Materials and methods for treating EIF2B5 mutations and diseases resulting therefrom

Abstract

Gene therapy vectors, such as adeno-associated viruses (AAVs), designed for the treatment of mutations in the eukaryotic translation initiation factor 2B subunit epsilon (EIF2B5) gene are provided. The EIF2B5 gene provides instructions for making one of the five subunits of the eIF2B protein, specifically the epsilon subunit of the protein. Such mutations are associated with diseases or disorders such as leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-associated disease, oculodentodigital dysplasia, and / or giant axonal neuropathy. Such leukoencephalopathy or leukodystrophies include, but are not limited to, vanishing white matter (VWM). The disclosed gene therapy vector provides EIF2B5 cDNA to a subject in need, which results in the expression of wild-type or functional EIF2B5 protein. Also provided is a new promoter, designated gfa1405, designed to target astrocytes and neurons. Thus, compositions, nanoparticles, extracellular vesicles, exosomes, or vectors comprising the gfa1405 promoter, and methods of use thereof are also provided.

Description

[Technical field]

[0001] This application contains a sequence listing in computer readable form, which is incorporated by reference in its entirety as a separate part of this disclosure and is identified as follows: 56440_Seqlisting.XML, Size: 70,395 bytes, Created: February 16, 2023.

[0002] The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), designed for the treatment of mutations in the gene encoding the eukaryotic translation initiation factor 2B subunit epsilon 5 (EIF2B5) protein, which are associated with leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, giant axonal neuropathy, or vanishing white matter (VWM) disease. The disclosed gene therapy vectors provide EIF2B5 cDNA or wild-type EIF2B5 cDNA to a subject in need thereof, which results in expression of functional or wild-type EIF2B5 protein. The disclosure also provides novel promoters designed to target expression in astrocytes and / or neurons. [Background technology]

[0003] Vanishing white matter (VWM) is a devastating leukodystrophy (1-3). VWM is caused by autosomal recessive mutations in the five subunit genes of the eukaryotic initiation factor 2B (EIF2B) complex, which is required for the first step of protein translation. Currently, there is no cure for VWM disease.

[0004] Studies have shown that the majority of VWM mutations lie within the EIF2B5 gene, followed by the EIF2B2 gene (1, 4). Although the exact mechanism of pathology remains unclear, it is clear that glial cell dysfunction is the basis of pathophysiology. Established cell cultures from the brains of individuals with EIF2B5 VWM revealed that healthy oligodendrocytes were readily generated, but astrocytes were almost absent. Furthermore, astrocyte induction was significantly reduced, and the few astrocytes that were generated were not uniform. In vivo lesions also lacked glial fibrillary acidic protein (GFAP) positive (GFAP+) astrocytes. Furthermore, targeting eIF2B5 by RNA interference significantly impaired induction of GFAP+ astrocytes from normal human glial precursor cells (5). Currently, three mouse models of eIF2B5 VWM exist (6-8). Impaired maturation of white matter astrocytes was presymptomatic and corresponded with disease severity and progression in two models. In cocultures, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes (7). Collectively, these studies from human patients and mouse models demonstrate that astrocytes are central to VWM pathology and constitute a potential therapeutic target.

[0005] Advances in adeno-associated viral (AAV) vectors have resulted in safer and more efficient viral vehicles for delivering therapeutic transgenes with a single injection, and gene therapy is now the preferred therapeutic intervention for monogenic diseases. AAV serotype 9 has become the most widely used vector for neurological indications and has established a safety profile in the clinic. Intrathecal administration of AAV9 allows for the seeding of transgenes throughout the nervous system and is currently in clinical trials for the treatment of neuronal ceroid lipofuscinosis 3 (CLN3, NCT03770572), CLN6 (NCT02725580), spinal muscular atrophy (SMA, NCT03381729), and giant axonal neuropathy (GAN, NCT02362438). However, AAV9 has been shown to target primarily neurons and not effectively glia, which are clear therapeutic targets for VWM disease and other leukodystrophies, across species including mice (9), dogs (10), and non-human primates (11, 12). Nevertheless, it is now possible to achieve strong cell-specific targeting by driving transgene expression with a selectable promoter. In particular, GFAP promoter-driven transgene expression has been shown to be highly specific for astrocytes following AAV injection into newborn and adult mouse brains (13).

[0006] Patients with mutations in the eIF2B5 gene suffer from or are at risk of developing leukoencephalopathy with white matter loss, and new therapeutic options are urgently needed. Furthermore, mutations in eIF2B5 have been implicated in other diseases or disorders, such as macrocephalic leukoencephalopathy, leukodystrophies, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, and giant axonal neuropathy. The present disclosure provides replacement eIF2B5 nucleic acids and EIF2B2 gene replacement as a viable therapeutic strategy for treating mutations in the EIF2B2 gene and treating, preventing, or ameliorating leukodystrophies, leukoencephalopathy, and / or VWM diseases caused by such mutations. Thus, the present disclosure provides nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, recombinant AAV particles, and compositions comprising the eIF2B5 gene for treating mutations in eIF2B5. The disclosed products, methods, and uses provide a viable approach for robust and long-term expression of eIF2B5 protein, or functional eIF2B5, in cells of the human brain. Summary of the Invention

[0007] Provided herein are products, methods and uses for treating mutations in the gene encoding "eukaryotic translation initiation factor 2B subunit epsilon 5 (EIF2B5)" and in treating, ameliorating, delaying progression and / or preventing diseases caused by mutations in the EIF2B5 gene.

[0008] The present disclosure provides a nucleic acid comprising a polynucleotide comprising (a) one or more regulatory control elements and (b) an EIF2B5 cDNA sequence. In some embodiments, the EIF2B5 cDNA comprises a nucleotide sequence encoding EIF2B5 comprising (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:1, (b) the nucleotide sequence set forth in SEQ ID NO:1, or (c) the amino acid sequence set forth in SEQ ID NO:2.

[0009] In some aspects, the one or more regulatory control elements are a CAG promoter, a gfaABC1D promoter, a GFAP promoter, or a functional fragment of any of the CAG promoter, the gfaABC1D promoter, or the GFAP promoter. In some aspects, the regulatory control element comprises (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3, 4, 5, or 15, or (b) a nucleotide sequence set forth in SEQ ID NO: 3, 4, 5, or 15.

[0010] In some embodiments, the nucleic acid further comprises an SV40 intron and a post-transcriptional polyadenylation (polyA) sequence. In some embodiments, the nucleic acid comprises (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6-8 and 16, or (b) a nucleotide sequence set forth in any one of SEQ ID NOs: 6-8 and 16.

[0011] In some embodiments, the nucleic acid further comprises an inverted terminal repeat sequence. In some embodiments, the nucleic acid comprises (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-11 and 17, or (b) a nucleotide sequence set forth in any one of SEQ ID NOs: 9-11 and 17.

[0012] In some embodiments, the nucleic acid further comprises additional sequences of the AAV genome. In some embodiments, the nucleic acid comprises (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-14 and 18, or (b) a nucleotide sequence set forth in any one of SEQ ID NOs: 12-14 and 18.

[0013] In some aspects, a nucleic acid of the present disclosure comprises (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-14 and 18, or (b) a nucleotide sequence having at least 80% sequence identity to any one of (1) the 5'ITR, (2) the CAG, gfaABC(1), GFAP, or gfa1405 promoter, (3) the SV40 intron, (4) the eIF2B5 ORF, (5) polyA, (6) the F1 origin, (7) the kanamycin resistance gene, and (8) the pMB1 origin nucleotide sequences set forth in any one of SEQ ID NOs: 12-14 and 18, and Figures 12-14 and 21.

[0014] The present disclosure provides a nanoparticle, extracellular vesicle, exosome, or vector comprising any of the nucleic acids of the present disclosure, or any one or more combinations thereof. In some aspects, the vector is a viral vector. In some aspects, the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus. In some aspects, the viral vector is an AAV. In some aspects, the AAV comprises rep and cap genes. In some aspects, the AAV lacks rep and cap genes. In some aspects, the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV). In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAV2 / 1, AAV2 / 8, AAV2 / 9, or any of their derivatives. In some embodiments, the AAV is AAV9. In some embodiments, the ITR sequences present in the AAV are from AAV2. In some specific embodiments, the AAV is a ssAAV or ssrAAV.

[0015] The present disclosure provides rAAV particles comprising any of the AAVs of the present disclosure. The present disclosure provides compositions comprising any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles of the present disclosure and a pharma- ceutically acceptable carrier. In some aspects, the compositions are formulated for intrathecal, intraventricular, intracerebral, intravenous, intracisternal (or intracisternomagna (ICM)), or aerosol delivery.

[0016] The present disclosure provides a method of increasing expression of EIF2B5 gene or EIF2B5 protein in a cell, the method comprising contacting the cell with any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, or compositions of the present disclosure. In some embodiments, the cell is an astrocyte, a neuron, or a glial cell. In some embodiments, the cell is an astrocyte. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a human subject.

[0017] The present disclosure provides a method of treating a subject with a mutation in the EIF2BB5 gene, comprising administering to the subject an effective amount of any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, or compositions of the present disclosure. In some embodiments, the subject is a human subject. In some embodiments, the mutation in the EIF2BB5 gene causes the subject to suffer from or be at risk of suffering from leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodactyl dysplasia, giant axonal neuropathy, or vanishing white matter (VWM) disease. In some embodiments, the method further comprises a combination therapy. In some embodiments, the method further comprises administering to the subject any one or more of a corticosteroid, rituximab, and rapamycin. In some embodiments, the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, rAAV particle, or composition is administered intrathecally, intracerebroventricularly, intracerebrally, intravenously, intracisternally (or intracisternomagna (ICM)), or by aerosol delivery.

[0018] The present disclosure provides for the use of any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, or compositions of the present disclosure for the preparation of a medicament for increasing expression of the EIF2B5 gene or protein in a cell. In some embodiments, the cell is in a human subject. In some embodiments, the subject suffers from a mutation in EIF2B5, and in some embodiments, the mutation is associated with leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, giant axonal neuropathy, or vanishing white matter (VWM) disease. In some specific aspects, the leukoencephalopathy or leukodystrophy is vanishing white matter (VWM). In some aspects, the medicament is administered in combination with another medicament. In some aspects, the medicament is administered with any one or more of a corticosteroid, rituximab, and rapamycin. In some aspects, the medicament is formulated for intrathecal, intraventricular, intracerebral, intravenous, intracisternal (or intracisternomagna (ICM)), or aerosol delivery.

[0019] The present disclosure provides the use of any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, or compositions of the present disclosure in treating a subject with a mutated EIF2B5 gene. In some aspects, the cell is in a human subject. In some aspects, the subject suffers from a mutation in EIF2B5, a leukoencephalopathy, or a leukodystrophy. In some aspects, the leukoencephalopathy or leukodystrophy is vanishing white matter disease (VWM). In some aspects, the medicament is administered in combination with another medicament. In some aspects, the medicament is administered with any one or more of a corticosteroid, rituximab, and rapamycin. In some aspects, the medicament is formulated for intrathecal, intracerebroventricular, intracerebral, intravenous, or aerosol delivery.

[0020] The present disclosure provides compositions for treating a mutation in the EIF2B5 gene, leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, giant axonal neuropathy, or vanishing white matter (VWM) disease in a subject, the compositions comprising any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, or compositions of the present disclosure. In some embodiments, the subject is a human subject. In some embodiments, the leukoencephalopathy or leukodystrophy is vanishing white matter (VWM).

[0021] The present disclosure provides any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, compositions, methods, uses, or medicaments described herein. In some aspects, such nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, compositions, or medicaments are formulated for intrathecal (direct injection into the CSF), intravenous injection into the bloodstream, intracerebral injection, intraventricular injection, intracisternal (or intracisternomagna (ICM)), or aerosol delivery or administration.

[0022] The disclosure also provides a nucleic acid comprising a novel gfa1405 promoter comprising (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 15, or (b) the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid further comprises an inverted terminal repeat. Thus, in some embodiments, the nucleic acid comprises (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 17 or 18, or (b) the nucleotide sequence set forth in SEQ ID NO: 17 or 18.

[0023] The present disclosure provides nanoparticles, extracellular vesicles, exosomes, or vectors comprising such nucleic acids or any one or more combinations thereof. In some aspects, the vector is a viral vector. In some aspects, the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus. In some aspects, the viral vector is an AAV. In some aspects, the AAV lacks rep and cap genes. In some aspects, the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV). In some specific aspects, the AAV is a ssAAV or a ssrAAV. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAV2 / 1, AAV2 / 8, AAV2 / 9, or any of their derivatives. In some embodiments, the AAV is AAV9.

[0024] The present disclosure provides rAAV particles comprising any of the AAVs of the present disclosure. The present disclosure provides compositions comprising any one or more of a nucleic acid, a nanoparticle, an extracellular vesicle, an exosome, a vector, a viral vector, or an rAAV particle, and a pharma- ceutically acceptable carrier. In some aspects, the compositions are formulated for intrathecal, intraventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0025] The present disclosure provides a method of increasing gene or protein expression in a cell, the method comprising contacting the cell with any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles, or compositions of the present disclosure. In some embodiments, the cell is an astrocyte or a neuron. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a human subject.

