Aav vectors for treatment of dominant retinitis pigmentosa

Abstract

To provide compositions and methods for treating retinitis pigmentosa (for example, dominant retinitis pigmentosa) in a subject.SOLUTION: The present invention provides compositions and methods for delivering an interfering nucleic acid (for example, an interfering RNA) to a subject in order to reduce expression of one or both alleles of an endogenous rho gene (for example, a mutant rho allele associated with retinitis pigmentosa) in the subject. In some embodiments, a replacement rho gene that is resistant to the interfering nucleic acid is also delivered to the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 302,122, filed March 1, 2016, and U.S. Provisional Patent Application No. 62 / 398,451, filed September 22, 2016, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Government support This invention was made with Government support under Grant No. R24-EY022012 awarded by the National Institutes of Health. [Background technology]

[0003] background Autosomal dominant retinitis pigmentosa (adRP) is a blinding disease that affects 1 in 12,000 people. A significant percentage of these individuals have a mutation in the gene for rhodopsin (rho), a light-harvesting pigment protein in the photoreceptor cells of the retina. The disease is dominant because inheritance of a mutated gene from either parent leads to retinal degeneration and eventual blindness. Over 100 different mutations in rho have been identified that lead to blindness. There are currently no approved drug or gene therapy treatments for adRP. Therefore, effective treatment options are needed for all possible causes of adRP and associated symptoms. Summary of the Invention

[0004] overview Aspects of the present application relate to compositions and methods for treating retinitis pigmentosa (e.g., dominant retinitis pigmentosa) in a subject (e.g., in a human). In some embodiments, one or both alleles of a subject's rhodopsin gene (rho gene) are silenced by administering an interfering RNA molecule to the subject (e.g., a subject with retinitis pigmentosa, for example, a human with dominant retinitis pigmentosa). In some embodiments, a replacement rho gene is also administered to the subject to provide the subject with a functional RHO protein that restores photoreceptor cell function. In some embodiments, the replacement rho gene has one or more nucleotide substitutions compared to an allele of the endogenous gene, which makes the replacement gene resistant to the effects of interfering RNA. In some embodiments, the replacement rho gene is a human rho gene (e.g., a wild-type human rho gene) that contains one or more (e.g., 1, 2, 3, 4, 5 or more) substitutions that make the rho gene resistant (also referred to as "hardened") to degradation mediated by an interfering RNA molecule. In some embodiments, the one or more nucleotide substitutions are present in the coding sequence of the rho gene. In some embodiments, the one or more nucleotide substitutions are silent (e.g., do not change the amino acid sequence of the RHO protein). In some embodiments, the one or more substitutions introduce an amino acid change, but the resulting RHO protein functions sufficiently to be therapeutically effective (to restore or maintain at least partial or normal vision).

[0005] In some embodiments, the interfering RNA and / or replacement gene can be delivered to the subject using any suitable technique. In some embodiments, the interfering RNA is provided to the subject in the form of a gene encoding the interfering RNA. In some embodiments, the gene encoding the interfering RNA is provided to the subject in a recombinant adeno-associated virus (rAAV). In some embodiments, the replacement gene is provided to the subject in a rAAV. In some embodiments, the genes encoding the interfering RNA and the replacement gene are provided in the same rAAV (for example, they are both encoded on the same recombinant genome flanked by AAV inverted terminal repeats (ITRs)). In some embodiments, both genes are under the control of the same promoter. In some embodiments, these genes are under the control of two different promoters. In some embodiments, the genes encoding the interfering RNA and the replacement gene are provided in different rAAVs.

[0006] In some embodiments, the interfering RNA is: a) a sense strand of the sequence CUGCCUACAUGUUUCUGCU (SEQ ID NO: 1) and an antisense strand of the sequence AGCAGAAACAUGUAGGCAG (SEQ ID NO: 2); b) a sense strand with the sequence CCUACAUGUUUCUGCUGAU (SEQ ID NO: 3) and an antisense strand with the sequence AUCAGCAGAAACAUGUAGG (SEQ ID NO: 4); c) a sense strand of the sequence GCAUGGUCAUCAUCAUGGU (SEQ ID NO: 5) and an antisense strand of the sequence ACCAUGAUGAUGACCAUGC (SEQ ID NO: 6); or d) the sense strand of the sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 7) and the antisense strand of the sequence UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 8): It is a synthetic ribonucleic acid (RNA) molecule comprising:

[0007] In some embodiments, the synthetic RNA molecule is a small interfering RNA (siRNA). In some embodiments, the interfering RNA is a small hairpin RNA (shRNA). In some embodiments, the shRNA comprises a loop having the RNA of sequence UCAAGAG (SEQ ID NO: 9) or the RNA of sequence UGUGCUU (SEQ ID NO: 10).

[0008] In some embodiments, the synthetic RNA molecule is an artificial microRNA (miRNA). In some embodiments, the artificial miRNA is UGCUGUUGACAGUGAGCGA(X) n UAGUGAAGCCACAGAUGUA(Y) n CUGCCUACUGCCUCGGA (SEQ ID NO: 19) It has an RNA sequence of: a) (X)n comprises SEQ ID NO:1 and (Y)n comprises SEQ ID NO:2; b) (X)n comprises SEQ ID NO:3 and (Y)n comprises SEQ ID NO:4; c) (X)n comprises SEQ ID NO:5 and (Y)n comprises SEQ ID NO:6; or d) (X)n comprises SEQ ID NO:7 and (Y)n comprises SEQ ID NO:8.

[0009] In some embodiments, the synthetic RNA described above or elsewhere herein further comprises an unpaired overhang sequence at the 5' end and / or the 3' end. In some embodiments, the unpaired overhang sequence comprises a repeated base sequence. In some embodiments, the repeated base sequence comprises repeated uracil (U) bases. In some embodiments, the unpaired overhang sequence is UU.

[0010] In some embodiments, a composition (e.g., a composition for administration to a subject) comprises one or more (e.g., two, three, or four) of the interfering RNAs (e.g., synthetic RNA molecules) described above or elsewhere herein. In some embodiments, a composition (e.g., a composition for administration to a subject) comprises a nucleic acid (e.g., DNA) encoding one or more (e.g., two, three, or four) of the interfering RNAs (e.g., synthetic RNA molecules) described above or elsewhere herein.

[0011] In some embodiments, the compositions also include one or more physiologically acceptable carriers, and / or one or more physiologically acceptable adjuvants.

[0012] In some embodiments, the vector encodes one or more (one, two, three, or more) shRNAs and / or artificial miRNAs (e.g., as described above or elsewhere herein). In some embodiments, the shRNA has the sequence of one or more of SEQ ID NOs: 11-18.

[0013] In some embodiments, the vector encodes a replacement rho gene.

[0014] In some embodiments, the vector encodes a replacement rho gene and / or one or more shRNAs (eg, as described above or elsewhere herein) and / or artificial miRNAs.

[0015] In some embodiments, the vector is an expression plasmid. In some embodiments, the vector is a recombinant viral genome (e.g., a rAAV genome). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector comprises a rAAV genome.

[0016] In some embodiments, a method for reducing RHO expression in a subject includes administering to the subject one or more interfering RNAs and / or one or more vectors each encoding (e.g., capable of expressing) one or more interfering RNAs described above or elsewhere herein.

[0017] In some embodiments, a method for treating retinitis pigmentosa (RP) in a subject includes administering a composition comprising an interfering RNA or a vector expressing an interfering RNA, together with a composition comprising a recombinant rho gene (e.g., a vector encoding a recombinant rho gene), where the rho gene is resistant to targeting by the interfering RNA.

[0018] In some embodiments, the recombinant rho gene is delivered using rAAV. In some embodiments, the interfering RNA and the recombinant rho gene are delivered using the same rAAV. In some embodiments, the interfering RNA and the recombinant rho gene are both under the expression control of a single promoter sequence. In some embodiments, the interfering RNA and the recombinant rho gene are each under the expression control of another promoter sequence (e.g., either a constitutive promoter or an inducible promoter). In some embodiments, the interfering RNA and / or the modified rho gene are under the expression control of (e.g., operably linked to) a human promoter or a promoter of a different species (e.g., a viral promoter, a prokaryotic promoter, or a eukaryotic promoter, such as a promoter from a non-human primate, rodent, canine, feline, porcine, or other species). In some embodiments, the promoter is an RNA polymerase III promoter (e.g., an H1 RNA polymerase III promoter), or an RNA polymerase II promoter, or an RNA polymerase I promoter. In some embodiments, the interfering RNA is an shRNA, and the shRNA is under the expression control of an RNA polymerase III promoter (e.g., an H1 RNA polymerase III promoter). In some embodiments, the interfering RNA is an artificial miRNA, and the artificial miRNA is under the expression control of an RNA polymerase II promoter. In some embodiments, the recombinant rho gene is under the expression control of a constitutive promoter or an inducible promoter (e.g., a human promoter, an eye-specific promoter). In some embodiments, the constitutive promoter or the inducible promoter is a mouse promoter (e.g., a mouse opsin (MOPS) promoter).

[0019] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a rodent or a dog. In some embodiments, the mammal is a human (e.g., a human having or known to have, e.g., diagnosed with, retinitis pigmentosa, e.g., dominant retinitis pigmentosa). These and other aspects are described in the figures, examples, and claims below. [Brief description of the drawings]

[0020] The figures and the brief description that follows provide non-limiting examples of aspects of the compositions and methods described herein.

