerebellum of knockout control and mice treated with high dose of AAV9-JeT-hEPM2B.

[0054] FIG. 13K shows quantification of PASD staining in hippocampus of knockout control and mice treated with high dose of AAV9-JeT-hEPM2B.DETAILED DESCRIPTION

[0055] In some aspects, the current disclosure is based on the surprising discovery that strong transgene expression of certain genes mutated in patients with Lafora disease or other neurodegenerative diseases (e.g., EPM2A or NHLRC1 (EPM2B) genes) can be toxic. Accordingly, the disclosure herein provides new gene therapy vectors for expression of two Lafora disease genes, EPM2A and NHLRC1 under the control of weak promoters (e.g., JET or JET-I). These new vectors are designed specifically for weak expression and can also include codon optimized EPM2A or NHLRC1 as well as mRNA stabilizing elements. Further aspects of the disclosure are directed to methods of delivering the vectors using adeno- associated virus as a treatment mechanism for Lafora disease to allow for ubiquitous longterm expression.I. Definitions

[0056] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5thEd., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0057] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0058] Herein the term “active ingredient” refers to an isolated nucleic acid, gene construct or vector (e.g., AAV vector) that can be used to deliver and express a laforin or malin polypeptide or fragment thereof in a patient.

[0059] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0060] “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.

[0061] As used herein, the term “patient”, “subject”, or “test subject” refers to any organism to which provided compound or compounds described herein are administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and / ortherapeutic purposes. Typical subjects include animals (e g., mammals such as mice, rats, rabbits, dogs, cats, horses (and other farm animals), non-human primates, humans). In an aspect, a subject is a human. In some aspects, a subject may be suffering from, and / or susceptible to a disease, disorder, and / or condition (e.g., Lafora Disease)

[0062] The term “effective amount” as used herein is defined as the amount of the molecules of the present disclosure that are necessary to result in the desired physiological change in the cell or tissue to which it is administered. The term “therapeutically effective amount” as used herein is defined as the amount of the molecules of the present disclosure that achieves a desired effect with respect to whatever condition is being treated. For example, a desired effect in a method for treating pain could be a reduction or amelioration of pain in the subject while a desired effect in a method of treating cancer could be a reduction in tumor size or an arrest in tumor growth. A skilled artisan readily recognizes that in many cases the molecules may not provide a cure but may provide a partial benefit, such as alleviation or improvement of at least one symptom or parameter. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of molecules that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount.”

[0063] As used herein, the disclosure of numerical ranges by numerical endpoints includes all numbers encompassed by that range (e.g., “1 to 5” includes but is not limited to 1 , 1.25, 1.5, 1.75, 2, 2.3, 2. 5, 2.8, 3, 3.1 ,3.3, 3.8, 3.9, 4, 4.25, 4.5, 4.75 and 5). Unless otherwise indicated, all numbers used herein to express quantities, amounts, dimensions, measurements, and the like should be understood as encompassing the specific quantities, amounts, dimensions, measurements and so on, and also as encompassing such instances modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical descriptions set forth herein may vary while remaining well within the teachings of the present disclosure. At the very least, each numerical value should be construed in view of the number of significant digits and by applying routine rounding techniques. As various changes could be made in the above-described cells and methods without departing from the scope of thedisclosure, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense. As various changes could be made in the above-described cells and methods without departing from the scope of the disclosure, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.II. Compositions(a) Isolated Nucleic Acids

[0064] In various aspects of the present disclosure, an isolated nucleic acid (e.g., a gene expression construct or vector) containing at least one transgene is provided. In various aspects, the transgene comprises a Lafora disease gene (e.g., EPM2A or NHLRC1 (EPM2B)). In various aspects, the transgene encodes a laforin polypeptide or fragment thereof (e.g., the protein encoded by the EPM2A gene). In various aspects, the transgene encodes a malin polypeptide or fragment thereof (e.g., the protein encoded by the NHLRC1 (EPM2B) gene).

[0065] In some aspects, a transgene nucleic acid molecule can comprise a nucleic acid sequence encoding a laforin polypeptide, or at least one fragment thereof. In some aspects, a transgene nucleic acid molecule can comprise a nucleic acid sequence encoding a biological equivalent of a laforin polypeptide.

[0066] In some aspects, a laforin polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence put forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof. In some aspects, the laforin polypeptide comprises, consists essentially of, or consist of an amino acid sequence of SEQ ID NO: 1.

[0067] In various aspects, a nucleic acid sequence encoding a laforin polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence put forth in any one of SEQ ID NO: 3-6. In some aspects, a nucleic acid sequence encoding a laforin polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequences put forth in SEQ ID NO: 3.

[0068] In some aspects, the nucleic acid sequence encoding a laforin polypeptide can be a codon optimized nucleic acid sequence that encodes for a laforin polypeptide. A codon optimized nucleic acid sequence encoding a laforin polypeptide can comprise, consist essentially of, or consist of a nucleic acid sequence that is no more than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (or any percentage in between) identical to the wildtype human nucleic acid sequence encoding the laforin polypeptide. As used herein, the “wildtype human nucleic acid sequence encoding the laforin polypeptide” refers to the nucleic acid sequence that encodes the laforin polypeptide in a human genome, as put forth in SEQ ID NO: 13. SEQ ID NOs: 3-6 are unique codon optimized nucleic acid sequences that encode for a laforin polypeptide.

[0069] In some aspects, a codon optimized nucleic acid sequence encoding a laforin polypeptide, such as those put forth in SEQ ID NOs: 3-6, can comprise no donor splice sites. In some aspects, a codon optimized nucleic acid sequence encoding a laforin polypeptide can comprise no more than about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight, or about nine, or about ten donor splice sites. In some aspects, a codon optimized nucleic acid sequence encoding a laforin polypeptide comprises at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten fewer donor splice sites as compared to the wildtype human nucleic acid sequence encoding the laforin polypeptide. Without wishing to be bound by theory, the removal of donor splice sites in the codon optimized nucleic acid sequence can unexpectedly and unpredictably increase expression of the laforin polypeptide in vivo, as cryptic splicing is prevented. Moreover, cryptic splicing may vary between different subjects, meaning that the expression level of the laforin polypeptide comprising donor splice sites may unpredictably vary between different subjects.

