Materials and methods for neurofibromin 2 / merlin (NF2) gene therapy

Abstract

The present disclosure relates to methods of treating conditions associated with a requirement for merlin protein, for example due to a defective neurofibromin 2 / merlin (NF2) gene, such as in neurofibromatosis type 2 (NF2). Specifically, the present disclosure provides gene therapy vectors that specifically treat loss of expression of merlin protein or reduced levels of merlin protein.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 174,803, filed April 14, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to methods of treating conditions associated with a requirement for merlin protein, for example due to a defective neurofibromin 2 / merlin (NF2) gene, such as in neurofibromatosis type 2 (NF2). Specifically, the present disclosure provides gene therapy vectors that specifically treat loss of expression of merlin protein or reduced levels of merlin protein.

[0003] Incorporation by reference of sequence listing This application contains, as a separate part of the disclosure, a sequence listing in computer readable form (Filename: 54446_SeqListing.txt, 79,842 bytes, ASCII text file created on April 13, 2022), which is incorporated by reference in its entirety herein. [Background technology]

[0004] Neurofibromatosis type 2 (NF2) is an autodominant genetic disorder affecting 1 in 40,000 people worldwide, resulting from loss of the NF2 gene. The NF2 gene encodes the moesin, ezrin, and radixin-like protein, termed "merlin." Merlin functions as a tumor suppressor in tissues of the nervous system. Merlin functions in contact-dependent inhibition of cell proliferation. NF2 patients have loss-of-function mutations in one allele throughout the body, and tumor formation in patients is associated with loss of heterozygosity in the remaining NF2 allele. This loss is often due to second-hit mutations, but loss of expression or reduced levels of the NF2 tumor suppressor protein can also be caused by epigenetic or post-translational modifications in the absence of mutations in the NF2 gene.

[0005] NF2 is a severely debilitating condition characterized by bilateral vestibular schwannomas along with other intracranial, intraspinal, and peripheral tumors such as multiple meningiomas, spinal schwannomas, and ependymomas. It results in increased morbidity and reduced life expectancy. Symptoms may appear in early childhood and worsen over time. Apart from the tumors, NF2 causes significant morbidity including hearing loss, tinnitus (ringing in the ears), balance problems, facial palsy and other cranial nerve dysfunction, cataracts, seizures, and brainstem compression.

[0006] There is no cure for NF2, and current "gold standard" treatment options include surgery and radiation therapy. Surgical removal of the tumor often causes nerve damage, resulting in hearing and visual impairment, as well as facial nerve palsy. Radiation therapy is a less invasive option, but is believed to result in subsequent malignant transformation and a significant increase in secondary malignancies in additional tissue types. Furthermore, as the genetic contribution to tumorigenesis is emphasized, successful treatments are short-lived due to tumor regrowth and new tumors arising from other affected cells. Thus, complications, side effects, inefficiencies in tumor control, and associated loss of neurological abilities make these treatments inadequate. The significant emotional and financial costs of repeated tumor treatments without a therapy that can actually halt the progression of the disease state highlight the urgent need of this patient population.

[0007] In addition to NF2-associated tumors, vestibular schwannomas and meningiomas can occur sporadically in the general population. Most, if not all, vestibular schwannomas harbor inactivating NF2 mutations, and approximately half of sporadic meningiomas harbor NF2 loss.

[0008] Thus, there remains a need in the art for treatments for both NF2-associated and sporadic vestibular schwannomas and meningiomas, as well as other conditions, including cancers, where merlin protein tumor suppressor activity is required. Summary of the Invention

[0009] The present disclosure provides gene therapy vectors that express a functional merlin protein (sometimes referred to herein as "NF2 protein"), which are useful for delivering a transgene encoding a merlin protein to a subject in need of merlin protein tumor suppressor activity (i.e., activity that reduces tumor growth or the formation of new tumors in vivo or in vitro).

[0010] The provided methods treat conditions associated with reduced levels of merlin protein, including, but not limited to, NF2, as well as cancers associated with reduced levels of merlin protein that are not due to pathological mutations in the NF2 gene, and also include, but are not limited to, reduced merlin protein tumor suppressor activity due to transcriptional or post-translational regulation of NF2.

[0011] The present disclosure provides methods of treatment that include administering a gene therapy vector by systemic delivery routes such as direct injection into a tumor, intravenous delivery, intrathecal delivery, or any other delivery method used to apply the vector directly to a tumor or cerebrospinal fluid. Treatment stops the growth of existing tumors, shrinks existing tumors, and / or reduces or prevents the formation of new tumors.

[0012] The gene therapy vector is useful for delivering a transgene to cells that have reduced merlin protein levels in a subject. The cells include, but are not limited to, Schwann cells and meningeal cells. The cells include, but are not limited to, tumor cells, such as schwanomas, meningiomas, and glioblastomas.

[0013] The gene therapy vector is, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, or Anc80, AAV7m8, and their derivatives.The gene therapy vector is, for example, AAV2, AAV5, AAV6, AAV9, AAV8, or AAV10.The gene therapy vector is, for example, AAV9, AAV8, or AAV10.The gene therapy vector is, for example, AAV2, AAV5, or AAV6.

[0014] Transgenes in gene therapy vectors include, for example, the 2.3 kb NF2 promoter or a reduced version of the NF2 promoter, the CB promoter, the P546 promoter, the MBP promoter, or the CMV promoter driving expression of the merlin protein with tumor suppressor activity.

[0015] The gene therapy vector contains, for example, an NF2 / merlin isoform cDNA lacking exon 16, referred to herein as NF2 isoform I. The merlin protein encoded by NF2 isoform I has tumor suppressor activity.

[0016] As another example, the gene therapy vector comprises a polynucleotide encoding a merlin protein having a mutation that destroys a phosphorylation site of the merlin protein, while the merlin protein retains tumor suppressor activity.

[0017] An exemplary gene therapy vector includes, in sequence, an AAV2 internal repeat (ITR), an NF2 promoter, an SV40 intron, an NF2 polynucleotide encoding a protein with tumor suppressor activity, a synthetic polyA signal, and a second AAV2 ITR.

[0018] The gene therapy vector is administered to a subject in need thereof using, for example, intrathecal delivery, and the subject is placed in the Trendelenburg position after administration of the gene therapy vector. [Brief description of the drawings]

[0019] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication containing such color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0020] [Figure 1] 1 provides a schematic diagram of an exemplary construct design for NF2 gene therapy. [Diagram 2] FIG. 1 shows NF2 promoter-dependent differential GFP expression in mammalian cells. [Diagram 3] NF2 promoter-dependent differential NF2 expression in mammalian cells. [Figure 4] Figure 2 shows NF2 promoter-dependent differential GFP expression in vivo. [Diagram 5] NF2 promoter-dependent differential GFP expression in the vestibular nuclei. [Figure 6] NF2 promoter-dependent GFP expression in the sciatic nerve. [Figure 7] NF2 promoter-dependent GFP expression in the sciatic nerve. [Figure 8] Promoter-dependent GFP expression in the sciatic nerve. [Figure 9] Promoter-dependent GFP expression in the sciatic nerve. [Figure 10] NF2 promoter-dependent differential expression of NF2 in vivo. [Figure 11] NF2 promoter-dependent differential NF2 expression in vivo. [Figure 12] NF2 promoter-dependent differential NF2 expression in vivo. [Figure 13] NF2 promoter-dependent differential NF2 expression in vivo. [Figure 14]FIG. 1 shows NF2 promoter-dependent differential NF2 S518A expression in mammalian cells. [Figure 15] Reprogrammed induced Schwann cells (iSCs) express Schwann cell markers (S100 and Krox20) after 9 (A) and 24 (B) days of reprogramming. Fibroblast surface markers (FSM) are absent in both conditions. [Figure 16] We demonstrate the direct conversion of patient skin-derived fibroblasts into induced Schwann cells (iSCs) to model NF2 disease in vitro. [Figure 17] 1 shows reduced NF2 expression in patient-derived human iSCs. [Figure 18] 1 shows reduced NF2 expression in patient-derived human iSCs. [Figure 19] 1 shows reduced NF2 expression in patient-derived human iSCs. [Figure 20] 1 shows increased Phosph-NF2S518 expression in patient-derived human iSCs. [Figure 21] 1 shows reduced expression of Schwann KROX20 and MBP cell maturation markers in NF2 patient iSCs. [Figure 22] 1 shows increased expression of the c-Myc stem cell marker in NF2 patient-derived iSCs. [Figure 23] 1 shows increased expression of SOX2 stem cell marker in NF2 patient-derived iSCs. [Figure 24] 13 shows successful transduction of healthy control iSCs with the AAV9.NF2.GFP vector. [Diagram 25] 1 shows successful transduction of NF2 patient-derived iSCs with the AAV9.NF2.GFP vector. [Figure 26] 1 shows the reduction of c-Myc stem cell marker expression in NF2 patient iSCs by AAV9.NF2 treatment. [Figure 27] 1 shows the reduction of SOX2 stem cell marker expression in NF2 patient iSCs by AAV.NF2 treatment. [Figure 28]Sorted iSCs that were direct hits with shNF2 show reduced levels of NF2. [Figure 29] 1 shows reduced NF2 expression in primary human vestibular schwannoma (hVS) tumor cells. [Diagram 30] 1 shows increased phospho-NF2 S518 expression in hVS tumor cells. [Diagram 31] This shows that hVS tumor cells are more distant from mature Schwann cells. [Diagram 32] High proliferation rates in monoallelic mutant NF2 patient iSCs and NF2 double knockdown patient iSCs. [Diagram 33] 1 shows increased expression of c-Myc and SOX2 stem cell markers in hVS tumor cells. [Diagram 34] 1 shows a decrease in c-Myc stem cell marker expression by AAV.NF2 treatment in hVS cells. [Diagram 35] 1 shows a decrease in SOX2 stem cell marker expression by AAV.NF2 treatment in hVS cells. [Diagram 36] 1 shows reduced NF2 protein expression in glioblastoma. [Figure 37] 1 shows reduced expression of NF2 mRNA in glioblastoma. [Figure 38] 14 shows that AAV9.NF2 treatment restores NF2 protein expression in glioblastoma. [Figure 39] 13 shows that AAV9.NF2 treatment restores NF2 mRNA expression in glioblastoma. [Diagram 40] 4 shows that phosphorylation-resistant AAV9.NF2 treatment better restores NF2 protein in glioblastoma. [Diagram 41] 4 shows that phosphorylation-resistant AAV9.NF2 treatment restores NF2 mRNA expression in glioblastoma. [Diagram 42] 13 shows that AAV9.NF2 treatment reduces cell proliferation in glioblastoma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Gene Therapy Vectors Adeno-associated virus (AAV) is a replication-defective parvovirus, whose single-stranded DNA genome is about 4.7 kb long and contains two 145-nucleotide inverted terminal repeats (ITRs), and may be used to refer to the virus itself or its derivatives. This term includes all subtypes, and both naturally occurring and recombinant forms, unless otherwise specified. There are multiple serotypes of AAV. Each serotype of AAV is associated with a specific clade, whose members share serological and functional similarities. Thus, AAV may also be referred to by clade. For example, the AAV9 sequence is referred to as a "clade F" sequence (Gao et al., J. Virol., 78:6381-6388 (2004). The present disclosure contemplates the use of any sequence within a particular clade, e.g., clade F. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al. al., J. Virol., 45:555-564 (1983). The complete genome of AAV-3 is provided under GenBank accession number NC_1829. The complete genome of AAV-4 is provided under GenBank accession number NC_001829. The genome of AAV-5 is provided under GenBank accession number AF085716. The complete genome of AAV-6 is provided under GenBank accession number NC_001862. At least portions of the genomes of AAV-7 and AAV-8 are provided under GenBank accession numbers AX753246 and AX753249, respectively. The genome of AAV-9 is described in Gao et al. al., J. Virol., 78:6381-6388 (2004). The genome of AAV-10 is provided in Mol. Ther., 13(1):67-76 (2006). The genome of AAV-11 is provided in Virology, 330(2):375-383 (2004). A portion of the genome of AAV-12 is provided in Genbank accession number DQ813647.A portion of the AAV-13 genome is provided in Genbank Accession No. EU285562. The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928 (incorporated herein by reference). The sequence of the AAV-B1 genome is provided in Choudhury et al., Mol. Ther., 24(7):1247-1257 (2016). Anc80 is an AAV vector that is AAV1, AAV2, AAV8, and AAV9. The sequence of Anc80 is provided in Zinn et al., Cell Reports 12:1056-1068, 2015, and Vandenberghe et al., PCT / US2014 / 060163 (all of which are incorporated herein by reference in their entirety), and Genbank Accession Nos. KT235804-KT235812.

