Novel capsid protein variants and uses thereof

Abstract

1. A capsid protein variant, a recombinant AAV viral particle comprising the same, and a method of using the recombinant AAV viral particle for the treatment of an ocular disease, wherein the capsid protein variant comprises the following substitutions compared to the parent AAV capsid protein VP1: Q464V, A467P, D469N, I470M, R471A, D472V, S474G, optionally Y500F and / or S501A; and wherein the ocular disease is X-linked hereditary retinoschisis.

Description

[Technical Field]

[0001] The present invention relates to capsid protein variants, recombinant AAV viral particles comprising the same, and methods of using the recombinant AAV viral particles to treat ocular diseases. [Background technology]

[0002] Currently, there are approximately 15 million people worldwide who are blind due to inherited retinal diseases, accounting for approximately 0.02% of the total population. There are many types of inherited retinal diseases (IRDs), mainly including non-syndromic retinitis pigmentosa (40%), Usher syndrome (10%), cone-rod dystrophy (10%), Leber congenital amaurosis (5%), Leber hereditary optic neuropathy, and major subtypes.

[0003] The leading cause of macular degeneration in adolescent males is X-linked hereditary retinoschisis. As early as 1898, German ophthalmologist Hass described two cases of typical radiocystoid macular degeneration in men, later identified as X-linked hereditary retinoschisis. The clinical manifestations of XLRS are highly variable, but are primarily characterized by microcystic, axial changes in the macula, accompanied by peripheral bullous lesions in approximately 50% of cases due to inner retinal breaks. Most patients develop progressive visual impairment during school age. Later in the disease, vision may decline significantly or even lead to blindness due to complications such as vitreous hemorrhage, choroidal sclerosis, retinal detachment, and retinal atrophy. Electroretinogram (ERG) studies reveal a decrease in the b-wave arising from the inner retina, while the a-wave representing the photoreceptors remains relatively normal or is slightly reduced, resulting in a decrease in the b / a ratio, a characteristic electrophysiological change of this disease. In addition, optical coherence tomography (OCT) reveals fissures in the nerve fiber layer, and fundus photography and fluorescein angiography (FFA) are also useful for diagnosing this disease. This disease occurs worldwide, with a prevalence of 1:5,000–1:25,000. It is an X-linked recessive genetic disorder. Males are affected, while females are asymptomatic carriers. The RS1 gene was identified by positional cloning in 1997 by Professor Bernard Weber (Sauer et al., Nat Genet). The human RS1 protein, containing only 224 amino acids, is an extracellular matrix molecule with a secretory signal peptide. The C-terminal discoid domain of the RS1 protein exerts an "adhesion function" between cells.

[0004] The successful construction of the human genetic map and the rapid development of molecular biology techniques have led to the successful application of viral vectors, which has led to significant progress in gene therapy for XLRS. Many experimental studies have been conducted on the basis of basic research and preliminary studies for the clinical application of gene therapy for XLRS.

[0005] However, currently, there are no specific drugs or treatments for juvenile XLRS retinoschisis in clinical practice. Most existing XLRS gene therapy technologies use the CMV promoter. In primate and clinical trials, the CMV promoter is at risk of being silenced and is toxic to photoreceptors and retinal pigment epithelial cells in the eye. Meanwhile, the serotypes disclosed in the prior art for XLRS gene therapy products are generally AAV2, AAV8, and AAV2.7m8. When administered through the vitreous cavity, the serotypes disclosed in the prior art cannot efficiently penetrate the retinal internal limiting membrane, resulting in most rAAV remaining in the vitreous, inducing intraocular inflammation and affecting the clinical treatment efficacy of XLRS. The serotypes disclosed in the present invention can efficiently penetrate the internal limiting membrane and secrete and express sufficient RS1 multimeric protein to ultimately reach photoreceptor cells and act as a scaffold to treat retinoschisis. Regarding XLRS gene therapy, not only the efficacy of the treatment but also the long-term effects of the therapeutic drug should be considered. Since most patients develop the disease after childhood, achieving long-term efficacy of drug treatment and resolving the convenience of drug administration in clinical trials are key to improving patient compliance, and are also issues that need to be addressed urgently.

[0006] AAV is a small, non-enveloped, single-stranded DNA virus. The AAV genome is 4.7 kb and is characterized by two inverted repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. These two ITRs are the only cis-elements required for AAV replication, packaging, and integration. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins primarily play roles in regulating AAV replication and integrating AAV into host cell chromosomes. The Cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). More than 80% of the total proteins in AAV virions contain VP3; in mature virions, VP1, VP2, and VP3 are found in relative abundances of approximately 1:1:10. In vitro, these three proteins spontaneously assemble to form virosome-like structures, such as viral capsids. Therefore, viral capsid formation in infected cells appears to proceed independently of viral DNA synthesis.

[0007] Unlike other viral vectors, AAV has not been shown to be associated with any known human disease and is not generally considered pathogenic, making it a promising vector for human gene therapy. Furthermore, AAV can safely transduce postmitotic tissues with relatively low immunogenicity, can integrate into host chromosomes in a site-specific manner, and can integrate into chromosome 19 in tissue culture cells when Rep proteins are supplied in trans. The integrated genome of AAV has been shown to enable long-term gene expression in many tissues, including muscle, liver, and brain.

[0008] There is an urgent need in the art for the development of novel AAVs, as well as safe and reliable therapeutic methods or drugs based thereon, such as therapeutic vectors or compositions for gene therapy, for the effective treatment of XLRS. Summary of the Invention [Means for solving the problem]

[0009] The present invention relates to gene therapy agents (such as AAV viral particles) that are based on directed evolution variants of recombinant adeno-associated virus serotype 2 and are used as vectors carrying the human RS1 gene (hRS1).

[0010] In the present invention, due to its unique capsid and optimized RS1 gene, the AAV viral particles showed efficient transduction activity in photoreceptor cells in vitro. At the same time, compared with the reference product, the AAV viral particles showed significantly improved transduction activity in various layers of retinal tissue in mice under different in vivo administration formats. At the same time, compared with the existing serotype AAV8 in terms of efficacy under the same intravitreal administration (IVT) format, the viral particles of the present invention showed significant improvements in in vivo transduction activity and efficacy of gene therapy drugs.

[0011] Thus, the present invention relates to AAV capsid protein variants comprising, or consisting of, mutations relative to a parent AAV capsid protein variant selected from the group consisting of Q464V, A467P, D469N, I470M, R471A, D472V, and S474G.

[0012] The present invention further comprises: (i) an AAV capsid comprising an engineered capsid protein VP1 that contains, or contains only, the following amino acid mutation sites relative to the parent AAV capsid protein VP1: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G; (ii) a nucleic acid of interest, e.g., a nucleic acid encoding RS1, packaged within the AAV capsid; The present invention relates to a recombinant AAV viral particle comprising the

[0013] The present invention also relates to pharmaceutical preparations, compositions or medicaments comprising the recombinant AAV viral particles of the present invention.

[0014] The present invention also relates to restoring vision and / or treating or preventing eye diseases in a subject, comprising administering to the subject a recombinant AAV viral particle of the present invention, or a pharmaceutical formulation, composition or drug comprising same.

[0015] In one aspect, the present invention relates to the following embodiments: 1. An AAV capsid protein variant comprising a modified capsid protein VP1 comprising the following amino acid mutations relative to the parent AAV capsid protein VP1: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G, the positions of which are determined with reference to the amino acid sequence positions of SEQ ID NO: 51. 2. The AAV capsid protein variant of embodiment 1, wherein the parent AAV capsid protein VP1 is derived from AAV serotype 2 (AAV2) or AAV2 modified 7m8 (AAV2.7m8), preferably AAV2.7m8. 3. AAV capsid protein variants are: (i) comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1, and containing the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G; (iii) encoded by the nucleic acid sequence set forth in SEQ ID NO:2; or (iv) The AAV capsid protein variant of embodiment 1, encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO:2, and comprising the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G. 4. An isolated nucleic acid comprising a nucleotide sequence encoding an AAV capsid protein variant according to any one of embodiments 1 to 3. 5. The nucleotide sequence is (i) comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO:2; or (iv) The isolated nucleic acid of embodiment 4, wherein the capsid protein comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO:2, and wherein the capsid protein variant encoded thereby comprises the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G. 6. (i) An AAV capsid protein variant according to any one of embodiments 1 to 5; (ii) a nucleic acid of interest packaged within an AAV capsid, the nucleic acid of interest comprising a nucleic acid molecule encoding RS1; A recombinant AAV viral particle (rAAV) comprising: 7. The recombinant AAV viral particle of embodiment 6, wherein the RS1 is human RS1. 8. Human RS1 protein is (i) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15; or (ii) A recombinant AAV viral particle as described in embodiment 7, comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15. 9.RS1, (i) comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 16 to 18 or a complementary sequence thereof; or (ii) A recombinant AAV viral particle described in any one of embodiments 6 to 8, encoded by a coding sequence comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 16 to 18, or a complementary sequence thereof. 10. A recombinant AAV viral particle described in any of embodiments 6 to 9, wherein the nucleic acid of interest is contained in an expression cassette and then packaged within the AAV capsid. 11. The recombinant AAV viral particle of embodiment 10, wherein the expression cassette is single-stranded DNA, double-stranded DNA, or single- or double-stranded RNA. 12. The recombinant AAV viral particle of embodiment 10 or 11, wherein the expression cassette comprises at least one ITR (e.g., L-ITR or R-ITR) sequence flanking the nucleic acid of interest. 13. A recombinant AAV viral particle according to any one of embodiments 10 to 12, wherein the expression cassette comprises one or more regulatory sequences selected from one or more or all of the following: a promoter, an inverted repeat, an intron, an enhancer, a post-transcriptional regulatory sequence, a polyadenylation region, a selection marker, or a reporter gene. 14. A method for producing recombinant AAV viral particles, comprising culturing packaging cells under conditions sufficient for the production of recombinant AAV viral particles, wherein the packaging cells contain a nucleic acid encoding a capsid protein variant of any one of embodiments 1 to 3, or a plasmid comprising the nucleic acid of embodiment 4 or 5. 15. The method of embodiment 14, wherein the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleic acid of interest. 16. The method of embodiment 14 or 15, further comprising isolating from the culture supernatant containing self-complementary recombinant AAV viral particles. 17. The method of any one of embodiments 14 to 16, further comprising lysing the packaging cells and isolating the recombinant AAV viral particles from the cell lysate. 18. The following steps: a. removing necrotic debris; b. treating the supernatant containing the recombinant AAV viral particles with benzonase nuclease; c. Concentrating the recombinant AAV viral particles; d. Purifying the recombinant AAV viral particles; 17. The method of any one of embodiments 14 to 16, further comprising one or more of: 19. A recombinant AAV viral particle produced according to the method of any one of embodiments 14 to 18. 20. A packaging cell for producing recombinant AAV viral particles, comprising a plasmid containing a nucleic acid encoding a capsid protein variant described in any one of embodiments 1 to 3, or a nucleic acid described in embodiment 4 or 5. 21. A formulation or composition or medicament comprising a recombinant AAV viral particle according to any one of embodiments 6 to 13 or 19, optionally with pharmaceutically acceptable auxiliary substances, such as pharmaceutically acceptable carriers, excipients such as buffers known in the art. 22. A combination product comprising a recombinant AAV viral particle according to any one of embodiments 6 to 13 or 19, and one or more additional therapeutic agents, for example immunomodulatory agents such as immunosuppressants. 23. A method of treating an ocular disease in an individual, comprising administering to the individual a recombinant AAV viral particle of any one of embodiments 6 to 13 or 19, or a formulation or composition of embodiment 21, or a combination product of embodiment 22. 24. The method of embodiment 23, wherein the ocular disease is selected from the group consisting of retinopathy or chorioretinopathy, age-related macular degeneration, diabetic retinopathy, and other non-genetic ocular diseases such as physically or chemically induced retinal damage, for example diseases caused by X-linked retinoschisis or retinitis pigmentosa. 25. The method of embodiment 23 or 24, wherein administering is via intraocular administration, such as intraretinal or intravitreal administration, such as subretinal or intravitreal administration (IVT). 26. The method of embodiment 25, wherein administering is by injection. 27. Recombinant AAV viral particles are expressed in approximately 10 3 vg / eye / time, 10 4 vg / eye / count, 10 5 vg / eye / count, 10 6 vg / eye / count, 10 7 vg / eye / count, 10 8 vg / eye / count, 10 9 vg / eye / count, 10 10 vg / eye / count, 10 11 vg / eye / number of times, or 10 12 vg / eye / time or more, preferably 10 12 vg / eye / times or less, e.g., 2 x 10 8 vg / eye / count, 3 x 10 8 vg / eye / count, 4 x 10 8 vg / eye / count or 5 x 10 8 vg / eye / times or more, e.g., about 10 7 ~Approx. 5×10 8 vg / eye / count, 1×10 7 ~Approx. 5×10 8 vg / eye / times, e.g., approximately 2 × 10 8 vg / eye / count, 3 x 10 8 vg / eye / count, 4 x 10 8 vg / eye / count or 5 x 10 8 The method of any one of embodiments 23-26, wherein the dose is administered in a dose of vg / eye / time. [Brief explanation of the drawings]

