ES3011359T3 - Adeno-associated viral vectors for treating myocilin (myoc) glaucoma - Google Patents

Abstract

Described are methods for treating myocilin-like glaucoma (MYOC) using adeno-associated viral (AAV) vectors. In some aspects, the AAV vectors encode R-spondin 1 (RSPO1), R-spondin 2 (RSPO2), R-spondin 3 (RSPO3), or R-spondin 4 (RSPO4) and/or RNAi targeting myocilin (MYOC). In one aspect, viral particles are administered to the eye of a human subject. Viral particles encoding RSPO1, RSPO2, RSPO3, and/or RSPO4 and/or MYOC RNAi are contemplated. In some aspects, variant AAV2 particles that transduce the trabecular meshwork are provided.

Description

DESCRIPTION

Adeno-associated viral vectors for the treatment of myocilin glaucoma (MYOC)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority over U.S. Provisional Application No. 62/051,299, filed September 16, 2014.

FIELD OF INVENTION

The present invention relates generally to AAV vectors and methods of using AAV vectors to treat myocilin glaucoma (MYOC).

BACKGROUND OF THE INVENTION

Myocilin (MYOC) mutations account for approximately 2%–4% of primary open-angle glaucoma (POAG; ~90,000 US patients). In particular, glaucomatous MYOC P370L or Y437H mutations account for 10%–30% of juvenile-onset POAG (JOAG; ~6,000 US patients) and are associated with increased intraocular pressure (IOP), retinal ganglion cell death, and optic nerve head (ONH) damage (Shimizu et al. (2000) Am. J. Ophthalmol. 130:165–77; Fan and Wiggs (2010) J. Clin. Invest. 120:3064–72).

Despite the association between MYOC mutations and glaucoma, the effect of MYOC mutants on ocular function remains unclear. Therefore, further understanding of MYOC and mutant MYOC function is needed to develop new therapeutic strategies for treating myocilin-like glaucoma (MYOC).

Pechan et al. (2015) Investigative Opthalmology & Visual Science 56(7) concerns the role of myocilin mutants and Wnt signaling in glaucoma. Kim et al. (2008) Molecular Biology of the Cell 19(6), 2588-2596, concerns a comparison of R-spondin family members in the regulation of the Wnt pathway. Kwon et al. (2009) Molecular and Cellular Biology 29(8), 2139-2154, concerns a study of the functions of human myocilin and its two proteolytic fragments. Buie et al. (2010) Investigative Opthalmology & Visual Science 51 (1), 236-248, relates to an investigation of the ability of AAV vectors to induce safe long-term delivery of transgenes to the trabecular meshwork of living animals. WO 2009/046059 A1 relates to methods of delivering interfering RNA molecules using a self-complementary AAV vector to an eye of a patient to treat ocular disorders. WO 2005/079815 A2 relates to treatment for an autosomal dominant disease of the eye such as retinitis pigmentosa or glaucoma comprising means for suppressing substantially all alleles of a gene associated with the disease. WO 2005/117938 A2 relates to siRNA specific for myocilin and mutant myocilin, and to methods of treating ocular conditions and/or diseases.

SUMMARY OF THE INVENTION

The invention is set forth in the appended set of claims.

The invention provides a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding R-spondin 1 (RSPO1), R-spondin 2 (RSPO2), or R-spondin 4 (RSPO4) for use in a method of treating myocilin-like glaucoma (MYOC) in a mammal, said method comprising administering said rAAV particle to the eye of the mammal, and wherein RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein: (i) the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the eye of the mammal; and/or (ii) the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in trabecular meshwork cells.

The invention also provides a recombinant AAV particle comprising an AAV vector, wherein the AAV vector comprises nucleic acid encoding RSPO1, RSPO2 or r>S<p>O4, and wherein RSPO1, RSPO2> or RSPO4 is operably linked to a promoter, wherein: (i) the promoter is capable of expressing RSPO1, RSPO2 or RSPO4 in the eye of the mammal; and/or (ii) the promoter is capable of expressing RSPO1, RSPO2 or RSPO4 in trabecular meshwork cells, optionally wherein the nucleic acid further encodes an inhibitory nucleic acid that targets the expression of a myocilin (MYOC) in a mammal.

DETAILED DESCRIPTION

The technical information set forth below may, in some respects, go beyond the scope of the invention, which is defined by the appended claims. Additional technical information is provided to place the present invention in a broader technical context and to illustrate possible related technical advances.

Any incidental reference to methods of treatment of the human or animal body by surgery or therapy and diagnostic methods carried out on the human or animal body should not be construed as claiming protection for such methods as such, but should instead be construed as referring to products, in particular substances or compositions, for use in any of those methods.

The disclosure relates to methods of treating myocilin-like glaucoma (MYOC) in a mammal, comprising administering to the eye of the mammal an agent that increases Wnt signaling in the mammalian eye. Accordingly, the disclosure provides an agent that increases Wnt signaling in the eye of a mammal, for use in methods of treating myocilin-like glaucoma (MYOC) in the mammal. In some embodiments, the agent increases Wnt signaling in a trabecular meshwork (TM) cell of the mammalian eye. In some embodiments, the agent increases Wnt signaling in a trabecular meshwork (TM) cell of the mammalian eye.

agent increases the activity of R-spondin 1 (RSPO1), R-spondin 2 (RSPO2), R-spondin 3 (RSPO3), and/or R-spondin 4 (RSPO4) in the mammalian eye. In some embodiments, the agent is used in combination with one or more additional agents that increase one or more RSPO activities in the mammalian eye. In some embodiments, the agent increases RSPO1 in the TM of the mammalian eye. In some embodiments, the agent is RSPO1 or a functional variant thereof. In some embodiments, the agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1 or a functional variant thereof. In some embodiments, RSPO1 is a truncated RSPO1. In some embodiments, the agent increases RSPO2 in the TM of the mammalian eye. In some embodiments, the agent is RSPO2 or a functional variant thereof. In some embodiments, the agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO2 or a functional variant thereof. In some embodiments, RSPO2 is a truncated RSPO2. In some embodiments, the agent increases RSPO3 in the TM of the mammalian eye. In some embodiments, the agent is RSPO3 or a functional variant thereof. In some embodiments, the agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO3 or a functional variant thereof. In some embodiments, RSPO3 is a truncated RSPO3. In some embodiments, the agent increases RSPO4 in the TM of the mammalian eye. In some embodiments, the agent is RSPO4 or a functional variant thereof. In some embodiments, the agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO4 or a functional variant thereof. In some embodiments, RSPO4 is a truncated RSPO4. According to the invention, the agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, or RSPO4, wherein RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein: (i) the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the mammalian eye; and/or (ii) the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in trabecular meshwork cells.

In some aspects, the invention provides administering a second agent that increases Wnt signaling in the eye of the mammal. In some embodiments, the second agent increases Wnt signaling in the TM of the mammalian eye. In some embodiments, the second agent reduces or inhibits the expression of myocilin (MYOC) in the eye of the mammal. In some embodiments, the second agent reduces or inhibits the expression of MYOC in the TM of the mammalian eye. In some embodiments, the second agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding an inhibitory nucleic acid that targets MYOC expression. In some embodiments, the inhibitory nucleic acid is a MYOC RNAi that targets MYOC expression. In some embodiments, the MYOC RNAi is MYOC shRNA that targets MYOC expression.

In some aspects, the agent reduces or inhibits the expression of myocilin (MYOC) in the mammalian eye. In some cases, the agent reduces or inhibits the expression of MYOC in the TM of the mammalian eye. In some cases, the agent is

a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding an inhibitory nucleic acid that targets MYOC expression. In some cases, the inhibitory nucleic acid is a MYOC RNAi that targets MYOC expression. In additional cases, the MYOC RNAi is a MYOC shRNA that targets MYOC expression.

In some embodiments of the invention, the methods further comprise administering a second agent that increases Wnt signaling in the mammalian eye. In some embodiments, the second agent increases Wnt signaling in the TM of the mammalian eye. In some embodiments, the second agent increases R-spondin 1 (RSPO1), R-spondin 2 (RSPO2), R-spondin 3 (RSPO3), or R-spondin 4 (RSPO4) activity in the mammalian eye. In some embodiments, the second agent increases RSPO1 in the TM of the mammalian eye. In some embodiments, the second agent is RSPO1 or a functional variant thereof. In some embodiments, the second agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1 or a functional variant thereof. In some embodiments, RSPO1 is a truncated RSPO1. In some embodiments, the second agent increases RSPO2 in the TM of the mammalian eye. In some embodiments, the second agent is RSPO2 or a functional variant thereof. In some embodiments, the second agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO2 or a functional variant thereof. In some embodiments, RSPO2 is a truncated RSPO2. In some embodiments, the second agent increases RSPO3 in the TM of the mammalian eye. In some embodiments, the second agent is RSPO3 or a functional variant thereof. In some embodiments, the second agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO3 or a functional variant thereof. In some embodiments, RSPO3 is a truncated RSPO3. In some embodiments, the second agent increases RSPO4 in the TM of the mammalian eye. In some embodiments, the second agent is RSPO4 or a functional variant thereof. In some embodiments, the second agent is a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO4 or a functional variant thereof. In some embodiments, RSPO4 is a truncated RSPO4.

In some embodiments, the invention relates to methods of treating myocilin (MYOC) glaucoma in a mammal, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof. In accordance with the invention, the rAAV particle for use in a method of treating myocilin (MYOC) glaucoma in a mammal comprises a vector encoding RSPO1, RSPO2, or RSPO4. In some embodiments, the invention relates to methods of treating myocilin (MYOC) glaucoma in a mammal, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding a MYOC RNAi that targets the expression of a myocilin (MYOC) RNA.

in the mammal. In other embodiments, the disclosure relates to methods of treating myocilin-like glaucoma (MYOC) in a mammal, comprising administering to the eye of the mammal an agent that increases Wnt signaling in the eye of the mammal and an agent that reduces or inhibits myocilin expression in the mammal. In other embodiments, the disclosure relates to methods of treating myocilin-like glaucoma (MYOC) in a mammal, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and an rAAV particle comprising a vector encoding a MYOC RNAi that targets expression of a myocilin in the mammal. In still other embodiments, the disclosure relates to methods of treating myocilin (MYOC) glaucoma in a mammal, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and encoding a MYOC shRNA that targets expression of a myocilin (MYOC shRNA) in the mammal. In some embodiments, the RNAi is an shRNA that targets MYOC. In some embodiments, the shRNA reduces or inhibits expression of MYOC.

In some embodiments, the disclosure relates to methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof. In some embodiments, the disclosure relates to methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding an inhibitory nucleic acid that targets the expression of a myocilin (MYOC) in the mammal. In other embodiments, the disclosure relates to methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding a MYOC RNAi that targets expression of a myocilin (MYOC) in the mammal. In other embodiments, the disclosure relates to methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and an rAAV particle comprising a vector encoding a MYOC RNAi that targets expression of a myocilin in the mammal. In other cases, the disclosure relates to methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising

administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and encoding a MYOC RNAi that targets the expression of a myocilin in the mammal. In some cases, the ocular disorder is myocilin (MYOC) glaucoma.

In some embodiments, the mammal is a human. In some embodiments of the invention, myocilin glaucoma (MYOC) is associated with a mutation in a myocilin. In some embodiments, myocilin glaucoma (MYOC) is associated with a mutation in a human myocilin. In some embodiments, the myocilin mutation comprises one or more amino acid substitutions selected from E323K, K398R, Q368X, G364V, P370L, D380A, K423E, Y437H, and I477S. In some embodiments, the myocilin mutation comprises a P370L amino acid substitution. In some embodiments, the myocilin mutation comprises a Y437H amino acid substitution. In some embodiments, the myocilin glaucoma (MYOC) is primary open angle glaucoma (POAG). In some embodiments, myocilin glaucoma (MYOC) is the juvenile form of primary open angle glaucoma (JOAG). In some embodiments of the invention, the treatment reduces a symptom of myocilin glaucoma (MYOC). In some embodiments, the reduction of a symptom of myocilin glaucoma (MYOC) is a reduction in intraocular pressure, a reduction in MYOC accumulation in the trabecular meshwork, a reduction in ocular hypertension, or an increase in aqueous outflow from the trabecular meshwork.

In some embodiments, RSPO1 is a human RSPO1. In some embodiments, RSPO1 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to human RSPO1. In some embodiments, RSPO1 comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, RSPO1 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:8. In some embodiments, RSPO1 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, RSPO1 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11. In some embodiments, RSPO1 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, RSPO1 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12. In some embodiments, RSPO2 is a human RSPO2. In some embodiments, RSPO2 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to human RSPO2. In some embodiments, RSPO2 comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, RSPO2 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:9. In some embodiments, RSPO2 comprises the amino acid sequence of SEQ ID NO:9.

of SEQ ID NO: 13. In some embodiments, RSPO2 has greater than about 80%, 85%, 90%, 91%, <92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ i>D<NO: 13. In some embodiments, RSPO2> comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, RSPO2 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ iD NO: 14. In some embodiments, RSPO3 is a human RSPO3. In some embodiments, RSPO3 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to human RSPO3. In some embodiments, RSPO3 comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, RSPO3 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1. In some embodiments, RSPO3 comprises the amino acid sequence of SEQ ID NO: 15. In some cases, RSPO3 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. In some embodiments, RSPO3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, RSPO3 comprises the amino acid sequence of SEQ ID NO: 17. In some cases, RSPO3 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17. In some embodiments, RSPO4 is a human RSPO4. In some embodiments, RSPO4 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to human RSPO4. In some embodiments, RSPO4 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, RSPO4 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:10. In some embodiments, RSPO4 comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, RSPO4 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18. In some embodiments, RSPO4 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, RSPO4 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12. In some instances, RSPO1, RSPO2, RSPO3, RSPO4, and/or functional variant thereof is operably linked to a promoter. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, and/or a functional variant thereof in the mammalian eye. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, and/or a functional variant thereof in trabecular meshwork cells. According to the invention, RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the mammalian eye and/or the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin (CBA) hybrid promoter.

In some embodiments, the MYOC RNAi targeting MYOC expression used in the invention targets human MYOC. In some embodiments, the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA). In some embodiments, the MYOC RNAi is a mY<o>C shRNA. In some embodiments, the shRNA targets the amino acid sequence of MYOC set forth in SEQ ID NO:6. In some embodiments, the shRNA comprises the loop sequence of SEQ ID NO:7. In some embodiments, the MYOC RNAi (e.g., shRNA) is operably linked to a promoter. In some embodiments, the promoter is capable of expressing the MYOC RNAi (e.g., shRNA) in the eye of the mammal. In further embodiments, the promoter is capable of expressing MYOC RNAi (e.g., shRNA) in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin hybrid (CBA) promoter. In some embodiments, the promoter is an RNA polymerase III promoter. In some embodiments, expression of MYOC RNAi (e.g., shRNA) reduces or inhibits MYOC expression in the mammalian eye. In some embodiments, expression of MYOC RNAi (e.g., shRNA) reduces or inhibits MYOC expression in the mammalian trabecular meshwork cells. In some embodiments, the MYOC is a wild-type MYOC. In some embodiments, the MYOC is a mutant MYOC. In some embodiments, the MYOC is both a wild-type MYOC and a mutant MYOC. In further embodiments, the mutant MYOC comprises amino acid substitutions corresponding to P370L and/or Y437H amino acid substitutions of human MYOC. In some embodiments, the myocilin mutation comprises one or more amino acid substitutions selected from E323K, K398R, Q368X, G364V, P370L, D380A, K423E, Y437H, and I477S.

In some embodiments of the aspects and embodiments described above, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid, such as ShH10, as described in the US pre-grant publication.

2012/0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S. pre-grant publication 2013/0323226), AAV10, AAVrh10, AAV11, AAV12, a capsid tyrosine mutant, a capsid heparin binding mutant, an AAV2R471A capsid, an AAAV2/2-7m8 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids described in the pre-grant publication of US<u>.<2012/0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2/HBoV1 capsid, or an AAV capsid described in U.S. Patent No. 8,283,151 or International Publication No. WO/2003/042397. In some embodiments, the AAV viral particle comprises an AAV capsid comprising an amino acid substitution in one or more of

positions R484, R487, K527, K532, R585, or R588, numbering based on AAV2 VP1. In further embodiments, an AAV particle comprises capsid proteins from an AAV serotype from clades A-F. In some embodiments, the rAAV viral particle comprises an AAV serotype 2 capsid. In further embodiments, the AAV serotype 2 capsid comprises the AAV2 capsid protein comprising an R471A amino acid substitution, numbering relative to AAV2 VP1. In some embodiments, the vector comprises inverted terminal repeats (ITRs) of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, goat AAV, bovine AAV, or mouse AAV. In some embodiments, the vector comprises ITRs from AAV serotype 2. In some embodiments, the AAV viral particle comprises one or more ITRs and capsid derived from the same AAV serotype. In other embodiments, the AAV viral particle comprises one or more ITRs derived from a different AAV serotype than the capsid of the rAAV viral particles. In some embodiments, the rAAV viral particle comprises an AAV2 capsid, and wherein the vector comprises AAV2 ITRs. In further embodiments, the AAV2 capsid comprises the AAV2 capsid protein comprising an amino acid substitution R471A, numbering relative to AAV2 VP1.

In some embodiments, at least 1 x 109 genome copies of the rAAV particles are administered to the mammal. In some embodiments, the AAV is administered to the cornea, retina, and/or sclera of the mammal's eye. In some embodiments, the AAV particle is administered by intravitreal injection and/or intracameral injection. In some embodiments, the rAAV is administered to more than one location in the eye.

In some embodiments, the invention relates to the treatment of myocilin glaucoma (MYOC) in a mammal, wherein the mammal is a human. In some embodiments, the myocilin glaucoma (MYOC) is primary open-angle glaucoma (POAG). In some embodiments, the myocilin glaucoma (MYOC) is a juvenile form of primary open-angle glaucoma (JOAG).

In some embodiments of the invention, the rAAV viral particles are in a pharmaceutical composition. In additional embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In some embodiments of the above methods, the agent (e.g., the AAV particle) is used in combination with one or more additional agents that increase the activity of an R-spondin (e.g., RSPO1, RSPO2, RSPO3, and/or RSPO4).

In some embodiments, the invention provides recombinant AAV particles comprising an AAV vector, wherein the AAV vector comprises nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof. According to the invention, the AAV vector comprises nucleic acid encoding RSPO1, RSPO2, or RSPO4. In other embodiments, the invention provides rAAV particles comprising a vector encoding an inhibitory nucleic acid that targets expression of a myocilin (MYOC) in the mammal. In other embodiments, the invention provides rAAV particles comprising a vector encoding a MYOC RNAi that targets expression of a myocilin (MYOC) in the mammal. In still other instances, the disclosure provides rAAV particles comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and encoding a MYOC RNAi that targets the expression of a myocilin in the mammal.

In some embodiments, the AAV vector comprises nucleic acid encoding RSPO1 or a functional variant thereof, and the RSPO1 or functional variant thereof is a human RSPO1. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO1 or a functional variant thereof, and the RSPO1 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 8, 11, and/or 12. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO1 or a functional variant thereof, and the RSPO1 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8, 11, and/or 12. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO2 or a functional variant thereof, and the RSPO2 or functional variant thereof is a human RSPO2. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO2 or a functional variant thereof, and the RSPO2 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 9, 13, and/or 14. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO2 or a functional variant thereof, and the RSPO2 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 9, 13, and/or 14. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO3 or a functional variant thereof, and the RSPO3 or functional variant thereof is a human RSPO3. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO3 or a functional variant thereof, and the RSPO3 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 1 and/or 15-17. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO3 or a functional variant thereof, and the RSPO3 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1 and/or 15-17. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO4, and the RSPO4 is a human RSPO4. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO4 or a functional variant thereof, and the RSPO4 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 10, 18, and/or 19. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO4 or a functional variant thereof, and the RSPO4 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10, 18, and/or 19. In additional embodiments, RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, is operably linked to a promoter. In additional embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof in the mammalian eye. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof in trabecular meshwork cells. According to the invention, RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the mammalian eye and/or the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin (CBA) hybrid promoter.

In some embodiments, the inhibitory nucleic acid that targets expression of a myocilin (MYOC) in the mammal is an RNAi. In some embodiments, the MYOC RNAi (e.g., shRNA) that targets MYOC expression of the disclosure targets human MYOC. In some embodiments, the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA). In some embodiments, the MYOC RNAi is an shRNA. In some embodiments, the RNAi (e.g., shRNA) targets the amino acid sequence of MYOC set forth in SEQ ID NO:6. In some embodiments, the RNAi (e.g., shRNA) comprises the loop sequence of SEQ ID NO:7. In some embodiments, the MYOC RNAi (e.g., shRNA) is operably linked to a promoter. In some embodiments, the promoter is capable of expressing MYOC RNAi (e.g., shRNA) in the eye of the mammal. In further embodiments, the promoter is capable of expressing MYOC RNAi (e.g., shRNA) in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin hybrid (CBA) promoter. In some embodiments, the promoter is an RNA polymerase III promoter. In some embodiments, expression of MYOC RNAi (e.g., shRNA) reduces or inhibits MYOC expression in the eye of the mammal. In some embodiments, expression of MYOC RNAi (e.g., shRNA) reduces or inhibits MYOC expression in the trabecular meshwork cells of the mammal. In some embodiments, the MYOC is a wild-type MYOC. In some embodiments, the MYOC is a mutant MYOC. In some embodiments, the MYOC is both a wild-type MYOC and a mutant MYOC. In further embodiments, the mutant MYOC comprises amino acid substitutions corresponding to the E323K, K398R, Q368X, G364V, P370L, D380A, K423E, Y437H, and I477S amino acid substitutions of human MYOC. In some embodiments, the mutant MYOC comprises amino acid substitutions corresponding to P370L and/or Y437H amino acid substitutions of human MYOC. In some embodiments, the myocilin mutation is associated with

with primary open-angle glaucoma (POAG). In some embodiments, the myocilin mutation is associated with juvenile-onset primary open-angle glaucoma (JOAG).