[0022] Cis acting sequences that direct viral DNA replication, encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 after their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3). Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0023] AAV has unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, offering the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifetime of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The native AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. In some cases, the rep and cap proteins are provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can also be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0024] As used herein, the term "AAV" refers to a wild-type AAV virus or virus particle. The terms "AAV", "AAV virus", and "AAV virus particle" are used interchangeably herein. The term "rAAV" refers to a recombinant virus, infectious virus, encapsulated virus, or virus particle. The terms "rAAV", "rAAV virus", and "rAAV virus particle" are used interchangeably herein.

[0025] The term "rAAV genome" refers to a polynucleotide sequence derived from a modified native AAV genome. rAAV genomes are provided that have been modified to remove the native AAV cap and rep genes. The rAAV genome contains at least one or both endogenous 5' and 3' inverted terminal repeats (ITRs). The rAAV genome may contain ITRs from an AAV serotype different from the AAV serotype from which the AAV genome was derived.

[0026] Provided herein is a rAAV genome that contains a transgene flanked at the 5' and 3' ends by ITRs.

[0027] SEQ ID NO:1 shows the polynucleotide sequence of the NF2 / merlin isoform lacking exon 16, designated "NF2 isoform I." The NF2 isoform I amino acid sequence encoded by SEQ ID NO:1 is shown in SEQ ID NO:15.

[0028] Transgenes provided herein include, but are not limited to, transgenes comprising a polynucleotide encoding an NF2 isoform I polynucleotide of SEQ ID NO:1, or a polynucleotide encoding a merlin protein having tumor suppressor activity, the polynucleotide being 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide of SEQ ID NO:1.

[0029] Transgenes provided herein include, but are not limited to, transgenes set forth in SEQ ID NOs: 6-9, each of which comprises the NF2 isoform I polynucleotide of SEQ ID NO: 1. Provided herein are transgenes encoding merlin proteins having tumor suppressor activity that are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 6, 7, 8, or 9.

[0030] The transgenes provided herein can encode a merlin protein having tumor suppressor activity that is, for example, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to NF2 isoform I of SEQ ID NO:15.

[0031] The transgenes provided herein include polynucleotides that encode a merlin protein having tumor suppressor activity and that hybridize under stringent conditions to a transgene comprising SEQ ID NO: 1, or a transgene of SEQ ID NO: 6, 7, 8, or 9, or its complement.

[0032] The term "stringent" is used to refer to conditions generally understood in the art as stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing include, but are not limited to, 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY1989).

[0033] Merlin proteins with tumor suppressor activity can be resistant to post-translational negative regulation such as phosphorylation. Phosphorylation-resistant merlin proteins with tumor suppressor activity provided herein include, but are not limited to, merlin proteins in which one or more phosphorylation sites are removed by replacing one or more of the following: serine at position 10 of SEQ ID NO: 15, threonine at position 230 of SEQ ID NO: 15, serine at position 315 of SEQ ID NO: 15, and serine at position 518 of SEQ ID NO: 15 with another amino acid such as alanine to prevent phosphorylation at / around that position. Amino acid substitutions can be achieved, for example, by altering the polynucleotide sequence encoding the merlin protein. For example, altering the thymidine at position 1609 of SEQ ID NO: 1 to guanosine results in the substitution of an alanine residue for the serine residue in the encoded merlin protein. SEQ ID NO: 16 shows a polynucleotide with a guanosine at that position to encode a phosphorylation-resistant merlin protein. SEQ ID NOs: 17 to 20 show transgenes containing a polynucleotide encoding a phosphorylation-resistant merlin protein.

[0034] Exemplary promoters are the 2.3 kb NF2 promoter (SEQ ID NO:2), truncated NF2 promoter [including, but not limited to, the 400 bp NF2 promoter (SEQ ID NO:3), the 610 bp NF2 promoter (sometimes referred to herein as 609 bp) (SEQ ID NO:4), and the 2.1 kb NF2 promoter (SEQ ID NO:5)], the CMV promoter, the chicken β-actin promoter (CB), the P546 promoter, and the Myo7A promoter. Additional promoters are contemplated herein, including, but not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1a promoter, the hemoglobin promoter, and the creatine kinase promoter. Additionally, provided herein are NF2 promoters, truncated NF2 promoters, CMV promoters, CB promoters, P546 promoters, and Myo7A promoters that are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to their respective native promoter nucleotide sequences that have transcription-promoting activity.

[0035] Other examples of transcriptional control elements are tissue-specific control elements, such as promoters that allow expression specifically in neurons, or specifically in astrocytes, or specifically in Schwann cells. Examples include neuron-specific enolase, astrocyte-specific glial fibrillary acidic protein, and Schwann cell-specific myelin protein P0 promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters. The gene cassette may also include an intron sequence that facilitates processing of the transgene RNA transcript when expressed in mammalian cells. One example of such an intron is the SV40 intron.

[0036] "Packaging" refers to the series of intracellular events that result in the assembly and encapsidation of AAV particles. The term "production" refers to the process of production of rAAV (infectious encapsulated rAAV particles) by the packed cells.

[0037] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins, respectively, of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0038] "Helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to replicate and be packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenovirus, herpesvirus, and poxvirus such as vaccinia. Adenoviruses can encompass several different subgroups, but adenovirus type 5 of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as ATCC. Herpes family viruses include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV), and pseudorabies virus (PRV), which are also available from depositories such as ATCC.

[0039] "Helper virus functions" refers to functions encoded in the helper virus genome that enable AAV replication and packaging (in conjunction with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in a number of ways, such as by providing a helper virus or, for example, by providing polynucleotide sequences encoding the necessary functions to the producer cell in trans.

[0040] The rAAV genome provided herein lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome (e.g., ITR) contemplated herein can be derived from any AAV serotype suitable for deriving recombinant virus, including, but not limited to, AAV serotypes Anc80, Anc80L65, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV7mb, AAV-8, AAV-9, AAV-10, AAV-RH10, AAV-11, AAV-12, AAV-13, AAV rh.74, and AAV-B1, and derivatives thereof. As mentioned above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. rAAVs with capsid mutations are also contemplated. See, e.g., Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Modified capsids herein are also contemplated, including capsids with various post-translational modifications, such as glycosylation and deamidation. Deamidation of asparagine or glutamine side chains to convert asparagine residues to aspartic acid or isoaspartic acid residues, and conversion of glutamine to glutamic acid or isoglutamic acid are contemplated in the rAAV capsids provided herein. See, e.g., Giles et al., Molecular Therapy, 26(12):2848-2862 (2018). Modified capsids herein are also contemplated to include targeting sequences that direct the rAAV to diseased tissues and organs in need of treatment.

[0041] The DNA plasmids provided herein contain the rAAV genome described herein. The DNA plasmids can be transferred into cells permissive for infection with AAV helper viruses (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles with AAV9 capsid proteins. Techniques for producing rAAV that provide the packaged rAAV genome, rep and cap genes, and helper virus functions to cells are standard in the art. The production of rAAV particles requires that the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions are present in a single cell (referred to herein as a packaging cell). The AAV rep and cap genes may be from any AAV serotype from which a recombinant virus may be derived, or may be from an AAV serotype different from the rAAV genome ITRs. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, the entire disclosure of which is incorporated herein by reference. AAV capsid proteins can be modified to enhance the delivery of recombinant rAAV. Modifications to capsid proteins are generally known in the art. See, for example, US2005 / 0053922 and US2009 / 0202490, the entire disclosures of which are incorporated herein by reference.

[0042] The method for generating packaging cells is to create a cell line that stably expresses all the components required for the production of rAAV. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selection marker (e.g., a neomycin resistance gene) can be integrated into the genome of the cell. The rAAV genome can be introduced into a bacterial plasmid by procedures such as GC tailing [Samulski et al., Proc. Natl. Acad. S6. USA, 79:2077-2081 (1982)], the addition of a synthetic linker containing a restriction endonuclease cleavage site [Laughlin et al., Gene, 23:65-73 (1983)], or direct blunt-end ligation [Senapathy and Carter, J. Biol. Chem., 259:4661-4666 (1984)]. The packaging cell line may then be infected with a helper virus, such as adenovirus. The advantage of this method is that the cells are selectable and are suitable for large-scale production of rAAV. Other non-limiting examples of suitable methods use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0043] The general principles of rAAV particle production are reviewed in, for example, Carter, Current Opinions in Biotechnology, 1533-539 (1992) and Muzyczka, Curr. Topics in Microbial. and Immunol., 158:97-129 (1992). Various approaches have been proposed by Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mo1. Cell. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988), Samulski et al. al., J. Virol., 63: 3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658.776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine, 13: 1244-1250 (1995), Paul et al. al., Human Gene Therapy, 4:609-615 (1993), Clark et al., Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis on the sections of the documents relating to rAAV particle production.

[0044] Further provided herein are packaging cells that produce infectious rAAV particles. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (synonymous 293 cells). In another embodiment, the packaging cells can be cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblast cells), WI-38 cells (human fetal fibroblast cells), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0045] Also provided herein are rAAVs (e.g., infectious, encapsidated rAAV particles) comprising the rAAV genome of the present disclosure. The genome of the rAAV lacks AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the rAAV genome. The rAAV genome can be a self-complementary (sc) genome. An rAAV having a sc genome is referred to herein as a scAAV. The rAAV genome can be a single-stranded (ss) genome. An rAAV having a single-stranded genome is referred to herein as a ssAAV.

[0046] rAAV can be purified by methods standard in the art (e.g., by column chromatography or cesium chloride gradients, etc.) Methods for purifying rAAV from helper viruses are known in the art and can include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0047] composition Compositions comprising rAAV are also provided. The compositions include rAAVs encoding polypeptides of interest, including but not limited to merlin proteins. The compositions may include two or more rAAVs encoding different polypeptides of interest.

