Compositions and methods for treating neurofibromatous disorders

Abstract

For example, compositions and methods are provided herein for expressing Merlin protein or its functional fragment from a viral vector to treat neurofibromatosis disorder.In some embodiments, compositions are provided that comprise AAV as provided herein.In some embodiments, pharmaceutical compositions are provided that comprise AAV as provided herein and a pharma-ceutically acceptable carrier.In some embodiments, a method of delivering Merlin protein to cells, comprising contacting the cells with AAV as provided herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 202,359, filed June 8, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] background Neurofibromatosis type 2 (NF2) is a rare genetic disorder caused by germline mutations in the NF2 gene, which encodes Merlin, a tumor suppressor protein. These mutations result in a deficiency of the Merlin protein, which regulates cellular processes including contact inhibition, proliferation, and apoptosis. NF2 is characterized by slowly growing tumors, such as schwannomas (arising from Schwann cells), meningiomas (arising from arachnoid cells), and ependymomas (arising from ependymal cells), as well as juvenile cataracts, and retinal hamartomas.

[0003] Typical symptoms of NF2 include bilateral vestibular schwannomas, which are multi-lobular masses arising in the eighth cranial nerve. Vestibular schwannomas cause hearing loss, tinnitus, defness, dizziness, and loss of balance. Patients typically become deaf due to either the effects of tumor growth (including ototoxic effects on the ear leading to death of cells in the inner ear involved in the sense of sound) or interventions to manage the tumor (including partial or complete removal of the auditory nerve). If left unmanaged, these tumors can eventually compress the brain stem and cause death. Additional symptoms may include disfigurement, facial weakness, headaches, and loss of vision.

[0004] Nearly all affected individuals develop these schwannomas by age 30. For those with sporadic NF2 (slightly more than half), the average age of diagnosis of NF2 is between 18 and 24 years. The average life expectancy of patients with NF2 is 69 years, 11 years shorter than the life expectancy of the general population.

[0005] Patients with NF2 have an 80% lifetime chance of developing a meningioma. Meningiomas arise in the meninges, the lining of the brain or spinal cord, and originate from arachnoid cells. The median number of meningiomas these patients develop is 3. More than 33% of the tumors are characterized as significantly growing (>1 mm / year). In addition, meningiomas also occur in sporadic (non-NF2) patients due to somatic mutations in the NF2 gene. Sporadic meningiomas are the most common brain tumor (about 35% of all brain tumors) and can cause neurological disability due to compression of the brain or spinal cord. The development of meningiomas is the largest driver of permanent morbidity and mortality in NF2 patients.

[0006] There are currently no FDA-approved drugs for meningiomas. The current standard of care includes surgery and radiation therapy. Recurrence is rare for meningiomas that have been completely removed. In contrast, less than complete resection is associated with a high recurrence rate. Radiation therapy is a reasonable option for patients with non-surgical meningiomas. However, radiation is associated with an increased rate of secondary cancers, which is particularly problematic in the context of tumor suppressor syndrome. For this reason, many clinicians minimize the use of radiation in NF2 patients, especially younger patients.

[0007] Patients with NF2 are at increased risk of developing spinal ependymomas. These tumors arise from ependymal cells and typically occur within the brainstem or spine. Ependymomas often occur in a "pearls in a string" pattern with multiple lesions in the cervical spine. A small subset of ependymomas require treatment. The standard approach is surgical resection, with radiation reserved for lesions that recur after surgical resection.

[0008] In addition to the 8th cranial nerve (CN8), or vestibulocochlear nerve, patients with NF2 commonly develop schwannomas in other cranial nerves, including: CN3: oculomotor nerve, CN4: trochlear nerve, CN5: trigeminal nerve, CN6: abducens nerve, CN7: facial nerve, CN9: glossopharyngeal nerve, CN10: vagus nerve, CN11: accessory nerve, CN12: hypoglossal nerve. Damage or loss of function in any of these nerves can cause a wide variety of conditions in the head and neck.

[0009] NF2 patients can also develop a number of eye conditions, including: cataracts, retinal detachment, damage to the nerves of the eye, papilledema (optic disc edema), ocular migraine (retinal migraine), retinitis pigmentosa (RP) (retinal degeneration), mixed hamartomas of the retina and RPE, retinal microaneurysms, preretinal conjunctivitis, physiopedia (severe dry eye), nystagmus-oscillopsia (ocular flutter / cross), diplopia (double vision), and gaze-evoked tinnitus (GET).

[0010] Thus, there is a need for compositions and methods for treating NF2 patients. The present embodiments meet these and other needs. Summary of the Invention [Means for solving the problem]

[0011] Abstract In some embodiments, an adeno-associated virus (AAV) is provided that includes an adeno-associated virus capsid protein and a transgene encoding a full-length Merlin protein or one or more active fragments thereof (e.g., but not limited to, residues 1-359 of Merlin isoform 1, residues 1-313 of Merlin isoform 1, residues 1-219 of Merlin isoform 1, residues 1-73 of Merlin isoform 1, residues 312-595 of Merlin isoform 1, residues 479-595 of isoform 1, residues 503-595 of Merlin isoform 1, or any combination thereof).

[0012] In some embodiments, a composition is provided comprising an AAV as provided herein.

[0013] In some embodiments, a pharmaceutical composition is provided comprising an AAV as provided herein and a pharma- ceutically acceptable carrier.

[0014] In some embodiments, a method of delivering a Merlin protein to a cell, the method comprising contacting the cell with an AAV as provided herein.

[0015] In some embodiments, a method of treating a subject having NF2, the method comprising administering to the subject having NF2 an AAV as provided herein.

[0016] In some embodiments, a method of inhibiting the growth of a schwannoma, meningioma, or ependymoma in a subject, the method comprising administering an AAV as provided herein.

[0017] In some embodiments, a method for preventing the growth or formation of a schwannoma, ependymoma, or meningioma in a subject, the method comprising administering to the subject an AAV as provided herein.

[0018] In some embodiments, a method of treating a subject having or at risk for a disorder associated with merlin deficiency (e.g., neurofibromatosis type 2, schwannomatosis, or cancer), comprising administering to the subject an AAV as provided herein.

[0019] In some embodiments, provided herein is a nucleic acid molecule comprising a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, or a nucleic acid sequence encoding a Merlin protein or an active fragment thereof.

[0020] In some embodiments, a composition is provided that includes a nucleic acid molecule provided herein and a carrier.

[0021] In some embodiments, a method is provided of providing an AAV as provided herein, the method comprising contacting a cell with a nucleic acid molecule as provided herein to produce the AAV. [Brief description of the drawings]

[0022] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of the patent or application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] [Figure 1]FIG. 1 illustrates a non-limiting vector (plasmid) map of a recombinant DNA plasmid that can be used to generate AAV particles containing a nucleic acid molecule encoding a Merlin protein under a CAG promoter to express the Merlin protein.

[0024] [Diagram 2] Figure 2 illustrates a non-limiting vector (plasmid) map of a recombinant DNA plasmid that can be used to generate AAV particles containing a nucleic acid molecule encoding a Merlin protein under the control of a CAG promoter to express the Merlin protein. This example includes an HPRT stuffer sequence, a HBV post-transcriptional regulatory element, and a modified left AAV2 ITR, as compared to the plasmid illustrated in Figure 4. Additionally, the bacterial backbone has been modified with a kanamycin selection gene to facilitate testing in human clinical trials.

[0025] [Diagram 3] FIG. 3 shows an exemplary Western blot demonstrating that HEK 293T cells transduced with AAV9-CAG-Merlin-v2 can overexpress Merlin (NF2).

[0026] [Figure 4] FIG. 4 illustrates exemplary cerebellar tissue sections obtained from cynomolgus monkeys 28 days after administration of AAV9-CAG-Merlin-v2 (2TX-G38) or AAV9-CAG-eGFP (2TX-C10) by intracisternal magna (ICM) injection and stained for eGFP by immunohistochemistry, clearly demonstrating cerebellar biodistribution of AAV9-CAG-eGFP.

[0027] [Diagram 5]5 shows exemplary enhanced immunofluorescence micrographs of cervical DRG tissue sections stained for the presence of eGFP from 2-month-old Postn-Cre;Nf2flox / flox mice one month after administration of AAV9-CAG-GFP by ICM injection, clearly demonstrating prominent biodistribution of eGFP in the DRG of Postn-Cre;Nf2flox / flox mice injected with AAV9-CAG-GFP into the cisterna magna.

[0028] [Figure 6] 6 shows exemplary enhanced immunofluorescence micrographs of cervical DRG tissue sections stained for the presence of eGFP from 2-month-old Postn-Cre;Nf2flox / flox mice 4 weeks after administration of a low dose (group 3; 5.6 E12vg / kg) of AAV9-CAG-GFP by ICM injection, clearly showing GFP expression in the SC surrounding axonal cells in the DRG of these mice.

[0029] [Figure 7] 7 shows exemplary enhanced immunofluorescence micrographs of tissue sections with nerves distal to the DRG stained for the presence of eGFP from 2-month-old Postn-Cre;Nf2flox / flox mice 4 weeks after administration of a low dose (group 3; 5.6 E12vg / kg) of AAV9-CAG-GFP by ICM injection, clearly demonstrating that eGFP is also observed in nerves distal to the DRG of NF2 knockout mice administered ICM.

[0030] [Figure 8]8 is a graph showing the density of Schwann cell (SC) nuclei (nuclei / μm2) in sections of dorsal root ganglia (DRG) from 6-month-old Postn-Cre;Nf2flox / flox mice 5 months after AAV9-CAG-Merlin or AAV9-CAG-GFP administration by ICM injection, clearly showing that there may be a significant reduction in SC proliferation in NF2 knockout mice administered AAV9-CAG-Merlin compared to mice injected with the AAV9-CAG-GFP control vector.