In some embodiments of the aspects and embodiments described above, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid, such as ShH10, as described in the US pre-grant publication.

2012/0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S. pre-grant publication 2013/0323226), AAV10, AAVrh10, AAV11, AAV12, a tyrosine capsid mutant, a heparin-binding capsid mutant, an AAV2R471A capsid, an AAAV2/2-7m8 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids described in the pre-grant publication of EE.>U<u>.<2012/0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid,>AAV V708K capsid, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, Mouse AAV, rAAV2/HBoV1 capsid, or an AAV capsid described in US Patent No. 8,283,151 or International Publication No. WO/2003/042397. In some embodiments, the AAV viral particle comprises an AAV capsid comprising an amino acid substitution at one or more of positions R484, R487, K527, K532, R585, or R588, numbering based on VP1 of AAV2. In further embodiments, an AAV particle comprises capsid proteins from an AAV serotype from clades A-F. In some embodiments, the rAAV viral particle comprises an AAV serotype 2 capsid. In further embodiments, the AAV serotype 2 capsid comprises the AAV2 capsid protein comprising an R471A amino acid substitution, numbering relative to VP1 of AAV2. In some embodiments, the vector comprises inverted terminal repeats (ITRs) of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV. In some embodiments, the vector comprises ITRs from AAV serotype 2. In some embodiments, the AAV viral particle comprises one or more ITRs and capsid derived from the same AAV serotype. In other embodiments, the AAV viral particle comprises one or more ITRs derived from a different AAV serotype than the capsid of the rAAV viral particles. In some embodiments, the rAAV viral particle comprises an AAV2 capsid, and wherein the vector comprises AAV2 ITRs. In further embodiments, the AAV2 capsid comprises the AAV2 capsid protein comprising an amino acid substitution R471A, numbering relative to AAV2 VP1.

The disclosure provides pharmaceutical compositions comprising any of the recombinant AAV particles described herein. The disclosure also provides pharmaceutical compositions that are suitable for any of the methods described herein. The disclosure provides uses of a pharmaceutical composition and recombinant AAV particles described herein in the manufacture of a medicament for treating myocilin glaucoma (MYOC) in a mammal. In some embodiments, the mammal is a human. In some embodiments, the myocilin glaucoma (MYOC) is primary open angle glaucoma (POAG). In some embodiments, the myocilin glaucoma (MYOC) is a juvenile form of primary open angle glaucoma (JOAG).

In some embodiments, the disclosure provides kits for the treatment of myocilin (MYOC) glaucoma in a mammal, wherein the kit comprises an rAAV viral particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof; an rAAV viral particle comprising an AAV vector, wherein the AAV vector comprises nucleic acid encoding an inhibitory nucleic acid (e.g., MYOC RNAi including shRNA) that targets expression of a myocilin (MYOC) in the mammal; and/or an rAAV viral particle comprising an AAV vector, wherein the AAV vector comprises nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and encoding a MYOC RNAi (e.g., shRNA) that targets expression of a MYOC in the mammal. The invention provides a kit for treating myocilin-like glaucoma (MYOC) in a mammal, wherein the kit comprises an rAAV particle of the invention. In some embodiments, the kit further comprises instructions for use in treating myocilin-like glaucoma (MYOC). In some embodiments, the kit further comprises pharmaceutically acceptable buffers and/or excipients.

In some embodiments, the kits of the invention comprise nucleic acid encoding a MYOC RNAi (e.g., shRNA) that targets expression of a MYOC in the mammal. In some embodiments, the MYOC RNAi targets expression of a human MYOC. In some embodiments, the MYOC RNAi targets the MYOC amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA). In some embodiments, the RNAi is an shRNA. In some embodiments, the MYOC shRNA comprises the loop sequence of SEQ ID NO:7. In some embodiments, the kits of the invention comprise an AAV vector, wherein the AAV vector comprises nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO1 or a functional variant thereof, and the RSPO1 or functional variant thereof is a human RSPO1. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO1 or a functional variant thereof, and the RSPO1 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 8, 11, and/or 12. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO1 or a functional variant thereof, and the RSPO1 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8, 11, and/or 12. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO2 or a functional variant thereof, and the RSPO2 ...

It is a human RSPO2. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO2 or a functional variant thereof, and the RSPO2 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 9, 13, and/or 14. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO2 or a functional variant thereof, and the RSPO2 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 9, 13, and/or 14. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO3 or a functional variant thereof, and the RSPO3 or functional variant thereof is a human RSPO3. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO3 or a functional variant thereof, and the RSPO3 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 1 and/or 15-17. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO3 or a functional variant thereof, and the RSPO3 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1 and/or 15-17. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO4 or a functional variant thereof, and the RSPO4 or functional variant thereof is a human RSPO4. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO4 or a functional variant thereof, and the RSPO4 or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 10, 18, and/or 19. In some embodiments, the AAV vector comprises nucleic acid encoding RSPO4 or a functional variant thereof, and the RSPO4 or functional variant thereof comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10, 18, and/or 19. In some instances, RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, is operably linked to a promoter. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof in the mammalian eye. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof in trabecular meshwork cells. According to the invention, RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the mammalian eye; and/or the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin (CBA) hybrid promoter. In some embodiments, the MYOC RNAi is operably linked to a promoter. In some embodiments, the promoter is capable of expressing the MYOC RNAi in the mammalian eye. In some embodiments, the promoter is capable of expressing MYOC RNAi in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin hybrid (CBA) promoter. In some cases, the promoter is an RNA polymerase III promoter. In some embodiments, expression of MYOC RNAi reduces or inhibits MYOC expression in the mammalian eye. In some embodiments, expression of MYOC RNAi

reduces or inhibits MYOC expression in mammalian trabecular meshwork cells.

In some embodiments, the AAV particles described herein may be used in combination with one or more additional agents that increase the activity of an R-spondin (e.g., RSPO1, RSPO2, RSPO3, and/or RSPO4).

In some embodiments, the kits of the invention comprise an AAV viral particle comprising a vector and an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid such as ShH10, as described in U.S. pre-grant publication 2012/0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S. pre-grant publication 2013/0323226), AAV10, AAVrh10, AAV11, AAV12, a capsid tyrosine mutant, a capsid heparin binding mutant, a AAV2R471A capsid, an AAVAAV2/2-7m8 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids described in U.S. pre-grant publication 2012/0066783), A<a>V2<N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV>V708K capsid, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2/HBoV1 capsid, or an AAV capsid described in U.S. patent 2012/0066783. United States No. 8,283,151 or International Publication No. WO/2003/042397. In some embodiments, the AAV viral particle comprises an AAV capsid comprising an amino acid substitution at one or more of positions R484, R487, K527, K532, R585, or R588, numbering based on VP1 of AAV2. In further embodiments, an AAV particle comprises capsid proteins from an AAV serotype from clades A-F. In some embodiments, the rAAV viral particle comprises an AAV serotype 2 capsid. In some embodiments, the AAV serotype 2 capsid comprises the AAV2 capsid protein comprising an R471A amino acid substitution, numbering relative to VP1 of AAV2. In some embodiments, the vector comprises inverted terminal repeats (ITRs) of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV. In some embodiments, the vector comprises ITRs from AAV serotype 2. In some embodiments, the AAV viral particle comprises one or more ITRs and capsid derived from the same AAV serotype. In some embodiments, the AAV viral particle comprises one or more ITRs and capsid derived from a different AAV serotype than the capsid of the rAAV viral particles. In some embodiments, the rAAV viral particle comprises an AAV2 capsid, and the vector comprises AAV2 ITRs. In some embodiments, the AAV2 capsid comprises an AAV2 capsid protein comprising an amino acid substitution R471A, numbering relative to AAV2 VP1.

In some embodiments of the above kits, the AAV particle of the kit is used in combination with one or more additional agents that increase the activity of an R-spondin (e.g., RSPO1, RSPO2, RSPO3, and/or RSPO4). In some embodiments, the kits of the invention comprise an AAV particle as described herein and one or more additional agents that increase the activity of an R-spondin (e.g., RSPO1, RSPO2, RSPO3, and/or RSPO4).

The disclosure provides kits suitable for use in any one of the methods described herein. The disclosure provides kits comprising any of the recombinant AAV particles described herein. In some aspects, the kits described herein further comprise instructions for use in the treatment of myocilin glaucoma (MYOC). In some aspects, the kits described herein further comprise pharmaceutically acceptable buffers and/or excipients.

In some embodiments, the disclosure provides methods of delivering nucleic acid (e.g., a nucleic acid encoding a therapeutic transgene) to the trabecular meshwork of a mammal's eye, comprising delivering an AAV serotype 2 (AAV2) particle comprising an rAAV vector to the mammal's eye, wherein the rAAV vector comprises the nucleic acid, and wherein the AAV2 particle comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on AAV2 VP1. In some embodiments, the disclosure provides methods of treating an ocular disorder in a mammal comprising administering an AAV2 particle comprising an rAAV vector to the eye of the mammal, wherein the rAAV vector comprises nucleic acid encoding a therapeutic transgene, and wherein the AAV2 particle comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on VP1 of AAV2. In some embodiments, the rAAV particle is administered intravitreally and/or intracamerally. In some embodiments, the rAAV particle transduces cells of the trabecular meshwork of the eye. In some embodiments, the therapeutic transgene is expressed in the trabecular meshwork of the eye. In some embodiments, the therapeutic transgene encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the ocular disorder is a disorder associated with the trabecular meshwork of the eye. In some embodiments, the ocular disorder is myocilin glaucoma (MYOC). In some cases, the mammal is a human being.

In some cases, the disclosure provides a recombinant AAV2 particle for delivering nucleic acid (e.g., nucleic acid encoding a therapeutic transgene) to the trabecular meshwork of the eye of a mammal, wherein the AAV2 particle comprises an rAAV vector, wherein the rAAV vector comprises the nucleic acid, and wherein the AAV2 particle comprises the AAV2 capsid protein comprising an amino acid substitution R471A, numbering based on VP1 of

AAV2. In some embodiments, the invention provides a recombinant AAV2 particle for treating an ocular disorder in a mammal, wherein the AAV2 particle comprises an rAAV vector, wherein the rAAV vector comprises nucleic acid encoding a therapeutic transgene, and wherein the AAV2 particle comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on VP1 of AAV2. In some embodiments, the rAAV particle transduces cells of the trabecular meshwork of the eye. In some embodiments, the therapeutic transgene is expressed in the trabecular meshwork of the eye. In some embodiments, the therapeutic transgene encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the ocular disorder is a disorder associated with the trabecular meshwork of the eye. In some embodiments, the ocular disorder is myocilin glaucoma (MYOC). In some embodiments, the mammal is a human.

In some embodiments, the invention provides methods of a recombinant AAV2 particle for delivering nucleic acid (e.g., nucleic acid encoding a therapeutic transgene) to the trabecular meshwork of a mammalian eye, wherein the AAV2 particle comprises an rAAV vector, wherein the rAAV vector comprises the nucleic acid, and wherein the AAV2 particle comprises the AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on AAV2 VP1. In some embodiments, the invention provides a method of treating an ocular disorder in a mammal, wherein the AAV2 particle comprises an rAAV vector, wherein the rAAV vector comprises nucleic acid encoding a therapeutic transgene, and wherein the AAV2 particle comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on AAV2 VP1. In some embodiments, the rAAV particle is administered intravitreally and/or intracamerally. In some embodiments, the rAAV particle transduces cells of the trabecular meshwork of the eye. In some embodiments, the therapeutic transgene is expressed in the trabecular meshwork of the eye. In some embodiments, the therapeutic transgene encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some embodiments, the ocular disorder is a disorder associated with the trabecular meshwork of the eye. In some cases, the eye disorder is myocilin-induced glaucoma (MYOC). In some cases, the mammal is a human.

In some cases, the disclosure provides kits that deliver nucleic acid (e.g., a nucleic acid encoding a therapeutic transgene) to the trabecular meshwork of the eye of a mammal, comprising an rAAV2 particle comprising an rAAV vector, wherein the rAAV vector comprises the nucleic acid, and wherein the AAV2 particle comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on VP1 of AAV2. In some cases, the disclosure provides kits for treating an ocular disorder in a mammal comprising an rAAV2 particle comprising a rAAV vector, wherein the rAAV vector comprises nucleic acid encoding a therapeutic transgene, and wherein the AAV2 particle comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbering based on VP1 of AAV2. In some cases, the rAAV particle is administered intravitreally and/or intracamerally. In some cases, the rAAV particle transduces cells of the trabecular meshwork of the eye. In some cases, the therapeutic transgene is expressed in the trabecular meshwork of the eye. In some cases, the therapeutic transgene encodes a therapeutic polypeptide or a therapeutic nucleic acid. In some cases, the ocular disorder is a disorder associated with the trabecular meshwork of the eye. In some cases, the ocular disorder is myocilin-induced glaucoma (MYOC). In some cases, the mammal is a human.

Brief description of the drawings

FIG. 1 demonstrates that the MYOC mutants P370L and Y437H are not secreted and block secretion of wild-type MYOC ("wtMYOC"). Cell culture medium or cell lysates from 293 cells transfected with constructs expressing wtMYOC and/or MYOC mutants (as labeled) were probed by Western blotting using anti-human MYOC antibody.

FIG. 2 shows that the MYOC P370L mutant is not secreted and blocks secretion of wild-type MYOC ("wtMYOC") in T antigen-transformed human trabecular meshwork cells ("hTM-T") in both 293T and SV40. Cell culture medium or cell lysates from 293T or hTM-T cells transfected with constructs expressing wtMYOC and/or MYOC P370L (as labeled) were probed by Western blotting using anti-human MYOC antibody.

FIG. 3 depicts the effect of wtMYOC, MYOC P370L, or Y437H expression on Wnt signaling. For each experiment, the "without mWnt3a" bar is on the left, and the "with mWnt3a - 400 ng/ml" bar is on the right.

FIG. 4 shows that RSPO3 expression can restore Wnt signaling upon coexpression with MYOC P370L or Y437H. For each experiment, the "no mWnt3a" bar is on the left, and the "mWnt3a - 400 ng/ml" bar is on the right.

FIG. 5 shows that RSPO3 expression can restore Wnt signaling in hTM-T cells upon coexpression with MYOC P370L. For each experiment, the "no mWnt3a" bar is on the left, and the "400 ng/ml hWnt3a" bar is on the right.

FIG. 6 shows the effect of MYOC shRNA on MYOC expression in 293T cells. Cell culture medium or cell lysates from 293T cells were probed by Western blotting with anti-human MYOC antibody. Cells were transfected with plasmids expressing wtMYOC (lane 1); wtMYOC and MYOC #79 shRNA (2); wtMYOC and MYOC #93 shRNA (3); wtMYOC and shuffled shRNA control (4); or EGFP (5). The 55/57 kD bands represent glycosylated (57 kD) and non-glycosylated (55 kD) forms of a full-length MYOC protein. The 22 kD band represents the N terminus of a calpain II cleavage product.

FIG. 7 shows the effect of MYOC shRNA on MYOC expression in hTM-T cells. Cell culture medium or cell lysates from hTM-T cells were probed by Western blotting with anti-human MYOC antibody. Cells were transfected with plasmids expressing wtMYOC (lane 1); MYOC P370L (2); wtMYOC and MYOC P370L (3); wtMYOC and MYOC P370L and Grp94 shRNA #1 (4); wtMYOC and MYOC P370L and Grp94 shRNA #2 (5); wtMYOC and MYOC P370L and MYOC shRNA #253 (6); wtMYOC and MYOC P370L and MYOC pGIPZ shRNA #79 (7); wtMYOC and MYOC P370L and MYOC shRNA pGIPZ #93 (8); wtMYOC and MYOC P370L and shRNA control mixed (9); or EGFP (10).

FIG. 8 shows that RSPO3 expression and MYOC silencing synergistically restore Wnt signaling upon coexpression with MYOC P370L. 293T cells were cotransfected with the TOP-Flash reporter construct and wtMYOC (“MYOC”), plus MYOC P370L, Grp94 shRNA, MYOC pGIPZ shRNA #79 (the first) and #93 (the second), and/or RSPO3 plasmids, as labeled. Wnt signaling was amplified after the addition of recombinant mouse Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity (mean ± SD, n = 1-3 duplicate wells) was measured after transfection and normalized to the constitutively expressed Renilla luciferase level transfection control. For each experiment, the "no Wnt added" bar is on the left, and the "400 ng/ml Wnt3a added" bar is on the right.

FIG. 9 shows that MYOC silencing restores Wnt signaling upon coexpression with either P370L or Y437H MYOC. 293T cells were cotransfected with the TOP-Flash reporter construct and wtMYOC (“MYOC”), plus scrambled P370L MYOC, Y437H MYOC, MYOC shRNA, and/or control shRNA (“pGIPZ-null”), as labeled. Wnt signaling was amplified after the addition of recombinant mouse Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity (mean ± SD, n = 1–3 duplicate wells) was measured posttransfection and normalized to the constitutively expressed Renilla luciferase level transfection control. For each experiment, the "no Wnt added" bar is on the left, and the "400 ng/ml Wnt3a added" bar is on the right.

FIG. 10 shows in vitro (left panels) and in vivo (right panels) transduction of trabecular meshwork cells by wild-type AAV2 viral particles (top panels) and AAV2 particles comprising an R471A amino acid substitution of the capsid protein.

FIG. 11 shows a domain diagram of human RSPO family proteins representing furin-like Cys-rich domains, the thrombospondin type 1 domain, and the carboxy-terminal positively charged domain, as labeled (figure adapted from Kim, K.A. et al. (2008) Mol. Biol. Cell. 19:2588-2596).

FIG. 12 shows a domain diagram of human RSPO family genes representing the protein domains listed in FIG. 11. Amino acid sequence numbering is depicted, and truncation mutants tested for each family member are as labeled (figure adapted from Kim, K.A. et al. (2006) Cell Cycle 5:23-26).

FIG. 13A shows the full-length human RSPO3 sequence (SEQ ID NO: 1) with the signal sequence domains, FU1, FU2, and TSP1, labeled.

FIG. 13B shows the sequence of an active human RSPO3 fragment (SEQ ID NO: 16) with the signal sequence domains, FU1 and FU2, labeled. The fragment used, which lacks the signal peptide, corresponds to amino acids 22-146 of SEQ ID NO: 16, and is 15 kDa including the His tag.

FIG. 13C depicts the structure of the full-length hRSPO3 domain with the signal peptide domains, FU1, FU2, TSP1, and BR, labeled. Possible functions for each domain are listed below.

FIG. 13D shows a Western blot of full-length hRSPO3 and the hRSPO3 fragment.

FIG. 14 depicts the hRSPO3 fragments tested. The domain structure of full-length hRSPO3 with the signal peptide domains, FU1, FU2, TSP1, and BR, marked and possible functions for each domain are also provided below.

FIG. 15 shows that expression of RSPO3 fragments and full-length RSPO3 can restore Wnt signaling upon coexpression with MYOC Y437H.

FIG. 16 shows that expression of RSPO family members can induce Wnt signaling upon coexpression with MYOC Y437H even without the addition of Wnt3a.

ADDITIONAL DETAILED DESCRIPTION

The present disclosure provides methods of treating myocilin (MYOC) glaucoma in a mammal, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) viral particle. In some cases, wnt signaling in the eye of the mammal is increased; for example, by expression of R-spondin 1 (RSPO1), R-spondin 2 (RSPO2), R-spondin 3 (RSPO3), and/or R-spondin 4 (RSPO4). In some cases, myocilin (MYOC) expression is inhibited (e.g., mutant myocilin); for example, by the use of RNAi that targets MYOC expression. In some cases, the rAAV particle comprises a vector encoding RSPO1, RSPO2, RSPO3, and/or RSPO4, and/or a functional variant thereof. In accordance with the invention, the AAV particle comprises a vector encoding RSPO1, RSPO2, or RSPO4. In other embodiments, the rAAV particle comprises a vector encoding a MYOC RNAi (e.g., shRNA) that targets expression of a myocilin (MYOC) in the mammal. In other embodiments, the disclosure provides methods of treating myocilin (MYOC) glaucoma in a mammal, comprising administering to the eye of the mammal a mixture of rAAV particles comprising a vector encoding RSPO1, RSPO2, RSPO3, and/or RSPO4, and/or a functional variant thereof, and rAAV particles comprising a vector encoding a MYOC RNAi (e.g., shRNA) that targets expression of a myocilin in the mammal. In other embodiments, the disclosure provides methods of treating myocilin (MYOC) glaucoma in a mammal, comprising administering to the eye of the mammal an rAAV particle comprising a vector encoding RSPO1, RSPO2, RSPO3, and/or RSPO4, and/or a functional variant thereof, and encoding a MYOC RNAi (e.g., shRNA) that targets the expression of a myocilin (MYOC shRNA) in the mammal. The disclosure also provides compositions and kits for treating myocilin (MYOC) glaucoma using the rAAV vectors encoding RSPO1, RSPO2, RSPO3, and/or RSPO4, and/or a functional variant thereof and/or MYOC RNAi (e.g., shRNA). The disclosure also provides recombinant AAV particles, compositions, and kits.

