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Crispr/cas9-based compositions and methods for treating retinal degenerations — The Johns Hopkins University (US20240336934A1)

ABSTRACT

Abstract

Described herein are methods for treating a retinal degeneration in a subject, such as Leber's congenital amaurosis (LCA), retinitis pigmentosa (RP), and glaucoma. Also provided herein are methods of altering expression of one or more gene products in a cell, such as a retinal ganglion cell. Such methods may comprise utilizing a modified nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising a bidirectional HI promoter and gRNAs directed to retinal degeneration related genes, packaged in a single, compact adeno-associated virus (AAV) particle.

Description

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No. 62/358,337, filed Jul. 5, 2016, the entirety of which is hereby incorporated by reference.

BACKGROUND

Retinal degenerations are a group of disorders which include Leber's congenital amaurosis (LCA), retinitis pigmentosa (RP), and glaucoma, among others. LCA is a heritable form of retinal degeneration characterized by severe retinal dysfunction and severe visual impairment during the first months of life. LCA is an orphan disease (one that affects fewer than 200,000 Americans), but the 18 subtypes of LCA are together the most common cause of inherited blindness. The subtype designated LCA10, which is the most common subtype, accounting for >20% of all LCA cases. Some forms of LCA are amenable to treatment by recombinant adeno-associated viruses (AAVs) engineered to deliver a functional copy of the defective cellular gene. In 2008, a transgene that complemented the mutation in RPE65 was successfully delivered by AAV to LCA2 patients in a Phase I Clinical trial (Maguire A M et al. N Engl J Med. 2008; 358(21): 2240-2248). Some responses were noted, but these were not durable because transgene expression was eventually lost (Schimmer J et al. Hum Gene Ther Clin Dev. 2015; 26(4): 208-210; Azvolinsky A. Nat Biotechnol. 2015; 33(7): 678-678). Furthermore, some of the genes that cause the different LCA subtypes are simply too large for AAV delivery. These subtypes of LCA therefore remain untreatable.

The ADRP constitutes approximately 30-40% of all cases of RP, and among ADRP patients the most commonly mutated RP associated gene is the one that encodes the rod visual pigment rhodopsin (Dryja, T. P. et al. The New England journal of medicine 323, 1302-1307 (1990); Dryja, T. P. et al. Nature 343, 364-366 (1990)). At the moment, there are no FDA approved treatments for ADRP patients; however, a number of approaches are being developed. Most of these approaches are variations on the theme of “suppression and replacement.” In this approach, one knocks down expression of the gene responsible for degeneration, for example knocking down levels of rhodopsin RNA with a ribozyme or via RNA interference (RNAi) (both shRNAs and siRNA methodologies are being explored), and then replaces expression of the endogenous alleles with a “hardened” gene that is not susceptible to knock down by the ribozyme or RNAi agent. The variant of this theme that is perhaps closest to the clinic is the RhoNova agent being developed by Genable Technologies Limited. RhoNova employs an siRNA to knock down endogenous rhodopsin expression (both mutant and wild-type) combined with an AAV-delivered cDNA that encodes a modified but functional rhodopsin that is not susceptible to siRNA knock down (http://www.genable.net).

Glaucoma, the leading cause of irreversible blindness worldwide (Levkovitch-Verbin H et al. iovsorg 44, 3388-3393 (2003)), is an optic neuropathy in which progressive damage of retinal ganglion cell (RGC) axons at the lamina cribosa of the optic nerve head leads to axon degeneration and cell death (Howell G R et al. J Cell Biol 179, 1523-1537 (2007)). Currently, the only treatment, whether by eye drops, lasers or incisional surgery, is to lower intraocular pressure (IOP) and reduce the injury at the optic nerve head. Unfortunately, this is difficult in some patients while in others, the disease can continue to worsens despite aggressive IOP-lowering. The field has long needed an alternative therapeutic strategy that could complement IOP-lowering by mitigating the RGC response to residual axon injury. Moreover, the NEI has listed optic nerve regeneration amongst its Audacious Goals, and any regenerative therapy necessarily needs to tackle the issue of axotomized RGC survival. To this end, there is a great need to develop a neuroprotective that might directly interfere with the active genetic programs of RGC axon degeneration and/or axon injury-related cell death (Adalbert R et al. Science (2012), doi:10.1126/science.1223899; Yang J et al. Cell 160, 161-176 (2015); Welsbie D S et al. Proc Nat Acad Sci USA 110, 4045-4050 (2013); Watkins T A et al. Proc Nat Acad Sci USA 110, 4039-4044 (2013)).

Thus there is a great need for novel and improved therapies for treating retinal degenerations, like LCA, ADRP, and glaucoma.

SUMMARY

The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science , and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning. A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange 10 th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, available on the World Wide Web: http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), available on the World Wide Web: http://omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

Described herein are methods for treating retinal degenerations, such as optic neuropathies including Leper's congenital amaurosis (e.g., Leber's congenital amaurosis 10 CEP290 mutation (LCA)), retinitis pigmentosa (e.g., Rhodopsin R135 mutations), or glaucoma. The methods use a modified nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (e.g. CRISPR associated (Cas) 9 (CRISPR-Cas9, non-Cas9 CRISPR systems, CRISPR-Cpf-1 system, and the like), to cut and/or repair genomic DNA or RNA (e.g., Cas13a/C2c2 system). The CRISPR-system-based gene editing can be used to inactivate or correct gene mutations causing optic neuropathies and retinal degenerations (e.g., LCA and rhodopsin mutations), thereby providing a gene therapy approach for these groups of diseases. In some embodiments, the CRISPR system is used to introduce a mutation that will inactivate a normal gene (e.g. DLK and/or LZK) causing retinal degeneration (e.g. glaucoma). Because these genes play roles in damage-sensing and cell-survival, the resulting effect is cell survival. The “neuroprotective” approach is not a mutually exclusive approach as there are genetic mutations that could lead to glaucoma as well, and these would be the same as the retinal degenerations. In some embodiments, the mutation targets of glaucoma include, but not limited to, OPTN, TBK1, TMCO1, PMM2, GMDS, GAS7, FNDC3B, TXNRD2, ATXN2, CAV1/CAV2, p16INK4a, SIX6, ABCA1, AFAP1 and CDKN2B-AS.