[0026] The present disclosure provides a method of treating a subject comprising a mutation in a gene normally expressed in astrocytes or neurons of the subject, the method comprising administering to the subject an effective amount of any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles, or compositions of the present disclosure. In some embodiments, the subject is a human subject. In some embodiments, the mutation in the gene causes the subject to suffer from or be at risk for an astrocyte or neuron disorder or disease. In some embodiments, the disorder or disease is leukoencephalopathy or leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodactyl dysplasia, giant axonal neuropathy, or macrocephalic leukoencephalopathy. In some embodiments, the leukoencephalopathy or leukodystrophy is vanishing white matter disease (VWM). In some embodiments, the methods of the present disclosure further comprise administering to the subject any one or more of a corticosteroid, rituximab, and rapamycin. In some embodiments, the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, rAAV particle, or composition is administered intrathecally, intracerebroventricularly, intracerebrally, intravenously, intracisternally (or intracisternomagna (ICM)), or by aerosol delivery.

[0027] The present disclosure provides for the use of any one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles, or compositions of the present disclosure for the preparation of a medicament for increasing expression of a gene or protein in a cell. In some aspects, the cell is in a human subject.

[0028] The present disclosure provides the use of one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles, or compositions of the present disclosure for treating a subject with a mutated gene. In some embodiments, the subject is a human subject. In some embodiments, the subject suffers from a genetic mutation, or a disorder or disease that affects the central nervous system and / or the brain. In some embodiments, the subject suffers from a genetic mutation, or a disorder or disease that affects astrocytes or neurons of the brain. In some embodiments, the subject suffers from leukoencephalopathy or leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodactyl dysplasia, giant axonal neuropathy, or macrocephalic leukoencephalopathy. In some embodiments, the leukoencephalopathy or leukodystrophy is vanishing white matter disease (VWM). In some embodiments, the medicament is administered with any one or more of a corticosteroid, rituximab, and rapamycin. In some embodiments, the medicament is formulated for intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal (or intracisternomagna (ICM)), or aerosol delivery.

[0029] The present disclosure provides a composition for treating a genetic mutation or a disease or disorder in the central nervous system and / or brain of a subject, the composition comprising one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles or compositions of the present disclosure. In some embodiments, the subject is a human subject. In some embodiments, the subject suffers from a genetic mutation or a disorder or disease that affects the central nervous system and / or the brain. In some embodiments, the subject suffers from a genetic mutation or a disorder or disease that affects astrocytes or neurons of the brain. In some embodiments, the subject suffers from leukoencephalopathy or leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, giant axonal neuropathy, and / or macrocephalic leukoencephalopathy. In some embodiments, the leukoencephalopathy or leukodystrophy is vanishing white matter (VWM).

[0030] The disclosure provides one or more of a nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, rAAV particle, composition, or medicament, wherein the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, or rAAV particle, composition, or medicament is formulated for intrathecal injection into cerebrospinal fluid (CSF), intravenous injection into the bloodstream, intracerebral injection, intraventricular injection, intracisternal injection, or aerosol administration.

[0031] Other features and advantages of the present disclosure will become apparent from the following description of the drawings and detailed description, but it should be understood that the drawings, detailed description and examples, while indicating embodiments of the disclosed subject matter, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure may become apparent from said drawings, detailed description and examples.

[0032] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0033] [Figure 1] Four AAV constructs designed are shown. These constructs include promoters that target astrocytes: full-length GFAP and truncated version gfaABC(1)D, the novel gfaABCD1405 (or gfa1405) promoter, and the ubiquitous promoter (also called CAG) consisting of the CBA promoter and CMV enhancer. Other important components of the vector include the AAV9 capsid for efficient targeting of the CNS, the AAV2 inverted terminal repeats (ITRs) that create single-stranded constructs with greater packaging capacity, the SV40 intron, the human eIF2B5 coding sequence, and the posttranscriptional polyadenylation (polyA) sequence. [Diagram 2]In vitro expression of GFP in transfected HEK293T cells is shown for each of the three eGFP constructs (AAV9-CAG-GFP, AAV9-gfaABC(1)D-GFP, and AAV9-CBA-GFP vectors). Specifically, HEK293 cells were transfected by lipofectamine, and cells were harvested 72 hours post-transfection for eGFP mRNA expression analysis. Although HEK cells were not an ideal cell model since astrocytes are the target cells for these constructs, HEK293 cells allowed for rapid in vitro analysis to determine whether the plasmids expressed the genes of interest. 72 hours post-transfection, GFP-positive cells were detectable by microscopy (left panel). In addition, quantitative PCR of mRNA expression on cell pellets showed increased expression, approximately 3500-fold for CAG, 1200-fold for GFAP, and 900-fold for GfaABC(1)D, over non-transfected control cells (right panel). These results demonstrated the expression of transgenes driven by various promoters in cells in vitro. [Diagram 3] Shown are fibroblast-derived astrocytes stained with glial fibrillary acidic protein (GFAP), a marker for astrocytes. Astrocytes were established as a relevant in vitro system for testing the gene therapy constructs of the present disclosure. Astrocytes were derived from fibroblasts cultured from skin biopsies of patients with confirmed eIF2B5 VMD disease. This provides an in vitro system of VWM astrocytes to study disease mechanisms and test therapeutics. [Figure 4] Shown are the results of an experiment with wild-type mice injected with AAV9-CAG-GFP at postnatal day 1 (PND1) and sacrificed 28 days after injection. Sagittal sections of the brain show the distribution of GFP (green). DAPI (blue) indicates nuclei. Insets 1-6 show higher magnifications of relevant voxels in sagittal sections. [Diagram 5]1 shows the results of an experiment with wild-type mice injected with AAV9-CAG-GFP at PND1 and sacrificed 28 days after injection. Sagittal sections of the brain show the distribution of GFP (green). DAPI (blue) indicates nuclei. Inset shows higher magnification of GFP-positive glial and neuronal cells. [Figure 6] Shown are the results of an experiment with four wild-type mice injected with AAV9-CAG-GFP at PND1 and sacrificed 28 days after injection. Sagittal sections of each mouse brain show the distribution of GFP (green). Injection of newborn wild-type mice with AAV9-gfaABC(1)D-GFP resulted in greater distribution in the white matter and more widespread distribution throughout the central neuraxis compared to AAV9-CBA-GFP. Particularly notable is the increase in GFP expression in caudal brain regions, including the white matter regions of the cerebellum in box 2. Furthermore, expression was primarily within astrocytes, as shown by the morphology in box 4. [Figure 7] Shown are the results of an experiment with wild-type mice injected with AAV9-gfaABC(1)D-GFP at PND1 and sacrificed 28 days after injection. Sagittal sections of the brain show the distribution of GFP (green). Insets 1-3 show higher magnifications of relevant voxels in sagittal sections. Inset 4 shows a higher magnification of inset 3 to demonstrate astrocyte morphology. [Figure 8] Shown are the results of an experiment with three wild-type mice injected with AAV9-gfaABC(1)D-GFP at PND1 and sacrificed 28 days post-injection. Sagittal sections of the brain show the distribution of GFP (green). [Figure 9] Shown are the results of an experiment with wild-type mice injected with AAV9-GFAP-GFP at PND1 and sacrificed 28 days after injection. Sagittal sections of the brain show the distribution of GFP (green). The inset shows a magnification of the cerebellar white matter indicated by the red arrow. Thus far, injection of newborn wild-type mice with AAV9-GFAP-GFP has resulted in a more widespread distribution and greater expression compared to both the AAV9-gfaABC(1)D-GFP and AAV9-CBA-GFP vectors. Expression remains retained for astrocytes. [Figure 10]Results from an experiment with four wild-type mice injected with AAV9-GFAP-GFP at PND1 and sacrificed 28 days after injection are shown. Sagittal sections of the brain show the distribution of GFP (green). Similar biodistribution patterns were seen between the injected mice. [Figure 11] Results of an experiment using wild-type mice injected with AAV9-GFAP-GFP at PND1 and sacrificed 28 days after injection are shown. Co-labeling shows that GFP (green) expression co-localizes with the astrocyte marker (GFAP, purple) but not with the neuronal marker (NeuN, red). GFP expression in astrocytes was confirmed by co-labeling with antibodies specific for astrocytes (GFAP, purple) and neurons (NeuN, red). GFP expression after treatment with AAV9-GFAP-GFP (shown) and AAV9-gfaABC(1)D-GFP (not shown) was clearly present in astrocytes. Thus, it was confirmed that expression occurs primarily in the target cells, the astrocytes. [Figure 12-1] 1 provides the annotated sequence of AAV.CAG.eIF2B5 (SEQ ID NO:12). [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 13-1] 1 provides the annotated sequence of AAV.gfaABC1D.eIF2B5 (SEQ ID NO:13). [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 14-1] 1 provides the annotated sequence of AAV.GFAP.eIF2B5 (SEQ ID NO:14). [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15] The sequence of the novel gfa1405 promoter (also called the gfaABCD1405 promoter) is provided (SEQ ID NO:15). [Figure 16-1] The annotated sequence of the complete ITR-gfa1405-eIF2B5-polyA-ITR sequence is provided (SEQ ID NO: 17). [Figure 16-2]Same as above. [Figure 17] 1 shows the design and components of the novel gfa1405 promoter. [Figure 18] 1 shows the design and components of the complete ITR-gfa1405-eIF2B5-polyA-ITR sequence. [Figure 19] 1 shows the plasmid map of pAAV.gfa1405.EIF2B5 (7086 bp of SEQ ID NO:18). [Figure 20A] We show that GFP expression in HEK293T cells can be achieved by both ubiquitous and astrocyte-specific promoters, including the novel gfa1405 promoter. Figure 20A shows GFP expression in HEK293T cells 72 hours after transfection with the four various promoters discussed herein, namely, CAG, GFAP, gfaABC(1)D, and gfaABCD1405 (or gfa1405). [Figure 20B] Figure 20B shows the quantification of GFP expression in percent of CAG expression after triplicate Western blots of all constructs compared to the expression of the housekeeping protein GAPDH. GFP protein expression was higher in the gfaABCD1405 (gfa1405) construct than in the GFAP and gfaABC(1)D constructs, but not as high as that of the ubiquitous promoter CAG construct. [Figure 21-1] The annotated sequence of AAV.gfa1405.eIF2B5 (SEQ ID NO:18) (7086 bp) is provided. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 22] FIG. 1 shows how gene therapy constructs were designed to assess cell-specific expression with different AAV reporter constructs. [Figure 23]Figure 1 shows GFP expression in mouse brain 4 weeks (top row) and 8 weeks (bottom row) after intracerebroventricular (ICV) delivery of AAV9-CAG-GFP on PND1. Green staining represents GFP and blue represents DAPI. This data reinforces the data shown in Figures 4-6. [Figure 24] Figure 1 shows GFP expression in mouse brain 4 weeks (top) and 8 weeks (bottom) after intracerebroventricular (ICV) delivery of AAV9-CAG-GFP on PND1. Green staining represents GFP and blue represents DAPI. This data reinforces the data shown in Figures 9-11. [Diagram 25] GFP expression in mouse brain 4 weeks (top) and 8 weeks (bottom) after intracerebroventricular (ICV) delivery of AAV9-gfABC(1)D-GFP on PND1. Green staining represents GFP and blue represents DAPI. This data reinforces the data shown in Figures 7 and 8. [Figure 26] IF quantification of GFP constructs, i.e., GFP-positive area percentage in whole mouse brain and GFP intensity in GFP-positive areas, 4 and 8 weeks after intracerebroventricular (ICV) delivery of AAV9-GFAP-GFP, AAV9-gfABC(1)D-GFP, and AAV9-CAG-GFP at PND1. [Figure 27] Figure 1 shows GFP expression in whole brain 4 weeks after ICV delivery of AAV9-GFAP-GFP, AAV9-gfABC(1)D-GFP, and AAV9-CAG-GFP. The bottom panel shows Western blot analysis of GFP protein relative to the housekeeping gene GAPDH at 4 and 8 weeks post-treatment. [Figure 28] We show colocalization of GFP with astrocytic (GFAP) and neuronal (NeuN) markers, demonstrating that expression using the GFAP and gfaABC(1)D promoters was restricted to astrocytes, whereas CAG conferred GFP expression in neurons and astrocytes. [Figure 29]GFP expression in mouse brain 4 weeks after intracerebroventricular (ICV) delivery of AAV9-gfa1405-GFP on PND1. [Diagram 30] FIG. 1 illustrates various methods used to evaluate the efficacy of cell-specific gene therapy constructs containing the EIF2B5 transgene in treating a model of VWM disease (VWM mice). [Diagram 31] Therapeutic efficacy in treating VWM mice. Rotarod data in two different mouse models of EIF2B5 (Eif2b5I98M at day 50, and Eif2b5R191H at 6 months). Both models show normalized latency (Eif2b5I98M) or RPM to fall (Eif2b5R191H) after treatment with AAV9-gfABC(1)D-GFP. Lesser improvements were seen with the AAV9-GFAP-GFP and AAV9-CAG-GFP promoters. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present disclosure provides eukaryotic translation initiation factor 2B subunit epsilon 5 (EIF2B5) gene replacement as a viable therapeutic strategy to treat mutations in the gene encoding EIF2B5 and thereby treat, ameliorate, slow progression, or prevent diseases or disorders resulting from such mutations, including, but not limited to, leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, giant axonal neuropathy, or vanishing white matter (VWM) disease. The disclosed products, methods and uses provide a feasible approach for robust and long-term expression of the EIF2B5 gene in human neurons and glial cells in the treatment of leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophies, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2 related diseases, oculodentodigital dysplasia, giant axonal neuropathy, or vanishing white matter (VWM) diseases.