[0031] [Figure 9] 9 is a non-limiting embodiment of a vector map for pAAV_CAG_Merlin_Kan (SEQ ID NO: 7) that can be used to generate AAV particles containing a nucleic acid molecule encoding Merlin under the control of a CAG promoter. This vector map is similar to FIG. 2 and is illustrated with a kanamycin resistance gene to aid in selection, although other selection genes can be used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Enumerated Embodiments An adeno-associated virus (AAV), comprising a transgene encoding an AAV capsid protein and a full-length Merlin protein (e.g., Merlin isoform 1 or Merlin isoform 2), or one or more active fragments thereof, such as, but not limited to, residues 1-359 of Merlin isoform 1, residues 1-313 of Merlin isoform 1, residues 1-219 of Merlin isoform 1, residues 1-73 of Merlin isoform 1, residues 312-595 of Merlin isoform 1, residues 479-595 of Merlin isoform 1, residues 503-595 of Merlin isoform 1, or combinations thereof.

[0033] 2 The AAV of embodiment 1, wherein the transgene is within an AAV inverted terminal repeat.

[0034] 3. The AAV of embodiment 1 or 2, wherein the transgene is operably linked to a regulatory sequence that directs expression of a heterologous gene in a host cell.

[0035] 4. The AAV of embodiment 3, wherein the regulatory sequence comprises a promoter.

[0036] 5. The AAV of embodiment 4, wherein the promoter is a constitutive or tissue-specific promoter, such as, but not limited to, a neuron-specific or neural tissue-specific promoter, including, but not limited to, those provided herein.

[0037] 6. The AAV of embodiment 5, wherein the promoter is a CAG promoter, a CMV promoter, a CBA promoter, or an SV40 promoter.

[0038] 7. The AAV of any of the preceding embodiments, wherein the capsid protein is an AAV9 capsid protein.

[0039] 8 The AAV9 capsid protein has the following amino acid sequence: Amino acid residues 1 to 736 of SEQ ID NO:3; Amino acid residues 138 to 736 of SEQ ID NO:3; or Amino acid residues 203 to 736 of SEQ ID NO:3; The AAV of embodiment 7, comprising a protein having the formula:

[0040] 9. The AAV of embodiment 7, wherein the AAV9 capsid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 203-736 of SEQ ID NO:3.

[0041] 10. The AAV of any of the preceding embodiments, wherein the Merlin protein encoded by the transgene comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or homologous to the sequence of SEQ ID NO:1 or SEQ ID NO:8.

[0042] 11. The AAV of any of the preceding embodiments, wherein the Merlin protein encoded by the transgene comprises the sequence of SEQ ID NO:1 or SEQ ID NO:8, or an active fragment thereof.

[0043] 12. The AAV of any of the preceding embodiments, wherein the transgene comprises a nucleic acid molecule encoding a protein comprising the sequence of SEQ ID NO:1 or SEQ ID NO:8, or an active fragment thereof.

[0044] 13. The AAV of embodiment 12, wherein the nucleic acid molecule encoding the Merlin protein comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 9, or a codon-optimized version of each of the foregoing.

[0045] 14. The AAV of embodiment 2, wherein the AAV inverted terminal repeat sequence is an AAV-2 inverted terminal repeat sequence.

[0046] 15. The AAV described in embodiment 14, wherein the AAV2 inverted terminal repeat sequence comprises the sequence of SEQ ID NO:4.

[0047] 16. The AAV of any of the preceding embodiments, wherein the AAV comprises a nucleic acid molecule stuffer sequence upstream of the transgene and downstream of the 5' (left) AAV ITR.

[0048] 17. The AAV of embodiment 16, wherein the stuffer sequence comprises the sequence of SEQ ID NO:6.

[0049] 18. The AAV of any of the preceding embodiments, wherein the AAV comprises a nucleotide intron sequence downstream of the transgene and upstream of the right (3') AAV (e.g., AAV2) ITR.

[0050] 19. The AAV of embodiment 18, wherein the intron sequence is an HPRE sequence.

[0051] 20 The AAV described in embodiment 19, wherein the HPRE sequence comprises the sequence of SEQ ID NO:5.

[0052] 21. A composition comprising an AAV according to any one of embodiments 1 to 20 and a physiologically compatible carrier.

[0053] 22. A pharmaceutical composition comprising an AAV according to any one of embodiments 1 to 20 and a pharma- ceutically acceptable carrier.

[0054] 23. A method for delivering Merlin protein to a cell, the method comprising contacting the cell with an AAV described in any one of embodiments 1 to 20 or a composition described in embodiment 21 or 22.

[0055] 24 A method of treating a subject having NF2, the method comprising administering to the subject having NF2 an AAV described in any one of embodiments 1 to 20 or a composition described in embodiment 21 or 22.

[0056] 25. The method of embodiment 24, wherein said administration is intracerebroventricular, intracisternal, intrathecal, intrastriatal, intrapleural, intramuscular, intravitreal, intravenous, or intratumor.

[0057] 26. A method for inhibiting the growth of a schwannoma, meningioma, or ependymoma in a subject, the method comprising administering to the subject an AAV described in any one of embodiments 1 to 20 or a composition described in embodiment 21 or 22.

[0058] 27. The method of embodiment 26, wherein the subject is a subject with NF2 or NF2 deficiency, such as schwannomatosis.

[0059] 28. The method of embodiment 26, wherein said administration is intrathecal, intravenous, intracerebroventricular, intrastriatal, intrapleural, intramuscular, intracisternal, or intratumor.

[0060] 29. A method for preventing the growth or formation of a schwannoma, ependymoma or meningioma in a subject, said method comprising administering to said subject an AAV described in any one of embodiments 1 to 20 or a composition described in embodiment 21 or 22.

[0061] 30. The method of embodiment 29, wherein the subject is a subject with NF2 or NF2 deficiency, such as schwannomatosis.

[0062] 31 The method of embodiment 30, wherein said administration is intrathecal, intravenous, intracisternal, or intratumoral.

[0063] 32 A method for treating a subject having or at risk of a disorder associated with merlin deficiency (e.g., neurofibromatosis type 2, schwannomatosis, or cancer), the method comprising administering to the subject an AAV described in any one of embodiments 1 to 20 or a composition described in embodiment 21 or 22.

[0064] 33 The disorder may be, for example, neurofibromatosis type 2, schwannomatosis, schwannoma (e.g., vestibular schwannoma); cancer (e.g., hematological cancer (e.g., juvenile myelomonocytic leukemia), leukemia (e.g., adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, or hairy cell leukemia); lymphoma (e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, , adult Hodgkin lymphoma, childhood Hodgkin lymphoma, adult non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, cutaneous Waldenstrom's macroglobulinemia); chronic myeloproliferative disorders; Langerhans cell histiocytosis; multiple myeloma / plasma cell neoplasms; myelodysplastic syndromes; myelodysplastic / myeloproliferative neoplasms); ovarian cancer (e.g., ovarian serous carcinoma); breast cancer, invasive breast cancer; or neurocutaneous disorders; (e.g., mesothelioma, peritoneal mesothelioma, cutaneous squamous cell carcinoma, cancer of the urinary tract, thyroid cancer, gastric cancer, schwannoma, renal cell carcinoma, cancer of the pituitary gland, ovarian cancer, meningioma, melanoma, lung cancer (e.g., squamous cell carcinoma, non-small cell lung cancer, mixed lung cancer, lung adenocarcinoma), liver cancer, colon cancer, hepatocellular carcinoma, acute myeloid leukemia (AML), cancer of the aerodigestive tract (squamous cell carcinoma), bladder cancer, bone cancer (e.g., bone sarcoma), colorectal cancer, 33. The method of embodiment 32, wherein the tumor is ependymoma, colorectal carcinoma, endometrium (mixed adenosquamous carcinoma), or glioma, or cataract, retinal detachment, damage to the nerves of the eye, papilledema (optic disc edema), ocular migraine (retinal migraine), retinitis pigmentosa (RP) (retinal degeneration), mixed hamartoma of the retina and RPE, retinal microaneurysm, preretinal conjunctivitis, physiopedia (severe dry eye), nystagmus-oscillopsia (ocular flutter / chiasm), diplopia (double vision), or gaze-induced tinnitus (GET).

[0065] 34. The method of any of embodiments 23-33, wherein the AAV is administered at least every 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 2 years, 3 years, 4 years, 5 years, 6 years, or longer.

[0066] 35. The method of any of embodiments 23-34, wherein the AAV is administered intravenously, intradermally, subcutaneously, intrathecally, systemically, intracisternally, intracerebroventricularly, intraparenchymally, intrapleurally, intrastriatally, intramuscularly, intravitreally, or locally.

[0067] 36 A nucleic acid molecule comprising a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, or is identical to SEQ ID NO:2.

[0068] 37. The nucleic acid molecule of embodiment 30, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

[0069] 38. The nucleic acid molecule of embodiment 36 or 37, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO:2.

[0070] 39. The nucleic acid molecule of any one of embodiments 36 to 39, wherein the nucleic acid molecule comprises an AAV inverted terminal repeat sequence.

[0071] 40. The nucleic acid molecule of embodiment 39, wherein the sequence of SEQ ID NO:2 is linked within the AAV inverted terminal repeat sequence.

[0072] 41. The nucleic acid molecule of embodiment 39 or 40, wherein the AAV inverted terminal repeat sequence is an AAV2 inverted terminal repeat sequence.

[0073] 42. The nucleic acid molecule of embodiment 41, wherein one of the AAV2 inverted terminal repeat sequences (e.g., the 3' AAV ITR) comprises the sequence of SEQ ID NO:4.

[0074] 43. The nucleic acid molecule according to any one of embodiments 36 to 42, wherein the nucleic acid comprising SEQ ID NO:2 is operably linked to a regulatory sequence directing expression of the protein encoded by SEQ ID NO:2.

[0075] 44. The nucleic acid molecule of embodiment 44, wherein the regulatory sequence comprises a promoter.

[0076] 45. The nucleic acid molecule of embodiment 44, wherein the promoter is a constitutive promoter or a tissue-specific promoter.

[0077] 46. ​​The nucleic acid molecule of embodiment 37 or 38, wherein the promoter is a CAG promoter, a CMV promoter, or an SV40 promoter.

[0078] 47. The nucleic acid molecule of any one of embodiments 36 to 46, wherein the nucleic acid molecule comprises a stuffer sequence upstream of the transgene of SEQ ID NO:2 and downstream of the 5' (left) AAV ITR.

[0079] 48. The nucleic acid molecule of embodiment 47, wherein the stuffer sequence comprises the sequence of SEQ ID NO:6.