[0016] [Figure 1] A comparison of the expression efficiency of different expression cassettes is shown. [Figure 1A] The structure of each GOI plasmid is shown. [Figure 1B] Expression of hRS1 in corresponding cell lysates and cell culture supernatants of 293T cells is shown. [Figure 1C-1D] Expression of RS1 from each plasmid in cells (C) and supernatant (D) is shown. [Figure 1E] Expression of hRS1 in cell lysates and supernatants for HEK293T cell expression is shown. [Figure 1F-1G] The relative expression levels of RS1 in the lysates (F) and supernatants (G) of 293T cells infected with each rAAV virus are shown in Table 2. The number of GOI plasmids corresponding to each plasmid is shown. Mock / M: blank control; RS1-HA: human RS1 protein with an HA tag; NC: untreated cells. [Figure 2] 1 shows a comparison of the in vitro expression efficiency of RS1 after codon optimization. [Figure 2A] The structure of each GOI plasmid is shown. [Figure 2B] 1 shows the expression of hRS1 in cells and supernatants of HEK293T cells infected with recombinant AAV. [Figure 2C-2D] The relative expression of RS1 for each plasmid in cells (C) and supernatant (D) is shown. [Figure 2E] The correspondence between the GOI plasmid and the expression cassette is shown. [Figure 2F] 1 shows the expression of hRS1 in cell lysates and supernatants from ARPE19 cells. [Figure 2G] The relative expression of RS1 of each plasmid in the supernatant of APRE19 cells is shown. [Figure 2H] 1 shows the expression of hRS1 in cell lysates and supernatants from 661W cells. [Figure 2I] The relative expression of RS1 for each plasmid in the supernatant of 661W cells is shown, and the GOI plasmid number corresponding to each plasmid is shown in Table 2, where NC means untreated cells. [Figure 3] 1 shows a comparison of hRS1 multimeric protein levels secreted in vitro by self-complementary AAV (scAAV) and single-stranded AAV (ssAAV). [Figure 3A] The structures of ssAAV and scAAV are shown. [Figure 3B]WB images of hRS1 octamer expressed by each packaging virus in the supernatant and cells of HEK293T are shown. [Figure 3C-3D] The relative expression levels of hRS1 octamer in the supernatant and cells are shown. Markers: protein size indicators; ssAAV8-200: single-stranded DNA (GOI-C59) & AAV8 serotype (multiplicity of infection 200); scAAV8-200: self-complementary DNA (GOI-C21) & AAV8 serotype (multiplicity of infection 200), etc., with a maximum multiplicity of infection (MOI) of 2000; mock: blank control; ssAAV8-CAG-hRS1: AAV8 serotype & GOI-C59; ssRC-C08-CAG-hRS1: RC-C08 serotype & GOI-C59; ssAAV8-CAG-hRS1: AAV8 serotype & GOI-C59; scAAV8-CAG-hRS1: AAV8 serotype & GOI-C21; NC supernatant: supernatant of untreated cells; NC cells: untreated cell lysate. [Figure 4] 1 shows a comparison of target protein transcription and protein expression levels by ssAAV and scAAV in 293T cells. [Figure 4A] Figure 1 shows the relative transcription levels of RS1 mRNA in HEK293T cells infected with RC-C08 capsid and GOI-C59 packaged viruses (ssAAV) at different MOIs. [Figure 4B] Figure 1 shows the relative transcription levels of RS1 mRNA in HEK293T cells infected with RC-C08 capsid and GOI-C21 packaged viruses (scAAV) at different MOIs. [Figure 4C] 1 shows the relative transcription levels of RS1 mRNA at different time points after infection of HKE293T cells with ssAAV and scAAV. [Figure 4D] Shown is the expression of RS1 octamer in the supernatant (D) and cells (F) at different time points detected by nondenaturing WB after infection of HKE293T cells with ssAAV (also called ssRC-C08) and scAAV (also called scRC-C08). [Figure 4E]Expression of RS1 octamer in supernatants (D) and cells (F) at different time points as detected by non-grayscale scanning statistics after infection of HKE293T cells with ssAAV and scAAV is shown. [Figure 4F] Shown is the expression of RS1 octamer in the supernatant (D) and cells (F) at different time points detected by nondenaturing WB after infection of HKE293T cells with ssAAV (also called ssRC-C08) and scAAV (also called scRC-C08). [Figure 4G] Expression of RS1 octamer in supernatants (D) and cells (F) at different time points as detected by non-grayscale scanning statistics after infection of HKE293T cells with ssAAV and scAAV is shown. [Figure 5] 1 shows the transcription level and protein expression intensity of RS1 by ssRC-C08&GOI-C59 and ssAAV2.7m8&GOI-C59 in photoreceptor cells. [Figure 5A] This shows the transcription level of RS1 mRNA in 293T infected with virus particles produced by AAV2.7m8 serotype capsid and RC-C08 serotype capsid at different MOIs (100, 1000, 10000). [Figure 5B] A comparison of RS1 mRNA transcription levels at two time points, 48 ​​hours and 72 hours, after infection of 293T with the two serotypes at different MOIs (100, 1000, 10000) is shown. [Figure 5C] 1 shows a dose-dependent curve of the transcription level of ssAAV2.7m8&GOI-C59 versus the MOI of the virus in 293T cells. [Figure 5D] The dose-dependent curves of transcribed mRNA of the target protein against the virus MOI at 48 hours and 72 hours after cell infection with RC-C08 & GOI-C59 are shown. [Figure 5E]1 shows the expression of monomeric RS1 in 661W cells measured at 48 and 72 hours after infection with viral particles produced by RC-C08 capsid and AAV2.7m8 capsid together with GOI-C59. [Figure 5F] Histogram statistics of data obtained from ImageJ grayscale scanning analysis of the WB photograph in Figure 5E are shown. [Figure 6] This shows a comparison of the expression intensity of target proteins by ssRC-C08 and ssAAV8 in the retinal tissue and vitreous humor of knockout mice. [Figure 6A] Detection of RS1 protein in retinal tissue at different time points (day 14 / day 21 / day 28) after administration of ssAAV8&GOI-C59 (Group A, OD administration to the right eye) and ssRC-C08&GOI-C59 (Group B, OD administration to the right eye) is shown (in terms of mouse numbering, A for Group A or B for Group B, B353, etc.). [Figure 6B] 1 shows detection of RS1 protein in the vitreous humor within 3 weeks after administration of ssAAV8&GOI-C59 (Group A, OD administration to the right eye) and ssRC-C08&GOI-C59 (Group B, OD administration to the right eye). [Figure 6C] ImageJ grayscale scanning analysis data of the Western blot detection results of Figure 6A is shown. [Figure 6D] ImageJ grayscale scanning analysis data of the Western blot detection results in Figure 6B is shown. [Figure 6E] Comparison of RS1 polymer expression by ssAAV8&GOI-C59 and ssRC-C08&GOI-C59 (2 MOIs) in 293T (WB). [Figure 7] Study of RS1 protein expression following IVT and SR administration of ssrC-C08&GOI-C59 in photoreceptor cells and model mouse ocular tissues. [Figure 7A] 1 shows RS1 polymer expression by ssRC-C08&GOI-C59 in 661w cells. [Figure 7B]Statistical analysis of RS1 polymer expression levels after in vitro transduction of cells with ssRC-C08 & GOI-C59 (3 gradient MOI). [Figure 7C] Statistical comparison of the intensity of secreted RS1 polymer in the supernatant after infection with ssRC-C08&GOI-C59 at different multiplicities of infection (MOI 100, 1000, and 10000) is shown. [Figure 7D] Statistical expression of intracellular and extracellularly secreted RS1 polymer protein in the supernatant after transduction of 293T cells with ssRC-C08&GOI-C59 at two multiplicities of infection at different time points is shown. [Figure 7E] Figure 1 shows the expression of RS1 polymer protein in Rs1 knockout mice administered two doses of ssRC-C08 & GOI-C59 in different ways (A-E represent mice in group A who received IVT administration (2E9 vg / OD); mice in group B who received IVT administration (4E8 vg / OD); mice in group C who received subretinal administration (2E9 vg / OD); mice in group D who received subretinal administration (4E8 vg / OD); mice in group E who did not receive intraretinal administration (control group); B407 represents the numbering of mice; OD represents the right eye; OS represents the left eye; M represents male; F represents female). [Figure 7F] 7E shows the grayscale scan semi-quantitative statistics. [Figure 7G] Figure 1 shows the expression of RS1 polymer protein in Rs1 knockout mice following IVT administration of two doses of ssRC-C08&GOI-C59. [Figure 7H] Semi-quantitative statistical graphs of the WB results of Figure 7G after grayscale scanning are shown. [Figure 8] Figure 1 shows the in vivo efficacy of ssRC-C08&GOI-C59 in an Rs1 knockout mouse model. [Figure 8A-B]Figure 1 shows the retinal tissue morphology away from the optic nerve head in a mouse model treated with a drug candidate (2 doses; 4 weeks of IVT administration) as assessed by OCT examination (E346 etc. is the numbering of the mice). Figure 2 shows the effect of a drug candidate (ssRC-C08 & GOI-C59) on retinal tissue morphology changes near the optic nerve head in a mouse model as assessed by OCT tomography. [Figure 8C] 8B shows semi-quantitative data statistics of grayscale scans of the cavitated region of FIG. 8A. [Figure 8D] A semi-quantitative bar graph of a grayscale scan of the cavitated area of ​​FIG. 8B is shown. [Figure 9] Evaluation of dose efficacy of ssrC-C08&GOI-C59 in Rs1 knockout model mice. [Figure 9A] 1 shows the grouping and dosing schedule of drug candidates in a drug efficacy evaluation model using a mouse model. [Figure 9B] The hollowed areas of retinal tissue in mice in each treatment group evaluated by OCT tomography (I-IV) are shown. [Figure 9C] Photographs of immunofluorescence staining (IF) of retinal tissues of mice in the administration group are shown. [Figure 9D] 1 shows healing of retinal inner nuclear layer (INL) cavitation in different groups of mice after treatment based on immunofluorescence photographs. [Figure 9E] Paraffin sections of retinal tissues from mice in the I-IV (four doses, IVT) administration groups (HE staining) are shown. [Figure 9F] Statistical bar graphs of healing of INL cavitation in Rs1 knockout model mice (HE, IVT). [Figure 9G] Graphs of immunofluorescent staining of microglia in retinal tissue from each treatment group (IVT) are shown. [Figure 10] Shows the long-term efficacy of candidate XLRS gene therapy drugs in the RS1 knockout mouse model (OCT & ERG). [Figure 10A] Semi-quantitative statistics of the cavitated area distal to the optic disc in the left and right eyes (OS and OD) of model mice 6 weeks after administration are shown (OCT examination). [Figure 10B] Semiquantitative statistics of the cavitated area away from the optic disc in the treated eyes (OD) in each dose group are shown. [Figure 10C] Statistics of values ​​from ERG tests (b-wave / a-wave) in the left and right eyes of the Rs1 knockout model 6 weeks after administration are shown. [Figure 10D] Statistics of b-wave / a-wave values ​​in the treated eye of the Rs1 knockout model 6 weeks after treatment are shown, with WT representing normal C57Balb / c mice. DETAILED DESCRIPTION OF THE INVENTION