[0070] In some aspects, a codon optimized nucleic acid sequence encoding a laforin polypeptide, such as those put forth in SEQ ID NOs: 3-6, can have a GC content that differs from the GC content of the wildtype human nucleic acid sequence encoding the laforin polypeptide. In some aspects, the GC content of a codon optimized nucleic acid sequence encoding a laforin polypeptide is more evenly distributed across the entire nucleic acid sequence, as compared to the wildtype human nucleic acid sequence encoding the laforin polypeptide. Without wishing to be bound by theory, by more evenly distributing the GC content across the entire nucleic acid sequence, the codon optimized nucleic acid sequence exhibits a more uniform melting temperature (“Tm”) across the length of the transcript.

[0071] In some aspects, a laforin polypeptide can further comprise a protein tag. Without wishing to be bound by theory, the inclusion of a protein tag can allow for the detection and / orvisualization of the exogenous laforin polypeptide. As would be appreciated by the skilled artisan, non-limiting examples of protein tags include Myc tags, poly-histidine tags, FLAG- tags, HA-tags, SBP-tags or any other protein tag known in the art.

[0072] In some aspects, a transgene nucleic acid molecule can comprise a nucleic acid sequence encoding a malin polypeptide, or at least one fragment thereof. In some aspects, a transgene nucleic acid molecule can comprise a nucleic acid sequence encoding a biological equivalent of a malin polypeptide.

[0073] In some aspects, a malin polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence put forth in SEQ ID NO: 7 or SEQ ID NO: 8, or a fragment thereof.

[0074] In some aspects, a malin polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to at least one portion of the amino acid sequence put forth in SEQ ID NO: 7, or a fragment thereof.

[0075] In some aspects, a nucleic acid sequence encoding a malin polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence put forth in any one of SEQ ID NO: 9-12. In some aspects, a nucleic acid sequence encoding a malin polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequences put forth in SEQ ID NO: 9. In some aspects, a nucleic acid sequence encoding a malin polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequences put forth in SEQ ID NO: 11 .

[0076] In some aspects, the nucleic acid sequence encoding a malin polypeptide can be a codon optimized nucleic acid sequence that encodes for a malin polypeptide. A codon optimized nucleic acid sequence encoding a malin polypeptide can comprise, consist essentially of, or consist of a nucleic acid sequence that is no more than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (or any percentage in between) identical to the wildtype human nucleic acid sequence encoding the malin polypeptide. As used herein, the “wildtype human nucleic acid sequence encoding the malin polypeptide” refers to the nucleic acid sequence that encodes the malin polypeptide in a human genome, as put forth in SEQ ID NO: 14.

[0077] SEQ ID NOs: 9-12 are unique codon optimized nucleic acid sequences that encode for a malin polypeptide.

[0078] In some aspects, a codon optimized nucleic acid sequence encoding a malin polypeptide, such as those put forth in SEQ ID NOs: 9-12, can comprise no donor splice sites. In some aspects, a codon optimized nucleic acid sequence encoding a malin polypeptide can comprise no more than about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight, or about nine, or about ten donor splice sites. In some aspects, a codon optimized nucleic acid sequence encoding a malin polypeptide comprises at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten fewer donor splice sites as compared to the wildtype human nucleic acid sequence encoding the malin polypeptide. Without wishing to be bound by theory, the removal of donor splice sites in the codon optimized nucleic acid sequence can unexpectedly and unpredictably increase expression of the malin polypeptide in vivo, as cryptic splicing is prevented. Moreover, cryptic splicing may vary between different subjects, meaning that the expression level of the malin polypeptide comprising donor splice sites may unpredictably vary between different subjects.

[0079] In some aspects, a codon optimized nucleic acid sequence encoding a malin polypeptide, such as those put forth in SEQ ID NOs: 9-12, can have a GC content that differs from the GC content of the wildtype human nucleic acid sequence encoding the malin polypeptide. In some aspects, the GC content of a codon optimized nucleic acid sequence encoding a malin polypeptide is more evenly distributed across the entire nucleic acid sequence, as compared to the wildtype human nucleic acid sequence encoding the malin polypeptide. Without wishing to be bound by theory, by more evenly distributing the GC content across the entire nucleic acid sequence, the codon optimized nucleic acid sequence exhibits a more uniform melting temperature (“Tm”) across the length of the transcript.

[0080] In some aspects, a malin polypeptide can further comprise a protein tag. Without wishing to be bound by theory, the inclusion of a protein tag can allow for the detection and / or visualization of the exogenous malin polypeptide. As would be appreciated by the skilled artisan, non-limiting examples of protein tags include Myc tags, poly-histidine tags, FLAG- tags, HA-tags, SBP-tags or any other protein tag known in the art.

[0081] In some aspects, a transgene nucleic acid molecule can comprise a nucleic acid sequence encoding a laforin polypeptide, or at least one fragment thereof, and a nucleic acid sequence encoding a malin polypeptide, or at least one fragment thereof. The nucleic acid sequence encoding a laforin polypeptide and the nucleic acid sequence encoding a malinpolypeptide can be positioned in any order. In a non-limiting example, the nucleic acid sequence encoding a laforin polypeptide can be positioned 5' to the nucleic acid sequence encoding a malin polypeptide. In another non-limiting example, the nucleic acid sequence encoding a malin polypeptide can be positioned 5' to the nucleic acid sequence encoding a laforin polypeptide. In some aspects, the nucleic acid sequence encoding a laforin polypeptide and the nucleic acid sequence encoding a malin polypeptide can be immediately adjacent to each other. In some aspects, the nucleic acid sequence encoding a laforin polypeptide and the nucleic acid sequence encoding a malin polypeptide can be separated by an intervening nucleic acid sequence. In some aspects, the intervening nucleic acid sequence can comprise at least one self-cleaving polypeptide sequence. In some aspects, the intervening nucleic acid sequence can comprise at least two self-cleaving polypeptide sequences.(b) Gene Expression Constructs

[0082] In various aspects, the isolated nucleic acids herein are provided as gene expression constructs comprising, at minimum the nucleic acid encoding a laforin or malin polypeptide or fragment thereof operably linked to a promoter sequence. The term “promoter” and “promoter sequence” as used herein means a control sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or a transgene, are controlled. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. Promoters may contain genetic elements at which regulatory proteins and molecules such as RNA polymerase and transcription factors may bind. As used throughout the disclosure, the term “operably linked” refers to the expression of a gene (i.e. a transgene) that is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. A promoter can be positioned 5’(upstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.