Thus, one aspect of the invention relates to a method for treating a disorder (e.g., retinal degenerations) affecting a retina area of a subject, the method comprising administering to the retina area of the subject a therapeutically effective amount of a nuclease system comprising a genome targeted nuclease and a guide DNA comprising at least one targeted genomic sequence.

Another aspect of the invention provides methods for treating retinal degenerations utilize a composition comprising a modification of a non-naturally occurring CRISPR system previously described in WO2015/195621 (herein incorporated by reference in its entirety). Such a modification uses certain gRNAs that target retinal degeneration-related genes, such as, but not limited, to LCA10 CEP290 gene, rhodopsin, Dual Leucine Zipper Kinase (DLK), Leucine Zipper Kinase (LZK), JNK1-3, MKK4, MKK7, ATF2, JUN, MEF2A, SOX11, or PUMA. In some embodiments, the composition comprises (a) a non-naturally occurring nuclease system (e.g., CRISPR) comprising one or more vectors comprising: i) a promoter (e.g., bidirectional H1 promoter) operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease (e.g., Cas9 protein), wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products. In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle. In some embodiments, the promoter comprises: a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a gRNA; and b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a genome-targeted nuclease.

Another aspect of the invention provides methods of altering expression of one or more gene products in a eukaryotic cell, wherein the cell comprises a DNA molecule encoding the one or more gene products, the method comprising introducing into the cell a modified non-naturally occurring CRISPR system previously described in WO2015/195621 (herein incorporated by reference in its entirety). Such a modification uses certain gRNAs that target retinal degeneration-related genes, such as, but not limited, to LCA10 CEP290 gene, rhodopsin, Dual Leucine Zipper Kinase (DLK), Leucine Zipper Kinase (LZK), JNK1-3, MKK4, MKK7, ATF2, JUN, MEF2A, SOX11, or PUMA. In some embodiments, the method comprising introducing into the cell a composition comprising (a) a non-naturally occurring nuclease system (e.g., CRISPR) comprising one or more vectors comprising: i) a promoter (e.g., bidirectional H1 promoter) operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease (e.g., Cas9 protein), wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products. In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle. In some embodiments, the promoter comprises: a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a gRNA; and b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a genome-targeted nuclease.

One aspect of the invention, relates to a method for treating a retinal degeneration in a subject in need thereof, the method comprising: (a) providing a non-naturally occurring nuclease system comprising one or more vectors comprising: i) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease, wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products; and (b) administering to the retinal area of the subject a therapeutically effective amount of the system.

In some embodiments, the system is CRISPR.

In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle.

In some embodiments, the system inactivates one or more gene products.

In some embodiments, the nuclease system excises at least one gene mutation.

In some embodiments, the promoter comprises a bidirectional promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 739-787. In some embodiments, the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 739-787.

In some embodiments, the bidirectional promoter is H1 (SEQ ID NO: 787). The H1 promoter is both a pol II and pol III promoter. In some embodiments, the promoter is orthologous to the H1 promoter.

In some embodiments, the orthologous H1 promoter is derived from eutherian mammals.

In some embodiments, the orthologous H1 promoter is derived from Ailuropoda melanoleuca, Bos taurus, Callithrix jacchus, Canis familiaris, Cavia porcellus, Chlorocebus sabaeus, Choloepus hofmanni, Dasypus novemcinctus, Dipodomys ordii, Equus caballus, Erinaceus europaeus, Felis catus, Gorilla gorilla, Homo sapiens, Ictidomys tridecemlineatus, Loxodonta africana, Macaca mulatta, Mus musculus, Mustela putorius furo, Myotis lucifugus, Nomascus leucogenys, Ochotona princeps, Oryctolagus cuniculus, Otolemur garnettii, Ovis aries, Pan troglodytes, Papio anubis, Pongo abelii, Procavia capensis, Pteropus vampyrus, Rattus norvegicus, Sus scrofa, Tarsius syrichta, Tupaia belangeri, Tursiops truncatus, Vicugna pacos.

In some embodiments, the orthologous H1 promoter is derived from mouse or rat.

In some embodiments, the orthologous H1 promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NOs: 752-786.

In some embodiments, the orthologous H1 promoter comprises a nucleotide sequences set forth in the group consisting of SEQ ID NOs: 752-786.

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CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No. 62/358,337, filed Jul. 5, 2016, the entirety of which is hereby incorporated by reference.

BACKGROUND

Retinal degenerations are a group of disorders which include Leber&#39;s congenital amaurosis (LCA), retinitis pigmentosa (RP), and glaucoma, among others. LCA is a heritable form of retinal degeneration characterized by severe retinal dysfunction and severe visual impairment during the first months of life. LCA is an orphan disease (one that affects fewer than 200,000 Americans), but the 18 subtypes of LCA are together the most common cause of inherited blindness. The subtype designated LCA10, which is the most common subtype, accounting for &gt;20% of all LCA cases. Some forms of LCA are amenable to treatment by recombinant adeno-associated viruses (AAVs) engineered to deliver a functional copy of the defective cellular gene. In 2008, a transgene that complemented the mutation in RPE65 was successfully delivered by AAV to LCA2 patients in a Phase I Clinical trial (Maguire A M et al. N Engl J Med. 2008; 358(21): 2240-2248). Some responses were noted, but these were not durable because transgene expression was eventually lost (Schimmer J et al. Hum Gene Ther Clin Dev. 2015; 26(4): 208-210; Azvolinsky A. Nat Biotechnol. 2015; 33(7): 678-678). Furthermore, some of the genes that cause the different LCA subtypes are simply too large for AAV delivery. These subtypes of LCA therefore remain untreatable.