[0021] [Figure 1] Figure 1A-1C show knockdown of GFP-tagged human rhodopsin as measured by FACS (fluorescence activated cell sorting). The experiment was performed in 293T cells in three biologic replicates. 500 ng of GFP-tagged human RHO cDNA was co-transfected with different siRNAs. Transfection was performed using LIPOFECTAMINE® 2000 transfection reagent. The control was a non-targeting siRNA purchased from Dharmacon. Samples were incubated for 72 hours and then analyzed by flow cytometry, first gated on forward and side scatter to exclude non-viable particles, and then gated on GFP expression above autofluorescence. The number of GFP-positive cells treated with control siRNA was set to 100%. [Diagram 2] Figure 2 shows knockdown of RHO by shRNA. The experiment was performed in 293T cells with three biological replicates. 200 ng of GFP-tagged human rhodopsin was co-transfected with pUC57 containing either shRNA131, 765, or 820 driven by the H1 promoter. Samples were incubated for 72 hours, and RHO knockdown was measured by flow cytometry as in Figure 1. [Diagram 3]Figure 3 shows that shRNA cleaves both mutant and wild-type RHO RNA. The experiment was performed in 293T cells with two biological replicates and three qRT-PCR replicates. 200 ng of GFP-labeled human rhodopsin (WT, T17M, or P23H) was co-transfected with rAAV-H1-shRNA plasmid (131 (SEQ ID NO: 11), 765 (SEQ ID NO: 15), or 820 (SEQ ID NO: 17)) using LIPOFECTAMINE® 2000. Non-targeting shRNA was designed to degrade an unrelated phototransduction protein, a subunit of the rod cyclic GMP-gated ion channel. Samples were incubated for 48 hours and then processed for qRT-PCR analysis. [Figure 4] Figure 4 shows the siRNA131 sense strand (SEQ ID NO: 1) and antisense strand (SEQ ID NO: 2) in the sequence context of miR30 (SEQ ID NO: 28). After expression from an RNA polymerase II promoter, siRNAs are excised from their precursors by the enzyme Drosha in the nucleus and by Dicer in the cytoplasm. [Diagram 5] Figures 5A-C show immunoblots for rhodopsin in biopsy punches taken from bleb and non-bleb regions of dog retina (Figure 5A). Figure 5B shows quantification of rhodopsin monomer amounts normalized to histone. Figure 5C is a table showing the normalized amounts. [Figure 6] Figures 6A-6B show absolute canine RHO RNA counts: Figure 6A is a plot of canine RHO RNA absolute counts, and Figure 6B is a table showing absolute RNA counts. [Figure 7] Figures 7A-C show immunoblots for rhodopsin in biopsy punches taken from bleb and non-bleb regions of dog retina (Figure 7A). Figure 7B shows quantification of rhodopsin monomer amounts normalized to histones. Figure 7C is a table showing the normalized amounts. [Figure 8]Figures 8A-8B show absolute RHO RNA counts in dogs. Figure 8A is a plot of absolute RHO RNA counts in dogs. Figure 8B is a table showing absolute RNA counts. [Figure 9] Figure 9 shows exemplary base pairing between shRNA and the target sequence of endogenous human RHOmRNA or cured RHOmRNA. All shRNAs perfectly base pair with the target sequence of dog RHOmRNA. Open boxes: mismatches between shRNA and endogenous dog RHOmRNA as well as cured RHOmRNA. Dark grey boxes: weak wobble base pairing between guanosine and uracil of RNA. Light grey boxes: mismatches only between shRNA and cured RHOmRNA. Sequences correspond from top to bottom to SEQ ID NOs: 29-37, respectively. [Figure 10] Figure 10 shows exemplary base pairing between shRNA134 and the target sequence of endogenous human RHO131mRNA or cured RHO131mRNA. The proximity of the target sequences of shRNA131 and 134 allows the same cured RHO131 to be used. Dark grey boxes: weak wobble base pairing between guanosine and uracil of RNA. Light grey boxes: mismatch only between shRNA and cured RHOmRNA. Sequences correspond from top to bottom to SEQ ID NOs: 38-40, respectively. [Figure 11] FIG. 11 depicts an exemplary map of a plasmid encoding GFP-tagged human RHO. [Figure 12-1] 12A-12C show non-limiting examples of maps of a plasmid encoding an siRNA (FIG. 12A and FIG. 12B) and a plasmid encoding human RHO (FIG. 12C). [Figure 12-2] 12A-12C show non-limiting examples of maps of a plasmid encoding an siRNA (FIG. 12A and FIG. 12B) and a plasmid encoding human RHO (FIG. 12C). [Figure 12-3] 12A-12C show non-limiting examples of maps of a plasmid encoding an siRNA (FIG. 12A and FIG. 12B) and a plasmid encoding human RHO (FIG. 12C). [Figure 13-1] 13A-E show RNA and protein analysis of rhodopsin knockdown by subretinally injected AAV2 / 5-sc-H1-shRNA820 at different viral titers in wild-type RHO+ / + dogs. FIG. 13A shows a retinal map showing the location of the biopsy punch used for Western blot analysis and RNA quantification. Paired dark grey, grey, dotted circles indicate the location of the biopsy punch in the bleb / treated and non-bleb treated areas for each replicate of the Western blot, while the filled circles indicate the location of the biopsy punch for RNA quantification. FIG. 13B shows a bar graph showing the remaining canine rhodopsin RNA in the treated areas as a percentage of the levels measured in the non-treated areas of the same retina. [Figure 13-2] Figure 13C shows immunoblots showing the amount of rhodopsin in biopsy punches taken from treated (Tx) and untreated (UnTx) areas of dog retina. Histone H3 was used for normalization. Bar graphs show remaining dog rhodopsin protein as a percentage of levels measured in untreated areas of the same retina. Figure 13D is a table showing the values ​​for each experiment reported as percent RNA or protein remaining. [Figure 13-3] FIG. 13E is another table showing the values ​​for each experiment reported as percent RNA or protein knockdown. [Figure 14-1] Figures 14A-D show the assessment of ONL and ELM / IS / OS integrity after subretinal injection of different titers of AAV2 / 5-sc-H1-shRNA820 in wild-type dogs. Figure 14A shows a plot of ONL thickness. Figure 14B shows a plot of normalized intensity of ELM / IS / OS. [Figure 14-2] FIG. 14C shows the ONL thickness values. [Figure 14-3] FIG. 14D shows the normalized intensity values ​​of the ELM / IS / OS layers. [Figure 15-1]15A-E show RNA and protein analysis of rhodopsin knockdown by subretinally injected different viral titers of AAV2 / 5-sc-H1-shRNA820 in mutant RHOT4R / + dogs. FIG. 15A shows a retinal map showing the location of the biopsy punch used for Western blot analysis and RNA quantification. Paired dark grey, grey and dotted circles indicate the location of the biopsy punch in the bleb and non-bleb areas for each replicate of the Western blot, while the filled circles indicate the location of the biopsy punch for RNA quantification. FIG. 15B is a bar graph showing the amount of remaining canine rhodopsin RNA as a percentage of the levels measured in untreated areas of the same retina. [Figure 15-2] Figure 15C is an immunoblot showing the amount of canine rhodopsin in biopsy punches taken from treated (Tx) and untreated (UnTx) areas of canine retina. Histone H3 was used for normalization. The bar graph shows the amount of remaining canine rhodopsin protein as a percentage of the levels measured in untreated areas of the same retina. Figure 15D is a table showing the values ​​for each experiment reported as percent RNA or protein remaining. [Figure 15-3] FIG. 15E is another table showing the values ​​for each experiment reported as percent RNA or protein knockdown. [Figure 16] Figures 16A-D show OCT B-scans including treated (with different viral titers of AAV2 / 5-sc-H1-shRNA820) and non-treated retinal areas of RHOT4R / + dogs 2 weeks after exposure to a brief light exposure that induces acute retinal degeneration in mutant RHOT4R / + dogs. (Figure 16A) OCT scan of dog treated with 1x1012vg / ml. (Figure 16B) OCT scan of dog treated with 5x1011vg / ml. (Figure 16C) OCT scan of dog treated with 2.5x1011vg / ml. (Figure 16D) OCT scan of dog treated with 1x1011vg / ml. [Figure 17-1]17A-B show histograms of ONL thickness from RHOT4R / + treated with AAV2 / 5-sc-H1-shRNA820 showing protection from light-induced retinal degeneration. (FIG. 17A) ONL thickness of untreated wild-type control dogs (left panel) and EM411-OS treated with AAV2 / 5-sc-H1-shRNA820 at 5E+11vg / ml showing preservation of ONL thickness in treated / bleb areas for weeks after light-induced damage. Black and white curves indicate the borders of the blebs seen immediately after subretinal injection. The bottom panel shows OCT B-scans with the ONL colored dark gray (middle band) for visualization. [Figure 17-2] (FIG. 17B) The outer and inner sites of the bleb were selected for ONL thickness measurements. [Figure 18] Figure 18 shows histology (HE staining) and immunohistochemistry in treated (Tx) and untreated (UnTx) areas of mutant RHOT4R / + retinas subretinally injected with different viral titers of AAV2 / 5-sc-H1-shRNA820 (rhodopsin is stained green and appears as a faint stain in the lower panel of Figure 18; human cone arrestin is stained red and appears as a gray stain in the lower panel of Figure 18). [Figure 19]Figures 19A-F show RNA and protein analysis of rhodopsin knockdown and replacement by AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 injected subretinally at a titer of 5x1011 vg / ml in mutant RHOT4R / + dogs. Figure 19A shows a retinal map showing the location of the biopsy punches used for Western blot analysis and RNA quantification. Paired dark grey, grey, dotted circles indicate the location of the biopsy punches in treated (Tx) and untreated (UnTx) areas for each replicate of the Western blot, while filled circles indicate the location of the biopsy punches for RNA quantification. Figure 19B shows an immunoblot showing the total rhodopsin amount (dog + human RHO820) in biopsy punches taken from treated (Tx) and untreated (UnTx) areas of the dog retina. Histone H3 was used for normalization. The bar graph shows the percentage of canine rhodopsin protein remaining in treated and untreated areas. Note the disappearance of the lower molecular weight band (corresponding to the mutant T4R RHO protein) in the treated areas of EM424-OD and EM425-OD. Figure 19C is a table showing the values ​​for each pair of punches used for protein quantification. Figure 19D is a bar graph showing the remaining canine rhodopsin RNA in treated areas as a percentage of the canine RHO RNA measured in untreated areas. Figure 19E is a bar graph showing human RHO820 in treated areas as a percentage of the canine RHO RNA measured in untreated areas. Figure 19F is a table showing the values ​​for each pair of punches used for RNA quantification. [Figure 20]20A-C show in vivo retinal imaging demonstrating protection from light-induced retinal degeneration in areas of mutant RHOT4R / + retina treated with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at a titer of 5x1011 / vg / ml. FIG. 20A shows an en face cSLO composite image showing the retinal area protected from degeneration (boundary demarcated by white arrow) 2 weeks after light exposure. Light grey arrows indicate locations within the treated area of ​​the OCT B-scan shown in FIG. 20B, and dark grey arrows indicate locations within the untreated area of ​​the OCT B-scan shown in FIG. 20C. FIG. 20B shows OCT B-scans within the treated area pre-injection, 11 weeks after injection, and 13 weeks after injection / 2 weeks after light exposure. ONL thickness is preserved throughout the treated area at both time points after viral vector injection. Figures 20B and 30V show OCT B-scans within the untreated area before injection, 11 weeks after injection, and 13 weeks after injection / 2 weeks after light exposure. The ONL is preserved up to 11 weeks after injection, but is completely lost after 2 weeks of light exposure. [Figure 21] FIG. 21 shows the histology of ONL thickness from two RHOT4R / + mice treated with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at a titer of 5x1011 / vg / ml, demonstrating protection from light-induced retinal degeneration. [Figure 22] FIG. 22 shows immunohistochemistry in treated, transition zone, and non-treated regions of mutant RHOT4R / + retinas subretinally injected with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at a titer of 5x1011 / vg / ml (rhodopsin stains green and appears as a faint stain in the FIG. 22 image; human cone arrestin stains red and appears as a gray stain in the FIG. 22 image). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description Aspects of the present application provide methods and compositions useful for treating retinitis pigmentosa in a subject (eg, in a subject with dominant retinitis pigmentosa).