[0048] The compositions provided herein comprise rAAV and a pharma- ceutically acceptable excipient(s). Acceptable excipients are non-toxic to recipients, preferably inert at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, e.g., phosphate-buffered saline (PBS), citric acid, or other organic acids, antioxidants such as ascorbic acid, proteins such as low molecular weight polypeptides, 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 non-ionic surfactants such as Tween, copolymers such as poloxamer 188, Pluronic® (e.g., Pluronic F68), or polyethylene glycol (PEG). The compositions provided herein can include a pharma- ceutically acceptable aqueous excipient containing a non-ionic hypoosmolar compound or contrast agent, such as iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, and the aqueous excipient containing the non-ionic hypoosmolar compound can have one or more of the following characteristics: an osmolality of about 180 mgI / mL, about 322 mOsm / kg water by vapor pressure osmometry, an osmolality of about 273 mOsm / L, an absolute viscosity of about 2.3 cp at 20° C. and about 1.5 cp at 37° C., and a specific gravity of about 1.164 at 37° C. Exemplary compositions include about 20-40% non-ionic hypoosmolar compound, or about 25% to about 35% non-ionic hypoosmolar compound. An exemplary composition includes scAAV or rAAV viral particles formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% of a non-ionic low osmolarity compound. Another exemplary composition includes scAAV formulated in 1× PBS and 0.001% Pluronic F68.

[0049] For CSF delivery, including but not limited to intrathecal delivery, the viral vector can be mixed with a contrast agent (Omnipaque or similar). For example, the composition can include a non-ionic low osmolarity contrast agent, including but not limited to iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, ioxilan, or combinations thereof.

[0050] Dosages may be expressed in units of viral genomes (vg). Doses contemplated herein are approximately 1×10 7 vg, approx. 1×10 8 vg, approx. 1×10 9 vg, approx. 5×10 9 vg, approx. 6×10 9 vg, approx. 7×10 9 vg, approx. 8×10 9 vg, approx. 9×10 9 vg, approx. 1×10 10 vg, approx. 2×10 10 vg, approx. 3×10 10 vg, approx. 4×10 10 vg, approx. 5×10 10 vg, approx. 1×10 11 vg, approx. 1.1×10 11 vg, approx. 1.2×10 11 vg, approx. 1.3×10 11 vg, approx. 1.2×10 11 vg, approx. 1.3×10 11 vg, approx. 1.4×10 11 vg, approx. 1.5×10 11 vg, approx. 1.6×10 11 vg, approx. 1.7×10 11 vg, approx. 1.8×10 11 vg, approx. 1.9×10 11 vg, approx. 2×10 11 vg, approx. 3×10 11 vg, approx. 4×10 11 vg, approx. 5×10 11 vg, approx. 1×10 12 vg, approx. 1×10 13 vg, approx. 1.1×10 13 vg, approx. 1.2×10 13vg, approx. 1.3×10 13 vg, approx. 1.5×10 13 vg, approx. 2×10 13 vg, approx. 2.5×10 13 vg, approx. 3×10 13 vg, approx. 3.5×10 13 vg, approx. 4×10 13 vg, approx. 4.5×10 13 vg, approx. 5×10 13 vg, approx. 6×10 13 vg, approx. 1×10 14 vg, approx. 2×10 14 vg, approx. 3×10 14 vg, approx. 4×10 14 vg, approx. 5×10 14 vg, approx. 1×10 15 vg, ~approx. 1×10 16 Contains a total viral genome of 1×10 9 vg~approx. 1×10 10 vg, approx. 5×10 9 vg~approx. 5×10 10 vg, approx. 1×10 10 vg~approx. 1×10 11 vg, approx. 1×10 11 vg~approx. 1×10 15 vg, approx. 1×10 12 vg~approx. 1×10 15 vg, approx. 1×10 12 vg~approx. 1×10 14 vg, approx. 1×10 13 vg~approx. 6×10 14 vg, and approximately 6 × 10 13 vg~approx. 1.0×10 14 vg, 2.0×10 14 vg, 3.0×10 14 vg, 5.0×10 14 Also contemplated is a dose of 1.65×10 11 vg.

[0051] For example, CSF doses range from approximately 1×10 13 vg / patient ~ approx. 1×10 15 vg / patient. For example, an intravenously delivered dose can range from 1×10 13vg / kilogram (kg) body weight ~2×10 14 vg / kg range.

[0052] Treatment methods The methods of treatment herein target cells with reduced merlin protein tumor suppressor activity. The methods of treatment herein can target cells with a "defective" NF2 gene, i.e., a gene with at least one "defective" (i.e., mutated) allele that encodes a merlin protein lacking tumor suppressor activity. As understood in the art, diploid subjects, such as human subjects, generally have two copies of each gene, referred to as alleles. The methods of treatment herein can target cells with reduced merlin protein tumor suppressor activity other than reduced activity caused by a defective NF2 gene, e.g., reduced merlin protein tumor suppressor activity caused by epigenetic regulation or post-translational modifications (e.g., phosphorylation) that reduce merlin protein tumor suppressor activity. Methods of transducing target cells in a subject (e.g., human subject) are provided. Methods of transducing such Schwann and / or meningeal cells in a subject (e.g., human subject) are provided. Methods of transducing a schwannoma, meningioma, and / or glioblastoma in a subject (eg, a human subject) are provided.

[0053] Use of the methods of treatment described herein is indicated for example for reduced merlin protein tumor suppressor activity in NF2, acute myeloid leukemia, squamous cell carcinoma, bladder cancer, sarcoma, breast cancer, epithelial carcinoma, colorectal cancer, mixed adenosquamous carcinoma, glioma, hepatocellular carcinoma, intestinal cancer, liver cancer, adenocarcinoma, mixed lung cancer, melanoma, meningioma, mesothelioma, serous carcinoma, pituitary carcinoma, renal cell carcinoma, schwannoma (sporadic tumors in patients without NF2), gastric cancer, anaplastic thyroid cancer, endometrial cancer, prostate cancer, and urinary tract cancer.

[0054] The terms "transduce" and "transduction" are used to refer to the administration / delivery of a rAAV of the present disclosure that encodes a merlin protein having tumor suppressor activity to a target cell, either in vivo or in vitro, resulting in expression of a functional merlin protein by the target cell. Transduction of a cell with a rAAV of the present disclosure results in sustained expression of the polypeptide encoded by the rAAV.

[0055] The methods provided herein transduce target cells with one or more rAAVs described herein. In some embodiments, the rAAV viral particles containing the transgene are administered or delivered to the subject's brain and / or spinal cord, for example, by systemic administration (e.g., intravenous administration) or intrathecal administration. Intrathecal administration refers to delivery to the subarachnoid space of the brain or spinal cord. In particular, intrathecal administration to the brain can be performed by intraventricular injection. Areas of the brain contemplated for delivery include, but are not limited to, the motor cortex, the visual cortex, the cerebellum, and the brainstem.

[0056] For intrathecal administration, the subject may be held in Trendelenburg position (head position) after injection of the rAAV (e.g., for about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes). For example, the patient may be tilted in head position from about 1 degree to about 30 degrees, about 15 degrees to about 30 degrees, about 30 degrees to about 60 degrees, about 60 degrees to about 90 degrees, or about 90 degrees to about 180 degrees.

[0057] The therapeutic methods provided herein include administering an effective dose or effective doses of a composition comprising a rAAV provided herein to a subject (e.g., an animal, including, but not limited to, a human patient) in need thereof. If the dose is administered prior to the onset of symptoms of NF2, the administration is prophylactic. If the dose is administered after the onset of symptoms, the administration is therapeutic. An effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the condition, delays or prevents the progression of the condition, reduces the extent of the condition, results in remission (partial or complete) of the condition, and / or prolongs survival.

[0058] An effective dose for the treatment of schwanoma, meningioma, and / or glioblastoma is one that palliates (eliminates or reduces) schwanoma, meningioma, and / or glioblastoma having a defective NF2 gene, delays or prevents the onset of schwanoma, meningioma, and / or glioblastoma, brings about remission (partial or complete) of schwanoma, meningioma, and / or glioblastoma, and / or prolongs survival.

[0059] An effective dose for treating NF2 is one that alleviates (eliminates or reduces) at least one symptom associated with NF2 (e.g., alleviates at least one of tumors (reducing the size and / or number of tumors and / or reducing metastases that form new tumors), hearing loss, tinnitus (ringing in the ears), balance problems, facial weakness or numbness, visual problems, cataracts, seizures, and brain stem compression), slows or prevents the progression of NF2, reduces the extent of NF2, brings about remission (partial or complete) of NF2, and / or prolongs survival.

[0060] The present disclosure provides methods of treating hearing loss associated with NF2.

[0061] Tests to determine whether the methods of treatment described herein improve hearing loss or impairment include physiological tests that objectively determine the functional status of the auditory system, and audiometric tests that subjectively determine how an individual processes auditory information. Physiological tests include auditory brainstem response testing (ABR; also known as BAER, BSER), which uses stimuli (e.g., clicks) that elicit electrophysiological responses originating from the eighth cranial nerve and the auditory brainstem and recorded with surface electrodes. The "V-wave detection threshold" of the ABR correlates best with hearing sensitivity in the 1500-4000 Hz region in neurologically normal individuals. The ABR does not assess low-frequency (<1500 Hz) sensitivity.

[0062] The auditory steady-state response test (ASSR) uses an objective, statistically-based, mathematical detection algorithm to detect and define hearing thresholds. ASSR can be obtained using broadband or frequency-specific stimuli and can differentiate hearing thresholds in the severe-to-profound range. ASSR testing is frequently used to provide frequency-specific information that ABR does not provide. Test frequencies of 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz are commonly used.

[0063] Evoked otoacoustic emissions (EOAEs) are sounds generated within the cochlea, measured in the ear canal using a probe equipped with a microphone and a transducer. EOAEs primarily reflect the activity of the outer hair cells of the cochlea over a wide frequency range and are present in ears with hearing sensitivity better than 40-50 dB HL. Immittance tests (tympanometry, acoustic reflex threshold, acoustic reflex attenuation) evaluate the peripheral auditory system, including middle ear pressure, tympanic membrane mobility, Eustachian tube function, and ossicular mobility.

[0064] Hearing measurements include behavioral tests such as auditory behavioral response testing (BOA) and visual reinforcement audiometry (VRA). Pure tone audiometry (air and bone conduction) involves determining the lowest intensity at which an individual "hears" a pure tone as a function of frequency (or pitch). Octave frequencies from 250 (close to middle C) to 8000 Hz are tested using earphones. Intensity or loudness is measured in decibels (dB), defined as the ratio between two sound pressures. 0 dB HL is the average threshold for normal hearing adults, and 120 dB HL is loud enough to cause pain. Speech reception thresholds (SRT) and speech discrimination are assessed. For air conduction audiometry, sounds are presented through earphones. The threshold depends on the condition of the ear canal, middle ear, and inner ear. For bone conduction audiometry, sounds are presented through a vibrator placed on the mastoid or forehead, thus bypassing the outer and middle ear. Thresholds depend on the condition of the inner ear. Additional testing includes conditioned play audiometry, which develops a complete frequency-specific audiogram for each ear, and conventional audiometry, which shows when an individual hears a sound.

[0065] A hearing profile refers to the recording of several audiograms on a single graph. These audiograms may be from one individual at different times. By plotting multiple audiograms with age on the same graph, the age-related progression of hearing loss can be identified.

[0066] Other Terms and Disclosures Where a range of values ​​is provided herein, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed in the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed in the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

[0068] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0069] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible. This disclosure is intended to provide support for all such combinations.

