WO2024259064A1

WO2024259064A1 - Materials and methods for the treatment of neurofibromin 1 mutations and diseases resulting therefrom

Info

Publication number: WO2024259064A1
Application number: PCT/US2024/033750
Authority: WO
Inventors: Allison Marie BRADBURY; Miguel Sena-Esteves
Original assignee: Nationwide Childrens Hospital Inc; University of Massachusetts Amherst
Current assignee: Nationwide Childrens Hospital Inc; University of Massachusetts Amherst
Priority date: 2023-06-13
Filing date: 2024-06-13
Publication date: 2024-12-19
Anticipated expiration: 2025-12-13
Also published as: AU2024305531A1

Abstract

Provided are gene therapy vectors, such as adeno-associated virus (AAV), designed for treatment of mutations in the neurofibromin 1 (NF1) gene. The disclosed gene therapy vectors provide a mini-NF1 cDNA to a subject in need which results in expression of a functional NF1 protein. Also provided are compositions, nanoparticles, extracellular vesicles, exosomes, or vector comprising the NF1 gene with nerve-cell specific and Schwann-cell specific promoters and methods of using the mini-NF1 gene with nerve-cell specific and Schwann-cell specific promoters in treating neurofibromatosis type 1. Also provided are novel mini-NF1 gene constructs.

Description

MATERIALS AND METHODS FOR THE TREATMENT OF NEUROFIBROMIN 1 MUTATIONS AND DISEASES RESULTING THEREFROM

[0001] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: Filename: 59177_SeqListing.xml; Size: 98,544 bytes; Created: June 11 , 2024.

FIELD OF THE INVENTION

[0002] The disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), designed for treatment of mutations in the neurofibromin 1 (NF1 ) gene encoding the neurofibromin protein. More than 1 ,000 NF1 mutations that cause neurofibromatosis type 1 have been identified. Most of these mutations are unique to a particular family. Many NF1 mutations result in the production of an extremely short version of neurofibromin. This shortened neurofibromin protein cannot function normally in inhibiting cell division. When mutations occur in both copies of the NF1 gene in Schwann cells, the resulting loss of neurofibromin allows noncancerous tumors called neurofibromas to form along nerves throughout the body. Such mutations in the NF1 gene are associated with Neurofibromatosis type 1 (also known as von Recklinhausen disease). The disclosed gene therapy vectors provide a reduced size NF1 gene (miniNFI ), i.e., a reduced size NF1 cDNA, for delivery to a subject in need which results in expression of a functional NF1 protein.

BACKGROUND

[0003] Neurofibromatoses are a group of genetic disorders that cause tumors to form on nerve tissue. These tumors can develop anywhere in the nervous system, including the brain, spinal cord and nerves. There are three types of neurofibromatosis: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2) and schwannomatosis. Neurofibromatosis or NF1 is usually diagnosed in childhood, while NF2 and schwannomatosis are usually diagnosed in early adulthood.

[0004] The tumors in these disorders are usually benign, but sometimes can become cancerous. Symptoms are usually mild, but complications of neurofibromatosis can include hearing loss, learning impairment, cardiovascular problems, loss of vision, and severe pain. When neurofibromatosis causes large tumors or tumors that press on a nerve, such as neurofibromas, surgery can reduce symptoms but new treatments are desperately needed.

[0005] Neurofibromatosis Type 1 (NF1) has a complex pathologic burden and patients suffer from cognitive impairment and are plagued with neurofibromas, which affect nerves on multiple organ systems and are often extensive and debilitating. NF1 results from mutations in the NF1 gene which encodes neurofibromin, a tumor suppressor protein. While the genetic etiology of NF1 is clear, developing an efficacious gene therapy for NF1 faces three particular challenges in therapeutic development: 1 ) the coding sequence of NF1 is very large, about 8.5 kb, vastly exceeding the packaging capacity of adeno-associated virus (AAV) vectors making it too large to fit in the best gene therapy currently available; 2) the target cells in the body (Schwann cells), which have an important role in the working of nerves, are difficult to target because they reside inside the avascular endoneurium making access of AAV vectors to these cells challenging; and 3) nerve tumors appear in many places in the body. The disclosure provides novel gene therapy nucleic acid constructs and a gene therapy system that is designed to address these three challenges. The disclosed products, methods and uses provide a feasible approach for robust and long-term expression of a functional mini-NF1 protein in the treatment of NF1 mutations and/or in compositions for use as a medicament.

SUMMARY

[0006] Provided herein are products, methods, and uses for treating mutations in the gene encoding neurofibromin 1 (NF1 ) and in treating, ameliorating, delaying the progression of, and/or preventing diseases resulting from mutations in the NF1 gene.

[0007] The disclosure provides novel mini-NFI transgene constructs for use in treating, ameliorating, delaying the progression of, and/or preventing diseases resulting from mutations in the NF1 gene.

[0008] Thus, the disclosure provides a nucleic acid comprising a polynucleotide encoding a mini-neurofibromin 1 (mini-NF1 ) protein; and a polynucleotide encoding a gfa1405 promoter, a full length CAG (FLCAG) promoter, a PO promoter, a truncated CAG promoter, a gfaABCI D promoter, or a GFAP promoter. In some aspects, the polynucleotide encoding the mini-NF1 protein comprises (a) a nucleotide sequence comprising at least 80% identity to the nucleotide sequence of SEQ ID NO: 1 or 3; (b) the nucleotide sequence of SEQ ID NO: 1 or 3; (c) a nucleotide sequence encoding a mini-NF1 protein comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 2 or 4; or (d) a nucleotide sequence encoding a mini-NF1 protein comprising the amino acid sequence of SEQ ID NO: 2 or 4. In some aspects, the nucleotide sequence encoding the gfa1405 promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 5. In some aspects, the nucleotide sequence encoding the FLCAG promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 6. In some aspects, the nucleotide sequence encoding the P0 promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 7. In some aspects, the nucleotide sequence encoding the truncated CAG promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 8. In some aspects, the nucleotide sequence encoding the gfaABCI D promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 9. In some aspects, the nucleotide sequence encoding the GFAP promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 10.

[0009] In some aspects, the nucleic acid further comprises a polynucleotide encoding an SV40 intron and/or a synthetic polyadenylation signal sequence. In some aspects, the nucleotide sequence encoding the SV40 intron comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 11 . In some aspects, the nucleotide sequence encoding the synthetic polyadenylation signal sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 12.

[0010] In some aspects, the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein comprises (a) a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 17, 19, or 21 ; or (b) the nucleotide sequence of SEQ ID NO: 17, 19, or 21 .

[0011] In some aspects, the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein further comprises a polynucleotide encoding an inverted terminal repeat (ITR). In some aspects, the nucleic acid comprising a polynucleotide encoding a mini-neurofibromin 1 (mini-NF1 ) protein further comprises a polynucleotide encoding at least two inverted terminal repeats (ITRs). In some aspects, the nucleotide sequence encoding a 5’ ITR comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 13; and/or a nucleotide sequence encoding a 3’ ITR comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 14.

[0012] In some aspects, the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein comprises (a) a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 16, 18, or 20; or (b) the nucleotide sequence of SEQ ID NO: 16, 18, or 20.

[0013] In some aspects, the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein further comprises a polynucleotide encoding a stuffer sequence. In some aspects, the nucleotide sequence encoding the stuffer sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 15.

[0014] In some aspects, the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 22, 23, or 24, or the nucleotide sequence of SEQ ID NO: 22, 23, or [0015] The disclosure also provides a nanoparticle, extracellular vesicle, exosome, or vector comprising any nucleic acid of the disclosure or a combination of any one or more thereof. In some aspects, the vector is a viral vector. In some aspects, the viral vector is an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, 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 singlestranded recombinant AAV (ssAAV). In some aspects, the AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1 A, AAVMYO, or AAV-B1 , AAV2/1 , AAV2/8, AAV2/9, or any of their derivatives. In some aspects, the AAV is AAV9. The disclosure likewise comprises an rAAV particle comprising any AAV described herein.

[0016] The disclosure provides a composition comprising: (a) the nucleic acid of any one of claims 1-19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c) the viral vector of any one of claims 21-27; or (d) the rAAV particle of claim 28; and a pharmaceutically acceptable carrier. In some aspects, the composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0017] The disclosure provides a method of increasing the expression of a mini-NF1 gene and/or a mini-NF1 protein in a cell comprising contacting the cell with (a) the nucleic acid of any one of claims 1 -19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c) the viral vector of any one of claims 21-27; (d) the rAAV particle of claim 28; or (e) the composition of claim 29 or 30. In some aspects, the cell is a nerve cell, an oligodendrocyte, and/or a Schwann cell. In some aspects, the cell is a human cell. In some aspects, the cell is in a human subject.

[0018] The disclosure provides a method of treating a subject comprising a neurofibromin 1 (NF1) gene mutation comprising administering to the subject an effective amount of (a) the nucleic acid of any one of claims 1-19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c) the viral vector of any one of claims 21-27; (d) the rAAV particle of claim 28; or (e) the composition of claim 29 or 30. In some aspects, the subject is a human subject. In some aspects, the NF1 gene mutation causes a subject to suffer from or be at risk of suffering from a tumor or a cancer. In some aspects, the tumor is a neurofibroma. In some aspects, the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer. In some aspects, the method of treatment further comprises administering any one or more of a corticosteroid, rituximab, and rapamycin to the subject. In some aspects, the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, rAAV particle, or composition is administered by intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0019] The disclosure provides a use of (a) the nucleic acid of any one of claims 1-19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c) the viral vector of any one of claims 21-27; (d) the rAAV particle of claim 28; or (e) the composition of claim 29 or 30 for the preparation of a medicament for increasing expression of the neurofibromin 1 (NF1 ) gene and/or protein in a cell. In some aspects, the cell is in a human subject. In some aspects, the subject suffers from a lesion, a tumor or a cancer. In some aspects, the lesion, tumor, or cancer is associated with aberrant NF1 expression. In some aspects, the tumor is a neurofibroma or a glioma. In some aspects, the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer. In some aspects, the nucleic acid is administered with any one or more of a corticosteroid, rituximab, and rapamycin. In some aspects, the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, or composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0020] The disclosure provides a use of (a) the nucleic acid of any one of claims 1-19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c) the viral vector of any one of claims 21-27; (d) the rAAV particle of claim 28; or (e) the composition of claim 29 or 30 in treating a subject comprising a mutant neurofibromin 1 (NF1) gene. In some aspects, the subject is a human subject. In some aspects, the subject suffers from a lesion, a tumor or a cancer. In some aspects, the lesion, tumor, or cancer is associated with aberrant NF1 expression. In some aspects, the tumor is a neurofibroma or a glioma. In some aspects, the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer. In some aspects, the nucleic acid is administered with any one or more of a corticosteroid, rituximab, and rapamycin. In some aspects, the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, or composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0021] The disclosure provides a composition for treating a neurofibromin 1 (NF1) gene mutation in a subject, wherein the composition comprises the nucleic acid of any one of claims 1-19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c) the viral vector of any one of claims 21-27; (d) the rAAV particle of claim 28; or (e) the composition of claim 29 or 30. In some aspects, the subject is a human subject. In some aspects, the NF1 gene mutation is associated with the risk or presence of a lesion, a tumor, or a cancer. In some aspects, the tumor is a neurofibroma or a glioma. In some aspects, the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.