The ADRP constitutes approximately 30-40% of all cases of RP, and among ADRP patients the most commonly mutated RP associated gene is the one that encodes the rod visual pigment rhodopsin (Dryja, T. P. et al. The New England journal of medicine 323, 1302-1307 (1990); Dryja, T. P. et al. Nature 343, 364-366 (1990)). At the moment, there are no FDA approved treatments for ADRP patients; however, a number of approaches are being developed. Most of these approaches are variations on the theme of “suppression and replacement.” In this approach, one knocks down expression of the gene responsible for degeneration, for example knocking down levels of rhodopsin RNA with a ribozyme or via RNA interference (RNAi) (both shRNAs and siRNA methodologies are being explored), and then replaces expression of the endogenous alleles with a “hardened” gene that is not susceptible to knock down by the ribozyme or RNAi agent. The variant of this theme that is perhaps closest to the clinic is the RhoNova agent being developed by Genable Technologies Limited. RhoNova employs an siRNA to knock down endogenous rhodopsin expression (both mutant and wild-type) combined with an AAV-delivered cDNA that encodes a modified but functional rhodopsin that is not susceptible to siRNA knock down (http://www.genable.net).

Glaucoma, the leading cause of irreversible blindness worldwide (Levkovitch-Verbin H et al. iovsorg 44, 3388-3393 (2003)), is an optic neuropathy in which progressive damage of retinal ganglion cell (RGC) axons at the lamina cribosa of the optic nerve head leads to axon degeneration and cell death (Howell G R et al. J Cell Biol 179, 1523-1537 (2007)). Currently, the only treatment, whether by eye drops, lasers or incisional surgery, is to lower intraocular pressure (IOP) and reduce the injury at the optic nerve head. Unfortunately, this is difficult in some patients while in others, the disease can continue to worsens despite aggressive IOP-lowering. The field has long needed an alternative therapeutic strategy that could complement IOP-lowering by mitigating the RGC response to residual axon injury. Moreover, the NEI has listed optic nerve regeneration amongst its Audacious Goals, and any regenerative therapy necessarily needs to tackle the issue of axotomized RGC survival. To this end, there is a great need to develop a neuroprotective that might directly interfere with the active genetic programs of RGC axon degeneration and/or axon injury-related cell death (Adalbert R et al. Science (2012), doi:10.1126/science.1223899; Yang J et al. Cell 160, 161-176 (2015); Welsbie D S et al. Proc Nat Acad Sci USA 110, 4045-4050 (2013); Watkins T A et al. Proc Nat Acad Sci USA 110, 4039-4044 (2013)).

Thus there is a great need for novel and improved therapies for treating retinal degenerations, like LCA, ADRP, and glaucoma.

SUMMARY

The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science , and Current Protocols in Cell Biology, all John Wiley &amp; Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning. A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley &amp; Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman&#39;s The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton &amp; Lange 10 th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, available on the World Wide Web: http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), available on the World Wide Web: http://omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

Described herein are methods for treating retinal degenerations, such as optic neuropathies including Leper&#39;s congenital amaurosis (e.g., Leber&#39;s congenital amaurosis 10 CEP290 mutation (LCA)), retinitis pigmentosa (e.g., Rhodopsin R135 mutations), or glaucoma. The methods use a modified nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (e.g. CRISPR associated (Cas) 9 (CRISPR-Cas9, non-Cas9 CRISPR systems, CRISPR-Cpf-1 system, and the like), to cut and/or repair genomic DNA or RNA (e.g., Cas13a/C2c2 system). The CRISPR-system-based gene editing can be used to inactivate or correct gene mutations causing optic neuropathies and retinal degenerations (e.g., LCA and rhodopsin mutations), thereby providing a gene therapy approach for these groups of diseases. In some embodiments, the CRISPR system is used to introduce a mutation that will inactivate a normal gene (e.g. DLK and/or LZK) causing retinal degeneration (e.g. glaucoma). Because these genes play roles in damage-sensing and cell-survival, the resulting effect is cell survival. The “neuroprotective” approach is not a mutually exclusive approach as there are genetic mutations that could lead to glaucoma as well, and these would be the same as the retinal degenerations. In some embodiments, the mutation targets of glaucoma include, but not limited to, OPTN, TBK1, TMCO1, PMM2, GMDS, GAS7, FNDC3B, TXNRD2, ATXN2, CAV1/CAV2, p16INK4a, SIX6, ABCA1, AFAP1 and CDKN2B-AS.

Thus, one aspect of the invention relates to a method for treating a disorder (e.g., retinal degenerations) affecting a retina area of a subject, the method comprising administering to the retina area of the subject a therapeutically effective amount of a nuclease system comprising a genome targeted nuclease and a guide DNA comprising at least one targeted genomic sequence.

Another aspect of the invention provides methods for treating retinal degenerations utilize a composition comprising a modification of a non-naturally occurring CRISPR system previously described in WO2015/195621 (herein incorporated by reference in its entirety). Such a modification uses certain gRNAs that target retinal degeneration-related genes, such as, but not limited, to LCA10 CEP290 gene, rhodopsin, Dual Leucine Zipper Kinase (DLK), Leucine Zipper Kinase (LZK), JNK1-3, MKK4, MKK7, ATF2, JUN, MEF2A, SOX11, or PUMA. In some embodiments, the composition comprises (a) a non-naturally occurring nuclease system (e.g., CRISPR) comprising one or more vectors comprising: i) a promoter (e.g., bidirectional H1 promoter) operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease (e.g., Cas9 protein), wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products. In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle. In some embodiments, the promoter comprises: a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a gRNA; and b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a genome-targeted nuclease.