[0070] As used herein, "may," "may comprise," "may be," "can," "can comprise," and "can be" all refer to things contemplated by the inventors as functional and usable as part of the subject matter provided. EXAMPLES

[0071] Various aspects and advantages of the present disclosure are illustrated by the following non-limiting examples.

[0072] Example 1 Production of rAAV expressing NF2 isoform I rAAV were produced using transgenes expressing reporters such as green fluorescent protein (GFP) (SEQ ID NO: 14) or expressing human NF2 cDNA (NF2 isoform I cDNA of SEQ ID NO: 1) for use in gene therapy re-expression approaches to NF2 loss-of-function pathologies.

[0073] The transgenes contained various NF2 promoters: 2.3 kb, 2.1 kb, 610 bp (sometimes referred to herein as 609 bp), or 400 bp (FIG. 1) driving expression of GFP or merlin protein. The transgene of SEQ ID NO:6 contains a 400 bp promoter, the transgene of SEQ ID NO:7 contains a 610 bp promoter, the transgene of SEQ ID NO:8 contains a 2.1 kb promoter, and the transgene of SEQ ID NO:9 contains a 2.3 kb promoter. The promoters contemplated for use in the methods herein are of a size that allows the transgene construct to fit well into the rAAV and also drives transgene expression to increase merlin protein expression by at least 10% in transduced cells, focusing expression in Schwann cells and meningeal cells, the cell types most frequently affected in NF2.

[0074] HEK293 cells were transiently transfected with the pAAV.NF2.GFP reporter plasmid and analyzed for GFP expression 72 h after transfection. Figure 2, (a) Fluorescence imaging of transfected cells showed NF2 promoter-dependent differential expression of GFP based on different intensities compared to the control chicken β-actin (CB) promoter driving GFP. (b) Western blot analysis of transfected HEK293 cell lysates confirms NF2 promoter-dependent expression of GFP in transfected cells without affecting endogenous NF2 protein levels between untransfected and transfected cells.

[0075] HEK293 cells were transiently transfected with the pAAV.NF2.NF2 CDS plasmid and NF2 expression was analyzed 72 hours post-transfection. Figure 3, (a) HEK293 cells treated with the pAAV.CB.GFP plasmid used as a transfection control showed robust GFP fluorescence confirming transfection efficiency, (b) Western blot analysis of transfected HEK293 cells confirms NF2 promoter-dependent differential overexpression of NF2 compared to pAAV.CB.GFP transfected and untransfected cells.

[0076] The transgenes were subcloned into AAV9 production plasmids, and scAAV and ssAAV were produced by transient triple transfection of 293 cells using a double-stranded AAV2-ITR-based production plasmid, a plasmid encoding the Rep2Cap9 sequence, and the adenovirus helper plasmid pHelper, as described in Foust et al., Nat Biotechnol., 27(1):59-65 (2009). The merlin protein-encoding scAAVs were designated scAAV9.NF2-2.3kb.NF2, scAAV9.NF2-2.1kb.NF2, scAAV9.NF2-610bp.NF2 (sometimes referred to herein as scAAV9.NF2-609bp.NF2), and scAAV9.NF2-400bp.NF2. The merlin protein-encoding ssAAVs were designated ssAAV9.NF2-2.3kb.NF2, ssAAV9.NF2-2.1kb.NF2, ssAAV9.NF2-610bp.NF2 (sometimes referred to herein as ssAAV9.NF2-609bp.NF2 since the promoter is actually 609bp), and ssAAV9.NF2-400bp.NF2.

[0077] To test different NF2 promoters, mice were injected with scAAVs driving GFP expression from the 400bp or 610bp NF2 promoter. Immunofluorescence showed that both promoters expressed GFP at various levels in NF2-positive cells in the brain (mainly in the cerebral cortex, hippocampus, basal forebrain, vestibular cerebellum, and vestibular nuclei), Schwann cells, and sciatic nerve, but injection of scAAVs with the 400bp promoter resulted in higher and more widespread GFP expression than scAAVs with the 610bp promoter. Similarly, mice were injected with ssAAVs driving GFP expression from the 2.3kb or 2.1kb NF2 promoter, and expression was assessed by immunofluorescence.

[0078] In separate experiments testing different promoters, scAAV9.NF2-400bp.GFP, scAAV9.NF2-609bp.GFP, ssAAV9.NF2-2.1kb GFP, and ssAAV9.NF2-2.3kb GFP vectors were delivered via the intracerebroventricular (ICV) route at the same dose (5.0 × 10 10 Neonatal mice were administered 100 mg / animal (vg) and sacrificed one month after injection. Different brain regions (basal forebrain, cortex, and hippocampus) examined for GFP expression by immunohistochemical fluorescence analysis showed that the 400bp.GFP vector had higher overall GFP expression and colocalization with S100β, followed by the 609bp.GFP, 2.1kb.GFP, and 2.3kb.GFP (Figure 4).

[0079] In yet another experiment, scAAV9.NF2-400bp.GFP, scAAV9.NF2-609bp.GFP, ssAAV9.NF2-2.1kb GFP, and ssAAV9.NF2-2.3kb GFP vectors were delivered via the ICV route at the same dose (5.0 × 10 10The 400bp.GFP vector showed higher overall GFP expression and colocalization with S100β in the vestibular nucleus, followed by the 2.3kb.GFP and 2.1kb.GFP vectors (Figure 5).

[0080] In a separate experiment, scAAV9.NF2-400bp.GFP and scAAV9.NF2-609bp.GFP vectors were delivered via the ICV route at the same dose (5.0 × 10 10 The 400bp.GFP vector was administered at 1000 x 1000 ng / animal (vg / animal) to neonatal mice and the animals were sacrificed one month after injection. The sciatic nerves of the mice were harvested and examined for GFP expression by immunohistochemical fluorescence analysis. The 400bp.GFP vector showed significantly higher expression of GFP in the sciatic nerve and co-localization with NF2 expressing cells compared to the 609bp.GFP vector (Figure 6).

[0081] In a separate experiment, scAAV9.NF2-400bp.GFP and scAAV9.NF2-609bp.GFP vectors were delivered via the ICV route at the same dose (5.0 × 10 10 The 400bp.GFP vector was administered at 100x the dose of 0.1vg / animal to neonatal mice and the animals were sacrificed one month after injection. The sciatic nerves of the mice were harvested and examined for GFP expression by immunohistochemical fluorescence analysis. The 400bp.GFP vector showed higher expression of GFP in the sciatic nerve and co-localization with S100β expressing cells compared to the 609bp.GFP vector (Figure 7).

[0082] Mice were also injected with scAAVs bearing the chicken beta-actin ubiquitous promoter or the myelin basic protein promoter. GFP expression from the beta-actin promoter in NF2-positive cells was observed to be greater than that from the myelin basic protein promoter.

[0083] scAAV9.CB.GFP and scAAV9.MBP.GFP vectors were delivered via the ICV route at the same dose (5.0 × 10 10The scAAV9.CB.GFP vector was administered at 1000 x 1000 mg / animal (vg / animal) to neonatal mice and the animals were sacrificed one month after injection. The sciatic nerves of the mice were harvested and examined for GFP expression by immunohistochemical fluorescence analysis. The scAAV9.CB.GFP vector showed significant expression of GFP in the sciatic nerve and co-localization with NF2 expressing cells compared to the MBP (myelin basic protein) driving GFP vector (Figure 8).

[0084] In a separate experiment, scAAV9.CB.GFP and scAAV9.MBP.GFP vectors were delivered via the ICV route at the same dose (5.0 × 10 10 The scAAV9.CB.GFP vector was administered at 1000 x 1000 mg / animal (vg / animal) to neonatal mice and the animals were sacrificed one month after injection. The sciatic nerves of the mice were harvested and examined for GFP expression by immunohistochemical fluorescence analysis. The scAAV9.CB.GFP vector showed significant expression of GFP in the sciatic nerve and co-localization with S100β expressing cells compared to the MBP (myelin basic protein) driving GFP vector (Figure 9).

[0085] Thus, colocalization of the reporter GFP with S100β was observed in all regions (basal forebrain, cortex, hippocampus, vestibular nuclei) for the 400bp, 609bp, and 2.1kB constructs. More colocalization of 400bp.GFP and 609bp.GFP was observed in the basal forebrain, cortex, and hippocampus than 2.1kB.GFP. The 400bp.GFP vector colocalizes better in the vestibular nuclei than any other construct. The 400bp.GFP and 609bp.GFP constructs colocalize in the sciatic nerve, with more colocalization for the 400bp.GFP.

[0086] In another experiment, scAAV9.NF2-400bp.NF2 and scAAV9.NF2-609bp.NF2 vectors were administered to neonatal mice via the ICV route at doses of 2.10E+10vg and 3.50E+10vg per animal, respectively. Animals were sacrificed one month after injection. Western blot analysis of whole brain lysates from animals showed moderate overexpression of NF2 protein in both 400bp.NF2 and 609bp.NF2 treated mice compared to uninjected controls (Figure 10).

[0087] In another experiment, scAAV9.NF2-400bp.NF2, scAAV9.NF2-609bp.NF2, and ssAAV9.NF2-2.1kb.NF2 vectors were administered to neonatal mice at various doses via the ICV route and sacrificed one month after injection. Sagittal brain sections were examined for NF2 expression by immunohistochemical fluorescence analysis, which demonstrated that all treated mice exhibited NF2 overexpression. Notably, the scAAV9.NF2 400bp.NF2 vector resulted in extensive NF2 overexpression when administered at lower doses compared to the 609bp.NF2 and 2.1kb.NF2 vectors (Figure 11).

[0088] In another experiment, scAAV9.NF2-400bp.NF2, scAAV9.NF2-609bp.NF2, and ssAAV9.NF2-2.1kb.NF2 vectors were administered at various doses via the ICV route to neonatal mice and sacrificed one month after injection. Different brain regions (hippocampus, anterior and posterior cortex, basal forebrain, cerebellum, vestibular nuclei) were examined for NF2 expression by immunohistochemical fluorescence analysis. Mice treated with all three vectors showed NF2 overexpression in all regions with the 400bp.NF2 vector resulting in robust NF2 overexpression when administered at lower doses compared to the 609bp.NF2 and 2.1kb.NF2 vectors (Figure 12).

[0089] In another experiment, scAAV9.NF2-400bp.NF2, scAAV9.NF2-609bp.NF2, and ssAAV9.NF2-2.1kb.NF2 vectors were administered at various doses via the ICV route to neonatal mice and sacrificed one month after injection. Different brain regions (hippocampus, anterior and posterior cortex, basal forebrain, cerebellum, vestibular nuclei) were examined for NF2 expression by immunofluorescence analysis. Mice treated with all three vectors showed NF2 overexpression in all regions with the 400bp.NF2 vector resulting in robust NF2 overexpression when administered at lower doses compared to the 609bp.NF2 and 2.1kb.NF2 vectors (Figure 13).

[0090] The results demonstrate which promoter constructs are most effective at targeting NF2-associated cell types and provide an indication of the potential expression levels of the transgene.

[0091] Example 2 Postn-Cre;Nf2 flox / flox Efficacy of AAV.NF2 in mice The rAAV AAV.NF2 produced according to Example 1 was injected into the model Postn-Cre;Nf2, which develops multiple spinal and cranial nerve tumors that are histologically identical to human schwannomas with 100% penetrance. flox / flox (Clapp et al., Hum Mol Genet 2015, 24:1-8) in mice. Mice also exhibit impairments in hearing and balance due to the development of cranial nerve VIII tumors.