In some embodiments, the disclosure provides methods of targeting AAV2 to transduce trabecular meshwork cells. In some embodiments, the disclosure provides rAAV2 particles comprising an R471A mutation, AAV2 VP1-based numbering. In some embodiments, the disclosure provides methods and compositions for treating ocular diseases associated with the trabecular meshwork (e.g., myocilin glaucoma (MYOC)) using AAV2 viral particles comprising mutated capsid protein (e.g., an R471A amino acid substitution).

I. General techniques

The techniques and procedures described or referred to herein are generally well understood and typically employed using standard methodology by those skilled in the art, such as, for example, the widely used methodologies described in Molecular Cloning: A Laboratory Manual (Sambrooket., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003); the Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J.P. Mather and PE. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mulliset al.,eds., 1994); Current Protocols in Immunology (J.E. Coliganet al.,eds., 1991); Short Protocols in Molecular Biology (Ausubelet al.,eds., J. Wiley and Sons, 2002); Immunobiology (C.A. Janewayet al.,2004); Antibodies (P Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVitaet al.,eds., J.B. Lippincott Company, 2011).

II. Definitions

A "vector," as used herein, refers to a plasmid or recombinant virus comprising a nucleic acid to be delivered to a host cell, either in vitro or in vivo.

The term "polynucleotide" or "nucleic acid," as used herein, refers to a polymeric form of nucleotides of any length, ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The nucleic acid backbone may comprise sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups.

Alternatively, the nucleic acid backbone may comprise a polymer of synthetic subunits such as phosphoramidates and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded nucleic acid may be obtained from the single-stranded polynucleotide product of chemical synthesis by synthesizing the complementary strand and annealing the strands under appropriate conditions or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain naturally occurring or non-naturally occurring amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include modifications after expression of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally of a conservative nature), to the naturally occurring sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or they may be accidental, such as through mutations in host proteins or errors due to PCR amplification.

A "recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one, preferably two, inverted terminal repeat (ITR) sequences.

A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence that is not of AAV origin) that are flanked by at least one, preferably two, AAV inverted terminal repeat (ITR) sequences. Such rAAV vectors are capable of replicating and encapsidating into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or is expressing suitable helper functions) and that is expressing the AAV rep and cap gene products (i.e., the AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome or another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a "provector" that can be "rescued" by replication and encapsidation in the presence of suitable AAV packaging functions and helper functions. An rAAV vector may be in any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and, in embodiments, encapsidated into a viral particle, particularly an AAV particle. An rAAV vector may be packaged into an AAV virus capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)." AAV helper functions (i.e., functions that allow a host cell to replicate and encapsidate the AAV) can be provided in any of several forms, including, but not limited to, a helper virus or helper virus genes that assist in AAV replication and encapsidation. Other AAV helper functions are known in the art.

An "rAAV virus" or "rAAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

"Heterologous" means derived from an entity genotypically distinct from the rest of the entity with which it is compared or into which it is introduced or incorporated. For example, a nucleic acid introduced by genome engineering into a different cell type is a heterologous nucleic acid (and, when expressed, may encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or part thereof) that is incorporated into a viral vector is a nucleotide sequence heterologous to the vector.

The term "transgene" refers to a nucleic acid that is introduced into a cell and can be transcribed into RNA and, optionally, translated and/or expressed under appropriate conditions. In some aspects, it confers a desired property to a cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as siRNA.

The terms "genomic particles (gp)", "genomic equivalents" or "genome copies", as used in reference to a viral titer, refer to the number of virions contained in the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures, such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

The terms "infection unit (iu)", "infectious particle" or "replication unit", as used in reference to a viral titer, refer to the number of infectious, replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

The term "transducing unit (tu)", as used in reference to a viral titer, refers to the number of infectious recombinant AAV vector particles that cause the production of a functional transgene product, as measured in functional assays, such as those described in the Examples herein or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

An "inverted terminal repeat" or "ITR" sequence is a well-understood term in the art and refers to relatively short sequences found at the ends of viral genomes that are in the opposite orientation.

An "AAV inverted terminal repeat (ITR)" sequence, a term well understood in the art, is a sequence of approximately 145 nucleotides that is present at both ends of the naturally occurring single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (referred to as the A, A', B, B', C, C', and D regions), which allow intrastrand base pairing to occur within this portion of the ITR.

A "terminal resolution sequence" or "trs" is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is resistant to cleavage by AAV rep proteins.

A "helper virus" for AAV refers to a virus that enables AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. Several such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia virus. Adenoviruses encompass several different subgroups, although adenovirus type 5 subgroup C (Ad5) is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and are available from repositories such as the ATCC. Viruses in the herpes family, which are also available from repositories such as the ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV).

"Percent (%) sequence identity" to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be accomplished in a variety of ways within the skill of the art, e.g., by using publicly available computer programs, e.g., those described in Current Protocols in Molecular Biology (Ausubelet et al., eds., 1987), Supp. 30, section 7.7.18, table 7.7.1, and including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. For the purposes of this document, the % amino acid sequence identity of a given amino acid sequence A with respect to, with, or versus a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a given % amino acid sequence identity with respect to, with, or versus a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues judged to be identical matches by the sequence alignment program in the alignment of A and B of that program, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. For the purposes of this document, the % nucleic acid sequence identity of a given nucleic acid sequence C with respect to, with, or versus a given amino acid sequence B ... given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity to, with or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z, where W is the number of nucleotides judged to be identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid sequence identity of D to C.

An "isolated" molecule (e.g., nucleic acid or protein) or cell means that it has been identified and separated and/or recovered from a component of its natural environment.

An "effective amount" is an amount sufficient to achieve beneficial or desired results, including clinical outcomes (e.g., symptom improvement, attainment of clinical endpoints, and the like). An effective amount can be administered in one or more administrations. In terms of a pathology, an effective amount is an amount sufficient to ameliorate, stabilize, or delay the development of a disease. For example, an effective amount of an rAAV particle expresses a desired amount of heterologous nucleic acid, such as a therapeutic polypeptide or therapeutic nucleic acid.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

As used herein, "treatment" is a strategy for obtaining beneficial or desired clinical outcomes. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, decrease in the extent of the disease, stabilized state (e.g., no worsening) of the disease, prevention of spread (e.g., metastasis) of the disease, delay or slowing of disease progression, improvement or palliation of pathology, and remission (either partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if untreated.

The term "trabecular meshwork," as used herein, refers to a sponge-like tissue located near the cornea and iris that functions to drain aqueous humor from the eye into the blood. A sponge-like tissue located near the cornea and iris that functions to drain aqueous humor from the eye into the blood. The trabecular meshwork contains endothelium-lined spaces (the intertrabecular spaces) through which aqueous humor passes into Schlemm's canal. It is normally divided into two parts: the sclerocorneal meshwork, which contacts the cornea and sclera and opens into Schlemm's canal, and the uveal meshwork, which faces the anterior chamber.

The term "central retina," as used herein, refers to the outer macula and/or inner macula and/or the fovea. The term "central retinal cell types," as used herein, refers to cell types of the central retina, such as, for example, RPE and photoreceptor cells.

The term "macula" refers to the region of the central retina in primates that contains a higher relative concentration of photoreceptor cells, specifically rods and cones, compared to the peripheral retina. The term "outer macula," as used herein, may also be referred to as the "peripheral macula." The term "inner macula," as used herein, may also be referred to as the "central macula."

The term "fovea" refers to a small region in the central retina of primates approximately equal to or less than 0.5 mm in diameter that contains a higher relative concentration of photoreceptor cells, specifically cones, when compared to the peripheral retina and macula.

The term "subretinal space," as used herein, refers to the location in the retina between the photoreceptor cells and the retinal pigment epithelial cells. The subretinal space may be a potential space, such as prior to any subretinal fluid injection. The subretinal space may also contain a fluid that is injected into the potential space. In this case, the fluid is "in contact with the subretinal space." Cells that are "in contact with the subretinal space" include cells that border the subretinal space, such as RPE cells and photoreceptors.

The term "bleb," as used herein, refers to a fluid space within the subretinal space of an eye. A bleb of the disclosure may be created by a single injection of fluid into a single space, by multiple injections of one or more fluids into the same space, or by multiple injections into multiple spaces, which when repositioned create a total fluid space useful for achieving a therapeutic effect on the desired portion of the subretinal space.

"Chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from a chicken β-actin gene (e.g., Gallus gallus beta actin, represented by GenBank Entrez Gene ID 396526). As used herein, "chicken β-actin promoter" may refer to a promoter containing a cytomegalovirus (CMV) early enhancer element, the promoter and the first exon and intron of the chicken β-actin gene, and the splice acceptor of the rabbit beta-globin gene, such as the sequences described in Miyazaki, J., et al. (1989) Gene 79(2):269-77. As used herein, the term "CAG promoter" may be used interchangeably. As used herein, the term "CMV early enhancer/chicken beta actin (CAG) promoter" may be used interchangeably.

"Myocilin (MYOC)" refers to a protein (or gene encoding such a protein) involved in cytoskeletal function, cell adhesion, cell signaling, and cell migration, also known as trabecular meshwork-inducible glucocorticoid response (GIFR), GPOA, TIGR, GLC1A, JOAG, and JOAG1. Myocilin is expressed as a secreted protein by many different cell types. In the eye, myocilin is thought to be secreted into the aqueous humor by the trabecular meshwork, a tissue that is critical in the regulation of intraocular pressure (IOP). As described above, mutations in myocilin are thought to account for a subset of primary cases of open-angle glaucoma, particularly the juvenile form of the disorder.

As used herein, "myocilin" may refer to a full-length precursor as well as any processed form of the protein (e.g., a mature protein secreted from a cell). Examples of myocilin proteins may include, but are not limited to, human, mouse, dog, and cat myocilin, e.g.,

NCBI reference sequences NP_000252, NP_034995, NP_001041495, and NP_001265779. Examples of myocilin genes may include, but are not limited to, human, mouse, dog, and cat myocilin genes, e.g., GenBank Entrez Gene ID 4653 (MYOC, also known as GPOA, JOAG, TIGR, GLC1A, and JOAGl), GenBank Entrez Gene ID 17926 (Myoc, also known as TIGR, GLC1A, and AI957332), GenBank Entrez Gene ID 490344, and GenBank Entrez Gene ID 101087632.

"R-spondin 1 (RSPO1)" refers to a member of the R-spondin family involved in the modulation of Wnt signaling. The term "RSPO1" can refer to either an RSPO1 protein or a gene encoding an RSPO1 protein. Members of a superfamily of thrombospondin type 1 (TSR-1) repeat-containing proteins, R-spondins include a signal peptide, a TSR-1 domain, and two furin-like repeats. Although the exact mechanism is unclear, R-spondin family polypeptides are thought to activate Wnt signaling. For further discussion of the connections between R-spondins and Wnt signaling, see, e.g., Kim, K.A. et al. (2006) Cell Cycle 5:23-26; Kim, K.A. et al. (2008) Mol. Biol. Cell. 19:2588-2596; Jin, Y.R. and Yoon, J.K. (2012) Int. J. Biochem. Cell Biol 44:2278-2287; and de Lau, W.B., et al. (2012) Genome Biol. 13(3):242.

As used herein, "RSPO1" can refer to a full-length precursor as well as any processed form of the protein (e.g., a mature protein secreted from a cell). Examples of RSPO1 proteins can include, but are not limited to, human, mouse, dog, and cat RSPO1, e.g., NCBI reference sequences NP_001229837, NP_619624, XP_00562890, and XP_003989918. Examples of RSPO1 genes may include, without limitation, human, mouse, dog, and cat RSPO1 genes, e.g., GenBank Entrez Gene ID 284654 (RSPO1, also known as Rs Po and CRISTIN3), GenBank Entrez Gene ID 192199 (Rspol, also known as Rspondin and R-spondin), GenBank Entrez Gene ID 608179, and GenBank Entrez Gene ID 101091033. In some embodiments, RSPO1 is a functional variant of an RSPO1. In some embodiments, a functional variant of RSPO1 may include one or more amino acid substitutions, insertions, and/or deletions (e.g., truncations), but retain some or all activity with respect to one or more activities of the full-length RSPO1 (e.g., Wnt signaling activity, assays for which are described and/or exemplified herein). In some embodiments, the functional variant of RSPO1 is a truncated RSPO1. Examples of truncated RSPO1 polypeptides include, but are not limited to, SEQ ID NO: 11 and 12, or processed forms of SEQ ID NO: 11 and 12 lacking the signal peptide.

"R-spondin 2 (RSPO2)" refers to a member of the R-spondin family involved in the modulation of Wnt signaling. The term "RSPO2" can refer to either an RSPO2 protein or a gene encoding an RSPO2 protein. Members of a superfamily of thrombospondin type 1 (TSR-1) repeat-containing proteins, R-spondins include a signal peptide, a TSR-1 domain, and two furin-like repeats. Although the exact mechanism is unclear, R-spondin family polypeptides are thought to activate Wnt signaling. For further discussion of the connections between R-spondins and Wnt signaling, see, e.g., Kim, K.A. et al. (2006) Cell Cycle 5:23-26; Kim, K.A. et al. (2008) Mol. Biol. Cell. 19:2588-2596; Jin, Y.R. and Yoon, J.K. (2012) Int. J. Biochem. Cell Biol 44:2278-2287; and de Lau, W.B., et al. (2012) Genome Biol. 13(3):242.

As used herein, “RSPO2” can refer to a full-length precursor as well as any processed form of the protein (e.g., a mature protein secreted from a cell). Examples of RSPO2 proteins can include, but are not limited to, human, mouse, dog, and cat RSPO2, e.g., NCBI reference sequences<n>P_848660, NP_766403, XP_005627927, and XP_004000104. Examples of RSPO2 genes may include, but are not limited to, human, mouse, dog, and cat RSPO2 genes, e.g., GenBank Entrez Gene ID 340419 (RSPO2, also known as CRISTIN2), GenBank Entrez Gene<i>D 239405 (Rspo2, also known as .ftls, AA673245, D430027K22, and 2610028F08Rik), GenBank Entrez Gene ID 482004, and GenBank Entrez Gene ID 101087380. In some embodiments, RSPO2 is a functional variant of an RSPO2. In some embodiments, a functional variant of RSPO2 may include one or more amino acid substitutions, insertions, and/or deletions (e.g., truncations), but retain some or all activity relative to one or more activities of the full-length RSPO2 (e.g., Wnt signaling activity, assays for which are described and/or exemplified herein). In some embodiments, the functional variant of RSPO2 is a truncated RSPO2. Examples of truncated RSPO2 polypeptides include, without limitation, SEQ ID NO: 13 and 14, or processed forms of SEQ ID NO: 13 and 14 lacking the signal peptide.

"R-spondin 3 (RSPO3)" refers to a member of the R-spondin family involved in the modulation of Wnt signaling. The term "RSPO3" can refer to either an RSPO3 protein or a gene encoding an RSPO3 protein. Members of a superfamily of thrombospondin type 1 (TSR-1) repeat-containing proteins, R-spondins include a signal peptide, a TSR-1 domain, and two furin-like repeats. Although the exact mechanism is unclear, RSPO3 is thought to activate Wnt signaling, and loss of RSPO3 function in mice and Xenopus results in Wnt loss-of-function phenotypes (Kazanskaya, O., et al. (2008) Development 135:3655-64). For further discussion of the connections between R-spondins and Wnt signaling, see, e.g., De Lau, W.B., et al. (2012) Genome Biol. 13(3):242.

As used herein, "RSPO3" can refer to a full-length precursor as well as any processed form of the protein (e.g., a mature protein secreted from a cell). Examples of RSPO3 proteins can include, but are not limited to, human, mouse, dog, and cat RSPO3, e.g., NCBI reference sequences NP_116173, NP_082627, XP_005615677, and XP_003986583.

Examples of RSPO3 genes may include, but are not limited to, human, mouse, dog, and cat RSPO3 genes, for example, GenBank Entrez Gene ID 84870 (RSPO3, also known as PWTSR, THSD2, and CRISTIN1), GenBank Entrez Gene ID 72780 (Rspo3, also known as Thsd2, Cristinl, AW742308, and 2810459H04Rik), GenBank Entrez Gene ID 476287, and GenBank Entrez Gene ID 101085635. In some instances, RSPO3 is a functional variant of an RSPO3. In some cases, a functional RSPO3 variant may include one or more amino acid substitutions, insertions, and/or deletions (e.g., truncations), but retain some or all activity relative to one or more activities of the full-length RSPO3 (e.g., Wnt signaling activity, for which assays are described and/or exemplified herein). In some cases, the functional RSPO3 variant is a truncated RSPO3. Examples of truncated RSPO3 polypeptides include, without limitation, SEQ ID NOs: 15-17, or processed forms of SEQ ID NOs: 15-17 lacking the signal peptide.

"R-spondin 4 (RSPO4)" refers to a member of the R-spondin family involved in the modulation of Wnt signaling. The term "RSPO4" can refer to either an RSPO4 protein or a gene encoding an RSPO4 protein. Members of a superfamily of thrombospondin type 1 (TSR-1)-containing proteins, R-spondins include a signal peptide, a TSR-1 domain, and two furin-like repeats. Although the exact mechanism is unclear, R-spondin family polypeptides are thought to activate Wnt signaling. For further discussion of the connections between R-spondins and Wnt signaling, see, e.g., Kim, K.A. et al. (2006) Cell Cycle 5:23-26; Kim, K.A. et al. (2008) Mol. Biol. Cell. 19:2588-2596; Jin, Y.R. and Yoon, J.K. (2012) Int. J. Biochem. Cell Biol 44:2278-2287; and de Lau, W.B., et al. (2012) Genome Biol. 13(3):242.

As used herein, “RSPO4” can refer to a full-length precursor as well as any processed form of the protein (e.g., a mature protein secreted from a cell). Examples of RSPO4 proteins can include, but are not limited to, human, mouse, dog, and cat RSPO4, e.g., NCBI reference sequences NP_001025042, NP_001035779, XP_542937, and XP_011279253. Examples of RSPO4 genes may include, but are not limited to, human, mouse, dog, and cat RSPO4 genes, e.g., GenBank Entrez Gene ID 343637 (RSPO4, also known as CRISTIN4 and C20orf182), GenBank Entrez Gene ID 228770 (Rspo4, also known as A730099F22 and A930029K19Rik), GenBank Entrez Gene ID 485813, and GenBank Entrez Gene ID 101091527. In some embodiments, RSPO4 is a functional variant of an RSPO4. In some embodiments, a functional variant of RSPO4 may include one or more amino acid substitutions, insertions, and/or deletions (e.g., truncations), but retain some or all of the activity relative to one or more activities of the full-length RSPO4 (e.g., Wnt signaling activity, assays for which are described and/or exemplified herein). In some embodiments, the functional variant of RSPO4 is a truncated RSPO4. Examples of truncated RSPO4 polypeptides include, but are not limited to

SEQ ID NO: 18 and 19, or processed forms of SEQ ID NO: 18 and 19 lacking the signal peptide.

As used herein, "RNA interference (RNAi)" is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Examples of RNAi include small inhibitory RNA (siRNA), microRNA (miRNA), and small hairpin RNA (shRNA).

As used herein, a "small hairpin RNA" or "short hairpin RNA" (shRNA) is an RNA molecule that makes a tight hairpin turn that can be used to silence target gene expression; for example, by RNA interference.

"Wnt signaling" refers to a group of related cellular signaling pathways that are regulated by the interaction between a Wnt protein and a Frizzled (Fz) family receptor (for review, see, e.g., Logan, C.Y., & Nusse, R. (2004) Annu. Rev. Cell Dev. Biol. 20:781-810). These pathways have been implicated in a wide range of pathogenic and developmental processes. As used herein, unless otherwise specified, the term "Wnt signaling" may refer to part or all of the canonical Wnt pathway, the Wnt/planar cell polarity (PCP) pathway, and/or the Wnt/calcium pathway. For example, in the canonical Wnt pathway, binding of Wnt to the Frizzled/LRP receptor complex results in the modulation of Dishevelled (DSH), Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase (GSK-3) activity, ultimately inhibiting beta-catenin degradation. Beta-catenin is then able to translocate to the nucleus and regulate gene transcription, e.g., in conjunction with lymphoid enhancer-binding factor 1/T cell-specific transcription factor (LEF/TCF) transcription factors. In some embodiments, beta-catenin activity can be assayed as a readout for Wnt signaling (e.g., by a TOP-Flash assay, such as characterized in Molenaar, M., et al. (1996) Cell 86(3):391-9).

A reference to "approximately" a value or parameter in this document includes (and describes) embodiments that refer to that value or parameter per se. For example, a description that refers to "approximately X" includes a description of "X".

As used herein, the singular forms of the articles "un/o", "una" and "el/la" include plural references unless otherwise indicated.

The aspects and embodiments of the invention described herein are understood to include "comprising", "consisting of" and/or "consisting essentially of" aspects and embodiments.