[0035] Leukodystrophies are a heterogeneous group of disorders with highly diverse clinical manifestations and pathological mechanisms. They are usually loosely grouped based on the initial findings of white matter abnormalities in the central nervous system (CNS), historically based on gross pathology, and now often based on neuroimaging. However, no formal definition or classification exists for this group of disorders. The term leukodystrophies technically refers to disorders involving the weakening (dystrophy) of the white matter (leuko) of the brain, and is traditionally reserved for genetic disorders, but there is a lack of consensus on how the term should be applied.

[0036] Further complicating the definition of leukodystrophies, a related but distinct term "leukoencephalopathy" exists in the literature. This term has been characteristically applied to disorders seen in the context of toxic, acquired vascular or infectious insults, and genetic disorders. In addition, different terms such as hypomyelination, demyelination, and dysmyelination are used, contributing to confusion.

[0037] VWM is a type of leukodystrophy caused by a mutation in one of the five genes EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 that code for the five subunits of a protein called eukaryotic initiation factor 2B (eIF2B). This protein is necessary to regulate the production of all other proteins in the body, and the rate of protein production, especially the decrease in protein synthesis during stress conditions such as fever and infection. It is very important that no one can live if any of these genes are fully non-functional or absent. VWM is caused by small changes in these genes that reduce the function of eIF2B, and certain cells in the brain are particularly vulnerable to the loss of this function. The decrease in function is particularly problematic during episodes of fever, infection, or head trauma, with accelerated deterioration following such episodes.

[0038] VWM is inherited in an autosomal recessive manner. Other clinical names for VWM include, but are not limited to, childhood ataxia with diffuse CNS hypomyelination (CACH), vanishing white matter leukodystrophy, Cree leukoencephalopathy, leukodystrophy with ovarian failure, ovarian leukodystrophy, and eIF2B-related disorder. VWM is a leukodystrophy and the most commonly occurring neurodegenerative white matter disorder in children. Clinically, it presents with ataxia, spasticity, neurological decline, and seizures that lead to early death. Currently, there is no treatment for VWM. VWM is caused by autosomal recessive loss-of-function mutations in a subunit of eukaryotic initiation factor 2B (EIF2B), with pathological variants in EIF2B5 being the most common. Due to the monogenic nature of VWM, it is a good candidate for adeno-associated virus (AAV)-mediated gene replacement therapy. VWM pathology suggests that astrocytes are important targets for therapy, as their differentiation, morphology, and function are compromised, mediating disease progression. Thus, the present disclosure provides gene replacement constructs to compare astrocyte-specific or ubiquitous expression of transgenes.

[0039] The EIF2B1 gene provides the instructions for making one of the five parts of a protein called eIF2B, specifically the alpha subunit of this protein. The eIF2B protein helps regulate overall protein production (synthesis) in cells by interacting with another protein, eIF2. The eIF2 protein is called an initiation factor because it is involved in starting (initiating) protein synthesis. Under some conditions, eIF2B increases protein synthesis by helping to recycle a molecule called GTP, which carries energy to initiation factors. Under other conditions, it slows down protein synthesis by binding tightly to initiation factors, which converts the eIF2B protein into an inactive form and prevents the recycling of GTP. Proper regulation of protein synthesis is essential to ensure that the correct levels of protein are available for cells to deal with changing conditions. For example, cells must synthesize proteins much faster when they are proliferating than when they are at rest.

[0040] The EIF2B1 gene (HGNC:3257 NCBI Entrez Genes:1967 Ensemble:ENSG00000111361 OMIM®:606686 UniProtKB / Swiss-Prot:Q14232) encodes one of the five subunits of eukaryotic translation initiation factor 2B (EIF2B), a GTP exchange factor for eukaryotic initiation factor 2, and an essential regulator for protein synthesis. Mutations in this gene and in genes encoding other EIF2B subunits are associated with leukoencephalopathy with white matter loss.

[0041] The present disclosure is focused on providing an EIF2B replacement gene or "transgene" to express normal or functionally active EIF2B protein. To accomplish this, specifically designed EIF2B replacement genes or "transgenes" are provided.

[0042] In some aspects, the nucleic acid of the EIF2B replacement gene comprises the nucleotide sequence set forth in SEQ ID NO: 1, or a codon optimized variant of the nucleotide sequence set forth in SEQ ID NO: 1. In various aspects, the nucleic acid is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 1. In some aspects, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence set forth in SEQ ID NO: 1.

[0043] In some embodiments, the polypeptide is an EIF2B polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In various embodiments, the polypeptide is an isoform or variant of an EIF2B polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0044] In some embodiments, the polynucleotide sequence of the transgene is operably linked to transcriptional control elements (including, but not limited to, promoters, enhancers and / or polyadenylation signal sequences) that are functional in the target cell. In some embodiments, the promoter is selected to target astrocytes. In some embodiments, the promoter is a CAG promoter, a GFAP promoter, a gfaABC1D promoter, or a gfa1405 (also called gfaABCD1405) promoter.

[0045] The gfa1405 or gfaABCD1405 promoter is a novel promoter described herein for the first time (more details included in Example 5) and comprises the nucleotide sequence of SEQ ID NO: 15. The gfa1405 promoter is designed to specifically target astrocytes and neurons and can be used to express any gene desired to be expressed in astrocytes or neurons. Thus, in some embodiments, the gfa1405 promoter is designed and used to express eIF2B5. Figure 19 provides a schematic diagram of the plasmid pAAV.gfa1405.eIF2B5, which comprises the gfa1405 promoter together with the eIF2B5 transgene sequence in an AAV vector, i.e., 7086 bp comprising SEQ ID NO: 15 (SEQ ID NO: 18). Figure 21 provides a detailed description of the 7086 bp sequence of SEQ ID NO: 18.

[0046] The CAG promoter is a ubiquitous promoter that targets neurons and astrocytes. The gfaABC1D and GFAP promoters drive transgene expression primarily in astrocytes.

[0047] Thus, in some embodiments, the promoter is a CAG promoter, a gfaABC1D promoter, a GFAP promoter, or a novel gfa1405 promoter. Although the CAG promoter is generally referred to simply as a "CAG promoter," it is not a promoter in the strict sense because it includes both a promoter and an enhancer. The CAG promoter of SEQ ID NO: 3 includes a CMV enhancer (nucleotides 1 to 306 of SEQ ID NO: 3) and a CBA promoter (nucleotides 307 to 581 of SEQ ID NO: 3).

[0048] In some aspects, the CAG promoter comprises the nucleotide sequence set forth in SEQ ID NO:3. In some aspects, the gfaABC1D promoter comprises the nucleotide sequence set forth in SEQ ID NO:4. In some aspects, the GFAP promoter comprises the nucleotide sequence set forth in SEQ ID NO:5. In some aspects, the gfa1405 promoter comprises the nucleotide sequence set forth in SEQ ID NO:15. Thus, in some exemplary aspects, the nucleic acid comprises a promoter comprising the nucleotide sequence set forth in any one of SEQ ID NOs:3-5 and 15. In various aspects, the nucleic acid is an isoform or variant of a nucleic acid comprising the nucleotide sequence set forth in any one of SEQ ID NOs:3-5. In some embodiments, an isoform or variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence set forth in any one of SEQ ID NOs:3-5 and 15.

[0049] [Table 1-1]

[0050] [Table 1-2]

[0051] [Table 1-3]

[0052] [Table 1-4]

[0053] [Table 1-5]

[0054] In some embodiments, the nucleic acids of the disclosure comprise a promoter, an SV40 intron, an eIF2B5 open reading frame, and a polyA tail (Table 2). The sequences in Table 2 are the nucleotide sequences of the eIF2B5 transgene sequences without the 5' and 3' ITR sequences, since the transgene sequences are used in various embodiments in self-complementary and / or single stranded AAV viral vectors.

[0055] In some aspects, a nucleic acid of the present disclosure comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 6-8 and 16 (Table 2). In various aspects, the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 6-8 and 16. In some aspects, the variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 6-8 and 16.

[0056] [Table 2-1]

[0057] [Table 2-2]

[0058] [Table 2-3]

[0059] [Table 2-4]

[0060] [Table 2-5]

[0061] [Table 2-6]

[0062] In some embodiments, the nucleic acids of the disclosure comprise a 5'ITR, a promoter, an SV40 intron, an eIF2B5 open reading frame, a polyA tail, and a 3'ITR (Table 3). In some embodiments, the nucleic acids of the disclosure comprise a nucleotide sequence that comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to a nucleotide sequence comprising the 5'ITR, a promoter, an SV40 intron, an eIF2B5 open reading frame, a polyA tail, and a 3'ITR set forth in Table 3.

[0063] In some aspects, the nucleic acid of the disclosure comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 9-11 and 17 (Table 3). The sequences in Table 3 are nucleotide sequences of eIF2B5 transgene sequences including 5' and 3' ITR sequences. In various aspects, the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 9-11 and 17. In some aspects, the variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 9-11 and 17.

[0064] [Table 3-1]

[0065]

Table 3-2

[0066]

Table 3-3

[0067]

Table 3-4

[0068]

Table 3-5

[0069]

Table 3-6

[0070]

Table 3-7

[0071] In some embodiments, the nucleic acid of the present disclosure comprises an AAV9 vector comprising a 5'ITR, a promoter, an SV40 intron, an eIF2B5 open reading frame, a polyA tail, and a 3'ITR, as depicted in Figures 12-14, 16, and 19. In some specific embodiments, the AAV is a ssAAV or a ssrAAV. In some embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence comprising, from 5' to 3', a promoter, an SV40 intron, an eIF2B5 open reading frame, and a polyA tail, as depicted in any one of Figures 12-14, and 19. In some embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence comprising, from 5' to 3', a 5'ITR, a promoter, an SV40 intron, an eIF2B5 open reading frame, a polyA tail, and a 3'ITR, as depicted in any one of Figures 12-14, 16, and 19. In some embodiments, the nucleic acid may comprise additional elements, or some elements may be modified or eliminated. In some embodiments, the nucleotide sequence of any of the 5'ITR, promoter, SV40 intron, eIF2B5 open reading frame, polyA tail, or 3'ITR shown in Figures 12-14, 16, and 19 may be a variant that comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence of any of the 5'ITR, promoter, SV40 intron, eIF2B5 open reading frame, polyA tail, or 3'ITR sequences shown in Figures 12-14, 16, and 19.

[0072] In some embodiments, the nucleic acids of the present disclosure comprise a nucleotide sequence set forth in any one of SEQ ID NOs: 12-14 and 18 (i.e., see Figures 12-14, 16, and 19). In various embodiments, the nucleic acid is a variant of a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 12-14 and 18. In some embodiments, the variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 12-14 and 18.

[0073] [Table 4]

[0074] A sequence index table (Table 5) is provided below for referencing the sequences provided in the sequence listing.

[0075] [Table 5]

[0076] In some embodiments, the present disclosure includes nanoparticles, extracellular vesicles, exosomes, or vectors comprising any of the nucleic acids of the present disclosure, or any one or more combinations thereof, to provide EIF2B5 gene replacement. In some embodiments, one or more copies of these sequences are combined into a single nanoparticle, extracellular vesicle, exosome, or vector.

[0077] Thus, the present disclosure includes vectors comprising a nucleic acid of the present disclosure or a combination of nucleic acids of the present disclosure.Embodiments of the present disclosure utilize vectors (e.g., viral vectors, such as adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine associated virus, alphavirus, poxvirus, herpes virus, herpes simplex virus, poliovirus, sindbis virus, vaccinia virus, or synthetic viruses, such as chimeric viruses, mosaic viruses, or pseudotyped viruses, and / or viruses that include foreign proteins, synthetic polymers, nanoparticles, or small molecules) to deliver the nucleic acids disclosed herein.

[0078] The present disclosure provides a recombinant (r)AAV vector comprising a nucleic acid comprising a polynucleotide encoding an EIF2B5 protein for use in treating a subject comprising a mutation in the EIF2B5 gene. In some specific aspects, the AAV is a ssAAV or ssrAAV. In some aspects, the nucleic acid of the present disclosure comprises an AAV vector comprising a nucleotide sequence set forth in SEQ ID NO: 1 (Table 1). In some aspects, the nucleic acid of the present disclosure comprises an AAV vector comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 6-8 and 16 (Table 2). In some aspects, the nucleic acid of the present disclosure comprises an AAV vector comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 9-11 and 17 (Table 3). In various aspects, the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 6-11, 16, and 17. In some embodiments, the variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity to the nucleotide sequence set forth in any one of SEQ ID NOs:1, 6-11, 16, and 17.

[0079] As a viral genome, AAV is unique in its safety profile; once transduced into its carrier cells, it remains stably expressed as episomal DNA and only rarely integrates into the host genome.

[0080] Thus, in some aspects, the present disclosure utilizes AAV to deliver EIF2B5 transgene, such as DNA encoding EIF2B5 protein.As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus.As used herein, "AAV vector" refers to a vector that comprises one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs).