[0023] In some embodiments, expression of endogenous rhodopsin (e.g., mutant or normal) is reduced or suppressed using RNA interference, and the missing protein is replaced by delivering the gene for the normal protein engineered to remove the target site for the RNA inhibitor.

[0024] In some embodiments, a single adeno-associated virus (AAV) vector is used to deliver both the RNA agent (eg, small hairpin RNA or artificial microRNA) and the recombinant rho gene.

[0025] In some embodiments, small hairpin RNAs, artificial microRNAs (a-miRs) and / or RNA enzymes (ribozymes) can be designed to degrade rhodopsin mRNA by targeting sequences common to mice, humans and dogs. These molecules can be useful for testing inhibition in cell cultures, mice and / or dogs to develop inhibitors that can function in human patients.

[0026] In some embodiments, small interfering nucleic acids (e.g., RNAs) are provided that target both human and canine rhodopsin mRNA. In some embodiments, four small interfering RNAs are provided that digest human and canine rhodopsin (RHO) mRNA, with the aim of depleting endogenously produced rhodopsin in humans and animals. In some embodiments, these interfering RNAs target the expression of rhodopsin in subjects with a dominant genetic form of retinitis pigmentosa caused by mutations in RHO. Some of these mutations result in toxic forms of the protein, the synthesis of which must be silenced to prevent retinal degeneration.

[0027] In some embodiments, the interfering nucleic acid (e.g., RNA) is designed to target a sequence specific to a mutant rho gene (e.g., the interfering nucleic acid is complementary to a sequence present in the mutant rho gene and not present in the wild-type rho gene). However, in some embodiments, the interfering nucleic acid is designed to target a wild-type sequence of a mutant endogenous rho gene (e.g., having one or more mutations at other positions not targeted by the interfering nucleic acid). In further embodiments, a functional (e.g., wild-type) rho gene that is resistant to the interfering nucleic acid is provided to restore RHO activity in a subject, thereby treating one or more symptoms of a disease or disorder associated with the mutant endogenous rho allele targeted by the interfering nucleic acid.

[0028] In some embodiments, one or more interfering RNAs may be delivered using an adeno-associated virus (AAV) either as small hairpin RNAs (shRNAs) driven by a promoter (e.g., an RNA polymerase III promoter or other suitable constitutive or inducible promoter) or as artificial microRNAs (miRNAs) driven by a promoter (e.g., an RNA polymerase II promoter or other suitable constitutive or inducible promoter).

[0029] In some embodiments, the same vector expresses a gene (cDNA) that encodes normal rhodopsin but is resistant to the action of virally expressed siRNA.

[0030] Non-limiting examples of interfering RNA are provided in Tables 1-4. [Table 1] [Table 2] [Table 3]

Table 4

[0031] In some embodiments, the hardened, normal (e.g., wild-type) rhodopsin (rho) gene may have a sequence based on the human rho gene (e.g., having a sequence set forth in Accession Number NG_009115.1; also set forth in SEQ ID NO:41), or may have a sequence based on an mRNA or protein coding portion thereof (e.g., an mRNA encoded by nucleotides 5001-5456 linked to 7238-7406 linked to 8613-8778 linked to 8895-9134 linked to 9970-11706 of SEQ ID NO:41, or a protein coding portion thereof, such as a coding sequence consisting of nucleotides 5096-5456 linked to 7238-7406 linked to 8613-8778 linked to 8895-9134 linked to 9970-10080 of SEQ ID NO:41). In some embodiments, the sequence of the normal rho gene is modified to include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that render the replacement rho gene resistant to one or more interfering RNAs used as knockdown agents to reduce the expression of the mutant endogenous rho gene in the subject being treated. However, in some embodiments, a recombinant rho gene with an unmodified normal (e.g., wild-type) sequence may be used if the recombinant rho gene has a sequence different from the endogenous rho gene or allele targeted in the subject being treated, and if the knockdown agent used is designed to target the endogenous rho gene or allele and not the recombinant rho gene provided. In some embodiments, a rho gene from a species different from the subject being treated may be used. However, in some embodiments, the rho gene (e.g., the modified rho gene) may be from the same species as the subject being treated.In some embodiments, the one or more modifications in the recombinant rho gene alter the nucleic acid sequence of the coding sequence but not the encoded protein (e.g., they are silent mutations, e.g., mutations at the third position of a codon that may include one of two or more different nucleotides without changing the encoded amino acid). In some embodiments, the recombinant rho gene does not include the intron sequence of the wild-type rho gene. In some embodiments, the recombinant rho gene encodes an mRNA (or a protein-coding portion thereof) of a rho gene that has been modified to be resistant to interfering RNA. In some embodiments, the recombinant rho gene includes a wild-type coding sequence that has been modified to be resistant to interfering RNA. In some embodiments, the modified wild-type coding sequence is provided with upstream and / or downstream mRNA sequences that are not derived from the wild-type rhomRNA. In some embodiments, the recombinant replacement rho gene includes SEQ ID NO: 42. SEQ ID NO: 42 (shown below) is resistant to an example of an interfering RNA called 820 (siRNA820 or shRNA820 described herein). The 820 interfering RNA sequences described herein target the corresponding sequences of endogenous human rhomRNA as shown in FIG. 9, however, SEQ ID NO:42 contains four substitutions (underlined and bolded below, corresponding to positions 9014, 9017, 9020, and 9023 of SEQ ID NO:41) that make it resistant to targeting by 820 interfering RNA. The coding sequence of SEQ ID NO:42 starts at position 88 of SEQ ID NO:42. In some embodiments, the recombinant rho gene delivered can include the coding sequence of SEQ ID NO:42 (e.g., starts at position 88 of SEQ ID NO:42), but can be accompanied by a different upstream mRNA sequence. In some embodiments, the recombinant rho gene can have one or more other sequence modifications (in addition or alternatively) to be resistant to additional or alternative interfering RNAs (e.g., other interfering RNAs whose sequences are provided herein). In some embodiments, one or more interfering RNAs that target different regions of the endogenous rho coding sequence in the subject can be used.

[0032] SEQ ID NO: 42 (Non-limiting example of a modified "hardened" recombinant human rho gene) [Table 5]

[0033] Thus, the compositions herein can be administered to a subject in need of treatment. In some embodiments, the subject has or is suspected of having one or more brain and / or eye symptoms, diseases, or disorders. In some embodiments, the subject has or is suspected of having one or more of the symptoms, diseases, and disorders disclosed herein. In some embodiments, the subject has one or more endogenous mutant rho alleles (e.g., associated with or causing an eye or retinal disease or disorder). In some embodiments, the subject has at least one dominant mutant rho allele (e.g., causing dominant retinitis pigmentosa). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus monkeys), marmosets, tamarins, spider monkeys, night monkeys, savannah monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domesticated animals, such as dogs and cats; farm animals, such as horses, cows, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters.

[0034] In some embodiments, the dose of rAAV particles administered to a cell or subject is 10 6 ~10 14 particles / mL or 10 3 ~10 15 The range may be on the order of 10 particles / mL or any value between any of the ranges, for example, about 10 6 , 10 7 , 10 8 , 109 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In one embodiment, 10 13 In some embodiments, the dose of rAAV particles administered to a subject is greater than 10 particles / mL. 6 ~10 14 Vector genomes (vgs) / mL or 10 3 ~10 15 vgs / mL, or any value between any of the ranges, for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In one embodiment, 10 13 More than vgs / mL of rAAV particles are administered. The rAAV particles may be administered as a single dose or may be divided into two or more doses, as may be required to achieve treatment of the particular disease or disorder being treated. In some embodiments, 0.0001 mL to 10 mL (e.g., 0.0001 mL, 0.001 mL, 0.01 mL, 0.1 mL, 1 mL, 10 mL) is delivered to the subject in one dose.

[0035] In some embodiments, the rAAV virus titer is 1x10 10 ~5x10 13 In some embodiments, the rAAV virus titer is in the range of 1x10 10 , 2.5x10 10 , 5x10 10 , 1x10 11 , 2.5x10 11 , 5x10 11 , 1x10 12 , 2.5x10 12 , 5x10 12, 1x10 13 , 2.5x10 13 , or 5x10 13 In some embodiments, the viral titer may be 1x10 vg / mL. 10 In some embodiments, the rAAV viral titer is less than 1x10 15 In one embodiment, the rAAV particles exceed 5x10 13 In some embodiments, the rAAV viral titer is administered (e.g., subretinally or intravitreally) by methods further described herein.

[0036] The rAAV particles may be administered as a single dose or may be divided into two or more doses, as may be required to achieve treatment of the particular disease or disorder being treated. In some embodiments, 1-500 microliters of the compositions described herein are administered to one or both eyes of a subject. By way of example, in some embodiments, about 1, about 10, about 50, about 100, about 200, about 300, about 400, or about 500 microliters may be administered to each eye. However, it should be understood that in some embodiments, smaller or larger amounts may be administered.

[0037] In some embodiments, the disclosure provides formulations of one or more rAAV-based compositions disclosed herein in a pharma- ceutically acceptable solution for administration to a cell or animal, either alone or in combination with one or more other therapeutic modalities, particularly for treating human cells, tissues, and diseases that occur in humans.

[0038] If necessary, rAAV particles or nucleic acid vectors can be administered in combination with other substances, such as, for example, proteins, or polypeptides, or various pharmacologic active substances, including systemic or local administration of one or more therapeutic polypeptides, bioactive fragments, or variants thereof.In fact, there is virtually no limit to the other components that can be included, provided that the additional substances do not cause significant adverse effects in contact with target cells or host tissues.Thus, rAAV particles can be delivered with various other substances as required in a particular case.These compositions can be purified from host cells or other biological sources, or can be chemically synthesized as described herein.

[0039] The formulation of pharma- ceutically acceptable excipients and carrier solutions is well known to those of skill in the art, as is the development of suitable dosing and treatment regimens for use with the particular compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, intraarticular, and intramuscular administration, and formulations.

[0040] Typically, these compositions may contain at least about 0.1% or more of the therapeutic agent (e.g., rAAV particles or host cells), and may conveniently be from about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation, although the percentage of active ingredient may of course vary. Of course, the amount of therapeutic agent (e.g., rAAV particles) in each therapeutically useful composition may be prepared to obtain a suitable dosage in a given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, etc., as well as other pharmacological considerations, will be taken into account by those skilled in the art of preparing these pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0041] In certain situations, it may be desirable to deliver the rAAV particles or host cells in a suitably formulated pharmaceutical composition disclosed herein subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection into one or more cells, tissues, or organs.