III. Treatment methods

The invention relates to gene therapy for myocilin glaucoma (MYOC), wherein rAAV particles comprising therapeutic vectors are administered to the eye of a mammal. The rAAV particle for use in the invention comprises a vector encoding RSPO1, RSPO2, or RSPO4, wherein RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein the promoter is capable of expressing RSPO1, RSPO4, or RSPO4 in the mammalian eye and/or in trabecular meshwork cells. In some embodiments, myocilin glaucoma (MYOC) is primary open angle glaucoma (POAG). In some embodiments, myocilin glaucoma (MYOC) is the juvenile form of primary open angle glaucoma (JOAG). In some embodiments, the mammal is a human (e.g., a human with POAG or a human with JOAG). In some embodiments, the mammal with myocilin glaucoma (MYOC) has a mutated MYOC. In some embodiments, the mutated MYOC comprises one or more amino acid substitutions corresponding to E323K, K398R, Q368X, G364V, P370L, D380A, K423E, Y437H, and I477S of human MYOC. In some embodiments, the mutated MYOC gene comprises one or more amino acid substitutions corresponding to P370L and/or Y437H amino acid substitutions of human MYOC. In some embodiments, the invention provides methods of treating myocilin-like glaucoma (MYOC) in a human, comprising administering to the eye of the human an effective amount of rAAV particles comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof and/or MYOC RNAi (e.g., shRNA). The rAAV particle for use in the invention comprises a vector encoding RSPO1, RSPO2, or RSPO4. In some embodiments, the rAAV particle for use in the invention is used to reduce a symptom of myocilin-like glaucoma (MYOC) in a mammal; for example, reducing intraocular pressure, reducing MYOC accumulation in the trabecular meshwork, reducing ocular hypertension, or increasing aqueous outflow from the trabecular meshwork.

In some embodiments, the disclosure provides methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof. In some embodiments, the disclosure provides methods of enhancing Wnt signaling in trabecular meshwork cells in a mammal having an ocular disorder, comprising administering to the eye of the mammal a recombinant adeno-associated virus (rAAV) particle comprising a vector encoding a MYOC RNAi that targets expression of a myocilin (MYOC) in the mammal. In some embodiments, Wnt signaling is enhanced using one or more viral particles expressing RSPO1, RSPO2, RSPO3, rSpO4, or a functional variant thereof and/or MYOC RNAi; For example, RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof and the MYOC Rn can be expressed from rAAV vectors with different recombinant viral genomes or from the same rAAV viral genome.

Therapeutic vectors

The invention relates to gene therapy for myocilin glaucoma (MYOC), wherein rAAV particles comprising therapeutic vectors are administered to the eye of a mammal; for example, the therapeutic vector may encode a therapeutic nucleic acid and/or a therapeutic polypeptide. The rAAV particle for use in the invention comprises a vector encoding RSPO1, RSPO2, or RSPO4. A therapeutic AAV vector encoding a therapeutic nucleic acid and/or therapeutic polypeptide can be generated using methods known in the art, using standard synthetic and recombinant methods. In some cases, the therapeutic polypeptide is a polypeptide that stimulates Wnt signaling. In some cases, the therapeutic polypeptide stimulates Wnt signaling in the presence of a mutant MYOC. In some cases, the therapeutic polypeptide stimulates Wnt signaling in the presence of a mutant human MYOC. In some cases, the therapeutic polypeptide stimulates Wnt signaling in the presence of a mutant human MYOC associated with glaucoma. In some cases, the mutant MYOC comprises a P370L and/or Y437H amino acid substitution. In some cases, the mutated MYOC comprises one or more amino acid substitutions corresponding to E323K, K398R, Q368X, G364V, P370L, D380A, K423E, Y437H, and I477S of human MYOC.

In some embodiments, the disclosure provides rAAV vectors for the treatment of myocilin glaucoma (MYOC), wherein the rAAV vectors encode an R-spondin (RSPO) polypeptide (e.g., RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof). In some embodiments, the RSPO1 polypeptide is a human RSPO1. In some embodiments, the RSPO1 comprises the amino acid sequence of SEQ ID NO:8, or a functional variant thereof. An example of a functional RSPO1 variant includes an RSPO1 having one or more amino acid substitutions, additions, and/or deletions from the amino acid sequence of SEQ ID NO: 8. In some embodiments, the RSPO1 variant comprises one, two, three, four, five, six, seven, eight, nine, ten, or more than 10 substitutions, additions, and/or deletions from the amino acid sequence of SEQ ID NO: 8 while maintaining the ability to stimulate Wnt signaling (e.g., in the presence of a mutant MYOC). In some embodiments, the RSPO1 variant has greater than about 80%, 85%, <90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8. In some embodiments, the RSPO1 is a truncated RSPO1. In some embodiments, the truncated RSPO1 may include one or more furin-like Cys-rich domains (e.g., FU1 and/or FU2), but lack one or more of: a signal peptide, a thrombospondin type 1 domain (e.g., TSR-1 or TSP1), and/or a positively charged carboxy-terminal domain (e.g., including a bipartite NLS and/or BR domain; for reference, see FIGS. 11-13C).

In certain embodiments, the truncated RSPO1 may comprise SEQ ID NO: 11 and/or 12, or processed forms of SEQ ID NO: 11 and/or 12 lacking the signal peptide. In certain embodiments, the truncated RSPO1 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11 and/or 12. In some embodiments, the RSPO2 polypeptide is a human RSPO2. In some embodiments, the RSPO2 comprises the amino acid sequence of SEQ ID NO:9, or a functional variant thereof. An example of a functional RSPO2 variant includes an RSPO2 having one or more amino acid substitutions, additions, and/or deletions from the amino acid sequence of SEQ ID NO:9. In some embodiments, the RSPO2 variant comprises one, two, three, four, five, six, seven, eight, nine, ten, or more than 10 substitutions, additions, and/or deletions from the amino acid sequence of SEQ ID NO:9, while maintaining the ability to stimulate Wnt signaling (e.g., in the presence of a mutant MYOC). In some embodiments, the RSPO2 variant has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:9. In some embodiments, RSPO2 is a truncated RSPO2. In some embodiments, the truncated RSPO2 may include one or more furin-like Cys-rich domains (e.g., FU1 and/or FU2), but lack one or more of: a signal peptide, a thrombospondin type 1 domain (e.g., TSR-1 or TSP1), and/or a positively charged carboxy-terminal domain (e.g., including a bipartite NLS and/or BR domain; for reference, see FIGS. 11-13C). In certain embodiments, the truncated RSPO2 may comprise SEQ ID NO: 13 and/or 14, or processed forms of SEQ ID NO: 13 and/or 14 lacking the signal peptide. In certain embodiments, the truncated RSPO2 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13 and/or 14. In some embodiments, the RSPO3 polypeptide is a human RSPO3. In some embodiments, RSPO3 comprises the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof. An example of a functional RSPO3 variant includes an RSPO3 having one or more amino acid substitutions, additions, and/or deletions from the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RSPO3 variant comprises one, two, three, four, five, six, seven, eight, nine, ten, or more than 10 substitutions, additions, and/or deletions from the amino acid sequence of SEQ ID NO: 1 while maintaining the ability to stimulate Wnt signaling (e.g., in the presence of a mutant MYOC). In some embodiments, the RSPO3 variant has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. In some embodiments, RSPO3 is a truncated RSPO3. In some embodiments, the truncated RSPO3 may include one or more furin-like Cys-rich domains (e.g., FU1 and/or FU2), but lack one or more of: a signal peptide, a thrombospondin type 1 domain (e.g., TSR-1 or TSP1), and/or a positively charged carboxy-terminal domain (e.g., including a bipartite NLS and/or BR domain; for reference, see FIGS. 11-13C). In certain embodiments, the truncated RSPO3 may comprise SEQ ID NO: 15, 16, and/or 17, or processed forms of SEQ ID NO: 15, 16, and/or 17 lacking the signal peptide. In certain instances, the truncated RSPO3 has more than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15,

16 and/or 17. In some embodiments, the RSPO4 polypeptide is a human RSPO4. In some embodiments, the RSPO4 comprises the amino acid sequence of SEQ ID NO:9, or a functional variant thereof. An example of a functional RSPO2 variant includes an RSPO2 having one or more amino acid substitutions, additions, and/or deletions of the amino acid sequence of SEQ ID NO:10. In some embodiments, the RSPO4 variant comprises one, two, three, four, five, six, seven, eight, nine, ten, or more than 10 substitutions, additions, and/or deletions of the amino acid sequence of SEQ ID NO:10 while maintaining the ability to stimulate Wnt signaling (e.g., in the presence of a mutant MYOC). In some embodiments, the RSPO4 variant has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, <95%, 96%, 97%, 98%, or 99% identity to SEQ>I<d NO: 10. In some embodiments, RSPO4 is a truncated RSPO4>. In some embodiments, the truncated RSPO4 may include one or more furin-like Cys-rich domains (e.g., FU1 and/or FU2), but lack one or more of: a signal peptide, a thrombospondin type 1 domain (e.g., TSR-1 or TSP1), and/or a positively charged carboxy-terminal domain (e.g., including a bipartite NLS and/or BR domain; for reference, see FIGS. 11-13C). In certain embodiments, the truncated RSPO4 may comprise SEQ ID NO: 18 and/or 19, or processed forms of SEQ ID NO: 18 and 19 lacking the signal peptide. In certain embodiments, the truncated RSPO4 has greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18 and/or 19.

In some embodiments, the rAAV vector comprises nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof operably linked to a promoter. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof in the mammalian eye. In some embodiments, the promoter is capable of expressing RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof in trabecular meshwork cells. In the rAAV particle of the invention, RSPO1, RSPO2, or RSPO4 is operably linked to a promoter wherein the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the mammalian eye and/or in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin (CBA) hybrid promoter.

The disclosure provides methods of gene therapy for myocilin-like glaucoma (MYOC), wherein rAAV particles comprising therapeutic vectors are administered to the eye of a mammal; for example, the therapeutic vector may encode a therapeutic nucleic acid and/or a therapeutic polypeptide. A therapeutic AAV vector encoding a therapeutic nucleic acid and/or therapeutic polypeptide can be generated using methods known in the art, using standard synthetic and recombinant methods. In some cases, the therapeutic nucleic acid encodes an RNA that targets the expression of MYOC. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits the expression of MYOC. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits the expression of a mutant

MYOC. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits the expression of a mutant human MYOC. In some cases, the mutant human MYOC comprises a P370L amino acid substitution and/or a Y437 amino acid substitution. Non-limiting examples of therapeutic nucleic acids include RNAi, small inhibitory RNA (siRNA), microRNA (miRNA), small hairpin RNA (shRNA), and/or ribozymes (such as hammerhead and hairpin ribozymes). In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits the expression of MYOC or a shRNA that reduces or inhibits the expression of MYOC (e.g., wild-type and mutant MYOC).

In some cases, the disclosure provides methods of gene therapy for myocilin-like glaucoma (MYOC), wherein rAAV particles comprising therapeutic vectors are administered to the eye of a mammal, wherein the vectors comprise nucleic acid encoding one or more therapeutic polypeptides. The rAAV particles comprising therapeutic vectors can be generated using methods known in the art, using standard synthetic and recombinant methods. In some cases, the vector encodes a therapeutic polypeptide. In some cases, the therapeutic polypeptide targets Wnt signaling. In some cases, the therapeutic polypeptide stimulates Wnt signaling.

In some cases, the disclosure provides methods of gene therapy for myocilin glaucoma (MYOC), wherein rAAV particles comprising therapeutic vectors are administered to the eye of a mammal, wherein the vectors comprise nucleic acid encoding one or more therapeutic polypeptides and one or more therapeutic nucleic acids. The rAAV particles comprising therapeutic vectors can be generated using methods known in the art, using standard synthetic and recombinant methods. In some cases, the therapeutic polypeptide targets Wnt signaling and the therapeutic nucleic acid targets MYOC expression. In some cases, the therapeutic polypeptide stimulates Wnt signaling. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits MYOC expression. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits the expression of a mutant MYOC. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits the expression of a mutant human MYOC. In some embodiments, the mutant human MYOC comprises a P370L amino acid substitution and/or a Y437 amino acid substitution. Non-limiting examples of therapeutic nucleic acids include RNAi, siRNA, miRNA, shRNA, and/or ribozymes.

In some cases, the disclosure provides methods of gene therapy for myocilin glaucoma (MYOC) in a mammal, wherein rAAV particles comprising vectors encoding one or more therapeutic polypeptides are administered to the mammal and rAAV particles comprising vectors encoding one or more therapeutic nucleic acids are administered to the mammal. In some cases, the therapeutic polypeptide targets Wnt signaling and the therapeutic nucleic acid targets MYOC expression. In some cases, the therapeutic polypeptide stimulates Wnt signaling. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits MYOC expression. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits expression of a mutant MYOC. In some cases, the heterologous nucleic acid encodes an RNA that reduces or inhibits expression of a mutant human MYOC. In some embodiments, the mutant human MYOC comprises a P370L amino acid substitution and/or a Y437 amino acid substitution. Non-limiting examples of therapeutic nucleic acid include RNAi, siRNA, miRNA, shRNA, and/or ribozymes. The rAAV particles comprising vectors encoding one or more therapeutic polypeptides and the rAAV particles comprising vectors encoding one or more therapeutic nucleic acids can be administered to the mammal simultaneously or sequentially. In some embodiments, the rAAV particles comprising vectors encoding one or more therapeutic polypeptides are administered before the rAAV particles comprising vectors encoding one or more therapeutic nucleic acids are administered. In some embodiments, the rAAV particles comprising vectors encoding one or more therapeutic polypeptides are administered after the rAAV particles comprising vectors encoding one or more therapeutic nucleic acids are administered.

The nucleic acids of the invention may encode polypeptides that are intracellular proteins, anchored to the cell membrane, remain within the cell, or are secreted by the cell transduced with the vectors of the invention. For polypeptides secreted by the cell receiving the vector; preferably the polypeptide is soluble (i.e., not bound to the cell). For example, soluble polypeptides are devoid of a transmembrane region and are secreted from the cell. Techniques for identifying and eliminating nucleic acid sequences encoding transmembrane domains are known in the art.

Vectors that can be administered in accordance with the present disclosure also include vectors comprising a nucleic acid encoding an RNA (e.g., shRNA, RNAi, ribozymes, miRNA, siRNA, antisense RNA) that, when transcribed from the vector nucleic acids, can treat myocilin glaucoma (MYOC) by interfering with the translation or transcription of an abnormal or excess protein associated with a disease state of the invention; e.g., MYOC. In some examples, the nucleic acids of the disclosure can encode an RNA that treats a disease by highly specific elimination or reduction of mRNA encoding the abnormal and/or excess proteins. Therapeutic RNA sequences include small hairpin RNA (shRNA), RNAi, small inhibitory RNA.

(siRNA), microRNA (miRNA), and/or ribozymes (such as hammerhead and hairpin ribozymes) that can treat diseases by highly specific elimination or reduction of mRNA encoding abnormal and/or excess proteins, such as those that occur in various forms of inherited retinal degeneration. Examples of therapeutic RNA sequences and nucleic acids encoding these sequences that can be used in the invention include those described, for example, in U.S. Patent No. 6,225,291.

In some embodiments of the invention, the therapeutic RNA sequence is an RNAi sequence (e.g., shRNA) that targets MYOC expression. In some embodiments, the expression-targeting MYOC RNAi sequence (e.g., shRNA) is an RNAi sequence (e.g., shRNA) that reduces or inhibits MYOC expression. In some embodiments, the RNAi (e.g., shRNA) reduces or inhibits expression of a human MYOC. In some embodiments, the RNAi (e.g., shRNA) reduces or inhibits expression of a MYOC comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the MYOC RNAi (e.g., shRNA) targets an amino acid sequence of QAMSVIH (SEQ ID NO:6) of MYOC. In some embodiments, the rAAV particles encode a vector comprising more than one RNAi (e.g., shRNA) that targets (e.g., reduces or inhibits) MYOC expression. In some embodiments, the loop sequence of the MYOC RNAi (e.g., shRNA) comprises the nucleic acid sequence AATAGTGAAGCCACAGATGTATT (SEQ ID NO:7). In some embodiments, the rAAV particles encode a vector comprising one, two, three, four, five, or more RNAi (e.g., shRNA) that targets (e.g., reduces or inhibits) MYOC expression.

In some embodiments, the rAAV vector comprises nucleic acid encoding a MYOC RNAi (e.g., shRNA) operably linked to a promoter. In some embodiments, the promoter is capable of expressing the MYOC RNAi (e.g., shRNA) in the eye of the mammal. In some embodiments, the promoter is capable of expressing the MYOC RNAi (e.g., shRNA) in trabecular meshwork cells. In some embodiments, the promoter is a chicken β-actin hybrid (CBA) promoter. In some embodiments, the promoter is an RNA polymerase III promoter.

In some embodiments, the rAAV vector comprises nucleic acid encoding any RSPO1, RSPO2, RSPO3, RSPO4, or functional variant thereof as described herein and nucleic acid encoding any MYOC RNAi (e.g., shRNA) as described herein. In some embodiments, the nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and the nucleic acid encoding MYOC RNAi (e.g., shRNA) are in different rAAV genomes. In some embodiments, the nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and the nucleic acid encoding MYOC RNAi (e.g., shRNA) are in the same rAAV genome. In some embodiments, the nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and the nucleic acid encoding MYOC RNAi (e.g., shRNA) are operably linked to the same promoter. In some embodiments, the nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and the nucleic acid encoding MYOC RNAi (e.g., shRNA) are operably linked to different promoters. In some embodiments, the nucleic acid encoding the RSPO1, RSPO2, RSPO3, RSPO4, or functional variant thereof is 5' to the nucleic acid encoding the MYOC RNAi (e.g., shRNA). In some embodiments, the nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof is 3' to the nucleic acid encoding the MYOC RNAi (e.g., shRNA). In some embodiments, the nucleic acid encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and the nucleic acid encoding MYOC RNAi (e.g., shRNA) are operably linked to the same promoter, wherein the nucleic acid includes an internal ribosome entry site (IRES) of the RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and the MYOC RNAi nucleic acids (e.g., shRNA).

rAAV compositions

In some embodiments, the disclosure provides compositions comprising any of the rAAV particles described herein. The rAAV particle for use in the invention may be in a pharmaceutical composition. Generally, compositions for use in the methods and systems of the disclosure comprise an effective amount of rAAV particles comprising rAAV vectors encoding a polypeptide and/or RNA, preferably in a pharmaceutically acceptable excipient. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and may be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use. For example, an excipient may provide shape or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for different osmolarities, encapsulating agents, pH buffering substances, and buffers. Such excipients include any pharmaceutical agent suitable for direct administration to the eye that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included therein, for example, salts of mineral acids such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like. A comprehensive discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

Generally, these compositions are formulated for administration by ocular injection (e.g., intravitreal, intracameral, subretinal). Accordingly, these compositions are preferably combined with pharmaceutically acceptable carriers, such as saline, Ringer's balanced salt solution (pH 7.4), and the like. Although not required, the compositions may optionally be administered in unit dosage form suitable for administration of a precise quantity.

In some cases, the disclosure provides rAAV pharmaceutical formulations for the treatment of myocilin glaucoma (MYOC). In some cases, the formulation comprises rAAV particles comprising a rAAV vector encoding an RSPO1, RSPO2, RSPO3, and/or RSPO4 polypeptide, or a functional variant thereof. In some cases, the formulation comprises rAAV particles comprising a rAAV vector encoding a MYOC RNAi (e.g., shRNA). In some cases, the formulation comprises rAAV particles comprising a rAAV vector encoding an RSPO1, RSPO2, RSPO3, and/or RSPO4 polypeptide, or a functional variant thereof, and a MYOC RNAi (e.g., shRNA). In some embodiments, the formulation comprises rAAV particles comprising a rAAV vector encoding an RSPO1, RSPO2, RSPO3, and/or RSPO4 polypeptide, or a functional variant thereof, and rAAV particles comprising a rAAV vector encoding a MYOC RNA (e.g., shRNA).

Ocular delivery methods of rAAV

In some aspects, the invention relates to the treatment of myocilin glaucoma (MYOC) in a mammal, the method comprising administering rAAV particles to the eye of the mammal. In some embodiments disclosed herein, the rAAV particles comprise a rAAV vector encoding an RSPO1, RSPO2, RSPO3, and/or RSPO4 polypeptide, or a functional variant thereof, and/or a rAAV vector encoding a MYOC RNAi (e.g., shRNA). In the invention, the rAAV particle comprises a vector encoding RSPO1, RSPO2, or RSPO4. In some embodiments, the rAAV particles are administered to the eye by intravitreal and/or intracameral injection. Methods of administering rAAV particles to the eye are known in the art.

In some embodiments, rAAV particles comprising rAAV vectors encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and/or MYOC RNAi (e.g., shRNA) are administered to the eye of a mammal where RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, and/or MYOC RNAi (e.g., shRNA) are expressed in the trabecular meshwork of the eye. In some embodiments, rAAV particles comprising rAAV vectors encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof, are administered to the eye of a mammal where other parts of the eye (e.g., retinal ganglion cells) are transduced. The use of rAAV particles comprising the AAV2 capsid comprising an R471A amino acid substitution may facilitate transduction of trabecular meshwork cells.

By safely and effectively transducing ocular cells (e.g., trabecular meshwork cells) with a vector comprising a therapeutic polypeptide or nucleic acid sequence, the methods of the disclosure can be used to treat an individual; e.g., a human, having myocilin glaucoma (MYOC), wherein the transduced cells produce the therapeutic polypeptide or RNA sequence in an amount sufficient to treat the myocilin glaucoma (e.g., POAG or JOAG). In some embodiments, ocular cell transduction is enhanced using rAAV2 particles comprising an R471A amino acid substitution of AAV capsid proteins, numbering based on VP1 of AAV2. In some embodiments, the rAAV particles demonstrate increased transduction of trabecular meshwork cells; for example, transduction of more than about 10%, 25%, 50%, 75%, 100%, or any number in between of trabecular meshwork cells.