[0017] I. Recombinant AAV viral particles The present invention provides (i) an AAV capsid protein variant, comprising a genetically engineered capsid protein VP1 containing the following amino acid mutation sites relative to the parent AAV capsid protein VP1: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G; or an AAV capsid protein variant, further comprising an engineered capsid protein VP1 comprising the following amino acid mutation sites relative to the parent AAV capsid protein VP1: Q464V, A467P, D469N, I470M, R471A, D472V, S474G, and Y500F; (iii) a nucleic acid of interest packaged within an AAV capsid, the nucleic acid of interest comprising a nucleic acid molecule encoding RS1; The present invention relates to a recombinant AAV viral particle (rAAV) comprising:

[0018] (I) AAV capsid protein variants Thus, the present invention also relates to AAV capsid protein variants, i.e., engineered capsid protein variants with multiple glycan attachment sites.

[0019] In some embodiments, the AAV capsid protein variant comprises one or more amino acid substitutions that introduce a new glycan binding site into the AAV capsid protein, hi some embodiments, the substitution introduces a new glycan binding site from a first AAV serotype into the capsid protein of a second AAV serotype that is different from the first AAV serotype.

[0020] In some embodiments, an AAV capsid protein variant suitable for the present invention comprises the following substitutions relative to the parent capsid protein, for example, relative to the parent AAV capsid protein VP1, an AAV capsid protein VP1 variant suitable for the present invention comprises the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G, and said AAV capsid protein VP1 further comprises Y500F and / or S501A.

[0021] The parent capsid protein, such as the parent capsid protein VP1, can be derived from a particular AAV serotype (AAV serotypes 2 through 12) or a modified version of any of these serotypes (including AAV4YF and AAV2.7m8 vectors), such as AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV2 modified 7m8 (AAV2.7m8), AAV serotype 3a (AAV3a), AAV serotype 3b (AAV3b), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or AAV serotype 10 (AAV10).

[0022] In exemplary embodiments, the parent capsid protein is a capsid protein of an AAV2 or AAV2.7m8 serotype. In exemplary embodiments, the parent capsid protein VP1 is a capsid protein VP1 of an AAV2 or AAV2.7m8 serotype. In exemplary embodiments, the capsid protein VP1 of the parent AAV2 serotype comprises or consists of the amino acid sequence set forth in SEQ ID NO:51. In exemplary embodiments, the capsid protein VP1 of the parent AAV2.7m8 serotype comprises or consists of the amino acid sequence set forth in SEQ ID NO:19.

[0023] In some embodiments, the galactose binding site of AAV9 can be introduced into the parent AAV2.

[0024] In some embodiments, the AAV capsid protein variants of the present invention comprise the following substitutions relative to the parent AAV2 or AAV2.7m8: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G, where the amino acid positions are determined with respect to the amino acid sequence of the AAV2.7m8 capsid protein (SEQ ID NO: 19).

[0025] In some embodiments, the AAV capsid protein variants of the invention comprise the following substitutions relative to the parent AAV2 or AAV2.7m8: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G, where the amino acid positions are determined with respect to the AAV2 capsid protein (SEQ ID NO: 51).

[0026] In some embodiments, the AAV capsid protein variants of the invention comprise: (i) comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:1, and containing the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G; (iii) encoded by the nucleic acid sequence set forth in SEQ ID NO:2; or (iv) is encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO:2 and contains the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G.

[0027] In some embodiments, the present invention further relates to a nucleic acid comprising a nucleotide sequence encoding a capsid protein variant of the present invention.

[0028] In some embodiments, the nucleic acid of the invention is (i) comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO:2; or (ii) a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO:2, and wherein the capsid protein variant encoded thereby contains the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G.

[0029] The capsid protein variants of the present invention may contribute to increased transduction activity of viral particles, and even when the mutations are particularly small, the transduction activity of viral particles (e.g., in vivo and / or in vitro transduction activity) may still be increased.

[0030] (II) the nucleic acid of interest The nucleic acid of interest contained in the viral particle of the present invention encodes an RS1 protein, such as the human RS1 protein. In a preferred embodiment, the human RS1 protein is (i) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15; or (ii) comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0031] In one embodiment, the human RS1 protein is encoded by an optimized RS1 gene coding sequence.

[0032] In one embodiment, the optimized RS1 gene coding sequence of the present invention comprises or consists of a nucleotide sequence set forth in any one of SEQ ID NOs: 16 to 18 or a complementary sequence thereof.

[0033] In one embodiment, the optimized RS1 gene coding sequence of the present invention comprises a nucleotide sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 16 to 18 or its complementary sequence, and encodes a human RS1 protein.

[0034] In one embodiment, the optimized RS1 gene coding sequence of the present invention can efficiently express RS1 protein in mammalian (e.g., human) cells (e.g., photoreceptor cells, optic nerve cells) compared to known RS1 gene coding sequences (e.g., the sequence set forth in SEQ ID NO: 20).

[0035] The nucleic acid of interest can be included in an expression cassette and thus packaged within the AAV capsid.

[0036] In some embodiments, the expression cassette contains at least one ITR sequence (e.g., one or two) flanking the nucleic acid of interest, thereby enabling the vector genome to be packaged by the AAV capsid. The expression cassette can be single-stranded DNA, double-stranded DNA, or single- or double-stranded RNA.

[0037] In some embodiments, the expression cassette can include one or more regulatory sequences that direct expression of the RS1 gene coding sequence in target cells (e.g., retinal target cells such as photoreceptor cells or optic nerve cells). The regulatory sequences can be selected from the group consisting of transcription initiation sequences, termination sequences, promoters, and enhancer sequences operably linked to the coding sequence; efficient RNA processing signals, such as splicing and polyadenylation (polyA) regions, including the human growth hormone polyadenylation region; inverted repeat sequences (e.g., L-ITR or R-ITR); selectable markers or reporter genes, such as resistance genes; microRNAs; post-transcriptional regulatory sequences, such as the woodchuck hepatitis virus post-transcriptional regulator (WPRE); sequences that stabilize cytoplasmic mRNA; nucleic acid restriction sites; homologous recombination sequences; sequences that increase translation efficiency (e.g., Kozak consensus sequences); sequences that increase protein stability; and, if desired, sequences that increase secretion of the encoded product.

[0038] In some preferred embodiments, the regulatory sequence is located in the 5'UTR or 3'UTR. In some preferred embodiments, the regulatory sequence is selected from one or more of the group consisting of a promoter, an inverted repeat, an intron, an enhancer, a post-transcriptional regulatory sequence, a polyadenylation region, a selectable marker, or a reporter gene.

[0039] Examples of promoters suitable for the present invention include, but are not limited to, promoters derived from bacteria, yeast, plants, viruses, and mammals (such as Simians and humans). Promoters can be constitutive or inducible. Constitutive promoters initiate RNA synthesis independent of regulatory influences.

[0040] In a preferred embodiment of the invention, the promoter is the CAG promoter, which is a fusion of the cytomegalovirus (CMV) early enhancer and the chicken β-actin promoter (eg, via a 1-3 nucleotide linker such as 1, 2, or 3G).

[0041] In one embodiment, the chicken β-actin promoter can comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the chicken β-actin promoter comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 6.

[0042] In one embodiment, the cytomegalovirus (CMV) early enhancer comprises a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the promoter comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 5.

[0043] In one embodiment, the CAG promoter can comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 32. In some embodiments, the promoter comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 32.

[0044] In one embodiment, the promoter is a CMV promoter. For example, the CMV promoter can comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 29. In some embodiments, the promoter comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 29.

[0045] The expression cassette of the present invention may further comprise a selectable marker or reporter gene, for example, to determine expression of the vector in a growth system (e.g., bacterial cells) or target cells in the retina. The "selectable marker" or "reporter gene" of the present invention may be selected from those known in the art. Suitable reporter genes include, but are not limited to, enhanced green fluorescent protein, red fluorescent protein, luciferase, and secreted embryonic alkaline phosphatase (seAP), and may include sequences encoding geneticin, hygromycin, or puromycin resistance, etc. To signal the presence of the plasmid in bacterial cells, such a selectable marker or reporter gene (which may or may not be located outside the viral genome so as to be packaged within the viral particle) may be used, for example, an antibiotic resistance marker gene such as ampicillin or tetracycline resistance or kanamycin resistance. In one embodiment, a selectable marker of the invention is a kanamycin resistance marker, e.g., comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the selectable marker comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 9.

[0046] A "post-transcriptional regulatory sequence" of the present invention is a DNA sequence that, when transcribed, enhances expression of one or more transgenes or fragments thereof delivered by a viral vector of the present invention. Post-transcriptional regulatory sequences include, but are not limited to, the Hepatitis B virus post-transcriptional regulatory element (HPRE) and the Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE). The WPRE is a tripartite cis-acting element that has been shown to enhance transgene expression driven by certain, but not all, promoters.

[0047] The expression cassette or expression vector of the present invention may further comprise a polyadenylation region, such as hGHpA (human growth hormone polyadenylation region) or SV40pA (SV40 poly(A) signal polyadenylation region). In one embodiment, the polyadenylation region of the present invention comprises a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO:8. In some embodiments, the polyadenylation region comprises or consists of the nucleotide sequence set forth in SEQ ID NO:8. In one embodiment, the polyadenylation region of the present invention comprises a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO:12. In some embodiments, the polyadenylation region comprises or consists of the nucleotide sequence set forth in SEQ ID NO:12.

[0048] The expression cassettes or expression vectors of the present invention contain introns, such as the chimeric intron derived from the chicken (Gallus gallus) cytoplasmic β-actin gene, sequence IDX00182.1. The use of chicken β-actin intron 1 or its functional equivalent as an enhancer element or to express a "hot spot sequence" is disclosed in a patent relating to the construction or engineering of mammalian expression vectors for very high-level recombinant protein expression, see CN200780052196.7 for details. Suitable introns for use in the expression cassettes of the present invention are known in the art. In some embodiments, the introns of the present invention comprise a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 7, 24, 28, or 31. In some embodiments, the intron sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 7, 24, 28, or 31. In some embodiments, an intron of the present invention comprises a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the intron sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 7.

[0049] In one embodiment, an L-ITR of the invention comprises a nucleic acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 11. In some embodiments, an L-ITR comprises, or consists of, the nucleotide sequence set forth in SEQ ID NO: 4 or 11. In one embodiment, an L-ITR of the invention comprises a nucleic acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10 or 13. In some embodiments, an R-ITR comprises, or consists of, the nucleotide sequence set forth in SEQ ID NO: 10 or 13.