[0083] In preferred aspects, the promoter sequence used in the gene expression constructs herein may considered a “weak promoter”. As used herein the term “weak promoter” refers to a promoter that induces little or infrequent RNA polymerase binding, resulting in low levels of transcription and correspondingly low levels of the corresponding protein. Use of a weak- promoter has been shown to have surprisingly beneficial effects over conventional stronger promoters (such as a chicken p-actin hybrid (CBh) promoter) because it prevents overexpression and resulting toxicity of the target protein (e.g., laforin or Malin).

[0084] In various aspects, the promoter sequence can comprise a JeT promoter sequence, a JeT-1 promoter sequence, a UBC promoter sequence, or a phosphoglycerate kinase (PGK) promoter sequence. In various aspects, the promoter sequence can comprise a JeT promoter sequence or a JeT-1 promoter sequence. Suitable JeT promoters are described in US20020098547A1 which is incorporated herein by reference in its entirety.

[0085] In some aspects, the weak promoter sequence can comprise, consist essentially of, or consist of a JeT promoter sequence. A JeT promoter sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence put forth in SEQ ID NO: 15.

[0086] In some aspects, the weak promoter sequence can comprise, consist essentially of, or consist of a UBC promoter sequence. A UBC promoter sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence put forth in SEQ ID NO: 16.

[0087] In some aspects, the weak promoter sequence can comprise, consist essentially of, or consist of a UBC promoter sequence. A UBC promoter sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence put forth in SEQ ID NO: 17.

[0088] In various aspects, other elements that control gene expression (e.g., enhancers or repressors) may be included in the gene expression construct to control level of expression. In various aspects, the gene expression constructs can comprise nucleic acids encoding for one or more mRNA stabilizing elements (e.g., a polyA tail).(c) Vectors

[0089] The isolated nucleic acids described above, including the gene expression constructs, can include one or more vectors. The term “vector” as used herein can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in thevector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell. Recombinant expression vectors can include a nucleic acid of the present disclosure in a form suitable for expression of the nucleic acid in a host cell, can mean that the recombinant expression vectors include one or more regulatory elements, which can be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.

[0090] In some embodiments, any of the gene expression constructs herein (e.g., encoding laforin or malin polypeptides) can be delivered to a cell using an adeno-associated virus (AAV). AAVs are small viruses which integrate site-specifically into the host genome and can therefore deliver a transgene. Inverted terminal repeats (ITRs) are present flanking the AAV genome and / or the transgene of interest and serve as origins of replication. Also present in the AAV genome are rep and cap proteins which, when transcribed, form capsids which encapsulate the AAV genome for delivery into target cells. Surface receptors on these capsids which confer AAV serotype, which determines which target organs the capsids will primarily bind and thus what cells the AAV will most efficiently infect. There are twelve currently known human AAV serotypes. In some embodiments, any mammalian AAV serotypes can be used herein for delivering the encoding nucleic acids described herein. Adeno-associated viruses are among the most frequently used viruses for gene therapy for several reasons. First, AAVs do not provoke an immune response upon administration to mammals, including humans. Second, AAVs are effectively delivered to target cells, particularly when consideration is given to selecting the appropriate AAV serotype. Finally, AAVs have the ability to infect both dividing and non-dividing cells because the genome can persist in the host cell without integration. This trait makes them an ideal candidate for gene therapy.

[0091] In some embodiments, polynucleotides disclosed herein can be delivered to a cell using at least one AAV vector. An AAV vector typically comprises a protein-based capsid, and a nucleic acid encapsidated by the capsid. The nucleic acid may be, for example, a vector genome comprising a transgene flanked by inverted terminal repeats. The AAV “capsid” is a near-spherical protein shell that comprises individual “capsid proteins” or “subunits.” AAV capsids typically comprise about 60 capsid protein subunits, associated and arranged with T=1 icosahedral symmetry. When an AAV vector is described herein as comprising an AAV capsid protein, it will be understood that the AAV vector comprises a capsid, wherein the capsid comprises one or more AAV capsid proteins (i.e., subunits). Also described herein are “viral-like particles” or “virus-like particles,” which refers to a capsid thatdoes not comprise any vector genome or nucleic acid comprising a transgene. The virus vectors of the present disclosure can further be “targeted” virus vectors (e.g., having a directed tropism) and / or a “hybrid” parvovirus (i.e., in which the viral TRs and viral capsid are from different parvoviruses) as described in international patent publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619. The virus vectors of the present disclosure can further be duplexed parvovirus particles as described in international patent publication WO 01 / 92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, double stranded (duplex) genomes can be packaged into the virus capsids of the present disclosure. Further, the viral capsid or genomic elements can contain other modifications, including insertions, deletions and / or substitutions.

[0092] In some embodiments, the isolated nucleic acids encoding the laforin or malin polypeptides herein may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof.

[0093] Exemplary AAV vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the transgene. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and / or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129,130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147,148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119,120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137,138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 ± 10 base pairs. In some embodiments, the ITRs have a length of130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 ± 10 base pairs. In some embodiments, the ITRs have a length of 150 ± 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs.

[0094] In some embodiments, the AAV vector may comprise additional elements to facilitate packaging of the vector and expression of the laforin or malin polypeptide. In some embodiments, the AAV vector may comprise a polyA sequence. In some embodiments, the polyA sequence may be a bgHi-polyA sequence. In some embodiments, the AAV vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor. In some embodiments, a regulator element is a posttranscriptional regulatory element (e.g., WPRE-3 -Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element- 3)

[0095] In some embodiments, the AAV vector may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the transgene (e.g., Lafora protein) or another element encoded by the vector. In some embodiments, the one or more promoters are considered “weak promoters”. Exemplary promoters include JeT or JeT-1 promoters. Other exemplary weak promoters include UBC and PGK promoters, as described above.

