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WO2024259064A1 - Matériaux et méthodes pour le traitement de mutations de la neurofibromine 1 et de maladies qui en résultent - Google Patents

Matériaux et méthodes pour le traitement de mutations de la neurofibromine 1 et de maladies qui en résultent Download PDF

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Publication number
WO2024259064A1
WO2024259064A1 PCT/US2024/033750 US2024033750W WO2024259064A1 WO 2024259064 A1 WO2024259064 A1 WO 2024259064A1 US 2024033750 W US2024033750 W US 2024033750W WO 2024259064 A1 WO2024259064 A1 WO 2024259064A1
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nucleotide sequence
nucleic acid
seq
aav
vector
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Allison Marie BRADBURY
Miguel Sena-Esteves
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Research Institute At Nationwide Children's Hospital
University Of Massachusetts
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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.
  • AAV adeno-associated virus
  • NF1 gene 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.
  • miniNFI reduced size NF1 gene
  • 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.
  • NF1 neurofibromatosis 1
  • NF2 neurofibromatosis 2
  • schwannomatosis schwannomatosis
  • 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 urgent needed.
  • NF1 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.
  • NF1 NF1
  • AAV adeno-associated virus
  • 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.
  • NF1 neurofibromin 1
  • 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.
  • 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.
  • mini-NF1 mini-neurofibromin 1
  • 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.
  • the nucleotide sequence encoding the gfa1405 promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 5.
  • the nucleotide sequence encoding the FLCAG promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleotide sequence encoding the P0 promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 7.
  • the nucleotide sequence encoding the truncated CAG promoter comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • 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.
  • the nucleic acid further comprises a polynucleotide encoding an SV40 intron and/or a synthetic polyadenylation signal sequence.
  • the nucleotide sequence encoding the SV40 intron comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 11 .
  • the nucleotide sequence encoding the synthetic polyadenylation signal sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 12.
  • 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 .
  • the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein further comprises a polynucleotide encoding an inverted terminal repeat (ITR).
  • 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).
  • 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.
  • 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.
  • the nucleic acid comprising a polynucleotide encoding a mini- neurofibromin 1 (mini-NF1) protein further comprises a polynucleotide encoding a stuffer sequence.
  • the nucleotide sequence encoding the stuffer sequence comprises at least 80% identity to the nucleotide sequence of SEQ ID NO: 15.
  • 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.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, or a synthetic virus.
  • AAV adeno-associated virus
  • the viral vector is an AAV.
  • the AAV lacks rep and cap genes.
  • the AAV is a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), or a singlestranded recombinant AAV (ssAAV).
  • the AAV is AAV9.
  • the disclosure likewise comprises an rAAV particle comprising any AAV described herein.
  • the disclosure provides a composition
  • 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.
  • the composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.
  • 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.
  • the cell is a nerve cell, an oligodendrocyte, and/or a Schwann cell.
  • the cell is a human cell. In some aspects, the cell is in a human subject.
  • 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.
  • the subject is a human subject.
  • the NF1 gene mutation causes a subject to suffer from or be at risk of suffering from a tumor or a cancer.
  • the tumor is a neurofibroma.
  • the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.
  • the method of treatment further comprises administering any one or more of a corticosteroid, rituximab, and rapamycin to the subject.
  • the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, rAAV particle, or composition is administered by intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.
  • 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.
  • the cell is in a human subject.
  • the subject suffers from a lesion, a tumor or a cancer.
  • the lesion, tumor, or cancer is associated with aberrant NF1 expression.
  • the tumor is a neurofibroma or a glioma.
  • the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.
  • the nucleic acid is administered with any one or more of a corticosteroid, rituximab, and rapamycin.
  • the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, or composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.
  • 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.
  • the subject is a human subject.
  • the subject suffers from a lesion, a tumor or a cancer.
  • the lesion, tumor, or cancer is associated with aberrant NF1 expression.
  • the tumor is a neurofibroma or a glioma.
  • the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.
  • the nucleic acid is administered with any one or more of a corticosteroid, rituximab, and rapamycin.
  • the nucleic acid, nanoparticle, extracellular vesicle, exosome, vector, viral vector, or composition is formulated for intrathecal intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.
  • 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.
  • the subject is a human subject.
  • the NF1 gene mutation is associated with the risk or presence of a lesion, a tumor, or a cancer.
  • the tumor is a neurofibroma or a glioma.
  • the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.
  • 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.