[0022] The disclosure provides (a) the nucleic acid of any one of claims 1-19; (b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20; (c)the viral vector of any one of claims 21-27; (d) the rAAV particle of claim 28; (e) the composition of claim 29 or 30; (f) the method of any one of claims 31-41 ; or (g) the use of any one of claims 42-50, wherein the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, composition, or medicament is formulated for intrathecal injection into the cerebrospinal fluid (CSF), intravenous injection into the blood stream, intracerebral injection, intracerebroventricular injection, intracisternal injection, or for aerosol administration.

[0023] The disclosure provides one or more of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, rAAV particles, compositions, or medicaments, wherein the nucleic acids, nanoparticles, extracellular vesicles, exosomes, vectors, viral vectors, or rAAV particles, compositions or medicaments is/are formulated for intrathecal injection into the cerebrospinal fluid (CSF), intravenous injection into the blood stream, intracerebral injection, intracerebroventricular injection, intracisternal injection, or for aerosol administration.

[0024] Other features and advantages of the disclosure will become apparent from the following description of the drawings and the detailed description. It should be understood, however, that the drawings, detailed description, and the examples, while indicating embodiments of the disclosed subject matter, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent from the drawing, detailed description, and the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 shows an AAV9.flCAG.miniNF1 sequence (SEQ ID NO: 22) of the disclosure.

[0026] Fig. 2 shows an AAV9.P0.miniNF1 sequence (SEQ ID NO: 23) of the disclosure.

[0027] Fig. 3 shows an AAV9.gfa1405.miniNF1 sequence (SEQ ID NO: 24) of the disclosure.

[0028] Fig. 4 shows transfection of AAV9-CAG-eGFP and AAV9-P0-eGFP constructs in a Schwannoma cell line.

[0029] Fig. 5 shows Western blot of GFP protein expression after 48 hour transfection in Schwannoma cell line. GAPDH is a loading control and GFP is the protein of interest. The first 3 lanes are a non-transfected control (NTC), the next 3 lanes pAAV-CAG-GFP, and the final 3 lanes pAAV-PO-GFP. The 3 lanes represent the experiment run in triplicate.

[0030] Fig. 6 shows Western blot of miniNFI protein expression after 48 hour transfection in Schwannoma cell line. GAPDH was used as a loading control and HA was the protein of interest (tagged to miniNFI). The first 3 lanes show an untransfected control, the next 3 lanes show pAAV-CAG-miniNF1 -HA, and the final 3 lanes show pAAV-PO- miniNFI HA. The 3 lanes represent the experiment run in triplicate.

[0031] Fig. 7 shows eGFP mRNA expression in NF1 -tumor related tissues 4 weeks after ICV delivery to mouse pup as measured by TaqMan qPCR.

[0032] Fig. 8A-B shows results of immunofluorescence experiments carried out on CNS and PNS tissues to visualize GFP expression. Staining of the brain (Fig. 8A) and peripheral nerve (Fig. 8B) was complete. GFP expression was striking in the brain and sciatic nerve, particularly with the ubiquitous CAG promoter. Fig. 8A shows a sagittal section of the brain from mice (n=3 each) treated at PND1 by ICV delivery with AAV9-1405-GFP (left column) or AAV9-CAG-GFP (right column) 4 weeks after injection. Fig. 8B shows a longitudinal section of sciatic nerve from mice injected (ICV) with ssAAV9. CAG. eGFP stained for neurofilament, myelin basic protein (MBP), green fluorescent protein (GFP), and DAPI.

DETAILED DESCRIPTION

[0033] The disclosure provides neurofibromin 1 (NF1 ) gene replacement as a feasible therapeutic strategy to treat a mutation(s) in the gene that encodes NF1 by delivering a nucleic acid encoding a functional NF1 protein (e.g., a mini-NF1 protein) and, as a result, treat, ameliorate, delay the progression of, or prevent a disease or disorder resulting from an NF1 gene mutation including, but not limited to, skin lesions, neurofibromas, optic pathway gliomas, and malignant peripheral nerve sheath tumors (MPNST) associated with neurofibromatosis type I (NFI). The disclosed products, methods and uses provide a feasible approach for robust and long-term expression of a functional NF1 protein (e.g., a mini-NF1 protein) in human neurons and glia in the treatment of patients suffering from an NF1 mutation resulting in the loss of a functional NF1 protein.

[0034] Neurofibromatosis type I is caused by sporadic or inherited germline mutations in the neurofibromin 1 gene (NF1 gene). Sporadic loss of the remaining wild-type NF1 allele is associated with skin lesions and benign neurofibromas, which develop along peripheral nerves. Malignant complications include conditions such as optic pathway gliomas and malignant peripheral nerve sheath tumors (MPNST). In addition, NF1 haploinsufficiency can cause cognitive deficits in NF1 patients. NF1 deficiency plays an important supporting role in tumor formation. The NF1 protein is a GTPase-activating protein (GAP) that inactivates Ras through activation of GTP to GDP hydrolysis. Loss of NF1 GAP function leaves Ras in the activated state (Ras-GTP) with resulting over-activation of this signaling pathway (RAF- MEK-ERK) (see, e.g., Johnson et al., Neurofibromin 1 inhibits Ras-dependent growth by a mechanism independent of its GTPase-accelerating function, Mol Cell Biol. 1994 January; 14(1 ): 641-645). Ras activation stimulates cell growth and formation of benign tumors which may progress to malignancies (e.g., MPNSTs and optic gliomas). NF1 patients may also show cognitive deficits, suggesting that NF1 plays an important role in normal neuronal function. Reconstitution of normal NF1 function (e.g., by rAAV mediated gene therapy) is capable of repressing RAS over-activation and treating Neurofibromatosis type I and associated conditions. However, the NF1 coding sequence is 8,540 bp, far exceeding the packaging capacity of recombinant AAV vectors. Thus, the disclosure provides a mini-NF1 gene (or an NF1 mini-gene) which can be packaged in an AAV vector and be delivered to a subject to express a functional mini-NF1 protein.

[0035] The disclosure therefore focuses on providing a mini-NF1 replacement gene or “transgene” in order to express normal or functionally active mini-NF1 protein. To accomplish this, specifically designed mini-NF1 replacement genes or “transgenes” are provided. Thus, the disclosure provides isolated nucleic acids and, in some aspects, vectors comprising the transgene for delivery to a cell or to a subject suffering from a mutation in the NF1 gene.

[0036] In some aspects, the nucleic acid of the mini-NF1 replacement gene comprises the nucleotide sequence set forth in SEQ ID NO: 1 or 3, or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 1 or 3. In various aspects, the nucleic acid is an isoform or variant of the nucleotide sequence nucleotide sequence set forth in set forth in SEQ ID NO: 1 or 3. 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 or 3. See Table 1 .

[0037] In some aspects, the polypeptide is a mini-NF1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4. In various aspects, the polypeptide is an isoform or variant of the mini-NF1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4. 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 amino acid sequence set forth in SEQ ID NO: 2 or 4. See Table 1 . [0038] In some aspects, the transgene polynucleotide sequence is operatively linked to transcriptional control elements (including, but not limited to, promoters, enhancers and/or polyadenylation signal sequences) that are functional in target cells.

[0039] In some aspects, the promoter is specifically selected to target cells in the central nervous system (CNS), including the brain, and in the peripheral nervous system (PNS). The CNS is composed of two major cell types: neurons and glia. The glia comprises astrocytes and oligodendrocytes. Astrocytes, through an intricate network surrounding blood vessels, play an important role in supplying food, water and ions from periphery to the CNS and maintain CNS homeostasis. Astrocytes also play an active role in neurogenesis. However, under inflammatory or neurodegenerative conditions, astrocytes produce proinflammatory mediators and take active part in the ongoing events. Neurons in the CNS are covered by a myelin sheath that maintains conduction of nerve impulse. Consistently, the CNS houses oligodendrocytes for myelin synthesis. Schwann cells are the myelinating cells in the PNS. Balanced expression of several genes and activation of transcription factors critically regulate the entire complicated functional network of astrocytes, oligodendrocytes and Schwann cells.

[0040] In some aspects of the disclosure, promoters are selected to target the CNS and the PNS. In some aspects, the promoter targets astrocytes or Schwann cells. In some aspects, the promoter is a gfa1405 (also called gfaABCD1405) promoter, a myelin protein zero (MPZ, P0) promoter (Ahmed et al. J Neurosci Methods. 2019 Jul 15; 323: 77-81 ; published online 2019 May 22. doi: 10.1016/j.jneumeth.2019.05.007), an FLCAG promoter, a gfaABCI D promoter, or a GFAP promoter.

[0041] The gfa1405 (also identified interchangeably as the gfaABCD1405 promoter) is a novel promoter, first described in International Patent Application No. PCT/US2023/063676. The gfa1405 promoter was designed to specifically target astrocytes and neurons and can be used to express any gene desired to be expressed in astrocytes or neurons. In some aspects, therefore, the gfa1405 promoter is designed and used to express mini-NF1 . In some aspects, the gfa1405 promoter comprises the nucleotide sequence of SEQ ID NO: 5 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 5. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 5. 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: 5. See Table 1 . [0042] The full length CAG (FLCAG) promoter comprises the nucleotide sequence of SEQ ID NO: 6 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 6. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 6. 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: 6. See Table 1 .

[0043] The P0 promoter (Ahmed et al., supra) comprises the nucleotide sequence of SEQ ID NO: 7 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 7. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in set forth in SEQ ID NO: 7. 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: 7. See Table 1 .

[0044] The CAG promoter is a ubiquitous promoter which targets neurons and astrocytes. In some aspects, the CAG promoter comprises the nucleotide sequence of SEQ ID NO: 8 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 8. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 8. 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: 8. Although the CAG promoter is commonly referred to only as the "CAG promoter", it is not a promoter in a strict sense, as it comprises both a promoter and an enhancer. The CAG promoter of SEQ ID NO: 8 comprises a CMV enhancer (nucleotides 1-306 of SEQ ID NO: 8) and a CBA promoter (nucleotides 307-581 of SEQ ID NO: 8). See Table 1.

[0045] The gfaABCI D promoter and the GFAP promoter drive transgene expression primarily toward astrocytes. In some aspects, the gfaABCI D promoter comprises the nucleotide sequence of SEQ ID NO: 9 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 9. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 9. 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: 9. See Table 1 . [0046] In some aspects, the GFAP promoter comprises the nucleotide sequence of SEQ ID NO: 10 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 10. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 10. 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: 10. See Table 1 .

[0047] In some aspects, a nucleic acid of the disclosure further comprises an SV40 intron and/or a poly(A) tail (or poly(A) sequence). Thus, in some aspects, a nucleic acid of the disclosure comprises a promoter, an SV40 intron, a mini-NF1 open reading frame, and/or a polyA tail.

[0048] In some aspects, the SV40 intron comprises the nucleotide sequence of SEQ ID NO: 11 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 11 . In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 11 . 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: 11 . See Table 1 .

[0049] In some aspects, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 12 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 12. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 12. 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: 12. See Table 1 .

[0050] In some aspects, a nucleic acid of the disclosure further comprises inverted terminal repeats(ITR) sequences (e.g., 5’ ITR sequence and/or 3’ ITR sequence, or 5’ or 3’ ITRs). In some aspects, ITR sequences are important for intermolecular recombination and circularization of adeno-associated virus (AAV) genomes. Thus, in some aspects, a nucleic acid of the disclosure comprises a promoter, an SV40 intron, a mini-NF1 open reading frame, a polyA tail, and a 5’ ITR and 3’ ITR. Thus, in some aspects, the transgene is flanked by AAV ITRs. In some aspects, the ITRs are AAV ITRs of a serotype including, but not limited to, AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR. [0051] In some aspects, the ITRs are AAV2 ITRs. In some aspects, the 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 13 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 13. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 13. 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: 13. In some aspects, the 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 14 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 14. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 14. 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: 14. See Table 1 .