An effective amount of rAAV (according to the invention, in particulate form) is administered depending on the treatment objectives. For example, when a low percentage of transduction can achieve the desired therapeutic effect, then the treatment objective in general is to meet or exceed this level of transduction. In some cases, this level of transduction can be achieved by transducing as few as about 1 to 5% of the target cells (e.g., trabecular meshwork cells), in some embodiments at least about 20% of the cells of the desired tissue type, in some embodiments at least about 50%, in some embodiments at least about 80%, in some embodiments at least about 95%, in some embodiments at least about 99% of the cells of the desired tissue type. As a guide, the number of particles administered per injection is generally between about 1 x 10 to about 1 x 10 particles, between about 1 x 10 to 1 x 10 particles, between about 1 x 10 to 1 x 10 particles, or about 1 x 10 particles, about 1 x 10 particles, or about 1 x 10 particles. The rAAV composition may be administered by one or more ocular injections, either during the same procedure or spaced by days, weeks, months, or years. In some embodiments, multiple vectors may be used to treat the human.

Methods for identifying ocular cells transduced by AAV viral particles are known in the art; for example, immunohistochemistry or the use of a marker, such as enhanced green fluorescent protein, can be used to detect transduction of viral particles; for example, viral particles comprising an rAAV capsid with one or more amino acid substitutions.

In some embodiments of the invention, the methods comprise intravitreal and/or intracameral administration of an effective amount of AAV viral particles to the mammal for treating an individual with myocilin glaucoma (MYOC); e.g., a human with POAG or JOAG. In some embodiments, the composition is injected into one or more locations in the eye to allow expression of a heterologous nucleic acid in cells of the eye (e.g., trabecular meshwork cells). In some embodiments, the composition is injected into any one of one, two, three, four, five, six, seven, eight, nine, ten, or more than ten locations in the eye.

In some embodiments, the rAAV viral particles comprising a rAAV capsid are administered to more than one location simultaneously or sequentially. In some embodiments, multiple injections of rAAV viral particles are spaced no more than one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours, or 24 hours apart.

Subretinal administration methods

Subretinal delivery methods are known in the art. For example, see patent document WO 2009/105690 . Briefly, the general method for delivering rAAV particles (e.g., rAAV2 particles) to the subretinal region of the macula and fovea can be illustrated by the following brief diagram. This example is intended merely to illustrate certain features of the method and is not intended to be limiting in any way.

In general, the rAAV vector can be administered as a composition injected intraocularly (subretinal) under direct observation using a surgical microscope. In some embodiments, the vector is encapsidated in an rAAV particle wherein the rAAV particle comprises an rAAV capsid comprising rAAV capsid proteins comprising one or more amino acid substitutions at one or more positions that interact with a heparan sulfate proteoglycan (e.g., reduces or inhibits or suppresses HSPG binding), and the rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat. This procedure may involve vitrectomy followed by injection of rAAV vector suspension using a fine cannula through one or more small retinotomies into the subretinal space.

Briefly, an infusion cannula can be sutured in place to maintain a normal globe volume by infusion (e.g., of saline) throughout the operation. A vitrectomy is performed using a cannula of appropriate bore size (e.g., 20- to 27-gauge), wherein the volume of vitreous gel that is removed is replaced by infusion of saline or other isotonic solution from the infusion cannula. Vitrectomy is advantageously performed because (1) removal of its cortex (the posterior hyaloid membrane) facilitates penetration of the retina by the cannula; (2) its removal and replacement with fluid (e.g., saline) creates space to accommodate intraocular injection of the vector, and (3) its controlled removal reduces the possibility of retinal tears and unplanned retinal detachment.

In some embodiments, the rAAV composition is injected directly into the subretinal space outside the central retina, using a cannula of the appropriate bore size (e.g., 27-45 gauge), thereby creating a bleb in the subretinal space. In other embodiments, the subretinal injection of the rAAV composition is preceded by the subretinal injection of a small volume (e.g., about 0.1 to about 0.5 ml) of an appropriate fluid (such as saline or Ringer's solution) into the subretinal space outside the central retina. This initial injection into the subretinal space establishes an initial fluid bleb within the subretinal space, causing a localized retinal detachment at the location of the initial bleb. This initial fluid bleb may facilitate targeted delivery of the rAAV composition to the subretinal space (by defining the injection plane prior to rAAV administration), and minimize potential delivery of rAAV into the choroid and the possibility of rAAV injection or reflux into the vitreous cavity.

In some embodiments, this initial fluid vial may be further injected with fluids comprising one or more rAAV compositions and/or one or more additional therapeutic agents by administering these fluids directly to the initial fluid vial with the same cannula or with additional fine bore cannulas.

Intraocular administration of the rAAV compositions and/or the small initial volume of fluid can be performed using a fine-bore cannula (e.g., 27-45 gauge) attached to a syringe. In some embodiments, the plunger of this syringe can be actuated by a mechanized device, such as by depressing a foot pedal. The fine-bore cannula is advanced through the sclerotomy, through the vitreous cavity, and into the retina at a predetermined site in each subject according to the area of the retina to be targeted (but outside the central retina). Under direct visualization, the vector suspension is mechanically injected under the neurosensory retina, causing a localized retinal detachment with a non-expandable, self-sealing retinotomy. As indicated above, the rAAV composition can be injected directly into the subretinal space, creating a bleb outside the central retina, or the vector can be injected into an initial bleb outside the central retina, causing it to expand (and expanding the area of retinal detachment). In some embodiments, the injection of the rAAV composition is followed by the injection of another fluid into the bleb.

Without wishing to be bound by theory, the rate and location of the subretinal injection(s) may result in localized shear forces that may damage the macula, fovea, and/or underlying RPE cells. Subretinal injections may be performed at a rate that minimizes or avoids shear forces. In some embodiments, the rAAV composition is injected over about 15-17 minutes. In some embodiments, the vector is injected over about 17-20 minutes. In some embodiments, the rAAV composition is injected over about 20-22 minutes. In some embodiments, the rAAV composition is injected at a rate of about 35 to about 65 µl/min. In some embodiments, the rAAV composition is injected at a rate of about 35 µl/min. In some embodiments, the rAAV composition is injected at a rate of about 40 µl/min. In some embodiments, the rAAV composition is injected at a rate of about 45 µl/min. In some

In some embodiments, the rAAV composition is injected at a rate of about 50 µl/min. In some embodiments, the rAAV composition is injected at a rate of about 55 µl/min. In some embodiments, the rAAV composition is injected at a rate of about 60 µl/min. In some embodiments, the rAAV composition is injected at a rate of about 65 µl/min. One of skill in the art would recognize that the bleb injection rate and timing may be directed, for example, by the volume of the rAAV composition or the size of the bleb needed to create a retinal detachment sufficient to access the central retinal cells, the size of the cannula used to deliver the rAAV composition, and the ability to securely maintain the position of the cannula of the invention.

In some embodiments of the invention, the volume of the composition injected into the subretinal space of the retina is more than about any one of 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, or 1 ml, or any amount therebetween.

One or multiple (e.g., 2, 3, or more) blebs may be created. In general, the total volume of blebs or blisters created by the methods and systems of the invention cannot exceed the fluid volume of the eye, e.g., about 4 ml in a typical human subject. The total volume of each individual bleb is preferably at least about 0.3 ml, and more preferably at least about 0.5 ml in order to facilitate a retinal detachment of sufficient size to expose the central retinal cell types and create a sufficiently large bleb for optimal manipulation. One of skill in the art will appreciate that in creating the bleb in accordance with the methods and systems of the invention, appropriate intraocular pressure must be maintained to prevent damage to the ocular structures. The size of each individual ampoule may be, for example, about 0.5 to about 1.2 ml, about 0.8 to about 1.2 ml, about 0.9 to about 1.2 ml, about 0.9 to about 1.0 ml, about 1.0 to about 2.0 ml, about 1.0 to about 3.0 ml. Thus, in one example, to inject a total of 3 ml of rAAV composition suspension, 3 ampoules of about 1 ml each may be provided. The total volume of all ampoules combined may be, for example, about 0.5 to about 3.0 ml, about 0.8 to about 3.0 ml, about 0.9 to about 3.0 ml, about 1.0 to about 3.0 ml, about 0.5 to about 1.5 ml, about 0.5 to about 1.2 ml, about 0.9 to about 3.0 ml, about 0.9 to about 2.0 ml, about 0.9 to about 1.0 ml.

In order to safely and effectively transduce target retinal areas (e.g., the central retina) outside the border of the original bleb location, the bleb can be manipulated to reposition the bleb to the target area for transduction. Bleb manipulation can occur by the bleb dependence created by the bleb volume, repositioning of the eye containing the bleb, repositioning of the human head with an eye or eyes containing a bleb, or by repositioning the bleb to the target area.

or more blebs, and/or by fluid-air exchange. This is particularly relevant for the central retina, as this area normally resists detachment by subretinal injection. In some embodiments, fluid-air exchange is used to relocate the bleb; the fluid in the infusion cannula is temporarily replaced by air, for example, by blowing air over the retinal surface. As the volume of air displaces vitreous cavity fluid from the retinal surface, fluid in the vitreous cavity can flow out of a cannula. The temporary lack of vitreous cavity fluid pressure causes the bleb to move and gravitate to a dependent portion of the eye. By appropriately positioning the eyeball, the bleb of the subretinal rAAV composition is manipulated to involve adjacent areas (e.g., the macula and/or fovea). In some cases, the mass of the bleb is sufficient to cause it to gravitate, even without the use of fluid-air exchange. Movement of the bleb to the desired location can be further facilitated by altering the subject's head position to allow the bleb to gravitate to the desired location on the eye. Once the desired bleb configuration is achieved, the fluid is returned to the vitreous cavity. The fluid is an appropriate fluid, e.g., freshly prepared saline. In general, the subretinal rAAV composition can be left in situ without retinopexy to the retinotomy and without intraocular tamponade, and the retina will spontaneously reattach within approximately 48 hours.

By safely and effectively transducing ocular cells (e.g., trabecular meshwork cells) with a vector comprising a therapeutic polypeptide or RNA sequence, the methods of the invention can be used to treat an individual; e.g., a human, having myocilin-like glaucoma (MYOC), wherein the transduced cells produce the therapeutic polypeptide or RNA sequence in an amount sufficient to treat myocilin-like glaucoma (MYOC).

An effective amount of rAAV (in some embodiments in particulate form) is administered depending on the treatment goals. For example, when a low percentage of transduction can achieve the desired therapeutic effect, then the treatment goal in general is to meet or exceed this level of transduction. In some cases, this level of transduction can be achieved by transducing as few as about 1 to 5% of the target cells, in some embodiments at least about 20% of the cells of the desired tissue type, in some embodiments at least about 50%, in some embodiments at least about 80%, in some embodiments at least about 95%, in some embodiments at least about 99% of the cells of the desired tissue type. As discussed above, substituting one or more amino acids in the rAAV capsid that interacts with HSPGs improves rAAV transduction. As a guide, the number of particles delivered per injection is generally between about 1 x 106 and about 1 x 1014 particles, between about 1 x 107 and 1 x 1013 particles, between about 1 x 109 and 1 x 1012 particles, or about 1 x 1011 particles. The rAAV composition may be delivered by one or more subretinal injections, either during

the same procedure or spaced out by days, weeks, months, or years. In some embodiments, multiple vectors may be used to treat the human.

In some embodiments, administration to the eye of an effective amount of rAAV viral particles results in greater than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% transduction or any intermediate % of ocular cells. In some embodiments, from about 5% to about 100%, from about 10% to about 50%, from about 10% to about 30%, from about 25% to about 75%, from about 25% to about 50%, or from about 30% to about 50% of the ocular cells were transduced. Methods for identifying ocular cells transduced by AAV viral particles comprising an rAAV capsid are known in the art; for example, immunohistochemistry or the use of a marker, such as enhanced green fluorescent protein, can be used to detect viral particle transduction.

In some embodiments, administration to the trabecular meshwork of an effective amount of rAAV viral particles results in greater than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 100% transduction or any intermediate % of trabecular meshwork cells. In some embodiments, from about 5% to about 100%, from about 10% to about 50%, from about 10% to about 30%, from about 25% to about 75%, from about 25% to about 50%, or from about 30% to about 50% of the trabecular meshwork cells are transduced. Methods for identifying ocular meshwork cells transduced by AAV viral particles comprising an rAAV capsid are known in the art; for example, immunohistochemistry or the use of a marker, such as enhanced green fluorescent protein, can be used to detect viral particle transduction.

In some embodiments of the invention, the methods comprise administering to the eye of a mammal an effective amount of AAV viral particles for treating an individual with myocilin-like glaucoma (MYOC); e.g., a human with myocilin-like glaucoma (MYOC). In some embodiments, the composition is injected into one or more locations in the eye to allow expression of a heterologous nucleic acid in ocular cells. In some embodiments, the composition is injected into any one of one, two, three, four, five, six, seven, eight, nine, ten, or more than ten locations in the eye.

In some embodiments of the invention, the methods comprise administering to the trabecular meshwork of a mammal an effective amount of AAV viral particles to treat an individual with myocilin-like glaucoma (MYOC); e.g., a human with myocilin-like glaucoma (MYOC). In some embodiments, the composition is injected into one or more locations in the trabecular meshwork to allow expression of a heterologous nucleic acid in the trabecular meshwork cells.

In some embodiments, the composition is injected into any one of one, two, three, four, five, six, seven, eight, nine, ten, or more than ten locations in the trabecular meshwork.

In some embodiments, the rAAV viral particles are administered to more than one location simultaneously or sequentially. In some embodiments, multiple injections of rAAV viral particles are spaced no more than one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours, or 24 hours apart.

Intravitreal injection methods

The general method for intravitreal injection can be illustrated by the following brief outline. This example is intended merely to illustrate certain features of the method, and is in no way intended to be limiting. Procedures for intravitreal injection are known in the art (see, e.g., Peyman, G.A., et al (2009) Retina 29(7):875-912 and Fagan, X.J. and Al-Qureshi, S. (2013) Clin. Experiment. Ophthalmol.

41 (5):500-7).

Briefly, a subject for intravitreal injection can be prepared for the procedure by pupillary dilation, sterilization of the eye, and administration of anesthetic. Any suitable mydriatic agent known in the art can be used for pupillary dilation. Adequate pupillary dilation can be confirmed prior to treatment. Sterilization can be achieved by applying a sterilizing ocular treatment, for example, an iodide-containing solution such as povidone-iodine (BETADINE®). A similar solution can also be used to clean the eyelid, eyelashes, and any other nearby tissue (e.g., skin). Any suitable anesthetic, such as lidocaine or proparacaine, can be used at any suitable concentration. The anesthetic can be administered by any method known in the art, including, but not limited to, topical drops, gels, or jellies, and subconjunctival application of anesthetic.

Before the injection, a sterile blepharostat can be used to clean the eyelashes in the area. The injection site can be marked with a syringe. The injection site can be chosen based on the patient's lens. For example, the injection site can be 3–3.5 mm from the limbus in pseudophakic or aphakic patients, and 3.5–4 mm from the limbus in phakic patients. The patient can look in a direction opposite the injection site.

During injection, the needle can be inserted perpendicular to the sclera and pointed toward the center of the eye. The needle can be inserted so that the tip ends in the vitreous, rather than the subretinal space. Any suitable volume known in the art for injection can be used. After injection, the eye can be treated with a sterilizing agent such as an antibiotic. The eye can also be rinsed to remove excess sterilizing agent.

Intracameral injection methods

Methods of intracameral injection into the eye are known in the art. Buie, et al., (2010) IOVS 51 (1):236-248 provides a non-limiting example of intracameral injection.

The efficacy of rAAV administration by intravitreal or intracameral injection can be monitored by various criteria as described herein. For example, following treatment in a subject using methods of the present invention, the subject can be evaluated for, for example, an improvement and/or stabilization and/or delay in the progression of one or more signs or symptoms of the disease state by one or more clinical parameters, including those described herein. Examples of such tests are known in the art, and include objective as well as subjective (e.g., subject-reported) measures. For example, to measure the efficacy of a treatment on a subject's visual function, one or more of the following may be assessed: the subject's subjective quality of vision or improved central vision function (e.g., an improvement in the subject's ability to read fluently and recognize faces), the subject's visual mobility (e.g., a decrease in the time taken to navigate a maze), visual acuity (e.g., an improvement in the subject's LogMAR score), microperimetry (e.g., an improvement in the subject's dB score), dark-adapted perimetry (e.g., an improvement in the subject's dB score), fine array mapping (e.g., an improvement in the subject's dB score), Goldmann perimetry (e.g., a reduced size of scotomatous area (i.e., areas of blindness) and improved ability to resolve smaller targets), flicker sensitivities (e.g., an improvement in hertz), autofluorescence, and electrophysiological measurements (e.g., improvement in ERG). In some embodiments, visual function is measured by the subject's visual mobility. In some embodiments, visual function is measured by the subject's visual acuity. In some embodiments, visual function is measured by microperimetry. In some embodiments, visual function is measured by dark-adapted perimetry. In some embodiments, visual function is measured by ERG. In some embodiments, visual function is measured by the subject's subjective quality of vision.

For any of the methods or compositions described herein, a medical test for myocilin glaucoma (MYOC) can be used to evaluate the efficacy of a treatment described herein or diagnose a patient who may benefit from a treatment described herein. Numerous medical tests are known in the art for diagnosing or monitoring myocilin glaucoma (MYOC). For example, ophthalmoscopy, laser polarimetry, ocular coherence tomography, and/or scanning laser tomography can be used to inspect the optic nerve, which may be damaged by myocilin glaucoma (MYOC). Intraocular pressure can be measured by tonometry. A pachymeter can be used to measure central corneal thickness (e.g., thin central corneal thickness can be

myocilin glaucoma predictor (MYOC). A visual field test can be used to assess the visual field.

As described above, myocilin mutations have been implicated in primary open-angle myocilin glaucoma (MYOC). Therefore, a medical test for diagnosing POAG can be used to evaluate the efficacy of a treatment described herein or to diagnose a patient who may benefit from a treatment described herein. Any medical test for diagnosing POAG known in the art can be used, for example, to distinguish POAG from another form of myocilin glaucoma (MYOC) (such as angle-closure glaucoma). For example, gonioscopy can be used to provide an evaluation that aids in the diagnosis of POAG.

The efficacy of myocilin-like glaucoma (MYOC) treatments can be evaluated in an animal model. Animal models for myocilin-like glaucoma (MYOC) are known in the art. For example, mice expressing human MYOC Y437H or mouse MYOC Y423H have been shown to develop myocilin-like glaucoma (<m>Y<o>C) symptoms similar to POAG (see Zode et al. (2011) J. Clin. Invest. 121(9):3542-53 and Senatorov, V., et al. (2006) J. Neurosci. 26(46):11903-14). Additionally, mice lacking the soluble guanylate cyclase alpha subunit of the nitric oxide receptor are another model of POAG (Buys, E.S., et al. (2013) PLoS ONE 8(3):e60156). Rat models have also been developed; rats expressing human TGF-beta delivered by adenoviral gene transfer show increased IOP (Shepard, A.R., et al. (2010) Invest. Ophthalmol. 51(4):2067-76). A further description of other animal models for various aspects of POAG, including primate, dog, and zebrafish models, can be found in Bouhenni, R.A., et al. (2012) J. Biomed. Biotechnol. 2012:692609).

In some ocular disorders, there is a "nurse cell" phenomenon, in which improving the function of one cell type improves the function of another. For example, transduction of the RPE of the central retina by an rAAV of the invention can then improve rod function, and in turn, the improved rod function results in improved cone function. Accordingly, treatment of one cell type can result in improved function in another. In myocilin glaucoma (MYOC), reducing IOP by transducing tM will reduce the degeneration of ganglion cell structure and function.

The selection of a particular rAAV vector and composition depends on several different factors, including, but not limited to, the individual's medical history and the characteristics of the condition and individual being treated. The evaluation of these characteristics and the design of an appropriate therapeutic regimen are ultimately the responsibility of the prescribing physician.

The compositions of the disclosure (e.g., AAV viral particles encoding RSPO1, RSPO2, RSPO3, RSPO4, or a functional variant thereof and/or MYOC RNAi (e.g., shRNA)) can be used alone or in combination with one or more additional therapeutic agents to treat ocular disorders. The interval between sequential administrations can be in terms of at least (or, alternatively, less than) minutes, hours, or days.

In some embodiments, one or more additional therapeutic agents may be delivered to the trabecular meshwork. Non-limiting examples of the additional therapeutic agent include prostaglandins such as Xalatan, Lumigan, Travatan Z, and Rescula; beta-blockers including Timoptic XE, Istalol, and Betoptic S; alpha-adrenergic agonists including Iopidine, Alphagan, and Alphagan-P; carbonic anhydrase inhibitors including Trusopt and Azopt, Diamox, Neptazane, and Daranide; parasympathomimetics including pilocarpine, carbachol, echothiophate, and demecarium; epinephrines including Propine; or combination therapies including Cosopt, Combigan, and DuoTrav.