  • CSF cerebrospinal fluid
  • 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.
  • CSF cerebrospinal fluid
  • Fig. 1 shows an AAV9.flCAG.miniNF1 sequence (SEQ ID NO: 22) of the disclosure.
  • Fig. 2 shows an AAV9.P0.miniNF1 sequence (SEQ ID NO: 23) of the disclosure.
  • FIG. 3 shows an AAV9.gfa1405.miniNF1 sequence (SEQ ID NO: 24) of the disclosure.
  • FIG. 4 shows transfection of AAV9-CAG-eGFP and AAV9-P0-eGFP constructs in a Schwannoma cell line.
  • 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.
  • 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.
  • Fig. 7 shows eGFP mRNA expression in NF1 -tumor related tissues 4 weeks after ICV delivery to mouse pup as measured by TaqMan qPCR.
  • 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. 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.
  • MBP myelin basic protein
  • GFP green fluorescent protein
  • 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).
  • NFI neurofibromatosis type I
  • 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.
  • a functional NF1 protein e.g., a mini-NF1 protein
  • 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.
  • GAP GTPase-activating protein
  • 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.
  • malignancies e.g., MPNSTs and optic gliomas
  • NF1 coding sequence is 8,540 bp, far exceeding the packaging capacity of recombinant AAV vectors.
  • 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.
  • the disclosure therefore focuses on providing a mini-NF1 replacement gene or “transgene” in order to express normal or functionally active mini-NF1 protein.
  • specifically designed mini-NF1 replacement genes or “transgenes” are provided.
  • 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.
  • 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.
  • 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.
  • 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 .
  • the polypeptide is a mini-NF1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 6.
  • 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 .
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in set forth in SEQ ID NO: 7.
  • 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 .
  • the CAG promoter is a ubiquitous promoter which targets neurons and astrocytes.
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 8.
  • 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.
  • the gfaABCI D promoter and the GFAP promoter drive transgene expression primarily toward astrocytes.
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 9.
  • 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 .
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 10.
  • 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 .
  • a nucleic acid of the disclosure further comprises an SV40 intron and/or a poly(A) tail (or poly(A) sequence).
  • a nucleic acid of the disclosure comprises a promoter, an SV40 intron, a mini-NF1 open reading frame, and/or a polyA tail.
  • 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 .
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 11 .
  • 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 .
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 12.
  • 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 .
  • 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).
  • ITR sequences are important for intermolecular recombination and circularization of adeno-associated virus (AAV) genomes.
  • 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.
  • the transgene is flanked by AAV ITRs.
  • 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.
  • the ITRs are AAV2 ITRs.
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 13.
  • 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.
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 14.
  • 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 .
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 16, 18, or 20.
  • 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 .
  • 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.
  • the disclosure provides a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 17, 19, or 21 .
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 17, 19, or 21 .
  • 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 .
  • 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.
  • 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.
  • 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.
  • the nucleotide sequence is an isoform or variant of the nucleotide sequence set forth in SEQ ID NO: 15.
  • 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 .
  • 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.
  • 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.
  • 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.
  • Table 1 Sequences of the human mini-NF1 gene, mini-NF1 protein, and CNS- and PNS-specific promoters in various aspects of the disclosure.
  • a sequence index table (Table 2) is provided below for reference to sequences provided in the sequence listing.
  • 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.
  • one or more copies of these sequences are combined into a single nanoparticle, extracellular vesicle, exosome, or vector.
  • 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.
  • viral 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
  • 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.
  • the AAV is an ssAAV or an ssrAAV.
  • 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).
  • the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 1 -24.
  • 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.
  • 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).
  • the nucleic acid is a variant of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-24.
  • 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.
  • 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.
  • the disclosure utilizes AAV to deliver a mini-NF1 transgene, such as DNA encoding a mini-NF1 protein.
  • 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).
  • 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).
  • ITRs nucleotide inverted terminal repeat
  • 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.
  • nucleotide sequences of the genomes of the AAV serotypes are known.
  • 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.
  • 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).
  • 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).
  • 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.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo.
  • 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.
  • AAV genome encapsidation and integration
  • some or all of the internal approximately 4.3 kb of the genome encoding replication and structural capsid proteins, rep-cap
  • the rep and cap proteins may be provided in trans.
  • AAV 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.
  • the AAV lacks rep and cap genes.
  • the AAV is a recombinant linear AAV (rAAV), a single-stranded AAV, or a recombinant self- complementary AAV (scAAV).