[0042] Suitable pharmaceutical forms of rAAV particles or host cell compositions for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the forms are sterile and fluid to the extent that easy syringability exists. In some embodiments, the forms are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0043] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with rAAV particles or host cells. These pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum oils such as mineral oils, vegetable oils such as peanut oil, soybean oil, and sesame oil, animal oils, or synthetic oils. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers.

[0044] The compositions of the present disclosure may be administered to the subject to be treated by standard routes, including, but not limited to, pulmonary, intranasal, oral, inhalation, parenteral (e.g., intravenous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intravitreal, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection).

[0045] The compositions of the present disclosure may be delivered to the eye by various routes. They may be delivered into the eye by topical application to the eye or by intraocular injection, for example, intraocular injection into the inter-photoreceptor space in the vitreous (intravitreal injection) or subretinal (subretinal injection). In some embodiments, they are delivered to rod photoreceptor cells. Alternatively, they may be delivered locally by insertion or injection into tissues surrounding the eye. They may be delivered systemically by oral route or subcutaneous, intravenous or intramuscular injection. Alternatively, they may be delivered by catheter or implant, which is made of porous, non-porous or gelatinous materials, including membranes such as silastic membranes or fibers, biodegradable polymers, or proteinaceous materials. They may be administered prior to the onset of symptoms, for example, during eye surgery, or shortly after the onset of a pathological condition, or during the onset of acute or long-term symptoms, to prevent their onset.

[0046] For administration of injectable aqueous solutions, for example, the solution may be suitably buffered if necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, intravitreal, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous solvents that can be used will be known to those skilled in the art in light of this disclosure. For example, a dose is dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion or injected at the proposed injection site (see, for example, pages 1035-1038 and 1570-1580 of "Remington's Pharmaceutical Sciences" 15th Edition). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person administering will determine the appropriate dosage for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards (eg, in accordance with FDA Office of Biologics standards).

[0047] Sterile injection solution is prepared by adding rAAV particles or host cells in a required amount in a suitable solvent together with some other components as listed above as necessary, and then sterilizing by filtration as necessary.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary components other than those listed above.In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0048] The amount of rAAV particles, nucleic acid vectors, or host cell compositions and the time of administration of the compositions are within the expertise of a person skilled in the art who has the benefit of the present teachings. However, administration of a therapeutically effective amount of the disclosed compositions can be achieved by a single administration to a patient undergoing the treatment, such as by a single injection of a sufficient number of infectious particles to provide a therapeutic benefit. Alternatively, in some situations, it may be desirable to provide multiple or continuous administrations of rAAV particles or host cell compositions over a relatively short or long period of time, which can be determined by the physician supervising the administration of these compositions.

[0049] In some embodiments, rod cells remain structurally intact and / or viable upon silencing of cellular rhodopsin gene expression. In some embodiments, rod cells in which cellular rhodopsin gene expression has been silenced have shortened outer segments that normally contain rhodopsin. In some embodiments, the length of the outer segments is maintained or can be restored (e.g., partially or completely) using an exogenously added (hardened) rhodopsin gene whose expression is resistant to silencing using the compositions described in this application.

[0050] The compositions can include rAAV particles or host cells, alone or in combination with one or more additional active ingredients, which can be obtained from natural or recombinant sources or can be chemically synthesized. In some embodiments, the rAAV particles are administered in combination with a proteasome inhibitor, such as bortezomib or hydroxyurea, in the same composition or as part of the same therapeutic regimen.

[0051] As used herein, "treating" a disease means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.The above-mentioned composition is usually administered to a subject in an effective amount, i.e., an amount that can produce a desired result.The desired result will depend on the active agent administered.As an example, an effective amount of rAAV particles can be an amount that can transfer heterologous nucleic acid into a host organ, tissue, or cell.

[0052] The toxicity and efficacy of the compositions utilized in the methods of the present disclosure can be determined by standard pharmaceutical procedures, using either cultured cells or experimental animals to determine the LD50 (the dose lethal to 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compositions that exhibit large therapeutic indices are preferred. Those that exhibit toxic side effects may be used, but care should be taken to design a delivery system that minimizes the potential damage of these side effects. The dosage of the compositions described herein is generally within a range that includes the ED50 with little or no toxicity. Dosages can vary within this range, depending on the dosage form employed and the route of administration utilized.

[0053] Aspects of the present disclosure relate to recombinant adeno-associated virus (rAAV) particles for delivery of one or more nucleic acid vectors comprising a gene of interest to various tissues, organs and / or cells. In some embodiments, the rAAV particles comprise a rAAV capsid protein as described herein, e.g., comprising one or more amino acid substitutions. In some embodiments, the gene of interest encodes a polypeptide or protein of interest (e.g., a therapeutic polypeptide or protein). In some embodiments, the gene of interest encodes an RNA of interest (e.g., a therapeutic mRNA, siRNA, shRNA, microRNA, antisense RNA, tRNA, rRNA, or ribozyme). In some embodiments, the gene of interest is a replacement gene (e.g., an eye-specific gene, a functional gene, a functional rho gene). In some embodiments, the gene of interest comprises or encodes an RNA of interest (e.g., a microRNA, siRNA, shRNA) and a replacement gene of interest (e.g., an eye-specific gene, a functional gene, a functional rho gene). In some embodiments, a functional rho gene comprises a rho gene that contains a silent nucleotide substitution that prevents it from being degraded by an RNA of interest (e.g., microRNA, siRNA, shRNA). In some embodiments, the RNA of interest and the replacement gene of interest are under the control of the same promoter. In some embodiments, the RNA of interest and the replacement gene of interest are under the control of another promoter. Any suitable promoter can be used, including, but not limited to, viral promoters (e.g., CMV or other viral promoters), microbial (e.g., yeast or bacterial), or eukaryotic (e.g., mammalian) promoters.

[0054] A recombinant AAV (rAAV) particle can include, at a minimum, (a) one or more heterologous nucleic acid regions that include a sequence encoding a gene of interest (e.g., an RNA of interest and / or a replacement gene of interest), and (b) one or more regions that include inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences, or engineered ITR sequences) flanking the one or more heterologous nucleic acid regions. In some embodiments, the nucleic acid vector is 4 kb to 5 kb in size (e.g., 4.2 to 4.7 kb in size). The nucleic acid vector can be encapsidated by a viral capsid, e.g., an AAV1, AAV2, AAV3, AAV4, or AAV5 capsid, which can include a modified capsid protein described herein. In some embodiments, the nucleic acid vector is circular. In some embodiments, the nucleic acid vector is single-stranded. In some embodiments, the nucleic acid vector is double-stranded. In some embodiments, the double-stranded nucleic acid vector may be, for example, a self-complementary vector that contains a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector, initiating the formation of a double strand of the nucleic acid vector.

[0055] The rAAV particles may be any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2 / 1, 2 / 5, 2 / 8, or 2 / 9), including any derivative or pseudotype. As used herein, the serotype of an rAAV viral vector (e.g., an rAAV particle) refers to the serotype of the capsid protein of the recombinant virus. In some embodiments, the rAAV particle is not AAV2. In some embodiments, the rAAV particle is AAV2. In some embodiments, the rAAV particle is AAV6. In some embodiments, the rAAV particle is an AAV6 serotype that includes the rAAV capsid protein described herein. Non-limiting examples of derivatives and pseudotypes include rAAV2 / 1, rAAV2 / 5, rAAV2 / 8, rAAV2 / 9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAVrh. These include AV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2(Y->F), AAV8(Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. These AAV serotypes and derivatives / pseudotypes, as well as methods for producing these derivatives / pseudotypes, are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A1, Schaffer DV, Samulski RJ.).In some embodiments, the rAAV particles are pseudotyped rAAV particles that comprise (a) a nucleic acid vector that includes ITRs from one serotype (e.g., AAV2) and (b) a capsid composed of capsid proteins from another serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0056] Methods for producing rAAV particles and nucleic acid vectors are also known in the art and commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; U.S. Patent Application Publication Nos. US20070015238 and US20120322861, which are incorporated herein by reference, as well as plasmids and kits available from ATCC and Cell Biolabs, Inc.). By way of example, a plasmid containing the nucleic acid vector may be transfected into a producer cell line in combination with one or more helper plasmids containing, for example, rep genes (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and cap genes (e.g., encoding VP1, VP2, and VP3, including the modified VP3 described herein), allowing the rAAV particles to be packaged and subsequently purified.

[0057] In some embodiments, the one or more helper plasmids include a first helper plasmid that includes a rep gene and a cap gene (e.g., encoding a rAAV capsid protein described herein) and a second helper plasmid that includes the E1a, E1b, E4, E2a, and VA genes. In some embodiments, the rep gene is from AAV2 or AAV6, and the cap gene is from AAV2 or AAV6 and can include modifications to the gene to produce a modified capsid protein described herein. Helper plasmids and methods for producing these plasmids are known in the art and commercially available (e.g., pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al.(See, e.g., Gao, G.P. et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0058] An exemplary, non-limiting method for producing rAAV particles is described below. One or more helper plasmids are produced or obtained, containing the rep and cap ORFs of the desired AAV serotype, as well as the adenovirus VA, E2A (DBP) and E4 genes under the transcriptional control of the native promoter. The cap ORF also contains one or more modifications to produce the modified capsid proteins described herein. HEK293 cells (available from ATCC®) are transfected with the helper plasmid and a plasmid containing the nucleic acid vector described herein via CaPO4-mediated transfection, lipid or polymer molecules such as polyethyleneimine (PEI). The HEK cells are then cultured for at least 60 hours to allow for the production of AAV particles. Alternatively, in another example, an Sf9-based stable producer cell line is infected with a single recombinant baculovirus containing the nucleic acid vector. In yet another example, HEK293 or BHK cell lines are infected with HSV containing a nucleic acid vector and, optionally, one or more helper HSV containing the rep and cap ORFs described herein and the adenovirus VA, E2A (DBP), and E4 genes under the transcriptional control of a native promoter. HEK293, BHK, or Sf9 cells are then cultured for at least 60 hours to allow for the production of rAAV particles. The rAAV particles can then be purified using any method known in the art or described herein, for example, by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0059] The present disclosure also contemplates host cells comprising at least one of the disclosed rAAV particles or nucleic acid vectors. These host cells include mammalian host cells, preferably human host cells, and can be isolated in either cell culture or tissue culture. In the case of transgenic animal models (e.g., mice), the transformed host cells can be contained within the non-human animal itself. In some embodiments, the host cells express CD36 +A cell of the erythroid lineage, such as a burst forming cell-erythroid (BFU-E) or a colony forming unit-erythroid (CFUE-E) precursor cell.