[0080] 49. The nucleic acid molecule of any one of embodiments 36-48, wherein the nucleic acid molecule comprises a nucleotide intron sequence downstream of SEQ ID NO:2 and upstream of the right (3') AAV (e.g., AAV2) ITR.

[0081] 50. The nucleic acid molecule of embodiment 49, wherein the intron sequence is an HPRE intron sequence.

[0082] 51. The nucleic acid molecule of embodiment 50, wherein the HPRE intron sequence comprises the sequence of SEQ ID NO:5.

[0083] 52. The nucleic acid molecule of any one of embodiments 36 to 51, wherein the molecule is a plasmid.

[0084] 53. A composition comprising a nucleic acid molecule according to any one of embodiments 36 to 52 and a carrier.

[0085] 54. The composition of embodiment 53, wherein the carrier is a transfection reagent.

[0086] 55. A method for producing an AAV particle described in any one of embodiments 1 to 20, the method comprising contacting (e.g., transfecting or electroporating) a cell with a nucleic acid molecule described in any one of embodiments 36 to 52 or a composition described in embodiment 53 or 54 to produce the AAV.

[0087] 56. The method of embodiment 55, wherein the cell is a cell of an AAV packaging cell line.

[0088] 57. The method of embodiment 56, wherein the AAV packaging cell line is an AAV9 packaging cell line.

[0089] 58 A cultured host cell comprising a recombinant nucleic acid molecule encoding an AAV capsid protein and a recombinant nucleic acid molecule encoding a Merlin protein.

[0090] 59. The cell of embodiment 58, wherein the capsid protein is an AAV9 capsid protein.

[0091] 60. The cell of embodiment 58 or 59, wherein the nucleic acid molecule encoding the capsid protein encodes a protein that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, a protein having an amino acid sequence of amino acid residues 1 to 736 of SEQ ID NO:3; amino acid residues 138 to 736 of SEQ ID NO:3; or amino acid residues 203 to 736 of SEQ ID NO:3.

[0092] 61. The cell of any one of embodiments 58 to 60, wherein the recombinant nucleic acid molecule encoding the Merlin protein encodes a protein that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or homologous to the sequence of SEQ ID NO:1, SEQ ID NO:8, or an active fragment thereof, or is identical to said sequence.

[0093] 62. The cell of any one of embodiments 58 to 61, wherein the recombinant nucleic acid molecule encoding the Merlin protein encodes a protein comprising the sequence of SEQ ID NO:1, SEQ ID NO:8, or an active fragment thereof.

[0094] 63. The cell of any one of embodiments 58 to 62, wherein the recombinant nucleic acid molecule encoding the Merlin protein comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 or SEQ ID NO:9.

[0095] 64. The cell of any one of embodiments 58 to 63, wherein the recombinant nucleic acid molecule encoding the Merlin protein comprises the sequence of SEQ ID NO:2 or SEQ ID NO:9.

[0096] 65. The cell of any one of embodiments 58 to 64, wherein the recombinant nucleic acid molecule is a plasmid.

[0097] Detailed Description The embodiments provided herein relate, in part, to compositions, methods, and other embodiments for treating NF2 disease or conditions, tumors, or disorders related to Merlin protein deficiency.

[0098] Reduction of functional Merlin, which plays an important role as a tumor suppressor protein, results in neurofibromatosis type 2 or Merlin deficiency related conditions, tumors, or disorders, and restoration of Merlin activity by delivering a functional copy of the NF2 gene could provide an effective treatment.

[0099] Thus, in some embodiments, a recombinant virus is provided that contains a transgene encoding a Merlin protein. The type of virus used can be any virus that, when used to infect a cell or subject, results in the expression of the transgene and the Merlin protein, or any part thereof, in the cell or subject. In some embodiments, the part is an N-terminal fragment of the Merlin protein (Tikoo et. al., An Anti-Ras Function of Neurofibromatosis Type 2 Gene Product (NF2 / Merlin), The Journal of Biological Chemistry, Vol. 269, No 38, Issue of September 23, 1994 pp. 23387-23390; Cui et. al., The NF2 tumor suppressor merlin interacts with Ras and RasGAP, which may modulate Ras signaling, Oncogene, Vol. 38, 2019, pp. 6370-6381). In some embodiments, the encoded N-terminal fragment of Merlin comprises or consists of amino acid residues 1-359 of Merlin isoform 1. In some embodiments, the encoded N-terminal fragment of Merlin comprises or consists of amino acid residues 1-313 of Merlin isoform 1. In some embodiments, the encoded N-terminal fragment of Merlin comprises or consists of amino acid residues 1-219 of Merlin isoform 1. In some embodiments, the encoded N-terminal fragment of Merlin comprises or consists of amino acid residues 1-73 of Merlin isoform 1.In some embodiments, the portion is a C-terminal fragment of Merlin protein (Cui et. al., The NF2 tumor suppressor merlin interacts with Ras and RasGAP, which may modulate Ras signaling, Oncogene, Vol. 38, 2019, pp. 6370-6381). In some embodiments, the encoded C-terminal fragment of Merlin comprises or consists of amino acid residues 312-595 of Merlin isoform 1. In some embodiments, the encoded C-terminal fragment of Merlin comprises or consists of amino acid residues 479-595 of Merlin isoform 1. In some embodiments, the encoded C-terminal fragment of Merlin comprises or consists of amino acid residues 503-595 of Merlin isoform 1.

[0100] In some embodiments, an "active fragment" as used herein is a fragment of wild-type Merlin that has biological activity. In some embodiments, an "isoform" as used herein is an alternative splice variant of wild-type Merlin.

[0101] In some embodiments, the virus is an adeno-associated virus (AAV). AAV has many naturally occurring serotypes that can be used. In some embodiments, the AAV serotype is AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ. Serotype refers to the capsid protein encoded by a particular strain of AAV. Furthermore, proteins including capsid proteins of different AAV serotypes can be engineered to have enhanced properties or modified from naturally occurring serotypes. Enhanced properties as a result of capsid engineering can include, but are not limited to, easily scalable manufacturing of AAV, reduced immunogenicity, altered tissue tropism, and / or providing advantageous properties for therapeutic applications. Thus, in some embodiments, AAV is provided that includes a transgene encoding AAV capsid protein and Merlin protein. Methods for making AAV are known in the art and can be found, for example, in U.S. Patent No. 7,906,111, which is incorporated herein by reference in its entirety.The transgene can be incorporated into what can be called a minigene, which comprises the AAV inverted terminal repeat sequence and the sequence encoding the Merlin protein.The sequence of the transgene can also be operably linked to a regulatory element or sequence that induces the expression of the Merlin protein in host cells.

[0102] The regulatory elements may include conventional control elements that are operably linked to the transgene in a manner that allows transcription, translation and / or expression of the transgene in cells transfected with the plasmid vector or infected with the virus. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0103] Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals (e.g., splicing signals and polyadenylation (polyA) signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be utilized.

[0104] Examples of constitutive promoters include, but are not limited to, the CAG promoter (Miyazaki et al, Gene. 79 (2): 269-77; Niwa et al., Gene. 108 (2): 193-9), the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the simian virus 40 (SV40) promoter, the dihydrofolate reductase promoter, the beta actin promoter, the phosphoglycerol kinase (PGK) promoter, the human elongation factor 1 alpha (EF1) promoter (Qin, JY, Zhang, L., Clift, KL, Hulur, I., Xiang, AP, Ren, BZ, et al. (2010). Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5 (5), e10611. doi: 10.1371 / journal.pone.0010611), and the human ubiquitin C promoter (UBC) (Qin, JY, Zhang, L., Clift, KL, Hulur, I., Xiang, AP, Ren, BZ, et al. (2010). Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5 (5), e10611. doi: 10.1371 / journal.pone.0010611). Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors (e.g., temperature), or the presence of certain physiological conditions (e.g., acute phase), a certain differentiation state of cells, or only in replicating cells.Inducible promoters and inducible systems are available from a variety of commercial sources. Many other systems have been described and could be readily selected by one of skill in the art. Examples of inducible promoters that are regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (International Patent Publication No. WO 98 / 10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repressible system (Gossen et al, Proc. Natl. Acad Sci. USA, 89:5547-5551 (1992)), the tetracycline inducible system (Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem. BioL, 2:512-518 (1998)), the tetracycline-in ... (1998)), the RU486 inducible system (Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al, J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions (e.g., temperature, acute phase, specific differentiation states of cells, or only in replicating cells).

[0105] In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is an SV40 promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is not a tissue-specific or cell-specific promoter. In some embodiments, the promoter is a tissue-specific or cell-specific promoter. In some embodiments, the promoter is not an inducible promoter.

[0106] In some embodiments, the native promoter of the Merlin product is used. The native promoter can be used when it is desired that the expression of Merlin should mimic the native expression. The native promoter can be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In some embodiments, other native expression control elements (e.g., enhancer elements, polyadenylation sites, or Kozak consensus sequences) can be included in the promoter sequence to more closely mimic the native expression.