[0050] In one embodiment, an L-ITR of the invention comprises a nucleic acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4, and an R-ITR of the invention comprises a nucleic acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the L-ITR comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 4, and the R-ITR comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 10.

[0051] In one embodiment, the L-ITR of the invention comprises a nucleic acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 11, and the R-ITR comprises a nucleic acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the L-ITR comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 11, and the R-ITR comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 13.

[0052] In one embodiment, the expression cassette of the invention comprises, in the 5' to 3' direction, the following elements: It comprises an L-ITR sequence, a CMV enhancer, a chicken β-actin promoter sequence, a chimeric intron sequence, an optimized nucleic acid molecule of the present invention encoding the RS1 protein, and a downstream hGHpA sequence, and an R-ITR sequence.

[0053] In one embodiment, the expression cassette of the present invention comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 3 or 14, or comprises a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 or 14.

[0054] II. Preparation method The present invention relates to a method for producing recombinant AAV viral particles (rAAV). Many methods for packaging and producing rAAV are known in the art. Currently, the commonly used rAAV packaging systems mainly include a three-plasmid co-transfection system, a system with adenovirus as a helper virus, a packaging system with herpes simplex virus type 1 (HSV1) as a helper virus, and a baculovirus-based packaging system. Each packaging system has its own unique characteristics, and those skilled in the art can make an appropriate selection according to their needs.

[0055] All rAAV production cultures for the production of rAAV viral particles require: 1) a suitable host cell, such as a human-derived cell line such as HEK-293T cells, or an insect-derived cell line (in the case of a baculovirus production system); 2) appropriate helper virus functions provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct to provide helper functions; 3) AAV rep and cap genes and gene products; 4) a gene of interest flanked by at least one AAV ITR sequence and preferably driven by an operably linked promoter; and 5) a suitable culture system that supports rAAV production.

[0056] In some embodiments, the present invention relates to a method for producing recombinant AAV viral particles, comprising culturing packaging cells under conditions sufficient for the production of recombinant AAV viral particles, wherein the packaging cells comprise a nucleic acid encoding a capsid protein variant according to the present invention or a plasmid comprising a capsid protein variant-encoding nucleic acid of the present invention.

[0057] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleic acid of interest.

[0058] In some embodiments, the method further comprises isolating the self-complementary recombinant AAV viral particles from the culture supernatant.

[0059] In some embodiments, the method further comprises lysing the packaging cells and isolating the recombinant AAV viral particles from the cell lysate.

[0060] In some embodiments, the method further comprises the steps of: a. removing necrotic debris; b. treating the supernatant containing the recombinant AAV viral particles with benzonase nuclease; c. Concentrating the recombinant AAV viral particles; d. Purifying the recombinant AAV viral particles; It includes one or more of the following.

[0061] Thus, the present invention also relates to a packaging cell for producing recombinant AAV viral particles, the packaging cell comprising a nucleic acid encoding a capsid protein variant of the present invention, or a capsid protein-encoding nucleic acid of the present invention, or a plasmid comprising an expression cassette of the present invention.

[0062] III. Compositions, Drugs, or Formulations The invention provides formulations, compositions, or medicaments containing (a) an rAAV of the invention, and (b) pharmaceutically acceptable adjuvants, e.g., pharmaceutically acceptable carriers and excipients such as buffers known in the art.

[0063] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonicity agents, absorption delaying agents, etc. See also Handbook of Pharmaceutical Excipients, 8th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago, for a discussion of pharmaceutically acceptable excipients and their uses.

[0064] In some embodiments, pharmaceutically acceptable adjuvants include, but are not limited to, one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficiently high purity and sufficiently low toxicity. "Compatible," as used herein, means that the components of the composition can be mixed with and among the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients. Suitable pharmaceutically acceptable adjuvants are known to those skilled in the art. Examples of pharmaceutically acceptable carrier ingredients include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0065] The formulation or composition or medicament of the present invention can be liquid or solid, such as powder, gel or paste. Preferably, the formulation or composition or medicament of the present invention is a liquid, preferably an injectable liquid. Preferably, the injectable liquid is provided as a capsule or in a syringe.

[0066] The rAAV of the present invention, or a formulation, composition, or drug comprising the same, can be administered intravenously, intramuscularly, subcutaneously, orally, by mucosal contact, intraperitoneally, or intralesionally, and preferably by local administration to the eye, for example, intraretinal or intravitreal administration, for example, intravitreal injection, subretinal injection, or suprachoroidal injection. In some embodiments, the rAAV of the present invention, or a formulation, composition, or drug comprising the same, can be administered intraretinaly or intravitreally, for example, by intravitreal or subretinal administration, for example, intravitreal administration (IVT administration). In any administration format, the formulation, composition, or drug of the present invention is preferably provided as an injectable liquid.

[0067] The compositions or formulations or medicaments may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, or emulsions, and sterile powders to be reconstituted into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0068] The compositions, eg, pharmaceutical compositions or formulations, of the present invention may further comprise additional active ingredients, eg, one or more additional therapeutic agents, eg, immunomodulatory agents such as immunosuppressants.

[0069] IV. Combination Products In one aspect, the present invention also provides a combination product (e.g., a pharmaceutical combination product) comprising an rAAV of the present invention and one or more additional therapeutic agents. The combination product of the present invention can be used in the therapeutic methods of the present invention.

[0070] The present invention also provides a pharmaceutical kit comprising the combination product, for example, the pharmaceutical kit comprises the following in the same package: - a first container containing the rAAV of the invention or a drug comprising said rAAV; - a second container containing a pharmaceutical composition comprising one or more additional therapeutic agents, e.g., an immunomodulatory agent; Equipped with.

[0071] In some embodiments, the additional therapeutic agent is an immunomodulatory agent, e.g., an immunosuppressant, to reduce an immune response, such as an immune inflammatory response, produced by the rAAV particles.

[0072] V. Therapeutic method In one embodiment, the rAAV, formulation or composition or drug of the invention is used to treat ocular diseases such as, but not limited to, diseases caused by retinopathy or chorioretinopathy, age-related macular degeneration, diabetic retinopathy, and other non-genetic ocular diseases such as physically or chemically induced retinal damage.

[0073] In another preferred embodiment, the rAAV, formulation, composition, or drug of the present invention is used to treat X-linked retinoschisis.

[0074] In another preferred embodiment, the rAAV, formulation or composition or drug of the present invention is used to treat retinitis pigmentosa.

[0075] In one embodiment, the rAAV, formulation, composition, or drug of the invention is administered intraocularly, for example by intraretinal or intravitreal administration, for example by subretinal or intravitreal administration (IVT administration). In one embodiment, the administration is by injection.

[0076] In one embodiment, the rAAV of the present invention is about 10 3 vg / eye / dose, 10 4 vg / eye / dose, 10 5 vg / eye / dose, 10 6 vg / eye / dose, 10 7 vg / eye / dose, 10 8 vg / eye / dose, 10 9 vg / eye / dose, 10 10 vg / eye / dose, 10 11 vg / eye / dose, or 10 12 vg / eye / dose or more, preferably 10 12 vg / eye / dose or less, e.g., 2 x 10 8 vg / eye / dose, 3×10 8 vg / eye / dose, 4×10 8 vg / eye / dose or 5 × 10 8 vg / eye / dose or more, e.g., about 10 7 ~Approx. 5×10 8 vg / eye / dose, 1×10 7 ~Approx. 5×10 8 vg / eye / dose, e.g., about 2 x 10 8 vg / eye / dose, 3×10 8 vg / eye / dose, 4×10 8 vg / eye / dose or 5 × 10 8 It is administered at a dose of vg / eye / dose.

[0077] In one embodiment, for intravitreal administration, the dose of the rAAV of the present invention is about 10 6 vg / eye / dose, 10 7 vg / eye / dose, 10 8 vg / eye / dose, 10 9 vg / eye / dose, 10 10 vg / eye / dose, 10 11 vg / eye / dose, or 10 12 vg / eye / dose or more, preferably 10 12 vg / eye / dose or less, e.g., 2 × 10 8 vg / eye / dose, 3×10 8 vg / eye / dose, 4×10 8 vg / eye / dose or 5 × 10 8 vg / eye / dose or more, e.g., about 10 7 ~Approx. 5×10 8 vg / eye / dose, 1×10 7 ~Approx. 5×10 8 vg / eye / dose, e.g., about 2 x 10 8 vg / eye / dose, 3×10 8 vg / eye / dose, 4×10 8 vg / eye / dose or 5 × 10 8 vg / eye / dose.

[0078] In one embodiment, the present invention also relates to the use of a recombinant AAV viral particle, a formulation or composition or combination product comprising same in the manufacture of a medicament for the treatment of an ocular disease according to the present invention.

[0079] VI.Definition For the purposes of interpreting this specification, the following definitions will be used, and where appropriate, terms used in the singular may also include the plural and vice versa. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0080] The term "about" or "approximately" encompasses a statistically significant range of values. Such a range can be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, also more preferably within 10%, and even more preferably within 5% or 1%. The acceptable variation encompassed by the term "about" or "approximately" will depend on the particular system under study and can be readily discerned by one of ordinary skill in the art.

[0081] As used herein, the term "and / or" means any one of the alternatives or more than one or all of the alternatives.

[0082] As used herein, the term "comprising" or "including" means the inclusion of the stated elements, integers, or steps, but does not mean the exclusion of any other elements, integers, or steps. When the term "comprising" or "comprising" is used herein, unless otherwise specified, it also encompasses situations consisting of the specified elements, integers, or steps. For example, reference to an antibody variable region "comprising" a particular sequence is also intended to encompass an antibody variable region consisting of that particular sequence.

[0083] As used herein, the terms "RS1 gene," "RS1," and "XLRS1" are used interchangeably herein to refer to the congenital retinoschisis gene. The RS1 gene (retinoschisin 1) is a causative gene for XLRS located on Xp22 in humans, consisting of six exons, and has GenBank accession number NC_000023. The expression product encoded by the RS1 gene is called the "RS1 protein" and consists of 224 amino acids. The protein contains a discoidin domain (encoded by exons 4 to 6), which is present in many families of secreted or membrane-bound proteins. The RS1 protein is involved in cell adhesion and cell interaction and is highly conserved across species. The RS1 protein is primarily secreted in the outer layer of the retina, and its reduction leads to retinal cavitation, synaptic dysfunction, vision loss, and easy retinal detachment. In one embodiment, the RS1 protein is human. The RS1 protein described herein also encompasses its naturally occurring variants. In a preferred embodiment, the RS1 protein comprises the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0084] The adeno-associated virus (AAV), also known as adeno-associated virus, described herein, belongs to the Dependovirus genus in the Parvoviridae family. It is a single-stranded DNA-defective virus with the simplest structure currently known and requires a helper virus (usually an adenovirus) for replication. It encodes the cap and rep genes in inverted repeats (ITRs) at both ends. The ITRs are important for viral replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells. Adeno-associated viruses are smaller than other viral vectors, are nonpathogenic, and can transfect both dividing and nondividing cells. Therefore, AAV vector-based gene therapy for ocular diseases, particularly inherited retinal degeneration, has attracted widespread attention. Recombinant adeno-associated virus (rAAV), derived from the nonpathogenic wild-type adeno-associated virus (WAV), is considered one of the most promising gene transfer vectors due to its excellent safety profile, broad host cell range (dividing and non-dividing cells), low immunogenicity, and long in vivo exogenous gene expression. It has been widely applied in gene therapy and vaccine research worldwide. Over the past decade, the biological properties of rAAV have been well understood, and extensive data has been accumulated on the effects of rAAV in various cells, tissues, and in vivo experiments. In medical research, rAAV is used in gene therapy studies for various diseases (including in vivo and in vitro experiments); meanwhile, gene transfer vectors are used as intrinsic gene transfer vectors and are widely applied in technical aspects such as gene function studies, disease model construction, and gene knockout mouse generation.