[0052] Thus, in some aspects, the disclosure provides a nucleic acid (herein also interchangeably referred to as a nucleic acid construct, or construct) comprising a 5’ ITR, a promoter, an SV40 intron, a mini-NF1 open reading frame, a polyA tail, and a 3’ ITR comprising the nucleotide sequence set forth in SEQ ID NO: 16, 18, or 20. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 16, 18, or 20. 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: 16, 18, or 20. See Table 1 .

[0053] In some other aspects, the disclosure provides a nucleic acid comprising a promoter, an SV40 intron, a mini-NF1 open reading frame, and/or a polyA tail without the 5’ and 3’ ITR sequences because the transgene sequences, in various aspects, are used in self-complementary and/or single-stranded AAV viral vectors. Thus, in some aspects, the disclosure provides a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 17, 19, or 21 . In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 17, 19, or 21 . 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: 17, 19, or 21 . See Table 1 . [0054] In some aspects, a nucleic acid of the disclosure further comprises a stuffer sequence to improve transgene expression including, but not limited to, preventing reverse packaging of the transgene construct in the AAV vector. In some aspects, therefore, a nucleic acid of the disclosure comprises a 5’ ITR, a promoter, an SV40 intron, a mini-NF1 open reading frame, a polyA tail, a 3’ ITR, and a stuffer sequence.

[0055] In some aspects, the stuffer sequence comprises the nucleotide sequence of SEQ ID NO: 15 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 15. In various aspects, the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 15. 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: 15. See Table 1 .

[0056] In some aspects, therefore, a nucleic acid of the disclosure comprises a 5’ ITR, a promoter, an SV40 intron, the mini-NF1 open reading frame, a polyA tail, a 3’ ITR, and a stuffer sequence. In some aspects, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 22, 23, or 24 or a codon-optimized variant of the nucleotide sequence set forth in SEQ ID NO: 22, 23, or 24. 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: 22, 23, or 24. See Table 1 and Figs. 1-3.

[0057] Table 1. Sequences of the human mini-NF1 gene, mini-NF1 protein, and CNS- and PNS-specific promoters in various aspects of the disclosure.

[0058] A sequence index table (Table 2) is provided below for reference to sequences provided in the sequence listing.

[0059] Table 2 - Sequence index.

[0060] In some aspects, the disclosure includes a nanoparticle, extracellular vesicle, exosome, or vector comprising any of the nucleic acids of the disclosure or a combination of any one or more thereof for providing mini-NF1 gene replacement. In some aspects, one or more copies of these sequences are combined into a single nanoparticle, extracellular vesicle, exosome, or vector.

[0061] The disclosure therefore includes vectors comprising a nucleic acid of the disclosure or a combination of nucleic acids of the disclosure. Embodiments of the disclosure utilize vectors (for example, viral vectors, such as adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox virus, herpes virus, herpes simplex virus, polio virus, sindbis virus, vaccinia virus or a synthetic virus, e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and/or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule) to deliver the nucleic acids disclosed herein.

[0062] The disclosure provides a recombinant (r) AAV vector comprising the nucleic acid comprising a polynucleotide encoding the mini-NF1 protein for use in treating a subject comprising a mutation in the mini-NF1 gene. In some particular aspects, the AAV is an ssAAV or an ssrAAV. In some aspects, a nucleic acid of the disclosure comprises an AAV vector comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1-24 (Table 1). In various aspects, the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 1 -24. 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: 1-24. In some aspects, a nucleic acid of the disclosure comprises an AAV vector comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 16-24 (Table 1). In various aspects, the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-24. 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: 16-24.

[0063] AAV is unique in its safety profile, as the viral genome, once transduced into its carrier cell, remains stably expressed as an episomal DNA and only very rarely ever integrates into the host genome.

[0064] In some aspects, therefore, the disclosure utilizes AAV to deliver a mini-NF1 transgene, such as DNA encoding a mini-NF1 protein. As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. An "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs).

[0065] Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. AAV is a single-stranded replication-deficient DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. The genome of AAV is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1 , pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, 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 all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous selfannealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0066] There are multiple serotypes of AAV. In some aspects, 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, for example rAAV with capsid mutations, are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11): 1900-1909 (2014). As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.

[0067] The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV1 is provided in GenBank Accession No.

NC_002077; the complete genome of AAV2 is provided in 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_00 1862; at least portions of AAV7 and AAV8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent 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); the AAV10 genome is provided in Mol. Then, 13(1): 67-76 (2006); and the AAV11 genome is provided in Virology, 330(2): 375-383 (2004). Information regarding MyoAAV 1A is provided by Tabebordbar et al. (Cell 184(19): 4919-38 (2021 )).

Information regarding AAVMYO is provided by 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 also are known in the art. Cis- acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1 , VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related 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).

[0068] Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromo-some integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1 , VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related 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).

[0069] AAV possesses 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 noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and nondividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus, making cold preservation of AAV less critical. AAV may be lyophilized and AAV-infected cells are not resistant to superinfection.

[0070] In some aspects, the AAV lacks rep and cap genes. In some aspects, the AAV is a recombinant linear AAV (rAAV), a single-stranded AAV, or a recombinant self- complementary AAV (scAAV). The self-complementary (sc) technology allows for binding of the single-stranded viral DNA genome onto itself, thereby priming second strand DNA synthesis. This sc element both quickens and strengthens gene expression relative to constructs lacking the sc element. In some particular aspects, the AAV is an ssAAV or an ssrAAV.

[0071] Advances in AAV vectors have led to safer and more efficient viral vehicles to deliver therapeutic transgenes in a single injection, and gene therapy is now a favorable therapeutic intervention for monogenic diseases. AAV vectors can provide long-term expression of gene products in post-mitotic target tissues. Thus, current AAV-based strategies may only require one-time vector administration.

[0072] Recombinant AAV genomes of the disclosure comprise one or more AAV ITRs flanking a polynucleotide encoding, for example, one or more mini-NF1 polynucleotides, including any one or more of the sequences set out in SEQ ID NO: 1 or 3, or any one or more of the sequences set out in SEQ ID NOs: 16-24. Provided herein are rAAV, each comprising one or more mini-NF1 genes. An rAAV comprising one or more mini-NF1 genes can encode one, two, three, four, five, six, seven or eight mini-NF1 proteins.

[0073] In some aspects, therefore, the viral vector is an AAV, such as an AAV1 (i.e., an AAV containing AAV1 inverted terminal repeats (ITRs) and AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., an AAV containing AAV9 ITRs and AAV9 capsid proteins), AAV10 (i.e., an AAV containing AAV10 ITRs and AAV10 capsid proteins), AAV11 (i.e., an AAV containing AAV11 ITRs and AAV11 capsid proteins), AAV12 (i.e., an AAV containing AAV12 ITRs and AAV12 capsid proteins), AAV13 (i.e., an AAV containing AAV13 ITRs and AAV13 capsid proteins), AAVanc80 (i.e., an AAV containing AAVanc80 ITRs and AAVanc80 capsid proteins), AAVrh.74 (i.e., an AAV containing AAVrh.74 ITRs and AAVrh.74 capsid proteins), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid proteins), AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid proteins), MyoAAV 1 A, AAVMYO, or AAV-B1 , or pseudotyped AAV, such as AAV2/1 , AAV2/8, or AAV2/9, or AAVMYO, or any of their derivatives.

[0074] In various aspects, the AAV is AAV9. AAV9 has become the most widely used vector for muscular and/or neurological indications with an established safety profile in the clinic. Intrathecal administration of AAV9 permits dissemination of transgenes throughout the nervous system and is currently approved by FDA for spinal muscular atrophy (SMA, NCT03381729), and in trials for the treatment of neuronal ceroid lipofuscinosis 3 (CLN3, NCT03770572), CLN6 (NCT02725580), giant axonal neuropathy (GAN, NCT02362438), mucopolysaccharidoses types 3A (NCT02716246) and 3B (NCT03315182), and exon 2 duplications in the DMD gene (NCT04240314). Such features make AAV9 an ideal gene delivery method for treatment of genetic disorders, such as mutations in EIF2B5, which result in white matter abnormalities in the central nervous system. It has been shown that AAV9 can also target Schwann cells, and other peripheral neuropathies. More importantly, AAV9 was reported to transduce Schwann cells in large animals and non-human primates, indicating that it is a desirable viral vector for clinical applications requiring delivery of therapeutic genes into the human Schwann cells. Finally, data from studies in other models of CNS disease show that an AAV9 vector efficiently transfects CNS (Lukashchuk et al., Molecular Therapy 3:15055, 2016; doi.org/10.1038/mtm.2015.55).

[0075] DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1 -deleted adenovirus or herpes virus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than 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 aspects, AAV DNA in the rAAV genomes is from any AAV serotype for 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 1 A, AAVMYO, or AAV- B1 . Other types of rAAV variants, for example rAAV with capsid mutations, are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11 ): 1900- 1909 (2014). As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety. An "AAV virion" or "AAV viral particle" or “AAV particle” or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector". Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than 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 their derivatives. In some aspects, AAV DNA in the rAAV genomes is from any AAV serotype for 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 their derivatives. Other types of rAAV variants, including those for example with capsid mutations, are also included in the disclosure. Such variants include, but are not limited to, MyoAAV or AAVMYO, and other variants as 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 Tabebordbar, M. et al., Cell 184(19): 4919-4938 e22 (2021 ), which are incorporated for use herein by reference in their entirety. As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.

[0076] In some embodiments, the viral vector is a pseudotyped AAV, containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudo-typed AAV is AAV2/9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2/8 (i.e., an AAV containing AAV2 ITRs and AAV8 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2/1 (i.e., an AAV containing AAV2 ITRs and AAV1 capsid proteins).

[0077] In some embodiments, the AAV contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more of capsid proteins from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, MyoAAV 1 A, AAVMYO, or AAV-B1 , AAVAnc80, AAV7m8, AAV2/1 , AAV2/8, or AAV2/9 and their derivatives. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, 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.

[0078] Multiple studies have demonstrated long-term (>1 .5 years) recombinant AAV- mediated protein expression. See, Clark etal., Hum Gene Ther, 8: 659-669 (1997)³²;

Kessler etal., Proc Nat. Acad Sc. USA, 93 14082-14087 (1996); and Xiao eta!., 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).

[0079] Recombinant AAV genomes, in various aspects, comprise nucleic acids of the disclosure and one or more AAV ITRs flanking the nucleic acid. AAV DNA in the rAAV genomes may be from any AAV serotype for 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 1 A, AAVMYO, or AAV-B1 , AAVAnc80, AAV7m8, AAV2/1 , AAV2/8, or AAV2/9 and their derivatives). Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11 ): 1900-1909 (2014)²⁹. As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0080] The provided recombinant AAV {i.e., infectious encapsidated rAAV particles) comprise a rAAV genome. The term “rAAV genome” refers to a polynucleotide sequence that is 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 the 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 on the 5’ and 3’ ends by inverted terminal repeat (ITR). In some embodiments, the rAAV genome comprises a “gene cassette.” In exemplary embodiments, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes.

[0081] DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1 -deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (/.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than 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 their derivatives. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.