[0107] In some embodiments, the transgene comprises a gene operably linked to a tissue-specific promoter, hi some embodiments, the promoter is a neuronal or neural tissue specific promoter. Examples of such promoters include, but are not limited to, the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene (Piccioli et al., Proc. Natl. Acad Sci. USA, 88:5611-5 (1991)), the neuron-specific VGF gene (Piccioli et al., Neuron, 15:373-84 (1995)), or the human synapsin promoter (Syn1) (Kugler, S., Kilic, E., and Bahr, M. (2003). Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area.(Gene Ther. 10 (4), 337-347. doi: 10.1038 / sj.gt.3301905 , Kugler, S., Meyn, L., Holzmuller, H., Gerhardt, E., Isenmann, S., Schulz, JB, et al. (2001). Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors. Mol. Cell Neurosci. 17 (1), 78-96. doi: 10.1006 / mcne.2000.0929 , McLean, JR, Smith, GA, Rocha, EM, Hayes, MA, Beagan, JA, Hallett, PJ, et al. (2014). Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection.. Neurosci. Lett. 576, 73-78. doi: 10.1016 / j. neulet.2014.05.044 ), or chicken β-actin (CBA), or CBh (a hybrid of CBA) (Gray et. al., Optimizing Promoters for Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors, HUMAN GENE THERAPY 22:1143-1153 (September 2011) a Mary Ann Liebert, Inc.), or MeCp2 (Gray et. al., Optimizing Promoters for Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors, HUMAN GENE THERAPY 22:1143-1153 (September 2011) a Mary Ann Liebert, Inc.), or PDGFβ (Ingusci et. al., Gene Therapy Tools for Brain Diseases, Selene Ingusci, Frontiers in Pharmacology, July 1, 2019), astrocyte-specific (e.g., GFAP) (Smith-Arica, JR, Morelli, AE, Larregina, AT, Smith, J., Lowenstein, PR, and Castro, MG (2000). Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression. Mol. Ther. 2 (6), 579-587. doi: 10.1006 / mthe.2000.0215 , ; Lee, Y., Messing, A., Su, M., and Brenner, M. (2008). GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56 (5), 481- 493. doi: 10.1002 / glia.20622), oligodendrocyte-specific (e.g., human myelin-associated glycoprotein (MAG)) (von Jonquieres, G., Mersmann, N., Klugmann, CB, Harasta, AE, Lutz, B., Teahan, O., et al. (2013). Glial promoter selectivity following AAV-delivery to the immature brain. PLoS One 8 (6), e65646. doi: 10.1371 / journal.pone.0065646 ), or the myelin basic promoter (MBP) (von Jonquieres, G., Frohlich, D., Klugmann, CB, Wen, X., Harasta, AE, Ramkumar, R., et al. (2016).Recombinant human myelin-associated glycoprotein promoter drives selective AAV-mediated transgene expression in oligodendrocytes. Front. Mol. Neurosci. 9, 13. doi: 10.3389 / fnmol.2016.00013), microglia-specific (e.g., F4 / 80) (Rosario, AM, Cruz, PE, Ceballos-Diaz, C., Strickland, MR, Siemienski, Z., Pardo, M., et al. (2016). Microglia-specific targeting by novel capsid-modified AAV6 vectors. Mol. Ther. Methods Clin. Dev. 3, 16026. doi: 10.1038 / mtm.2016.26) or CD68 (Rosario, AM, Cruz, PE, Ceballos-Diaz, C., Strickland, M.R. Siemienski, Z., Pardo, M., et al. (2016). Microglia-specific targeting by novel capsid-modified AAV6 vectors. Mol. Ther. Methods Clin. Dev. 3, 16026. doi: 10.1038 / mtm.2016.26), glutamatergic neuron-specific (e.g., phosphate-activated glutaminase promoter (PAG)) (Rasmussen, M., Kong, L., Zhang, GR, Liu, M., Wang, X., Szabo, G., et al. (2007).Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter. Brain Res. 1144, 19-32. doi: 10.1016 / j. brainres.2007.01.125), or vascular glutamate transporter promoter (vGLUT), (Rasmussen, M., Kong, L., Zhang, GR, Liu, M., Wang, X., Szabo, G., et al. (2007). Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter. promoter. Brain Res. 1144, 19-32. doi: 10.1016 / j. brainres.2007.01.125), and GABAergic neuron-specific (e.g., glutamic acid decarboxylase promoter (GAD)) (Rasmussen, M., Kong, L., Zhang, GR, Liu, M., Wang, X., Szabo, G., et al. (2007).Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter. Brain Res. 1144, 19-32. doi: 10.1016 / j. brainres.2007.01.125). Other promoters can also be used (for example, but not limited to, if expression in skeletal muscle is desired, a promoter active in muscle should be used). These include promoters derived from genes encoding skeletal P-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with higher activity than naturally occurring promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Other tissue-specific promoters include promoters known to be tissue specific for the liver (albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), lymphocyte (CD2, Hansal et al., J. Immunol., 161:1063-8 (1996)), and IL-11 (IL-11). (1998); immunoglobulin heavy chain; T cell receptor chain).

[0108] The combination of the transgene product, the promoter / enhancer regulatory sequences, and 5' and 3' AAV ITRs may be collectively referred to herein as a "minigene" for ease of reference.

[0109] In some embodiments, the capsid used is derived from AAV9. In some embodiments, the AAV9 capsid comprises a group of viral proteins (VP1, VP2, VP3, etc.) having the following amino acid sequences: amino acid residues 1 to 736 of SEQ ID NO:3; amino acid residues 138 to 736 of SEQ ID NO:3; or amino acid residues 203 to 736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises a group of viral proteins (VP1, VP2, VP3, etc.) having the amino acid sequence of amino acid residues 1 to 736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises a group of viral proteins (VP1, VP2, VP3, etc.) having the amino acid sequence of amino acid residues 138 to 736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises a group of viral proteins (VP1, VP2, VP3, etc.) having the amino acid sequence of amino acid residues 203 to 736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-736 of SEQ ID NO:3; amino acid residues 138-736 of SEQ ID NO:3; or amino acids 203-736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 138-736 of SEQ ID NO:3. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 203-736 of SEQ ID NO:3.

[0110] In some embodiments, the recombinant AAV genome encodes a Merlin protein. In some embodiments, the Merlin is isoform 1. In some embodiments, the Merlin is isoform 2.

[0111] In some embodiments, the Merlin protein encoded by the transgene comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or homologous to the sequence of SEQ ID NO:1 (isoform 1). In some embodiments, the Merlin protein encoded by the transgene comprises the sequence of SEQ ID NO:1. In some embodiments, the recombinant AAV genome encodes one or more active fragments of a Merlin protein. In some embodiments, the one or more active fragments include, but are not limited to, residues 1-359 of SEQ ID NO:1, residues 1-313 of SEQ ID NO:1, residues 1-219 of SEQ ID NO:1, residues 1-73 of SEQ ID NO:1, residues 312-595 of SEQ ID NO:1, residues 479-595 of SEQ ID NO:1, residues 503-595 of SEQ ID NO:1, or any combination thereof. In some embodiments, the one or more active fragments include, but are not limited to, residues 1-359 of SEQ ID NO:1. In some embodiments, the one or more active fragments include, but are not limited to, residues 1-313 of SEQ ID NO:1. In some embodiments, the one or more active fragments include, but are not limited to, residues 1-219 of SEQ ID NO:1. In some embodiments, the one or more active fragments include, but are not limited to, residues 1-73 of SEQ ID NO:1. In some embodiments, the one or more active fragments include, but are not limited to, residues 312-595 of SEQ ID NO:1. In some embodiments, the one or more active fragments include, but are not limited to, residues 479-595 of SEQ ID NO:1. In some embodiments, the one or more active fragments include, but are not limited to, residues 503-595 of SEQ ID NO:1.

[0112] The active fragments may, in some embodiments, comprise a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or homologous to such a fragment.

[0113] In some embodiments, the transgene comprises a nucleic acid molecule encoding a protein or an active fragment thereof comprising the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 2, or a sequence encoding said active fragment thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or to a sequence encoding said active fragment thereof.

[0114] Variants of the polypeptides or nucleic acid molecules described herein are also provided herein, including polypeptides or nucleic acid molecules having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences or nucleic acid molecules provided herein, or polypeptides or nucleic acid molecules of the wild-type sequence. For example, variants of Merlin (NF2) include polypeptides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or sequence homology to the amino acid sequence of SEQ ID NO: 1, or an active fragment thereof. For example, variants of nucleic acid molecules encoding Merlin (NF2) include nucleic acid molecules having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2, or encoding an active fragment thereof.

[0115] In some embodiments, the Merlin protein encoded by the transgene comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or homologous to the sequence of SEQ ID NO: 8 (isoform 2). In some embodiments, the Merlin protein encoded by the transgene comprises the sequence of SEQ ID NO: 8. In some embodiments, the recombinant AAV genome encodes one or more active fragments of a Merlin protein. The active fragments may, in some embodiments, comprise a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical or homologous to such fragments.

[0116] In some embodiments, the transgene comprises a nucleic acid molecule encoding a protein comprising the sequence of SEQ ID NO: 8, or an active fragment thereof. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence encoding the active fragment thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, or to a sequence encoding the active fragment thereof.

[0117] Variants of the polypeptides or nucleic acid molecules described herein are also provided herein, including polypeptides or nucleic acid molecules having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences or nucleic acid molecules provided herein, or to the wild-type sequences thereof. For example, variants of Merlin (NF2) include polypeptides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or sequence homology to the amino acid sequence of SEQ ID NO: 8, or to an active fragment thereof. For example, variants of nucleic acid molecules encoding Merlin (NF2) include nucleic acid molecules having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9, or to those encoding an active fragment thereof.

[0118] Calculation of "identity" or "sequence homology" between two sequences (the terms are used interchangeably herein) is performed as follows: The sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0119] A variant of the polypeptide described herein may have an amino acid modification (e.g., deletion, addition, or substitution (e.g., conservative substitution)) from the wild-type amino acid sequence of the polypeptide or any active fragment thereof as described above. For example, a variant of Merlin may differ from Merlin (SEQ ID NO:1 or SEQ ID NO:8) by at least 1, 2, 3, 4, 5, but not more than 50, 40, 30, 20, 15, or 10 amino acids. The sequence may also have conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0120] Also provided herein is an isolated nucleic acid molecule. An "isolated" protein or nucleic acid molecule refers to a purified protein or nucleic acid molecule that is removed from at least 90% of at least one component of the natural sample from which the isolated protein or nucleic acid molecule can be obtained. A protein can be "at least" of some degree of purity when the target species or population of target species is at least 5%, 10%, 25%, 50%, 75%, 80%, 90%, 92%, 95%, 98%, or 99% pure on a weight-weight basis.

[0121] As provided herein, the recombinant AAV can comprise AAV inverted terminal repeat (ITR).In some embodiments, the inverted terminal repeat used is AAV-2 inverted terminal repeat.ITR from other AAV serotypes can also be used, this is merely a non-limiting example.

[0122] In addition to AAV being a vehicle for delivering Merlin protein (e.g., by delivering DNA encoding Merlin protein), other vehicles can be used (e.g., adenovirus, retrovirus, lentivirus, etc.). Thus, in some embodiments, a recombinant virus for gene delivery is provided that encodes Merlin protein, or an active fragment thereof, and expresses Merlin or an active protein fragment thereof in infected cells. The AAV can also be pseudotyped with other viral structural or capsid proteins (non-AAV) to help specify the cell type that the AAV infects. Thus, in some embodiments, the AAV is a pseudotyped AAV.