IV. Expression constructions

The invention provides methods of delivering heterologous nucleic acid to the eye by subretinal delivery of an rAAV vector comprising the heterologous nucleic acid, wherein the rAAV vector is encapsidated in an rAAV capsid comprising one or more amino acid substitutions that interact with HSPGs. In some embodiments, the heterologous nucleic acid (e.g., a transgene) is operably linked to a promoter. In the invention, the rAAV particle comprises a vector encoding RSPO1, RSPO2, or RSPO4, wherein RSPO1, RSPO2, or RSPO4 is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40 (SV40) promoter, and a CK6 promoter, a transthyretin (TTR) promoter, a TK promoter, a tetracycline-responsive (TRE) promoter, an HBV promoter, an hAAT promoter, a LSP promoter, liver-specific chimeric (LSP) promoters, the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer/chicken beta-actin/rabbit p-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9) and the elongation factor 1-alpha (EFL-alpha) promoter (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a human p-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken p-actin (CBA) promoter. The promoter may be a constitutive, inducible, or repressible promoter. In some cases, the promoter is capable of expressing the heterologous nucleic acid in a cell of the eye. In the invention, RSPO1, RSPO2 or RSPO4 is operably linked to a promoter, wherein the promoter is capable of expressing RSPO1, RSPO2 or RSPO4 in the mammalian eye and/or in trabecular meshwork cells. In some cases, the promoter is capable of expressing the heterologous nucleic acid in photoreceptor or RPE cells. In some cases, the promoter is a rhodopsin kinase (RK) promoter; for example, a human RK promoter. In some

In some cases, the promoter is an opsin promoter; for example, a human opsin promoter or a mouse opsin promoter. In some cases, the promoter is an RNA polymerase III promoter. In some cases, the disclosure provides methods of treating myocilin-like glaucoma (MYOC) in a mammal (e.g., a human) by administering to the eye of the mammal an rAAV particle comprising an rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, under the control of a CBA promoter. In some embodiments, the disclosure provides methods of treating myocilin glaucoma (MYOC) in a mammal (e.g., a human) by administering to the eye of the mammal an rAAV particle comprising an rAAV vector encoding an RNAi (e.g., shRNA) that targets (e.g., reduces or inhibits) a MYOC (e.g., a human MYOC) under the control of a CBA promoter. In some embodiments, the disclosure provides methods of treating myocilin glaucoma (MYOC) in a mammal (e.g., a human) by administering to the eye of the mammal an rAAV particle comprising an rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, under the control of a CBA promoter and a rAAV particle comprising a rAAV vector encoding an RNAi (e.g., shRNA) that targets (e.g., reduces or inhibits) a MYOC (e.g., a human MYOC) under the control of a cBa promoter. In some embodiments, the disclosure provides methods of treating myocilin glaucoma (MYOC) in a mammal (e.g., a human) by administering to the eye of the mammal an rAAV particle comprising an rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, under the control of a CBA promoter and an RNAi (e.g., shRNA) that targets (e.g., reduces or inhibits) a MYOC (e.g., a human MYOC) under the control of a CBA promoter.

The present application contemplates the use of a recombinant viral genome for the introduction of one or more nucleic acid sequences encoding a therapeutic polypeptide and/or nucleic acid to be encapsidated into a rAAV viral particle. The recombinant viral genome may include any element for establishing expression of the therapeutic polypeptide and/or nucleic acid, for example, a promoter, ITR, ribosome-binding element, terminator, enhancer, selectable marker, intron, polyA signal, and/or origin of replication.

In some embodiments, the disclosure provides viral particles comprising a recombinant self-complementary genome. AAV viral particles with self-complementary genomes and methods of using self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,765,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111. An rAAV comprising a self-complementary genome will rapidly form a double-stranded DNA molecule by virtue of its partially complementary sequences (e.g., complementary coding and non-coding strands of a transgene). In some instances, the first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence are joined by a mutated ITR (e.g., the right ITR). In some instances, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACG CCCGGGCTTTGCCCGGGGCG-3' (SEQ ID NO:20). The mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence. As a result, upon replicating an AAV viral genome, the rep proteins will not cleave the viral genome at the mutated ITR and as such a recombinant viral genome comprising the following in 5' to 3' order will be encapsidated into a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, the mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide, and a third AAV ITR.

VI. Viral particles and methods of producing viral particles

rAAV viral particles

The invention relates to the use of rAAV particles for treating myocilin glaucoma (MYOC) and compositions comprising rAAV particles are disclosed herein. In some embodiments, the viral particle is a recombinant AAV particle comprising a nucleic acid comprising a sequence encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and/or a MYOC RNAi (e.g., shRNA) described herein flanked by one or two ITRs. The nucleic acid is encapsidated in the AAV particle. The AAV particle also comprises capsid proteins. In some embodiments, the nucleic acid comprises coding sequence(s) for operably linked components of interest (e.g., nucleic acid encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and/or a MYOC RNAi (e.g., shRNA))) in the direction of transcription, control sequences including transcription initiation and termination sequences, thereby forming an expression cassette. The expression cassette is flanked at the 5' and 3' ends by at least one functional AAV ITR sequence. By "functional AAV ITR sequences" is meant that the ITR sequences function as intended for AAV virion rescue, replication, and encapsidation. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16. According to the invention, the viral particle is a rAAV particle comprising a vector encoding RSPO1, RSPO2 or RSPO4, wherein RSPO1, RSPO2 or RSPO4 is operably linked to a promoter. For practicing some aspects of the invention, the recombinant vectors comprise at least all of the AAV sequences essential for encapsidation and the physical structures for rAAV infection. AAV ITRs for use in the vectors of the invention need not have a naturally occurring nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801), and may be altered by nucleotide insertion, deletion, or substitution, or the AAV ITRs may be derived from any of several AAV serotypes. Over 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossiset al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered within the scope of the present invention. In some embodiments, an rAAV vector is a vector derived from an AAV serotype including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV or the like. In some embodiments, the nucleic acid in the AAV comprises an inverted terminal repeat (ITR) ITR of serotype AAV1, A<a>V2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, A<a>V11, AAV12, AAV2R471A, A<a>V<DJ, A<a>V<goat, bovine AAV, or mouse AAV, or the like. In some embodiments, the nucleic acid in the AAV further encodes an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide or a functional variant thereof; MYOC RNAi (e.g., shRNA); or a RSPO1, RSPO2, RSPO3, RSPO4 polypeptide or a functional variant thereof, and MYOC as described herein. For example, the nucleic acid in the AAV may comprise at least one ITR from any AAV serotype contemplated herein and may further encode a nucleic acid encoding a MYOC RNAi (e.g., shRNA) targeting SEQ ID NO:6 and comprising the loop sequence of SEQ ID NO:7 and/or one or more of: an RSPO1 comprising SEQ ID NO:8, 11 and/or 12; an RSPO2 comprising SEQ ID NO:9, 13 and/or 14; and/or an SPO3, comprising SEQ ID NO:1, 15, 16 and/or 17; and a R<s>P<o>4 comprising SEQ ID NO: 10, 18, and/or 19. In some embodiments, the nucleic acid encodes an RSPO1 that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, 11, or 12; an RSPO2 that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, 13, or 14; an RSPO3 that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 15-17; or an RSPO4 that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, 18, or 19.

In further embodiments, the rAAV particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 capsid proteins (e.g., a wild-type AAV6 capsid, or a variant AAV6 capsid such as ShH10, as described in US pre-grant publication 2012/0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in US pre-grant publication 2013/0323226), AAV10, AAVRH10, AAV11, AAV12, a tyrosine capsid mutant, a heparin-binding capsid mutant, one AAV2R471A capsid, one AAVAAV2/2-7m8 capsid, one AAV DJ capsid

(e.g., an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids described in <pre-grant publication>U.S. 2012/0066783), AAV2 N587A capsid, AAV2 E548A capsid, A<a>V2< N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2/HBoV1 capsid, or an AAV capsid described in U.S. Patent No. 8,283,151 or International Publication No. WO/2003/042397. In some embodiments, the AAV viral particle comprises an AAV capsid comprising an amino acid substitution at one or more of positions R484, R487, K527, K532, R585, or R588, numbering based on VP1 of AAV2. In further embodiments, an rAAV particle comprises capsid proteins from an AAV serotype from clades A-F. In some embodiments, a mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the rAAV particles comprise a capsid protein that permits transduction of the trabecular meshwork. In some embodiments, the rAAV particles comprise a mutant capsid protein that permits transduction of the trabecular meshwork. In some embodiments, the rAAV particle comprises AAV2 capsid proteins, wherein the capsid protein comprises an R471A amino acid substitution, numbering based on VP1 of AAV2 (Lochrie et al., J Virol (2006) 80(2):821-834). In some embodiments, the disclosure provides rAAV particles comprising a vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof; an AAV2 capsid comprising an R471A amino acid substitution, numbering based on VP1 of AAV2; and/or a vector encoding MYOC RNAi (e.g., shRNA).

In some embodiments, the disclosure provides compositions and methods for treating myocilin glaucoma (MYOC) in a mammal, wherein a rAAV2 viral particle comprising an rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, is administered to the eye of the mammal where different parts of the eye (e.g., the retina) can be transduced, and a rAAV2 R471A viral particle comprising a rAAV vector encoding a MYOC RNAi is administered to the eye of the mammal where trabecular meshwork cells are transduced. In some embodiments, the disclosure provides compositions and methods for treating myocilin glaucoma (MYOC) in a mammal, wherein a rAAV2 R471A viral particle comprising a rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and a rAAV2 R471A viral particle comprising a rAAV vector encoding a MYOC RNAi are administered to the eye of the mammal where trabecular meshwork cells are transduced. In some embodiments, the disclosure provides compositions and methods for treating myocilin glaucoma (MYOC) in a mammal, wherein an R471A rAAV2 viral particle comprising an rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and encoding a MYOC RNAi is administered to the eye of the mammal where trabecular meshwork cells are transduced.

In some embodiments, the invention provides compositions and methods for delivering a transgene (e.g., a therapeutic transgene to the trabecular meshwork of the eye). In some embodiments, the compositions and methods use an rAAV2 particle comprising a mutant capsid wherein the capsid comprises an R471A amino acid substitution, numbering relative to VP1 of AAV2. Such compositions and methods may be used in the treatment of ocular diseases; e.g., trabecular meshwork-associated ocular disease, such as myocilin-like glaucoma (MYOC).

Different AAV serotypes are used to optimize transduction of particular target cells or to target specific cell types within a particular target tissue (e.g., a diseased tissue). An rAAV particle may comprise viral proteins and viral nucleic acids of the same serotype or a mixed serotype.

Self-complementary viral genomes of AAV

In some embodiments, the disclosure provides viral particles comprising a recombinant self-complementary genome. AAV viral particles with self-complementary genomes and methods of using self-complementary AAV genomes are described in U.S. Patent Nos. 6,596,535; 7,125,717; 7,765,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10:2105-2111. An rAAV comprising a self-complementary genome will rapidly form a double-stranded DNA molecule by virtue of its partially complementary sequences (e.g., by complementing coding and non-coding strands of a transgene). In some embodiments, the disclosure provides an AAV viral particle comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., miR-708 and/or a rhodopsin coding strand) and a second heterologous polynucleotide sequence (e.g., miR-708 antisense strand and/or a rhodopsin antisense or non-coding strand), wherein the first heterologous polynucleotide sequence can form intra-strand base pairs with the second polynucleotide sequence over most or all of its length. In some cases, the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a sequence that facilitates intrastrand base pairing; for example, a hairpin DNA structure. Hairpin structures are known in the art, for example, in siRNA molecules. In some cases, the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a mutated ITR (e.g., the right-hand ITR). In some cases, the ITR comprises the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC ACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO:20). The mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence. As a result, upon replicating an AAV viral genome, the rep proteins will not cleave the viral genome at the mutated ITR and as such a recombinant viral genome comprising the following in 5' to 3' order will be encapsidated into a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, the mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide, and a third AAV ITR. In some embodiments, the invention provides AAV viral particles comprising a recombinant viral genome comprising a functional AAV2 ITR, a first polynucleotide sequence encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and/or a MYOC RNAi (e.g., shRNA), a mutated AAV2 ITR comprising a deletion of the D region and lacking a functional terminal resolution sequence, a second polynucleotide sequence comprising the sequence complementary to the sequence encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and/or a MYOC RNAi (e.g., shRNA), the first polynucleotide sequence, and a functional AAV2 ITR.

Production of AAV particles

rAAV particles can be produced using methods known in the art. See, e.g., U.S. Patent Nos. 6,566,118; 6,989,264; and 6,995,006. In the practice of the invention, host cells for producing rAAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeast. Host cells can also be encapsidating cells in which the AAV rep and cap genes are stably maintained in the host cell or producer cells in which the AAV vector genome is stably maintained. Exemplary encapsidating and producer cells are derived from 293, A549, or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

In some aspects, a method for producing any rAAV particle as disclosed herein is disclosed comprising (a) culturing a host cell under a condition wherein rAAV particles are produced, wherein the host cell comprises (i) one or more AAV encapsidation genes, wherein each of said AAV encapsidation genes encodes an AAV replication and/or encapsidation protein; (ii) a rAAV provector comprising a nucleic acid encoding a therapeutic polypeptide and/or nucleic acid as described herein flanked by at least one AAV ITR, and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell.

In additional cases, the rAAV particles are purified. The term "purified," as used herein, includes a preparation of rAAV particles devoid of at least some of the other components that may also be present when rAAV particles are naturally produced or initially prepared from them.

Thus, for example, isolated rAAV particles can be prepared using a purification technique to enrich them from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRP) or genomic copies (gc) present in a solution, or by infectivity, or it can be measured with respect to a second, potentially interfering substance present in the source mixture, such as contaminants, including production culture contaminants, or process contaminants, including helper viruses, medium components, and the like.

Also disclosed herein are pharmaceutical compositions comprising an rAAV particle comprising a heterologous nucleic acid encoding a therapeutic polypeptide and/or therapeutic nucleic acid, wherein the rAAV particle comprises a rAAV capsid comprising one or more HSPG-interacting amino acids or substitutions, and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be suitable for any mode of administration described herein; for example, by subretinal administration.

In some cases, pharmaceutical compositions comprising a rAAV described herein and a pharmaceutically acceptable carrier are suitable for administration to humans. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). In some cases, pharmaceutical compositions comprising a rAAV described herein and a pharmaceutically acceptable carrier are suitable for ocular injection. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous solutions of dextrose, polyethylene glycol (PEG), and glycerol can also be employed as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may further comprise additional ingredients, for example, preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like. The pharmaceutical compositions described herein may be packaged in single unit doses or in multi-dose forms. The compositions are generally formulated as a sterile, substantially isotonic solution.

VII. Systems and kits

The rAAV compositions of the invention may be contained within a system designed for use in one of the methods of the invention. In some embodiments, the invention provides a system for delivering a vector to an eye of an individual, comprising a) a composition comprising an effective amount of rAAV particles, wherein the vector comprises a heterologous nucleic acid encoding a therapeutic polypeptide and/or therapeutic RNA and at least one AAV terminal repeat; and b) a device for ocular delivery of the rAAV. In some embodiments, the rAAV particles comprise a rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof. In some embodiments, the rAAV particles comprise a rAAV vector encoding one or more MYOC RNAi (e.g., shRNA) that target (e.g., reduce or inhibit) MYOC expression. In some embodiments, the rAAV particles comprise a rAAV vector encoding an RSPO1, RSPO2, RSPO3, rSpO4 polypeptide, or a functional variant thereof, and one or more MYOC RNAi(s) (e.g., shRNA) that target (e.g., reduce or inhibit) MYOC expression. In some embodiments, the kit or system comprises rAAV particles comprising a rAAV vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide, or a functional variant thereof, and rAAV particles comprising a rAAV vector encoding one or more MYOC RNAi (e.g., shRNA) that target (e.g., reduce or inhibit) MYOC expression.

In general, the system comprises a fine bore cannula, wherein the cannula is 27 to 45 gauge, one or more syringes (e.g., 1, 2, 3, 4 or more) and one or more fluids (e.g., 1, 2, 3, 4 or more) suitable for use in the methods of the invention.

The fine-bore cannula is suitable for subretinal injection of the vector suspension and/or other fluids to be injected into the subretinal space. In some cases, the cannula is 27 to 45 gauge. In some cases, the fine-bore cannula is 35 to 41 gauge. In some cases, the fine-bore cannula is 40 or 41 gauge. In some cases, the fine-bore cannula is 41 gauge. The cannula may be any suitable type of cannula, for example, a de-John® cannula or an Eagle® cannula.

The syringe can be any suitable syringe, as long as it can be connected to the needle for fluid delivery. In some cases, the syringe is an Accurus® Syringe System. In some cases, the system has one syringe. In some cases, the system has two syringes. In some cases, the system has three syringes. In some cases, the system has four or more syringes.

The system may further comprise an automated injection pump, which can be activated, for example, by a foot pedal.

Suitable fluids for use in the methods of the invention include those described herein, for example, one or more fluids, each comprising an effective amount of one or more vectors as described herein, one or more fluids for creating an initial blister (e.g., saline or other appropriate fluid), and one or more fluids comprising one or more therapeutic agents.

Suitable fluids for use in the methods of the invention include those described herein, for example, one or more fluids, each comprising an effective amount of one or more vectors as described herein, one or more fluids for creating an initial blister (e.g., saline or other appropriate fluid), and one or more fluids comprising one or more therapeutic agents.

In some embodiments, the volume of the fluid comprising an effective amount of the vector is greater than about 0.8 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is at least about 0.9 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is at least about 1.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is at least about 1.5 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is at least about 2.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is greater than about 0.8 to about 3.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is greater than about 0.8 to about 2.5 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is greater than about 0.8 to about 2.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is greater than about 0.8 to about 1.5 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is greater than about 0.8 to about 1.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is from about 0.9 to about 3.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is from about 0.9 to about 2.5 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is from about 0.9 to about 2.0 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is from about 0.9 to about 1.5 ml. In some embodiments, the volume of the fluid comprising an effective amount of the vector is from about 0.9 to about 1.0 ml. In some embodiments, the volume of fluid comprising an effective amount of the vector is from about 1.0 to about 3.0 ml. In some embodiments, the volume of fluid comprising an effective amount of the vector is from about 1.0 to about 2.0 ml.

The fluid to create the initial blister may be, for example, about 0.1 to about 0.5 ml. In some embodiments, the total volume of all fluids in the system is about 0.5 to about 3.0 ml.

In some embodiments, the kit comprises a single fluid (e.g., a fluid comprising an effective amount of the vector). In some cases, the kit comprises two fluids. In some cases, the kit comprises three fluids. In some cases, the kit comprises four or more fluids.

The systems of the disclosure may further be packaged in kits, wherein the kits may further comprise instructions for use. In some cases, the kits further comprise a device for subretinal delivery of rAAV particle compositions. In some cases, the instructions for use include instructions according to one of the methods described herein. In some cases, the instructions for use include instructions for intravitreal and/or intracameral delivery of rAAV particles comprising a vector encoding an RSPO1, RSPO2, RSPO3, RSPO4 polypeptide or a functional variant thereof, and/or MYOC RNAi (e.g., shRNA).

EXAMPLES

The invention will be more fully understood by reference to the following examples. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art.

Example 1: Glaucomatous MYOC mutations (e.g., P370L and Y437H) block MYOC secretion

Understanding how MYOC mutants affect ocular function, particularly cells such as trabecular meshwork cells that may contribute to IOP, will provide insight into the pathogenesis of myocilin-like glaucoma (MYOC). Understanding MYOC function may also help uncover potential therapeutic strategies for myocilin-like glaucoma (MYOC). The results described here demonstrate that MYOC mutants reduce wild-type MYOC expression and block Wnt signaling. Furthermore, these results indicate that expression of R-spondin 3 (RSPO3) and/or silencing MYOC can restore Wnt signaling blocked by mutant MYOC expression.

Methods

Plasmid vectors

For MYOC and RSPO3 plasmids, MYOC cDNA was provided by Clone DB-Sanofi Oncology. RSPO3 cDNA was provided by Clone DB-Sanofi Oncology.

For the construction of pCBA2 in MYOC P370L, the QUIKCHANGE® II kit (Agilent, Santa Clara) was used to introduce the desired single base substitution following the manufacturer's recommendations and the primers 5'-ACCACGGACAGTTCCTGTATTCTTGGGGTGG-3' (SEQ ID NO:21) and 5'-CCACCCCAAGAATACAGGAACTGTCCGTGGT-3' (SEQ ID NO:22).

For the construction of pCBA2 in MYOC Y437H, the QUIKCHANGE® Lightning kit (Agilent, Santa Clara) was used to introduce the desired single base substitution following the manufacturer's recommendations and the primers 5'-TCTGTGGCACCTTGCACACCGTCAGCAGC-3' (SEQ ID NO:23) and 5'-GCTGCTGACGGTGTGCAAGGTGCCACAGA-3' (SEQ ID NO:24).

Grp94 shRNA plasmids were obtained from OriGene Technologies, Inc. (Cat. No. TR312309). pGIPZ-MYOC plasmids (Dharmacon GE Life Sciences) were provided by Clone DB- Sanofi Oncology. The GIPZ microRNA-tailored shRNA collection (Stegmeier, et al. (2005) Proc. Natl. Acad. Sci. USA. 102:13212-7). GIPZ shRNA designs are based on the native miR-30 primary transcript to allow processing by the endogenous RNAi pathway and result in specific gene silencing with minimized cellular toxicity. The pGIPZ-null plasmid, a constitutive miRNA shRNA vector expressing null, non-targeting miRNA, was provided by Clone DB-Sanofi Oncology.

Cell culture and recombinant proteins

HEK293 cells (Microbix Biosystems Inc.) were cultured in DMEM, 10% FCS, and 5% CO2. The HEK293T cell line (293T) was obtained from A<t>CC and cultured in DMEM, 10% FCS, and 5% CO2.