[0060] In some embodiments, the compositions described herein (e.g., siRNAs, shRNAs, and / or replacement genes of interest) are formulated into nanoparticles. In some embodiments, the compositions described herein (e.g., siRNAs, shRNAs, and / or replacement genes of interest) are formulated into lipid nanoparticles. In some embodiments, the compositions described herein (e.g., the siRNA, shRNA, and / or replacement gene of interest) are formulated into lipid-polycation complexes, referred to as cationic lipid nanoparticles. The formation of lipid nanoparticles may be accomplished by methods known in the art and / or described in U.S. Patent Application Publication No. 20120178702, the entirety of which is incorporated herein by reference. By way of non-limiting example, the polycation may include cationic peptides or polypeptides (such as, but not limited to, polylysine, polyornithine, and / or polyarginine), and cationic peptides described in International Publication No. WO2012013326, or U.S. Patent Application Publication No. US20130142818, each of which is incorporated herein by reference in its entirety. In some embodiments, the compositions described herein (e.g., the siRNA, shRNA, and / or replacement gene of interest) are formulated into lipid nanoparticles comprising a non-cationic lipid (such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE)). EXAMPLES

[0061] Example 1: Identification of small interfering RNA (siRNA) that knocks down RHO The design principle of small interfering RNA (siRNA) described by Khvorova et al. 4、5Using NCBI, 14 siRNAs were designed to specifically cleave canine siRNAs; 12 of them also target the human mRNA for rhodopsin. Before proceeding, siRNA positions 2-19 were screened against the NCBI human RefSeq database using the NCBI Blast utility (blast.ncbi.nlm.nih.gov / Blast.cgi). Two of the potential siRNAs matched well with other genes that may be expressed in the retina and were therefore excluded. RNA versions of the 10 siRNAs were ordered from GE Healthcare Dharmacon, along with a non-targeting siRNA to be used as a control. These were tested in cells expressing human RHO fused to green fluorescent protein (GFP, an exemplary plasmid map is shown in Figure 11), and the reduction in green fluorescent cells was measured by fluorescence-activated cell sorting (FACS). The principle was that cleavage of RHO mRNA would reduce the production of GFP. Although all siRNAs were designed based on the latest siRNA design principles, only three of the ten tested led to a reduction of 30% or more (Figure 1A-C).

[0062] To confirm that these siRNAs are effective as short hairpin RNAs expressed from an RNA polymerase III promoter and delivered by AAV, we cloned the DNA sequences of the shRNAs and expressed three siRNAs (Figure 2). Interestingly, although siRNA131 (SEQ ID NO: 1) was the most effective siRNA, the shRNA tested was the least effective, so the relative knockdown of RHO with siRNAs did not accurately predict the relative suppression of RHO with shRNAs. Simple transfection of siRNAs as RNAs would not accurately predict the effectiveness of shRNAs designed to produce the same siRNAs by transcription and processing.

[0063] To confirm that the shRNAs were effective at cleaving both wild-type (normal) and mutant RHO, the same three shRNAs cloned into an AAV vector were tested for their ability to digest wild-type and two different mutant RHO mRNAs, using quantitative reverse transcription PCR (qRT-PCR) as an assay (Figure 3).

[0064] Table 5 shows the RNA sequences corresponding to the shRNA sequences identified as capable of cleaving RHO, including the sense strand, loop, antisense strand and overhang. Each sequence shows two alternative loop sequences highlighted in bold and underlined, and the overhangs highlighted in italics and underlined. [Table 6]

[0065] siRNA can also be used as an artificial microRNA having the structure shown in FIG. 7 The exemplary microRNA in Figure 4 comprises the sense strand (100) and antisense (101) of RHO131 (SEQ ID NOs: 1 and 2, respectively). The advantage of this expression mode is that the production of siRNA can be made cell type specific by the use of specific promoter sequences. In this case, the proximal promoter of the human rhodopsin gene or the human rhodopsin kinase promoter would be used to restrict expression to photoreceptor cells.

[0066] Example 2: RHO + / + Analysis of RHO knockdown (KD) in dogs Dog 2190 (rcd1 carrier) received subretinal injections of AAV2 / 5-sc-H1-shRNA-Rho13 at the concentrations shown in Table 6. The dog was sacrificed 8 weeks after injection. Several 3 mm neural retinal biopsy punches were collected from each eye, from both the bleb and non-bleb areas. [Table 7]

[0067] A non-limiting example of the AAV2 / 5-sc-H1-shRNA construct described herein can also be referred to as AAV2 / 5-sc-MOP500 rGFP-shRNA construct. AAV2 / 5 refers to the nucleic acid encoding the shRNA being provided in a rAAV particle that is flanked by AAV2 ITRs and includes AAV5 capsid protein. In some embodiments, any interfering RNA described herein and any recombinant rho gene described herein can be provided on the same AAV nucleic acid (e.g., flanked by AAV2 ITRs) within a rAAV particle (e.g., including AAV5 capsid protein). In some embodiments, any of the interfering RNAs described herein and any of the recombinant rho genes described herein can be provided encapsidated in different rAAV particles (e.g., those comprising AAV5 capsid proteins or those having capsid proteins from a different AAV serotype), on different AAV nucleic acids (e.g., those flanked by AAV2 ITRs or those flanked by ITRs from a different AAV serotype).

[0068] Western Blot Analysis: Two biopsy punches from each eye (representing either bleb or non-bleb areas) were incubated in 50 μl of Lewin's buffer A (containing protease inhibitors) for 15 min on ice. Samples were sonicated at 40% amplitude, 15 sec on / 10 sec off x 8 pulses. Samples were then centrifuged and the pellet was discarded. Protein concentration in the supernatant was measured by Bradford method. 1 μg of total protein was immunoblotted to visualize rhodopsin (antibody used: Millipore MAB5356, diluted 1:1000 in ODYSSEY® blocking buffer) and anti-histone antibody (Abcam ab1791, diluted 1:3000) was used as a loading control (Figure 5A) and to normalize the signal (Figure 5B). Comparison of RHO protein amount between bleb and non-bleb areas was not consistent with the experimental design (Figure 5C).

[0069] Absolute quantification of canine rhodopsin RNA in the canine retina: To quantify the absolute amount of rhodopsin RNA present in the retina after ribozyme treatment, absolute quantification was performed using the Q-PCR standard curve method. A standard curve was constructed using a dilution series of known amounts of canine RHO cDNA. The total amount of canine RHO RNA in each sample was calculated based on this standard curve (Figure 6A).

[0070] Partial knockdown of canine RHO is seen with AAV2 / 5-sc-H1-shRNA-Rho131. At the highest concentration used, protein levels are reduced by 37% and RNA by almost 50% (Figure 6B).

[0071] Example: RHO + / + Further analysis of RHO knockdown in dogs Dog 2194 (rcd1 carrier) received a subretinal injection of AAV2 / 5-sc-H1-shRNA-Rho810 at the concentrations shown in Table 7. The dog was sacrificed 8 weeks after injection. Several 3 mm neural retinal biopsy punches were collected from each eye, from both the bleb and non-bleb areas. [Table 8]

[0072] Western Blot Analysis: Two biopsy punches from each eye (representing either bleb or non-bleb areas) were incubated in 50 μl of Lewin's buffer A (containing protease inhibitors) for 15 min on ice. Samples were sonicated at 40% amplitude, 15 sec on / 10 sec off x 8 pulses. Samples were then centrifuged and the pellet was discarded. Protein concentration in the supernatant was measured by Bradford method. 1 μg of total protein was immunoblotted to visualize rhodopsin (antibody used: Millipore MAB5356, diluted 1:1000 in ODYSSEY® blocking buffer) and anti-histone antibody (Abcam ab1791, diluted 1:3000) was used as a loading control (Figure 7A) and to normalize the signal (Figure 7B). Comparison of RHO protein amount between bleb and non-bleb areas was not consistent with the experimental design (Figure 7C).

[0073] Absolute quantification of canine rhodopsin RNA in the canine retina: To quantify the absolute amount of rhodopsin RNA present in the retina after ribozyme treatment, absolute quantification was performed using the Q-PCR standard curve method. A standard curve was constructed using a dilution series of known amounts of canine RHO cDNA. The total amount of canine RHO RNA in each sample was calculated based on this standard curve (Figure 8A).

[0074] Complete knockdown of canine RHO RNA and protein was observed at 1 × 10 12 This was also seen with AAV2 / 5-sc-H1-shRNA-Rho820 at lower doses of vg / ml ( Fig. 8B ).

[0075] Example 4: Cured mRNA of replacement genes In some embodiments, the replacement gene (e.g., replacement RHO) mRNA may be modified at one or more positions to "harden" it (i.e., to make it resistant to degradation by siRNA). In some embodiments, one or more silent nucleotide substitutions may be included in the replacement gene associated with the siRNA provided to knock down the endogenous gene (e.g., rho gene). Figures 9 and 10 provide non-limiting examples of nucleotide substitutions that may be introduced into the replacement RHO mRNA. It is understood that the replacement rho gene may include one or more of these substitutions.

[0076] Figure 9 shows a depiction of the base pairing that occurs between each shRNA and the target sequence of endogenous human RHOmRNA or cured RHOmRNA. All shRNAs are perfectly base-paired with the target sequence of canine RHOmRNA. Open boxes: mismatches between the shRNA and endogenous canine RHOmRNA as well as cured RHOmRNA. Dark grey boxes: weak wobble base pairing that occurs between guanosine and uracil of the RNA. Light grey boxes: mismatches only between the shRNA and cured RHOmRNA.

[0077] Figure 10 shows a depiction of base pairing occurring between shRNA134 and the target sequence of endogenous human RHO131mRNA or cured RHO131mRNA. The proximity of the target sequences of shRNA131 and 134 allows the same cured RHO131 to be used. Dark grey box: Weak wobble base pairing occurring between guanosine and uracil of the RNA. Light grey box: Mismatch only between shRNA and cured RHOmRNA.

[0078] Example 5: siRNA and replacement gene coding In some embodiments, it may be desirable to provide siRNA encoded by DNA vector. Figure 12A and Figure 12B show non-limiting examples of siRNA (e.g., shRNA) encoded by DNA vector. In some embodiments, as further shown in the exemplary plasmid map shown in Figure 12A and Figure 12B, the DNA vector can further code for an inverted terminal repeat (ITR) sequence flanking the siRNA (e.g., shRNA). In some embodiments, the DNA vector that codes for siRNA flanked by ITR sequences can be used in the production of recombinant AAV particles that contain siRNA.