Another aspect of the invention provides methods of altering expression of one or more gene products in a eukaryotic cell, wherein the cell comprises a DNA molecule encoding the one or more gene products, the method comprising introducing into the cell a modified non-naturally occurring CRISPR system previously described in WO2015/195621 (herein incorporated by reference in its entirety). Such a modification uses certain gRNAs that target retinal degeneration-related genes, such as, but not limited, to LCA10 CEP290 gene, rhodopsin, Dual Leucine Zipper Kinase (DLK), Leucine Zipper Kinase (LZK), JNK1-3, MKK4, MKK7, ATF2, JUN, MEF2A, SOX11, or PUMA. In some embodiments, the method comprising introducing into the cell a composition comprising (a) a non-naturally occurring nuclease system (e.g., CRISPR) comprising one or more vectors comprising: i) a promoter (e.g., bidirectional H1 promoter) operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease (e.g., Cas9 protein), wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products. In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle. In some embodiments, the promoter comprises: a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a gRNA; and b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a genome-targeted nuclease.

One aspect of the invention, relates to a method for treating a retinal degeneration in a subject in need thereof, the method comprising: (a) providing a non-naturally occurring nuclease system comprising one or more vectors comprising: i) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease, wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products; and (b) administering to the retinal area of the subject a therapeutically effective amount of the system.

In some embodiments, the system is CRISPR.

In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle.

In some embodiments, the system inactivates one or more gene products.

In some embodiments, the nuclease system excises at least one gene mutation.

In some embodiments, the promoter comprises a bidirectional promoter. In some embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 739-787. In some embodiments, the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 739-787.

In some embodiments, the bidirectional promoter is H1 (SEQ ID NO: 787). The H1 promoter is both a pol II and pol III promoter. In some embodiments, the promoter is orthologous to the H1 promoter.

In some embodiments, the orthologous H1 promoter is derived from eutherian mammals.

In some embodiments, the orthologous H1 promoter is derived from Ailuropoda melanoleuca, Bos taurus, Callithrix jacchus, Canis familiaris, Cavia porcellus, Chlorocebus sabaeus, Choloepus hofmanni, Dasypus novemcinctus, Dipodomys ordii, Equus caballus, Erinaceus europaeus, Felis catus, Gorilla gorilla, Homo sapiens, Ictidomys tridecemlineatus, Loxodonta africana, Macaca mulatta, Mus musculus, Mustela putorius furo, Myotis lucifugus, Nomascus leucogenys, Ochotona princeps, Oryctolagus cuniculus, Otolemur garnettii, Ovis aries, Pan troglodytes, Papio anubis, Pongo abelii, Procavia capensis, Pteropus vampyrus, Rattus norvegicus, Sus scrofa, Tarsius syrichta, Tupaia belangeri, Tursiops truncatus, Vicugna pacos.

In some embodiments, the orthologous H1 promoter is derived from mouse or rat.

In some embodiments, the orthologous H1 promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NOs: 752-786.

In some embodiments, the orthologous H1 promoter comprises a nucleotide sequences set forth in the group consisting of SEQ ID NOs: 752-786.

In some embodiments, the H1 promoter comprises: a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a gRNA; and b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a genome-targeted nuclease.

In some embodiments, the genome-targeted nuclease is Cas9 protein.

In some embodiments, the Cas9 protein is codon optimized for expression in the cell.

In some embodiments, the promoter is operably linked to at least one, two, three, four, five, six, seven, eight, nine, or ten gRNA.

In some embodiments, the retinal area is the retina.

In some embodiments, the cell is a retinal photoreceptor cell.

In some embodiments, the cell is a retinal ganglion cell.

In some embodiments, the retinal degeneration is selected from the group consisting of Leber&#39;s congenital amaurosis (LCA), retinitis pigmentosa (RP), and glaucoma.

In some embodiments, the retinal degeneration is LCA1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

In some embodiments, the retinal degeneration is LCA10.

In some embodiments, the target sequence is in the LCA10 CEP290 gene.

In some embodiments, the target sequence is a mutation in the CEP290 gene.

In some embodiments, the target sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 1-109, 164-356, 735-738, or combinations thereof.

In some embodiments, the target sequence comprises SEQ ID NOs: 1, 2, 3, and 4 operably linked.

In some embodiments, the vector comprises the nucleotide sequence set forth in SEQ ID NO: 110.

In some embodiments, the retinal degeneration is an autosomal dominant form of retinitis pigmentosa (ADRP).

In some embodiments, the one or more gene products are rhodopsin.

In some embodiments, the target sequence is a mutation in the rhodopsin gene.

In some embodiments, the target sequence is a mutation at R135 of the rhodopsin gene.

In some embodiments, the mutation at R135 is selected from the group consisting of R135G, R135W, R135L.

In some embodiments, the target sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 111-126, or combinations thereof.

In some embodiments, the gRNA sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 127-142, or combinations thereof.

In some embodiments, the retinal degeneration is glaucoma.

In some embodiments, the one or more gene products are Dual Leucine Zipper Kinase (DLK), Leucine Zipper Kinase (LZK), ATF2, JUN, sex determining region Y (SRY)-box 11 (SOX11), myocyte enhancer factor 2A (MEF2A), JNK1-3, MKK4, MKK7, SOX11, or PUMA, or combinations thereof.

In some embodiments, the one or more gene product are members of the DLK/LZK, MKK4/7, JNK1/2/3 or SOX11/ATF2/JUN/MEF2A pathway.

In some embodiments, the target sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 143-163, or combinations thereof.

In some embodiments, administering to the subject occurs by implantation, injection, or virally.

In some embodiments, administering to the subject occurs by subretinal injection.

In some embodiments, the subject is human.

Another aspect of the invention relates to a method of altering expression of one or more gene products in a cell, wherein the cell comprises a DNA molecule encoding the one or more gene products, the method comprising introducing into the cell a non-naturally occurring nuclease system comprising one or more vectors comprising: a) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of the DNA molecule; and

b) a regulatory element operable in the cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease,

wherein components (a) and (b) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products.

In some embodiments, the system is CRISPR.

In some embodiments, the system is packaged into a single adeno-associated virus (AAV) particle.

In some embodiments, the system inactivates one or more gene products.

In some embodiments, the nuclease system excises at least one gene mutation.

In some embodiments, the promoter is a bidirectional promoter.