[0092] Postn-Cre;Nf2 flox / flox Mice are allowed to grow for 6 months to develop tumors. Postn-Cre;Nf2 flox / flox Mice are treated with AAV.NF2 by intravenous or intrathecal injection either before or after tumor development. Mice receive approximately 1×10 11 vg~approx. 5×10 11A single intrathecal (IT) injection of 1000 mg of rAAV AAV.NF2 (exemplarily formulated in 1× PBS and 0.001% Pluronic F68 (referred to as PBS / F68)) is administered. An injection of AAV-GFP vector is used as a control. Auditory brainstem response measurements are performed to assess hearing impairment in these mice at 8 months for mice treated pre-symptomatically or 3 months after injection for mice treated after the onset of tumor formation, and compared to separate mouse cohorts. Dorsal root ganglia and tumors from AAV.NF2 and AAV.GFP treated mice are then sectioned to compare treatment effects.

[0093] Experiments demonstrate that re-expression of NF2 can prevent schwannoma development and hearing loss in this genetically engineered mouse model.

[0094] Example 3 A murine in vivo NF2 model demonstrating gene therapy in the context of various human NF2 mutations Gene replacement therapy is demonstrated in a human patient in vitro model using patient fibroblasts reprogrammed into induced Schwann cells (iSCs). The reprogramming method utilizes a chemically defined medium to induce expression of Schwann cell markers (S100 and Krox20) within 9 days. Furthermore, the reprogrammed cells downregulate fibroblast surface markers (Figure 15A). The cells can be further grown and still maintain expression of S100 and Krox20 (Figure 15B), allowing for expansion of the cells and use in xenograft models.

[0095] iSC cells are used in patient-specific xenograft mouse models to evaluate the efficacy of gene therapy approaches in vivo. 5000 human patient iSC cells are injected directly into the sciatic nerve of mice as previously described in Fuse et al., Mol Cancer Ther 2017,16:2387-2398. Mice are sacrificed and tumor formation is assessed 2 weeks to 2 months after injection. Alternatively, patient-derived immortalized tumor cells (such as sporadic schwannoma cell lines or NF2 patient schwannoma and meningioma cell lines) are injected instead of patient-derived iSC cells.

[0096] Apart from induced iSCs, patient-derived immortalized human tumor cells are also used in xenograft models, where immortalized sporadic schwannoma cells, as well as NF2 patient schwannoma and meningioma cells, are injected into mice and further evaluated for tumor formation.

[0097] It is contemplated that mutations in human patient fibroblast NF2 gene will generate tumor formation in vivo in mice.In addition, to further reduce NF2 gene expression to promote tumor growth, lentivirus NF2 shRNA is used to reduce NF2 gene expression before Schwann cell differentiation to mimic the "second hit" that tumorigenic cells experience in human patients.The ability of Schwann cells generated from these NF2 shRNA-expressing fibroblasts to generate tumors in vivo in mice is compared with that of non-transduced fibroblasts.

[0098] The resulting mice carrying different patient NF2 gene mutations are used to demonstrate the efficacy of the rAAV AAV.NF2 in preventing tumor formation or reducing tumor growth in the context of the patient mutation. Mice are treated with AAV.NF2 either 24 hours or 2 weeks after injection of iSC / immortalized human tumor cells into the sciatic nerve and / or tumor of the mice. Mice are injected with approximately 1×10 11 vg~approx. 5×10 11A single intrathecal (IT) or intratumoral injection of 1000 μg of rAAV AAV.NF2 (formulated, by way of example, in 1× PBS and 0.001% Pluronic F68 (referred to as PBS / F68)) is administered. Mice are sacrificed and tumor number and size are assessed.

[0099] guided iSC Three healthy control (wild type) and three patient (NF2 mutant) fibroblast cell lines were successfully converted into induced Schwann cells (iSCs) using sequential treatment with six small molecules. Brightfield images of the fibroblasts and respective iSCs showed differences in cell morphology (Figure 16).

[0100] Western blot analysis of patient-derived iSCs showed significantly reduced expression of NF2 protein compared to healthy iSCs (Figure 17). NF2 mRNA expression analysis by qRT-PCR of patient-derived iSCs also showed significantly reduced expression of NF2 protein compared to healthy iSCs (Figure 18). Immunofluorescence analysis of patient-derived iSCs showed reduced expression of total NF2 protein staining compared to healthy iSCs (Figure 19).

[0101] Immunofluorescence analysis of patient-derived iSCs showed increased expression of phospho-S518 merlin (an inactive tumor suppressor) compared to healthy control iSCs. Increased phospho-S518 merlin expression appears to correlate with the severity of the disease phenotype (patient 207 being associated with a more severe phenotype). In addition, phospho-S518 merlin is mislocalized in patient-derived iSCs compared to healthy (wild-type NF2) control iSCs (Figure 20).

[0102] Figure 21 shows reduced Schwann cell maturation markers in NF2 patient iSCs. a) Immunofluorescence analysis showed significantly reduced expression of mature myelinating Schwann cell markers (KROX20 and MBP) in NF2 patient-derived iSCs compared to healthy (wild-type NF2) control iSCs. b) mRNA expression analysis by qRT-PCR showed significantly reduced expression of KROX20 and MBP mRNA in NF2 patient iSCs compared to healthy control iSCs.

[0103] Figure 22 shows increased stem cell marker expression in NF2 patient iSCs. a) Immunofluorescence analysis showed increased expression of the stem cell marker c-Myc in two of three NF2 patients compared to healthy (wild type NF2) control iSCs. b) Image-J analysis of c-Myc positive cells across the total number of cells measured by DAPI showed a significant increase in the percentage of high c-Myc positive in the corresponding patient-derived iSCs. It is therefore contemplated that c-Myc may be a potential biomarker for evaluating therapeutic strategies in vitro.

[0104] Figure 23 shows increased stem cell marker expression in NF2 patient iSCs. a) Immunofluorescence analysis showed increased expression of stem cell marker SOX2 in two of three NF2 patients compared to healthy (wild type NF2) control iSCs. b) Image-J analysis of SOX2 positive cells across the total number of cells measured by DAPI showed a significant increase in the percentage of high SOX2 positivity in the corresponding patient-derived iSCs. Therefore, it is contemplated that SOX2 may be a potential biomarker for evaluating therapeutic strategies in vitro.

[0105] Healthy control iSCs were treated with neuraminidase (NA), transduced with the AAV9.NF2.GFP vector (MOI 300K) and analyzed for GFP fluorescence 72 hours after transduction. All four vectors showed GFP expression in AAV9.CB.GFP vector-treated cells with varying intensity, with treated cells showing high overexpression compared to untreated control iSCs (Figure 24).

[0106] NF2 patient-derived iSCs were treated with neuraminidase (NA), transduced with AAV9.NF2.GFP vector (MOI 300K) and analyzed for GFP fluorescence 72 hours after transduction. All four vectors showed GFP expression at various intensities compared to non-transduced iSC controls. AAV9.CB.GFP vector-treated cells with GFP overexpression serve as transduction controls. (Figure 25)

[0107] Healthy control and NF2 patient derived iSCs were treated with neuraminidase (NA) and transduced with scAAV9.NF2-400bp.NF2, scAAV9.NF2-609bp.NF2, or ssAAV9.NF2-2.1kb.NF2 (MOI 300K) (seeding 25K, transduction for 72 hours) and analyzed for c-Myc expression 72 hours post-transduction. Figure 26, a) Immunofluorescence analysis showed that all three vectors reduced expression of the stem cell marker c-Myc in NF2 patient derived iSCs and healthy (wild type NF2) control iSCs, b) Image-J analysis of high c-Myc positive cells across the total number of cells measured by DAPI showed that scAAV9.NF2-400bp.NF2 treatment resulted in the highest reduction of high c-Myc positive cells compared to the other two vectors.

[0108] Healthy control and NF2 patient-derived iSCs were treated with neuraminidase (NA) and transduced with scAAV9.NF2 400bp.NF2, scAAV9.NF2 609bp.NF2, or ssAV9.NF2 2.1kb.NF2 (MOI 300K) and analyzed for SOX2 expression 72 hours after transduction (n=2). Immunofluorescence analysis showed that all three vectors reduced the expression of the stem cell marker SOX2 in NF2 patient-derived iSCs and healthy (wild-type NF2) control iSCs (Figure 27).

[0109] Thus, all patient iSCs have reduced NF2 levels when normalized to alpha-tubulin. Patient iSCs appear to have reduced SC maturation capacity. Patient iSC lines 205 and 207 show elevated c-Myc and Sox2 levels. Transduction of AAV9.400bp.NF2 and AAV9.609bp.NF2 can reduce c-MYC levels, with the greatest effect observed with the AAV9.400bp.NF2 vector.

[0110] As mentioned above, to further reduce NF2 gene expression to promote tumor growth, lentiviral NF2 shRNA can be used to reduce NF2 gene expression prior to Schwann cell differentiation to mimic the "second hit" experienced by tumor-forming cells in human patients. In addition, the efficacy of NF2 downregulation after Schwann cell differentiation was also tested by using the same lentiviral NF2 shRNA construct to transduce healthy control and NF2-derived induced Schwann cells.

[0111] To reduce NF2 expression, shRNA (shNF2) targeting the NF2 sequence 5'-GGATGAAGCTGAAATGGAATA-3' (SEQ ID NO: 47) was designed and cloned into LV-H2B-RFP (Addgene 26001) under the control of the H1 promoter. The resulting vector expresses shNF2 from the H1 promoter and H2B-RFP from the hPGK promoter, resulting in nuclear RFP expression. iSCs were transduced with LV-shNF2 and LV-RFP (transduction control) (100 MOI) and expanded until there were enough cells to be sorted using RFP-based FACS. After sorting, cells were re-seeded, expanded, and maintained for subsequent analysis of the sorted transduced cells (Western blot, qPCR and MTT cell proliferation assays, antiretroviral drug treatment).

[0112] Sorted iSCs directly hit with shNF2 showed reduced NF2 levels. Figure 28, (A) Fluorescence imaging showed sorted iSCs (black) expressing RFP and shNF2. (B, C, D) Western blot data showed that NF2 protein levels were reduced by 40-50% in sorted healthy and patient iSCs transduced with shNF2 compared to RFP control. T-test: *P≦0.05, **P≦0.01, ***P≦0.00.

[0113] Cell proliferation (MTT) assays analyzed 48 hours after seeding showed higher proliferation rates in monoallelic mutant NF2 patient iSCs and NF2 double knockdown patient iSCs. Figure 29, (A) Two of the three monoallelic mutant NF2 iSCs proliferate 1.5 times faster than healthy iSCs. (B) shNF2 knockdown patient iSCs proliferate 2-3 times faster than healthy shNF2 knockdown iSCs. (C) RFP patient iSCs (monallelic mutant) and shNF2 (NF2 double knockdown) proliferation is reduced to approach healthy levels after antiretroviral drug treatment. T-test: *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001.

[0114] Thus, shRNA-based reduction of NF2 in induced Schwann cells mimics the "second hit" experienced by tumor-forming cells in patients. It is contemplated that this further reduction of NF2, which mimics the second hit, is a suitable tumor modeling approach in vitro, as shown by proliferation assays. In addition to the use of gene therapy constructs, it is contemplated that these cells can be used for drug screening of antiretroviral and other small molecule drugs for treatment options in NF2 patients.