  • rAAV recombinant linear AAV
  • scAAV self- complementary AAV
  • 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.
  • the AAV is an ssAAV or an ssrAAV.
  • AAV vectors can provide long-term expression of gene products in post-mitotic target tissues.
  • current AAV-based strategies may only require one-time vector administration.
  • 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.
  • 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.
  • 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
  • AAV1 i.e
  • 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).
  • 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).
  • 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.
  • helper virus of AAV e.g., adenovirus, E1 -deleted adenovirus or herpes virus
  • rAAV 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.
  • 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.
  • 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.
  • AAV vector particle a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell
  • production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.
  • rAAV 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.
  • AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrh.74, AAVrh.8,
  • 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.
  • 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.
  • 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.
  • 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.
  • a 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, AAV
  • 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.
  • 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).
  • rAAV 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 .
  • the nucleotide sequences of the genomes of various AAV serotypes are known in the art.
  • 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.
  • 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.
  • helper virus of AAV e.g., adenovirus, E1 -deleted adenovirus or herpesvirus
  • rAAV 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.
  • a method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production.
  • a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing, addition of synthetic linkers containing restriction endonuclease cleavage sites 41 or by direct, blunt-end ligation.
  • the packaging cell line is then infected with a helper virus such as adenovirus.
  • 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.
  • 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.
  • packaging cells that produce infectious rAAV.
  • packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line).
  • 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).
  • 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.
  • compositions comprising the nucleic acids and viral vectors of the disclosure are provided.
  • Compositions comprising delivery vehicles (such as rAAV) described herein are provided.
  • delivery vehicles such as rAAV
  • such compositions also comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is a diluent, excipient, or buffer.
  • the compositions may also comprise other ingredients, such as adjuvants.
  • 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).
  • buffers such as phosphate, citrate, or other organic acids
  • antioxidants such
  • 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.
  • 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.
  • 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.
  • 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 x10 6 , about 1 x10 7 , about 1 x10 8 , about 1 x10 9 , about 1 x10 10 , about 1 x10 11 , about 1 x10 12 , about 1 x10 13 to about 1 x10 14 or more DNase resistant particles (DRP) per ml.
  • DNase resistant particles DNase resistant particles
  • 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 x10 7 , about 1 x10 8 , about 1 x10 9 , about 5x10 9 , about 6x10 9 , about 7x10 9 , about 8x10 9 , about 9x10 9 , about 1 x10 10 , about 2x10 10 , about 3x10 10 , about 4x10 10 , about 5x10 10 , about 6x1 O 10 , about 7x1 O 10 , about 8x1 O 10 , about 9x1 O 10 , about 1 x10 11 , about 2x10 11 , about 3x10 11 , about 4x10 11 , about 5x10 11 , about 6x10 11 , about 7x10 11 , about 8x10 11 , about 9x10 11 , about 1x10 12 , about 2x10 12 , about 3x10 12 , about 4x10 12 , about 5x10 12 , about 6x10 12 , about
  • Dosages of about 1 x10 9 to about 1x10 10 , about 5x10 9 to about 5x10 10 , about 1 x10 10 to about 1 x10 11 , about 1 x10 11 to about 1 x10 15 vg, about 1 x10 12 to about 1 x10 15 vg, about 1 x10 12 to about 1 x10 14 vg, about 1 x10 13 to about 6x10 14 vg, about 1 x10 13 to about 1 x10 15 vg and about 6x10 13 to about 1 .0x10 14 vg are also contemplated.
  • One dose exemplified herein is 1 x10 13 vg administered via intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.
  • 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 x10 7 vg/kg, about 1x10 8 vg/kg, about 1 x10 9 vg/kg, about 5x10 9 vg/kg, about 6x10 9 vg/kg, about 7x10 9 vg/kg, about 8x10 9 vg/kg, about 9x10 9 vg/kg, about 1x10 10 vg/kg, about 2x10 10 vg/kg, about 3x10 10 vg/kg, about 4x10 10 vg/kg, about 5x10 10 vg/kg, about 1x10 11 vg/kg, about 5x10 11 vg/kg, about 1 x10 12 vg/kg, about 2x10 12 vg/kg, about 3x10 12 vg/kg, about 4x10 12 v
  • Some doses exemplified herein are about 1 .5x10 11 vg/kg or about 3x10 13 vg/kg administered via intrathecal, intracerebroventricular, intracerebral, intravenous, intracisternal, or aerosol delivery.