[0085] The term "capsid protein" encompasses proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are commonly referred to as VP1, VP2, and / or VP3, each encoded by a single cap gene. For AAV, all three proteins share a common stop codon, but these three AAV capsid proteins are produced in an overlapping manner from the cap open reading frame (ORF) using mRNA alternative splicing and / or alternative translation start codon usage. Warrington et al., (2004) J. Virol. 78:6595, is incorporated herein by reference in its entirety. AAV2 VP1 is generally translated from the ATG start codon (amino acid M1) on the 2.4 kb mRNA, while AAV2 VP2 and VP3 arise from the smaller 2.3 kb mRNA using read-through translation of the weaker ACG start codon for VP2 (amino acid T138) and the next available ATG codon for the most abundant capsid protein, VP3 (amino acid M203). The amino acid sequences of adeno-associated virus capsid proteins are well known in the art and are generally conserved, particularly among parvoviruses. See Rutledge et al. (1998) J. Virol. 72:309-19. Thus, the amino acid positions provided herein may be provided with respect to the AAV VP1 capsid protein, and unless otherwise specified, the amino acid positions provided herein are determined relative to the amino acid positions of AAV2 VP1 set forth in SEQ ID NO:51. Those skilled in the art will be able to individually and readily determine the same amino acid positions within the VP2 and / or VP3 capsid proteins of AAV, as well as the corresponding amino acid positions in different serotypes. Furthermore, those skilled in the art will be able to make corresponding mutations between capsid proteins of different serotypes to form "capsid protein variants."

[0086] As used herein, the phrase "operably linked" includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that directly or indirectly interact with each other or otherwise coordinate with each other to participate in a biological event, which juxtaposition achieves or enables such interaction and / or coordination. In some embodiments, "operably linked" refers to the covalent attachment of the associated components or elements to each other. However, one of skill in the art will recognize that in some embodiments, covalent attachment is not required to achieve effective operable attachment.

[0087] The term "capsid protein variant" encompasses capsid proteins that have at least one mutation (eg, a substitution, deletion, or insertion) compared to the corresponding parent capsid protein.

[0088] The terms "transduction" or "infection" and the like refer to the introduction of nucleic acid into a target cell by a viral vector. The term "transduction efficiency" refers to the fraction (e.g., percentage) of cells that express a nucleotide of interest after incubation with a certain number of viral vectors containing the nucleotide of interest. Known methods for determining transduction efficiency include fluorescence-activated cell sorting of cells transduced with a fluorescent reporter gene, PCR for expression of the nucleotide of interest, and the like.

[0089] "Percent identity" of an amino acid sequence or nucleic acid sequence refers to the percentage of amino acid residues / nucleotides in a candidate sequence that are identical to the amino acid residues / nucleotides of a specific sequence set forth herein after aligning the candidate sequence with the specific sequence set forth herein and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not take into account conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the proteins or polypeptides or nucleic acids of the invention having a significant degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more identity, to the polypeptides or proteins or nucleic acids specifically disclosed herein. Variants may include conservative changes.

[0090] The terms "individual" or "subject" are used interchangeably herein to refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0091] The term "treating" includes the administration of a composition or hybrid polypeptide to delay or prevent the onset of symptoms, complications, or biochemical indicators of a disease, to alleviate symptoms, or to arrest or prevent further development of a disease, condition, or disorder. The term "preventing" includes the suppression of the appearance or development of a disease or disorder, or the symptoms of a particular disease or disorder.

[0092] The term "pharmaceutically acceptable adjuvant" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, with which an active agent is administered.

[0093] The term "pharmaceutical composition" means a composition that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0094] The term "effective amount" refers to the amount or dosage of a hybrid protein or a nucleic acid encoding same, or a composition or combination of the invention, that, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0095] By "therapeutically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic effect. A therapeutically effective amount is also one in which the toxic or detrimental effects of the hybrid protein or composition or combination are less than the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% relative to untreated subjects.

[0096] By "prophylactically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, since a prophylactic dose is used prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0097] Any documents mentioned herein, including patents, patent applications, and literature, are incorporated herein by reference in their entirety.

[0098] Any or all of the features discussed above and throughout the present invention may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, but the examples are set forth by way of illustration and not by way of limitation, and are not intended to, and should not be construed to, limit the scope of the present invention, and it will be understood that various modifications may be made by those skilled in the art. [Example]

[0099] Example 1: Construction of GOI plasmid and comparison of expression efficiency at the cellular level for different expression cassettes Cloning process of GOI plasmids: The first GOI-E04 (see SEQ ID NO: 21 for the sequence) was synthesized by using gene synthesis technology as a base vector linearized with NotI at the N-terminus (nt position 143) and XhoI at the C-terminus (nt position 3243); different promoter elements CAG-intron-hRS1-1 (CAG-intron template from GOI-E04 plasmid, CAG promoter: SEQ ID NO: 32), EF1n-intron-hRS1-1 (EF1n promoter: SEQ ID NO: 22) were inserted. ; intron: SEQ ID NO: 24; EF1n-intron: SEQ ID NO: 23; hRS1-1: SEQ ID NO: 16), Ubc-intron-hRS1-1 (Ubc-intron: SEQ ID NO: 26; intron: SEQ ID NO: 28; Ubc-intron: SEQ ID NO: 27; hRS1-1: SEQ ID NO: 16), CMV-intron-hRS1-1 sequences (CMV-promoter: SEQ ID NO: 29; intron: SEQ ID NO: 31; CMV-intron: SEQ ID NO: 30; hRS1-1: SEQ ID NO: 16) were enriched by PCR and then subjected to homologous recombination to replace the GOI-E04 nt 143 to 3243 sequence, resulting in the clones GOI-C59, GOI-C03, GOI-C24, GOI-C26 shown in Figure 1A, using the primer sequences shown in Table 1.

[0100] GOI-C26 was used as a base vector, which was linearized with EcoRI at the N-terminus (nt position 1323) and BamHI at the C-terminus (nt position 2010); different hRS1 CDS sequences (hRS1-2 (SEQ ID NO: 17), hRS1-3 (SEQ ID NO: 18), hRS1-WT (SEQ ID NO: 20)) were amplified by PCR, enriched, and then subjected to homologous recombination to replace the GOI-C26 nt 2030-2749 sequence, resulting in clones of GOI-C45, GOI-C46, and GOI-E11-V1, respectively, using the primer sequences shown in Table 1.

[0101] GOI-C59 was used as the base vector, which was linearized with BglII at the N-terminus (nt position 3226) and FspI at the C-terminus (nt position 3518); a synthetic 3'-terminal ITR TRS binding site deletion fragment was enriched by PCR as a template and then subjected to homologous recombination to replace the GOI-C59 nt 3226-3518 sequence, using the primer sequences shown in Tables 1 and 2, to obtain GOI-C21, the backbone structure of which is scAAV.

[0102] [Table 1]

[0103] [Table 2]

[0104] Four GOI plasmids (GOI-C26, GOI-C24, GOI-C59, and GOI-C03) were transfected into 6-well plates using 500 ng of plasmid. After 72 h, cells and supernatants were collected, respectively. RS1 expression in the four expression cassettes was detected by Western blot as shown in Figures 2B-2D. The highest expression in the supernatant and cells was observed in GOI-C26 (CMV-hRS1 cassette), while the amount of protein transiently expressed by the other three cassettes showed no significant difference. To further verify the efficiency of in vivo expression of target proteins by recombinant AAVs packaged with different GOIs, we followed the triple-plasmid AAV packaging standard protocol (see Example 2) for preparation, with the corresponding capsid plasmid being RC-C08. As shown in Figures 1E-1G, at an MOI of 200, RC-C08&GOI-C59 (ss-CAG-opti-hRS1) and RC-C08&GOI-C26 (ss-CMV-opti-hRS1) showed higher expression of RS1 protein in 293T supernatants and cells than the other two recombinant viruses. Published data from clinical trials have shown that the CMV promoter element poses a risk of oncogenicity. Therefore, the CAG element is preferred for broad-spectrum expression cassettes in the development of ocular gene therapy products.

[0105] Example 2: Comparison of transcription and translation efficiency of humanized codon-optimized RS1 The known construction scheme and process of the RC-C08 plasmid was as follows: The sequence of the capsid protein VP1 of the RC-C08 serotype was the sequence set forth in SEQ ID NO: 1, and the sequence of the corresponding cap gene containing seven mutation sites Q464V, A467P, D469N, I470M, R471A, D472V, and S474G was the sequence set forth in SEQ ID NO: 2. Furthermore, the capsid protein of the RC-C08V2 serotype (VP1) was a mutant in which Y500F was added to VP1 of RC-C08.

[0106] To obtain the above-mentioned engineered viruses, Shanghai Langjing Biotechnology Co., Ltd. was commissioned to carry out gene synthesis of the Cap genes of the RC-C08 and RC-C08V2 serotypes, and the upstream and downstream cloning sequences were simultaneously added and cloned into the pUC57 vector using TA cloning as the amplification template. The resulting vectors were designated pUC-RC-C08-VP1 and pUC-RC-C08V2-VP1, respectively.

[0107] 2.1 Amplification of target fragment genes The PCR reaction system shown in Table 3 was used to amplify the genes of the fragments of interest for the subsequent cloning step.

[0108] [Table 3]

[0109] If the forward primer sequence is 5'-gacgtcagacgcggaagcttcgatc-3' (SEQ ID NO: 49), the reverse primer sequence is 5'-gctgtttaaacgcccgggctgtag-3' (SEQ ID NO: 50).

[0110] The PCR amplification procedure is shown in Table 4.

[0111] [Table 4]

[0112] 1) The PCR product was confirmed to be correct by electrophoresis using a 1% agarose gel and the corresponding DNA marker as a control. The gel band at the predicted position of approximately 2.2 kbp was excised to recover the remaining PCR product using the QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer's instructions, and the product concentration was determined to be 86 ng / μl using a Nano-300.

[0113] 2) In the following reaction system shown in Table 5, the pRC2 vector (all genes synthesized by Genescript) was subjected to double digestion (2 hours at 37°C).

[0114] [Table 5]

[0115] 3) The PCR product was confirmed to be correct by electrophoresis using a 1% agarose gel and the corresponding DNA marker as a control. To recover the remaining PCR product, the gel band at the predicted position of approximately 2.2 kbp was excised according to the QIAquick Gel Extraction Kit (QIAGEN) instructions, and the product concentration was determined to be 20 ng / μl using a Nano-300.

[0116] 4) The purified products were ligated (50°C for 1 hour) according to the reaction system shown in Table 6 below.

[0117] [Table 6]

[0118] 5) The ligation product was transformed into Stbl3 chemically competent cells (Shanghai Weidi Biotechnology Co., Ltd.) according to the manufacturer's instructions.

[0119] 6) A single clone on the LB (Kana) plate was picked and placed in a 1.5 mL sterile tube to which 200 μl of LB (Kana) medium had been added, followed by incubation at 37°C and 250 rpm for 3 hours. Then, colony PCR screening was performed for positive clones according to the reaction system shown in Table 7 below.

[0120] [Table 7]

[0121] The colony PCR procedure is shown in Table 8.

[0122] [Table 8]

[0123] 7) Identification by agarose gel electrophoresis Three recombinant clones (five single colonies / clone) were identified by PCR using bacterial suspensions, and all colonies were positive.

[0124] 8) Positive clones (#1, #2, and #3) were selected and sequenced.

[0125] 9) After accurate alignment of the sequencing results, the plasmid was extracted using the TIANGEN EndoFree Maxi Plasmid Kit (TIANGENDP117) according to the manufacturer's instructions.