[0115] Patient-derived immortalized human tumor cells Other experiments showed reduced NF2 expression in hVS tumor cells. Figure 30, a) Western blot analysis showed reduced expression of NF2 in hVS tumor cells compared to healthy iAstrocytes and iSCs. b) NF2 mRNA expression analysis by qRT-PCR also showed a corresponding reduction in expression of NF2 in hVS tumor cells compared to healthy iAstrocytes, iSCs, and fibroblasts.

[0116] Immunofluorescence analysis of hVS tumor cells showed (a) reduced overall expression of total NF2 compared to healthy fibroblasts. Importantly, when analyzed for the inactive form of the tumor suppressor (phosphorylated form), hVS tumor cells showed (b) increased expression of phospho-S518 merlin compared to healthy control fibroblasts (Figure 31).

[0117] Analysis of mRNA expression by qRT-PCR showed reduced expression of mature Schwann cell markers (Krox20 and MBP) but increased expression of fibroblast markers (FN1 and Col3A) in hVS tumor cells compared to healthy control fibroblasts, suggesting that hVS tumor cells are far removed from mature, and therefore functional, Schwann cells (Figure 32).

[0118] Stem cell marker expression was increased in hVS tumor cells. FIG. 33, a) Immunofluorescence analysis showed increased expression of stem cell markers c-Myc and SOX2 in hVS tumor cells compared to healthy (wild type NF2) control iSCs. b) Image-J analysis of c-Myc or SOX2 positive cells across the total number of cells measured by DAPI showed a higher percentage of c-Myc / SOX2 positivity among hVS tumor cells compared to healthy iSCs. Thus, c-Myc and SOX2 are contemplated as biomarkers for evaluating therapeutic strategies in vitro.

[0119] hVS tumor cells were treated with neuraminidase (NA), transduced with scAAV9.NF2 609bp.NF2 or ssAAV9.NF2 2.1kb.NF2 (MOI 300K) and analyzed for c-Myc expression 72 hours after transduction. Figure 34, a) Immunofluorescence analysis showed that scAAV9.NF2 609bp.NF2 treatment reduced expression of the stem cell marker c-Myc in hVS tumor cells. b) Image-J analysis of high c-Myc positive cells across the total number of cells measured by DAPI showed that scAAV9.NF2 609bp.NF2 treatment resulted in a greater reduction in high c-Myc positive cells compared to ssAAV9.NF2 2.1kb.NF2 treatment.

[0120] hVS tumor cells were treated with neuraminidase (NA), transduced with scAAV9.NF2 609bp.NF2 or ssAAV9.NF2 2.1kb.NF2 (MOI 300K) and analyzed for c-Myc expression 72 hours after transduction. Figure 35, a) Immunofluorescence analysis showed that both vectors reduced expression of the stem cell marker SOX2 in hVS tumor cells. b) Image-J analysis of high c-Myc positive cells across the total number of cells measured by DAPI showed that scAAV9.NF2 609bp.NF2 treatment resulted in a higher reduction in high SOX2 positive cells compared to ssAAV9.NF2 2.1kb.NF2 treatment.

[0121] Thus, hVS tumor cells have reduced NF2 levels. hVS tumor cells appear to have reduced SC maturation capacity. hVS tumor cells showed elevated c-Myc and Sox2 levels. Transduction of AAV9.609bp.NF2 and AAV9.2.1kb.NF2 can reduce c-MYC levels.

[0122] summary The above model does not require the generation of a new mouse model for each mutation, but demonstrates the efficacy of gene therapy using the rAAV gene therapy vectors described herein against specific NF2 mutations. Furthermore, the model allows for the simultaneous evaluation of gene therapy and complementary therapeutic combination approaches in a highly variable patient population.

[0123] Example 4 Production of rAAV expressing phosphorylation-resistant merlin protein Phosphorylation of the merlin protein can reduce its tumor suppressor activity by converting it to an open, inactive conformation, inhibiting its association with other cellular proteins and the cytoskeleton, and / or leading to its degradation by ubiquitination. Thus, phosphorylation-resistant merlin proteins are also provided herein for use, optionally, in gene therapy.

[0124] Merlin proteins with tumor suppressor activity can be resistant to post-translational negative regulation such as phosphorylation. The phosphorylation-resistant merlin proteins with tumor suppressor activity provided herein have one or more phosphorylation sites removed by replacing one or more of the following: serine at position 10 of SEQ ID NO: 15, threonine at position 230 of SEQ ID NO: 15, serine at position 315 of SEQ ID NO: 15, and serine at position 518 of SEQ ID NO: 15 with another amino acid such as alanine to prevent phosphorylation at / around that position. Amino acid substitutions are achieved, for example, by altering the polynucleotide sequence encoding the merlin protein. For example, changing the thymidine at position 1609 of SEQ ID NO: 1 to guanosine results in the substitution of an alanine residue for the serine residue in the encoded merlin protein. SEQ ID NO: 16 shows a polynucleotide with a guanosine at that position to encode a phosphorylation-resistant merlin protein. SEQ ID NOs: 17-20 show transgenes containing the polynucleotides.

[0125] HEK293 cells were transiently transfected with pAAV.NF2.NF2 S518A plasmid and NF2 expression was analyzed 72 hours after transfection. (a) qRT-PCR analysis of HEK293 cells transfected with pAAV.NF2.NF2 S518A plasmid showed all four promoters driving overexpression of NF2 S518A compared to pAAV.CB.GFP transfected and untransfected cells. (b) Western blot analysis of transfected HEK293 cells confirms NF2 promoter-dependent overexpression of NF2 S518A relative to pAAV9.CB.GFP transfected and untransfected cells. Historically, expression of NF2 mRNA has not directly correlated with NF2 protein levels, suggesting further post-transcriptional regulation of NF2.

[0126] rAAVs encoding phosphorylation-resistant merlin proteins were produced as described in Example 1 using transgenes shown in SEQ ID NOs: 17-20, each transgene encoding an S518A substitution (substitution of alanine for serine at position 518). As in Example 1, the four transgenes contain different NF2 promoters: 2.3 kb, 2.1 kb, 610 bp (actually 609 bp), and 400 bp (Figure 1).

[0127] Example 5 In vitro targeting efficiency of AAV9 vectors for NF2 re-expression in glioblastoma Glioblastoma is a highly malignant tumor derived from astrocytes. The U87 human glioblastoma cell line was derived from a 44-year-old Caucasian female with stage 3 glioblastoma. Merlin is downregulated in U87 high-grade glioblastoma.

[0128] Western blot analysis of U87 glioblastoma cells showed lower NF2 protein expression compared to healthy control lines (Figure 36a-b). mRNA expression analysis by qRT-PCR showed lower expression of NF2 isoform 1 and total NF2 in U87 glioblastoma cells compared to healthy control lines (Figure 37a-b).

[0129] U87 glioblastoma cells were treated with neuraminidase (NA), transduced with scAAV9.NF2-609bp.NF2 or ssAAV9.NF2-2.1kb.NF2 (MOI 300K) and analyzed for NF2 expression 72 hours post-transduction. Figure 38, a) Western blot analysis showed that all three vectors restored NF2 expression in treated U87 glioblastoma cells. b) Quantification of the Western blot analysis reveals that scAAV9.NF2-400bp.NF2 and scAAV9.NF2-609bp.NF2 treatment resulted in a higher increase in NF2 expression in U87 glioblastoma cells compared to ssAAV9.NF2-2.1kb.NF2 treatment.

[0130] U87 glioblastoma cells were treated with neuraminidase (NA), transduced with scAAV9.NF2-609bp.NF2 or ssAAV9.NF2-2.1kb.NF2 (MOI 300K) and analyzed for NF2 expression 72 hours post-transduction. mRNA expression analysis was performed to determine the specific NF2 isoform. Figure 39, a-b) mRNA expression analysis using primers specific for NF2 isoform 1 and total NF2 showed a corresponding increase in AAV9.NF2 treated U87 glioblastoma cells. c) Analysis of vector-derived NF2 transcripts showed a significant increase in vector-specific NF2 in treated U87 cells with higher NF2 expression in scAAV9.NF2-400bp.NF2 and scAAV9.NF2-609bp.NF2 treated U87 glioblastoma cells compared to ssAAV9.NF2-2.1kb.NF2 treatment. However, high RNA expression with lower protein expression may suggest that the NF2 protein may be degraded.

[0131] U87 glioblastoma cells were treated with neuraminidase (NA) and transduced with scAAV9.NF2-400bp.NF2 (wild type NF2 isoform 1) or scAAV9.NF2-400bp.NF2 S518A (phospho-resistant NF2 isoform 1) (MOI 300K) and analyzed for NF2 expression 72 hours post-transduction. Figure 40, a) Western blot analysis showed that both vectors restored NF2 expression in treated U87 glioblastoma cells. b) Quantification of the Western blot analysis reveals that scAAV9.NF2-400bp.NF2 S51A treatment resulted in a higher increase in NF2 expression in U87 glioblastoma cells compared to scAAV9.NF2-400bp.NF2 treatment.

[0132] U87 glioblastoma cells were treated with neuraminidase (NA) and transduced with scAAV9.NF2-400bp.NF2 (wild type NF2 isoform 1) or scAAV9.NF2-400bp.NF2 S518A (phospho-resistant NF2 isoform 1) (MOI 300K) and analyzed for NF2 expression 72 hours post-transduction. mRNA expression analysis was performed to determine the specific NF2 isoform. mRNA expression analysis using primers specific for NF2 isoform 1 (a), total NF2 (b), and vector-derived NF2 transcript (c) showed significantly higher expression in U87 glioblastoma cells treated with both scAAV9.NF2-400bp.NF2 and scAAV9.NF2-400bp.NF2 S518A. Importantly, scAAV9.NF2-400bp.NF2 S518A treatment resulted in high RNA and protein expression in U87 glioblastoma cells (Figure 41).

[0133] U87 glioblastoma cells were treated with neuraminidase (NA) and transduced with scAAV9.NF2-400bp.NF2 (wild type NF2 isoform 1), scAAV9.NF2-400bp.NF2 S518A (phospho-resistant NF2 isoform 1), or scAAV9.NF2-400bp.GFP) transduction control only) (MOI 300K) and analyzed for cell proliferation by MTT assay 144 hours post-transduction. Figure 42, a) Brightfield and fluorescent images of control U87 glioblastoma cells after NA treatment and scAAV9.NF2-400bp.GFP showed cell density and efficient tAAV9 transduction as evidenced by GFP expression. b) U87 glioblastoma cells transduced with scAAV9.NF2-400bp.NF2 and scAAV9.NF2-400bp.NF2 S518A showed a decreased proliferation rate compared to NA-only treated and untreated glioblastoma cells.

[0134] Thus, AAV9.400bp.NF2 and AAV9.609bp.NF2 were the most effective in increasing NF2 levels. AAV9.400bp.NF2 cDNA and AAV9.400bp.NF2 S518A transduction reduce glioblastoma growth rates. Because NF2 mRNA levels are indeed high, but the amount of NF2 detected is low, it is also contemplated that phosphorylation-resistant NF2 protein would be useful in therapeutic methods.