[0082] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing, addition of synthetic linkers containing restriction endonuclease cleavage sites⁴¹ or by direct, blunt-end ligation. The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells. [0083] 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 are described in 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. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., Oct; 8(10):3988-96 (1988); Samulski etal., J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95/13365 and corresponding U.S. Patent No. 5,658.776 ; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. Vaccine 13:1244-1250 (1995); Paul et 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 hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production. The production and use of self-complementary (sc) rAAV are specifically contemplated and exemplified.

[0084] The disclosure thus provides packaging cells that produce infectious rAAV. In one embodiment, packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

[0085] In some aspects, 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 virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Then, 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.

[0086] Compositions comprising the nucleic acids and viral vectors of the disclosure are provided. Compositions comprising delivery vehicles (such as rAAV) described herein are provided. In various aspects, such compositions also comprise a pharmaceutically acceptable carrier. In some aspects, a pharmaceutically acceptable carrier is a diluent, excipient, or buffer. The compositions may also comprise other ingredients, such as adjuvants. [0087] Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to recipients and are 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, pluronics or polyethylene glycol (PEG).

[0088] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0089] Titers of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about 1 x10⁶, about 1 x10⁷, about 1 x10⁸, about 1 x10⁹, about 1 x10¹⁰, about 1 x10¹¹ , about 1 x10¹², about 1 x10¹³ to about 1 x10¹⁴ or more DNase resistant particles (DRP) per ml.

[0090] Dosages of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the time of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Dosages may be expressed in units of viral genomes (vg). Dosages contemplated herein include, but are not limited to, a dosage of about 1 x10⁷, about 1 x10⁸, about 1 x10⁹, about 5x10⁹, about 6x10⁹, about 7x10⁹, about 8x10⁹, about 9x10⁹, about 1 x10¹⁰ , about 2x10¹⁰, about 3x10¹⁰, about 4x10¹⁰, about 5x10¹⁰, about 6x1 O¹⁰, about 7x1 O¹⁰, about 8x1 O¹⁰, about 9x1 O¹⁰, about 1 x10¹¹ , about 2x10¹¹ , about 3x10¹¹ , about 4x10¹¹ , about 5x10¹¹ , about 6x10¹¹ , about 7x10¹¹, about 8x10¹¹, about 9x10¹¹, about 1x10¹², about 2x10¹², about 3x10¹², about 4x10¹², about 5x10¹², about 6x10¹², about 7x10¹², about 8x10¹², about 9x10¹², about 1 x10¹³, about 1 .1 x10¹³, about 1.2x10¹³, about 1.3x10¹³, about 1 .5x10¹³, about 2 x10¹³, about 2.5 x10¹³, about 3x10¹³, about 3.5x10¹³, about 4x10¹³, about 4.5x10¹³, about 5x10¹³, about 6x10¹³, about 7x10¹³, about 8x10¹³, about 9x10¹³, about 1x10¹⁴, about 2x10¹⁴, about 3x10¹⁴, about 4x10¹⁴, about 5x10¹⁴, about 1x10¹⁵, to about 1x10¹⁶, or more total viral genomes.

[0091] Dosages of about 1 x10⁹ to about 1x10¹⁰, about 5x10⁹ to about 5x10¹⁰, about 1 x10¹⁰ to about 1 x10¹¹ , about 1 x10¹¹ to about 1 x10¹⁵ vg, about 1 x10¹² to about 1 x10¹⁵ vg, about 1 x10¹² to about 1 x10¹⁴ vg, about 1 x10¹³ to about 6x10¹⁴ vg, about 1 x10¹³ to about 1 x10¹⁵ vg and about 6x10¹³ to about 1 .0x10¹⁴ vg are also contemplated. One dose exemplified herein is 1 x10¹³ vg administered via intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0092] Dosages of rAAV to be administered also, in various aspects, are expressed in units of vg/kg. Such dosages include, but are not limited to, a dosage of about 1 x10⁷ vg/kg, about 1x10⁸ vg/kg, about 1 x10⁹ vg/kg, about 5x10⁹ vg/kg, about 6x10⁹ vg/kg, about 7x10⁹ vg/kg, about 8x10⁹ vg/kg, about 9x10⁹ vg/kg, about 1x10¹⁰ vg/kg, about 2x10¹⁰ vg/kg, about 3x10¹⁰ vg/kg, about 4x10¹⁰ vg/kg, about 5x10¹⁰ vg/kg, about 1x10¹¹ vg/kg, about 5x10¹¹ vg/kg, about 1 x10¹² vg/kg, about 2x10¹² vg/kg, about 3x10¹² vg/kg, about 4x10¹² vg/kg, about 5x10¹² vg/kg, about 6x10¹² vg/kg, about 7x10¹² vg/kg, about 8x10¹² vg/kg, about 9x10¹² vg/kg, about 1x10¹³ vg/kg, about 1.1x10¹³ vg/kg, about 1.2x10¹³ vg/kg, about 1 .3x10¹³ vg/kg, about 1.5x10¹³ vg/kg, about 2x10¹³ vg/kg, about 2.5x10¹³ vg/kg, about 3x10¹³ vg/kg, about 3.5x10¹³ vg/kg, about 4x10¹³ vg/kg, about 4.5x10¹³ vg/kg, about 5x10¹³ vg/kg, about 6x10¹³ vg/kg, about 7x10¹³ vg/kg, about 8x10¹³ vg/kg, about 9x10¹³ vg/kg, about 1x10¹⁴ vg/kg, about 2x10¹⁴ vg/kg, about 3x10¹⁴ vg/kg, about 4x10¹⁴ vg/kg, about 5x10¹⁴ vg/kg, about 1 x10¹⁵ vg/kg, or about 1 x10¹⁶ vg/kg.

[0093] Dosages of about 1x10⁹ vg/kg to about 1 x10¹⁰ vg/kg, about 5x10⁹ vg/kg to about 5x1 O¹⁰ vg/kg, about 1 x10¹⁰ vg/kg to about 1 x10¹¹ vg/kg, about 1 x10¹¹ vg/kg to about 1 x10¹⁵ vg/kg, about 1 x10¹² vg/kg to about 1 x10¹⁵ vg/kg, about 1 x10¹² vg/kg to about 1 x10¹⁴ vg/kg, about 1 x10¹³ vg/kg to about 2x10¹⁴ vg/kg, about 1 x10¹³ vg/kg to about 1 x10¹⁵ vg/kg and about 6x10¹³ vg/kg to about 1.0x10¹⁴ vg/kg are also included in various aspects. Some doses exemplified herein are about 1 .5x10¹¹ vg/kg or about 3x10¹³ vg/kg administered via intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

[0094] Transduction or transfection of cells with rAAV of the disclosure results in sustained expression of the mini-NF1 gene/protein. As used herein, the terms “transduction” and “transfection” are used interchangeably. The term “transduction” or “transfection” is used to refer to, as an example, the administration/delivery of the mini-NF1 gene to a target cell either in vivo or in vitro, via a replication-deficient rAAV described herein resulting in the expression of the mini-NF1 gene/protein by the target cell. The disclosure thus provides methods of administering/delivering rAAV which express the mini-NF1 gene to a cell or to a subject. In some aspects, the subject is a mammal. In some aspects, 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 described herein. Transduction may be carried out with gene cassettes comprising cell-specific control elements.

[0095] Methods of transducing a target cell, such as an astrocyte, with a delivery vehicle (such as a nanoparticle, extracellular vesicle, exosome, or vector (e.g., rAAV)), in vivo or in vitro, are provided. The in vivo methods comprise the step of administering an effective dose, or effective multiple 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 prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development 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, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. Thus, methods are provided of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV described herein to a subject in need thereof.

[0096] Provided herein are nucleic acids (in some aspects, as a form of a medicament) and methods for treating, ameliorating, or preventing diseases associated with a mutant NF1 gene or aberrant NF1 gene expression. Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the methods. The level of human NF1 or mini-NF1 transcript in animals and/or in humans can be confirmed by RT-PCR and/or RNAseq. The level of human NF1 or mini-NF1 protein expression level in tissues and organs of interest can be assessed using western blotting. EIF2B5 localization can be confirmed by immunohistochemistry.

[0097] Combination therapies are also contemplated by the disclosure. Combination as used herein includes both simultaneous treatment and sequential treatments. Combinations of methods of the disclosure with standard medical treatments are specifically contemplated, as are combinations with novel therapies. In some embodiments, the combination therapy comprises administering an immunosuppressing agent in combination with the gene therapy disclosed herein.

[0098] The immunosuppressing agent may be administered before or after the onset of an immune response to the rAAV in the subject after administration of the gene therapy. In addition, the immunosuppressing agent may be administered simultaneously with the gene therapy or the protein replacement therapy. The immune response in a subject includes an adverse immune response or an inflammatory response following or caused by the administration of rAAV to the subject. The immune response may be the production of antibodies in the subject in response to the administered rAAV.

[0099] Exemplary immunosuppressing agents include glucocorticosteroids, janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, cyctostatic agents such as purine analogs, methotrexate and cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, biologies such as monoclonal antibodies or fusion proteins and polypeptides, and di peptide boronic acid molecules, such as Bortezomib.

[00100] The immunosuppressing agent may be an anti-inflammatory steroid, which is a steroid that decreases inflammation and suppresses or modulates the immune system of the subject. Exemplary anti-inflammatory steroid are glucocorticoids such as prednisolone, betamethasone, dexamethasone, methotrexate, hydrocortisone, methylprednisolone, deflazacort, budesonide or prednisone.

[00101] Janus kinase inhibitors are inhibitors of the JAK/STAT signaling pathway by targeting one or more of the Janus kinase family of enzymes. Exemplary janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.

[00102] Calcineurin inhibitors bind to cyclophilin and inhibits the activity of calcineurin Exemplary calcineurine inhibitors includes cyclosporine, tacrolimus and picecrolimus.

[00103] mTOR inhibitors reduce or inhibit the serine/threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include rapamycin (also known as sirolimus), everolimus, and temsirolimus.

[00104] The immunosuppressing agents include immune suppressing macrolides. The term “immune suppressing macrolides” refer to macrolide agents that suppresses or modulates the immune system of the subject. A macrolide is a class of agents that comprise a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desoamine, are attached. The lactone rings are usually 14-, 15-, or 16-membered.

Macrolides belong to the polyketide class of agents and may be natural products. Examples of immunosuppressing macrolides include tacrolimus, pimecrolimus, and rapamycin (also known as sirolimus).

[00105] Purine analogs block nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolate such as mycophenolate acid or mycophenolate mofetil and lefunomide.

[00106] Exemplary immunosuppressing biologies include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacep, and daclizumab.

[00107] In particular, the immunosuppressing agent is an anti-CD20 antibody. The term anti-CD20 specific antibody refers to an antibody that specifically binds to or inhibits or reduces the expression or activity of CD20. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab or ofatumumab.

[00108] Additional examples of immuosuppressing antibodies include anti-CD25 antibodies (or anti- 1 L2 antibodies or anti-TAC antibodies) such as basiliximab and daclizumab, and anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplizumab and visilizumab, anti-CD52 antibodies such as alemtuzumab.

[00109] One exemplary combination therapy is the delivery of rapamycin and rituximab prior to, or contemporaneous with, delivery of the AAV vector. Another exemplary combination therapy is the delivery of rapamycin, rituximab, and a corticosteroid, such as prednisone.