[0081] Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products. AAV is a single-stranded replication-defective DNA parvovirus that grows only in cells in which certain functions are provided by a coinfecting helper virus. The genome of AAV is approximately 4.7 kb in length, including an inverted terminal repeat (ITR) of 145 nucleotides. There are several AAV serotypes that have been characterized. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, so it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 in Parvoviruses and Human Disease, J.R. Puttson, ed., and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome, and by heteroduplex analysis, which reveals the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0082] There are multiple serotypes of AAV. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAV2 / 1, AAV2 / 8, AAV2 / 9, or any of their derivatives. Other types of rAAV variants, such as rAAVs with capsid mutations, are also included in the present disclosure. See, for example, Marsic et al., Molecular Therapy 22(11): 1900-1909(2014). The nucleotide sequences of the genomes of the various AAV serotypes shown above are known in the art. The use of cognate components is specifically contemplated. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated by reference in its entirety.

[0083] The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV1 is provided under GenBank Accession No. NC_002077, the complete genome of AAV2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 {1983), the complete genome of AAV3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV4 is provided in GenBank Accession No. NC_001829, the AAV5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV6 is provided in GenBank Accession No. NC_001862, at least portions of the AAV7 and AAV8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 relating to AAV8), the AAV9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004), and the AAV10 genome is provided in Mol. Ther., 13(1): The AAV genome is provided in Virology, 330(2): 375-383(2004). Information regarding MyoAAV 1A is provided in Tabeboldbar et al. (Cell 184(19): 4919-38(2021)). Information regarding AAVMYO is provided in Weinmann et al. (Nature Communications 11:5432(2020), doi.org / 10.1038 / s41467-020-19230). The genomes of AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, and AAV-B1 are also known in the art. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs.Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three associated capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0084] Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0085] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication and genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It readily survives the conditions used to inactivate adenovirus, making cryopreservation of AAV less important: AAV can be lyophilized, and AAV-infected cells are not resistant to superinfection.

[0086] In some embodiments, the AAV lacks the rep and cap genes. In some embodiments, the AAV is a recombinant linear AAV (rAAV), a single-stranded AAV, or a recombinant self-complementary AAV (scAAV). Self-complementary (sc) technology allows the single-stranded viral DNA genome to bind to itself, thereby priming second strand DNA synthesis. This sc element speeds up and enhances gene expression compared to constructs lacking the sc element. In some specific embodiments, the AAV is a ssAAV or ssrAAV.

[0087] Advances in AAV vectors have resulted in safer and more efficient viral vehicles for delivering therapeutic transgenes with a single injection, and gene therapy is now the preferred therapeutic intervention for monogenic diseases. AAV vectors can provide long-term expression of gene products in postmitotic target tissues. Thus, current AAV-based strategies may require only a one-time vector administration.

[0088] The recombinant AAV genome of the present disclosure comprises any one or more of the sequences set forth in SEQ ID NOs: 1 and 6-11, e.g., one or more AAV ITRs flanking a polynucleotide encoding one or more EIF2B5 polynucleotides. Provided herein are rAAVs each comprising one or more EIF2B5 genes. The rAAVs comprising one or more EIF2B5 genes may encode one, two, three, four, five, six, seven, or eight EIF2B5 proteins.

[0089] Thus, in some embodiments, the viral vector is an AAV, such as AAV1 (i.e., an AAV comprising AAV1 inverted terminal repeats (ITRs) and AAV1 capsid protein), AAV2 (i.e., an AAV comprising AAV2 ITRs and AAV2 capsid protein), AAV3 (i.e., an AAV comprising AAV3 ITRs and AAV3 capsid protein), AAV4 (i.e., an AAV comprising AAV4 ITRs and AAV4 capsid protein), AAV5 (i.e., an AAV comprising AAV5 ITRs and AAV5 capsid protein), AAV6 (i.e., an AAV comprising AAV6 ITRs and AAV6 capsid protein), AAV7 (i.e., an AAV comprising AAV7 ITRs and AAV7 capsid protein), AAV8 (i.e., an AAV comprising AAV8 ITRs and AAV8 capsid protein), AAV9 (i.e., an AAV comprising AAV9 ITRs and AAV9 capsid protein), AAV10 (i.e., an AAV comprising AAV10 ITRs and AAV10 capsid protein), AAV11 (i.e., an AAV comprising AAV10 ITRs and AAV10 capsid protein), AAV12 (i.e., an AAV comprising AAV10 ITRs and AAV10 capsid protein), AAV13 (i.e., an AAV comprising AAV10 ITRs and AAV10 capsid protein), AAV14 (i.e., an AAV comprising AAV14 ITRs and AAV10 capsid protein), AAV15 (i.e., an AAV comprising AAV15 ITRs and AAV10 capsid protein), AAV16 (i.e., an AAV comprising AAV16 ITRs and AAV16 capsid protein), AAV17 (i.e., an AAV comprising AAV17 AAV includes AAVs comprising AAV13 ITRs and AAV13 capsid protein, AAV14 (i.e., AAVs comprising AAV14 ITRs and AAV14 capsid protein), AAV15 (i.e., AAVs comprising AAV15 ITRs and AAV15 capsid protein), AAV16 (i.e., AAVs comprising AAV16 ITRs and AAV16 capsid protein), AAV17 (i.e., AAVs comprising AAV17 ITRs and AAV17 capsid protein), AAV18 (i.e., AAVs comprising AAV18 ITRs and AAV18 capsid protein), AAV19 (i.e., AAVs comprising AAV19 ITRs and AAV19 capsid protein), AAV10 (i.e., AAVs comprising AAV10 ITRs and AAV10 capsid protein), AAV11 (i.e., AAVs comprising AAV11 ITRs and AAV11 capsid protein), AAV12 (i.e., AAVs comprising AAV12 ITRs and AAV12 capsid protein), AAV13 (i.e., AAVs comprising AAV13 ITRs and AAV13 capsid protein), AAVanc80 (i.e., AAVs comprising AAVanc80 ITRs and AAVanc80 capsid protein), AAVrh.74 (i.e., AAVs comprising AAVrh.74 ITRs and AAVrh.74 capsid protein), AAVrh.8 (i.e., AAVs comprising AAVrh.8 ITRs and AAVrh.8 capsid protein), AAVrh.10 (i.e., AAVrh.10 ITRs and AAVrh.10 capsid protein), MyoAAV 1A, AAVMYO, or AAV-B1, or a pseudotyped AAV, such as AAV2 / 1, AAV2 / 8, or AAV2 / 9, or AAVMYO, or any of their derivatives.

[0090] In various embodiments, the AAV is AAV9. AAV9 has become the most widely used vector for muscle and / or neurological indications with an established safety profile in the clinic. Intrathecal administration of AAV9 allows for the seeding of transgenes throughout the nervous system and is currently approved by the FDA for spinal muscular atrophy (SMA, NCT03381729) and is under investigation for the treatment of neuronal ceroid lipofuscinosis 3 (CLN3, NCT03770572), CLN6 (NCT02725580), giant axonal neuropathy (GAN, NCT02362438), mucopolysaccharidosis type 3A (NCT02716246) and type 3B (NCT03315182), and exon 2 duplication in the DMD gene (NCT04240314). Such characteristics make AAV9 an ideal gene delivery method for the treatment of genetic disorders such as mutations in EIF2B5, which result in white matter abnormalities in the central nervous system. AAV9 has also been shown to be able to target Schwann cells and other peripheral neuropathies. More importantly, AAV9 has been reported to transduce Schwann cells in large animals and non-human primates, indicating that it is a desirable viral vector for clinical applications that require the delivery of therapeutic genes to human Schwann cells. Finally, data from studies in other models of CNS disease indicate that AAV9 vectors efficiently transfect the CNS (Lukashchuk et al., Molecular Therapy 3:15055, 2016, doi.org / 10.1038 / mtm.2015.55).

[0091] The DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell that is permissive for infection with a helper virus (e.g., adenovirus, E1 deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into an infectious viral particle. Techniques for producing rAAV particles are standard in the art, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell. Production of rAAV requires that the following components are present in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep gene may be derived from any AAV serotype from which a recombinant virus may be derived, or may be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1. In some embodiments, the AAV DNA of the rAAV genome is derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1. Other types of rAAV variants, such as rAAVs with capsid mutations, are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11):1900-1909(2014). The nucleotide sequences of the genomes of the various AAV serotypes shown above are known in the art. The use of cognate components is specifically contemplated.The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety. "AAV virion" or "AAV virus particle" or "AAV particle" or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, e.g., a transgene delivered to a mammalian cell), it is usually referred to as an "AAV vector particle" or simply an "AAV vector". Thus, the production of AAV vector particles necessarily includes the production of AAV vectors, since such vectors are contained within the AAV vector particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell, are standard in the art. Production of rAAV requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep gene may be from any AAV serotype from which a recombinant virus may be derived, and may be from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, or AAV2 / 9, and derivatives thereof.In some embodiments, the AAV DNA of the rAAV genome is derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, or AAV2 / 9, and derivatives thereof. Other types of rAAV variants (e.g., including those with capsid mutations) are also included in the disclosure. Such variants include, but are not limited to, MyoAAV or AAVMYO, as well as other variants described, for example, in Marsic et al., Molecular Therapy 22(11):1900-1909(2014), Weismann, J., et al., Nat Commun 11(1):5432(2020), and Tabeboldbar, M. et al., Cell 184(19):4919-4938 e22(2021), which are incorporated by reference in their entirety for use herein. The nucleotide sequences of the genomes of the various AAV serotypes shown above are known in the art. The use of cognate components is specifically contemplated. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated by reference in its entirety for use herein.

[0092] In some embodiments, the viral vector is pseudotyped AAV and comprises ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV comprising AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 8 (i.e., an AAV comprising AAV2 ITRs and AAV8 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., an AAV comprising AAV2 ITRs and AAV1 capsid proteins).

[0093] In some embodiments, the AAV comprises a recombinant capsid protein, such as a capsid protein comprising a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, or AAV2 / 9, and derivatives thereof. Other types of rAAV variants are also contemplated, such as rAAVs with capsid mutations. See, e.g., Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0094] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression. Clark et al., Hum Gene Ther, 8: 659-669 (1997) 32See, Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996), and Xiao et al., J Virol, 70: 8098-8108 (1996). See also Chao et al., Mol Ther, 2: 619-623 (2000) and Chao et al., Mol Ther, 4: 217-222 (2001).

[0095] The recombinant AAV genome, in various embodiments, comprises the nucleic acid of the present disclosure and one or more AAV ITRs flanking the nucleic acid. The AAV DNA of the rAAV genome may be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, or AAV2 / 9, and derivatives thereof). The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants are also contemplated, such as rAAVs with capsid mutations. For example, Marsic et al., Molecular Therapy, 22(11):1900-1909(2014) 29 See, The nucleotide sequences of the genomes of the various AAV serotypes, set forth in the background section above, are known in the art.

[0096] The provided recombinant AAV (i.e., infectious, encapsidated rAAV particles) comprise a rAAV genome. The term "rAAV genome" refers to a polynucleotide sequence derived from a native AAV genome that has been modified. In some embodiments, the rAAV genome has been modified to remove the native cap and rep genes. In some embodiments, the rAAV genome comprises endogenous 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises ITRs from an AAV serotype that is different from the AAV serotype from which the AAV genome was derived. In some embodiments, the rAAV genome comprises a transgene of interest flanked at the 5' and 3' ends by inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises a "gene cassette." In some exemplary embodiments, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep and cap DNA between the ITRs of the genome.

[0097] The DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell that is permissive for infection with a helper virus (e.g., adenovirus, E1 deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into an infectious viral particle. Techniques for producing rAAV particles are standard in the art, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell. Production of rAAV requires that the following components are present in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus may be derived, or may be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, or AAV2 / 9, and derivatives thereof. The production of pseudotyped rAAVs is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0098] Recombinant AAV genomes of the present disclosure include, for example, one or more AAV ITRs flanking a polynucleotide encoding one or more EIF2B5. Accordingly, embodiments of the present disclosure include an rAAV genome that includes a nucleic acid that includes a nucleotide sequence set forth in any one of SEQ ID NOs: 1, 6-11, 16, and 17, or a nucleotide sequence that includes at least, or about, or at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the sequence set forth in any one of SEQ ID NOs: 1, 6-11, 16, and 17.

[0099] Embodiments of the present disclosure include a nucleic acid comprising an rAAV genome comprising a nucleotide sequence set forth in any one of SEQ ID NOs:12-14 and 18, or a nucleotide sequence comprising at least, or about, or at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the sequence set forth in any one of SEQ ID NOs:12-14 and 18.

[0100] The method for generating packaging cells is to create a cell line that stably expresses all the components necessary for the creation of AAV particles. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separate from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome can be modified by GC tailing, the addition of synthetic linkers containing restriction endonuclease cleavage sites, or by other methods. 41The rAAV genome and / or rep and cap genes have been introduced into a bacterial plasmid by procedures such as direct blunt-end ligation. The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or rep and cap genes into the packaging cell.

[0101] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mol. Cell. al., J. Virol., 62:1963(1988), and Lebkowski et al., Mol. Cell. Biol., Oct;8(10):3988-96(1988), Samulski et al. al., J. Virol., 63: 3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658.776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13: 1244-1250 (1995), Paul et al. al. Human Gene Therapy 4:609-615 (1993), Clark et al., Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis on those portions of the documents that relate to rAAV production. The production and use of self-complementary (sc) rAAVs is specifically contemplated and exemplified.

[0102] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (synonymous 293 line). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0103] In some embodiments, the rAAV is purified by methods standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0104] Compositions are provided that include the nucleic acid and viral vector of the present disclosure. Compositions are provided that include the delivery vehicle (such as rAAV) described herein. In various aspects, such compositions also include a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier is a diluent, excipient, or buffer. The composition may also include other components, such as an adjuvant.