[0135] Various Materials and Methods Used in the Examples AAV Constructs and Vector Production: The final construct contains AAV2 inverted terminal repeats (ITRs) flanked by various promoters (CMV enhancer / beta-actin (CB) promoter, myelin basic protein (MBP) promoter, and NF2 promoter), SV40 intron sequence, and GFP, NF2, or NF2 S518A coding sequences followed by BGH polyA sequence. Four truncated NF2 promoter sequences were amplified by PCR from human genomic DNA using the primer combinations listed in Table 1. The amplicons were used to replace the CB promoter with KpnI and PstI flanking sequences by restriction enzyme digestion to generate the NF2 promoter driving GFP expression. To generate a construct expressing wild-type NF2 cDNA, the full-length wild-type NF2 isoform 1 sequence was amplified from a commercially available expression plasmid (Origene, SC124024) using the primers listed in Table 1 below. The NF2 isoform 1 sequence was then used to replace the GFP cDNA sequence in the four NF2.GFP constructs by restriction enzyme digestion with AgeI and BsrGI flanking sequences. To generate a construct expressing phosphorylation-resistant NF2 S518A, the NF2 cDNA sequence containing a point mutation conferring the S518A substitution was commercially synthesized (GenScript, Piscataway, NJ), amplified, and used to replace the GFP cDNA sequence in the four NF2.GFP constructs by restriction enzyme digestion with AgeI and BsrGI flanking sequences. The constructed NF2 promoter-NF2 cDNA and NF2 promoter-GFP cDNA expression cassettes were cloned into self-complementary and single-stranded AAV plasmids in forward or reverse orientation with ampicillin and kanamycin resistance genes in either orientation. All vector DNA constructs were sequence verified before packaging into AAV9 capsids (self-complementary or single-stranded) by small-scale vector production (Andelyn Biosciences, Columbus, OH).

[0136] Animals: All procedures performed were in accordance with National Institutes of Health guidelines and approved by the Research Institute at Nationwide Children's Hospital (Columbus, OH) Institutional Animal Care and Use Committees (IACUC). Experiments were performed on wild-type C57Bl / 6 mice (strain no. 000664; The Jackson Laboratory, ME, USA). P0-P2 pups were used for unilateral intracerebroventricular (ICV) injection of AAV9 vectors. Animals were evaluated daily to ensure health status and sacrificed 1 month after injection for analysis.

[0137] Injections: For ICV injections of mice at P0-P2, pups were anesthetized on ice for 10 min before injection. Injections were performed with a Hamilton syringe attached to a 33 G needle, as previously described. AAV9 vectors were diluted in phosphate-buffered saline (PBS) to reduce the dose. The total volume injected for each animal was 5 µL.

[0138] Cell Lines: Human dermal fibroblasts were isolated from skin punch samples obtained from NF2 patients. Informed consent was obtained from all subjects prior to sample collection. Receipt of human samples was approved through the Nationwide Children's Hospital Institutional Review Board. Heterozygous NF2 mutations present in each patient (P1-205, P2-207, and P3-201) were confirmed by amplicon sequencing using an Illumina HiSeq 2000. Established dermal fibroblast cell lines were cultured and grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Waltham, MA) supplemented with GlutaMAX™, 10% fetal bovine serum (FBS) (Gibco), and 1% antibiotic-antimycotic (Gibco).

[0139] The immortalized human vestibular schwannoma (hVS) cell line was previously described [Chang et al, PLoS ONE, 16(7):e0252048. https: / / doi.org / 10.1371 / journal.pone.0252048(2021)]. hVS cells were cultured in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) supplemented with GlutaMAX™, 10% FBS and 1% antibiotic-antimycotic, 100 μM recombinant human HRG1-beta1 (HRG; R&D Systems, Minneapolis, MN), and 10 mM forskolin (Peprotech, Cranbury, NJ). Tissue culture-treated plates were coated with 50 μg / mL poly-L-lysine (70–150 kDa) (Sigma-Aldrich, St. Louis, MO) and 0.1 M boric acid and 0.01% borate for 30 min at room temperature, washed twice with 1× PBS, and coated with 4 μg / mL laminin mouse protein (Gibco) for 30 min at room temperature before seeding with hVS cells.

[0140] U87 glioblastoma cell line (received from Dean Lee, Nationwide Children's Hospital, Columbus, Ohio) and HEK293 human embryonic kidney cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% antibiotic-antimycotic.

[0141] Direct conversion of fibroblasts into induced Schwann cells: Patient fibroblasts and healthy fibroblasts were directly converted into induced Schwann cells (iSCs) using a modified protocol previously described by Kitada et al. (2019). Briefly, fibroblasts between passages 5 and 9 were plated at 790 cells / cm in tissue culture-treated plates using DMEM supplemented with GlutaMAX™, 10% FBS, and 1% antibiotic-antimycotic. 2The cells were seeded at a density of 100-200 μM. 24 hours after seeding, the medium was replaced with DMEM without added serum and supplemented with 1 mM β-mercaptoethanol (BME; Sigma-Aldrich). The next day, the medium was replaced with DMEM supplemented with 10% FBS and 35 ng / ml retinoic acid (Sigma-Aldrich) and incubated in this medium for 72 hours. Afterwards, the cells were washed once with Dulbecco's phosphate-buffered saline (DPBS) and incubated in fresh iSC induction medium (DMEM supplemented with GlutaMAX™, 5% FBS, 1% antibiotic-antimycotic, and the following small amounts of chemicals: 5 μM forskolin (PeproTech), 10 ng / mL human basic fibroblast growth factor (bFGF; Peprotech), 10 ng / mL platelet-derived growth factor-AA (PDGF-AA; Peprotech), and 200 ng / mL HRG). Cells were incubated in this medium and split at 90% confluence for proliferation and then characterized by microscopy and molecular and cellular assays.

[0142] Transfection of HEK293 cells for evaluation of GFP and NF2 expression: 1.5 × 10 6 Cells / well in 6-well plates (2 x 10 5 cells / cm 2 Cells were seeded at 1000 x g (cell density of 100 x 1000). Cells were transfected at 80% confluence with 1.5 μg of purified plasmid constructs by calcium chloride treatment. Briefly, plasmid DNA was added to 250 mM CaCl2 and gently swirled before adding an equal volume of 2x HEPES-buffered saline (HBS) (pH 7.05) to each tube. 2 ml of transfection medium (DMEM 2% FBS) was added to the plasmid solution, mixed by pipette, and carefully added to the cells. After 12 hours, the transfection mixture was replaced with fresh medium and incubated for 72-96 hours to allow for transient expression of GFP or NF2 from the transfection constructs. Cells were harvested by trypsinization and used for subsequent analysis by Western blot for qPCR for GFP and NF2 expression.

[0143] AAV transduction of cells in culture: hVS tumor cells and iSCs were seeded in 24-well plates with glass coverslips at 25,000 cells / well for subsequent analysis by immunostaining. U87 glioblastoma cells were seeded in 12-well tissue culture plates at 80,000 cells / well for analysis by Western blot, qPCR, and MTT cell proliferation assay. All culture vessels were pretreated with 5 μg / mL human fibronectin in DPBS at room temperature for 30 min to enhance cell attachment. The next day, cells were carefully washed three times with 2% FBS medium (hVS), 5% FBS medium (iSC), or serum-free medium (U87), then incubated in 100 mU / mL neuraminidase (NA) (Sigma-Aldrich) for 2 h at 37 °C / 5% CO2. The NA was carefully removed and the cells were washed again three times with the respective medium above and treated with AAV vectors diluted in medium at 300,000 DRP / mL. The plates were incubated at 4 °C for 1 h and then transferred to a humidified incubator at 37 °C / 5% CO2. 48 h after transduction, fresh complete medium was added to the cells and incubation was continued for up to 72–96 h for subsequent analysis of the transduced cells.

[0144] Immunofluorescence staining: Fibroblasts, hVS tumor cells, and iSCs were seeded in 24-well plates with glass coverslips. The next day, cells were fixed with 4% paraformaldehyde at room temperature for 12 minutes, washed three times with DPBS, and blocked for 1 hour with DPBS containing 10% goat serum, 0.1% TritonX-100, and 0.1% Tween-20 at room temperature. Cells were then incubated in primary antibodies overnight at 4°C with gentle shaking. All primary antibodies were diluted in blocking solution. The respective dilutions and suppliers are listed in Table 2 below. The next day, cells were washed three times in DPBS and incubated in Alexa Fluor-conjugated secondary antibodies (Invitrogen) and DAPI (Thermo Fisher Scientific) diluted in blocking solution at room temperature for 1 hour. After washing three times with DPBS, coverslips were mounted and sealed on slides with Vectashield (Vector Labs, Burlingame, CA). Images were captured with a Nikon Eclipse Ti2-E motorized inverted microscope and NIS-Elements imaging software. Quantification of cells with high c-Myc or Sox2 staining was performed using ImageJ / Fiji image processing and analysis software (Schneider et al., 2012).

[0145] Immunohistochemistry: Mouse brains and sciatic nerves were sectioned from deeply anesthetized mice that were transcardially perfused with 10 mL of 0.9% saline. Brains were post-fixed in 4% PFA at 4°C until sectioning, mounted on a vibratome stage using 5% agarose in water, and cut sagittally at a thickness of 40 μm. Sections were sequentially placed in adjacent wells of a multi-well plate containing 0.1 M PBS and 0.2% sodium azide for storage at 4°C until immunostaining. Sections were then washed three times in PBS containing 0.1% Tween-20 (PBST) and then blocked for 2 hours at room temperature in DPBS containing 10% normal donkey serum, 1% Triton X-100, and 0.02% sodium azide. Sections were incubated in primary antibodies diluted in blocking solution for 48 hours at 4°C with gentle shaking. The respective dilutions and providers of all antibodies used are listed in Table 2. Before incubation in Alexa Fluor-conjugated secondary antibodies, washed three times with DPBS and diluted DAPI in blocking solution for 2 h at room temperature with gentle shaking, sections were washed again three times in PBS and mounted and sealed on slides using polyvinyl alcohol mounting medium with DABCO antifading (PVA-DABCO; Sigma-Aldrich).

[0146] Sciatic nerves were post-fixed in 4% PFA for 24 hours at 4°C and then transferred to PBS for 2 hours at room temperature. Nerves were incubated in sucrose gradients (5%, 10%, 20%) for 30 minutes, 5% and 10% at room temperature, and 20% sucrose in DPBS overnight at 4°C. Nerves were mounted in Tissue-Tek's optimal cooling temperature (OCT) mounting medium (Sakura Finetek, Torrence, CA), sectioned in a cryostat at -20°C at a thickness of 14 μm, mounted on microscope slides, and stored at -80°C until staining. Staining was performed by warming slides briefly at room temperature, washing with Tris-buffered saline (TBS), and then incubating primary antibodies diluted in antibody dilution buffer (ADB; TBS with 0.02% sodium azide and 0.2% Triton X-100) overnight at room temperature. Slides were washed three times in TBS and then incubated in Alexa Fluor-conjugated secondary antibodies diluted in ADB for 1 h at room temperature, washed again three times in TBS, mounted and sealed with PVA-DABCO.

[0147] Visualization and imaging of brain sections and sciatic nerves was performed using a Nikon Eclipse Ti2-E motorized inverted microscope and NIS-Elements imaging software, or a Zeiss LSM 800 confocal microscope and Zeiss ZEN imaging software.