[0126] 2.2 rAAV virus-adherent packaging process: The GOI (gene of interest) plasmid used in the packaging process is transcribed and translated into the RS1 protein (224 aa), and the expression cassette of the GOI plasmid contains an enhancer, promoter, intron, polyadenylation region, and also contains ITR (L-ITR and R-ITR) sequences on each side of the expression cassette, which further provide the genome of the rAAV vector.

[0127] As shown in Figure 2, differently optimized human wild-type RS1 and RS1 codon sequences were constructed into rAAV vectors controlled by a combined promoter and intron (Figure 2A). The tissue distribution of rAAV genetic material in patients with rare diseases needs to be studied after clinical administration of XLRS. Because the majority of patients have point mutations in the RS1 gene, the human RS1 gene can still be detected at the mRNA level. Therefore, codon optimization of the RS1 protein is necessary for product design, and protein translation efficiency must be evaluated after different codon optimizations. Subsequently, recombinant AAV viruses were packaged into HEK293T cells by cotransfecting a triple plasmid containing the RC-C08 capsid and the four different expression cassette GOI combinations described above (Figure 2A). After density gradient purification with iodixanol, HEK293T cells were infected with the packaged AAV viruses at a multiplicity of infection (MOI) of 2000 vg / cell, and supernatant and cell samples were collected 48 hours later.

[0128] Triple plasmid packaging AAV has the following working process: First, HEK293T cells were allowed to attach to the culture dish after recovery. When the confluency of the HEK293T cells in the culture dish reached 90%, the cell line was passaged at a ratio of 1:3, and then cultured for 24 hours until the cell confluence reached 80%, followed by plasmid transfection. Before transfection, an appropriate transfection system was prepared. For each 10 cm dish of the package system, the transfection mixture was preferably prepared according to the following system: 500 μl of serum-free medium Opti-MEM (Gibco), 15 μg of HLP plasmid (all genes synthesized by Genescript), 7.5 μg of the RC-C08 (or RC-C08V2) plasmid prepared as described above, 7.5 μg of the GOI plasmid (prepared in Example 1), and 22.5 μl of PEIpro (Polyplus). Third, the transfection process: the transfection mixture was dropped into different areas of a 10 cm culture dish, followed by gentle cross shaking; fourth, packaging and culturing: the transfected cells were transferred to a carbon dioxide incubator and cultured at 37°C for 72 hours; finally, the virus was harvested: 72 hours after transfection, the cells were pipetted with the cell supernatant and centrifuged at 1500 rpm to collect the cell pellet; the lysate was added to dissolve the cells in a shaker at 37°C for 1 hour, followed by centrifugation at 4000 rpm for 10 minutes using a horizontal rotor, the supernatant was collected, filtered through a 0.45 μm needle filter, and subpackaged in a volume of 10 μl, and the viral gene copy number (unit: vg) in the cells was detected by real-time quantitative PCR. The cell samples were lysed and subjected to non-denaturing WB analysis together with the supernatant (Figure 2B). The results showed that after infection of HEK293T cells with GOI-C59 virus, the expression level of RS1 polymer secreted protein was superior to that of other viruses, while the expression level of intracellular RS1 was weaker than that of other viruses. The Western blot results were subjected to grayscale scanning using tubulin as an internal standard and the quantitative relative value of RS1 / tubulin as the relative expression level of RS1 protein. The final results were consistent with the Western blot results (Figure 2C-D).

[0129] [Table 9]

[0130] As shown in Figure 2E: the expression cassette of GOI-C59 was further optimized: 1. GOI-C59V1: replacing the chimeric intron in the GOI-C59 plasmid expression cassette with an EF1n intron; 2. GOI-C59V2: replacing opti-hRS1-1 with opti-hRS1-3 in the GOI-C59 plasmid expression cassette; 3. GOI-C59V3: replacing the chimeric intron in the GOI-C59 plasmid expression cassette with opti-hRS1-4; 1. GOI-C59V1: hRS1-WT replaces opti-hRS1-1; 2. GOI-C59V2: hRS1-WT replaces opti-hRS1-1 in the expression cassette of the GOI-C59 plasmid; 3. GOI-C59V4: hRS1-WT replaces the chimeric intron in the expression cassette of the GOI-C59 plasmid; 4. GOI-C59V5: hRS1-WT replaces opti-hRS1-1 in the expression cassette of the GOI-C59 plasmid; 5. GOI-C59V6: hRS1-WT replaces opti-hRS1-3 in the expression cassette of the GOI-C59V4 plasmid. ARPE19 and 661W (purchased from YaJi Biological) were infected with the viruses corresponding to GOI-C59, GOI-C59V1, GOI-C59V2, GOI-C59V3, GOI-C59V4, and GOI-C59V5, respectively, and the supernatants were collected after 72 hours to detect the in vitro expression intensity of RS1 from the different plasmids by WB. As shown in Figures 2F-2I, the results indicated that changes in expression cassette elements affected the intensity of protein expression, with the Rs1 polymer of GOI-C59V5 having the highest expression level, suggesting that the 5-LTR modification element promoted the protein transcription and translation processes, and that the LTR-nIntron significantly increased protein expression by affecting the stability of RS1 mRNA, especially in retinal cells.

[0131] As shown in Figures 2G and 2I, when the same GOI (GOI-C59) and different capsids were subjected to rAAV retinal cell in vitro infection activity tests, the expression of RS1 polymer revealed that the in vitro transduction activity of RC-C08V2&GOI-C59 was stronger than that of RC-C08&GOI-C59, suggesting that the Y500F mutation in VP1 of the RC-C08 capsid significantly improved the viral transduction activity.

[0132] Example 3: Efficiency of in vitro secretory expression of hRS1 multimeric protein by different serotypes Viruses were packaged in HEK293T cells by triple-plasmid cotransfection of ssAAV (GOI-C59) and its double-stranded complementary form, scAAV (GOI-C21), together with AAV8 and RC-C08 capsids (Figure 3A), and HLP (see Example 2 for specific procedures). After density gradient purification using iodixanol, the packaged viruses were infected into HEK293T cells at an MOI of 200 and 2000, respectively, and the supernatants and cells were collected 48 hours later. Similarly, cells were lysed and subjected to a non-denaturing WB assay together with the supernatants to detect RS1 octamer expression (Figure 3B). There was no significant difference in RS1 octamer expression between ssAAV8 and scAAV8 in the supernatant and cells (Figure 3D); RC-C08 capsids had stronger transduction efficiency in HEK293T cells than AAV8 capsids, i.e., the RS1 octamer expression level of GOI-C59 packaged by RC-C08 capsids in the supernatant and cells was significantly higher than that of GOI-C59 packaged by AAV8 capsids (Figure 3C).

[0133] Example 4: Differences in target protein transcription levels and protein expression between ssAAV and scAAV (RC-C08 & GOI-C59) in cells For virus production, GOI-C59 (ssAAV) and GOI-C21 (scAAV) were packaged in RC-C08 capsids. After density gradient purification using iodixanol, the packaged viruses were infected into HEK293T cells at an MOI of 200 vg / cell, and supernatant and cell samples were collected at 6, 12, 24, 48, and 72 h.

[0134] The relative transcription levels of RS1 mRNA in cells were detected and calculated by qPCR in cell samples. GOI-C21 (scAAV) infected cells at a low MOI and reached its peak transcription level within 24 hours (Figure 4B), whereas GOI-C59 (ssAAV) required infection at a higher MOI (MOI > 100) for more than 48 hours to reach its peak transcription level (Figure 4A). As the MOI of ssAAV increased, RS1 transcription levels increased significantly, whereas scAAV production increased only slightly. Concurrently, scAAV transcription peaked at 24 hours and began to decrease significantly after 48 hours. However, at the same low MOI (MOI = 250), cells were collected at 6, 12, 24, 36, 48, and 72 hours after viral infection and subjected to qPCR assays. The intensity of hRS1 mRNA transcription levels in scAAV was found to be higher than that in ssAAV at various time points (Fig. 4C). All cell samples and supernatants were subjected to non-denaturing WB to detect RS1 octamer expression (Fig. 4D, F). Grayscale scanning statistics were used to obtain curves of RS1 octamer expression in cells and supernatants over time (Fig. 4E, G). Between 6 and 24 hours, the RS1 octamer expression level in cells infected with ssAAV was slightly higher than that in scAAV; between 24 and 72 hours, the RS1 octamer expression level in cells infected with scAAV was significantly higher than that in ssAAV. The RS1 octamer expression levels in scAAV- and scAAV-infected cells generally showed a positive correlation over time (Fig. 4E, G).

[0135] As shown in Figure 4, statistical results indicated that scAAV secreted more active hRS1 octamer at the late stage of infection in 293 cells compared with ssAAV.

[0136] Example 5: mRNA transcription levels and target protein expression capabilities of ssRC-C08 and ssAAV2.7m8 in photoreceptor cells RC-C08 capsid and AAV2.7m8 capsid (CN103561774B, AAV2.7m8 capsid protein sequence set forth in SEQ ID NO: 19) carrying the triple-plasmid GOI-C59 and pHLP (AAV packaging helper plasmid from Genescript (Nanjing) total gene synthesis) were respectively subjected to virus packaging and purified by iodixanol density gradient centrifugation (see Example 2). The two packaged viruses, RC-C08 and AAV2.7m8, were used to infect 661w cells at MOIs of 100, 1,000, and 10,000, respectively. Supernatant and cell samples were collected at 48 and 72 hours. Total RNA was extracted using TRIZOL. After reverse transcription into cDNA, hRS1 mRNA copy numbers were detected by RT-PCR and qPCR, thereby detecting RS1 mRNA levels, as shown in Figures 5A-5D.

[0137] FIG. 5 shows the intensity profile of hRS1 mRNA transcript levels over time following infection of 661W with AAV2.7m8 and RC-C08 virions at different MOIs.

[0138] As a result, MOI ≥ 10 3 It can be seen that the administration of the virus led to clear expression of hRS1 mRNA, and the hRS1 mRNA expression level increased significantly with increasing viral dose.

[0139] Western blotting was further applied, which included the following specific steps: 1. 48 hours after recombinant AAV virus infection, cells and supernatants were collected, and 120 μL of strong RIPA lysate (supplemented with protease inhibitors) was added to the cells, and RIPA lysate was also added to the supernatant. 2. Sample preparation: The total volume of loading buffer, ultrapure water (ddH2O), and sample was 50 μL, and the added sample was 33 μL. For the denaturing gel, NuPAGE™ LDS sample buffer (x4), NuPAGE™ sample reducing agent (x10), and non-denaturing gel buffer were used to prepare in-house. 3. Boiling of samples: For denaturing gels, samples were boiled at 98°C for 15 minutes, and for non-denaturing gels, samples were not boiled. 4. Loading: The gel was loaded onto the electrophoresis clip (ensure that the recessed line and short plate are tightly attached), and the running buffer was filled between the clips and added to the electrophoresis chamber up to a level higher than the bottom standard line. 15 μL of protein sample and 5 μL of marker were added to the lane in order. 5. Electrophoresis: The initial voltage of electrophoresis was 80 V, and after the sample entered the separation gel, the voltage was adjusted to 120 V. The electrophoresis time was controlled according to the molecular position of the pre-stained protein marker. 6. Transfer to membrane: After SDS-PAGE electrophoresis of the proteins, the PVDF membrane was activated with methanol and placed on an electroblotting apparatus (Genescript) in the following order: sponge plate - PVDF membrane - gel - sponge plate (careful not to introduce air bubbles), and then placed in an electrotransfer tank for 16 minutes for non-denaturing gels and 5 minutes for denaturing gels. 7. Blocking and antibody incubation: After electrophoretic transfer was completed, the PVDF membrane was removed and placed on a plate. Approximately 10 ml of TBST blocking solution containing 5% nonfat milk powder was added, followed by blocking for 60 minutes at room temperature. The blocking solution was discarded, and an appropriate dilution of RS1 antibody at 1:3000 was added and incubated overnight at 4°C with shaking. The membrane was washed three times with TBST for 5 minutes each time. 8. Secondary antibody incubation: Goat anti-rabbit secondary antibody diluted at 1:5000 was added, followed by incubation at normal temperature for 1 hour, and the membrane was washed with TBST three times for 10 minutes each. Color development: Solution A and solution B in the ECL color development substrate were mixed uniformly in a 1:1 ratio, and an appropriate amount of the uniformly mixed color development solution was coated onto a PVDF membrane, followed by exposure imaging using a Biorad automated chemiluminescence imaging system.