[0123] Also provided is a composition comprising the AAV or virus encoding Merlin, or an active fragment thereof. In some embodiments, the composition comprises a recombinant virus encoding Merlin, or an active fragment thereof, and a physiologically compatible carrier. In some embodiments, the composition is a pharmaceutical composition and comprises a pharma- ceutically acceptable carrier.

[0124] By "pharmacologically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the recombinant virus and not significantly deleterious to the recipient thereof.

[0125] In some embodiments, the composition or pharmaceutical composition comprises an effective amount of the virus. This can also be referred to as a therapeutically effective amount. "Therapeutically effective amount" refers to an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent to induce a desired response in the individual. The therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects resulting from the composition.

[0126] As provided herein, the composition can include pharma- ceutically acceptable vehicle, carrier, or excipient.The pharma-ceutically acceptable carrier (vehicle) useful in the present disclosure is conventional.Remington's Pharmaceutical Sciences (EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975)) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional agents.

[0127] Generally, the nature of carrier or vehicle suitable for delivery depends on the particular mode of administration used.For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, etc.) as vehicles.For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate).

[0128] In some embodiments, the compositions, whether they are solutions, suspensions, or other similar forms, may contain one or more of the following: DMSO, a sterile diluent (e.g., water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, a fixed oil that may serve as a solvent or suspending medium (e.g., synthetic mono- or diglycerides), polyethylene glycol, glycerin, propylene glycol or other solvents; an antibacterial agent (e.g., benzyl alcohol or methyl parabens); an antioxidant (e.g., ascorbic acid or sodium bisulfite); a chelating agent (e.g., ethylenediaminetetraacetic acid); a buffer (e.g., acetate, citrate or phosphate) and an agent for adjusting tonicity (e.g., sodium chloride or dextrose).

[0129] Also provided herein is a nucleic acid molecule comprising a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO:2. In some embodiments, SEQ ID NO:2 can be used to distinguish between endogenous mRNA encoding Merlin protein and mRNA generated from AAV encoding Merlin protein. SEQ ID NO:2 is a non-limiting example of a sequence that is different from the natural mRNA, and another sequence that can be distinguished from the natural sequence can be used. Thus, in some embodiments, a method is provided for detecting Merlin mRNA encoded by the AAV. In some embodiments, the method includes contacting a sample with a probe that is specific for the AAV encoding mRNA and does not bind to natural mRNA, and detecting the AAV mRNA when the probe binds and interacts with the AAV encoding mRNA. In some embodiments, the method includes performing RT-PCR on a sample with primers specific to the AAV encoding mRNA to detect the AAV-encoded mRNA. In some embodiments, the primers are specific to the AAV-encoded mRNA and do not bind or interact with natural mRNA. As used herein, the term "natural mRNA" refers to the RNA encoded by the original DNA of the cell or organism that is not modified (i.e., wild-type, not genetically engineered). This type of assay can be used, for example, in subjects that are not treated with the AAV composition provided herein, to determine the expression of the AAV encoding a Merlin protein that is different from the natural protein.

[0130] In some embodiments, the nucleic acid molecule comprises an AAV inverted terminal repeat. In some embodiments, the sequence of SEQ ID NO:2 is bound within the AAV inverted terminal repeat. In some embodiments, the AAV inverted terminal repeat is an AAV2 inverted terminal repeat. In some embodiments, the nucleic acid comprising SEQ ID NO:2 is operably linked to a regulatory sequence that directs the expression of the protein encoded by SEQ ID NO:2. In some embodiments, the regulatory sequence comprises a promoter. In some embodiments, the promoter is as described herein. In some embodiments, the promoter is a constitutive promoter or a tissue-specific promoter. In some embodiments, the promoter is a CAG promoter, a CMV promoter, or an SV40 promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is an SV40 promoter. In some embodiments, the nucleic acid molecule is a plasmid.

[0131] In some embodiments, a non-viral vector is used. The composition is provided that includes the nucleic acid molecule described herein. In some embodiments, the composition includes a carrier. In some embodiments, the carrier is a transfection reagent or a reagent that facilitates delivery of the nucleic acid molecule to cells. In some embodiments, the transfection reagent is a lipid-based transfection reagent. In some embodiments, the carrier is an electroporation agent.

[0132] The nucleic acid molecules provided herein can be used to generate AAV encoding the Merlin protein. Methods for generating AAV are known to those skilled in the art, and any method can be used. A non-limiting example is provided in U.S. Pat. No. 7,906,111, which is incorporated herein by reference. For example, the minigene can be carried in any suitable vector (e.g., plasmid) that is delivered to the host cell. The plasmids can be engineered so that they are suitable for replication and, if necessary, integration in prokaryotic cells, mammalian cells, or both. In some embodiments, these plasmids (or other vectors with the 5' AAV ITR-heterologous molecule-3' AAV ITR) contain sequences that allow the minigene to replicate in eukaryotes and / or prokaryotes, as well as selection markers for these systems. Selection markers or reporter genes can include sequences that code for geneticin, hygromycin, or puromycin resistance, among others. The plasmids can also contain certain selection reporter or marker genes that can be used to signal the presence of the vector in bacterial cells (e.g., ampicillin resistance). Other components of the plasmid may include an origin of replication and an amplicon (e.g., an amplicon system using Epstein-Barr virus nuclear antigen), which allows high-copy episomal replication in cells, or other similar amplicon components. In some embodiments, a molecule carrying the minigene is transfected into the cell, where it may be present transiently. Alternatively, the minigene (with the 5' AAV ITR-heterologous molecule-3' AAV ITR) may be stably integrated into the genome of the host cell, either chromosomally or episomally. In some embodiments, the minigene may be present in multiple copies, optionally in head-to-head, head-to-tail, or tail-to-tail concatemers.Suitable transfection techniques are known and can be readily utilized to deliver the minigene to the host cell.

[0133] In some embodiments, the AAV comprises a nucleic acid molecule stuffer sequence upstream of the transgene and downstream of the 5' (left) AAV ITR. In some embodiments, the stuffer sequence comprises the sequence of SEQ ID NO:6. In some embodiments, the AAV comprises a nucleotide intron sequence downstream of the transgene and upstream of the right (3') AAV (e.g., AAV2) ITR. In some embodiments, the intron sequence is an HPRE sequence (e.g., as described herein). In some embodiments, the HPRE sequence comprises the sequence of SEQ ID NO:5.

[0134] In some embodiments, when the vector containing the minigene is delivered by transfection, the vector is delivered in an amount of about 5 μg to about 100 μg DNA, about 10 μg to about 50 μg DNA, about 1×10 4 cells ~ approx. 1 x 10 13 cells, or approximately 1 x 10 5 However, the relative amounts of vector DNA to host cells may be adjusted, taking into account such factors as the selected vector, the delivery method, and the selected host cells.

[0135] The host cells themselves for producing the AAV can be selected from any organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. In some embodiments, the host cells are selected from any mammalian species, including, but not limited to, cells such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO. W138, HeLa, 293 cells (which express functional adenovirus E1), Saos-2, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts from mammals, including humans, monkeys, mice, rats, rabbits, and hamsters. A requirement of the cells used is that they do not carry any adenoviral genes other than E1, E2a and / or E4 ORF6, in order to avoid homologous recombination of contaminating viruses during the generation of AAV; and that they are capable of infection or transfection of DNA and expression of the transfected DNA.

[0136] In some embodiments, the host cell is stably transfected with the capsid protein (e.g., AAV9 capsid). It may also have the rep protein produced from the same cell. Alternatively, these proteins may be encoded on separate plasmids that encode the viral proteins and are transfected into the host cell simultaneously or sequentially to produce the AAV encoding Merlin. For example, one host cell that may be used is one that is stably transformed with sequences encoding rep and cap and transfected with a construct carrying adenovirus E1, E2a, and E4ORF6 DNA and a minigene as described above. Stable rep and / or cap expressing cell lines (e.g., B-50 (International Patent Application Publication No. WO 99 / 15685), or those described in U.S. Patent No. 5,658,785) may also be used similarly. Another example of a host cell is one that contains the minimal adenovirus DNA sufficient to express E4 ORF6. Yet other cell lines can be constructed using the AAV9 cap sequence provided herein.

[0137] The preparation of the host cell includes techniques such as the assembly of selected DNA sequences. This assembly can be achieved using conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and described in Sambrook et al. (cited above), the use of overlapping oligonucleotide sequences of adenovirus and AAV genomes, in combination with polymerase chain reaction, synthetic methods, and any other suitable method that provides the desired nucleotide sequence.

[0138] Introduction of molecules (as a plasmid or virus) into the host cell can also be accomplished using techniques known to those skilled in the art, as discussed throughout the specification. In some embodiments, standard transfection techniques are used (e.g., CaPO4 transfection, lipid-based transfection, or electroporation, and / or infection of cell lines such as the human embryonic kidney cell line HEK 293 (a human kidney cell line that contains a functional adenovirus E1 gene that provides E1 proteins acting in trans) with hybrid adenovirus / AAV vectors).

[0139] The AAV9 capsid protein can also be used to pseudotype other viruses that code for Merlin.Therefore, they can be used in other rAAV and non-rAAV vector systems.Such vector systems can include, for example, lentivirus, retrovirus, poxvirus, vaccinia virus, and adenovirus systems, among others.

[0140] Thus, in some embodiments, a method for producing AAV as provided herein is provided. In some embodiments, the method comprises contacting a cell with a nucleic acid molecule or composition thereof as provided herein to produce the AAV. In some embodiments, the cell is an AAV packaging cell line. In some embodiments, the AAV packaging cell line is an AAV9 packaging cell line (e.g., as described herein or as described in U.S. Patent No. 7,906,111, which is incorporated herein by reference in its entirety).

[0141] In some embodiments, a cultured host cell is provided that contains a recombinant nucleic acid molecule encoding an AAV capsid protein and a recombinant nucleic acid molecule encoding a Merlin protein. In some embodiments, the capsid protein is an AAV9 capsid protein or a variant thereof as described herein.

[0142] In some embodiments, a method for delivering Merlin protein to a cell is provided. In some embodiments, the method comprises contacting the cell with AAV or a virus (as described herein) encoding Merlin. In some embodiments, the method comprises a non-viral delivery method, such as needle injection, ballistic DNA injection, sonoporation, photoporation, magnetofection, hydro-poration, or electroporation, transfection, and any combination thereof. In some embodiments, the non-viral method comprises liposomes or polymer carriers, and combinations thereof, for delivery of genetic material encoding Merlin protein.