[0148] Western Blot: Cells were lysed in radioimmunoprecipitation (RIPA) lysis and extraction buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 1× cOmplete™, EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN). Tissue samples were incubated in T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific) containing 1× cOmplete™, EDTA-free protease inhibitor cocktail, then homogenized for 2 min at room temperature using a QIAGEN TissueLyser II bead mill (QIAGEN, Valencia, CA) set at 30 Hz. Lysates were sonicated for 3-5 s and then centrifuged at 5000×g for 20 min at 4°C. The supernatant containing total protein was quantified by detergent-compatible (DC) protein assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol. 40 μg of cell / tissue lysate was loaded onto a 4-12% Bis-Tris polyacrylamide gel (Invitrogen) and run at 120 V for 1 h. Proteins were transferred onto a PDVF membrane and blocked with Intercept® (PBS) blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature. Primary antibodies against NF2 (ABclonal A2456, 1:2000 dilution), GFP (Abcam ab13970, 1:1000 dilution), GAPDH (Millipore MAB374, 1:5000 dilution), and α-tubulin (Sigma-Aldrich T6074, 1:5000 dilution) were diluted in 1:1 blocking buffer / TBST with 1% Tween-20 (TBS-T) and incubated overnight at 4° C. Membranes were then washed three times with TBS-T and incubated with LI-COR IRDye 800 or 680 secondary antibodies (1:7000 dilution) in 1:1 blocking buffer / TBS-T containing 0.2% sodium dodecyl sulfate (SDS) for 1 h at room temperature.Blots were washed three times with TBS-T and then imaged using an Odyssey DLx Imaging System (LI-COR) and analyzed using Image Studio™ Lite Quantification Software (LI-COR). GAPDH or α-tubulin were used as housekeeping genes to normalize NF2 or GFP expression.

[0149] Quantitative PCR: Total RNA was isolated from cell pellets using TRIzol (Invitrogen) extraction protocol. RNA samples were then subjected to DNAse-I treatment using QIAGEN RNase-Free DNase Set. RNA quality and concentration were determined by NanoDrop spectrophotometry (Thermo Scientific). First strand cDNA was generated with 1000 μg RNA per setup using RevertAid RT Reverse Transcription Kit (Thermo Scientific) according to the manufacturer's protocol. Quantitative PCR (qPCR) was performed using 1 μL RT product, 500 nM primers, 1× Power SYBR® Green PCR Master Mix (Applied Biosystems), and nuclease-free water in a total volume of up to 20 μL. PCR was performed on a 96-well plate qPCR system (QuantStudio 3, Applied Biosystems) with cycling conditions of 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Gene-specific primers used are listed in Table 3 below. Each sample was run in triplicate and relative mRNA expression was calculated using the comparative Ct (ΔΔCT) method with endogenous GAPDH and RPL13A as housekeeping controls for normalization. At least three biological replicates were performed for each experiment.

[0150] Cell proliferation assay (MTT): U87 cells were replated in triplicate in 96-well plates at 1500 cells / well in DMEM supplemented with 5% FBS and 1% antibiotic-antimycotic at the time of AAV transduction as described above. The number of metabolically active cells per well was measured 48 h after seeding using the Cell Proliferation Kit I (MTT) (Roche) by adding 10 μL of MTT labeling reagent per well, incubating at 37°C for 4 h, and adding 10 μL of solubilization buffer and incubating at 37°C overnight. Absorbance values ​​at 575 nm were measured with a colorimetric plate reader (Synergy 2; BioTek, Winooski, VT) and analyzed using Gen5 data acquisition and analysis software (BioTek). The average absorbance was calculated for each setup and normalized to cells treated with NA only.

[0151] Statistical analysis: All statistical tests were performed by unpaired two-tailed t-test to measure differences between two setups. Data from all quantitative experiments are presented as mean ± standard error of the mean (SEM). In all tests, significance values ​​were defined as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Graphs were generated using GraphPad Prism 9.0 software. [Table 1] [Table 2] [Table 3]

[0152] While the present invention has been described with respect to specific embodiments, it is understood that variations and modifications thereof will occur to those skilled in the art, and therefore only such limitations as appear in the claims should be placed on the invention.

[0153] All documents referenced in this application are incorporated herein by reference in their entirety. The present invention provides, for example, the following items. (Item 1) A method for delivering a transgene encoding a merlin protein having tumor suppressor activity to a subject in need of merlin protein tumor suppressor activity, comprising administering a gene therapy vector containing the transgene to the subject by intravenous, intratumoral, or intrathecal delivery. (Item 2) 2. The method of claim 1, comprising delivering to a subject having at least one defective NF2 allele a transgene encoding a merlin protein having tumor suppressor activity, said method comprising administering to said subject a gene therapy vector comprising said transgene by intravenous, intratumoral, or intrathecal delivery. (Item 3) 3. The method of claim 2, wherein the transgene is delivered to Schwann cells or meningeal cells, or both, of the subject. (Item 4) 4. The method of claim 3, wherein said administering treats malignant glioblastoma in said subject. (Item 5) 4. The method of item 2 or 3, wherein the administration treats neurofibromatosis type 2 in the subject. (Item 6) 6. The method of claim 5, wherein said administering alleviates one or more of tumors, hearing loss, tinnitus, balance problems, facial weakness or numbness, visual impairment, cataracts, stroke, and brain stem compression in said subject. (Item 7) 7. The method of claim 6, wherein the tumor is one or more of schwannoma, meningioma, ependymoma, or other cranial nerve tumors. (Item 8) 8. The method according to any one of items 1 to 7, wherein the transgene comprises a polynucleotide of SEQ ID NO: 1, 6, 7, 8, or 9 encoding a merlin protein having tumor suppressor activity, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 6, 7, 8, or 9. (Item 9) 8. The method according to any one of items 1 to 7, wherein the transgene comprises a polynucleotide of SEQ ID NO: 16, 17, 18, 19, 20 encoding a phosphorylation-resistant merlin protein having tumor suppressor activity, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, 17, 18, 19, 20. (Item 10) 10. The method of any one of items 1 to 9, wherein the gene therapy vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, Anc80, or AAV7m8 serotype vector, or a derivative thereof. (Item 11) 11. The method of any one of items 1 to 10, wherein the gene therapy vector is administered by intrathecal delivery. (Item 12) 12. The method of any one of items 1-11, wherein the gene therapy vector is administered by intrathecal delivery, and the method further comprises placing the subject in Trendelenburg position after administration of the gene therapy vector. (Item 13) 11. The method of any one of items 1 to 10, wherein the gene therapy vector is administered by intratumoral delivery. (Item 14) 11. The method of any one of items 1 to 10, wherein the gene therapy vector is administered by intravenous delivery. (Item 15) 11. The method of any one of items 1 to 10, wherein the gene therapy vector is administered by intrathecal delivery. (Item 16) 16. The method of any one of items 1-15, wherein the gene therapy vector is administered by intrathecal delivery, and the method further comprises placing the subject in Trendelenburg position after administration of the gene therapy vector. (Item 17) A transgene comprising a polynucleotide of SEQ ID NO: 1, 6, 7, 8, or 9 encoding a merlin protein having tumor suppressor activity, or a polynucleotide which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 6, 7, 8, or 9. (Item 18) A transgene comprising a polynucleotide of SEQ ID NO: 16, 17, 18, 19, or 20 encoding a phosphorylation-resistant merlin protein having tumor suppressor activity, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, 17, 18, 19, or 20. (Item 19) A recombinant adeno-associated virus (rAAV) having a genome comprising a transgene comprising a polynucleotide of SEQ ID NO: 1, 6, 7, 8, or 9, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 6, 7, 8, or 9, encoding a merlin protein having tumor suppressor activity. (Item 20) A recombinant adeno-associated virus (rAAV) having a genome comprising a transgene comprising a polynucleotide of SEQ ID NO: 16, 17, 18, 19, 20, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, 17, 18, 19, 20, encoding a phosphorylation-resistant merlin protein having tumor suppressor activity. (Item 21) 21. The recombinant adeno-associated virus (rAAV) of item 19 or 20, wherein the recombinant adeno-associated virus is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, Anc80, or AAV7m8 serotype vector, or a derivative thereof. (Item 22) 22. The recombinant adeno-associated virus (rAAV) of item 21, which is AAV9.

Claims

1. 1. A composition for use in a method for delivering a transgene encoding a merlin protein having tumor suppressor activity to a subject in need of merlin protein tumor suppressor activity, said composition comprising a gene therapy vector comprising said transgene; the method comprising administering the composition to the subject by intravenous, intratumoral, or intrathecal delivery; The composition, wherein the transgene comprises a polynucleotide of SEQ ID NO: 1, 6, 7, 8, or 9 encoding a merlin protein having tumor suppressor activity, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 6, 7, 8, or 9, or the transgene comprises a polynucleotide of SEQ ID NO: 16, 17, 18, 19, or 20 encoding a phosphorylation-resistant merlin protein having tumor suppressor activity, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, 17, 18, 19, or 20.

2. The composition described in claim 1, wherein the method further comprises delivering the introduced gene to a subject having at least one defective NF2 allele.

3. The composition of claim 2, wherein the transgene is delivered to Schwann cells or meningeal cells, or both, of the subject.

4. The composition of claim 3 , wherein said administration treats malignant glioblastoma in said subject.

5. 3. The composition of claim 2, wherein said administration treats neurofibromatosis type 2 in said subject.

6. 6. The composition of claim 5, wherein said administration relieves one or more of tumors, hearing loss, tinnitus, balance problems, facial weakness or numbness, visual impairment, cataracts, stroke, and brain stem compression in said subject.

7. The composition of claim 6, wherein the tumor is one or more of a schwannoma, a meningioma, an ependymoma, or other cranial nerve tumor.

8. 2. The composition of claim 1, wherein the gene therapy vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, Anc80, or AAV7m8 serotype vector, or a derivative thereof.

9. The composition according to any one of claims 1 to 8, characterized in that the composition is administered by intrathecal delivery.

10. 10. The composition of claim 9, wherein the composition is administered by intrathecal delivery, the method further comprising placing the subject in Trendelenburg position after administration of the composition.

11. The composition according to any one of claims 1 to 8, characterized in that the composition is administered by intratumoral delivery.

12. The composition according to any one of claims 1 to 8, characterized in that the composition is administered by intravenous delivery.

13. The composition according to any one of claims 1 to 8, characterized in that the composition is administered by intrathecal delivery.

14. A transgene comprising a polynucleotide of SEQ ID NO: 1, 6, 7, 8, or 9 encoding a merlin protein having tumor suppressor activity, or a polynucleotide which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 6, 7, 8, or 9.

15. A transgene comprising a polynucleotide of SEQ ID NO: 16, 17, 18, 19, 20, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, 17, 18, 19, 20, encoding a phosphorylation-resistant merlin protein having tumor suppressor activity.

16. A recombinant adeno-associated virus (rAAV) having a genome comprising a transgene comprising a polynucleotide of SEQ ID NO: 1, 6, 7, 8, or 9, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 6, 7, 8, or 9, encoding a merlin protein having tumor suppressor activity.

17. A recombinant adeno-associated virus (rAAV) having a genome comprising a transgene comprising a polynucleotide of SEQ ID NO: 16, 17, 18, 19, 20, or a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16, 17, 18, 19, 20, encoding a phosphorylation-resistant merlin protein having tumor suppressor activity.

18. 18. The recombinant adeno-associated virus (rAAV) of claim 16 or 17, wherein the recombinant adeno-associated virus is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT, Anc80, or AAV7m8 serotype vector, or a derivative thereof.

19. The recombinant adeno-associated virus (rAAV) of claim 18, which is AAV9.

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