[0140] As shown in Figures 5E–5F, significant RS1 expression was observed in 661w cells infected with the recombinant viruses by WB assay (denaturing and non-denaturing gels) at both 48 and 72 hours postinfection, and at 72 hours, hRS1 protein expression after RC-C08 infection was significantly greater than that after AAV2.7m8 infection at the same viral MOI. In 661w photoreceptor cells, hRS1 protein expression mediated by the RC-C08 serotype was significantly stronger than that mediated by the AAV2.7m8 serotype.

[0141] Example 6: Comparison of the expression intensity of target proteins in the retina, choroid, and vitreous humor of knockout mouse models using ssRC-C08 and ssAAV8 Preparation of ssRC-C08 and ssAAV8 using 293F suspension cells: After culturing 293F suspension cells for 2-4 days, samples were taken and counted for cell density and viability. 6 When the cell density exceeded 10 cells / ml and the cell viability was greater than 90%, the cells were subjected to a dilution and passage treatment. The treated cells were diluted using fresh Dynamis™ medium (Gibco) pre-warmed in a 37°C water bath kettle until the cell density reached 0.N (N = 1–5) × 10 6 Cells were cultured in an incubator shaker at 37°C, 5% CO2 and 120 rpm; cells used for transfection were harvested and in logarithmic growth phase with a viability of over 90% and approximately 2 x 10 6The cells were grown for at least four generations, with a density of 1 x 10 cells / mL. On the day of transfection, a sample was removed and counted for cell density and viability. Preparation of transfection solution (30 mL system) using fresh Dynamis™ medium pre-warmed in a 37°C water bath kettle: (1) Plasmid (1 x 10) 6 (2) 60 μg of the diluted plasmid (60 μg cells / μg) was added to 1.5 mL of Dynamis™ medium at a ratio of 2:1:0.3 (helper plasmid (HLP plasmid): capsid plasmid (RC-C08 or AAV8): gene of interest plasmid (GOI plasmid)); (3) 60 μL of Fecto VIR-AAV was added to the diluted plasmid, mixed by rapid vortexing for 3 seconds, and then incubated at room temperature for 30 minutes. The transfection solution was added dropwise to cell culture medium containing 300 μl of PS (final concentration: 1%) and 30 μl of 10% P188 (final concentration: 0.01%), and the flask was returned to the shaker at 37°C, 5% CO2, and 120 rpm for further incubation. 72 hours after transfection, the cells were lysed. Consistent with the treatment of adherent cells, the cell samples were maintained in a processing mode, and the viral gene (vg) copy number was detected in the cells by real-time quantitative PCR. Ultracentrifugation and purification yielded the viruses ssRC-C08&GOI-C59 and ssAAV8&GOI-C59.

[0142] Administration to mice via intravitreal injection: The viruses ssRC-C08&GOI-C59 and ssAAV8&GOI-C59 obtained by ultracentrifugation and purification were administered intraocularly by IVT in C57 mice with the specific steps as follows: On the day of the experiment, mice of appropriate age (3-4 mice per group, 6-8 weeks old, C57 balb / c (purchased from Shanghai Model Organisms Center, Inc.)) were intraperitoneally injected with Zoletil® 50 60 mg / kg and xylazine hydrochloride 7.98 mg / kg, prepared to the desired concentration, in 0.9% sodium chloride injection. 0.5% tropicamide phenylephrine eye drops (Mydrin-p) were used for pupil dilation, and ofloxacin eye ointment (Dikeluo) was applied to the ocular surface. Under an operating microscope, a disposable needle was used to puncture the pleura inside the corneal scleral limbus, and a microsyringe with a 36G flat needle was used to avoid the lens and reach the vitreous body for intravitreal injection. After injection, the mice were placed in a warm environment for resuscitation. After awakening, they were returned to their cages. For each experimental group, injections were completed sequentially, and the time to start anesthesia and administration (the time when injection was completed) was recorded. Unless otherwise specified, the left eye (OS) was the unadministered eye, and the right eye (OD) was the administered eye. During intravitreal injection, any manual abnormalities, such as failed injections or intraocular injuries, were recorded and those with severe injuries were excluded.

[0143] Vitreous humor was extracted on days 14, 21, and 28 after IVT administration. The retina and choroid tissues were then isolated and pretreated before protein loading and subsequent WB analysis. The right eye (OD) of the mice was the treated eye, and the left eye (OS) served as a control. The results are shown in Figures 6A-D (B35X represents the mouse numbering). RS1 protein was strongly expressed in the retina, choroid, and vitreous humor of wild-type mice. RS1 protein expression was absent in the retina, choroid, and vitreous humor of RS1 knockout mice. Compared with the left eyes, the right eyes of RS1 knockout mice injected with ssRC-C08 and ssAAV8 had clear expression of RS1 protein in the retina, choroid, and vitreous humor, and the expression level of the target protein after IVT injection of ssRC-C08 was significantly higher than that of RS1 protein after IVT injection of ssAAV8, suggesting that the transduction efficiency of RC-C08 in vivo was stronger than that of AAV8.

[0144] As shown in Figures 6A-6B, WB analysis of non-denaturing mouse ocular tissues revealed that multimeric RS1 protein was expressed in virus-injected eyes. Multimeric RS1 expression was significantly stronger in Group B (RC-C08) than in Group A (AAV8) at day 28. WB analysis of the vitreous humor showed that the secreted multimeric RS1 protein in the vitreous humor of the RC-C08-treated group was significantly higher than that of the AAV8-treated group at days 14 and 21 after IVT administration. In vitro, there were differences in the intensity of hRS1 expression in HEK293T cells at different multiplicities of infection with ssRC-C08 / AAV8 (GOI-C59) (Figure 6E).

[0145] The above results indicated that the RC-C08 serotype has a stronger ability to express RS1 polymer in vivo and in vitro than the AAV8 serotype (same GOI).

[0146] Example 7: Comparison of RS1 protein expression after IVT administration of ssRC-C08 & GOI-C59 drugs in photoreceptor cells and model mouse ocular tissues First, 661w cells were infected with the purified virus ssRC-C08 (GOI-C59) at MOIs of 100, 1,000, and 10,000. Supernatant and cell samples were then collected at 48 and 72 hours, respectively, and WB assays were performed (see Example 5 for sample processing and detection methods). The results are shown in Figures 7A-D. WB results demonstrated that ssRC-C08rAAV infected photoreceptor cells in vitro, resulting in significant expression of hRS1 polymer protein (both in the cell supernatant and secreted supernatant). Secreted RS1 in the supernatant was significantly higher than that in the cells at 72 hours. RS1 polymer protein expression showed a significant dose-dependent effect with increasing viral MOI. Figures 7A-7D show that: 1) hRS1 polymer was significantly expressed in both the cells and supernatants of photoreceptors infected with ssRC-C08; 2) the level of biologically active functional protein (octameric form of hRS1) secreted into the supernatant was significantly higher than that in the cells; and 3) as the MOI of RC-C08 virus increased, the expression of hRS1 polymer protein showed a significant dose-dependent effect in both the cell samples and the supernatants and was positively correlated with the time of infection. The right eye (OD) of the mouse was intravitreally and subretinally administered with two injections (ssRC-C08 & GOI-C59), while the left eye (OS) served as a control and was not administered. Retina and choroid proteins from the mice were extracted separately for WB analysis (see Example 6 for mouse selection, sample processing, and detection methods).

[0147] The isolation method for mouse retinal tissue involved the following steps: the mouse from which the tissue was to be harvested was anesthetized, its cervical vertebrae dislocated, and the mouse's eyeball was removed under a microscope to separate the retina, choroid, and retinal pigment epithelium (RPE) complex. The tissue was then placed in a 1.5 ml EP tube, frozen on dry ice, and stored in a -80°C freezer until further processing.

[0148] As shown in Figures 7E-7H, RS1 protein was strongly expressed in both the retina and choroid of wild-type mice, but not in the retina or choroid of RS1 control knockout mice. Specifically, as shown in Figures 7E-7F, all virus-injected OD eyes in groups A, B, C, and D expressed high levels of RS1 octamer protein from non-denaturing gel mouse ocular tissue WB. Grayscale scan results show that octamer RS1 (in ocular tissue): hRS1 expression levels in ssRC-C08 (GOI-C59) were lower with IVT administration than with subretinal administration, but hRS1 expression with both administration methods was higher than that of WT mRS1. At the same dosage, subretinal administration had superior hRS1 protein expression than intravitreal administration. As shown in Figures 7G-7H, WB immunoblotting results showed that multimeric hRS1 protein was expressed in the retinal tissues of mice in the treatment groups (high-dose E9vg / OD in Group A and low-dose E8vg / OD in Group B). The expression of hRS1 multimers in the high-dose Group A was stronger than that in the low-dose Group A, and the expression level in the high-dose Group A was closer to that of normal mice. The results indicated that the expression of multimeric hRS1 protein in mouse tissues after IVT administration of ssRC-C08 drug was dose-dependent and related to the amount of virus injected.

[0149] The data in Example 7 show that the subretinal administration format results in significantly higher expression of the protein of interest than the intravitreal administration format, with both showing dose-dependent expression.

[0150] Example 8: In vivo efficacy of ssRC-C08 & GOI-C59 in RS1 knockout mouse model Optical coherence tomography (OCT) detection method in mice: Rs1 knockout mice (see CN112899311A for the preparation method) were intraperitoneally injected with 60 mg / kg of Zoletil® 50 and 7.98 mg / kg of xylazine hydrochloride, prepared with 0.9% sodium chloride injection to the desired concentration. 0.5% tropicamide phenylephrine eye drops (Mydrin-p) were used for pupil dilation, and ofloxacin eye ointment (Dikeluo) was applied to the ocular surface. Under an operating microscope, a disposable needle was used to puncture the inner pleura of the corneal scleral limbus, and a microsyringe with a 36G flat needle was used to avoid the lens and reach the vitreous or subretinal cavity for intravitreal injection. As shown in Table 3, mice in group I were intravitreally injected with ssRC-C08&GOI-C59 at a dose of 2E8vg, mice in group II were injected with ssRC-C08&GOI-C59 4E7vg into the subretinal space of the right eye, and mice in group III were injected with the control virus (ssRC-C08&GOI-E04) by IVT.

[0151] The mice were anesthetized with conventional anesthesia and administered tropicamide phenylephrine eye drops to dilate the pupils. Then, the mice were placed face down on the testing table and ofloxacin eye ointment was applied to protect the corneas of the mice. The integrated in vivo imaging device for the mice was turned on, the optical fiber and imaging lens were connected, the desktop software was opened, and the mouse position and lens angle were adjusted to capture fundus photography and OCT images.

[0152] During the OCT test, any abnormalities found in the mouse eyes, such as visible ocular surface damage, ocular collapse, etc., are recorded faithfully, and those with severe damage are excluded.