[0096] In some embodiments, the AAV vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV vector may be optimized for expression in human cells. In some embodiments, the AAV vector may be optimized for expression in a bacculovirus expression system.

[0097] In some embodiments of the gene editing constructs of the disclosure, the construct comprises or consists of a promoter and a nucleic acid encoding the laforin or malin polypeptide described herein. In some embodiments, the construct comprises or consists of a JeT promoter and a nucleic acid encoding a laforin polypeptide or fragment thereof. In some embodiments, the construct comprises or consists of a JeT promoter and a nucleic acid encoding a malin polypeptide or fragment thereof. An exemplary promoter that may be used in the AAV vectors herein can comprise SEQ ID NO: 15.

[0098] In some embodiments, the construct comprising a promoter and a transgene further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a promoter and a transgene further comprises at least two ITR sequences from isolated or derived from an AAV of serotype 9 (AAV9). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a weak promoter (e.g., a JeT promoter), optionally, a sequence encoding a nuclearlocalization signal, a sequence encoding a Lafora protein (e.g., laforin or malin) and a second ITR. Suitable ITRs for various AAV serotypes are known in the art.

[0099] In various aspects, the isolated nucleic acid, gene expression construct and / or vector provided herein can comprise one or more regulatory regions for efficient viral packaging and expression. For example, in some aspects, the nucleic acid (vector) can comprise one or more of a Kozak sequence, a polyA sequence (e.g., a bovine growth hormone (BGH) polyA sequence), a regulatory sequence, or a self-cleaving peptide sequence (e.g., a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide).

[0100] In various aspects, the rAAV vector provided herein may comprise, consist essentially of, or consists of a nucleic acid at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence put forth in SEQ ID NO: 18 or 19.AA V viral vectors

[0101] A “viral vector” is defined as a recombinantly produced virus or viral particle that contains a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, e.g., Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0102] An “AAV virion” or “AAV viral particle” or “AAV viral vector” or “rAAV viral vector” or “AAV vector particle” or “AAV particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. Thus, production of an rAAV viral vector necessarily includes production of an rAAV vector, as such a vector is contained within an rAAV vector.

[0103] As used herein, the term “viral capsid” or “capsid” refers to the proteinaceous shell or coat of a viral particle. Capsids function to encapsidate, protect, transport, and release into the host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of protein (“capsid proteins”). As used herein, the term “encapsidated” means enclosed within a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1 , VP2, and VP3. The mixture of VP1 , VP2 and VP3 contains 60 monomers that are arranged in a T =1 icosahedral symmetry in a ratio of 1 :1 :10 (VP1 :VP2:VP3) or 1 :1 :20 (VP1 :VP2:VP3) as described in Sonntag F et al., (June 2010). “A viral assembly factor promotes AAV2 capsid formation in the nucleolus”. Proceedings of theNational Academy of Sciences of the United States of America. 107 (22): 10220-5, and Rabinowitz JE, Samulski RJ (December 2000). “Building a better vector: the manipulation of AAV virions”. Virology. 278 (2): 301-8, each of which is incorporated herein by reference in its entirety.

[0104] The present disclosure provides an rAAV viral vector comprising: a) any of the rAAV vectors described herein, or complement thereof; and b) an AAV capsid protein.

[0105] The present disclosure provides an rAAV viral vector comprising: a) any of the rAAV vectors described herein; and b) an AAV capsid protein.

[0106] An AAV capsid protein can be any AAV capsid protein known in the art. An AAV capsid protein can be an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein or an AAVrh.10 capsid protein.

[0107] In some aspects, the AAV provides expression of protein (for e.g., Lafora) for a period of time in targeted tissues such as, but not limited to, nervous system tissues. Expression may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days,2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months,3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. Expression may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years or 5-10 years.

[0108] In some embodiments, AAV vectors herein may be packaged into virus particles which can be used to deliver the gene (for e.g., EPM2A or EPM2B) for transgene-encoding sequence expression in target cells. In some embodiments, AAV vectors disclosed herein can be packaged into particles by transient transfection, use of producer cell lines, combining viral features into Ad-AAV hybrids, use of herpesvirus systems, or production in insect cells using baculoviruses.

[0109] A method of generating a packaging cell for use herein can involve creating a cell line that stably expresses all of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing, addition of synthetic linkers containing restriction endonuclease cleavage sites, or by direct, blunt-end ligation. The packaging cell line is then infected with a helper virus, such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Examples of suitable methods herein employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and / or rep and cap genes into packaging cells.III. Pharmaceutical Formulations and Treatment Regimens

[0110] The nucleic acids, gene expression constructs and vectors disclosed herein for use according to the methods herein described may be provided per se or as part of a pharmaceutical composition, where the compounds can be mixed with suitable carriers or excipients.Pharmaceutically acceptable carriers and excipients

[0111] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0112] In various embodiments, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti- oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, and combinations thereof. Herein the term “excipient” refers to an inertsubstance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0113] In various embodiments, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In other embodiments, any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.

[0114] In various embodiments, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water- soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water- insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of polymers as suspending agent(s) by total weight of the composition.

[0115] In various embodiments, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of the composition may be increased by the addition of one or more gelling or thickening agents. In other aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In still other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In yet other aspects, suitable thickening agents can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose,microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVIWMA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or combinations thereof. In some embodiments, suitable thickening agent may be carboxymethylcellulose.

[0116] In various embodiments, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more agents by total weight of the composition. In other aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and / or other property of the muscarinic antagonist composition of the present disclosure. In s aspects, additives will be biocompatible, and will not be harsh, abrasive, or allergenic.

[0117] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition.

[0118] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In other aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition.

[0119] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition.

[0120] In other embodiments, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In another aspect, compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In other aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some embodiments, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In other embodiments, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater thanabout 7, greater than about 6.5, or greater than about 6. In a preferred embodiment, compositions disclosed herein may have a pH greater than about 6.8.