[0105] Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0106] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, with various other ingredients as listed above as necessary, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques, which produce a powder of active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0107] The titer of the rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per mL. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14, or even higher for DNase resistant particles (DRP).

[0108] The dosage of rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the time of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. Dosages may be expressed in units of viral genomes (vg). Dosages contemplated herein include doses of about 1×10 7 , about 1×10 8 , about 1×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13 , about 1.1×10 13 , about 1.2×10 13 , about 1.3×10 13 , about 1.5×10 13 , about 2×10 13, about 2.5×10 13 , about 3×10 13 , about 3.5×10 13 , about 4×10 13 , about 4.5×10 13 , about 5×10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , about 5×10 14 , about 1×10 15 ~Approx. 1×10 16 , or more total viral genome doses.

[0109] Approximately 1×10 9 ~Approx. 1×10 10 , about 5×10 9 ~Approx. 5×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 14 vg, approx. 1×10 13 ~about 6×10 14 vg, approx. 1×10 13 ~Approx. 1×10 15 vg, and approximately 6 × 10 13 ~Approx. 1.0×10 14 vg doses are also contemplated. One dose exemplified herein is 1x10 administered intrathecally, intracerebroventricularly, intracerebrally, intravenously, intracisternally, or via aerosol delivery. 13 vg.

[0110] The dose of rAAV administered is also expressed in units of vg / kg in various embodiments. Such a dose may be about 1×10 7 vg / kg, approximately 1×10 8 vg / kg, approximately 1×109 vg / kg, about 5×10 9 vg / kg, about 6×10 9 vg / kg, about 7×10 9 vg / kg, about 8×10 9 vg / kg, about 9×10 9 vg / kg, about 1×10 10 vg / kg, about 2×10 10 vg / kg, about 3×10 10 vg / kg, about 4×10 10 vg / kg, about 5×10 10 vg / kg, about 1×10 11 vg / kg, about 5×10 11 vg / kg, about 1×10 12 vg / kg, about 2×10 12 vg / kg, about 3×10 12 vg / kg, about 4×10 12 vg / kg, about 5×10 12 vg / kg, about 6×10 12 vg / kg, about 7×10 12 vg / kg, about 8×10 12 vg / kg, about 9×10 12 vg / kg, about 1×10 13 vg / kg, about 1.1×10 13 vg / kg, about 1.2×10 13 vg / kg, about 1.3×10 13 vg / kg, about 1.5×10 13 vg / kg, about 2×10 13 vg / kg, about 2.5×10 13 vg / kg, about 3×10 13 vg / kg, about 3.5×10 13 vg / kg, about 4×10 13 vg / kg, about 4.5×10 13 vg / kg, about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×1014 vg / kg, approx. 5×10 14 vg / kg, approximately 1×10 15 vg / kg, or approximately 1 × 10 16 These include, but are not limited to, dosages in vg / kg.

[0111] Approximately 1×10 9 vg / kg ~ approx. 1×10 10 vg / kg, approx. 5×10 9 vg / kg ~ approx. 5×10 10 vg / kg, approximately 1×10 10 vg / kg ~ approx. 1×10 11 vg / kg, approximately 1×10 11 vg / kg ~ approx. 1×10 15 vg / kg, approximately 1×10 12 vg / kg ~ approx. 1×10 15 vg / kg, approximately 1×10 12 vg / kg ~ approx. 1×10 14 vg / kg, approximately 1×10 13 vg / kg ~ approx. 2×10 14 vg / kg, approximately 1×10 13 vg / kg ~ approx. 1×10 15 vg / kg, and approximately 6 × 10 13 vg / kg ~ approx. 1.0×10 14 Doses of about 1.5×10 vg / kg are also included in various embodiments. Some doses exemplified herein are about 1.5×10 administered intrathecally, intracerebroventricularly, intracerebrally, intravenously, intracisternally, or via aerosol delivery. 11 vg / kg or approximately 3 × 10 13 vg / kg.

[0112] Transduction or transfection of cells with the rAAV of the present disclosure results in sustained expression of the EIF2B5 gene / protein. As used herein, the terms "transduction" and "transfection" are used interchangeably. The terms "transduction" or "transfection" are used to refer to administration / delivery of the EIF2B5 gene to a target cell, either in vivo or in vitro, via a replication-deficient rAAV as described herein, which results in expression of the EIF2B5 gene / protein by the target cell, for example. Thus, the present disclosure provides methods of administering / delivering an rAAV expressing the EIF2B5 gene to a cell or subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. These methods include transducing cells and tissues (including, but not limited to, astrocytes, neurons, glia, peripheral motor neurons, sensory motor neurons, neurons, Schwann cells, and other tissues or organs such as muscle, liver, and brain) with one or more rAAV as described herein. Transduction can be performed with a gene cassette that contains cell-specific regulatory elements.

[0113] Methods are provided for transducing target cells, e.g., astrocytes, in vivo or in vitro with a delivery vehicle (e.g., nanoparticles, extracellular vesicles, exosomes, or vectors (e.g., rAAV)). In vivo methods include administering an effective dose or effective doses of a composition comprising a delivery vehicle (such as rAAV) to an animal (including a human subject or patient) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. An effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the extent of the disease, causes remission (partial or complete) of the disease, and / or prolongs survival. Thus, methods are provided in which an effective dose (or doses administered essentially simultaneously, or doses administered at intervals) of a rAAV as described herein is administered to a subject in need thereof.

[0114] Provided herein are medicaments and methods for treating, ameliorating, or preventing diseases associated with mutant or abnormal EIF2B5 gene expression. Molecular, biochemical, histological, and functional outcome measures indicate the therapeutic efficacy of the method. The level of human EIF2B5 transcripts in animals and / or humans can be confirmed by RT-PCR and / or RNAseq. The expression level of EIF2B5 protein in tissues and organs of interest can be assessed using Western blotting. The localization of EIF2B5 can be confirmed by immunohistochemistry. To evaluate the efficacy of potential treatments in mice, measurements of function can be performed using various functional outcome measures, including but not limited to the rotarod test and other functional testing methods for leukodystrophies known in the art. In patients, various functional outcome measures may be used to evaluate successful treatments, including but not limited to gait analysis, MRI / DTI, seizure monitoring (EEG), and other testing methods for leukodystrophies known in the art. See also, e.g., Parikh et al., Mol Genet Metab 2015;114(4):501-515.

[0115] In the methods of the disclosure, expression of EIF2B5 protein is increased by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, at least 98 percent, at least 99 percent, or 100 percent.

[0116] Combination therapy is also contemplated by the present disclosure. Combination as used herein includes simultaneous or sequential therapy. Combination of the methods of the present disclosure with standard medical treatment is specifically contemplated, as is combination with novel therapies. In some embodiments, combination therapy includes administering an immunosuppressant in combination with the gene therapy disclosed herein.

[0117] The immunosuppressant may be administered before or after the initiation of an immune response to the rAAV in the subject following administration of gene therapy. Additionally, the immunosuppressant may be administered simultaneously with gene therapy or protein replacement therapy. The immune response in the subject includes an adverse immune response or inflammatory reaction following or caused by administration of the rAAV to the subject. The immune response may be the production of antibodies in the subject in response to the administered rAAV.

[0118] Exemplary immunosuppressants include glucocorticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, cyclostatic agents such as purine analogs, methotrexate, and cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, biologics such as monoclonal antibodies or fusion proteins and polypeptides, and dipeptide boronic acid molecules such as bortezomib.

[0119] The immunosuppressant may be an anti-inflammatory steroid, which is a steroid that reduces inflammation and suppresses or modulates the immune system in a subject. Exemplary anti-inflammatory steroids are glucocorticoids, such as prednisolone, betamethasone, dexamethasone, methotrexate, hydrocortisone, methylprednisolone, deflazacort, budesonide, or prednisone.

[0120] Janus kinase inhibitors are inhibitors of the JAK / STAT signaling pathway by targeting one or more of the enzymes of the Janus kinase family. Exemplary Janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.

[0121] Calcineurin inhibitors bind to cyclophilin and inhibit the activity of calcineurin. Exemplary calcineurin inhibitors include cyclosporine, tacrolimus, and picecrolimus.

[0122] mTOR inhibitors reduce or inhibit the serine / threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include rapamycin (also known as sirolimus), everolimus, and temsirolimus.

[0123] Immunosuppressants include immunosuppressant macrolides. The term "immunosuppressant macrolide" refers to a macrolide drug that suppresses or modulates the immune system of a subject. Macrolides are a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desoamine, are attached. The lactone ring is usually 14, 15, or 16 members. Macrolides belong to the polyketide class of drugs and can be natural products. Examples of immunosuppressant macrolides include tacrolimus, pimecrolimus, and rapamycin (also known as sirolimus).

[0124] Purine analogs block nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolates, such as mycophenolate acid or mycophenolate mofetil, and lefunomide.

[0125] Exemplary immunosuppressant biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacept, and daclizumab.

[0126] In particular, the immunosuppressant is an anti-CD20 antibody. The term anti-CD20 specific antibody refers to an antibody that specifically binds to CD20 or inhibits or reduces the expression or activity of CD20. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0127] Additional examples of immunosuppressive antibodies include anti-CD25 antibodies (or anti-IL2 antibodies or anti-TAC antibodies), such as basiliximab and daclizumab, and anti-CD3 antibodies, such as muromonab-CD3, otelixizumab, teplizumab, and vicilizumab, anti-CD52 antibodies, such as alemtuzumab.

[0128] One exemplary combination therapy is the delivery of rapamycin and rituximab prior to or concomitantly with the delivery of the AAV vector. Another exemplary combination therapy is the delivery of rapamycin, rituximab, and a corticosteroid, such as prednisone.

[0129] Administration of an effective dose of a nucleic acid, nanoparticle, extracellular vesicle, exosome, viral vector, or composition of the present disclosure may be by a route standard in the art, including, but not limited to, intrathecal, intracerebral, intraventricular, intracisternal, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. In various embodiments, an effective dose is delivered by a combination of routes. For example, in various embodiments, an effective dose is delivered intrathecal, intracerebral, intraventricular, intravenous, intracisternal, and / or intramuscular, or intrathecal and / or intravenous and / or intraventricular, etc. In some embodiments, an effective dose is delivered sequentially or consecutively. In some embodiments, an effective dose is delivered simultaneously. The route of administration and the serotype of the AAV components of the rAAV of the present disclosure (in particular the AAV ITRs and capsid proteins) may be selected and / or adapted by one skilled in the art taking into account the infection and / or disease condition to be treated and the target cells / tissues expressing the EIF2B5 gene.

[0130] In particular, the actual administration of the delivery vehicle (such as rAAV) may be accomplished by using any physical method that will transport the delivery vehicle (such as rAAV) to the target cells (i.e., astrocytes) of the subject. Administration includes, but is not limited to, injection into the cerebrospinal fluid (CSF) (intrathecal), intraventricular injection, intracerebral injection, injection into the bloodstream and / or directly into the nervous system, intracisternal injection (or intracisternal injection (ICM)), or intranasally. Simply resuspending rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for tissue expression, and there are no known limitations regarding carriers or other components that can be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner of using rAAV). The capsid protein of rAAV can be modified to target rAAV to a specific target tissue of interest, such as neurons. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation for intrathecal injection or as an aerosol formulation for inhalation. Numerous formulations for intrathecal, intraventricular, intracerebral, or intracisternal injection have been developed in advance and can be used in carrying out the method of the present disclosure. The delivery vehicle (such as rAAV) can be used with any pharma- ceutically acceptable carrier to facilitate administration and handling.

[0131] Dispersions of the delivery vehicle (such as rAAV) can be prepared in glycerol, sorbitol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily available by standard techniques well known to those skilled in the art.

[0132] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, sorbitol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, suMPZ or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0133] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, with various other ingredients as listed above as necessary, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques, which produce a powder of active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0134] The present disclosure also provides a kit for use in the treatment of a disease or disorder described herein. Such a kit comprises at least a first sterile composition comprising any of the nucleic acids described herein above or any of the viral vectors described herein above in a pharma-ceutically acceptable carrier. Another component is optionally a second therapeutic agent for the treatment of the disorder, together with a suitable container and vehicle for administration of the therapeutic composition. The kit optionally comprises a solution or buffer for suspending, diluting, or delivering the first and second compositions.

[0135] In one embodiment, such a kit includes the nucleic acid or vector in a diluent packaged in a container, such as a sealed bottle or vessel, with a label attached to the container or included in the package that describes the use of the nucleic acid or vector. In one embodiment, the diluent is in the container such that the amount of head space in the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of head space is negligible (i.e., almost nonexistent).

[0136] In some embodiments, the formulation comprises a stabilizer. The term "stabilizer" refers to a substance or excipient that protects the formulation from harmful conditions, such as those that occur during heating or freezing, and / or extends the stability or shelf life of the formulation in a stable state. Examples of stabilizers include, but are not limited to, stabilizers such as sucrose, lactose and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

[0137] In some embodiments, the formulation includes an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance added to the composition to inhibit the growth of microorganisms that may be introduced upon repeated puncturing of the vial or container used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0138] In some embodiments, the kits include labels and / or instructions that describe the use of the reagents provided in the kit. The kits also optionally include catheters, syringes, or other delivery devices for delivering one or more of the compositions used in the methods described herein.