  • Transduction or transfection of cells with rAAV of the disclosure results in sustained expression of the mini-NF1 gene/protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acids in some aspects, as a form of a medicament
  • 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.
  • 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.
  • the combination therapy comprises administering an immunosuppressing agent in combination with the gene therapy disclosed herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • anti-inflammatory steroid are glucocorticoids such as prednisolone, betamethasone, dexamethasone, methotrexate, hydrocortisone, methylprednisolone, deflazacort, budesonide or prednisone.
  • 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.
  • Calcineurin inhibitors bind to cyclophilin and inhibits the activity of calcineurin
  • Exemplary calcineurine inhibitors includes cyclosporine, tacrolimus and picecrolimus.
  • mTOR inhibitors reduce or inhibit the serine/threonine-specific protein kinase mTOR.
  • exemplary mTOR inhibitors include rapamycin (also known as sirolimus), everolimus, and temsirolimus.
  • the immunosuppressing agents include immune suppressing macrolides.
  • 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.
  • immunosuppressing macrolides include tacrolimus, pimecrolimus, and rapamycin (also known as sirolimus).
  • Purine analogs block nucleotide synthesis and include IMDH inhibitors.
  • Exemplary purine analogs include azathioprine, mycophenolate such as mycophenolate acid or mycophenolate mofetil and lefunomide.
  • Exemplary immunosuppressing biologies include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacep, and daclizumab.
  • the immunosuppressing agent is an anti-CD20 antibody.
  • 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.
  • 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.
  • anti-CD25 antibodies or anti- 1 L2 antibodies or anti-TAC antibodies
  • anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplizumab and visilizumab
  • anti-CD52 antibodies such as alemtuzumab.
  • 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.
  • an effective dose is delivered by a combination of routes.
  • an effective dose is delivered intrathecally, intracerebrally, intracerebroventricularly, intravenously, intracisternally, and/or intramuscularly, or intrathecally and/or intravenously and/or intracerebroventricularly, and the like.
  • an effective dose is delivered in sequence or sequentially.
  • 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.
  • actual administration of delivery vehicle 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.
  • CSF cerebrospinal fluid
  • ICM intracisterna magna
  • compositions can be prepared as injectable formulations for intrathecal injection or as aerosol formulations for inhalation.
  • the delivery vehicle (such as rAAV) can be used with any pharmaceutically acceptable carrier for ease of administration and handling.
  • 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.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cancer is breast cancer, leukemia, colorectal cancer, brain cancer, and/or soft tissue cancer.
  • 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).
  • GAP GTPase-activating protein
  • NF1 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.
  • 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.
  • 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.
  • a wildtype full-length NF1 coding sequence comprises 61 exons.
  • 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.
  • a target tissue is nervous system (e.g., neuron cells having loss of function of NF1 , etc.) tissue.
  • 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.
  • intrastromal injection of rAAV mediates efficient transduction of a nervous tissue (e.g., peripheral neuron, etc.).
  • the injection is intrastromal injection (IS).
  • the administration is via injection, optionally via intratumoral injection, etc.
  • the injection is topical administration (e.g., topical administration to the skin lesion). In some cases, multiple doses of a rAAV are administered.
  • 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).
  • a symptom of NF1 e.g., a disease associated with a mutation of NF1 gene.
  • mutations in NF1 gene include those described by The Human Gene Mutation Database (HGMD, Institute of Medical Genetics, Edinburgh, http://www.hgmd.org), by the Leiden Open Variation Database (LOVD), which are incorporated herein by reference.
  • 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 (2016) 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.
  • 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.
  • the control subject is a subject in need thereof who is not administered with the nucleic acid, the nanoparticle, the vector, or the rAAV.
  • the control subject is a healthy subject.
  • treating the subject or administering to the cell results in changes of molecular markers of NF1 signaling pathway.
  • the changes of the molecular markers of NF1 signaling pathway may reverse the pre-existing neurological deficits associated with NF1 .
  • the changes of the molecular markers of NF1 signaling pathway may prevent neurological deficits associated with NF1 .
  • the molecular markers of NF1 signaling pathway comprise at least pCREB, pSynapsinl, pERK1/2, pDARP32 and tyrosine hydroxylase (TH).
  • the administration results in an increase of pCREB.
  • the administration results in a decrease of pERK1/2.
  • the molecular markers of NF1 signaling pathway can comprise any biological markers that are known or unknown in the art.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • “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.