[0121] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition.

[0122] In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In other aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0123] In various embodiments, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition.

[0124] In other embodiments, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate,sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In preferred embodiments, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In other aspects, a composition herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, a pharmaceutical composition described herein has an osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In still other aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition.Dosage formulations

[0125] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially trans-nasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as, intravenous, intraperitoneal, intranasal injections, intraocular (e.g., via eye drops) or topical (e.g., creams or ointments). .

[0126] One may administer the pharmaceutical composition in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to the human patient via at least two administration routes. In some examples, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and intra-parenchymal injection and intravenous injection.

[0127] Pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0128] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0129] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0130] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0131] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0132] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0133] Pharmaceutical compositions suitable for use in context of the present disclosure include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients (i.e., a compound disclosed herein) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., a pain disorder, apsychiatric disorder, or other neurological / cognitive disorder brought about by disrupted synaptic activity) or prolong the survival of the subject being treated.

[0134] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0135] For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0136] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

[0137] Dosage amount and interval may be adjusted individually to brain or blood levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0138] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Effective doses may be extrapolated from dose- responsive curves derived from in vitro or in vivo test systems.IV. Methods of Treatment

[0139] Provided herein are methods for alleviating one or more symptoms and / or for treating a disease or a condition in a subject in need of the treatment (for e.g., Lafora Disease) by compositions disclosed herein, as well as a pharmaceutical compositionscomprising such. In some embodiments, a subject of the methods herein may be a human subject. In some embodiments, the subject may be a juvenile human subject, such a human subject under the age of 18 years. In some embodiments, the subject may be a human subject under the age of 5, 4, 3, 2 or 1 year. In some embodiments, the subject may be a subject that has not been previously exposed to wild-type AAV or a recombinant (rAAV) vector. In some embodiments, the subject may be a subject that has not been previously administered a rAAV vector. In some embodiments, the subject is a subject that has been previously administered a rAAV vector, e.g., a rAAV vector described herein. A subject that has been exposed or administered an AAV or rAAV can be identified using methods known in the art, e.g., by PCR detection of viral DNA or by measuring antibody titer to AAV or rAAV, either the capsid or the transgene. In some embodiments, the subject may be a subject that has not been administered an enzyme replacement therapy (e.g., by administration of the enzyme protein). A subject that has been administered an enzyme replacement therapy can be identified using methods known in the art, e.g., by measuring antibody titer to the enzyme. However, in some embodiments the subject has previously been treated with an enzyme replacement therapy. In some embodiments, the subject is a subject that has undergone one or more approaches to clear neutralizing antibodies (NAbs) (e.g., plasmapheresis, immunosuppression, enzymatic degradation). In some embodiments, a subject suitable of methods of use herein may not need to clear neutralizing antibodies (NAbs) before administration of any of the compositions (e.g., AAV vectors, AAV particles, AAV genomes) described herein.

[0140] In various embodiments, a subject in need thereof can be having, suspected of having, or at risk of being in pain, or having Lafora Disease. For example, the subject in need thereof can be having, suspected of having, or at risk of having neuropathic pain. For example, the subject in need thereof can be having, suspected of having, or at risk of having Lafora Disease.

[0141] A suitable subject includes a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non- limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, the subject may be a companion animal. Non- limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a specific embodiment, the animal is a laboratory animal. Non-limiting examples of a laboratory animalmay include rodents, canines, felines, and non-human primates. In certain embodiments, the animal is a rodent. Non-limiting examples of rodents may include mice, rats, guinea pigs, etc. In preferred embodiments, the subject is a human.

[0142] In certain embodiments, the present disclosure also provides for methods of introducing a nucleic acid molecule into a cell, comprising contacting the cell with a virus vector and / or composition disclosed herein. In some embodiments, methods herein can include delivering a nucleic acid molecule herein to a cell, comprising contacting the cell with a viral vector disclosed herein wherein the viral vector comprises the nucleic acid molecule of interest. In some embodiments of this method, a nucleic acid molecule of interest can encode laforin or malin. In some embodiments, nucleic acid molecules herein can be delivered to a specific tissue by administering AAV particles having one or more nucleic acid molecules herein to a tissue such as for e.g., CNS.

[0143] In some embodiments, methods of administering at least one AAV particle or at least one AAV vector having one or more nucleic acid molecules herein to a tissue substantially modulates expression of the at least one protein and / or gene (for e.g., EMP2A or EMP2B) as compared to baseline. As used herein, “baseline” refers to the expression of the at least one transgene (and the encoded product of the transgene) before the AAV particle or AAV vector having one or more nucleic acid molecules was administered. As used herein, “substantially modulates expression” refers to at least a 1-fold change in expression (e.g., increased expression, decreased expression) as compared to baseline. In some embodiments, methods of administering at least one AAV particle or AAV vector having one or more nucleic acid molecules herein to a tissue modulates expression of the at least one protein and / or gene as compared to baseline by at least about 2-fold to about 50-fold (e.g., about 2-, 4-, 6-, 8-, 10-, 20-, 30-, 40-, 50-fold). In some embodiments, methods of administering at least one AAV particle or AAV vector having one or more nucleic acid molecules herein to a tissue modulates expression of the at least one protein and / or gene as compared to baseline by at least about 2-fold to about 50-fold (e.g., about 2-, 4-, 6-, 8-, 10-, 20-, 30-, 40-, 50-fold) when the at least one AAV particle or AAV vector is delivered to a CNS tissue.

[0144] In any of the methods disclosed herein, an effective amount of the compositions (e.g., AAV vectors, AAV particles, AAV genomes) described herein can be given to a subject in need thereof to alleviate one or more symptoms associated with a disease and or condition. “An effective amount” as used herein refers to a dose of a disclosed composition which is sufficient to confer a therapeutic effect on a subject having a disease and or condition. In some embodiments, an effective amount can be an amount that reduces at least one symptom of disease or condition in the subject.