[0139] This entire document is intended to be related as a unified disclosure, and it is understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes all embodiments of the disclosure that are somewhat narrower in scope than, for example, the variations specifically mentioned above. With respect to aspects of the disclosure described as genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed with "a" or "an", these terms are understood to mean "one or more" unless the context clearly requires a more limited meaning. When aspects of the disclosure are described as "comprising" a feature, the embodiment is also contemplated as "consisting of" or "consisting essentially of" the feature.

[0140] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety, to the extent that it is not inconsistent with this disclosure.

[0141] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light of them will be suggested to those skilled in the art, but are within the spirit and scope of this application and the scope of the appended claims. Thus, the following examples are provided by way of illustration and not by way of limitation. EXAMPLES

[0142] Example 1 Gene Therapy Constructs Encoding EIF2B5 Protein, and Materials and Methods Initially, three AAV genome vector constructs encoding EIF2B5 were generated. These constructs contain promoters targeted to astrocytes: a ubiquitous promoter (referred to as CAG promoter) containing the CBA promoter and CMV enhancer (SEQ ID NO: 3), a full-length glial fibrillary acidic protein (GFAP) promoter (SEQ ID NO: 5), and a truncated version of the GFAP promoter, i.e., the gfaABC(1)D promoter (SEQ ID NO: 4). The gfaABC(1)D promoter is a compact GFAP promoter derived from the conventional approx. 2.2 kb human GFAP promoter. Other important components of the vector include the AAV9 capsid for efficient targeting of the CNS, the AAV2 inverted terminal repeats (ITRs) to create a single-stranded construct with a larger packaging capacity (for the gene of interest), the human eIF2B5 coding sequence, and a posttranscriptional polyadenylation (polyA) sequence (Figure 1).

[0143] Each of these sequences contains a full-length transcript of EIF2B5 cDNA under the control of the CAG promoter (SEQ ID NO: 3), the gfaABC(1)D promoter (SEQ ID NO: 4), or the GFAP promoter (SEQ ID NO: 5), and various other components, as shown in Figures 12-14. Each of these constructs was designed to test for restoration of EIF2B5 expression. The specific sequences for each of the three constructs are provided in Tables 1-3 and shown in Figures 12-14.

[0144] The GFAP, gfaABC(1)D, and CBA promoters were cloned into plasmids with eIF2B5, respectively. GFAP (2.2 kb) and eIF2B5 (2.2 kb), as well as a truncated gfABC(1)D promoter of only 681 bp, were evaluated. Each construct was sequenced, and eIF2B5 mRNA and protein levels were evaluated in vitro. Cloning, sequencing, and in vitro expression analysis were performed.

[0145] Materials and Methods Vector creation. Recombinant AAV (rAAV) vectors were produced at Andelyn Biosciences using calcium phosphate-mediated triple transfection in adherent HEK293 cells followed by purification. Briefly, harvested media was filtered, concentrated, and then purified by gradient ultracentrifugation followed by ion exchange chromatography. Vectors were formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, and 200 mM NaCl and 0.001% Pluronic F68 and sterile filtered. The AAV production process was developed using the method described by Rabinowotiz et al. (J.Virol.2002 76(2):791-801, doi: 10.1128 / jvi.76.2.791-801.2002). Physical titer determination was determined by ddPCR based on the degradation of non-encapsidated DNA after digestion of the viral capsid.

[0146] cell line. Human embryonic kidney 293 (HEK293) cells (ATCC catalog number CRL-1573) were used to confirm transfection of plasmid DNA.

[0147] To establish astrocyte cell lines and test the AAV constructs of the present disclosure as a relevant in vitro system to study disease mechanisms, skin biopsies were performed on patients with confirmed eIF2B5 VWM. Fibroblast cultures were established from the patient's skin biopsy and the fibroblasts were converted into the primary cell type of interest, astrocytes (Figure 3).

[0148] GFP expression in vitro. 90% confluent HEK293 cells in 6-well plates were transfected with 2.5ug of plasmid DNA using Lipofectamine 3000 according to the manufacturer's protocol. 72 hours after transfection, cells were imaged, harvested, and cell pellets were frozen at -80 C. One well of non-transfected cells was also harvested at the same time as a negative control.

[0149] RNA was isolated from cells using Trizol, DNAseI treated, and cDNA was generated using the Qiagen RT2 First Strand Synthesis Kit. Relative GFP mRNA levels were quantified by qPCR using SYBR green and the comparative CT method with primers specific for GFP and beta-actin as an endogenous control.

[0150] Mice and mouse sources. Wild-type (WT) mice. Newborn (postnatal day 1–3) C57BL / 6 mice were used in various experiments.

[0151] R191H VWM mice. R191H VWM mice (described in Wong et al. (eLife.2019;8:e42940, doi: 10.7554 / eLife.42940)) are used as an animal model of VWM. The mutation described corresponds to the human G584A mutation, Arg195H, in Cree leukoencephalitis (described in Fogli et al. (Annals of Neurology 52(4):506-10,2002), doi.org / 10.1002 / ana.10339). It is one of the most rapidly progressive leukoencephalopathies, with onset at 3-9 months and 100% mortality by 21 months. This mouse model has a survival time of approximately 8 months and various symptoms have been reported, including motor impairment at 5 months, decreased MBP expression by 28 days, axonal changes at 7 months, increased numbers of astrocytes from P14 onwards that increased with disease progression, and increased GFAPδ protein levels in forebrain lysates.

[0152] ICV injection. All animal procedures were approved by the Nationwide Children's Hospital Institutional Animal Care and Use Committee (IACUC). Newborn (postnatal day 1–3) mice (C57BL / 6) were cryo-anesthetized (~2 min) prior to intracerebroventricular (ICV) injection. ICV injections were performed in the left hemisphere at 2 / 5 of the distance from the lambdoid suture to the eye using a Hamilton syringe (Cal7635-01) and a 33GA 30° bevel needle (Hamilton, 7803-05). Neonates were injected with 7.50E+10vg ssAAV9 vectors encoding GFP under the GFAP, CAG, and truncated variant gfaABC(1)D (GFAP) promoters.

[0153] Assessment of GFP biodistribution in animal tissues following AAV9 ICV injection. Four or eight weeks after ICV injection, mice were terminally anesthetized with ketamine / xylazine (100 / 10 mg / kg ip) and perfused transcardially with ice-cold 0.9% heparinized saline. Tissues were dissected and post-fixed in 4% PFA in PBS for 12 hours. After fixation, the right hemisphere was cryoprotected in 30% sucrose in PBS for 3 days at 4°C. All specimens were embedded and frozen in OST compound (Tissue Plus, Fisher). Sagittal sections were cut at a thickness of 25 mm on a cryostat (1950 LEICA). Free-floating sections were washed in PBS and incubated with DAPI solution in PBS for 1 min at room temperature. To remove antigens from PFA-fixed tissues, sections were treated with 0.1% sodium borohydride in 1× PBS for 15 min at room temperature. For immunohistochemical analysis of GFP colocalization with specific cell markers, all sections were blocked and permeabilized in 10% normal goat serum in 1XPBS containing 0.3% Triton (PBST) for 1 hour at room temperature, followed by overnight incubation in fresh PBST containing chicken anti-GFAP (AbCam, 1:300) and rabbit anti-NeuN (Cell Signaling, 1:500) at 4C. Sections were washed in 1XPBS and incubated in PBST with donkey anti-chicken Cy5 (Jackson ImmunoResearch, 1:500) and donkey anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific, 1:500) secondary antibodies for 1 hour at room temperature in PBST containing 10% normal donkey serum. Sections were washed in 1XPBS and mounted on slides in ProLong Gold antifade reagent (Thermo Fisher Scientific). Images were acquired using a Nikon Ti2E fluorescence microscope and analyzed using NIS-Elements software (Nikon) and Prism (GraphPad). The percentage of GFP distribution was evaluated within the area covered by GFP and DAPI in each section. The intensity of the GFP signal was evaluated within all GFP-positive areas.

[0154] Protein extraction and Western blotting analysis. After mice were treated, protein extraction and Western blotting analysis were performed. Total protein was extracted from selected animal tissues using tissue protein extraction reagent (T-PER tissue protein extraction reagent; 78510, Thermo Scientific) and one tablet of protease inhibitor (Pierce protease inhibitor tablet, A32953, Thermo Scientific) per 10 mL of extraction reagent. Steel beads were added to the sample and extraction reagent, and the samples were homogenized using a TissueLyser II. The steel beads were then removed and the samples were sonicated twice for 15 seconds.

[0155] Total extracted protein was quantified using the DC protein assay (Bio-Rad). 25 μg samples were separated on 4–12% SDS-PAGE and transferred to nitrocellulose membranes using a wet transfer system. Ponceau staining was performed, after which the membrane was washed three times for 10 min each in PBS supplemented with 1% Tween. The membrane was blocked for 1 h at room temperature in Pierce protein-free blocking buffer. The nitrocellulose membrane was then incubated overnight at 4 C with the following antibodies: mouse monoclonal antibody against GAPDH (1:5000 in Pierce protein-free blocking buffer, 274102, Synaptic Systems), chicken polyclonal antibody against GFP (1:5000 in Pierce protein-free blocking buffer, ab13970, AbCam). The membrane was then washed three times for 10 min each in PBST buffer.

[0156] The membrane was then incubated with the following antibodies for 1 hour at room temperature: goat antibody AF488 against chicken (1:1000 in Pierce protein-free blocking buffer, ab150169, AbCam), donkey antibody AF568 against mouse (1:1000 in Pierce protein-free blocking buffer, A10037, Invitrogen), and then washed three times for 10 minutes each with PBST buffer.

[0157] Blots were then imaged using a blot imager. Quantification of bands was performed using a Bio-Rad ImageLab. Protein extraction and Western blotting analysis after transfection. Protein extraction and Western blotting analysis were performed after cells were harvested 72 h post-transfection, washed in PBS, centrifuged, and flash frozen. Total protein was extracted from 1-2 million HEK293T cells by first thawing the cell pellet on ice for 15 min, then using 30 uL of RIPA buffer (Pierce, RIPA Lysis and Extraction Buffer 89901, Thermo Scientific) per pellet and one tablet of protease inhibitor (Pierce Protease Inhibitor Tablet, A32953, Thermo Scientific) per 10 mL of extraction reagent. Samples were gently homogenized using a pipette, lysed on ice for 15 min at 4 C, sonicated briefly, and then centrifuged at 10,000 g for 10 min. The supernatant was transferred to a new tube and total protein was quantified.

[0158] Total extracted protein was quantified using the DC protein assay (Bio-Rad). 50 μg samples were separated on 4–12% SDS-PAGE and transferred to nitrocellulose membranes using a wet transfer system. Ponceau staining was performed, after which the membrane was washed three times for 10 min each in PBS supplemented with 1% Tween. The membrane was blocked for 1 h at room temperature in Pierce protein-free blocking buffer. The nitrocellulose membrane was then incubated overnight at 4 C with the following antibodies: mouse monoclonal antibody against GAPDH (1:5000 in Pierce protein-free blocking buffer, 274102, Synaptic Systems), chicken polyclonal antibody against GFP (1:5000 in Pierce protein-free blocking buffer, ab13970, AbCam). The membrane was then washed three times for 10 min each in PBST buffer.

[0159] The membrane was then incubated with the following antibodies for 1 hour at room temperature: goat antibody AF488 against chicken (1:1000 in Pierce protein-free blocking buffer, ab150169, AbCam), donkey antibody AF568 against mouse (1:1000 in Pierce protein-free blocking buffer, A10037, Invitrogen), and then washed three times for 10 minutes each with PBST buffer.

[0160] Blots were then imaged using a blot imager. Quantification of bands was performed using a Bio-Rad ImageLab. Example 2 EIF2B5 protein gene replacement in cells in vitro The following experiments were performed to confirm that AAV9 containing various promoters of interest could be used for successful and highly successful expression of EIF2B5. However, in this example, AAV containing the promoter of interest with GFP was transfected into HEK cells and astrocytes derived from fibroblasts in vitro. Expression analysis was performed for three eGFP constructs: AAV9-CAG-GFP, AAV9-gfaABC(1)D-GFP, and AAV9-GFAP-GFP in HEK cells (Figure 2) and astrocytes derived from fibroblasts (Figure 3).

[0161] Specifically, HEK293 cells were transfected by lipofectamine and cells were harvested 72 hours post-transfection for eGFP mRNA expression analysis. Although HEK cells were not an ideal cell model (i.e., astrocytes are the ultimate target cells for these constructs), HEK293 cells were used initially to observe expression because they allow for rapid in vitro analysis to determine whether the plasmids can successfully express the gene of interest.

[0162] 72 hours after transfection, GFP-positive cells were detectable by microscopy (Figure 2, left panel). In addition, quantitative PCR of mRNA expression on cell pellets showed increased expression, approximately 3500-fold for CAG, 1200-fold for GFAP, and 900-fold for GfaABC(1)D, over non-transfected control cells (Figure 2, right panel). These results demonstrated successful expression of transgenes driven by various promoters in HEK cells in vitro. Figure 3 shows astrocytes derived from fibroblasts stained for GFAP.

[0163] Example 3 EIF2B5 gene replacement in wild-type mice The following study was performed to confirm that the AAV9-eIF2B5 construct described herein is capable of delivering a transgene, in this example GFP, to cells of interest in the mouse brain and to compare cell-specific transduction and biodistribution of various promoters. Healthy C57 / BL6 wild-type mice (male and female) were injected with three reporter constructs: AAV9-CAG-GFP, AAV9-gfaABC(1)D-GFP, or AAV9-GFAP-GFP on postnatal day 1 (PND1) and sacrificed 28 days after injection. Mouse tissues were isolated and analyzed for the presence of GFP.