[0143] In some embodiments, a method of treating a subject having NF2 or other merlin deficiency is provided, the method comprising administering to the subject having NF2 or other merlin deficiency a virus or AAV encoding Merlin (e.g., as provided herein). The administration method can be any suitable method (e.g., as described herein). In some embodiments, the administration is intrathecal, intravenous, intrapleural, or intratumoral.

[0144] In some embodiments, a method is provided for inhibiting the growth of schwannoma, meningioma, mesothelioma, or ependymoma in a subject. In some embodiments, the method comprises administering to the subject a virus or AAV (e.g., as provided herein) encoding Merlin. The administration method can be any suitable method (e.g., as described herein). In some embodiments, the administration is intrathecal, intracisternal, intravenous, intrapleural, or intratumoral.

[0145] In some embodiments, a method of treating a subject with NF2 is provided. In some embodiments, the subject is a subject with NF2. In some embodiments, the subject is a subject with NF2 deficiency. As used herein, the term "NF2 deficiency" refers to a condition or disorder in a subject caused by mutated or inactivated Merlin protein. It can also be referred to as "Merlin deficiency". These conditions can be various types of tumors or disorders as provided herein. In some embodiments, the disorder is as provided herein. In some embodiments, the disorder is cataract, retinal detachment, damage to the nerves of the eye, papilledema (optic disc edema), ocular migraine (retinal migraine), retinitis pigmentosa (RP) (retinal degeneration), mixed hamartoma of the retina and RPE, retinal microaneurysm, preretinal conjunctivitis, physiopedia (severe dry eye), nystagmus-oscillopsia (eye flutter / chiasm), diplopia (double vision), or gaze-induced tinnitus (GET). In some embodiments, a method of treating a subject having or at risk for a disorder associated with merlin deficiency (e.g., neurofibromatosis type 2, schwannomatosis, or cancer) is provided, comprising administering to the subject an AAV (e.g., as provided herein).

[0146] In some embodiments, a method is provided for preventing the growth or formation of spinal schwannoma or meningioma in a subject. In some embodiments, the method comprises administering to the subject a virus or AAV (e.g., as provided herein) encoding Merlin. The administration method can be any suitable method (e.g., as described herein). In some embodiments, the administration is intrathecal, intravenous, or intratumoral. In some embodiments, the subject is a subject with NF2.

[0147] In some embodiments, methods of treating a subject having or at risk for a disorder associated with merlin (e.g., neurofibromatosis type 2, schwannomatosis, or cancer) are provided. In some embodiments, the method includes administering to the subject a virus or AAV encoding Merlin (e.g., as provided herein). The method of administration can be any suitable method (e.g., as described herein). In some embodiments, the administration is intrathecal, intravenous, intrapleural, or intratumor. In some embodiments, the subject is a subject with NF2.In some embodiments, the disorder is neurofibromatosis type 2, schwannomatosis, schwannoma (e.g., vestibular schwannoma); cancer (e.g., hematological cancer (e.g., juvenile myelomonocytic leukemia), leukemia (e.g., adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, or hairy cell leukemia); lymphoma (e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, adult Hodgkin's lymphoma, childhood Hodgkin's lymphoma, lymphoma, adult non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, cutaneous Waldenström's macroglobulinemia; chronic myeloproliferative disorders; Langerhans cell histiocytosis; multiple myeloma / plasma cell neoplasms; myelodysplastic syndromes; myelodysplastic / myeloproliferative neoplasms; ovarian cancer (e.g., ovarian serous carcinoma); breast cancer; invasive ductal carcinoma, or neurocutaneous disorders; (e.g., mesothelioma, pleural mesothelioma, pleural epithelioid mesothelioma epithelioid mesothelioma), skin melanoma, cancer of the urinary tract, thyroid cancer, anaplastic thyroid cancer, gastric cancer, schwannoma, renal cell carcinoma, papillary renal cell carcinoma, cancer of the pituitary gland, ovarian cancer, meningioma, melanoma, lung cancer (e.g., squamous cell carcinoma, mixed lung cancer, lung adenocarcinoma), liver cancer, colon cancer, hepatocellular carcinoma, acute myeloid leukemia (AML), cancer of the aerodigestive tract (squamous cell carcinoma), bladder cancer, bladder urothelial carcinoma, bone cancer (e.g., bone sarcoma), colorectal cancer, ependymoma, colorectal cancer, colorectal adenocarcinoma, pancreatic adenocarcinoma, endometrium (mixed adenosquamous carcinoma), endometrioid endometrioid carcinoma adenocarcinoma, or glioma, or conventional glioblastoma multiforme. In some embodiments, the disorder is cataract, retinal detachment, damage to the nerves of the eye, papilledema (optic disc edema), ocular migraine (retinal migraine), retinitis pigmentosa (RP) (retinal degeneration), mixed hamartoma of the retina and RPE, retinal microaneurysm, preretinal conjunctivitis, physiopedia (severe dry eye), nystagmus-oscillopsia (ocular flutter / chiasm), diplopia (double vision), or gaze-induced tinnitus (GET).

[0148] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0149] As used herein, the terms "comprise," "have," "has," and "include" and their variants mean "including but not limited to" as used herein. Although various compositions and methods are described in terms that "comprise" various components or steps (which are interpreted to mean "including but not limited to"), the compositions, methods, and devices can also "consist essentially of" or "consist of" the various components and steps, and such terminology should be interpreted to define an essentially closed group of members.

[0150] The term "co-administration" and the like is meant to encompass the administration of selected therapeutic agents to one patient, and is intended to include treatment regimens in which the agents are administered by the same or different routes of administration, or at the same time or at different times.

[0151] The term "subject" or "patient" as used herein includes, but is not limited to, humans and non-human vertebrates (e.g., wild animals, domesticated animals, and farm animals). In certain embodiments, the subjects or patients described herein are animals. In certain embodiments, the subject or patient is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject or patient is a non-human animal. In certain embodiments, the subject or patient is a non-human mammal. In certain embodiments, the subject or patient is a domesticated animal (e.g., a dog, cat, cow, pig, horse, sheep, or goat). In certain embodiments, the subject or patient is a companion animal (e.g., a dog or cat). In certain embodiments, the subject or patient is a farm animal (e.g., a cow, pig, horse, sheep, or goat). In certain embodiments, the subject or patient is a zoo animal. In another embodiment, the subject or patient is a research animal (e.g., a rodent, dog, or non-human primate). In certain embodiments, the subject or patient is a non-human transgenic animal (e.g., a transgenic mouse or a transgenic pig).

[0152] The terms "treat", "treated" or "treating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, where the purpose is to inhibit, prevent or slow (reduce) an undesirable physiological condition, disorder or disease, or to improve, inhibit or otherwise obtain a beneficial or desirable clinical outcome. For purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, amelioration or alleviation of symptoms; reduction in the extent of the condition, disorder or disease; stabilization (i.e., no worsening) of the condition, disorder or disease state; delay in onset or slowing of progression of the condition, disorder or disease; improvement of the condition, disorder or disease state; detectable or undetectable remission (whether partial or complete) or enhancement of the improvement of the condition, disorder or disease; and prevention of disorder or disease manifestation. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival in the absence of treatment.

[0153] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide (e.g., a gene, a cDNA, or an mRNA or viral RNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be said to code for the protein or other product of that gene or cDNA.

[0154] As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue or system.

[0155] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue or system.

[0156] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0157] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. In some embodiments, the expression vector is an alphavirus as described herein.

[0158] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease.

[0159] Formulations and Routes of Administration to Patients When a clinical application or method is contemplated, it is necessary to prepare the pharmaceutical composition--expression constructs, viruses, expression vectors, fused proteins, transfected or transduced cells, in a form appropriate for the intended application. Generally, this entails preparing a composition that is essentially free of pyrogens, except for the viruses or plasmids provided herein, and other impurities that may be harmful to humans or animals.

[0160] The recombinant nucleic acid, viral product or pharmaceutical composition thereof may be delivered in a dose of, for example, about 1 to 5 million particles per dose. The product may be provided in a vial or other container with a volume per vial of, for example, about 0.25 ml to about 10 ml (e.g., about 0.25 ml, 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml, 4.5 ml, 5 ml, 5.5 ml, 6 ml, 6.5 ml, 7 ml, 7.5 ml, 8 ml, 8.5 ml, 9 ml, 9.5 ml, or 10 ml (e.g., about 2 ml)).

[0161] In general, it may be desirable to use appropriate salts and buffers when recombinant nucleic acid, virus, or virus product is introduced into a patient. The phrase "pharmacologically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce significant adverse, allergic, or other untoward reactions when administered to animals or humans. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is known. Except insofar as any conventional media or agent is incompatible with the vector or cell, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0162] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may be readily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like. For parenteral administration in aqueous solutions, for example, the solutions may be suitably buffered, if necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, a sterile aqueous medium may be used. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations will meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.

[0163] The composition can be formulated for aerosolized delivery to a subject.For aerosolized delivery, the composition described can be formulated in aqueous solution (e.g., water) or physiologically compatible buffer (e.g., Hanks' solution, Ringer's solution, or physiological saline buffer).The solution can contain one or more formulatory agents (e.g., suspending agents, stabilizing agents, or dispersing agents).

[0164] The delivery system of the present disclosure that delivers the polynucleotide of the present disclosure to the desired cells of a subject is, but is not limited to, the virus of the present disclosure.In some embodiments, the delivery vector comprises the virus as provided herein.They can be used in the method of treating disorders or conditions (such as those described herein).

[0165] In some embodiments, the therapeutic agent is administered at least every 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 2 years, 3 years, 4 years, 5 years, 6 years, or longer. In some embodiments, the therapeutic agent is administered intravenously, intradermally, subcutaneously, intracerebroventricularly, intracisternally, or intrathecally, including lumbar injection, intracisternally, intrapleurally, intravitreally, subretinally, intramuscularly, systemically, or locally (e.g., intratumoral injection).