[0079] In some embodiments, it is desirable to provide a replacement RHO mRNA encoded by a DNA vector. Figure 12C represents a non-limiting example of a replacement RHO mRNA encoded by a DNA vector. In some embodiments, the DNA vector may further encode an ITR sequence flanking the replacement RHO mRNA (e.g., human RHO), as further represented in the exemplary plasmid map shown in Figure 12C. In some embodiments, a DNA vector encoding a replacement RHO mRNA flanked by ITR sequences may be used in the production of recombinant AAV particles containing human rho. In some embodiments, a similar DNA vector may be provided that includes both a replacement rho gene and one or more sequences encoding one or more interfering RNAs, flanked by ITR sequences. In some embodiments, the interfering RNA and / or replacement rho gene are operably linked to a promoter (e.g., an RNA polymerase III promoter, or an H1 RNA polymerase III promoter, or other promoters described in this application). The interfering RNA and / or replacement rho gene depicted in Figures 12A-12C are under the control of an H1 RNA polymerase III promoter. The constructs of Figures 12A-C also contain MOP500-rGFP (-385 / +86 portion of the mouse rod opsin promoter (MOP500) upstream of the reverse sequence of GFP (rGFP, therefore not expressed)) and may be referred to as H1 constructs or MOP500 constructs. However, the MOP500 (e.g., MOP500-rGFP) portion is not required. Thus, a plasmid or rAAV nucleic acid can encode an interfering RNA and / or modified rho gene as described herein, respectively, under the control of the same or a different promoter (e.g., the H1 promoter), without the mouse opsin promoter, and / or without any GFP coding sequence (in either orientation).

[0080] Example 6: Wild-type RHO + / + Analysis of RHO knockdown with shRNA820 in dogs Example 6 describes the determination of viral titers of AAV2 / 5 carrying shRNA820 knockdown reagent that efficiently suppresses rhodopsin expression after subretinal injection in wild-type dogs, the demonstration by in vivo retinal imaging in wild-type dogs that the photoreceptor cell-containing layer (ONL) is preserved but the rhodopsin-containing layer is reduced after subretinal injection with AAV2 / 5-sc-H1-shRNA820, and the identification of mutant RHO T4R / + Identification of viral titers of AAV2 / 5 carrying shRNA820 knockdown reagent that efficiently suppresses rhodopsin expression after subretinal injection in a spontaneous canine model of RHO-ADRP, a mutant RHO T4R / + These include identifying viral titers of AAV2 / 5-sc-H1-shRNA820 that confer protection to photoreceptor cells from light-induced retinal degeneration in dogs, and demonstrating that efficient knockdown of rhodopsin expression causes outer segment loss in rods. Because outer segment preservation is critical for phototransduction (the mechanism by which rods convert light into an electrical signal), an optimal treatment for RHO-ADRP should reduce native RHO but preserve outer segment structure. These results demonstrate that knockdown approaches are not sufficient and argue for the use of a combination of knockdown and replacement strategies.

[0081] 5 wild-type RHO + / + Dogs were dosed at 1x10 11 ~5x10 12 Both eyes received subretinal injections of AAV2 / 5-sc-H1-shRNA-Rho820 with viral concentrations ranging from . [Table 9]

[0082] Six to eight weeks after injection, in vivo retinal imaging (cSLO / OCT) was performed and then the dogs were sacrificed. Several 3 mm neural retinal biopsy punches were collected from both eyes (dog 2194) or only from the OS eye (other dogs) in both bleb / treated and non-bleb / non-treated regions to measure expression levels of canine rhodopsin by Western blot and RHO RNA by qPCR analysis. OD eyes were fixed, embedded in optimal cutting temperature medium, and processed for histology and immunohistochemical staining.

[0083] Western Blot Analysis: Up to three sets of biopsy punches (representing either bleb or non-bleb areas) were incubated in 50 μl of Lewin's buffer A (containing protease inhibitors) for 15 min on ice. Samples were sonicated at 40% amplitude, 15 sec on / 10 sec off × 8 pulses. Samples were then centrifuged and the pellet was discarded. Protein concentration in the supernatant was measured by Bradford method. Samples were stored at -20°C. To visualize rhodopsin, 1 μg of total protein was loaded onto the gel (antibody used: Millipore MAB5356, diluted 1:1000 in ODYSSEY blocking buffer). Anti-histone H3 antibody (Abcam ab1791, diluted 1:3000) was used as a loading control to normalize the signal.

[0084] Absolute quantification of canine rhodopsin RNA in the canine retina: To determine the absolute amount of rhodopsin RNA present in the retina after shRNA820 treatment, absolute quantification was performed using the Q-PCR standard curve method. A standard curve was constructed using a dilution series of known amounts of canine RHO cDNA. 0.1 nanograms of total cDNA was used for quantification. The total amount of canine RHO RNA in each sample was calculated based on this standard curve.

[0085] Wild-type RHOs injected with different viral titers of AAV2 / 5-sc-H1-shRNA820 + / +Assessment of retinal integrity in the dog eye by in vivo retinal imaging. In vivo retinal imaging by cSLO / OCT was used to assess the integrity of the ONL of the retina 6-8 weeks after subretinal injection of different titers of AAV2 / 5-sc-H1-shRNA820. ONL thickness histograms showed preservation of this layer at all titers. Segmentation of the outer limiting membrane (ELM), inner segment (IS) and outer segment (OS) was performed using 1x10 12 and 5x10 12 13A-13E show that wild-type RHO was significantly reduced in signal intensity in the area corresponding to the bleb region in eyes injected with a titer of 10 ... + / + RNA and protein analysis of rhodopsin knockdown in dogs by subretinally injected AAV2 / 5-sc-H1-shRNA820 at different viral titers. Figure 13A shows a retinal map showing the location of the biopsy punches used for Western blot analysis and RNA quantification. Paired dark grey, grey, dotted circles show the location of the biopsy punches in the bleb / treated and non-bleb treated areas for each replicate of the Western blot, while the filled circles show the location of the biopsy punches for RNA quantification. Figure 13B shows a bar graph showing the remaining canine rhodopsin RNA in treated areas as a percentage of the levels measured in untreated areas of the same retina. Figure 13C shows an immunoblot showing the amount of rhodopsin in biopsy punches taken from treated (Tx) and untreated (UnTx) areas of the canine retina. Histone H3 was used for normalization. Bar graphs show remaining canine rhodopsin protein as a percentage of levels measured in untreated areas of the same retina, and Figures 13D-E show tables showing the values ​​for each experiment (reported as percent RNA or protein remaining or percent RNA or protein knockdown, respectively).

[0086] Figures 14A-D show the evaluation of ONL and ELM / IS / OS integrity after subretinal injection of different titers of AAV2 / 5-sc-H1-shRNA820 in wild-type dogs. Figure 14A shows the ONL thickness plot; Figure 14B shows the ELM / IS / OS normalized intensity plot; Figure 14C shows the ONL thickness values; and Figure 14D shows the ELM / IS / OS layer normalized intensity values. These results suggest a shortening of photoreceptor cell OS and IS as a result of RHO silencing and can be used to evaluate the efficacy of RHO knockdown in vivo.

[0087] Complete knockdown of canine RHO RNA and protein was achieved at 1x10 12 This was seen in wild-type dogs subretinally injected with AAV2 / 5-sc-H1-shRNA820 at viral titers as low as 100 vg / ml. No reduction in ONL thickness was observed even at the highest viral concentration, suggesting that rhodopsin knockdown does not induce photoreceptor cell death. A reduction in OCT reflectance of the ELM / IS / OS layers is seen in the treatment / bleb region of eyes injected with high viral titers inducing 100% KD of rhodopsin. This finding is compatible with the thinness of these layers and can be used as an in vivo outcome measure of knockdown efficiency.

[0088] Example 7: RHO T4R / + Analysis of RHO knockdown by shRNA820 in mutant dogs 4 RHOs T4R / + Dogs received subretinal injections of AAV2 / 5-sc-H1-shRNA-Rho820 in both eyes at the virus concentrations shown in Table 9 below. [Table 10]

[0089] Eight weeks after injection, cSLO / OCT was performed and all dogs were exposed to light (1 mW / cm) on the OS eye to induce light-induced retinal degeneration. 2A 10-minute cSLO / OCT was performed on all dogs 2 weeks after light exposure to assess the rescue effect afforded by the treatment. Dogs were sacrificed and several 3 mm neural retinal biopsy punches were collected from OD eyes, both from bleb / treated and non-bleb / non-treated areas, to measure canine rhodopsin expression levels by Western blot and RHO RNA expression levels by qPCR analysis. OS eyes were fixed, embedded in optimal cutting temperature medium, and processed for histology and immunohistochemical staining.

[0090] Western Blot Analysis: Triplicate biopsy punches (representing either bleb / treated or non-bleb / non-treated retinal areas) from each OS eye were incubated in 50 μl of Lewin's buffer A (containing protease inhibitors) for 15 min on ice. Samples were sonicated at 40% amplitude, 15 sec on / 10 sec off x 8 pulses. Samples were then centrifuged and the pellet was discarded. Protein concentration in the supernatant was measured by Bradford method. Samples were stored at -20°C. To visualize rhodopsin, 1 μg of total protein was loaded onto the gel (antibody used: Millipore MAB5356, diluted 1:1000 in ODYSSEY blocking buffer). Anti-histone H3 antibody (Abcam ab1791, diluted 1:3000) was used as a loading control to normalize the signal.

[0091] Absolute quantification of canine rhodopsin RNA in the canine retina: To determine the absolute amount of rhodopsin RNA present in the retina after shRNA820 treatment, absolute quantification was performed using the Q-PCR standard curve method. A standard curve was constructed using a dilution series of known amounts of canine RHO cDNA. 0.1 nanograms of total cDNA was used for quantification. The total amount of canine RHO RNA in each sample was calculated based on this standard curve.

[0092] Mutant RHOs injected with different viral titers of AAV2 / 5-shRNA820 T4R / +Assessment of photoreceptor cell rescue from light-induced damage in the dog eye by in vivo retinal imaging. 1x10 12 From 1x10 11 Mutant RHO subretinally injected with virus titers in the range of vg / ml T4R / + Eyes from dogs were examined by cSLO / OCT imaging 8 weeks after injection (before light exposure). ONL thickness in the bleb / treated area was preserved, suggesting that shRNA820 did not cause photoreceptor cell loss during that time period. The intensity (1 mW / cm2) previously shown to cause acute retinal degeneration in RHO T4R mutants but not in wild-type dogs was used. 2 Illumination with white light for 1 min at 1x10 corneal irradiance was used to assess the level of protection afforded by AAV2 / 5-sc-H1-shRNA820 in the treated / bleb area. The surrounding non-bleb / untreated area was used as an internal control for each eye. Two weeks after light exposure, retinas were imaged again and 1x10 12 and 5x10 11 We demonstrated good preservation of ONL thickness in the bleb / treated area of ​​eyes injected with a titer of 2.5x10 vg / ml. 11 vg / ml titer of 1x10 11 No titers in vg / ml were seen.