In some embodiments, the bidirectional promoter is H1. The H1 promoter is both a pol II and pol III promoter.

In some embodiments, the H1 promoter comprises: a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a gRNA; and b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a genome-targeted nuclease.

In some embodiments, the genome-targeted nuclease is Cas9.

In some embodiments, the Cas9 protein is codon optimized for expression in the cell.

In some embodiments, the promoter is operably linked to at least one, two, three, four, five, six, seven, eight, nine, or ten gRNA.

In some embodiments, the cell is a eukaryotic or non-eukaryotic cell.

In some embodiments, the eukaryotic cell is a mammalian or human cell.

In some embodiments, the cell is a retinal photoreceptor cell.

In some embodiments, the cell is a retinal ganglion cell.

In some embodiments, the one or more gene products are LCA10 CEP290.

In some embodiments, the target sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 1-109, 164-356, 735-738, or combinations thereof.

In some embodiments, the target sequence comprises SEQ ID NOs: 1, 2, 3, and 4 operably linked.

In some embodiments, the vector comprises the nucleotide sequence set forth in SEQ ID NO: 110.

In some embodiments, the one or more gene products are rhodopsin.

In some embodiments, the target sequence is a mutation in the rhodopsin gene.

In some embodiments, the target sequence is a mutation at R135 of the rhodopsin gene.

In some embodiments, the mutation at R135 is selected from the group consisting of R135G, R135W, R135L.

In some embodiments, the target sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 111-126, or combinations thereof.

In some embodiments, the gRNA sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 127-142, or combinations thereof.

In some embodiments, the one or more gene products are Dual Leucine Zipper Kinase (DLK), Leucine Zipper Kinase (LZK), ATF2, JUN, sex determining region Y (SRY)-box 11 (SOX11), myocyte enhancer factor 2A (MEF2A), JNK1-3, MKK4, MKK7, SOX11, or PUMA, or combinations thereof.

In some embodiments, the one or more gene product are members of the DLK/LZK, MKK4/7, JNK1/2/3 or SOX11/ATF2/JUN/MEF2A pathway.

In some embodiments, the target sequence is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 143-163, or combinations thereof.

In some embodiments, the expression of the one or more gene products is decreased.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1 shows the platform technology. The genomic sequence of the H1 bidirectional promoter with a pol II transcript shown in blue and a pol III transcript shown in orange (left). Packaging of the spCas9 and a gRNA within a single AAV vector (right).

FIG. 2 shows delivery of AAV-H1-CRISPR to the mouse retina. Virus engineered to express GFP in place of Cas9 demonstrates both efficient and specific transduction of mouse photoreceptors in the outer nuclear layer (ONL) after sub-retinal injection. These are the cells that are affected by LCA mutations.

FIG. 3 shows an illustration of the Cas9 nickase approach, which require two closely opposed target sites (L and R) on opposite strands.

FIG. 4 shows the LCA10 mutation. The four identified gRNA sites that would result in ˜1 kb deletion, removing the cryptic Exon X from CEP290.

FIG. 5 shows the current SaCas9 approach delivering 2 gRNAs at 4550 bp (left) versus compact H1 system delivering 4 gRNAs using 4335 bp (right). The AAV packaging capacity is indicated by the dotted line.

FIG. 6 shows all SaCas9 sites (1 kb upstream and 1 kb downstream of the CEP290 mutation).

FIG. 7 shows the SaCas9 sites available for targeting (All start with 5′G).

FIG. 8 shows a much safer SaCas9 nickase approach.

FIG. 9 shows a SaCas9 nickase deletion (1078 bp).

FIG. 10 shows potential SpCas9 sites.

FIG. 11 shows the number of CRISPR sites for SaCas9 or SpCas9 by 5′ nucleotide in the CEP290 (LCA10) targeting region.

FIG. 12 shows cloned ˜4.2 kb SaCas9 construct with four gRNAs.

FIG. 13 shows the rhodopsin gene structure.

FIG. 14 shows mutation spectrum of the RHO gene worldwide (from http://www.hindawi.com/journals/bmri/2014/302487/)

FIG. 15 shows the rhodopsin Arg135 mutation.

FIG. 16 shows the R135W Pedigree of 2 French Families (from Audo I et al. Invest Ophthalmol Vis Sci . (2010) July; 51(7):3687-700).

FIG. 17 shows the R135W from 5-generation Sicilian Pedigree (from Pannarale M R et al. Ophthalmology . (1996) September; 103(9):1443-52).

FIG. 18 shows the Six generation Swedish Family with R135L (from Andréasson S et al. Ophthalmic Paediatr Genet . (1992) September; 13(3):145-53).

FIG. 19 shows sensitized kinome screen identifies LZK as cooperating with DLK to promote RGC cell death.

FIG. 20 shows whole genome siRNA screens identify ATF2, SOX11, and MEF2A as mediators of RGC cell death.

FIG. 21 shows downstream mediators of LZK/DLK-dependent RGC cell death.

FIG. 22 shows calcium-sensing motif in LZK is dispensable for toxicity.

FIG. 23 shows hammerhead ribozyme-sgRNA fusions to increase the number of targetable spCas9 sites.

FIG. 24 shows network-based siRNA and siPOOL screening has improved sensitivity and specificity.

FIG. 25 shows H1 promoter allows for bidirectional expression of Pol II and Pol III transcripts.

FIG. 26 shows flow cytometry-based quantification of RGCs.

FIG. 27 shows CRISPR targeting of DLK exon 1 (A) and exon 2 (B) in vitro. Exon 1 and Exon 2 of DLK with target sites shown in blue, and T7E1 primers shown in green.