[00110] Administration of an effective dose of a nucleic acid, nanoparticle, extracellular vesicle, exosome, viral vector, or composition of the disclosure may be by routes standard in the art including, but not limited to, intrathecal, intracerebral, intracerebroventricular, intracisternal, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. In various aspects, an effective dose is delivered by a combination of routes. For example, in various aspects, an effective dose is delivered intrathecally, intracerebrally, intracerebroventricularly, intravenously, intracisternally, and/or intramuscularly, or intrathecally and/or intravenously and/or intracerebroventricularly, and the like. In some aspects, an effective dose is delivered in sequence or sequentially. In some aspects, an effective dose is delivered simultaneously. Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure may be chosen and/or matched by those skilled in the art taking into account the infection and/or disease state being treated and the target cellsZtissue(s) that are to express the EIF2B5 gene.

[00111] In particular, actual administration of delivery vehicle (such as rAAV) may be accomplished by using any physical method that will transport the delivery vehicle (such as rAAV) into a target cell (i.e., an astrocyte) of a subject. Administration includes, but is not limited to, injection into the cerebrospinal fluid (CSF) (intrathecally), injection intracerebroventricularly, injection intracerebrally, injection into the bloodstream and/or directly into the nervous system, injection intracisternally (or via intracisterna magna ( ICM)), or nasally. Simply resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as neurons. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations for intrathecal injection or as aerosol formulations for inhalation. Numerous formulations for intrathecal, intracerebroventricular, intracerebral, or intracisternal injection have been previously developed and can be used in the practice of the methods of the disclosure. The delivery vehicle (such as rAAV) can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[00112] A dispersion of delivery vehicle (such as rAAV) can also be prepared in glycerol, sorbitol, liquid polyethylene glycols and mixtures thereof and 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 obtainable by standard techniques well-known to those skilled in the art.

[00113] The pharmaceutical forms suitable 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 must be 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 actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, 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, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The 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 isotonic agents, for example, suMPZ or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00114] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[00115] In some aspects, the disclosure relates to compositions and methods useful for treating certain genetic diseases, for example Neurofibromatosis type I (NF1) and/or conditions associated thereof. NF1 is caused by sporadic or inherited germline mutations in the Neurofibromin 1 gene (NF1 gene). Sporadic loss of the remaining wild-type NF1 allele is associated with skin lesions and benign neurofibromas, which develop along peripheral nerves. In some aspects, the NF1 deficiency also is associated with cognitive impairment and benign and malignant tumors. Malignant complications include conditions such as optic pathway gliomas and malignant peripheral nerve sheath tumors (MPNST). Likewise, insufficient NF1 expression is also associated with some other cancers. In some aspects, the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.

[00116] In addition, NF1 haploinsufficiency can cause cognitive deficits in Neurofibromatosis type I patients. NF1 deficiency plays an important supporting role in tumor formation. The NF1 protein is a GTPase-activating protein (GAP) that inactivates Ras through activation of GTP to GDP hydrolysis. Loss of NF1 GAP function leaves Ras in the activated state (Ras-GTP) with resulting over-activation of this signaling pathway (RAF- MEK-ERK) (see, e.g., Johnson et al., Neurofibromin 1 inhibits Ras-dependent growth by a mechanism independent of its GTPase-accelerating function, Mol Cell Biol. 1994 January; 14(1 ): 641-645). Ras activation stimulates cell growth and formation of benign tumors which may progress to malignancies (e.g., MPNSTs and optic gliomas). NF1 patients may also show cognitive deficits, suggesting that NF1 plays an important role in normal neuronal function. Reconstitution of normal NF1 function (e.g., by rAAV mediated gene therapy) is capable of repressing RAS over-activation and treating Neurofibromatosis type I and associated conditions. However, the NF1 coding sequence is 8,540 bp, far exceeding the packaging capacity of recombinant AAV vectors. In some embodiments, an NF1 protein coding sequence comprises the nucleic acid sequence set forth in NCBI Reference Sequence Accession Number NM 001042492.3, or splice variants thereof generated by incorporation of exons 9a, 23a, or 48a. In some embodiments, an NF1 gene encodes a protein having the amino acid sequence set forth in NCBI Reference Sequence Accession Number NP 001035957.1 , or protein isoforms with additional amino acids resulting from incorporation of exons 9a, 23a, and 48a in the NF1 mRNA. In some embodiments, a wildtype full-length NF1 coding sequence comprises 61 exons.

[00117] The disclosure, in some aspects, provides an “effective amount” of a nucleic acid, nanoparticle, vector, or composition in a sufficient amount to infect a sufficient number of cells of a target tissue in a subject. In some embodiments, a target tissue is nervous system (e.g., neuron cells having loss of function of NF1 , etc.) tissue. In some embodiments, a transgene is delivered to neurons (e.g., peripheral neurons, such as the optic nerve). An effective amount may also depend on the mode of administration. For example, targeting a nervous tissue (e.g., peripheral neuron, etc.) tissue by intrastromal administration or subcutaneous injection may require different (e.g., higher or lower) doses, in some cases, than targeting a nervous tissue (e.g., peripheral neuron, etc.) by another method (e.g., systemic administration, topical administration). In some embodiments, intrastromal injection (IS) of rAAV mediates efficient transduction of a nervous tissue (e.g., peripheral neuron, etc.). Thus, in some embodiments, the injection is intrastromal injection (IS). In some embodiments, the administration is via injection, optionally via intratumoral injection, etc. In some embodiments, the injection is topical administration (e.g., topical administration to the skin lesion). In some cases, multiple doses of a rAAV are administered.

[00118] An effective amount, in some aspects, is an amount sufficient to have a therapeutic benefit in a subject, e.g., to improve in the subject one or more symptoms of disease, e.g., a symptom of NF1 (e.g., a disease associated with a mutation of NF1 gene). Examples of mutations in NF1 gene include those described by The Human Gene Mutation Database (HGMD, Institute of Medical Genetics, Cardiff, http://www.hgmd.org), by the Leiden Open Variation Database (LOVD), which are incorporated herein by reference. In some embodiments, the mutations in the NF1 gene include those described in Wu-Chou et al, Genetic diagnosis of neurofibromatosis type 1 : targeted next-generation sequencing with Multiple Ligation-Dependent Probe Amplification analysis, Journal of Biomedical Science (2018) 25:72; Yang et al., The investigation for potential modifier genes in patients with neurofibromatosis type 1 based on next-generation sequencing, OncoTargets and Therapy 2018:11 919-932, which are incorporated herein by reference). The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue. In some aspects, an effective amount may also depend on the rAAV used.

[00119] In some embodiments, the administration results in reduction of a skin lesion or a tumor burden in a subject in need thereof by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20- fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. In some embodiments, the control subject is a subject in need thereof who is not administered with the nucleic acid, the nanoparticle, the vector, or the rAAV. In some embodiments, the control subject is a healthy subject.

[00120] In some aspects, treating the subject or administering to the cell results in changes of molecular markers of NF1 signaling pathway. In some embodiments, the changes of the molecular markers of NF1 signaling pathway may reverse the pre-existing neurological deficits associated with NF1 . In some embodiments, the changes of the molecular markers of NF1 signaling pathway may prevent neurological deficits associated with NF1 . In some embodiments, the molecular markers of NF1 signaling pathway comprise at least pCREB, pSynapsinl, pERK1/2, pDARP32 and tyrosine hydroxylase (TH). In some embodiments, the administration results in an increase of pCREB. In some aspects, the administration results in a decrease of pERK1/2. In some embodiments, the molecular markers of NF1 signaling pathway can comprise any biological markers that are known or unknown in the art.

[00121] In some embodiments, treating the subject or administering to the cell results in changes of molecular markers of NF1 signaling pathway in a subject in need thereof by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a cell culture or a subject in need thereof who is not administered.

[00122] Aspects of the disclosure relate to methods for treating NF1 in a subject in need thereof. In some aspects, a subject is a mammal, for example a human, mouse, rat, dog, cat, non-human primate, etc. In some aspects, the subject is a human.

[00123] As used herein, the term “treating” refers to the application or administration of a composition (e.g., an isolated nucleic acid, nanoparticle, rAAV, or composition as described herein) to a subject who exhibits one or more signs or symptoms of NF1 (e.g., skin lesions, bone deformities, benign neurofibroma, tumor on the optic nerve (e.g., optic glioma), malignant peripheral nerve sheath tumors (MPNST), cognitive impairment, cancer, and/or one or more mutations in an NF1 gene), with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward NF1.

[00124] Alleviating NF1 includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of NF1 , in some aspects, is a means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[00125] “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset.

[00126] The disclosure also provides kits for use in the treatment of a disease or disorder described herein. Such kits include 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 pharmaceutically acceptable carrier. Another component is optionally a second therapeutic agent for the treatment of the disorder along with suitable container and vehicles for administrations of the therapeutic compositions. The kits optionally comprise solutions or buffers for suspending, diluting or effecting the delivery of the first and second compositions.

[00127] In one embodiment, such a kit includes the nucleic acids or vectors in a diluent packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the nucleic acids or vectors. In one embodiment, the diluent is in a container such that the amount of headspace 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 headspace is negligible (i.e., almost none). [00128] In some aspects, the formulation comprises a stabilizer. The term "stabilizer" refers to a substance or excipient which protects the formulation from adverse conditions, such as those which occur during heating or freezing, and/or prolongs the stability or shelflife 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.

[00129] In some aspects, the formulation comprises an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance which is added to the composition that inhibits the growth of microorganisms that may be introduced upon repeated puncture of the vial or container being used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[00130] In some aspects, the kit comprises a label and/or instructions that describes use of the reagents provided in the kit. The kits also optionally comprise catheters, syringes or other delivering devices for the delivery of one or more of the compositions used in the methods described herein.

[00131] This entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described as a 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," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning. If aspects of the disclosure are described as "comprising" a feature, embodiments also are contemplated "consisting of" or "consisting essentially of" the feature.

[00132] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in its entirety to the extent that it is not inconsistent with the disclosure.

[00133] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Thus, the following examples are provided by way of illustration and not limitation.

EXAMPLES

Example 1 Materials and Methods

[00134] Design of gene therapy constructs encoding a mini-NF1 protein.

[00135] Because NF1 presents particular challenges in therapeutic development, including that the NF1 gene exceeds the physical limitations of gene delivery vehicles and necessitates targeting difficult to transduce cells (Schwann cells) within peripheral nerves, gene therapy constructs for use in adeno-associated virus (AAV) gene therapy to deliver a functioning copy of NF1 to Schwann cells were developed with an NF1 mini gene (“miniNFI” or “mini-NF1 ”).

[00136] Initially, three AAV genome vector constructs encoding a mini-NF1 promoter were constructed. These constructs include promoters targeting astrocytes: an FLCAG promoter (SEQ ID NO: 22) (Table 1 , Fig. 1 ) , a myelin protein zero (MPZ, P0) P0 promoter (SEQ ID NO: 23) (Table 1 , Fig. 2), and a gfa1405 promoter (SEQ ID NO: 24) (Table 1 , Fig. 3).

[00137] The gfa1405 promoter, described in International Patent Application No. PCT/US2023/063676, was designed in order to obtain a smaller astrocyte-specific promoter to be useful in the treatment of astrocytic and neuronal diseases. The gfa1405 promoter is 1405 base pairs (SEQ ID NO: 5) and when combined with the mini-NF1 gene (i.e., SEQ ID NO: 1 or 3) remains under the packaging threshold for AAV.