[0145] In various aspects, the methods herein comprise delivering a pharmaceutically effective dose of the AAV vectors to the subject. In some aspects, the AAV vectors may be delivered in a dose of about 1011to about 1018viral particles. In some aspects, the AAV vectors may be delivered in a dose of about 1011to about 1013viral particles. In some aspects, the AAV vectors may be delivered in a dose of about 1x1011viral particles. In some aspects, the AAV vectors may be delivered in a dose of about 5x1011viral particles.

[0146] In some embodiments, methods herein may include administering at least one AAV particle and / or at least one AAV vector to a subject at least once. In some embodiments, methods herein may include administering at least one AAV particle and / or at least one AAV vector to a subject more than once. In some embodiments, methods herein may include administering at least one AAV particle and / or at least one AAV vector to a subject between at least once to at least 10 times (e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 times). In some embodiments, methods herein may include administering at least one AAV particle and / or at least one AAV vector to a subject at least twice, at least 3 times, at least 4 times, or at least 5 times. In some embodiments, methods herein may include administering at least one AAV particle and / or at least one AAV vector to a subject once a day, once every other day, once a week, once every two weeks, once every three weeks, once a month, once every other month, once every three months, once every four months, once a year, or twice a year.V. Kits

[0147] The present disclosure provides kits for use in treating or alleviating Lafora Disease, as described herein. In some embodiments, the kit can include instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of a composition containing a vector (e.g., a vector encoding laforin or malin) disclosed herein and to treat, delay the onset, or alleviate a target disease as those described herein. The kit may further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease. In still other embodiments, the instructions can include a description of administering a compound to an individual at risk of the target disease.

[0148] The instructions relating to the use of a composition containing a vector for expressing laforin or malin, generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0149] The label or package insert indicates that the composition is used for treating, delaying the onset and / or alleviating the disease. Instructions may be provided for practicing any of the methods described herein.

[0150] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some examples, at least one active agent in the composition can be a compound (e.g., a CDK8 inhibitor) as described herein.

[0151] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above.EXAMPLES

[0152] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0153] The publications discussed throughout are provided solely fortheir disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0154] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0155] Unless specified, reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The examples illustrate various aspects of the disclosure and practice of the methods of the disclosure. The examples are not intended to provide an exhaustive description of the many different embodiments of the disclosure. Thus, although thedisclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.Example 1: Toxicity assessment of CBh driven EPM2A expression in wild type LD mouse model

[0156] LaFora disease (LD) is characterized by progressive myoclonus seizures, dementia, ataxia and visual manifestations and complete loss of autonomy. Data has shown that the mean age at disease onset is about 13.4 years, followed by death in a about 10 years after onset. Lafora Bodies (LB) are one of the pathological hallmark of LD. LBs usually form in neurola and glial cells which can drive neurodegeneration and neuroinflammation. FIGs. 1A- 1B show periodic acid Schiff diastase staining of LBs in hippocampus of Lafora Disease Mouse Model as compared to normal hippocampus tissue in a wildtype mouse.

[0157] Experiments were conducted to test whether gene replacement therapy with EPM2A and EPM2B be sufficient to reverse or prevent lafora body formation in the brain tissue and associated toxicity was tested in Lafora Disease wild type mouse model. A construct comprising human codon optimized EPM2A and a bGH Poly A Tail driven by the ubiquitous Chicken Beta hybrid intron promoter (CBh) was engineered in a self complementary AAV9 vector, to deliver EPM2A into the Central nervous system (CNS), as shown in FIG. 2A. Intracerebroventricular injection was conducted at a high dose of 4X1011vg in EPM2A wildtype mice at post natal day 10. As control, mice injected with PBS was used. The mice were sacrificed at 1 month old, to evaluate for the presence of lafora bodies. Following sacrifice, brain, spine, heart, muscle, liver, kidney, and spleen were harvested and several evaluations were further performed. Western blots were performed to analyze laforin expression, and Periodic acid Schiff diastase staining (PASD) and immunohistochemistry (IHC) were performed to determine laforin distribution (FIG. 2B). A shown in Table 1 , control wild type mice treated with PBS showed normal tissue histology. However, AAV9 treated wild type mice showed large intracytoplasmic vacuoles in liver and variably sized to oval vacuoles in neurons of spinal cord and dorsal root ganglion. Further, administration of AAV9-CBh- hEPM2A increased the likelihood for formation of Lafora body in tissue with proximity to high dose of the construct such as spinal column and dorsal root ganglion (FIG. 3 and FIG. 4), while tissue distal from the site of injection had decreased likelihood of formation of Lafora body such as in the cerebellum, dentate gyrus and skeletal muscle (FIG. 5A-5C).Table 1 : Toxicity assessment of PASD staining of AAV9-CBh-hEPM2A treated wild type and PBS treated wild type miceExample 2: Toxicity assessment of low expressing promoter JeT driven EPM2A expression wild type LD mouse model

[0158] Evaluation was undertaken to test toxicity of a construct comprising EPM2A driven by a low expressing promoter. A construct comprising human codon optimized EPM2A and a Poly A Tail driven by a JeT promoter was engineered in a self complementary AAV9 vector, as shown in FIG. 6A. Intracerebroventricular injection was conducted at a high dose of 5X1011vg in EPM2A wildtype mice at postnatal day 10. As control, mice injected with PBS was used. The mice were sacrificed at 1 month old, to evaluate for the presence of lafora bodies. Following sacrifice, brain, spine, heart, muscle, liver, kidney, and spleen were harvested and several evaluations were further performed. Western blots were performed to analyze laforin expression, and Periodic acid Schiff diastase staining (PASD), immunohistochemistry (IHC) and immunofluorescence were performed to determine laforin distribution (FIG. 6B). A shown in Table 2, control wild type mice treated with PBS showed normal tissue histology. However, AAV9-JeT-hEPM2A treated wild type mice showed large intracytoplasmic vacuoles in liver and variably sized to oval vacuoles in neurons of spinal column and dorsal root ganglion, comparable to high dose administration of AAV9-CBh- hEPM2A. Further, administration of AAV9-JeT-hEPM2A increased the likelihood for formation of Lafora body in tissue with proximity to high dose of the construct such as spinal column and dorsal root ganglion (FIG. 7A and FIG. 7B), while tissue distal from the site of injection had decreased likelihood of formation of Lafora body such as in the cerebellum, dentate gyrus and skeletal muscle (FIG. 8A-8C).Table 2: Toxicity assessment using PASD staining of AAV9-JeT-hEPM2A treated wild type and PBS treated wild type miceExample 3: Efficacy of gene therapy using scAAV9-JeT-hEPM2A in LD knockout mouse model