FIG. 28 comprises two panels, A and B, showing CRISPR targeting of DLK in vitro. FIG. 28 A shows screening gRNAs for their ability to target the DLK gene from mouse. Target site nomenclature is according to http://crispr.technology. FIG. 28 B shows in vitro cleavage using a bidirectional promoter to express Cas9 and a gRNA demonstrates efficient targeting of the DLK locus. The control is a standard 2-plasmid transfection for Cas9 and a gRNA. FIG. 28 B shows experiments testing the ability to drive both Cas9 and a gRNA from the H1 bidirectional promoter. Cells in culture were transfected with either the standard two plasmids (Cas9 and gRNA) or a single plasmid using the H1 bidirectional promoter. T7EI assay indicates comparable levels of cutting using either system.

FIG. 29 shows DLK targeting by AAV in vitro.

FIG. 30 comprises three panels, A, B, and C, showing bidirectional expression in RGCs in vivo. FIG. 30 A shows construct that was packaged into AAV. FIG. 30 B shows the cell-type expression of GFP in vivo is affected by the AAV serotype used. Top shows preferential expression in photoreceptors and the lower panel shows preferential expression in RGCs. The H1 promoter clearly expresses GFP in either photoreceptor cells delivered by AAV5, or in retinal ganglion cells by AAV2 (control). Both viruses were delivered by subretinal injection to P0.5 day mice. Both were delivered by sub-retinal injection of the reporter indicated in FIG. 30 A . FIG. 30 C shows GFP expression by flatmount following 15 days of AAV2 intravitreal delivery of the reporter construct.

FIG. 31 comprises three panels, A, B, and C, showing bidirectional targeting using self-complementary AAV viruses. FIG. 31 A shows self-complementary AAV construct that expresses a nuclear mCherry and a gRNA from a bidirectional promoter. This figure further shows experiments testing the ability to use self-complementary AAV (scAAV) to delivery a gRNA and a fluorescent reporter protein (H2B-mCherry). Cells were harvested from the Cas9 mouse, and transduced in vitro. The benefits of scAAV is the ability to test constructs much faster as expression occurs in days and not weeks. FIG. 31 A shows a construct was generated using the H1 promoter to express a gRNA (shown in black) and mCherry simultaneously. The gRNA targets the DLK mouse gene, a gene that when inactivated, results in enhanced retinal ganglion cell survival. The construct was packaged to produce a self-complementary AAV (scAAV). Retinal ganglion cells were harvested from the Cas9 transgenic mouse (which co-expresses GFP), and transduced with the scAAV virus; mCherry expression was apparent in all cells expressing GFP, indicating highly efficient transduction and expression from the construct. FIG. 31 A also shows testing the ability to use self-complementary AAV (scAAV) to delivery a gRNA and a fluorescent reporter protein (H2B-mCherry). Cells were harvested from the Cas9 mouse, and transduced in vitro. The benefits of scAAV is the ability to test constructs much faster as expression occurs in days and not weeks. FIG. 31 B shows in vitro expression of the scAAV reporter transducing RGCs in vitro; GFP expression is from the Cas9 mouse. FIG. 31 C shows highly-efficient targeting (essentially) 100% as detected by a BglII assay. The gRNA (mm079) was delivered by ssAAV and Cas9 was present from the mouse

FIG. 32 comprises five panels, A-E, showing bidirectional targeting using self-complementary AAV viruses. Titration of scAAV virus transducing either WT RGC or RGCs derived from the Cas9 mouse. FIG. 32 C shows that genome-editing occurs in RGCs when Cas9 is present. These in vitro experiments demonstrate very high levels (˜100%) cutting. In this assay, we are assaying for cutting by loss of a restriction enzyme site. In the WT animals, the PCR product is fully digested by BglII, however, in Cas9 mice transduced with AAV, there is essentially undetectable BglII cutting at the highest concentration, indicating ˜100% CRISPR cutting. FIGS. 32 D and 32 E show in vivo rescue of retinal ganglion cells following optic nerve crush in treated eyes. A construct was generated using the H1 promoter to express a gRNA (shown in black) and mCherry simultaneously. The gRNA targets the DLK mouse gene, a gene that when inactivated, results in enhanced retinal ganglion cell survival. The construct was packaged to produce a self-complementary AAV (scAAV). The virus was administered intravitreally into the Cas9 transgenic, or a WT mouse as control. Retinal ganglion cell survival was quantitated 14 days following optic nerve crush, indicating that CRISPR delivery resulted in RGC survival in the treated mouse, as compared to the control. (Both mice receive the CRISPR gRNA, but the difference between the mice is the presence of Cas9, which is required for genome-editing.) FIG. 33 comprises three panels, A, B, and C, showing CRISPR targeting of DLK results in RGC survival. Transduction of RGCs from the Cas9 mouse by lentivirus results in ˜100% cutting as measured by BglII assay. Disruption of DLK results in a significant increase in RGC survival, demonstrating the potential for DLK targeting as therapeutical target in optic neuropathies.

FIG. 34 comprises two panels, A and B, showing CRISPR targeting of LZK in vitro. FIG. 34 A shows exon 1 of LZK with target sites shown in blue, and T7E1 primers shown in green. FIG. 34 B shows exon 2 of LZK with target sites shown in blue, and T7E1 primers shown in green.

FIG. 35 comprises seven panels, A-H, showing sensitized siRNA screening of the kinome identifies LZK as a mediator of RGC cell death in vitro. FIG. 35 A shows survival of Dlk fl/fl RGCs transduced with Cre-expressing or control adenovirus and cultured in the presence of tozasertib (1 μM) or a vehicle control. FIG. 35 B depicts a histogram showing the normalized survival for all 1,869 siRNAs in the kinome library (transfected in the presence of Dlk siRNA). Arrows show the survival for each of the three siRNAs for Lzk. FIG. 35 C shows survival of WT RGCs transfected with control or Dlk siRNA, in combination with one of four independent Lzk siRNAs or the nontargeting control. FIG. 35 D shows capillary-based immunoassay of WT RGCs after transfection with control, Dlk, Lzk or both Dlk and Lzk siPOOLs. FIG. 35 E shows survival of WT RGCs transfected with increasing amounts control, Dlk, Lzk or both Dlk and Lzk siPOOLs. FIG. 35 F shows survival of WT RGCs transfected with Dlk siRNA and either control siRNAs or one of four independent siRNAs targeting the other members of the mixed-lineage kinase family of kinases. FIG. 35 G shows survival of WT RGCs transfected with control, Dlk, Lzk or both Dlk and Lzk siPOOLs and cultured in the presence of increasing doses of tozasertib. FIG. 35 H depicts Survival (±SD) of WT RGCs transfected with siPOOLs, in the presence or absence of neurotrophins (NTs, 50 ng/mL BDNF, 5 ng/mL GDNF, 5 ng/mL CNTF), two days after colchicine (1 μM) addition. *P&lt;0.05, Mann-Whitney U test. D/L, Dlk/Lzk.