[00138] The other key components of the vectors include AAV9 capsid for efficient targeting of the CNS, AAV2 inverted terminal repeats (ITRs) creating a single-stranded construct with a larger packaging capacity (for the gene of interest), a mini-NF1 gene coding sequence, a stuffer sequence to prevent reverse packaging, and an SV40 intron and an SV40 polyadenylation (polyA) sequence (see Table 1 and Figs. 1-3). Each of these constructs was designed for mini-NF1 gene expression so that a functional NF1 protein would be made. The specific sequences for each of the three constructs are provided in Table 1 and in Figs. 1-3.

[00139] Each of a gfa1405 promoter (SEQ ID NO: 5), a FLCAG promoter (SEQ ID NO: 6), and a P0 promoter (SEQ ID NO: 7) were cloned into a plasmid with a mini-NF1 gene (SEQ ID NO: 1 or 3). Each construct was sequenced and mRNA and protein levels of the mini-NF1 protein were evaluated in vitro.

[00140] Vector production.

[00141] Three different constructs were created by cloning the NF1 mini gene (mini-NF1 ; e.g. the polynucleotide of SEQ ID NO: 1 or 3) from the Sena-Esteves lab (see U.S. Patent No. 11 ,401 ,531 , incorporated by reference herein in its entirety), after three different promoters, i.e., a gfa1405 promoter, a ubiquitous promoter (FLCAG), and a Schwann cellspecific promoter (P0) into an AAV9 vector.

[00142] Recombinant AAV (rAAV) vectors were manufactured at Andelyn Biosciences using a calcium phosphate-mediated triple transfection in adherent HEK293 cells followed by purification. Briefly, harvested media was filtered and concentrated then purified by gradient ultracentrifugation followed by ion exchange chromatography. Vectors were formulated in 20mM T ris (pH8.0), 1 mM MgCl2 and 200mM NaCI and 0.001 % Pluronic F68 and sterile filtered. The AAV production process was developed using methods 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 based on degradation of non-encapsidated DNA following digestion of viral capsids and determined by ddPCR.

[00143] Cell lines.

[00144] A Schwannoma cell line (TCC cat# RT4-D6P2T-CRL-2768) was used in various experiments of the disclosure. The cell were cultured in complete media and standard cell culture plating was used.

[00145] In vitro GFP expression.

[00146] 70% confluent RT4-D6P2T-CRL-2768 Schwannoma cells in a 6-well plate were transfected with 2.ug of plasmid DNA using Lipofectamine 3000 following the manufacturer’s protocol. 48 hours post transfection, the cells were imaged and harvested, and cell pellets were frozen at -80C. One well of untransfected cells was also harvested at the same time as a negative control.

[00147] RNA was isolated from the cells using Trizol, DNAsel treated, and cDNA was generated using the Qiagen RT2 First Strand synthesis kit. The relative GFP mRNA levels were quantified via qPCR using the comparative CT method with SYBR green and primers specific to GFP, and primers specific to beta actin as the endogenous control.

[00148] TaqMan qPCR assay. [00149] A TaqMan qPCR assay (Thermo Fisher) was carried out to quantify mRNA expression according to manufacturer’s protocol.

[00150] Mice and source of mice.

[00151] Wild-type (WT) mice.

[00152] Neonatal (days 1-3 post-natal) C57BL/6 mice were used in various experiments.

[00153] Neurofibromin 1 mice.

[00154] Nf1 F/Arg681*; HoxB7-Cre mice (University of Alabama) are used in various experiments.

[00155] Nf1 F/Arg681*; HoxB7-Cre mice have paraspinal plexiform and cutaneous tumors. Mice are treated with mini-NF1 gene therapy vectors to treat and ameliorate tumors and/or prevent formation of tumors in neonatal mice.

[00156] ICV injections.

[00157] All animal procedures were approved by Nationwide Children’s Hospital Institutional Animal Care and Use Committee (IACUC). Neonatal (days 1-3 post-natal) mice (C57BL/6) were cryo-anesthetized (~2 min) prior to intracerebroventricular (ICV) injections. ICV injections were performed with a Hamilton syringe (Cal7635-01 ) and 33GA 30°beveled needles (Hamilton, 7803-05) into the left hemisphere at 2/5 of the distance from the lambda suture to the eye. Neonates were injected with 7.50E+10 vg of ssAAV9 vectors encoding GFP under the FLCAG, P0 and gfa1405 promoters.

[00158] Evaluation of GFP biodistribution in animal tissues after AA V9 ICV injections.

[00159] Four or eight weeks after ICV injections, mice were or are terminally anesthetized with Ketamine/Xylazine (100/10 mg/kg i.p.) and transcardially perfused with ice-cold 0.9% heparinized saline. Tissues were dissected and post-fixed in 4% PFA in PBS for 12 hr. After fixation, the right brain hemisphere was cryoprotected in 30% sucrose in PBS at 4°C for 3 days. All samples were embedded and frozen in OST compound (Tissue Plus, Fisher). Sagittal sections were cut at 25 mm thickness on a cryostat (1950 LEICA). Free- floating sections were washed in PBS and incubated with DAPI solution in PBS for 1 min at RT. To retrieve antigen from PFA fixed tissues, slices were treated with 0.1% Sodium borohydride in 1X PBS 15 min at RT. For immunohistochemical analysis of the GFP colocalization with specific cellular markers, all the slices were blocked and permeabilized in 10% normal goat serum in 1XPBS with 0.3% Triton (PBST) for 1 hr at RT followed by overnight incubation in fresh PBST with 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 in PBST with 10% normal donkey serum for 1 hr at RT. Sections were washed in 1XPBS and mounted on slides in Prolong Gold antifade reagent (Thermo Fisher Scientific). Images were acquired using a Nikon Ti2E fluorescent 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 on each section. The intensity of GFP signal was evaluated within all GFP positive area.

[00160] Protein extraction and Western blotting analysis.

[00161] Protein extraction and Western blotting analysis were carried out after mice were treated. Total protein was extracted from selected animal tissues using tissue protein extraction reagent (T-PER Tissue Protein Extraction Reagent; 78510; Thermo Scientific) and 1 tablet of protease inhibitor (Pierce Protease Inhibitor Tablets; A32953; Thermo Scientific) per 10 mL of extraction reagent. Steel bead was added to sample and extraction reagent, and samples were homogenized using TissueLyser II. Steel bead was then removed, and samples were sonicated twice for 15 seconds.

[00162] Total protein extracted was quantified using the DC Protein Assay (Bio-Rad). Twenty-five microgram samples were separated on 4-12% SDS-PAGE and transferred to nitrocellulose membrane using the wet transfer system. Ponceau staining was performed and membranes were subsequently washed in PBS supplemented with .1% Tween three times, 10 min each. Membranes were blocked for 1 hour at RT in Pierce Protein Free Blocking Buffer. Nitrocellulose membranes were then incubated with the following antibodies overnight at 4C: mouse monoclonal antibody to GAPDH (1 :5000 in Pierce Protein Free Blocking Buffer, 274102; Synaptic Systems); chicken polyclonal antibody to GFP (1 :5000 in Pierce Protein Free Blocking Buffer, ab13970; AbCam). Membranes were then washed three times, 10 min each with PBST buffer.

[00163] Membranes were then incubated for 1 hour at RT with the following antibodies: goat antibody to chicken AF 488 (1 :1000 in Pierce Protein Free Blocking Buffer, ab150169; AbCam); donkey antibody to mouse AF 568 (1 :1000 in Pierce Protein Free Blocking Buffer, A10037; Invitrogen). Membranes were then washed three times, 10 min each with PBST buffer.

[00164] Blots were subsequently imaged using blot imager. Quantification of bands was performed using Bio-Rad ImageLab. [00165] Protein extraction and Western blotting analysis after transfection.

[00166] Protein extraction and Western blotting analysis were carried out after the 72- hours post-transfection cells were collected, washed in PBS, centrifuged, and flash frozen. Total protein was extracted from 1-2 million HEK293T cells by first thawing the cell pellets on ice for 15 minutes, and then using 30 microliters of RIPA buffer per pellet (Pierce, RIPA lysis and Extraction Buffer 89901 ; Thermo Scientific) and 1 tablet of protease inhibitor (Pierce Protease Inhibitor Tablets; A32953; Thermo Scientific) per 10 mL of extraction reagent. Samples were gently homogenized using pipette, lysed on ice for 15 minutes at 4C, briefly sonicated, and then centrifuged at 10,000g for 10 minutes. The supernatant was transferred to a new tube, and total protein was quantified.

[00167] Total protein extracted was quantified using the DC Protein Assay (Bio-Rad). Fifty microgram samples were separated on 4-12% SDS-PAGE and transferred to nitrocellulose membrane using the wet transfer system. Ponceau staining was performed and membranes were subsequently washed in PBS supplemented with .1% Tween three times, 10 min each. Membranes were blocked for 1 hour at RT in Pierce Protein Free Blocking Buffer. Nitrocellulose membranes were then incubated with the following antibodies overnight at 4C: mouse monoclonal antibody to GAPDH (1 :5000 in Pierce Protein Free Blocking Buffer, 274102; Synaptic Systems); chicken polyclonal antibody to GFP (1 :5000 in Pierce Protein Free Blocking Buffer, ab13970; AbCam). Membranes were then washed three times, 10 min each with PBST buffer.

[00168] Membranes were then incubated for 1 hour at RT with the following antibodies: goat antibody to chicken AF 488 (1 :1000 in Pierce Protein Free Blocking Buffer, ab150169; AbCam); donkey antibody to mouse AF 568 (1 :1000 in Pierce Protein Free Blocking Buffer, A10037; Invitrogen). Membranes were then washed three times, 10 min each with PBST buffer.

[00169] Blots were subsequently imaged using blot imager. Quantification of bands was performed using Bio-Rad ImageLab.

[00170] This study aims to inform the selection of vector regulatory elements and viral administration route to develop a Schwann-cell targeted adeno-associated virus (AAV)- mediated gene replacement therapy for neurofibromatosis type 1 (NF1). NF1 often presents with debilitating peripheral nerve tumors that arise from Schwann cells. Due to the monogenic nature, NF1 is a promising candidate for AAV gene therapy, but the large gene size and difficult to target cell population have remained a challenge in developing a translatable therapy. [00171] First, we designed AAV reporter constructs expressing enhanced green fluorescent protein (eGFP) to compare the transcriptional activity of a promoter with high selectivity for Schwann cells, myelin protein zero (P0), and a ubiquitously expressing promoter, chicken beta actin with a CMV enhancer (CAG). Expression of the AAV constructs, pAAV-PO. eGFP and pAAV-CAG.eGFP, was verified in cultured Schwann cells and they were packaged into AAV9, a serotype with known tropism for central and peripheral nervous systems (CNS, PNS). To assess viral particle biodistribution and expression using clinically feasible administration routes, AAV9-P0. eGFP or AAV9-CAG. eGFP were injected into wildtype mice pups via intracerebroventricular (ICV) injection and wildtype mice weanlings via intrathecal lumbar (IT-L) injection.

[00172] By expression data from qPCR and Western blot, we show both pAAV-PO. eGFP and pAAV-CAG.eGFP drive robust eGFP expression in Schwann cells in vitro. In vivo, vector biodistribution and expression data by qPCR and immunohistochemistry demonstrate expression is driven in the peripheral nervous system (Figure 2). These findings are currently being validated by IT-L or subpial injections of both AAV9 vectors into wildtype minipigs. This proof-of-concept work demonstrates the capability of an AAV to drive expression to the Schwann cells of the peripheral nervous system, therein establishing a strong foundation for the development of an AAV-mediated gene replacement therapy for neurofibromatosis type 1 .