Immortalization of primary human trabecular meshwork (hTM) cells

SV40 large T antigen (SV40-tag) was used for immortalization by transduction with an AAV2-SV40 T antigen vector. Passage 7 hTM cells (ScienCell Research Laboratories, Carlsbad, CA) maintained in complete fibroblast growth medium (ScienCell) were seeded onto 10-cm cell culture dishes and transduced with 1 × 105 DRP of AAV2-SV40-tag (tagged "hTM-T") or AAAV2-EGFP (negative control, tagged "hTM-ENT") for 24 hours. Once cells reached confluence, they were passaged in 2 × 15-cm dishes (P8). Cells were passaged repeatedly approximately every 3-4 days. At passage 10, an aliquot was taken to determine a cell count. The total cell number of hTM-T cells was 5.2 x 106, compared with 2.5 x 105 total cells from hTM-ENT cells.

Western blotting was performed to determine the presence of SV40 T antigen. Briefly, a 500 ul suspension of cells was centrifuged, and the resulting cell pellet was lysed in 100 ul of RIPA buffer containing protease inhibitor mixture. 5 ul of cell lysates were analyzed by SDS-PAGE followed by immunoblotting using the iBlot Rapid Transfer System (Life Technologies). The blot was blocked using TBS protein-free blocker (Thermo Fisher Scientific, Waltham, MA) and incubated with an anti-SV40 T antigen monoclonal antibody (GeneTex, Irvine, CA). The blot was then incubated with an anti-mouse HRP-labeled antibody (R&D Systems, Minneapolis, MN). Immunoreactive bands were visualized using the Supersignal West Femto chemiluminescent substrate (Thermo Fisher). A prominent 80 kDa band corresponding to the SV40 T antigen was detected from hTM-T cells, but not hTM-ENT cells, indicating the presence and expression of the SV40 T antigen. 293T cell lysate served as a positive control that also contained the 80 kDa SV40 T antigen band. hTM-T cells were expanded, and cell banks were frozen in cell freezing medium (Life Technologies, Grand Island, NY) at passage 12 (10 vials at 1 x 10 cells) and later at passage 18 (46 vials at 10 cells).

Characterization of hTM-T.

Comparison of hTM-T cells and primary hTM cells showed a marked difference in cell morphology, population doubling times, and plasmid transfection efficiency. Primary hTM cells appeared larger and fibroblast-like with a long cell body, whereas immortalized hTM-T cells were smaller, cubical in shape, and relatively uniform in size. The hTM-T cell line demonstrated an increased growth rate with population doublings occurring approximately 3-4 times faster than primary cells. Furthermore, hTM-T cells continued to proliferate beyond 20 cell passages, whereas primary hTM cells displayed a reduced growth rate at passage 10 and eventual growth arrest at passage 12. Transfection efficiency was determined using an EGFP plasmid and Lipofectamine in both cell types of similar cell density. Briefly, subconfluent hTM-T or hTM cells were transfected with an EGFP plasmid using Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol. Although hTM-T cells had a higher number of cells per mm of cell culture surface, there was clearly a higher percentage of EGFP+ hTM-T cells (~50%) compared to primary hTM cells (~5%).

Western Blot

293T or hTM-T cells were transfected with plasmids expressing wtMYOC, P370L and Y437H MYOC mutants, RSPO3, and/or shRNA using Lipofectamine 2000 (Life Technologies). Briefly, cells were lysed in 50–100 gl of RIPA buffer containing protease inhibitor mixture. 10–13 gl of cell lysates were analyzed by SDS-PAGE followed by immunoblotting using the iBlot Rapid Transfer System (Life Technologies). The blot was blocked using Tris-buffered saline, 0.05% Tween 20 (TBST), 0.2% I-Block (Casein-based blocking reagent; Life Technologies), and incubated with a mouse anti-human MYOC antibody. The blot was then incubated with an HRP-labeled anti-mouse antibody (R&D Systems, Minneapolis, MN). Immunoreactive bands were visualized using the ECL chemiluminescent substrate (Thermo Fisher) and viewed on BioMax XAR film (Carestream Health) developed with a Kodak X-Omat 2000 processor.

Luciferase reporter assay

293T or hTM-T cells were seeded in Costar 96-well white or black wall plates at 2 x 104/well. Transfections were performed 1-2 days after cell seeding using Fugene HD transfection reagent (Promega, Madison, WI) according to the manufacturer's protocol.

Briefly, the Topflash reporter plasmid (Millipore, Billerica, MA) containing a 40:1 ratio of the TCF/LEF-regulated firefly luciferase reporter gene and the cytomegalovirus (CMV)-driven Renilla luciferase gene was mixed 1:1 with target plasmids. 8 µl of Fugene HD reagent was added, and samples were immediately vortexed and then incubated for 15 min at room temperature. Plasmid DNA complexes were added to cells and incubated at 37°C for 24 h. Samples were either unstimulated or stimulated with 400 ng/ml of recombinant human or mouse wnt3a protein (R&D Systems) and incubated for an additional 20–24 h. Wnt signaling was measured using the Dual Luciferase Assay System (Promega) according to the manufacturer's protocol. Absorbance values were measured on a Centro XS3 960 Microplate Luminometer (Berthold Technologies, Oak Ridge, TN) and reported as relative light units (RLUs). To monitor transfection efficiency, firefly luciferase RLUs were normalized to Renilla luciferase RLUs. All samples were run in triplicate wells.

Results

Wild-type MYOC (wtMYOC) is secreted from cultured cells, but little or no MYOC is secreted from cells expressing five different mutant forms of MYOC, and cotransfection of cultured cells with normal and mutant MYOC has been reported to suppress wtMYOC secretion (Jacobson et al. (2001) Hum. Mol. Genet. 10(2):117-25). In order to examine the effects of mutant MYOC expression on MYOC secretion, 293 cells were transfected with plasmids expressing wild-type MYOC, P370L mutant MYOC, or Y437H mutant MYOC.

As shown in FIG. 1, 293 cells expressing wild-type MYOC showed detectable expression of MYOC protein in both cell lysates (see lower blot marked “CELLS”) and were secreted into the cell culture medium (see upper blot “MEDIUM”). However, cells transfected with plasmids expressing either P370L or Y437H MYOC showed intracellular expression, but no secretion into the cell culture medium. Furthermore, co-transfection of 293 cells with plasmids expressing wild-type MYOC and either P370L or Y437H MYOC resulted in a lack of MYOC secretion into the cell culture medium. These results indicate that the P370L and Y437H mutants are not secreted from 293 cells and are also capable of blocking wild-type MYOC secretion.

Additional experiments were performed to determine whether these results are observed in human ocular cells. A human trabecular meshwork cell line was immortalized by AAV-mediated expression of the SV40 large T antigen (hTM-T cells), as described above. 293T and hTM-T cells were transfected with a plasmid expressing wild-type MYOC, a plasmid expressing MYOC P370L, or both plasmids. FIG. 2 shows Western blots probing for the presence of intracellular or secreted MYOC protein in these cells. Although wild-type MYOC was expressed and secreted by both 293T and hTM-T cells, MYOC P370L was expressed but not secreted by both 293T and hTM-T cells. MYOC P370L also blocked the secretion of wild-type MYOC in both 293T and hTM-T cells.

These results demonstrate that glaucomatous MYOC mutants (e.g., P370L and Y437H) are capable of blocking wild-type MYOC secretion in human cells. Furthermore, mutant MYOC is also capable of blocking MYOC secretion in hTM cells.

Example 2: Glaucomatous MYOC mutations (e.g., P370L and Y437H) block Wnt signaling

MYOC is thought to interact with components of Wnt signaling pathways, such as Wnt receptors of the Frizzled (FZD) family, Wnt antagonists of the secreted Frizzled-related protein (sFRP) family, and Wnt inhibitory factor 1 (WIF-1)), which modulate actin cytoskeletal organization by stimulating stress fiber formation (Kwon et al. (2009) Mod. Cell. Biol. 29:2139–54). Stress fiber formation is critical for trabecular meshwork (TM) contractility and IOP regulation. However, exactly how MYOC is connected to Wnt signaling, and how this connection affects IOP, are unclear. Based on cell biological experiments, a role for myocilin as a matricellular protein has been proposed (Resch and Fautsch, 2009; Kochet al.,2014). Other groups have shown that myocilin is a mediator of oligodendrocyte differentiation and is involved in optic nerve myelination in mice (Kwonet al.,2014).

It has been suggested that MYOC may serve as a modulator of Wnt signaling and that Wnt proteins may compensate for an absence of myocilin by performing its functions (Kwon et al. (2009) Mod. Cell. Biol. 29:2139-54). Several groups have reported similarities between the actions of myocilin and Wnt proteins acting through a b-catenin-independent mechanism (Kwon and Tomarev (2011) J. Cell. Physiol. 226(12):3392-402). Reduced Wnt signaling in glaucomatous TM (GTM) cells has been reported to be due to higher endogenous levels of sFRP1 (Wang et al. (2008) J. Clin. Invest. 118:1056-64; Lin and Hankenson (2011) J. Cell. Biochem.

112:3491-501). Another group has shown that the Wnt signaling pathway protects the retinal cell line RGC-5 from high blood pressure (Fragoso et al. (2011) Cell. Mol. Neurobiol. 31 (1) :163-73).

It is unclear from the literature whether glaucomatous MYOC mutations (e.g., P370L or Y437H) have any effect on Wnt signaling in the TM. One report states that the effect of glaucomatous MYOC mutations, which inhibit MYOC secretion from the TM, on Wnt signaling in the TM is unclear, as measured by the TOP-Flash Wnt signaling assay (Mao et al. (2012) Invest. Ophthalmol. Vis. Sci. 53(11):7043-51). Another group has reported that P370L had a stimulatory effect on Wnt signaling in Caco-2 cells, shown by the TOP-Flash Wnt signaling assay (Shen et al. (2012) PLoS ONE 7(9):e44902).

In contrast, the inventors have discovered that MYOC mutations (e.g., P370L and Y437H) have an inhibitory effect on Wnt signaling in 293 and TM cells, as shown by the TOP-Flash Wnt signaling assay, which reports beta-catenin activity.

To assess the effect of MYOC P370L and Y437H mutants on Wnt signaling, 293T cells were co-transfected with the TOP-Flash reporter construct and either wtMYOC (“MYOC”), MYOC P370L, or MYOC Y437H plasmids. Wnt signaling was amplified after the addition of recombinant mouse Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity (mean ± SD, n = 4) was measured after transfection and normalized to the constitutively expressed Renilla luciferase level transfection control.

As shown in FIG. 3 , stimulation of 293T cells with recombinant mouse Wnt3 resulted in an increase in the TOP-Flash reporter. Expression of wild-type MYOC did not interfere with Wnt signaling, as assayed by TOP-Flash. However, coexpression of wild-type MYOC with either MYOC P370L or MYOC Y437H blocked TOP-Flash activation in 293T cells. These results indicate that expression of glaucomatous MYOC mutants (e.g., P370L and Y437H) are capable of inhibiting Wnt signaling in human cells.

Example 3: Restoration of Wnt signaling blocked by glaucomatous MYOC mutations (e.g., P370L and Y437H)

The previous example demonstrates that the glaucomatous MYOC mutants P370L and Y437H act by blocking Wnt signaling in human cells. Additional experiments were performed to examine possible mechanisms by which Wnt signaling can be restored in cells expressing these MYOC mutants.

R-spondin 3 (RSPO3) is a protein encoded by the RSPO3 gene that activates Wnt signaling, and we examined whether RSPO3 expression was able to restore Wnt signaling after its inhibition by expression of mutant MYOC. For these experiments, similar to FIG. 3 above, 293T cells were co-transfected with the TOP-Flash reporter construct and wtMYOC ("MYOC"), MYOC P370L, MYOC Y437H, and/or RSPO3 plasmids, as labeled. Wnt signaling was amplified after the addition of recombinant mouse Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity (mean ± SD, n = 3) was measured after transfection and normalized to the constitutively expressed Renilla luciferase level transfection control.

As shown in FIG. 4 , RSPO3 expression caused an increase in Wnt signaling, as measured by TOP-Flash. Importantly, co-expression of RSPO3 and MYOC P370L or MYOC Y437H was able to restore Wnt signaling, compared to the inhibition of Wnt signaling observed upon expression of MYOC P370L or MYOC Y437H alone. 293T cells were co-transfected with the TOP-Flash reporter construct and wtMYOC ("MYOC"), MYOC P370L, MYOC Y437H, and/or RSPO3 plasmids, as labeled. Wnt signaling was amplified after the addition of recombinant mouse Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity was measured after transfection and normalized to the constitutively expressed Renilla luciferase level transfection control.

To assess whether a similar effect is observed in hTM cells, hTM-T cells were co-transfected with the TOP-Flash reporter construct and wtMYOC (“w.t. MYOC”), MyOC P370L, and/or RSPO3 plasmids. Wnt activity was measured by the TOP-Flash assay as described above (luciferase activity is shown as mean ± SD, n = 3). FIG. 5 shows that expression of MYOC P370L caused a reduction in Wnt signaling and was able to reduce Wnt signaling in hTM-T cells co-expressing wild-type MYOC. Expression of RSPO3 was able to increase Wnt signaling in hTM-T cells expressing MYOC P370L alone or MYOC P370L in combination with wild-type MYOC. The results are depicted in FIG. 4and 5 indicate that RSPO3 expression restores Wnt signaling in cells expressing glaucomatous MYOC mutants, such as 293T and hTM-T cells.

Surprisingly, Wnt inhibition by expression of glaucomatous MYOC mutants has also been found to be reversed by silencing MYOC (e.g., by RNAi). The effect of MYOC shRNA on MYOC expression was assessed in 293T cells. As shown in FIG. 6, MYOC shRNA reduced MYOC protein expression in cells expressing wild-type MYOC, compared to the scrambled shRNA control. This reduction was observed for both intracellular and secreted MYOC.

FIG. 7 shows the effect of MYOC shRNA on hTM-T cells. MYOC shRNA reduced MYOC protein expression in hTM-T cells co-expressing wild-type and P370L mutant MYOC. This reduction was observed for both intracellular and secreted MYOC. In contrast, shRNA targeting Grp94 had no effect on MYOC expression. Grp94 is a molecular chaperone that is involved in the processing and transport of secreted proteins, and was recently proposed as a therapeutic for patients suffering from some cases of MYOC glaucoma because Grp94 is thought to facilitate the clearance of MYOC mutants (Suntharalingam et al., (2012) J. Biol. Chem. 287(48):40661-9). Scrambled shRNA controls also had no effect on MYOC expression.

Since MYOC shRNA affected MYOC expression, its effects on Wnt signaling were next investigated. As shown in FIG. 8, MYOC P370L expression reduced Wnt signaling in 293T cells. Grp94 shRNA and scrambled shRNA controls were unable to restore MYOC P370L-inhibited Wnt signaling. In contrast, MYOC shRNA increased Wnt signaling in MYOC P370L-expressing cells to approximately wild-type levels (i.e., the level of Wnt signaling observed in control cells not expressing MYOC P370L, as measured by TOP-Flash). Expression of RSPO3 was also found to increase Wnt signaling in cells expressing MYOC P370L, and combination expression of RSPO3 with MYOC RNAi (e.g., shRNA) led to a synergistic increase in Wnt signaling in cells expressing MYOC P370L.

Although Grp94 inhibition has been proposed as a mechanism to reduce the effects of MYOC mutants, these results described here indicate that shRNA expression of RSPO3 and/or MYOC may be more effective in derepressing Wnt signaling in the presence of MYOC mutant expression.

The effect of MYOC shRNA on Wnt signaling in cells expressing MYOC Y437H was also examined. As shown in FIG. 9 , expression of MYOC P370L or Y437H reduced Wnt signaling in 293T cells. However, MYOC shRNA was able to restore Wnt signaling in cells expressing MYOC P370L or Y437H. This effect was not observed following expression of a scrambled shRNA control.

In summary, these results demonstrate that Wnt signaling blocked by MYOC mutants (e.g., P370L and Y437H) can be restored by expressing R-spondin 3 (RSPO3) and/or inhibiting MYOC (e.g., by RNAi).

Example 4: AAV2 R471A transduces trabecular meshwork cells

To determine whether AAV particles could transduce trabecular meshwork cells, AAV2 vectors encoding EGFP were encapsidated into wild-type AAV2 particles or AAV2 particles comprising an R471A amino acid substitution (VP1-based numbering). Viral particles were evaluated in vitro by treating hTM cells (described above) with AAV2 EGFP and AAV2 R471A EGFP. As shown in FIG.

10 (left panels), AAV2 R471A EGFP showed increased TM cell transduction compared to wild-type AAV2. To assess TM cell transduction in vivo, AAV2 EGFP and AAV2 R471A EGFP were injected into the eyes of mice. Mice were then sacrificed and analyzed for EGFP expression. As shown in FIG. 10 (right panels), AAV2 R471A EGFP showed increased TM cell transduction in vivo compared to wild-type AAV2.

Example 5: Expression of RSPO3 or MYOC shRNA in animal models of myocilin glaucoma (MYOC)

The above examples demonstrate that glaucomatous MYOC mutations (e.g., P370L and Y437H) block Wnt signaling, and that this inhibition of Wnt signaling can be reversed by expression of R-spondin 3 (RSPO3) or MYOC shRNA. Without wishing to be bound by any particular theory, it is thought that MYOC mutations (e.g., P370L and/or Y437H) may effect Wnt signaling in the TM, thereby modulating IOP and contributing to POAG. The following experiments test whether expression of R-spondin 3 (RSPO3) or MYOC shRNA, delivered via the AAV2 vector, is able to improve glaucoma symptoms in mouse models of the disease.

A mouse model of POAG is used to examine the efficacy of AAV-mediated delivery of R-spondin 3 (RSPO3) expression and/or MYOC shRNA to the eye in the treatment of myocilin glaucoma (MYOC). For example, a mouse model expressing MYOC Y437H can be used (see Zode et al. (2011) J. Clin. Invest. 121(9):3542-53). In this model, human MYOC Y437H is expressed under the control of the CMV promoter in a transgenic mouse. Using this system, MYOC Y437H is expressed in myocilin glaucoma (MYOC)-related tissues such as the trabecular meshwork and sclera. These mice present extremely normal ocular morphology, but begin to show myocilin-like glaucoma (MYOC) symptoms after three months of age, such as increased IOP and progressive axonal degeneration of the optic nerve.

Transgenes expressing GFP, mouse RSPO3, mouse MYOC-targeting shRNA (MYOC shRNA target and loop sequence from plasmid pGIPZ#93; Dharmacon, GE Healthcare), or scrambled shRNA are cloned into an AAV2 genome under the control of a hybrid p-chicken actin (CBA) promoter from plasmid pCBA(2)-int-BGH, which also contains the bovine growth hormone polyadenylation sequence (Xu, R., et al. (2001) Gene Ther. 8:1323-32). The expression cassette is then cloned into a previral plasmid vector pAAVSP70 containing AAV2 inverted terminal repeats (ITRs) (Ziegler, R.J., et al. (2004) Mod. Ther.

9:231-40). The total size of the resulting AAV genome in the sp70.BR/sFLT01 plasmid including the ITR-flanked region is 4.6 kb.

AAV2 genomes are encapsidated into R471A-mutated AAV2 capsids to allow infection of the trabecular meshwork or wild-type AAV2 capsids to allow infection of retinal ganglion cells. AAV2 genomes are encapsidated into either the wild-type or R471A AAV2 capsid using the "gutless" vector approach using a triple transfection method (see, e.g., Xiao et al. (1998) J. Virol., 3:2224-32). Briefly, the rep and cap genes are replaced by the therapeutic gene and its regulatory elements, both sandwiched between a 5' and 3' inverted terminal repeat (ITR). The rep and cap genes are provided in trans on a separate plasmid, and a third plasmid contributes the required adenoviral helper genes. Alternatively, the required helper genes are provided by a replication-deficient adenovirus and/or adenoviral helper genes that are stably integrated into the host cell genome. Without wishing to be bound by any particular theory, it is postulated that the viral capsids are fully assembled, and the ITR-flanked vector genome is then inserted into the capsid through a capsid pore (Myers & Carter (1980) Virology, 102:71-82). The genome-containing capsids are then formulated for injection.

Transgenic mice expressing human Y437H MYOC are cultured until approximately three months of age and then randomly assigned to treatment groups. Mice are anesthetized and injected by intravitreal or intracameral injection with AAV vectors encoding GFP, mouse RSPO3, mouse MYOC-targeting shRNA, or scrambled shRNA. In one treatment group, to test the effects on retinal ganglion cells, one mouse receives one injection of AAV2 vectors with mouse RSPO3 expressing the wild-type AAV2 capsid and one injection of AAV2 vectors with wild-type AAV2 capsid expressing GFP in the contralateral eye. In one treatment group, to test the effects on the trabecular meshwork, a mouse receives one injection of AAV2 vectors with the AAV2 R471A capsid expressing shRNA targeting mouse MYOC and one injection of AAV2 vectors with the AAV2 R471A capsid expressing scrambled shRNA in the contralateral eye. In one treatment group, a mouse receives one injection of a mixture of AAV vectors expressing RSPO3 vectors and mouse AAVs expressing shRNA targeting mouse MYOC in one eye and one injection of AAV vectors expressing GFP vectors and/or AAVs expressing scrambled shRNA in the contralateral eye. In one treatment group, a mouse receives one injection of AAV vectors expressing mouse RSPO3 and expressing shRNA targeting mouse MYOC in one eye and one injection of AAV vectors expressing GFP and expressing scrambled shRNA in the contralateral eye.