[0159] Further, experiments were conducted to determine the efficacy of JeT Driven EPM2A (laforin) in LD Knockout Mouse Model. Self complementary AAV9 vector comprising a construct with human codon optimized EPM2A and a Poly A Tail driven by a JeT promoter, as shown in FIG. 9A was injected intracerebroventricularly at a low dose of 1X1011and a high dose of 5X1011vg in EPM2AAmice at postnatal day 10. As control, EPM2A / ' mice injected with PBS was used. The mice were sacrificed at postnatal day 100, to evaluate for the presence of lafora bodies. Following sacrifice, brain, spine, heart, muscle, liver, kidney, and spleen were harvested and several evaluations were further performed. Western blots were performed to analyze laforin expression, and Periodic acid Schiff diastase staining (PASD), immunohistochemistry (IHC) and immunofluorescence were performed to determine laforin distribution (FIG. 9B). It was found that EPM2A_ / ‘ mice treated with high dose of AAV9- JeT-hEPM2A and control EPM2A / _mice showed enhanced distribution of Lafora body, while EPM2A / _mice treated with low dose of AAV9-JeT-hEPM2A showed reduced distribution of Lafora body in the cerebellum, hippocampus and cortex (FIG. 10A). Additionally, insoluble glycogen amount was evaluated in mice treated with low dose and high dose of AAV9-JeT- hEPM2A. When compared to the control mice, mice treated with either low or high dose of AAV9-JeT-hEPM2A did not have a statistically different insoluble glycogen amount (FIG. 10B-10C). However, the low dose treated mice showed an increasing trend in amount of insoluble glycogen. Quantification of PASD staining showed that when administered with the low dosage, there was no significant difference in the relative mask area of staining compared to control mice, in the cortex, cerebellum and hippocampus (FIG. 10D-10F). There was a significant difference in the relative mask of staining in cortex and hippocampus, but not in the cerebellum of mice treated with high dose, compared to the control mice (FIG. 10G-10I). These results suggested that administration of a low dose of AAV9-JeT-hEPM2A decreased likelihood of formation of Lafora body.Example 4: Toxicity assessment of JeT driven EPM2B (malin) expression in wild type LD mouse model

[0160] Experiments were further conducted to evaluate the in vivo toxicity of a construct comprising JeT driving the expression of EPM2B. A construct comprising human codon optimized EPM2B and a bGH Poly A Tail driven by JeT was engineered in a self complementary AAV9 vector, as shown in FIG. 11 A. Intracerebroventricular injection was conducted at a high dose of 5X1011vg in EPM2B wildtype mice at postnatal day 10. As control, wild type mice injected with PBS was used. The mice were sacrificed at 1 month old, to evaluate for the presence of lafora bodies. Following sacrifice, brain, spine, heart, muscle, liver, kidney, and spleen were harvested and several evaluations were further performed. Western blots were performed to analyze laforin expression, and Periodic acid Schiff diastase staining (PASD), immunohistochemistry (IHC) and Immunofluorescence were performed to determine malin distribution (FIG. 11 B). Histological analysis revealed that administration of AAV9-JeT-hEPM2B did not induce toxicity in tissue proximal to administration of high dose of virus, shown in Table 3 (FIG. 12).Table 3: Toxicity assessment using PASD staining of AAV9-JeT-hEPM2B treated wild type and PBS treated wild type miceExample 5: Efficacy of gene therapy using scAAV9-JeT-hEPM2B in LD knockout mouse model

[0161] Experiments were conducted to determine the efficacy of JeT Driven EPM2B in LD knockout Mouse Model. Self-complementary AAV9 vector comprising a construct with human codon optimized EPM2B and a Poly A Tail driven by a JeT promoter, as shown in FIG. 13A was injected intracerebroventricularly at a low dose of 1X1011and a high dose of 5X1011vg in LD knockout mice at post-natal day 10. As control, LD knockout mice injected with PBS was used. The mice were sacrificed at post-natal day 100, to evaluate for the presence of lafora bodies. Following sacrifice, brain, spine, heart, muscle, liver, kidney, and spleen were harvested and several evaluations were further performed. Western blots were performed to analyze laforin expression, and Periodic acid Schiff diastase staining (PASD), immunohistochemistry (IHC) and immunofluorescence were performed to determine malin distribution (FIG. 13B). Quantitative polymerase chain reaction (qPCR) was further used to determine relative expression of malin in wild type and knock out mice treated with low doseor high dose of AAV9-JeT-hEPM2B. High dose of AAV9-JeT-hEPM2B was found to significantly enhance malin expression in knockout mice (FIG.13C). Evaluation of amount of insoluble glycogen in mice treated with low dose and high dose of AAV9-JeT-hEPM2B revealed that treatment with high dose resulted in decrease of glycogen amount in the treated mice (FIG. 13D-13E). Quantification of PASD staining further showed that both low and high dose treatment in knockout mice resulted in statistically significant reduction in relative mask area in cerebellum and hippocampus (FIG. 13F-13K).Summary of Examples

[0162] Lafora Disease (LD) is a monogenic neurodegenerative disorder affecting adolescents and is associated with mutations in either EPM2A or EPM2B genes which encode for laforin and malin respectively. In the absence of either protein, there is an excessive accumulation of poorly branched glycogen which become aggregated and are called lafora bodies (LB). These lafora bodies are formed in neurons and glial cells and drive neurodegeneration and neuroinflammation. Following the recent success of gene therapy for treating spinal muscular atrophy (SMA), the objective of these experiments were to determine the feasibility of gene replacement therapy as a treatment strategy for Lafora Disease. It was hypothesized that the replacement of either EPM2A or EPM2B genes in their respective knockout mouse model would reduce or alleviate lafora body formation.