[00173] Next, the therapeutic efficacy of a truncated version of the Nf1 gene, miniNFI, generated by the Sena-Esteves lab and which fits in the confines of AAV is examined. This miniNFI transgene has been cloned into our P0- and CAG-driven AAV gene replacement constructs, packaged into AAV9, and is delivered to a murine model of NF1 ( Nf 1 F/Arg681*; HoxB7-Cre mice) to assess therapeutic efficacy. The lead candidate AAV vector from this study will be evaluated in a porcine model of NF1 .

Example 2 GFP Expression in Schwannoma Cells

[00174] Cloning, in vitro expression analysis, and viral packaging of AAV9-FLCAG-eGFP and AAV9-P0-eGFP constructs were carried out to allow for comparison of the biodistribution of two constructs to the peripheral nerves and Schwann cells using a reporter protein, green fluorescent protein (GFP). Schwannoma cells were transfected with both constructs and an untransfected control as described in Example 1 .

[00175] Both constructs were able to achieve robust GFP expression by 48 hours post transfection. Fig. 4 shows transfection of AAV9-CAG-eGFP and AAV9-P0-eGFP constructs in a Schwannoma cell line. Due to the extreme rapidity of growth by the cell line, the cells were harvested at 48 hours. GFP protein was quantified by Western blot and mRNA was quantified by qPCR. A TaqMan qPCR assay was used to quantify mRNA expression as well.

[00176] Western blot showed abundant and comparable levels of GFP from both CAG and P0 promoters. Fig. 5 shows Western blot of GFP protein expression after 48 hour transfection in Schwannoma cell line. GFP mRNA expression was also abundant and comparable between the two promoters, with mRNA expression being achieved by both the CAG and the P0 promoters.

[00177]

[00178] Robust levels of miniNFI protein expression were observed in cells which were transfected with CAG-miniNF1 . The levels of miniNFI protein expression were lesser in cells which were transfected with PO-miniNF1 , i.e.. driven by the Schwann cell specific P0 promoter (Figure 3). This is very likely due to the fact that the P0 promoter takes longer to initiate robust expression.

Example 3 NF1 mRNA Expression in Schwannoma Cells

[00179] To ensure that each promoter could drive miniNFI expression, each of the miniNFI expression constructs was transfected in a Schwannoma cell line (same experimental design as with the GFP reporter constructs above). After transfection of the CAG-miniNF1 and P0-miniNF1 constructs into the Schwannoma cell line, cells were harvested 48 hours post transfection and HA protein expression (HA is tagged to miniNFI for quantification and tracking) was analyzed by Western blot. Additionally, mRNA expression was analyzed by TaqMan qPCR.

[00180] Fig. 6 shows Western blot of miniNFI protein expression after 48 hour transfection in Schwannoma cell line. GAPDH was used as a loading control and HA was the protein of interest (tagged to miniNFI). The first 3 lanes show an untransfected control, the next 3 lanes show pAAV-CAG-miniNF1 -HA, and the final 3 lanes show pAAV-PO- miniNFI HA. The 3 lanes represent the experiment run in triplicate.

[00181] Messenger RNA (mRNA) expression was measured in the same Schwannoma cell line after transfection of the FLCAG-miniNF1 and P0-miniNF1 constructs. Cells were harvested 48 hours post transfection and HA protein expression (HA is tagged to miniNFI for quantification and tracking) was analyzed by Western blot. Additionally, mRNA expression was analyzed by TaqMan qPCR. [00182] Robust levels of miniNFI protein and mRNA expression were observed in cells which were transfected with both the PO-miniNF1 and the FLCAG-miniNF1 constructs as both showed substantially greater expression than in untransfected cells.

[00183] Because the CAG promoter and P0 promoter were observed to drive comparable levels of GFP reporter protein expression in Schwannoma cells, these experiments confirm the P0 promoter is functional in Schwannoma cells. Additional experiments are being carried out with the P0-miniNF1 construct to investigate observed expression prior to packaging into an AAV9 vector for delivery into NF1 mice. Likewise, because robust miniNFI expression was confirmed with the ubiquitous FLCAG promoter, the FLCAG-miniNF1 construct was packaged into an AAV9 vector so that efficacy could be evaluated in NF1 mice.

Example 4 GFP Expression in Wild-Type Mice

[00184] The following study was carried out to establish that the AAV9 constructs described herein could deliver the transgene, i.e., GFP in this example, to cells of interest in the brains of mice, and to compare cell-specific transduction and biodistribution of the various promoters. Healthy C57/BL6 wild-type mice (male and female) were injected at postnatal day 1 (PND1 ) (ICV; 5 microliters, 1 .05e11 vg) with the three reporter constructs: AAV9.gfa1405.GFP, AAV9.FLCAG-GFP, AAV9.P0. miniNFI .GFP, or control and sacrificed 28 days post-injection. Tissues of the mice were isolated and analyzed for the presence of GFP.

[00185] Injection of neonatal wild-type mice with AAV9.gfa1405.GFP,

AAV9. FLCAG. GFP, and AAV9.P0. miniNFI .GFP resulted in GFP expression in the brain. These experiments establish that the constructs and techniques used herein can be used for transfecting the mini-NF1 gene of interest into mouse brains.

Example 5

Evaluation of Toxicity of Injection of the Mini-NF1 Gene Expression in Wild-Type Mice

[00186] The following study was carried out to establish that the AAV9 constructs described herein could deliver the transgene to the brains of mice without toxicity. Healthy C57/BL6 wild-type mice (male and female) were injected (ICV; 5 microliters, 1 .05e11 vg) at postnatal day 1 (PND1) with one of the three mini-NF! Gene constructs, AAV9. FLCAG. miniNFI (SEQ ID NO: 22), AAV9.P0. miniNFI (SEQ ID NO: 23), or AAV9.gfa1405. miniNFI (SEQ ID NO: 24), or control. Additional mice (3 per cohort) were then also treated with AAV9.FLCAG.miniNF1 at a dose of 2.1 E10 vg, and AAV9-P0-miniNF1 at a dose of 7.5E10 vg.

[00187] All injected mice survived and did well through the predetermined end point of 6 weeks, demonstrating that the method is safe to carry out in the NF1 mouse model.

Example 6 Evaluation of GFP Expression after Injection of the Gene Expression Cassette in Wild- Type Mice

[00188] The following study was carried out to establish that the AAV9 constructs described herein could deliver the transgene to the brains of mice. Healthy C57/BL6 wildtype mice (male and female) were injected (ICV) at postnatal day 1 (PND1) with one of the three constructs, AAV9.CAG.GFP, AAV9.P0.GFP, or AAV9.gfa1405.GFP, or control and eGFP expression was evaluated in various tissues. Mice were sacrificed 4 weeks after injection to track protein and viral particle distribution. GFP mRNA expression was quantified in NF1 relevant tissues, including eye and optic nerve, cerebellum, cervical, thoracic, and lumbar spinal cord, and sciatic nerve by qPCR (Fig. 7). Fig. 8A-B shows results of immunofluorescence experiments carried out on CNS and PNS tissues to visualize GFP expression. Staining of the brain (Fig. 8A) and peripheral nerve (Fig. 8B) was complete. GFP expression was striking in the brain and sciatic nerve, particularly with the ubiquitous CAG promoter. Fig. 8A shows a sagittal section of the brain from mice (n=3 each) treated at PND1 by ICV delivery with AAV9-1405-GFP (left column) or AAV9-CAG-GFP (right column) 4 weeks after injection. Fig. 8B shows a longitudinal section of sciatic nerve from mice injected (ICV) with ssAAV9. CAG. eGFP stained for neurofilament, myelin basic protein (MBP), green fluorescent protein (GFP), and DAPI.

[00189] The construct with the CAG promoter, i.e., AAV9.CAG.GFP vector, achieved the greatest level of GFP mRNA expression in all tissues except for the sciatic nerve. The construct comprising the P0 promoter achieved greater GFP mRNA expression which was expected because P0 should drive expression to Schwann cells. The construct comprising the novel gfa1405 promoter resulted in GFP mRNA expression equivalent to or slightly lower than the CAG promoter in all brain and spinal cord tissues analyzed but achieved lower expression than CAG and P0 in the PNS (sciatic nerve) as was anticipated due to its role in targeting glial cells. Example 7

Mini-NF1 Gene Expression in a Mouse Model of NF1

[00190] The following study is carried out to evaluate CSF delivery of each of AAV9.FLCAG. miniNFI (SEQ ID NO: 22), AAV9.P0.miniNF1 (SEQ ID NO: 23), and AAV9.gfa1405.miniNF1 (SEQ ID NO: 24) in a mouse model of NF1 , Nf1 F/Arg681 *; HoxB7- Cre mice, and to determine the effects of the mini-NF1 transgene in preventing tumor formation in this mouse model of NF1 .

[00191] Nf1 F/Arg681*; HoxB7-Cre mice (male and female) are injected (ICV or by lumbar IT; 5 microliters, 1 .05e11 vg) at any of postnatal days 0-2 (PNDO-2) with one of the three constructs, AAV9.FLCAG.miniNF1 , AAV9.P0.miniNF1 , and AAV9.gfa1405.miniNF1 , or a control and monitored for at least 6-12 months or more after injection (since tumors normally form at 4-6 months), or until a humane or predetermined endpoint. Tissues of the mice are isolated and analyzed for the presence of mini-NF1 protein and mRNA and for tumor formation. Additionally, tumor size is measured.

[00192] It is expected that transfection of the transgene into this model of NF1 will prevent tumors from forming.

Example 8

Mini-NF1 Gene Expression in a Mouse Model of NF1

[00193] As explained herein above, NF1 presents particular challenges in therapeutic development including that the NF1 gene’s size exceeds the physical limitations of gene delivery vehicles and necessitates targeting difficult to transduce cells (Schwann cells) within peripheral nerves. Thus, the reduced size NF1 gene (miniNFI ) was developed with two different promoters, a ubiquitous promoter (CAG) and a Schwann cell specific promoter (P0), packaged inside of a AAV for treatment of NF1 mice.

[00194] The following study is carried out to evaluate CSF delivery of each of AAV9.FLCAG. miniNFI (SEQ ID NO: 22), AAV9.P0. miniNFI (SEQ ID NO: 23), and AAV9.gfa1405. miniNFI (SEQ ID NO: 24) in a mouse model of NF1 , Nf1 F/Arg681 *; HoxB7- Cre mice, and to determine the effects of the mini-NF1 transgene in this mouse model of NF1 after tumor formation.

[00195] Nf1 F/Arg681*; HoxB7-Cre mice (male and female) are injected after tumor formation has occurred in the mice with one of the three constructs, AAV9.FLCAG. miniNFI , AAV9.P0. miniNFI , and AAV9.gfa1405. miniNFI , or control. Mice are then monitored for at least 2-12 months or more after injection, or until a humane or predetermined endpoint. Tissues of the mice are isolated and analyzed for the presence of mini-NF1 protein and mRNA and for tumor reduction. Accordingly, tumor size is measured.

[00196] It is expected that transfection of the transgene into this model of NF1 will reduce tumor size and/or possibly eliminate the presence of the tumors in the mice. It is also expected that injection will increase survival of this mouse model and have an impact on the RAS pathway.

[00197] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

[00198] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will 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.