[0166] kit Additionally, certain components or embodiments of these recombinant nucleic acids, viruses, or pharmaceutical compositions may be provided in kits. For example, any of the recombinant nucleic acids or viruses may be frozen or refrigerated and packaged as a kit, either alone or with separate containers of other agents from the pre-conditioning or post-conditioning steps, and optional instructions for use.

[0167] Some embodiments also relate to any of the aforementioned compositions in a kit. In some embodiments, the kit may include ampoules, disposable syringes, capsules, vials, tubes, etc. In some embodiments, the kit may include a single-dose or multi-dose container containing the topical formulation of the embodiments herein. In some embodiments, each dose container may contain one or more unit doses. In some embodiments, the kit includes a pre-filled syringe containing a pharmaceutical composition. In some embodiments, the kit may include an applicator. In some embodiments, the kit includes all components required for the conditioning / treatment stage. In some embodiments, the cellular composition may have a preservative or may be preservative-free (e.g., in a single-use container). In some embodiments, the recombinant nucleic acid, viral product may be prepared and frozen or refrigerated at the desired stage suitable for transport to a hospital or treatment facility.

[0168] Various sequences are referred to herein and such sequences may, where applicable, include one or more of the following: MERLIN isoform 1 (SEQ ID NO:1): [ka] Nucleic acid sequence encoding MELRIN isoform 1 (SEQ ID NO:2) [ka] AAV9 capsid (SEQ ID NO:3) [ka] [ka] The left-ITR sequence that can be used as an alternative ITR replaces the original fragment in pAV-CAG-Merlin (SEQ ID NO: 4). [ka] HPRE fragment (SEQ ID NO:5) [ka] HPRT1 intron 1 DNA sequence (SEQ ID NO:6) that can be used as a stuffer sequence in the pAV-CAG-Merlin-V2 construct [ka] pAAV2_CAG_Merlin_Kan, DNA vector (SEQ ID NO: 7) [ka] [ka] [ka] MERLIN isoform 2 protein (SEQ ID NO:8): [ka] MERLIN isoform 2 coding region (SEQ ID NO:9): [ka] [ka]

[0169] While the above embodiments have been described in terms of various preferred embodiments, it is not intended to be limited thereto, but rather, those skilled in the art will recognize that variations and modifications may be made therein within the spirit of the above embodiments and the scope of the appended claims. EXAMPLES

[0170] Working Example

[0171] Example 1.

[0172] AAV design encoding Merlin isoform 1 An AAV plasmid was designed to generate an AAV vector encoding Merlin isoform 1. The transgene was subcloned into pAV-ACG (Vigene Biosciences) at the KpnI and XhoI sites. The entire AAV genome (excluding the ITRs) was sequence verified.

[0173] The sequence of Merlin is as follows: [ka]

[0174] The nucleotide sequences provided in the vectors are merely illustrative and due to the degeneracy of the genetic code, other nucleotide sequences may be used to encode the Merlin protein. In this example, the following nucleotide sequence may be used to encode the Merlin protein (stop codons not shown): [ka] Additionally, although this example, and the following examples, illustrate the use of isoform 1 of Merlin, isoform 2 of Merlin may also be used instead.

[0175] Example 2: Vectors capable of expressing Merlin protein

[0176] AAV9 vectors were used to transduce HEK293 cells. The vectors may be based on the plasmids shown in Figure 1 or Figure 2. The AAV9 vectors contained a transgene encoding the Merlin protein. The cells were analyzed for and detected for expression of the Merlin protein. The results are illustrated in Figure 3, which shows a Western blot detecting the expression of the protein.

[0177] Example 3. Viral delivery of Merlin to treat NF2

[0178] Adeno-associated virus (AAV) encoding normal (wild type) NF2 (Merlin) is administered to the central nervous system (CNS) of patients suffering from NF2. The AAV is administered by intrathecal injection, but can also be administered intracerebroventricularly or intravenously by multiple intratumoral injections. Expression of Merlin in the CNS stops tumor progression and / or causes tumor regression. The AAV comprises the AAV capsid and a NF2 transgene operably connected to a constitutive promoter. The AAV is based on AAV-DJ (Grimm D, et al. (2008) In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol. 82:5887-911.) or AAV-9 (Gao G. et al. (2004) Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol. 78:6381-8; Hocquemiller, M. et al. (2016) Adeno-Associated Virus-Based Gene Therapy for CNS Diseases. Hum Gene Ther. 7:478-496). The constitutive promoter is CAG (Fitzsimons et al., (2002) Promoters and regulatory elements that improve adeno-associated viral transgene expression in the brain. Methods 28:227-36.). Without being bound to any particular theory, the use of this promoter allows the transgene to be constitutively expressed in most cells. However, tissue-specific promoters can also be used. The NF2 transgene is a human sequence-optimized neurofibromatosis type 2 (NF2) gene, which can also be referred to as "h-soNF2".The isoform of NF2 used is the longest (595 amino acids), but other isoforms, or active fragments thereof, can be used instead.

[0179] Example 4. NF2 expression reduces spinal schwannoma in NF2 transgenic mice and meningioma xenografts in nude (SCID) mice. The AAV of Example 1 is delivered intrathecally to adult transgenic NF2 mice with spinal schwannoma or nude mice with meningioma xenografts. The mice are monitored and then, upon sacrifice, tumor cells and normal Schwann and arachnoid cells are harvested. The presence of full-length human merlin is confirmed via Western blot. Tumor volume measurements are performed and tumor burden is found to be reduced.

[0180] Example 5. AAV-NF2 is functional in a cell culture model of neurofibromatosis type 2. First, the AAV of Example 1 is used to deliver the merlin gene to cells. The gene is functional and produces the Merlin protein. Human merlin production from the AAV-delivered transgene is evaluated by Western blotting using a human merlin-specific antibody. The cell line can be, for example, the RT4 rat schwannoma cell line. Colony formation assays are performed as well, and the AAV-NF2 is found to reduce colony formation in soft agar assays.

[0181] Example 6. pAV-CAG-Merlin-V2 is an improved AAV gene therapy plasmid.

[0182] pAV-CAG-Merlin-V2 was generated by modifying sequence elements in the original Merlin construct (pAV-CAG-Merlin) as shown in Figure 1 and Figure 2. The cloning strategy is based on the nucleotide positions in the original Merlin construct. pAV-CAG-Merlin-V2 is a recombinant adeno-associated virus (AAV) DNA plasmid with a size of 6892 base pairs (bp) (Figure 2). It contains an inverted terminal repeat (ITR) from AAV serotype 2 (AAV2) and a transgene cassette expressing Merlin protein. In pAV-CAG-Merlin-V2, the left-ITR is modified to contain a stuffer sequence. This sequence can be used to confer optimal AAV packaging efficiency. An HPRE sequence is inserted downstream of the sequence encoding the Merlin protein to promote transgene expression. The vector was modified to contain a kanamycin resistance gene to facilitate the production of DNA products used in recombinant AAV production.

[0183] The expression cassette is composed of a CAG promoter linked to a sequence encoding a codon-optimized Merlin protein, followed by a Hepatitis B virus post-transcriptional regulatory element (HPRE), followed by a transcription-terminating SV40 polyadenylation (SV40-polyA) sequence. Without being bound to any particular theory, the HPRE element is a cis-acting sequence that facilitates cytoplasmic localization of intronless transcripts, contributing to higher gene expression. In addition, a 400bp stuffer sequence derived from the intron 1 region of the hypoxanthine phospho-ribosyl-transferase 1 (HPRT1) gene is inserted downstream of the left ITR and upstream of the CAG promoter to expand the AAV packaging size to 4487bp. A kanamycin resistance gene (KanR) is included in the plasmid backbone as a selection marker.

[0184] To generate a plasmid to increase AAV packaging efficiency, the left ITR sequence may be modified. The left ITR may include: [ka]

[0185] Vectors were generated to remove any artificial tags (e.g., histidine or FLAG tags). Transgenes carried by gene therapy vectors mostly lack intron sequences. Intronless transcripts may be less stable and more prone to accumulate inside the nucleus. Hepatitis B virus post-transcriptional regulatory elements (PRE) (HPRE) may increase the cytoplasmic mRNA accumulation of intronless genes by promoting mRNA export from the nucleus to the cytoplasm and enhancing 3'-end processing and stability. Thus, HPRE elements are added to the recombinant Merlin transcript to improve transgene mRNA stability and transgene expression. The HPRE fragment is synthesized and inserted between the XhoI site (2906) and the EcoRV site (3000). Hepatitis B virus post-transcriptional regulatory elements (PRE) (HPRE) may be as follows: [ka]

[0186] A stuffer sequence is inserted at the 5' end of the construct before the CAG promoter. The optimal packaging size of an AAV vector is 4.1-4.9 kb (Grieger et al. 2005). The original Merlin construct had a packaging size of just 3.5 kb. To increase the AAV vector packaging size, a 400 bp stuffer sequence from the intron 1 region of the HPRT1 gene is inserted downstream of the left ITR sequence and upstream of the CAG promoter sequence. With this sequence addition, the AAV is augmented to facilitate efficient packaging into the AAV capsid. The HPRT1 intron 1 sequence is used as the stuffer sequence, although other stuffer sequences can also be used: The stuffer sequence can be as follows: [ka]

[0187] Example 7. Merlin expressed in the cerebellum from an AAV vector in cynomolgus monkeys

[0188] A study was conducted to evaluate the safety and biodistribution of AAV9-CAG-Merlin-v2 and AAV9-CAG-eGFP for the treatment of neurofibromatosis type 2 (NF2) when given intracisternomajor (ICM) in cynomolgus monkeys. An additional objective was to confirm transgene delivery and expression from each of the above vectors in Schwann, arachnoid, and ependymal cells of the region of interest. Sections of each collected tissue were analyzed (via immunostaining) for green fluorescent protein (GFP; rabbit anti-GFP). Scans of each tissue sample were prepared for immunostaining and immunofluorescence for GFP. Figure 4 shows immunohistochemistry images of eGFP biodistribution in the cerebellum of cynomolgus monkeys administered AAV9-CAG-eGFP by ICM administration. These data using surrogate biomarker biodistribution clearly indicate that AAV9 vectors delivered by the ICM administration route can express proteins in the appropriate tissues, and possibly provide evidence that AAV9 vectors expressing Merlin protein can restore normal merlin function. eGFP expression was used as a surrogate marker for Merlin expression because the animals express native Merlin protein, which was not distinguishable from the merlin expressed from the AAV9 vector.