[0163] Recent studies have shown that malin, an E3 ubiquitin ligase, cannot localize on glycogen molecules and ubiquitinate glycogen metabolism enzymes in the absence of laforin. Hence, it was decided to begin this project by analyzing the feasibility of laforin as a target for gene replacement therapy. To achieve this, an AAV9 vector driven by the ubiquitous Chicken Beta hybrid intron promoter was designed to deliver EPM2A into the Central nervous system (CNS) using intracerebroventricular injection in EPM2A wildtype and knockout mouse model. Mouse Models were injected at post-natal day 1 and sacrificed at 3 months old, an age when lafora bodies are already present. Following sacrifice, western blots were used to analyze laforin expression, and Immunofluorescence imaging was used to determine laforin distribution in the brain. The reduction or alleviation of lafora bodies was the primary outcome of this study so enzymatic determination of glycogen and periodic acid schiff diastase staining (PASD) was used to measure lafora bodies.

[0164] Immunofluorescence imaging showed overexpression and widespread distribution of laforin across all brain regions, particularly in regions of the brains greatly affected by lafora bodies. Surprisingly, a dominant negative effect was observed when laforin was overexpressed in wild type mice; the brains of wild type mice form novel lafora bodies in regions of the brain which receive excessive amounts of laforin. Intrathecal and intramuscularinjections in wildtype mice also show that tissues proximal to high doses of overexpressed laforin protein are more prone to forming lafora bodies, the driver of Lafora Disease. It can be inferred from these results that laforin is a highly regulated protein and was probably needed in little amounts in any cell. In summary, it was hypothesized that laforin might be a target for gene replacement therapy when driven by a low expressing promoter. Further, both high and low doses of delivery of EPM2B driven by JeT promoter did not induce toxicity. In conclusion, gene replacement therapy for Lafora Disease would be evaluated by conducting toxicity and efficacy studies utilizing EPM2A and EPM2B genes as a target and delivered intrathecally using AAV serotype 9 driven under the low expressing promoter, JeT in Lafora Disease Mouse Model.*******

[0165] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present disclosure. Additionally, a number of well- known processes and elements have not been described in order to avoid unnecessarily obscuring the present disclosure. Accordingly, this description should not be taken as limiting the scope of the present disclosure.

[0166] Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.SEQUENCES

Claims

CLAIMS1. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid sequence encoding a laforin polypeptide or a malin polypeptide operably linked to a weak promoter sequence.

2. The rAAV vector of claim 1 , wherein the nucleic acid sequence encoding a laforin polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 3-6.

3. The rAAV vector of claim 2, wherein the nucleic acid sequence encoding a laforin polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 3.

4. The rAAV vector of claim 1 , wherein the nucleic acid sequence encoding a malin polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 9-12.

5. The rAAV vector of claim 4, wherein the nucleic acid sequence encoding a malin polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 9.

6. The rAAV vector of any one of claims 1 to 5, wherein the weak promoter sequence comprises a JeT promoter or a JeT-1 promoter.

7. The rAAV vector of claim 6, wherein the JeT promoter comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15.

8. The rAAV vector of claim 3, wherein the JeT promoter comprises SEQ ID NO: 15.

9. The rAAV vector of any one of claims 1 to 8, further comprising at least one other mRNA stabilizing element.

10. The rAAV vector of claim 9, wherein the mRNA stabilizing element comprises a polyA tail.11 . The rAAV vector of any one of claims 1 to 10, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18 or 19.

12. An rAAV viral vector comprising: a. an AAV capsid protein; andb. an rAAV vector of any one of claims 1 to 11 .

13. The rAAV viral vector of claim 12, wherein the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein or an AAVrh.10 capsid protein.

14. The rAAV viral vector of claim 13, wherein the AAV capsid protein is an AAV9 capsid protein.

15. A pharmaceutical composition comprising the rAAV viral vector of any one of claims 12 to 14 and at least one pharmaceutically appropriate carrier or excipient.

16. A method for treating a subject having a disease and / or disorder involving a mutation in a EPM2A gene and / or a NHLRC1 gene, the method comprising administering to the subject at least one therapeutically effective amount of the rAAV viral vector of any one of claims 12 to 14 or the pharmaceutical composition of claim 15.

17. The method of claim 16, wherein the disease and / or disorder is Lafora Disease.

18. The method of claim 16 or 17, wherein the rAAV viral vector or the pharmaceutical composition is administered to the subject at a dose ranging from about 1011to about 1018viral vector particles.

19. The method of claim 18, wherein the dose is about 1x 1011to about 1x1012viral particles.

20. The method of claim 19, wherein the dose is about 1x1011or about 5x1011viral particles.

21. The method of any one of claims 16 to 20, wherein the rAAV viral vector or the pharmaceutical composition is administered to the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intra- aurally, intra-ocularly, or peri-ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intranervally, intrapleurally, topically, intralymphatically, intracisternally or intranerve.

22. The method of claim 21 , wherein the rAAV viral vector or pharmaceutical composition is administered intrathecally.

23. The rAAV viral vector of any one of claims 12 to 14 orthe pharmaceutical composition of claim 15 for use in treating a disease and / or disorder involving an EPM2A gene and / or a NHLRC1 gene in a subject in need thereof24. The use of claim 23, wherein the disease and / or disorder is Lafora Disease.

25. The use of claim 23 or 24, wherein the rAAV viral vector or the pharmaceutical composition is for administration to the subject at a dose ranging from about 1011to about 1018viral vector particles.

26. The use of claim 25, wherein the dose is about 1x 1011to about 1x1012viral particles.

27. The use of claim 26, wherein the dose is about 1x1011or about 5x1011viral particles.

28. The use of any one of claims 23 to 27, wherein the rAAV viral vector or the pharmaceutical composition is for administration to the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intra- aurally, intra-ocularly, or peri-ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intranervally, intrapleurally, topically, intralymphatically, intracisternally or intranerve.

29. The use of claim 28, wherein the rAAV viral vector or pharmaceutical composition is for administration intrathecally.

30. The method or use of any one of claims 16 to 29, wherein the subject is human.

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