[0164] As expected, injection with AAV9-CBA-GFP resulted in both glial and neuronal transduction (Figures 4 and 5). Sagittal sections of the brain were prepared to show the distribution of GFP (green) and DAPI (blue) shows the nuclei. Insets 1-6 show higher magnification of relevant voxels in sagittal sections (Figure 4). Insets show higher magnification of GFP-positive glial and neuronal cells (Figures 5 and 28). Survey of multiple mice at both 4 and 8 weeks of age shows distribution throughout the central neuraxis (Figure 23). The % of positive GFP in total brain regions was quantified and was reduced compared to the GFAP and gfaABC(1)D promoters (Figure 26). Protein quantification by Western blot is shown in Figure 27.

[0165] Injection of newborn wild-type mice with AAV9-gfaABC(1)D-GFP resulted in greater distribution in white matter and more widespread distribution throughout the central neuraxis compared to AAV9-CBA-GFP (Figure 6). Particularly notable was the increase in GFP expression in caudal brain regions, including the cerebellar white matter region in box 2. Furthermore, expression was primarily in astrocytes, as shown by morphology in box 4 and Figure 28. Surveys of multiple mice at both 4 and 8 weeks of age showed moderate expression throughout the central neuraxis (Figure 25), which was quantified histologically (Figure 26) and by Western blot (Figure 27).

[0166] Newborn wild-type mice were injected with AAV9-gfaABC(1)D-GFP at PND1 and sacrificed 28 days after injection. The results are shown in Figure 7. Insets 1-3 show higher magnification of relevant voxels in sagittal sections. Inset 4 shows higher magnification of inset 3 to demonstrate astrocyte morphology. These results show that use of the astrocyte promoter increases distribution in white matter and expression in astrocytes. Figure 28 provides a comparison of co-labeling between cohorts.

[0167] Figure 9 shows results from wild-type mice injected with AAV9-GFAP-GFP at PND1 and sacrificed 28 days after injection. Thus far, injection of newborn wild-type mice with AAV9-GFAP-GFP has resulted in broader distribution and greater expression compared to both the AAV9-gfaABC(1)D-GFP and AAV9-CBA-GFP vectors. Transgene expression remains specific to astrocytes.

[0168] Figure 10 shows the results of an experiment with four wild type mice injected with AAV9-GFAP-GFP at PND1 and sacrificed 28 days after injection. Sagittal sections of the brain show the distribution of GFP (green). Similar biodistribution patterns were seen among the injected mice. Surveys of multiple mice at both 4 and 8 weeks of age showed robust expression throughout the central neuraxis (Figure 24), which was quantified histologically (Figure 26) and by Western blot (Figure 27).

[0169] Co-labeling after delivery of AAV9-GFAP-GFP (Figure 11) shows that GFP (green) expression colocalizes with the astrocyte marker (GFAP, purple) but not with the neuronal marker (NeuN, red). GFP expression in astrocytes was confirmed by co-labeling with antibodies specific for astrocytes (GFAP, purple) and neurons (NeuN, red). GFP expression after treatment with AAV9-GFAP-GFP (shown) and AAV9-gfaABC(1)D-GFP (not shown) was clearly present in astrocytes. Thus, transgene expression was confirmed to occur primarily in the target cells, the astrocytes. These data with AAV driving reporter proteins confirm that the use of astrocyte-specific promoters changes the tropism of AAV to greater expression in white matter and a broader distribution throughout the central neuraxis. Figure 28 provides a comparison of co-labeling between cohorts.

[0170] Example 4 EIF2B5 gene replacement in R191H VWM mice The following study was performed to evaluate CSF delivery of AAV9-eIF2B5 in a mouse model of eIF2B5, namely R191H VWM mice, and to determine the effect of the EIF2B5 transgene in this mouse model of VWM. As described herein, the AAV9-eIF2B5 construct was designed to target astrocytes and ameliorate oligodendrocyte pathology, which is due to downstream signaling effects secondary to astrocyte dysfunction. Additionally, more ubiquitous promoters were also evaluated to assess broader CNS transduction.

[0171] Presymptomatic eIF2B5 mice (i.e., R191H VWM mice) were treated by intracerebroventricular (ICV) injection with one of three AAV vector constructs, containing either an astrocyte-specific promoter, as described above: 1) AAV9-GFAP-eIF2B5, or 2) AAV9-gfaABC(1)-eIF2B5, containing a truncated form of GFAP, or a more ubiquitously expressed promoter: 3) AAV9-CBA-eIF2B5. Vectors were delivered in escalating doses. Based on the phenotypic characterization of untreated VWM mice, AAV-treated mice were evaluated accordingly. These evaluations included, but were not limited to, weight, motor testing, MRI, and survival. Additional postmortem evaluations included observation and quantification of eIF2B5, as well as select expression levels of ER stress markers. Histologically, myelination, ER stress, astrocytes, and oligodendrocytes were evaluated. The biodistribution of AAV9 is performed by qPCR.

[0172] Figure 31 shows the therapeutic efficacy in treating this VWM mouse model, as demonstrated by the rotarod data at 6 months (Figure 31, right). Six-month-old R191H mice were placed on an accelerating rotating rod (rotarod) and the number of revolutions per minute (RPM) at which the mouse fell was recorded. This assay can accurately measure motor function, which has been shown to be severely affected in this model of VWM, as well as in VWM patients. A higher RPM during the fall indicates better motility. The data show that while the R191H model treated with AAV9.GFAP.eIF2B5 showed a slight but significant improvement at 6 months, mice treated with AAV9.CAG.eIF2B5 had a better improvement in motility when compared to the aforementioned vectors. Finally, 6-month-old mice treated with AAV9.gfaABC(1)D.eIF2B5 had the greatest improvement and were not significantly different from wild-type mice, indicating the promise of treatment with this therapeutic vector.

[0173] Example 5 EIF2B5 gene replacement in I98M VWM mice The following study was performed to evaluate CSF delivery of AAV9-eIF2B5 in an additional mouse model of eIF2B5, i.e., I98M VWM mice, and to determine the effect of the EIF2B5 transgene in this mouse model of VWM. As described herein, the AAV9-eIF2B5 construct was designed to target astrocytes and ameliorate oligodendrocyte pathology, which is due to downstream signaling effects secondary to astrocyte dysfunction. Additionally, more ubiquitous promoters were also evaluated to evaluate broader CNS transduction.

[0174] Presymptomatic eIF2B5 mice (i.e., I98M VWM mice) were treated by intracerebroventricular (ICV) injection with one of three AAV vector constructs, containing either an astrocyte-specific promoter, as described above: 1) AAV9-GFAP-eIF2B5, or 2) AAV9-gfaABC(1)-eIF2B5, containing a truncated form of GFAP, or a more ubiquitously expressed promoter: 3) AAV9-CBA-eIF2B5. Vectors were delivered in escalating doses. Based on the phenotypic characterization of untreated VWM mice, AAV-treated mice were evaluated accordingly. These evaluations included, but were not limited to, weight, motor testing, MRI, and survival. Additional postmortem evaluations included observation and quantification of eIF2B5, as well as select expression levels of ER stress markers. Histologically, myelination, ER stress, astrocytes, and oligodendrocytes were evaluated. The biodistribution of AAV9 is performed by qPCR.

[0175] FIG. 31 shows therapeutic efficacy in treating this VWM mouse model, as demonstrated by rotarod data at 50 days (left). 50-day-old I98M mice were placed on an accelerating rotating rod (rotarod) and the duration (in seconds) that the mouse could remain on was recorded. This assay can accurately measure motor function, which has been shown to be severely affected in this mouse model, as well as in VWM patients. In this case, a longer latency to fall indicates better motor function. The data show that untreated I98M mice have a significantly shorter latency to fall than wild-type or heterozygous mice. However, I98M mice treated with AAV9.GFAP.eIF2B5, AAV9.CAG.eIF2B5, and AAV9.gfaABC(1)D.eIF2B5 at 50 days of age were statistically indistinguishable from wild-type mice, demonstrating the promise of these therapeutic vectors.

[0176] Example 6 Use of the novel promoter gfa1405 in EIF2B5 gene replacement Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is abundantly and almost exclusively expressed in astrocytes of the CNS. Thus, the GFAP promoter directs astrocyte-specific expression of genes (Brenner et al., J.Neurosci.1004 Mar;14(3 Pt 1):1030-7, doi: 10.1523 / JNEUROSCI.14-03-01030.1994). However, the GFAP promoter is rather large in size, constituting 2.2 kb. More specifically, due to the packaging size capacity of the single-stranded AAV genome (4.8 kb), the 2.2 kb astrocyte promoter of GFAP does not fit well with the EIF2B5 transgene (2.3 kb) together with the regulatory elements required for proper gene expression. Therefore, we performed a preliminary study of EIF2B5 expression utilizing a truncated version of GFAP, namely gfaABC(1)D, which contains only 681 bp. The gfaABC(1)D promoter also targets astrocytes, although not as strongly as the endogenous GFAP promoter.

[0177] Due to the aforementioned package size capability, a new promoter, the gfa1405 promoter, was designed to obtain a smaller astrocyte-specific promoter. The gfa1405 promoter was designed based on a publication (Lee et al., Glia. 2008;56:481-493) that described the design of a truncated gfaABC(1)D1 promoter by systematically restoring the important transcription factor binding regions of the C components (C3, C5, and C6) while leaving out the inhibitory region (C2). This new promoter, the gfa1405 promoter, is 1405 base pairs (SEQ ID NO: 15) and remains below the packaging threshold of AAV of 4.8 kb when combined with EIF2B5 (i.e., SEQ ID NO: 16 or 17) (see Figures 15 and 16, respectively). See also the schematic diagrams shown in Figures 17 and 18 showing the components of the new promoter (Figure 17) and the gfa1405 promoter with the EIF2B5 transgene (Figure 18).

[0178] [Table 6]

[0179] Figure 19 provides a schematic diagram of the plasmid pAAV.gfa1405.eIF2B5, which contains 7086 bp (SEQ ID NO: 18) containing the gfa1405 promoter together with the eIF2B5 transgene sequence in an AAV vector. Figure 21 provides a detailed description of the 7086 bp sequence of SEQ ID NO: 18. Thus, a fourth AAV genome vector construct encoding EIF2B5 was generated.

[0180] To test the efficacy of the novel promoter, the gfaABCD1405.eGFP plasmid was transfected into human embryonic kidney cells in parallel with the ubiquitous CAG.eGFP construct and two previously published astrocyte constructs, GFAP.eGFP and gfaABC(1)D.eGFP. Details of protein extraction and Western blotting methods are described in Example 1.

[0181] GFP expression was observed 72 hours after transfection (FIG. 20A), and it was clear that the novel gfaABCD1405 promoter drives GFP expression in HEK293 cells. 72 hours after transfection, cells were harvested and proteins were isolated for Western blot analysis. FIG. 20B shows the results of quantification in percent of CAG expression after Western blots were performed in triplicate for GFP expression of all constructs compared to the expression of the housekeeping protein GAPDH. Quantification of the Western blots (shown in the bar graph in FIG. 20B) shows that the expression of GFP protein of gfaABCD1405 (gfa1405) was greater than that of the GFAP and gfaABC(1)D constructs, but not as great as that of the ubiquitous promoter CAG construct. This suggests that GFAP, gfaABC(1)D and gfaABCD1405 are more specific to astrocytes, which may explain the lower expression in kidney cells. Nonetheless, the results show that the novel promoter gfa1405 drove greater amounts of protein expression than other previously published astrocyte-specific promoters.

[0182] As a result, the new promoter gfa1405 was designed to drive expression of genes in astrocytes, but not other cell types, making it useful for treating astrocyte and neuronal diseases. Because astrocytes also crosstalk with neurons, targeting astrocytes with this promoter in neuron-specific diseases may also have therapeutic effects in neuronal diseases. Thus, diseases or conditions that may benefit from therapeutic targeting of astrocytes and neurons using this promoter include, but are not limited to, vanishing white matter disease, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodactyl dysplasia, giant axonal neuropathy, and macrocephalic leukoencephalopathy (Ricci et al. J. Biol Phys 35,317-336, doi:10.1007 / s10867-009-9157-9 (2009); Phatnani, H. & Maniatis, T. Astrocytes in neurodegenerative disease. Cold Spring Harb Perspective Biol 7, doi:10.1101 / cshperspect.a020628(2015), Lanciotti et al., Int J Mol Sci 23, doi:10.3390 / ijms23010274(2021)).

[0183] As a result, the novel promoter, i.e., gfa1405 (also referred to herein as gfaABCD1405), is useful in a method of treating a subject containing a mutation in a gene normally expressed in astrocytes or neurons of a healthy subject, the method comprising administering to the subject an effective amount of a transgene linked to the gfa1405 promoter such that the transgene is expressed in the subject containing the mutated gene.

[0184] Example 7 Astrocyte-targeted gene therapy using the gfaABCD1405 promoter Wild-type (WT) mice were treated on PND0-1 with a dose of 1.2E11vg by intracerebroventricular (ICV) delivery with AAV9.gfaABCD1405-GFP. Robust GFP expression throughout the central neuraxis was seen 4 weeks after treatment. Expression was comparable to or higher than GFAP expression as measured histologically. The morphology of cells expressing GFP driven by the gfaABCD1405 promoter suggests robust targeting of astrocytes within the CNS while restricting gene expression in other CNS cell types such as neurons. See Figure 29. These experiments demonstrated that the astrocyte-specific reporter gfaABCD1405 described herein achieved adequate expression of transgenes in astrocytes leading to its utility in expressing genes including but not limited to eiF2B5.