Mice are examined at regular intervals after injection for symptoms of myocilin glaucoma (MYOC), comparing the eye receiving experimental treatment with the eye receiving control treatment. IOP is measured by tonometry (Kim, C.Y., et al. (2007) Eye (Lond.) 21(9):1202-9). Corneal thickness is measured by ultrasound pachymeter (Lively, G.D., et al. (2010) Physiol. Genomics 42(2):281-6). The iridocorneal angle is assessed by gonioscopy. Retinal ganglion cell function is measured by analyzing pattern electroretinography responses to visual stimuli using pattern electroretinography (PERG) (Zode et al. (2011) J. Clin. Invest. 121 (9):3542-53). Mice can be euthanized and their eyes dissected for further phenotypic characterization. For example, retinal ganglion cell number and/or morphology are assessed using immunofluorescence microscopy and/or transmission electron microscopy.

Example 6: Use of RSPO family proteins to restore Wnt signaling blocked by glaucomatous MYOC mutation

As demonstrated in Example 3, Wnt signaling blocked by MYOC mutants (e.g., P370L and Y437H) can be restored by expression of R-spondin 3 (RSPO3). To better understand the mechanisms underlying this restoration of Wnt signaling, the ability of different RSPO family members and variants to restore Wnt signaling was examined.

The human RSPO family proteins hRSPO1, 2, 3, and 4 share a similar domain structure that includes furin-like Cys-rich domains, a thrombospondin type I domain, and a carboxy-terminal positively charged domain, as illustrated in FIGS. 11 and 12. To examine the functional domains required for restoration of Wnt signaling, several truncated variants of human RSPO3 were generated. The variants, and the specific domains included and excluded in each variant, are shown in FIGS.

11, 12, 13A and 14.

To test the effect of RSPO3 variants on Wnt signaling, 293T cells were co-transfected with the TOP-Flash reporter construct and either wtMYOC (“MYOC”) or MYOC Y437H, and also transfected with full-length or partial RSPO3 plasmids, as labeled in FIG. 15. WNT signaling was amplified after the addition of recombinant human Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity (mean ± SD, n = 3) was measured after transfection and normalized to the constitutively expressed Renilla luciferase level transfection control.

As described in Example 3, mutant MYOC Y437H inhibits Wnt signaling in 293T cells as measured by the TOP-Flash assay. FIG. 15 shows the effect of the various truncated hRSPO3 variants on Wnt signaling in this assay. As shown in FIG. 15, all forms of hRSPO3 tested, both partial and full length, had Wnt restoration activity with full length RSPO3 exhibiting more potent activity than many of the truncated forms.

To test the effect of different RSPO family members on Wnt signaling, 293T cells were cotransfected with the TOP-Flash reporter construct and wtMYOC ("MYOC") or MYOC Y437H, and also transfected with full-length or partial RSPO1, RSPO2, or RSPO4 plasmids, as labeled in FIG. 16 (see FIG. 12 for representation of truncated forms of RSPO1, 2, 3, and 4). Wnt signaling was amplified after the addition of recombinant mouse Wnt3a (400 ng/ml) and measured by TOP-Flash assay. Luciferase activity (mean ± SD, n = 3) was measured after transfection and normalized to the transfection control level of constitutively expressed Renilla luciferase.

The results of these studies are shown in Fig. 16. These results indicate that full-length and truncated RSPO1, 2, and 4 also had Wnt restoration activity, with full-length RSPOs showing more potent activity than the truncated forms. All RSPO family members and forms worked with Wnt3a.

SEQUENCES

RSPO3 polypeptide sequence (signal sequence underlined)

MHLRLLSWLEIILNrMEYIGSONASRGRRORRMHPNVSOGCOGGCATCSDYNGCLSCKPRLFrALERIGM

KQIÜVCLSSCPSüYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYLIILGKCLDNCPEGLEANNIIT

MECVSIVHCEVSEWPWSPCTKKGKTCGFKRGTETRVREIIQHPSAKGNLCPPTNETRKCTVQRKKCQK

GERGKKGRERKRKKPNKGESKEAIPDSKSLESSKEIPEQRENKOÜQKKRKVQDKQKSVSVSTVII (SEQ

ID NO:l)

Polynucleotide sequence of RSPO3

ATGC ACTTGCG ACTG ATTTCTTGGCTTTTT ATC ATTTTG A ACTTT ATGG A ATACATCGGCAGCCAAAACGCCTCCCGGGGAAGGCGCCAGCGAAGAATGC ATCCT A ACGTT AGTC A AGGCTGCC A AGG AGGCTGTGC A AC ATGCTC AG AT T ACA ATGG ATGTTTGTCATGT AAGCCC AG ACT ATTTTTTGCTCTGG A A AG AATTGGCATGAAGCAGATTGGAGTATGTCTCTCTTCATGTCCAAGTGGAT ATT ATGG AACTCGATATCCAGATATAAATAAGTGTACAAAATGCAAAGCT G ACTGTG AT ACCTGTTTC A AC A A A A ATTTCTGC AC A A A ATGT A A A AGTGG ATTTT ACTT AC ACCTTGG A A AGTGCCTTG AC A ATTGCCC AG A AGGGTTGG

AGTGAATGGAATCCTTGGAGTCCATGCACGAAGAAGGGAAAAACATGTGG CTTCAAAAAGAGGGACTGAAACACGGGTCCGAGAAATACAGCATCCTT C AGC A A AGGGT A ACCTGTGTCCCCC A AC A A ATG AG AC A AG A A AGTGT ACA GTGCAAAGGAAGAAGTGTCAGAAGGGAGAACGAGGAAAAAAAGGAAGGGA G AGG A A A AG A A A A A ACCT A AT A A AGG AG A A AGT A A AG A AGC A AT ACCTG ACAGCAAAAGTCTGGAATCCAGCAAAGAAATCCCAGAGCAACGAGAAAAC A A AC AGC AGC AG A AG A AGCG A A A AGTCC A AG AT A A AC AG A A ATCGGT ATC AGTC AGC ACTGT AC ACT AG (SEQ ID NO:2)

MYOC polypeptide sequence

MRFFCARCCSFGPEMPAVQLLLLACLVWDVGARTAQLRKANDQSGRCQYTFSVASPNESSCPEQSQAM

SVIHNLQRDSSTQRLDLEATKARLSSLbSLLHQLTLDQAARPQETQEGLQRELGTLRRERDQLETQTREL p p g s r e v s t w n l

DTEAFQF.I XSFITFVPASRIIXFSPSGYI ,R SGFGDT GCGFI .VWVGEPT TERTAFTTTGKYGVWMRDPKPT

y p y t q e t t w r id t v g t d v r q v f e y d l is q e m q g y p s k v h il p r p l e st g a v v y s g s l y f q g a e s r t v ir y e l n t e t v k a e k e ip g a g y h g q f p y s w g g y t d id la v d e a g l w v iy st d e a k g a iv l s k l n p e n l e l e q t

WETN1RKQSVANAEIICGTLYTVSSYTSADATVNFAYDTGTG1SKTLT1PFKNRYKYSSMIDYNPLEKKLE

AWDNENMVTYDIKCSKM (SEQ 113 NO:3)

MYOC cDNA sequence

ATGAGGTTCTTCTGTGCACGTTGCTGCAGCTTTGGGCCTGAGATGCCAGCTGTCCAGCTGCTGCTTCT GGCCTGCCTGGTGTGGGATGTGGGGGCCAGGACAGCTCAGCTCAGGAAGGCCAATGACCAGAGTGG CCGATGCCAGTATACCTTCAGTGTGGCCAGTCCCAATGAATCCAGCTGCCCAGAGCAGAGCCAGGC CATGTCAGTCATCCATAACTTACAGAGAGACAGCAGCACCCAACGCTTAGACCTGGAGGCCACCAA AGCTCGACTCAGCTCCCTGGAGAGCCTCCTCCACCAATTGACCTTGGACCAGGCTGCCAGGCCCCAG GAGACCCAGGAGGGGCTGCAGAGGGAGCTGGGCACCCTGAGGCGGGAGCGGGACCAGCTGGAAAC CCAAACCAGAGAGTTGGAGACTGCCTACAGCAACCTCCTCCGAGACAAGTCAGTTCTGGAGGAAGA GAAGAAGCGACTAAGGCAAGAAAATGAGAATCTGGCCAGGAGGTTGGAAAGCAGCAGCCAGGAGG TAGCAAGGCTGAGAAGGGGCCAGTGTCCCCAGACCCGAGACACTGCTCGGGCTGTGCCACCAGGCT CCAGAGAGTTTCTACGTGGAATTTGGACACTTTGGCCTTCCAGGAACTGAAGTCCGAGCTAACTGA AGTTCCTGCTTCCCGAATTTTGAAGGAGAGCCCATCTGGCTATCTCAGGAGTGGAGAGGGAGACAC CGG ATGTGG AG A ACT AGTTTGGGT AGG AG AGCCTCTC ACGCTG AG A AC AGC AG A A AC A ATT ACTGG CAAGTATGGTGTGTGGATGCGAGACCCCAAGCCCACCTACCCCTACACCCAGGAGACCACGTGGAG AATCGACACAGTTGGCACGGATGTCCGCCAGGTTTTTGAGTATGACCTCATCAGCCAGTTTATGCAG GGCT ACCCTTCT A AGGTTC AC AT ACTGCCT AGGCC ACTGG A A AGC ACGGGTGCTGTGGTGT ACTCGG GGAGCCTCTATTTCCAGGGCGCTGAGTCCAGAACTGTCATAAGATATGAGCTGAATACCGAGACAG TGAAGGCTGAGAAGGAAATCCCTGGAGCTGGCTACCACGGACAGTTCCCGTATTCTTGGGGTGGCT ACACGGACATTGACTTGGCTGTGGATGAAGCAGGCCTCTGGGTCATTTACAGCACCGATGAGGCCA

AAGGTGCCATTGTCCTCTCCAAACTGAACCCAGAGAATCTGGAACTCGAACAAACCTGGGAGACAA AC ATCCGT A AGC AGTC AGTCGCC A ATGCCTTC ATC ATCTGTGGC ACCTTGT AC ACCGTC AGC AGCT A CACCTCAGCAGATGCTACCGTCAACTTTGCTTATGACACAGGCACAGGTATCAGCAAGACCCTGACC ATCCCATTCAAGAACCGCTATAAGTACAGCAGCATGATTGACTACAACCCCCTGGAGAAGAAGCTC TTTGCCTGGG AC A ACTTGA AC ATGGTC ACTT ATG AC ATC A AGCTCTCC A AG ATGT AG (SEQ ID NO:4)

MYOC shRNA target sequences

GGCCATGTCAGTCATCCAT (SEQ ID NO:5)

QAMSVIH (SEQ ID NO:6)

shRNA loop sequence

AATAGTGAAGCCACAGATGTATT (SEQ ID NO:7)

RSPO1 polypeptide sequence (signal sequence underlined)

MRIGrECVVALVESWTHLTISSRGIKGKRORRISAEGSOACAKGCEECSEVNGCLKCSPKEEIEEERNDIRQV C.VCLPSCPPGYEDARNPDMNKCIKCKIEHCEACESHNECTKCKEGLYLHKGRCYPACPEGSSAANGTMEC SSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLIIAPVGDIIAACSDTKETRRCTVRRVPCPEGQKR RKGÜQGRRENANRNEARKESKEAGAGSRRRKGQQQQQQGTVGPETSAGPA (SEQ ID NO:8)

RSPO2 polypeptide sequence (signal sequence underlined)

MQFRLFSFALIILNCMDYSIICOGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKLFFFLRREGMRQ YGECLHSOPSGYYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFAPLEETME CVEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKPVKDTILCPTIAESRRCKMTMRHCPGGKRTP KAKEKRNKKKKRKEIERAQEQHSVFT.ATDRANQ (SEQ ID NO:9)

RSPO4 polypeptide sequence (signal sequence underlined)

MRAPLCLLLLVAIIAVDMLALNRRKKOVGTGLGGNCTGCIICSEENGCSTCOORLFLFIRREGIROYGKCLII DCPPGYFGIRGQEVNRCKKCGATCESCFSQDFCIRCKRQFYLYKGKCLPTCPPGTLAHQNTRECQGECELG PWGGWSPCTHNGKTCGSAWÜLESRVREAGRAGHEEAATCQVLSESRKCPIQRPCPGERSPGQKKGRKDR RPRKDRKLDRREDVRPRQPGLQP (SEQ ID NO: 10)

RSPO1 truncation polypeptide sequence 1-135 (signal sequence underlined)

MRLGLCVVALVLSWTHLTISSRGIKGKRORRISAEGSOACAKGCELCSEVNGCLKCSPKLFILLERNDIROV GVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSA (SEQ ID NO: 11)

RSPO1 truncation polypeptide sequence 1-206 (underlined signal sequence)MREGECVVAEVESWTHETTSSRGIKGKRORRISAEGSOACAKGCFJ GSEVNGCJ .KCSPKT.FIT J ERNDTRQV GVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMEC SSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLIIAPVGDIIAAOSDTKETRRCTVRRVPC (SEQ ID NO: 12)

RSPO2 truncation polypeptide sequence 1-134 (signal sequence underlined) MOFRLFSFALIILNCMDYSHCOGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCOOKLFFFLRREGMRO YGECEIISCPSÜYYGIIRAPDMNRCARCRTENCDSCFSKDFCTKCKVGFYUIRGRCFDECPDÜFAP (SEQ TD NO: 13)

RSPO2 truncation polypeptide sequence 1-203 (underlined signal sequence)MOFREFSFAETTENCMDYSHCQGNRWRRSKRASYVSNPICKGCESCSKDNGCSRCQQKI.FFrERREGMRO YGECLHSCPSGYYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFAPLEETME CVEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQlVKKPVKÜTILCl’riAESRRCKMTMRHC (SEQ ID NO: 14)

RSPO3 truncation polypeptide sequence 1-135 (underlined signal sequence)MHLRLISWT FTTT NFMFYTGSONASRGRRORRMHPNVSOGrOGGrATSDYNGGT SGKPRT F F AT F.RTGMK QIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEA (SEQ ID NO: 15)

RSPO3 truncation polypeptide sequence 1-146 (underlined signal sequence)MHLRLISWT FTTT .NFMEYTGSONASRGRRQRRMHPNVSOGCOGGCATCSDYNGCT,SGKPRT FFAT ERTGMK QIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANNHTME CVSIV (SEQ ID NO: 16)

RSPO3 truncation polypeptide sequence 1-206 (signal sequence underlined)MHERETSWTEIIENFMEYTGSONASRGRRORRMHPNVSOGCOGGCATCSDYNGCESCKPREFrAT.ERIGMK QIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEC.LEANNHTME ( WSIVnCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQI IPSAKGNLCPPTNETRKCTVQRKKC (SEQ ID NO: 17)

RSPO4 truncation polypeptide sequence 1-128 (underlined signal sequence) T C E S C FS Q D F C IR C K R Q F Y L Y K G K C L PT C PP G T L A (SEQ ID NO: 18)RSPO4 truncation polypeptide sequence 1-195 (underlined signal sequence)MRAPLCLLLLVAHAVDMLALNRRKKOVGTGLGGNCTGCIICSEENGCSTCOQRLFLPIRREGIROYGKCLH DCPPGYFGIRGQEVNRCKKCGATCESCFSQDFCIRCKRQFYLYKGKCLPTCPPGTLAHQNTRECQGECELG PWGGWSPCT1INGKTCGSAWGEHSRVRKAGRAGI1KKAATCQVLSKSRKCPIQRP (SKQ ID NO: 19)Muted ITR polynucleotide sequence

C A C T C C C T C T C T G C G C G C T C G C T C G C T C A C T G A G G C C G G G C G A C C A A A G G T C G C C C A C G C C C G G G C T T T G C C C G G G C G (SEQ ID NO:20)

MYOC370L forward mutagenesis primer (substitution is underlined)ACCACGGACAGTTCCTGTATTCTTGGGGTGG (SEQ ID NO:21)

MYOC370L reverse mutagenesis primer (substitution is underlined)CCACCCCAAGAATACAGGAACTGTCCGTGT (SEQ ID NO:22)

MYOCY437H forward mutagenesis primer (substitution is underlined)TCTGTGGCACCTTGCACACCGTCAGCAGC (SEQ ID NO:23)

MYOCY437H reverse mutagenesis primer (substitution is underlined) GCTGCTGACGGTGTGCAAGGTGCCACAGA (SEQ ID NO:24)

Claims (13)

1. A recombinant adeno-associated virus (rAAV) particle comprising a vector encoding R-spondin 1 (RSPO1), R-spondin 2 (RSPO2), or R-spondin 4 (RSPO4) for use in a method of treating myocilin-induced glaucoma (MYOC) in a mammal, said method comprising administering said rAAV particle to the eye of the mammal, and wherein RSPO1, RSPO2, or RSPO4 is operably linked to a promoter, wherein: (i) the promoter is capable of expressing RSPO1, RSPO2, or RSPO4 in the eye of the mammal; and/or (ii) the promoter is capable of expressing RSPO1, RSPO2 or RSPO4 in trabecular meshwork cells. 2. The recombinant adeno-associated virus (rAAV) particle for use according to claim 1, wherein said method further comprises administering an rAAV particle comprising a vector encoding a MYOC RNAi that targets the expression of a myocilin in the mammal. 3. The recombinant adeno-associated virus (rAAV) particle for use according to claim 1, wherein the vector further encodes a MYOC RNAi that targets the expression of a myocilin in the mammal. 4. The AAV particle for use according to any one of claims 1-3, wherein (i) Myocilin glaucoma (MYOC) is a primary open angle glaucoma (POAG); (ii) MYOC glaucoma is the juvenile form of primary open-angle glaucoma; and/or (iii) the mammal is a human, and the MYOC glaucoma is associated with a mutation in a human myocilin comprising one or more amino acid substitutions selected from E323K, K398R, Q368X, G364V, P370L, D380A, K423E, Y437H, and I477S. 5. The AAV particle for use according to any one of claims 1-4, wherein (i) at least 1 x 109 copies of the rAAV particle genome are administered to the mammal; (ii) the rAAV particle is administered by intravitreal injection and/or intracameral injection; (iii) the rAAV particle is delivered to more than one location in the eye; (iv) the rAAV particle is in a pharmaceutical composition. 6. A recombinant AAV particle comprising an AAV vector, wherein the AAV vector comprises nucleic acid encoding RSPO1, RSPO2 or RSPO4, and wherein RSPO1, RSPO2 or RSPO4 is operably linked to a promoter, wherein: (i) the promoter is capable of expressing RSPO1, RSPO2 or RSPO4 in the mammalian eye; and/or (ii) the promoter is capable of expressing RSPO1, RSPO2 or RSPO4 in trabecular meshwork cells, optionally, wherein the nucleic acid further encodes an inhibitory nucleic acid that targets the expression of a myocilin (MYOC) in a mammal. 7. The rAAV particle for use according to any one of claims 1-5 or the rAAV particle according to claim 6, wherein: (i) RSPO1 is a human RSPO1 or has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to human RSPO1, optionally wherein RSPO1 comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 12 or an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 12; (ii) RSPO2 is a human RSPO2 or has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to human RSPO2, optionally wherein RSPO2 comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 14 or an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 14; or (iii) RSPO4 is a human RSPO4 or has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to human RSPO4, optionally wherein RSPO4 comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 18, or SEQ ID NO: 19 or an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 18, or SEQ ID NO: 19. 8. The rAAV particle for use according to any one of claims 1-5 or 7, or the rAAV particle according to claim 6 or claim 7, wherein the promoter is a chicken β-actin hybrid promoter (CBA). 9. The rAAV particle for use according to any one of claims 2-5 or 7-8, or the rAAV particle according to any one of claims 6-8, wherein: (i) the MYOC RNAi targets the amino acid sequence of MYOC set forth in SEQ ID NO: 6; (ii) the RNAi is a MYOC shRNA comprising the loop sequence of SEQ ID NO:7; and/or (iii) MYOC RNAi is operably linked to a chicken p-actin (CBA) hybrid promoter, or an NI RNA polymerase promoter. 10. The rAAV particle for use according to any one of claims 1-5 or 7-9, or the rAAV particle according to any one of claims 6-9, wherein: (i) the rAAV particle comprises a capsid of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6 ShH10, AaV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, bovine AAV, goat AaV or mouse AAV or a capsid of serotype AAV of clades A-F; (ii) the rAAV particle comprises a tyrosine capsid mutant, a heparin-binding capsid mutant, an AAV2R471A capsid, an AAVAV2/2-7m8 capsid, an AAV DJ capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, an AAV1/AAV2 chimeric capsid, or an AAV2/HboV1 capsid; and/or (iii) the rAAV particle comprises an AAV capsid comprising an amino acid substitution at one or more of positions R484, R487, K527, K532, R585, or R588, numbering based on VP1 of AAV2. 11. The rAAV particle according to any one of claims 1-5 or 7-10, or the rAAV particle according to any one of claims 6-10, wherein: (i) the vector comprises inverted terminal repeats (ITRs) of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV, or mouse AAV; and/or (ii) the vector comprises AAV serotype 2 ITRs, optionally wherein: (a) the rAAV particle comprises one or more ITRs and the capsid derived from the same AAV serotype; or (b) the rAAV particle comprises one or more ITRs derived from an AAV serotype different from the capsid of the rAAV particles. 12. The rAAV particle for use according to claim 10, or the rAAV particle according to claim 10, wherein the rAAV particle comprises an AAV serotype 2 capsid, and optionally wherein: (i) the AAV serotype 2 capsid comprises the AAV2 capsid protein comprising an amino acid substitution R471A, numbering relative to AAV2 VP1; and/or (ii) the vector comprises AAV2 ITR. 13. A kit for treating myocilin glaucoma (MYOC) in a mammal comprising the rAAV particle of any one of claims 6-12.