[0093] FIG. 15A to FIG. 15E show mutant RHO T4R / +RNA and protein analysis of rhodopsin knockdown in dogs by subretinally injected AAV2 / 5-sc-H1-shRNA820 at different viral titers. Figure 15A shows a retinal map showing the location of the biopsy punches used for Western blot analysis and RNA quantification. Paired dark gray, gray and dotted circles show the location of the biopsy punches in the bleb and non-bleb areas for each Western blot replicate, while the filled circle shows the location of the biopsy punch for RNA quantification. Figure 15B is a bar graph showing the amount of remaining canine rhodopsin RNA as a percentage of the levels measured in untreated areas of the same retina. Figure 15C is an immunoblot showing the amount of canine rhodopsin in biopsy punches taken from treated (Tx) and untreated (UnTx) areas of canine retina. Histone H3 was used for normalization. Bar graphs show the amount of dog rhodopsin protein remaining as a percentage of the levels measured in untreated areas of the same retina, and Figures 15D-E are tables showing the numerical values ​​for each experiment (reported as percent RNA or protein remaining or percent RNA or protein knockdown, respectively).

[0094] FIG. 16A to FIG. 16D show mutant RHO T4R / + RHO 2 weeks after exposure to brief light exposure that induces acute retinal degeneration in dogs T4R / + OCT B-scans of dogs including treated (with different viral titers of AAV2 / 5-sc-H1-shRNA820) and non-treated retinal areas are shown. (Figure 16A) 1x10 12 OCT scan of a dog treated with 5x10 vg / ml. (Figure 16B) 11 OCT scan of a dog treated with 2.5x10 vg / ml. (Figure 16C) 11 OCT scan of a dog treated with 1x10 vg / ml. (Figure 16D) 11 17A-B show protection from light-induced retinal degeneration in RHO dogs treated with AAV2 / 5-sc-shRNA820. T4R / +Figure 17 shows histology of ONL thickness from untreated wild-type control dogs (left panel) and EM411-OS treated with AAV2 / 5-sc-shRNA820 at 5E+11vg / ml, showing that ONL thickness is preserved in the treated / bleb area for several weeks after light-induced damage. Black and white curve indicates the border of the bleb seen immediately after subretinal injection. The bottom panel shows OCT B-scans with the ONL colored dark grey (middle band) for visualization. (Figure 17B) Sites on the outside and inside of the bleb were selected for ONL thickness measurement.

[0095] Complete knockdown of canine RHO RNA and protein (by Western blot) was observed in 1x10 12 and 5x10 1 Complete rescue of the outer nuclear layer (ONL), including photoreceptor cell bodies, was observed with AAV2 / 5-shRNA-Rho820 at a viral titer of 1x10 vg / ml. 12 and 5x10 11 A viral titer of 1000 vg / ml was achieved with AAV2 / 5-shRNA-Rho820.

[0096] Mutant RHOs injected with different viral titers of AAV2 / 5-sc-H1-shRNA820 T4R / + Histological and immunohistochemical evaluation of protection of photoreceptor cells from light-induced damage in the dog eye. 1x10 12 From 1x10 11 The virus was injected subretinal with titers ranging from 1000 to 10000 vg / ml, followed by a 10-μg pulse of 10000 vg / ml. 2 The same mutant RHO mice shown in the table above were exposed to white light for 1 min at a corneal irradiance of 100 nm (8 weeks after injection). T4R / +Eyes from dogs were examined. This dose of radiation has previously been shown to cause acute retinal degeneration in RHO T4R mutants but not in wild-type dogs. Histology and immunohistology were used to evaluate the level of protection conferred by AAV2 / 5-sc-H1-shRNA820 in the treated / bleb area. The surrounding non-bleb / non-treated area was used as an internal control for each eye. The integrity of rod outer segment and cone morphology was evaluated using antibodies against rhodopsin (RHO) and human cone arrestin (hCA). Figure 18 shows the results of subretinally injected AAV2 / 5-sc-H1-shRNA820 in mutant RHO dogs. T4R / + Histology (HE staining) and immunohistochemistry are shown for treated (Tx) and untreated (UnTx) areas of the retina (rhodopsin stained green and appears as a lighter stain in the lower panel of FIG. 18; human cone arrestin stained red and appears as a gray stain in the lower panel of FIG. 18). After 2 weeks of light exposure, 1x10 12 and 5x10 11 Good preservation of ONL thickness was seen in the bleb / treated area of ​​eyes injected with a titer of 2.5x10 11 In vg / ml, ONL thickness was slightly decreased, with 1x10 11 No protection was observed at titers of 1x10 vg / ml. 12 and 5x10 11 Consistent with highly efficient knockdown of rhodopsin expression at vg / ml titers, a reduction in rod cell OS length was seen in HE sections in combination with a reduction in rhodopsin expression.

[0097] Complete knockdown of canine RHO RNA and protein (by Western blot) was observed in 1x10 12 and 5x10 1 Complete protection of the outer nuclear layer (ONL), including the photoreceptor cell bodies, was observed with AV2 / 5-sc-H1-shRNA820 at viral titers of 1x10 vg / ml. 12 and 5x10 11This was achieved with AAV2 / 5-sc-H1-shRNA820 at a viral titer of 100 vg / ml. The loss of rod outer segment structure as a result of efficient RHO knockdown argues for the need for a combined knockdown and replacement strategy to ensure that processed rods retain their outer segments and remain functional.

[0098] Example 8 This example provides data supporting gene therapy using RHO-ADRP. An AAV2 / 5 vector construct (AAV2 / 5-sc-HOP-RHO820-H1-shRNA820) combining both the shRNA820 knockdown reagent and the replacement reagent RHO820 (=cured human RHO mRNA) was administered to the mutant RHO T4R / + To test in dogs, determine the viral titer of AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 that results in efficient knockdown of endogenous canine RHO and efficient expression of substituted sclerotic human RHO (RHO820), and to demonstrate that AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 inhibits the expression of mutant RHO T4R / + These include evidence that it confers protection to photoreceptor cells from light-induced retinal degeneration in dogs, and that preservation of rod outer segments is achieved in retinal regions treated with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820.

[0099] RHO T4R / + Analysis of RHO knockdown and replacement by shRNA820 and RHO820 in mutant dogs: Two RHOs T4R / + Dogs received subretinal injections of AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 in both eyes at the virus concentrations shown in the table below (Table 10). [Table 11]

[0100] Eleven weeks after injection, in vivo retinal imaging (cSLO / OCT) was performed and light irradiation (1 mW / cm) was administered to the OS eye in both dogs to induce light-induced retinal degeneration. 2 A 10-minute cSLO / OCT was performed on all dogs at 4°C for 1 min. Two weeks after irradiation, cSLO / OCT was repeated on all dogs to assess the rescue effect conferred by the treatment. Dogs were sacrificed and several 3 mm neural retinal biopsy punches were retrieved from OD eyes, both from bleb / treated and non-bleb / non-treated areas, to measure rhodopsin expression levels by Western blot and canine and human RHO RNA expression levels by qPCR analysis. OS eyes were fixed, embedded in optimal cutting temperature medium, and processed for histology and immunohistochemical staining.

[0101] Western Blot Analysis: Triplicate biopsy punches (representing either bleb / treated or non-bleb / non-treated retinal areas) from each OD eye were incubated in 50 μl of Lewin's buffer A (containing protease inhibitors) on ice for 15 min. Samples were sonicated at 40% amplitude, 15 sec / 10 sec off×8 pulses. Samples were then centrifuged and the pellet was discarded. Protein concentration in the supernatant was measured by Bradford method. Samples were stored at −20° C. To visualize rhodopsin, 1 μg of total protein was loaded onto the gel (antibody used: Millipore MAB5356, diluted 1:1000 in ODYSSEY blocking buffer). Note that the antibody detects both canine and human RHO. Due to the loss of glycosylation at Asn2, mutant T4R rhodopsin has a lower molecular weight (MW) that can be detected by immunoblotting and can therefore be distinguished from wild-type (canine or human) RHO protein. T4R / + RHO immunoblot from shows two bands corresponding to wild-type (high molecular weight) RHO protein and mutant T4R (low molecular weight) RHO protein. Anti-histone H3 antibody (Abcam ab1791, diluted 1:3000) was used as a loading control to normalize the signal.

[0102] Absolute quantification of canine and human rhodopsin RNA in the dog retina: To determine the absolute amount of endogenous canine rhodopsin RNA and human RHO820 RNA present in the retina after treatment with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820, absolute quantification was performed using the Q-PCR standard curve method. A dilution series of known amounts of canine and human RHO cDNA was used to construct the standard curve. 0.1 nanograms of total cDNA was used for quantification. The total amount of canine RHO and human RHO820 RNA in each sample was calculated based on this standard curve.

[0103] FIG. 19A to FIG. 19F show mutant RHO T4R / + In dogs, 5x10 11Figure 19B shows RNA and protein analysis of rhodopsin knockdown and replacement by AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 injected subretinally at a titer of 10 ... Figure 19C is a table showing the values ​​for each pair of punches used for protein quantification. Figure 19D is a bar graph showing remaining canine rhodopsin RNA in treated areas as a percentage of canine RHO RNA measured in untreated areas. Figure 19E is a bar graph showing human RHO820 in treated areas as a percentage of canine RHO RNA measured in untreated areas. Figure 19F is a table showing the values ​​for each pair of punches used for RNA quantification.

[0104] 5x10 11 Mutant RHO subretinally injected with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at 1000 mg / ml T4R / + Protection of photoreceptor cells from light-induced damage assessed by in vivo retinal imaging in the dog eye: 5x10 11 Two mutant RHO mice were injected subretinally with a virus titer of 1000 vg / ml. T4R / +Eyes from dogs were examined by cSLO / OCT imaging before injection, 11 weeks after injection (before light exposure), and 2 weeks after light damage. A 1-minute exposure to white light at an intensity (1 mW / cm2 corneal irradiance) previously shown to cause acute retinal degeneration in RHO T4R mutants but not in wild-type dogs was used to evaluate the level of protection afforded by the AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 vector in the treated / bleb area. The thickness of the ONL in the bleb / treated area was preserved at all time points after injection, suggesting that the vector construct was non-toxic and that there was no loss of photoreceptor cells during that time period. The surrounding non-bleb / non-treated area was used as an internal control for each eye. Two weeks after light exposure, the thickness of the ONL was completely preserved in the bleb / treated area of ​​both injected eyes.