FIG. 36 comprises six panels, A and F, showing RGCs with a targeted deletion of Dlk and Lzk are highly resistant to axon injury-induced cell death in vitro and in vivo. FIG. 36 A shows a diagram of the approach used to generate constitutive and conditional Lzk knockout mice. Inset shows a Southern blot confirming the presence of a single targeting construct in the heterozygous animals. FIG. 36 B shows a capillary-based immunoassay (top) and quantification (bottom) of RGCs isolated from WT vs. Lzk −/− mice, 0 or 24 hours after the immunopanning injury. FIG. 36 C shows a flow cytometry-based quantification of the number of surviving RGCs, normalized to the uninjured control, two weeks after optic nerve crush or a sham control. NS, nonsignificant, Mann-Whitney U test. FIG. 36 D shows a capillary-based immunoassay (top) and quantification (bottom) of RGCs isolated from WT or Lzk fl/fl mice and transduced with Cre-expressing or control adenovirus. FIG. 36 E shows survival WT, Dlk fl/fl , Lzk fl/fl or Dlk fl/fl Lzk fl/fl RGCs, transduced with increasing amounts of either Cre-expressing or control adenovirus. FIG. 36 F shows flow cytometry-based quantification of the number of surviving RGCs, normalized to the uninjured control, two weeks after optic nerve crush or a sham control. All eyes were injected with 10 9 vg AAV2-Cre two weeks prior to the surgery. *P&lt;0.05, Mann-Whitney U test.

FIG. 37 comprises five panels, A-E, showing LZK kinase signaling triggers RGC cell death via the MKK4/7 and JNK1-3 kinase cascade. FIG. 37 A shows survival of WT RGCs transfected with Dlk/Lzk siPOOL and then reconstituted with LZK signaling by transducing with adenovirus expressing WT or mutant, siPOOL-resistant, human LZK cDNA. FIG. 37 B-C show survival of WT (B-C), Jnk1 fl/fl Jnk2−/−Jnk3 −/− (B) or Mkk4 fl/fl Mkk7 fl/fl (C) RGCs transduced with increasing amounts of Cre-expressing or control adenovirus. FIG. 37 D-E show survival of WT (D-E), Jnk1 fl/fl Jnk2−/−Jnk3 −/− (D) or Mkk4 fl/fl Mkk7 fl/fl (E) RGCs transfected with Dlk/Lzk siPOOL, transduced with Cre-expressing or control adenovirus, and then, two days later, with reconstitution of LZK signaling by transducing with human LZK cDNA-expressing or control adenovirus.

FIG. 38 comprises three panels, A-C, showing whole-genome siRNA screen identifies ATF2, PUMA and MEF2A as mediators of RGC cell death. FIG. 38 A depicts a histogram showing the normalized, seed-adjusted survival for the median survival-promoting siRNA targeting each of the 17,575 genes in the whole-genome library. Arrows show the survival for the median survival-promoting siRNAs targeting Atf2, Puma and Mef2a. FIG. 38 B depicts survival of WT RGCs transfected with one of four independent siRNAs targeting Atf2, Puma or Mef2a or the nontargeting control. Dashed line shows the threshold of survival greater than 3SD from the negative control. FIG. 38 C shows survival of WT RGCs transfected with increasing amounts of control and either Atf2 (left), Puma (middle) or Mef2a (right) siPOOL.

FIG. 39 comprises seven panels, A-G, showing RGCs with a targeted disruption of the transcriptional regulatory domains of ATF2 and MEF2A are partially resistant to axon injury-induced cell death in vivo. FIG. 39 A shows survival of WT RGCs transfected with Dlk/Lzk or Puma siRNA and transduced with WT or KD human LZK. FIG. 39 B shows capillary-based immunoassay of Mef2a fl/fl RGCs transduced with Cre-expressing or control adenovirus. FIG. 39 C shows survival of WT or Mef2a fl/fl RGCs transduced with increasing amounts of Cre-expressing or control adenovirus. FIG. 39 D shows fold-change in survival with the transduction of Mef2a fl/fl versus Mef2a fl/fl Mef2c fl/fl Mef2d fl/fl RGCs with Cre-expressing or control adenovirus. FIG. 39 E shows flow cytometry-based quantification of the number of surviving RGCs, normalized to the uninjured control, two weeks after optic nerve crush or a sham control. All eyes were injected with 10 9 vg AAV2-Cre two weeks prior to the surgery. *P&lt;0.05, Mann-Whitney U test. FIG. 39 F shows flow cytometry-based quantification of the number of TUBB3/P-S408 MEF2A, expressed as a percentage of total, two days after an optic nerve crush or the sham control. FIG. 39 G shows survival of WT or Atf2 fl/fl RGCs transduced with increasing amounts of Cre-expressing or control adenovirus.