[0189] Example 8. In NF2 knockout mice administered AAV9-CAG-Merlin v2, there was a reduction in Schwann cell density compared to mice treated with AAV9-CAG-GFP.

[0190] Postn-Cre;Nf2 flox / floxMice were injected into the intracisternal cavity (ICM) with 5 μL or 10 μL of test article (AAV9-CAG-Merlin or AAV9-CAG-Merlin-v2) or negative control vector (AAV9-CAG-GFP). flox / flox In mouse models, SC hyperplasia is observed in Postn-Cre;Nf2 mice, similar to that observed in humans with NF2. flox / flox Observed in the dorsal root ganglion (DRG) of mice. At 6 months of age, Postn-Cre;Nf2 flox / flox Periostin-Cre:NF2 mice have significantly increased numbers of SCs (SC hyperplasia and multiple small schwannomas) in the DRG compared to control mice. flox / flox Mice were then transfected with NF2 flox / flox Mated with mice.

[0191] Study design: [Table 1]

[0192] Group 1a: 4 × 1-month-old pups were injected with AAV9-CAG-GFP (low-dose control, 5.6 E12 vg / kg)

[0193] Group 1b: 4 × 1-month-old pups were injected with AAV9-CAG-GFP (high-dose control, 4 E13 vg / kg).

[0194] Group 2a: 10× 1-month-old pups were injected with AAV9-CAG-Merlin v1 (low dose, 5.6 E12 vg / kg)

[0195] Group 2b: 10 × 1-month-old pups were injected with AAV9-CAG-Merlin v2 (high dose 7 vg / kg E13).

[0196] Group 3: 5× 1-month-old pups were injected with AAV-GFP (low dose control, 5.6 E12 vg / kg) and sacrificed 4 weeks post-injection for analysis of tissue distribution.

[0197] After administration, the mice were observed for 5 months (groups 1a, 1b, 2a, 2b) or 4 weeks (group 3). Two group 1b mice (AAV9-CAG-GFP) were sacrificed after 22 and 50 days, respectively, due to excessive weight loss. After the post-administration observation period, the mice were sacrificed for further analysis. To evaluate the anti-proliferative activity of AAV9-CAG-Merlin, the number of Schwann cells (SCs) in dorsal root ganglion (DRG) histology sections was counted as the number of SC nuclei / μm 2 and statistical analysis of values ​​for AAV9-CAG-Merlin-injected mice compared with AAV9-CAG-GFP-injected controls. Immunohistochemistry with GFP antibody was used to analyze AAV-GFP expression and tissue distribution (group 3), as well as the persistence of AAV-GFP expression at 6 months (group 2).

[0198] As a surrogate for the biodistribution of the AAV9-CAG-merlin construct, an AAV9-CAG-eGFP construct was developed, in which the Merlin transgene is replaced with a surrogate biomarker protein. eGFP expression from this construct was predicted to correlate with the expected biodistribution of similar tissues transduced with the Merlin construct. Prominent biodistribution of eGFP was observed in Postn-Cre;Nf2 mice injected with AAV9-CAG-GFP, as shown in the enhanced immunofluorescence micrographs in FIG. 5. flox / flox In the DRG of mice, eGFP expression was observed in the cells surrounding the axonal cells (Schwann cells) in mice that received a low dose (group 3; 5.6 E12 vg / kg) of AAV9-CAG-GFP via ICM administration and sacrificed 4 weeks later (Figure 6). The nerve distal to the DRG also showed eGFP expression in Schwann cells (Figure 7). Full-width sections of the DRG clearly showed eGFP expression distributed throughout the entire area of ​​the DRG.

[0199] To assess the antiproliferative activity of AAV9-CAG-Merlin and AAV9-CAG-Merlin-v2, the number of nuclei per square micron of tissue was counted in AAV9-CAG-Merlin and AAV9-CAG-Merlin-v2 treated animals and compared to mice receiving the negative control of AAV9-CAG-eGFP (number of SC nuclei / μm 2 A statistically significant reduction in the number of SC nuclei / μm was observed in the high dose arm of AAV9-CAG-Merlin-v2 compared to similar mice receiving an equal dose of AAV9-CAG-GFP. 2 A slight reduction (not statistically significant) in IL-1 expression was observed in the low dose arm of AAV9-CAG-Merlin v2 compared to AAV9-CAG-GFP. Figure 8 shows the effect of AAV9-CAG-Merlin administration on IL-1 expression in 6-month-old Postn-Cre;Nf2 mice 5 months after administration compared to AAV9-CAG-GFP-injected controls. flox / flox Schwann cell (SC) nuclei density (nuclei / μm ) in mouse DRG sections. 2 ) is shown to be significantly reduced.

[0200] All literature and similar materials cited in this application (including, but not limited to, patents, patent applications, articles, books, treatises, and web pages) are expressly incorporated by reference in their entirety, regardless of the format of such literature and similar materials. In the event that one or more of the incorporated literature and similar materials differs or contradicts this application (including, but not limited to, terms defined, usage of terms, techniques described, etc.), this application controls.

[0201] While compositions, methods, and the like have been described in conjunction with various embodiments and examples, it is not intended that they be limited to such embodiments and examples. On the contrary, the present disclosure encompasses various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0202] Although the compositions and methods have been shown and described with reference to specific illustrative non-limiting embodiments, it should be understood that various changes in form and details can be made without departing from the spirit and scope of the present disclosure. Accordingly, it is intended to claim all embodiments that fall within the scope and spirit of the present disclosure, and equivalents thereto. The claims, descriptions and figures of the compositions, methods, systems and assays of the present disclosure should not be read as limited to the described order of elements, unless otherwise stated to that effect.

Claims

1. An adeno-associated virus (AAV) comprising an AAV capsid protein and a transgene encoding a full-length Merlin protein or one or more of its active fragments.

2. The transgene is within the AAV inverted terminal repeat sequence, the transgene is operably linked to a regulatory sequence that induces the expression of a heterologous gene in a host cell, and the regulatory sequence comprises a constitutive promoter or a tissue-specific promoter. The AAV according to claim 1.

3. The promoter is a CAG promoter, a CMV promoter, a CBA promoter, or an SV40 promoter. The AAV according to claim 2.

4. The capsid protein is an AAV9 capsid protein. The AAV according to claim 1.

5. The AAV9 capsid comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 203-736 of SEQ ID NO:

3. The AAV according to claim 4.

6. The Merlin protein encoded by the transgene comprises a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

8. The AAV according to claim 1.

7. The Merlin protein encoded by the transgene comprises the sequence of SEQ ID NO:

1. The AAV according to claim 1.

8. The AAV inverted terminal repeat sequence is an AAV-2 inverted terminal repeat sequence. The AAV according to claim 2.

9. The AAV comprises a nucleic acid molecule stuffer sequence upstream of the transgene and downstream of the 5' AAV ITR. The AAV according to claim 1.

10. The AAV according to claim 1, wherein the AAV contains a nucleotide intron sequence that is downstream of the transgene and upstream of the 3' AAV ITR.

11. The AAV according to claim 10, wherein the intron sequence is an HPRE sequence.

12. A pharmaceutical composition comprising the AAV according to claim 1 and a pharmaceutically acceptable carrier.

13. A composition for use in a method of delivering Merlin protein to cells, comprising the AAV according to claim 1, wherein the method includes a step of contacting the cells with the AAV.

14. A composition for use in a method of treating a subject having NF2, comprising the AAV according to claim 1.

15. A composition for use in a method of inhibiting the growth of schwannoma, meningioma, or ependymoma in a subject, comprising the AAV according to claim 1.

16. A composition for use in a method of treating a subject having a Merlin protein deficiency disorder, comprising the AAV according to claim 1.

17. The disorder is neurofibromatosis type 2, schwannomatosis, schwannoma, cancer, leukemia, lymphoma, chronic myeloproliferative disorder, Langerhans cell histiocytosis, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, ovarian cancer, breast cancer, invasive breast cancer, or a neurocutaneous disorder, mesothelioma, peritoneal mesothelioma, cutaneous squamous cell carcinoma, urinary tract cancer, thyroid cancer, gastric cancer, schwannoma, renal cell carcinoma, pituitary cancer, ovarian cancer, meningioma, melanoma, lung cancer, liver cancer, colorectal cancer, hepatocellular carcinoma, acute myeloid leukemia (AML), airway gastrointestinal cancer, bladder cancer, bone cancer, colorectal cancer, epithelioma, colorectal cancer, endometrium, glioma, cataract, retinal detachment, damage to the nerves of the eye, papilledema, ophthalmic migraine, retinitis pigmentosa (RP), mixed errotic swelling of the retina and RPE, retinal microaneurysm, preretinal membrane conjunctivitis, physiopediatrics, nystagmus-oscillopsia, diplopia, or gaze-evoked tinnitus (GET), the composition according to claim 16.

18. A nucleic acid molecule comprising a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, or identical to SEQ ID NO:

2.

19. The nucleic acid comprising SEQ ID NO: 2 is operably linked to a regulatory sequence that induces the expression of the protein encoded by SEQ ID NO: 2, and the regulatory sequence Is a constitutive promoter or a tissue-specific promoter, A stuffer sequence upstream of the nucleic acid sequence of SEQ ID NO: 2 and downstream of the AAV ITR located on the 5' side of the nucleic acid sequence of SEQ ID NO: 2, and An intron sequence downstream of SEQ ID NO: 2 and upstream of the AAV ITR located on the 3' side of the nucleic acid sequence of SEQ ID NO: 2 The nucleic acid molecule according to claim 18.

20. A method for generating the AAV particles according to claim 1, the method comprising contacting a cell with the nucleic acid molecule according to claim 18 to generate the AAV. **Claim 21**: The AAV according to claim 1, wherein the active fragment consists essentially of residues 1 to 359 of SEQ ID NO: 1, residues 1 to 313 of SEQ ID NO: 1, residues 1 to 219 of SEQ ID NO: 1, residues 1 to 73 of SEQ ID NO: 1, residues 312 to 595 of SEQ ID NO: 1, residues 479 to 595 of SEQ ID NO: 1, or residues 503 to 595 of SEQ ID NO: 1, or a combination thereof.

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