[00199] Throughout the specification, where compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, it is contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless described otherwise. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[00200] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and/or with the aid of or automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various of the steps may be changed without departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[00201] All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control. References referred to herein with numbering are provided with the full citation as shown herein below. [00202] Additional References

1. Scherer, S.S. et al. Transgenic expression of human connexin32 in myelinating Schwann cells prevents demyelination in connexin32-null mice. J Neurosci 25, 1550- 9 (2005).

2. Kagiava, A. et al. AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy. Gene Ther (2021 ).

3. Li, K. et al. Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I. Dis Model Meeh 9, 759- 67 (2016).

4. Puttaraju, M., Jamison, S.F., Mansfield, S.G., Garcia-Blanco, M.A. & Mitchell, L.G. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat Biotechnol 17, 246-52 (1999).

5. Chao, H. et al. Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing. Nat Med 9, 1015-9 (2003).

6. Coady, T.H., Shababi, M., Tullis, G.E. & Lorson, C.L. Restoration of SMN function: delivery of a trans-splicing RNA re-directs SMN2 pre-mRNA splicing. Mol Ther 15, 1471 -8 (2007).

7. Shababi, M. & Lorson, C.L. Optimization of SMN trans-splicing through the analysis of SMN introns. J Mol Neurosci 46, 459-69 (2012).

8. Rindt, H. et al. Replacement of huntingtin exon 1 by trans-splicing. Cell Mol Life Sci 69, 4191 -204 (2012).

9. Rindt, H., Tom, C.M., Lorson, C.L. & Mattis, V.B. Optimization of trans-Splicing for Huntington's Disease RNA Therapy. Front Neurosci 11 , 544 (2017).

10. Sena-Esteves, M., Tebbets, J.C., Steffens, S., Crombleholme, T. & Flake, A.W. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods 122, 131 -9 (2004).

1 1. Toonen, J.A. et al. NF1 germline mutation differentially dictates optic glioma formation and growth in neurofibromatosis-1. Hum Mol Genet 25, 1703-13 (2016).

12. Ubogu, E.E. The molecular and biophysical characterization of the human blood-nerve barrier: current concepts. J Vase Res 50, 289-303 (2013).

13. Low, P.A. & Dyck, P.J. Increased endoneurial fluid pressure in experimental lead neuropathy. Nature 269, 427-8 (1977).

14. Myers, R.R., Heckman, H.M. & Powell, H.C. Endoneurial fluid is hypertonic. Results of microanalysis and its significance in neuropathy. J Neuropathol Exp Neurol 42, 217- 24 (1983).

15. Kristensson, K., Stromberg, E., Elofsson, R. & Olsson, Y. Distribution of protein tracers in the nervous system of the crayfish (Astacus astacus L.) following systemic and local application. J Neurocytol 1 , 35-48 (1972).

16. Pettersson, C.A., Sharma, H.S. & Olsson, Y. Vascular permeability of spinal nerve roots. A study in the rat with Evans blue and lanthanum as tracers. Acta Neuropathol 81 , 148-54 (1990).

17. Olsson, Y. Microenvironment of the peripheral nervous system under normal and pathological conditions. Crit Rev Neurobiol 5, 265-311 (1990). Isakson, S.H. et al. Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1. Commun Biol 1 , 158 (2018). Osum, S.H. et al. Selumetinib normalizes Ras/MAPK signaling in clinically relevant neurofibromatosis type 1 minipig tissues in vivo. Neurooncol Adv 3, vdab020 (2021).

Claims

Claims \Ne claim:

1 . A nucleic acid comprising: a polynucleotide encoding a mini-neurofibromin 1 (mini-NF1 ) protein; and a polynucleotide encoding a gfa1405 promoter, a full-length CAG (FLCAG) promoter, a PO promoter, a truncated CAG promoter, a gfaABCI D promoter, or a GFAP promoter.

2. The nucleic acid of claim 1 wherein the polynucleotide encoding the mini-NF1 protein comprises

(a) a nucleotide sequence comprising at least 80% identity to the nucleotide sequence of SEQ ID NO: 1 or 3;

(b) the nucleotide sequence of SEQ ID NO: 1 or 3;

(c) a nucleotide sequence encoding a mini-NF1 protein comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 2 or 4; or

(d) a nucleotide sequence encoding a mini-NF1 protein comprising the amino acid sequence of SEQ ID NO: 2 or 4.

3. The nucleic acid of claim 1 or 2 wherein the nucleotide sequence encoding the gfa1405 promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO:

5.

4. The nucleic acid of claim 1 or 2 wherein the nucleotide sequence encoding the FLCAG promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO:

6.

5. The nucleic acid of claim 1 or 2 wherein the nucleotide sequence encoding the PO promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 7.

6. The nucleic acid of claim 1 or 2 wherein the nucleotide sequence encoding the truncated CAG promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 8.

7. The nucleic acid of claim 1 or 2 wherein the nucleotide sequence encoding the gfaABCI D promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 9.

8. The nucleic acid of claim 1 or 2 wherein the nucleotide sequence encoding the GFAP promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 10.

9. The nucleic acid of any one of claims 1 -8 further comprising a polynucleotide encoding an SV40 intron and/or a synthetic polyadenylation signal sequence.

10. The nucleic acid of claim 9 wherein the nucleotide sequence encoding the SV40 intron comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 11 .

11 . The nucleic acid of claim 9 wherein the nucleotide sequence encoding the synthetic polyadenylation signal sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 12.

12. The nucleic acid of claim 1 or 2 comprising

(a) a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 17, 19, or 21 ; or

(b) the nucleotide sequence of SEQ ID NO: 17, 19, or 21 .

13. The nucleic acid of any one of claims 1 -12 further comprising a polynucleotide encoding an inverted terminal repeat (ITR).

14. The nucleic acid of claim 13 further comprising a polynucleotide encoding at least two inverted terminal repeats (ITRs).

15. The nucleic acid of claim 13 or 14 wherein a nucleotide sequence encoding a 5’ ITR comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 13; and/or a nucleotide sequence encoding a 3’ ITR comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 14.

16. The nucleic acid of any one of claims 13-15 comprising

(a) a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 16, 18, or 20; or

(b) the nucleotide sequence of SEQ ID NO: 16, 18, or 20.

17. The nucleic acid of any one of claims 1 -16 further comprising a polynucleotide encoding a stuffer sequence.

18. The nucleic acid of claim 17 wherein the nucleotide sequence encoding the stuffer sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 15.

19. The nucleic acid of any one of claims 1 -18, wherein the nucleotide sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 22, 23, or 24.

20. A nanoparticle, extracellular vesicle, exosome, or vector comprising the nucleic acid of any one of claims 1 -19 or a combination of any one or more thereof.

21 . The vector of claim 20, wherein the vector is a viral vector.

22. The viral vector of claim 21 , wherein the viral vector is an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, or a synthetic virus.

23. The viral vector of claim 21 or 22, wherein the viral vector is an AAV.

24. The viral vector of claim 23, wherein the AAV lacks rep and cap genes.

25. The viral vector of claim 23 or 24, wherein the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a single-stranded recombinant AAV (ssAAV).

26. The viral vector of any one of claims 23-25, wherein the AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVanc80, AAVrh.74, AAVrh.8, AAVrh.10, MyoAAV 1 A, AAVMYO, or AAV-B1 , AAV2/1 , AAV2/8, AAV2/9, or any of their derivatives.

27. The viral vector of any one of claims 23-26, wherein the AAV is AAV9.

28. An rAAV particle comprising the AAV of any one of claims 23-27.

29. A composition comprising:

(a) the nucleic acid of any one of claims 1 -19;

(b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) the viral vector of any one of claims 21 -27; or (d) the rAAV particle of claim 28; and a pharmaceutically acceptable carrier.

30. The composition of claim 28, wherein the composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

31 . A method of increasing the expression of a mini-NF1 gene and/or a mini-NF1 protein in a cell comprising contacting the cell with

(a) the nucleic acid of any one of claims 1 -19;

(b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) the viral vector of any one of claims 21-27;

(d) the rAAV particle of claim 28; or

(e) the composition of claim 29 or 30.

32. The method of claim 31 , wherein the cell is a nerve cell, an oligodendrocyte, and/or a Schwann cell.

33. The method of claim 31 or 32, wherein the cell is a human cell.

34. The method of claim 33, wherein the cell is in a human subject.

35. A method of treating a subject comprising a neurofibromin 1 (NF1) gene mutation comprising administering to the subject an effective amount of

(a) the nucleic acid of any one of claims 1 -19;

(b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) the viral vector of any one of claims 21-27;

(d) the rAAV particle of claim 28; or

(e) the composition of claim 29 or 30.

36. The method of claim 35, wherein the subject is a human subject.

37. The method of claim 35 or 36, wherein the NF1 gene mutation causes a subject to suffer from or be at risk of suffering from a tumor or a cancer.

38. The method of claim 37, wherein the tumor is a neurofibroma.

39. The method of claim 37, wherein the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.

40. The method of any one of claims 35-39, further comprising administering any one or more of a corticosteroid, rituximab, and rapamycin to the subject.

41 . The method of any one of claims 35-39, wherein the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, rAAV particle, or composition is administered by intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

42. Use of

(a) the nucleic acid of any one of claims 1 -19;

(b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) the viral vector of any one of claims 21-27;

(d) the rAAV particle of claim 28; or

(e) the composition of claim 29 or 30 for the preparation of a medicament for increasing expression of the neurofibromin 1 (NF1 ) gene and/or protein in a cell.

43. The use of claim 42, wherein the cell is in a human subject.

44. Use of

(a) the nucleic acid of any one of claims 1 -19;

(b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) the viral vector of any one of claims 21-27;

(d) the rAAV particle of claim 28; or

(e) the composition of claim 29 or 30 in treating a subject comprising a mutant neurofibromin 1 (NF1) gene.

45. The use of claim 44, wherein the subject is a human subject.

46. The use of any one of claims 42-45, wherein the subject suffers from a lesion, a tumor or a cancer.

47. The use of claim 46, wherein the tumor is a neurofibroma or a glioma.

48. The use of claim 46, wherein the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.

49. The use of any one of claims 42-48, wherein the medicament is administered with any one or more of a corticosteroid, rituximab, and rapamycin.

50. The use of any one of claims 42-49, wherein the medicament is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.

51 . A composition for treating a neurofibromin 1 (NF1) gene mutation in a subject, wherein the composition comprises

(a) the nucleic acid of any one of claims 1 -19;

(b) the nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) the viral vector of any one of claims 21-27;

(d) the rAAV particle of claim 28; or

(e) the composition of claim 29 or 30.

52. The composition of claim 51 , wherein the subject is a human subject.

53. The composition of claim 51 or 52, wherein the NF1 gene mutation is associated with the risk or presence of a lesion, a tumor, or a cancer.

54. The composition of claim 53, wherein the tumor is a neurofibroma or a glioma.

55. The composition of claim 53, wherein the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.

56. The

(a) nucleic acid of any one of claims 1-19;

(b) nanoparticle, extracellular vesicle, exosome, or vector of claim 20;

(c) viral vector of any one of claims 21 -27;

(d) rAAV particle of claim 28;

(e) composition of claim 29 or 30;

(f) method of any one of claims 31 -41 ; or

(g) use of any one of claims 42-50, wherein the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, composition, or medicament is formulated for intrathecal injection into the cerebrospinal fluid (CSF), intravenous injection into the blood stream, intracerebral injection, intracerebroventricular injection, intracisternal injection, or for aerosol administration.