  • kits for use in the treatment of a disease or disorder described herein 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.
  • 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.
  • 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).
  • the formulation comprises a stabilizer.
  • 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.
  • 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.
  • 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.
  • 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 ”).
  • miniNFI adeno-associated virus
  • 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).
  • FLCAG promoter SEQ ID NO: 22
  • MPZ, P0 myelin protein zero
  • SEQ ID NO: 23 a myelin protein zero
  • SEQ ID NO: 24 Table 1 , Fig. 3
  • 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.
  • 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).
  • ITRs inverted terminal repeats
  • polyA polyadenylation
  • gfa1405 promoter SEQ ID NO: 5
  • a FLCAG promoter SEQ ID NO: 6
  • a P0 promoter SEQ ID NO: 7
  • mini-NF1 NF1 mini gene
  • SEQ ID NO: 1 or 3 the polynucleotide of SEQ ID NO: 1 or 3
  • promoters i.e., a gfa1405 promoter, a ubiquitous promoter (FLCAG), and a Schwann cellspecific promoter (P0) into an AAV9 vector.
  • 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.
  • 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.
  • 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.
  • TaqMan qPCR assay [00149] A TaqMan qPCR assay (Thermo Fisher) was carried out to quantify mRNA expression according to manufacturer’s protocol.
  • mice and source of mice [00150] Mice and source of mice.
  • Neonatal (days 1-3 post-natal) C57BL/6 mice were used in various experiments.
  • Nf1 F/Arg681* HoxB7-Cre mice (University of Alabama) are used in various experiments.
  • 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.
  • Neonatal 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.
  • 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.
  • slices were treated with 0.1% Sodium borohydride in 1X PBS 15 min at RT.
  • PBST Triton
  • 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.
  • PBST Triton
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Blots were subsequently imaged using blot imager. Quantification of bands was performed using Bio-Rad ImageLab.
  • NF1 neurofibromatosis type 1
  • AAV adeno-associated virus
  • 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).
  • eGFP enhanced green fluorescent protein
  • CAG CMV enhancer
  • 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).
  • 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.
  • IMV intracerebroventricular
  • IT-L intrathecal lumbar
  • 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).
  • GFP green fluorescent protein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mice 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.
  • PND1 postnatal day 1
  • IMV postnatal day 1
  • AAV9.gfa1405.GFP AAV9.FLCAG-GFP
  • AAV9.P0. miniNFI .GFP miniNFI .GFP
  • AAV9. FLCAG. GFP, and AAV9.P0. miniNFI .GFP resulted in GFP expression in the brain.
  • mice 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.
  • 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.
  • 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. 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.
  • MBP myelin basic protein
  • GFP green fluorescent protein
  • the construct with the CAG promoter 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.
  • AAV9.FLCAG. miniNFI SEQ ID NO: 22
  • AAV9.P0.miniNF1 SEQ ID NO: 23
  • AAV9.gfa1405.miniNF1 SEQ ID NO: 24
  • 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.
  • 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.
  • miniNFI reduced size NF1 gene
  • CAG ubiquitous promoter
  • P0 Schwann cell specific promoter
  • miniNFI SEQ ID NO: 22
  • AAV9.P0. miniNFI SEQ ID NO: 23
  • AAV9.gfa1405. miniNFI SEQ ID NO: 24
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne des vecteurs de thérapie génique, tels qu'un virus adéno-associé (VAA), conçus pour le traitement de mutations dans le gène de neurofibromine 1 (NF1). Les vecteurs de thérapie génique divulgués fournissent un ADNc mini-NF1 à un sujet en ayant besoin, ce qui conduit à l'expression d'une protéine NF1 fonctionnelle. L'invention concerne également des compositions, des nanoparticules, des vésicules extracellulaires, des exosomes ou un vecteur comprenant le gène NF1 ayant des promoteurs spécifiques des cellules nerveuses et des promoteurs spécifiques des cellules de Schwann et des procédés d'utilisation du gène mini-NF1 ayant des promoteurs spécifiques des cellules nerveuses et spécifiques des cellules de Schwann dans le traitement de la neurofibromatose de type 1. L'invention concerne également de nouvelles constructions de gènes mini-NF1.
PCT/US2024/033750 2023-06-13 2024-06-13 Matériaux et méthodes pour le traitement de mutations de la neurofibromine 1 et de maladies qui en résultent WO2024259064A1 (fr)

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