[0105] Figures 20A to 20C are 5x10 11 Mutant RHO treated with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at a titer of 100 / vg / ml. T4R / + 20A shows an en face cSLO composite image showing a region of the retina protected from degeneration (boundary demarcated by white arrow) 2 weeks after light exposure. The light grey arrows indicate the location within the treated region of the OCT B-scan shown in FIG. 20B, and the dark grey arrows indicate the location within the non-treated region of the OCT B-scan shown in FIG. 20C. FIG. 20B shows OCT B-scans within the treated region before injection, 11 weeks after injection, and 13 weeks after injection / 2 weeks after light exposure. The thickness of the ONL is preserved throughout the treated region at both time points after viral vector injection. FIG. 20B and FIG. 30V show OCT B-scans within the non-treated region before injection, 11 weeks after injection, and 13 weeks after injection / 2 weeks after light exposure. The ONL is preserved up to 11 weeks after injection, but is completely lost after 2 weeks of light exposure. FIG. 21 shows protection from light-induced retinal degeneration. 11Two RHOs treated with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at a titer of 1 / vg / ml T4R / + 1 shows a histology map of ONL thickness derived from the grafts.

[0106] 5x10 11 Mutant RHO injected with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at a titer of vg / ml T4R / + Histological and immunohistochemical assessment of protection of photoreceptor cells from light-induced damage in the dog eye: 5x10 11 AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 was injected subretinally at a titer of 1000 mg / vg / ml, followed by stimulation with a certain intensity (1 mW / cm 2 The same mutant RHO mice shown in the table above were exposed to white light for 1 min at a corneal irradiance of 100 nm (11 weeks after injection). T4R / + Eyes from dogs were studied, and this dose of radiation has previously been shown to cause acute retinal degeneration in RHO T4R mutant but not wild-type dogs.

[0107] Immunohistology was used to assess the level of protection conferred by AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 in the treated / bleb area. The surrounding non-bleb / non-treated area was used as an internal control for each eye. The integrity of rod outer segment and cone morphology was assessed using antibodies against rhodopsin (RHO) and human cone arrestin (hCA). Two weeks after light exposure, 5x10 11 Excellent preservation of ONL thickness was achieved in the bleb / treated area of ​​eyes injected at a titer of 1000 vg / ml. Furthermore, rod outer segment preservation was achieved with the AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 construct in contrast to when the retina was treated with the knockdown reagent shRNA820.

[0108] Figure 22 shows a 5x10 11 AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 was injected subretinally at a titer of 100 / vg / ml.T4R / + Immunohistochemistry is shown in treated (Tx), transition zone, and untreated (UnTx) regions of the retina (rhodopsin stained green and appears as a lighter stain in the image in FIG. 22; human cone arrestin stained red and appears as a gray stain in the image in FIG. 22). In treated regions, the thickness of the outer nuclear layer (ONL) and rod outer segments (OS) are fully preserved, whereas in untreated regions, all rods are lost and cone cell bodies are reduced in line. A clear border between treated and untreated regions is seen in the transition zone.

[0109] Complete knockdown of canine RHO RNA and protein (by Western blot) was observed with a viral titer of 5x10 11 AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at 1000 ng / ml has been achieved. When compared to normal RHO levels in the canine retina, efficient replacement with sclerotic RHO820 is achieved at both the mRNA (118-130%) and protein (30-33%) levels. Complete protection of the outer nuclear layer (ONL), including the photoreceptor cell bodies, was achieved with a viral titer of 5 × 10 11 Complete preservation of rod outer segments as a result of efficient RHO replacement by RHO820 is achieved with AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at 1000 ng / ml. A viral titer of 5×10 11 This is achieved by AAV2 / 5-sc-HOP-RHO820-H1-shRNA820 at 1000 mg / ml.

[0110] References 1.Mao H, Gorbatyuk MS, Rossmiller B, Hauswirth WW, Lewin AS. Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23H RHO Transgenic Mice. Hum Gene Ther. 2012;23:356-366. 2.Rossmiller B, Mao H, Lewin AS. Gene therapy in animal models of autosomal dominant retinitis pigmentosa. Mol Vis. 2012;18:2479-2496. 3.Gorbatyuk M, Justilien V, Liu J, Hauswirth WW, Lewin AS. Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery. Vision Res. 2007;47:1202-1208. 4.Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003;115:209-216. 5. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004;22:326-330. 6.Jensen SMR, Schmitz A, Pedersen FS, Kjems J+, Bramsen JB. Functional Selection of shRNA Loops from Randomized Retroviral Libraries. PLoS ONE. 2012;7:e43095. 7.Zhou H, Xia XG, Xu Z. An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res. 2005;33:e62-e70.

[0111] Equivalent While several embodiments of the invention are described and illustrated herein, those skilled in the art can readily conceive of various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein. Moreover, these variations and / or modifications are deemed to be within the scope of the inventive embodiments described herein. Moreover, those skilled in the art will generally recognize that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will be based on the particular application for which the teachings of the invention are used. Those skilled in the art will recognize, or can ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, it should be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and equivalents thereof, the inventive embodiments may be practiced otherwise than as described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of these features, systems, articles, materials, kits, and / or methods is within the scope of the present disclosure, provided that the features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0112] All definitions defined and used herein should be understood to control for any dictionary definitions, definitions in documents incorporated herein by reference, and / or ordinary meanings of the defined terms.

[0113] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and may, in some cases, be included in their entirety.

[0114] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly stated to the contrary, should be understood to mean "at least one."

[0115] The term "and / or" as used herein and in the claims should be understood to mean "one or both" of the elements so conjunctively present, i.e., in some cases conjunctively and in other cases disjunctively present. Multiple elements described as "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjunctively present. Other elements than the elements specifically identified by the "and / or" clause may optionally be present, whether or not associated with the specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" may refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements), etc.

[0116] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of, but also including a plurality of, and possibly including additional unlisted items of, an element or list of elements. Only terms expressly to the contrary, such as "only one" or "exactly one" or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of an element or list of elements. In general, the term "or" as used herein will be interpreted to indicate exclusive alternatives (i.e., "either, but not both") only when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" will have its ordinary meaning as used in the field of patent law.

[0117] As used herein and in the claims, the phrase "at least one" with reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, and does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as non-limiting examples, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer to, in one embodiment, at least one (optionally including more than one) A and no B (optionally including elements other than B); in another embodiment, at least one (optionally including more than one) B and no A (optionally including elements other than A); in yet another embodiment, at least one (optionally including more than one) A and at least one (optionally including more than one) B (optionally including other elements), etc.

[0118] It should also be understood that, unless expressly stated to the contrary, in any method described herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0119] In the above specification and claims, all transitional phrases should be understood to be open-ended, such as, for example, "comprises," "having," and the like. Open-ended means including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, and are set forth in the United States Patent Office Manual of Patent Examining Procedure, Section 2111.03. In alternative embodiments, it should be recognized that embodiments described in this document using open-ended transitional phrases (e.g., "comprising") are also considered to "consist of" and "consist essentially of" the features described by the open-ended transitional phrases. For example, if the present disclosure describes "a composition comprising A and B," the present disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. a) a first nucleic acid sequence encoding a synthetic ribonucleic acid (RNA) molecule comprising a sense strand of the sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 7) and an antisense strand of the sequence UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 8); and b) a second nucleic acid sequence comprising a recombinant RHO gene that does not contain a sequence targeted by the synthetic RNA molecule; 13. A vector comprising:

2. The vector described in claim 1, wherein the synthetic RNA molecule further comprises a loop comprising the sequence UCAAGAG (sequence number 9) or the sequence UGUGCUU (sequence number 10).

3. A vector described in claim 1 or 2, wherein the synthetic RNA molecule is a small interfering RNA (siRNA).

4. The vector described in claim 1 or 2, wherein the synthetic RNA molecule is a small hairpin RNA (shRNA).

5. A vector described in claim 1 or 2, wherein the synthetic RNA molecule is an artificial microRNA (miRNA).

6. A synthetic RNA molecule having the sequence UGCUGUUGACAGUGAGCGA(X) ｎ UAGUGAAGCCACAGAUGUA(Y) ｎ CUGCCUACUGCCUCGGA (SEQ ID NO: 19), (X) ｎ comprises SEQ ID NO: 7, (Y) ｎ The vector of claim 1 or 5, wherein said vector comprises SEQ ID NO:

8.

7. A vector described in any one of claims 1 to 6, wherein the synthetic RNA molecule and the recombinant rho gene are both under the expression control of a single promoter sequence.

8. A vector described in any one of claims 1 to 6, wherein the synthetic RNA molecule and the recombinant rho gene are each under the expression control of a separate promoter sequence.

9. The vector described in claim 8, wherein the synthetic RNA molecule is an shRNA, and the shRNA is under the expression control of an RNA polymerase III promoter.

10. The vector described in claim 8, wherein the synthetic RNA molecule is an artificial miRNA, and the artificial miRNA is under the expression control of an RNA polymerase II promoter.

11. A vector described in any of claims 8 to 10, wherein the recombinant RHO gene is under the expression control of a constitutive promoter or an inducible promoter.

12. A vector described in any one of claims 1 to 11, wherein the recombinant RHO gene comprises the nucleotide sequence of SEQ ID NO:

42.

13. A vector described in any one of claims 1 to 12, wherein the synthetic RNA molecule further comprises an unpaired overhang sequence at the 5' end or 3' end.

14. The vector of claim 13 , wherein the unpaired overhang sequence comprises a repeated base sequence.

15. 15. The vector of claim 14, wherein the repeated base sequence comprises repeated uracil (U) bases.

16. 16. The vector of claim 14 or 15, wherein the unpaired overhang sequence is UU.

17. The vector according to any one of claims 1 to 16, wherein the vector is an expression plasmid.

18. The vector according to any one of claims 1 to 16, wherein the vector is a viral vector.

19. The vector of claim 18, wherein the viral vector is an adeno-associated viral (AAV) vector.

20. The vector described in claim 19, wherein the AAV vector is an AAV2, AAV2 / 1, AAV2 / 5, AAV2 / 8 or AAV2 / 9 vector.

21. A composition comprising the vector according to any one of claims 1 to 20.

22. 22. The composition of claim 21, further comprising one or more physiologically acceptable carriers or one or more physiologically acceptable adjuvants.

23. 23. The composition of claim 21 or 22 for reducing RHO expression in a subject.

24. The composition of claim 21 or 22 for treating retinitis pigmentosa (RP) in a subject.

25. The composition of claim 24, wherein treating RP comprises silencing one or both alleles of the RHO gene.

26. Use of a vector described in any one of claims 1 to 20 or a composition described in claim 21 or 22 in the manufacture of a medicament for treating retinitis pigmentosa (RP).

27. The use of claim 26, wherein treating RP comprises silencing one or both alleles of the RHO gene.

28. A composition described in claim 24 or 25 or a use described in claim 26 or 27, wherein RP is autosomal dominant retinitis pigmentosa (adRP).

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