FIG. 40 comprises six panels, A-G, showing sensitized whole-genome siRNA screen identifies JUN and SOX11 as mediators of RGC cell death. FIG. 40 A depicts a histogram showing the normalized survival for the siRNA minipool targeting each of the genes in the whole-genome library (transfected in the presence of Lzk siPOOL). Arrow shows the survival for the top siRNA minipool, targeting Dlk. FIG. 40 B shows quantitative PCR (qPCR) assay for Sox11 mRNA, normalized to GAPDH levels, in WT RGCs at the indicated time following immunopanning injury and transfected with either control siPOOLs or siPOOLs against Dlk/Lzk or Sox11. FIG. 40 C shows survival of WT RGCs transfected with increasing amounts of Lzk and/or Sox11 siPOOL. FIG. 40 D shows survival of WT RGCs transfected with Lzk or Lzk/Sox11 siPOOLs and reconstituted for SOX11 signaling by transducing with control or human SOX11 cDNA-expressing adenovirus. FIG. 40 E shows survival of WT or Sox11 fl/fl RGCs transduced with increasing amounts of Cre-expressing or control adenovirus. FIG. 40 F shows flow cytometry-based quantification of the number of surviving RGCs, normalized to the uninjured control, two weeks after optic nerve crush or a sham control. All eyes were injected with 10 9 vg AAV2-Cre two weeks prior to the surgery. *P&lt;0.05, Mann-Whitney U test. FIG. 40 G depicts QPCR assay of Sox11 mRNA, normalized to GAPDH levels, in Sox11fl/fl RGCs transduced with adenovirus.

FIG. 41 comprises seven panels, A-H, showing DLK/LZK-dependent cell death is mediated by a set of four transcription factors: JUN, ATF2, SOX11 and MEF2A. FIG. 41 A shows survival of WT RGCs transfected with the indicated siPOOLs. FIG. 41 B shows survival of WT RGCs transfected with Dlk/Lzk siPOOLs and either control or Jun/Atf2/Sox11/Mef2a siPOOLs, and then reconstituted with LZK signaling by transducing with siPOOL-resistant, human LZK cDNA-expressing or control adenovirus. FIG. 41 C shows survival of WT or SpCas9 knockin RGCs transfected with Lzk siPOOL and either tracrRNA or sgRNAs targeting Dlk. FIG. 41 D shows survival of WT or SpCas9 knockin RGCs transfected with Dlk siPOOL and either tracrRNA or sgRNAs targeting Lzk. FIG. 41 E shows survival of WT or SpCas9 knockin RGCs transfected with individual sgRNAs or pools of sgRNA targeting Dlk and/or Lzk. FIG. 41 F shows normalized survival (SpCas9-WT) conferred by transfecting increasing amounts of sgRNA targeting each of the four transcription factors (Jun, Atf2, Sox11, Mef2a) alone or in combination and compared to transfection with negative control tracrRNA or positive control sgRNAs targeting Dlk/Lzk. FIG. 41 H depicts survival (±

CLAIMS

Claims ( 27 )

1 . A method for treating a retinal degeneration in a subject in need thereof, the method comprising:

(a) providing a non-naturally occurring nuclease system comprising one or more vectors comprising:

i) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule in a cell of the subject, and wherein the DNA molecule encodes one or more gene products expressed in the cell, wherein the one or more gene products comprise Dual Leucine Zipper Kinase (DLK) and Leucine Zipper Kinase (LZK); and

ii) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease,

wherein components (i) and (ii) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves one or two strands of the DNA molecule to alter expression of the one or more gene products; and

(b) administering to a retinal area of the subject a therapeutically effective amount of the system.

2 . The method of claim 1 , wherein the system is CRISPR.

3 . The method of claim 1 , wherein the system is packaged into a single adeno-associated virus (AAV) particle.

4 . The method of claim 1 , wherein the system inactivates the one or more gene products.

5 . (canceled)

6 . The method of claim 1 , wherein the promoter is a bidirectional promoter.

7 . The method of claim 6 , wherein the bidirectional promoter is H1.

8 . The method of claim 7 , wherein the H1 promoter comprises:

a) control elements that provide for transcription in one direction of the at least one nucleotide sequence encoding the gRNA; and b) control elements that provide for transcription in the opposite direction of the nucleotide sequence encoding the genome-targeted nuclease.

9 . The method of claim 1 , wherein the genome-targeted nuclease is Cas9 protein.

10 . The method of claim 9 , wherein the Cas9 protein is codon optimized for expression in the cell.

11 . The method of claim 6 , wherein the promoter is operably linked to at least one, two, three, four, five, six, seven, eight, nine, or ten gRNA.

12 . The method of claim 1 , wherein the retinal area is the retina.

13 . (canceled)

14 . The method of claim 1 , wherein the cell is a retinal ganglion cell.

15 . The method of claim 1 , wherein the retinal degeneration is glaucoma.

16 - 29 . (canceled)

30 . The method of claim 15 , wherein the retinal degeneration is glaucoma.

31 - 32 . (canceled)

33 . The method of claim 1 , wherein the target sequence is selected from the group consisting of the nucleotide sequences set forth in any one of SEQ ID NOs: 143-163 or combinations thereof.

34 - 36 . (canceled)

37 . A method of altering expression of one or more gene products in a cell, wherein the cell comprises a DNA molecule encoding the one or more gene products, wherein the one or more gene products comprise Dual Leucine Zipper Kinase (DLK) and Leucine Zipper Kinase (LZK), the method comprising introducing into the cell a non-naturally occurring nuclease system comprising one or more vectors comprising:

a) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of the DNA molecule; and b) a regulatory element operable in the cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease,

wherein components (a) and (b) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule to alter expression of the one or more gene products.

38 - 47 . (canceled)

48 . The method of claim 37 , wherein:

(a) the cell is a eukaryotic or non-eukaryotic cell; (b) the system is CRISPR; (c) the system is packaged into a single adeno-associated virus (AAV) particle; (d) the system inactivates the one or more gene products; (e) the genome-targeted nuclease is Cas9 protein; and/or (f) the promoter is operably linked to at least one, two, three, four, five, six, seven, eight, nine, or ten gRNA.

49 . The method of claim 48 , wherein:

(a) the eukaryotic cell is a mammalian or human cell; and/or (b) the Cas9 protein is codon optimized for expression in the cell.

50 - 65 . (canceled)

66 . The method of claim 37 , wherein the promoter is a bidirectional promoter.

67 . The method of claim 66 , wherein the bidirectional promoter is H1.

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