[0185] Example 8 Astrocyte-targeted gene therapy slows disease progression, rescues body weight, and increases latency to fall in a model of VWM disease Intracerebroventricular (ICV) injection of vectors expressing the reporter protein GFP into wild-type mice revealed that the astrocyte-specific reporter constructs described herein (AAV9.GFAP.eGFP and AAV9.gfaABC(1)D.eGFP) achieved appropriate expression of the transgene in astrocytes, with the AAV9.GFAP.eGFP vector having significantly greater biodistribution throughout the central neuraxis compared to its truncated or ubiquitous counterparts (AAV9.gfaABC(1)D.eGFP and AAV9.CAG.eGFP).

[0186] To evaluate potential therapeutic efficacy, three constructs were initially generated driving expression of the EIF2B5 transgene. However, due to the size capacity of AAV (4.8 kb), the EIF2B5 transgene (2.2 kb) and the GFAP promoter (2.2 kb) combined with the necessary regulatory elements resulted in constructs that were too large, subsequent poor packaging, and low viral titers. Due to the significant difference in expression between the full-length and truncated astrocyte promoters in GFP reporter studies, studies were designed to increase biodistribution by generating a tailored promoter that restores critical sequences from the endogenous GFAP promoter to its truncated gfaABC(1)D counterpart. Thus, a moderate gfaABCD1405 promoter was designed and engineered, and studies have demonstrated expression comparable to full-length GFAP.

[0187] Four AAV constructs were administered to two mouse VWM models, Eif2b5 Arg191His and Eif2b5 Ile98Met These show significant gait defects, myelin loss, and shortened life span. Disease progression was monitored. Arg191His and Eif2b5 Ile98Met Mice were indistinguishable from normal mice on the rotarod when older than 50 days, and were significantly improved compared to untreated R191H diseased mice (P≦0.001) (FIG. 31). Treatment with vectors containing the CAG and GFAP promoters shows moderate efficacy in mice. Eif2b5 treated with AAV9-gfaABCD1405-EIF2B5 Arg191His and Eif2b5 Ile98Met have not yet reached 50 days of age. Data to date indicate that the astrocyte-targeted gene therapy described herein, specifically AAV9-gfaABC(1)D-EIF2B5, can significantly increase rotarod fall latency to a significantly greater extent than ubiquitous (AAV9-CAG-EIF2B5) gene therapy in both disease models (see FIG. 31).

[0188] The improved latency to fall on the rotarod caused by these therapies indicates increased coordination and motor function, which have been shown to be severely limited in both mouse models as well as VWM patients. Fall RPM specifically indicates how fast the spinning rod is accelerating. Thus, higher RPM measurements also indicate increased coordination and motor function. The difference between these two measurements is that RPM measures how fast the rotarod is spinning, while latency to fall measures duration (i.e., time on the rotarod). These two variables are related. Thus, longer duration (longer time) means greater RPM and vice versa. Significant improvements in both measurements after treatment indicate the promise of the disclosed therapeutic methods as potential therapies for VWM.

[0189] Since modifications within the scope of the invention may be apparent to those skilled in the art, the foregoing description is given only for clarity of understanding, and no unnecessary limitations should be understood therefrom. Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" should be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0190] Throughout this specification, when a composition is described as comprising a component or material, it is contemplated that the composition may consist essentially of, or consist of, any combination of the recited components or materials, unless otherwise stated. Similarly, when a method is described as comprising particular steps, it is contemplated that the method may also consist essentially of, or consist of, any combination of the recited steps, unless otherwise stated. The invention illustratively disclosed herein may suitably be practiced in the absence of any element or step not specifically disclosed herein.

[0191] The implementation of the methods disclosed herein, and their individual steps, can be performed manually and / or with the aid of automation provided by electronic devices. Although the process is described with reference to a particular embodiment, one skilled in the art will readily appreciate that other ways of performing the acts associated with the method may be used. For example, unless otherwise stated, the order of various steps may be altered without departing from the scope or spirit of the method. In addition, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0192] All patents, publications, and references cited herein are incorporated by reference in their entirety. In the event of a conflict between this disclosure and the incorporated patents, publications, and references, this disclosure controls. References mentioned herein with numbering are provided with full citations as set forth herein below.

[0193] References 1. Fogli A, Schiffmann R, Bertini E, Ughetto S, Combes P, Eymard-Pierre E, et al. The effect of genotype on the natural history of eIF2B-related leukodystrophies. Neurology. 2004;62(9):1509-17. 2.van der Knaap MS,Pronk JC,and Scheper GC. Vanishing white matter disease. Lancet Neurol. 2006;5(5):413-23. 3. Bonkowsky JL,Nelson C,Kingston JL,Filloux FM,Mundorff MB,and Srivastava R.The burden of inherited leukodystrophies in children. Neurology. 2010;75(8):718-25. 4. Zhou L,Zhang HH,Chen N,Zhang ZB,Liu M,Dai LF,et al.[Clinical features of 54 cases of leukoencephalopathy with vanishing white matter disease in children]. Zhonghua Er Ke Za Zhi.2019;57(11):837-43. 5. Dietrich J,Lacagnina M,Gass D,Richfield E,Mayer-Proschel M,Noble M,et al.EIF2B5 mutations compromise GFAP+ astrocyte generation in vanishing white matter leukodystrophy.Nat Med.2005;11(3):277-83. 6. Geva M,Cabilly Y,Assaf Y,Mindroul N,Marom L,Raini G,et al.A mouse model for eukaryotic translation initiation factor 2B-leucodystrophy reveals abnormal development of brain white matter.Brain. 2010;133(Pt 8):2448-61. 7. Dooves S,Bugiani M,Postma NL,Polder E,Land N,Horan ST,et al.Astrocytes are central in the pathomechanisms of vanishing white matter. J Clin Invest.2016;126(4):1512-24. 8. Terumitsu-Tsujita M,Kitaura H,Miura I,Kiyama Y,Goto F,Muraki Y,et al.Glial pathology in a novel spontaneous mutant mouse of the Eif2b5 gene:a vanishing white matter disease model.J Neurochem.2019. 9.Cearley CN,and Wolfe JH.Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7,8,9,and Rh10 in the mouse brain. Mol Ther. 2006;13(3):528-37. 10. Swain GP,Prociuk M,Bagel JH,O’Donnell P,Berger K,Drobatz K,et al.Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain. Gene Ther. 2014;21(1):28-36. 11. Samaranch L,Salegio EA,San Sebastian W,Kells AP,Foust KD,Bringas JR,et al.Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates.Human gene therapy.2012;23(4):382-9. 12. Hordeaux J,Hinderer C,Buza EL,Louboutin JP,Jahan T,Bell P,et al.Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys. Hum Gene Ther.2019;30(8):957-66. 13.von Jonquieres G,Mersmann N,Klugmann CB,Harasta AE,Lutz B,Teahan O,et al.Glial promoter selectivity following AAV-delivery to the immature brain.PLoS One. 2013;8(6):e65646. 14. Lee,Y.,Messing,A.,Su,M.& Brenner,M.GFAP promoter elements required for region-specific and astrocyte-specific expression.Glia 56,481-493,doi:10.1002 / glia.20622(2008). 15. Ricci,G.,Volpi,L.,Pasquali,L.,Petrozzi,L.& Siciliano,G.Astrocyte-neuron interactions in neurological disorders.J Biol Phys 35,317-336,doi:10.1007 / s10867-009-9157-9(2009). 16. Phatnani,H.& Maniatis,T.Astrocytes in neurodegenerative disease.Cold Spring Harb Perspect Biol 7,doi:10.1101 / cshperspect.a020628(2015). 17. Lanciotti,A.,Brignone,M.S.,Macioce,P.,Visentin,S.& Ambrosini,E.Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies. Int J Mol Sci 23,doi:10.3390 / ijms23010274(2021).

Claims

1. (a) one or more regulatory control elements, and (b) a nucleic acid comprising a polynucleotide comprising a eukaryotic translation initiation factor 2B subunit epsilon 5 (EIF2B5) cDNA sequence.

2. The EIF2B5 cDNA is (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:1; (b) the nucleotide sequence set forth in SEQ ID NO: 1; or (c) a nucleotide sequence encoding EIF2B5 comprising the amino acid sequence set forth in SEQ ID NO:

2.

3. 2. The nucleic acid of claim 1, wherein the one or more regulatory control elements are a CAG promoter, a gfaABC1D promoter, a GFAP promoter, or a gfa1405 promoter.

4. The regulatory control element (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3, 4, 5, or 15; or (b) the nucleic acid of claim 1, comprising the nucleotide sequence set forth in SEQ ID NO: 3, 4, 5, or 15.

5. The nucleic acid of claim 1, further comprising an SV40 intron and a post-transcriptional polyadenylation (polyA) sequence.

6. (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6-8 and 16; or (b) the nucleic acid according to claim 5, comprising a nucleotide sequence shown in any one of SEQ ID NOs: 6 to 8 and 16.

7. The nucleic acid of claim 1, further comprising an inverted terminal repeat sequence.

8. (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-11 and 17; or (b) the nucleic acid according to claim 7, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 9 to 11 and 17.

9. (a) a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-14 and 18; or (b) the nucleic acid according to claim 1, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 12 to 14 and 18.

10. A nanoparticle, extracellular vesicle, exosome, or vector comprising the nucleic acid of claim 1 or any one or more combinations thereof.

11. A viral vector comprising the nucleic acid of claim 1 or any one or more combinations thereof.

12. 12. The viral vector of claim 11, wherein the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus.

13. The viral vector of claim 11, wherein the viral vector is an AAV.

14. The viral vector of claim 13, wherein the AAV lacks the rep and cap genes.

15. The viral vector of claim 13, wherein the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).

16. The viral vector of claim 13, wherein the AAV is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1A, AAVMYO, or AAV-B1, AAV2 / 1, AAV2 / 8, AAV2 / 9, or derivatives thereof.

17. The viral vector of claim 13, wherein the AAV is AAV9.

18. An rAAV particle comprising the viral vector of claim 13.

19. (a) a nucleic acid according to any one of claims 1 to 9; (b) the nanoparticle, extracellular vesicle, exosome, or vector according to claim 10; (c) a viral vector according to any one of claims 11 to 17, or (d) the rAAV particle of claim 18, and a pharmaceutically acceptable carrier.

20. 20. The composition of claim 19, formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

21. 20. The composition of claim 19 for increasing the expression of the EIF2B5 gene or EIF2B5 protein in a cell.

22. 22. The composition of claim 21, wherein the cells are astrocytes.

23. 22. The composition of claim 21, wherein the cell is a human cell.

24. 23. The composition of claim 22, wherein the cell is in a human subject.

25. 20. The composition of claim 19 for treating a subject containing a mutation in the EIF2BB5 gene.

26. 26. The composition of claim 25, wherein the subject is a human subject.

27. 26. The composition of claim 25, wherein a mutation in the EIF2BB5 gene causes the subject to suffer from or be at risk of suffering from leukoencephalopathy, macrocephalic leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutieres syndrome, CLC-2-related disease, oculodentodigital dysplasia, and / or giant axonal neuropathy.

28. 28. The composition of claim 27, wherein the leukoencephalopathy or leukodystrophy is vanishing white matter (VWM).

29. 26. The composition of claim 25, wherein any one or more of a corticosteroid, rituximab, and rapamycin is further administered to the subject.

30. The composition of claim 25, administered intrathecally, intracerebroventricularly, intracerebrally, intravenously, intracisternally, or by aerosol delivery.

31. For the preparation of a medicament for increasing the expression of EIF2B5 gene or protein in a cell, (a) a nucleic acid according to any one of claims 1 to 9; (b) the nanoparticle, extracellular vesicle, exosome, or vector according to claim 10; (c) a viral vector according to any one of claims 11 to 17, or (d) Use of the rAAV particles described in claim 18.

32. 1. A composition for treating a mutation in the EIF2B5 gene, leukoencephalopathy, leukodystrophy, stroke, migraine, epilepsy, multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), astrogliosis in aging, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alexander disease, hepatic encephalopathy (HE), Aicardi-Goutières syndrome, CLC-2-related disease, oculodentodigital dysplasia, giant axonal neuropathy, and / or macrocephalic leukoencephalopathy in a subject, comprising: (a) a nucleic acid according to any one of claims 1 to 9; (b) the nanoparticle, extracellular vesicle, exosome, or vector according to claim 10; (c) a viral vector according to any one of claims 11 to 17, or A composition comprising (d) the rAAV particle of claim 18.

33. 33. The composition of claim 32, wherein the subject is a human subject.

34. 33. The composition of claim 32, wherein the leukoencephalopathy or leukodystrophy is vanishing white matter (VWM).

35. (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 15; or (b) a nucleic acid comprising a gfa1405 promoter comprising the nucleotide sequence set forth in SEQ ID NO:

15.

36. 36. The nucleic acid of claim 35, further comprising an inverted terminal repeat sequence.

37. (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 17; or (b) the nucleotide sequence set forth in SEQ ID NO:

17.

38. (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18; or (b) the nucleotide sequence set forth in SEQ ID NO:

18. The nucleic acid of claim 35 .

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