[0153] [Table 10]

[0154] OCT images (distal to the optic disc) in Figures 8A-8B show that the luminal size of the right eyes (OD) in Group I (IVT, 2E8 vg / eye) and Group II (subretinal, 4E7 vg / eye) was significantly smaller than that of the uninjected eyes (OS) at 28 days after injection. Before injection (Figure 8A) and at 28 days after injection (Figure 8B), the size of the cavitation (OD) in the injected eyes of Group III knockout mice was not different from that of the untreated eyes (OS), and the cavitation area in the control Group III eyes had a larger disruption area in both the left and right eyes.

[0155] When comparing administration groups I and II with group II (AAV-EGFP), OCT results of the distal and inferior edges of the optic disc showed that after 4 weeks, the size of retinal cavities in the injected eyes (OD) of groups I and II (ssRC-C08 & GOI-C59) was significantly smaller than that of the left eye control of the same mice and was close to the level of normal WT mice. Statistical results showed that group II (subretinal injection group) in particular had the most significant efficacy, and retinal breaks were fundamentally restored in this short-term efficacy study.

[0156] Example 9: Evaluation of dose effectiveness of ssRC-C08&GOI-C59 in Rs1 knockout model mice (OCT&IF&HE) As described in Example 6, ssRC-C08&GOI-C59 was injected into the eyes of Rs1 knockout model mice at a starting dose of 1.00E+7vg / eye (10-fold progressive increasing dose).

[0157] On the 29th day after the subretinal injection, the mice were placed in a completely dark environment overnight (>8 hours). On the 30th day after the subretinal injection, 60 mg / kg of Zoletil® 50 was intraperitoneally injected; the mice were anesthetized with 8 mg / kg of xylazine hydrochloride, 0.5% tropicamide phenylephrine ophthalmic solution (Mydrin-p) was used for pupil dilation, and 0.4% oxybuprocaine hydrochloride ophthalmic solution (Benoxil) was used for topical ocular anesthesia. Five minutes later, the mice were fully anesthetized and placed on the operating table with a ground electrode inserted into their tails, reference electrodes inserted into their left and right ears, and working electrodes placed in their left and right eyes, respectively. A Scotopic 3.0 test was performed, and after approximately 2 minutes of exposure to white light, a Photopic 3.0 test was performed to record and store the mouse ERG waveforms. During the ERG test, any abnormalities observed in the eyes of the mice, such as visible ocular surface damage, collapse of the eye, etc., are recorded faithfully, and those with severe damage are excluded.

[0158] The integrated in vivo imaging device for mice was turned on, the optical fiber and imaging lens were connected, the desktop software was opened, the mouse was anesthetized under conventional anesthesia, and tropicamide phenylephrine eye drops were administered to dilate the pupils. The mouse was then placed face down on the testing table, and ofloxacin eye ointment was applied to protect the mouse cornea. The mouse's position and lens angle were adjusted to capture fundus and OCT images. During the OCT test, any abnormalities observed in the mouse's eyes, such as visible ocular surface damage and ocular collapse, were faithfully recorded, and those with severe damage were excluded.

[0159] Immunofluorescence of frozen sections of retinal tissue from experimental mice is performed by the following method: Mice from which tissues were harvested were anesthetized, their cervical vertebrae dislocated, and their eyes removed, frozen, sliced, and placed in ocular fixative overnight for fixation. The mouse eyeballs were carefully removed from the ocular fixative with tweezers and placed in molds filled with optimal sectioning temperature (OCT) embedding medium (the corneal surface should be on the left side). The eyeball's position was carefully adjusted using a pipette tip. The molds containing the mouse eyeballs were placed in dry ice or a -80°C freezer for approximately 30 minutes to allow the optimal sectioning temperature (OCT) to completely solidify before removal from the mold and slicing. The freezing microtome was adjusted to thin-slicing mode up to 12 μm thick. The cut slices were attached to slides using the rolling collection method, i.e., the first slice was attached to the first slide, the second slice to the second slide, and so on. After 20 slides had been attached, the 21st slice was attached to the first slide. The number of sections that can be made from one slide can be roughly determined by the size of the sample, and generally five sections with excellent morphology were selected for immunofluorescence staining.

[0160] As shown in Figure 9A, the IV administration regimen was as follows: RS1 knockout model mice were administered intravitreal (IVT) at approximately 3 weeks of age. The right eye of each mouse was administered, and the left eye served as a control. Six mice were administered E10vg, E9vg, E8vg, and E7vg doses were administered in each group, ranging from high to low. OCT fundus examinations were performed at 2, 4, and 6 weeks after administration, respectively (Figure 10A). As shown in Figure 10C, immunofluorescence markers in retinal sections of mice in each group demonstrated that RC-C08 & GOI-C59 IVT injection could effectively transduce retinal tissue and express anaplerosis protein (RS1 protein, marked by a wavelength of 594 nm), and the protein expression intensity was positively correlated with the dosage.

[0161] As shown in Figure 9B, RC-C08 & GOI-C59 (IVT administration) significantly inhibited cavity formation in all layers of the mouse retina in a dose-dependent manner (administration groups I-III), with an effective dose of 1E8VG / eye (Figures 9C & 9D). IVT injection of RC-C08 & GOI-C59 (compared to subretinal administration) was also effective in reducing the formation of inner nuclear layer cavities and disease progression in a dose-dependent manner (Figures 9E & 9F). The formation of inner nuclear layer cavities was significantly suppressed in groups I-II. In group I, the inflammatory response decreased with increasing dose, and in group I, there were no tears in all layers of the retina, and the disease course was completely reversed; in the four groups (I-IV) of RC-C08&GOI-C59 administered by IVT, after 6 weeks of administration, the local inflammatory response in the retina was weak in all three groups of mice, except for the E10vg group (Figure 9G), indicating that the use of IVT administration of RC-C08&GOI-C59 is safe and effective.

[0162] Example 10: Evaluation of long-term efficacy of drug candidates in RS1 knockout mouse model (OCT & ERG) The long-term efficacy of the drug candidates RC-C08 & GOI-C59 was evaluated using the OCT and ERG testing procedures described in Examples 8 and 9. As can be seen from the OCT images (distal to the optic disc) at 6 weeks post-administration and 42 days post-injection in Figures 10A-10B, the tear counts in the right eyes (OD) in Group I (IVT, 2E8 vg / eye) and Group II (subretinal, 4E7 vg / eye) were not significantly reduced compared to control eyes, which correlated with the natural course of the disease in Rsl knockout mice. However, analysis of the data from the treated right eyes in Groups II and III revealed that the subretinal administration group still demonstrated significant efficacy in reducing or healing cavitation tears at 6 weeks post-administration (W6). Statistical results indicated that the drug candidate can be administered either subretinally or via IVT, with the effective dose for IVT being approximately 10 times higher than that for subretinal administration.

[0163] As shown in Figures 10C-10D, the ERG test results of the three treatment groups (I-III) and WT mice were further analyzed. Compared with group III (AAV-EGFP), the ERG visual function of treatment groups I and II was restored to a certain extent. The efficacy of the SR treatment group was stronger than that of the IVT treatment group, and the b / a values ​​tended to be normal for WT mice. The visual function of the mice was improved to a certain extent.

[0164] Specific sequence information is shown below.

[0165] [Table 11] JPEG2026503581000012.jpg255170JPEG2026503581000013.jpg255170JPEG202 6503581000014.jpg255170JPEG2026503581000015.jpg255170JPEG20265035810 00016.jpg255170JPEG2026503581000017.jpg255170JPEG2026503581000018.j pg255170JPEG2026503581000019.jpg255170JPEG2026503581000020.jpg255170 JPEG2026503581000021.jpg255170JPEG2026503581000022.jpg255170JPEG202 6503581000023.jpg255170JPEG2026503581000024.jpg255170JPEG20265035810 00025.jpg255170JPEG2026503581000026.jpg255170JPEG2026503581000027.j pg255170JPEG2026503581000028.jpg255170JPEG2026503581000029.jpg110170

Claims

1. 1. An AAV capsid protein variant comprising an engineered capsid protein VP1 comprising the following amino acid mutation sites relative to the parent AAV capsid protein VP1: Q464V, A467P, D469N, 1470M, R471A, D472V, and S474G, wherein said positions are determined with respect to the amino acid sequence positions of SEQ ID NO: 51, and optionally said VP1 further comprises Y500F and / or S501A.

2. 2. The AAV capsid protein variant of claim 1, wherein the parent AAV capsid protein VP1 is derived from AAV serotype 2 (AAV2) or AAV2 variant version 7m8 (AAV2.7m8), preferably AAV2.7m8.

3. The AAV capsid protein variant is (i) comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:1, and containing the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G; (iii) encoded by the nucleic acid sequence set forth in SEQ ID NO:2; or (iv) The AAV capsid protein variant of claim 1, encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO:2, and comprising the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G.

4. An isolated nucleic acid comprising a nucleotide sequence encoding the AAV capsid protein variant of any one of claims 1 to 3.

5. the nucleotide sequence (i) comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO:2; or (iv) The isolated nucleic acid of claim 4, encoding a capsid protein, comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO:2, and wherein the capsid protein variant encoded by the nucleic acid sequence comprises the following substitutions: Q464V, A467P, D469N, I470M, R471A, D472V, and S474G.

6. (i) an AAV capsid protein variant according to any one of claims 1 to 5; (ii) a nucleic acid of interest packaged within the AAV capsid, the nucleic acid of interest comprising a nucleic acid molecule encoding RS1; A recombinant AAV virus particle (rAAV) comprising:

7. The recombinant AAV virus particle of claim 6, wherein the RS1 is human RS1.

8. The human RS1 protein is (i) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15; or (ii) the recombinant AAV virus particle of claim 7, comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

15.

9. The RS1 is (i) comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 16 to 18 or a complementary sequence thereof; or (ii) The recombinant AAV virus particle of any one of claims 6 to 8, encoded by a coding sequence comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 16 to 18, or a complementary sequence thereof.

10. The recombinant AAV viral particle of any one of claims 6 to 9, wherein the nucleic acid of interest is contained in an expression cassette and is therefore packaged within the AAV capsid.

11. The recombinant AAV viral particle of claim 10, wherein the expression cassette is single-stranded DNA, double-stranded DNA, or single-stranded or double-stranded RNA.

12. The recombinant AAV virus particle of claim 10 or 11, wherein the expression cassette comprises at least one ITR (e.g., L-ITR or R-ITR) sequence flanking the nucleic acid of interest.

13. 13. The recombinant AAV viral particle of any one of claims 10 to 12, wherein the expression cassette comprises one or more regulatory sequences selected from one or more, or all, of a promoter, an inverted repeat, an intron, an enhancer, a post-transcriptional regulatory sequence, a polyadenylation region, a selection marker, or a reporter gene.

14. 10. A method for producing recombinant AAV viral particles, comprising culturing packaging cells under conditions sufficient for the production of recombinant AAV viral particles, wherein the packaging cells contain a plasmid comprising a nucleic acid encoding a capsid protein variant of any one of claims 1 to 3 or a nucleic acid of claim 4 or 5.

15. 15. The method of claim 14, wherein the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleic acid of interest.

16. The method of claim 14 or 15, further comprising isolating the self-complementary recombinant AAV virus particles from the culture supernatant containing the particles.

17. The method of any one of claims 14 to 16, further comprising lysing the packaging cells and isolating recombinant AAV viral particles from the cell lysate.

18. a. removing debris; b. treating the supernatant containing the recombinant AAV viral particles with benzonase nuclease; c. Concentrating the recombinant AAV viral particles; d. Purifying the recombinant AAV viral particles; The method of any one of claims 14 to 17, further comprising one or more of:

19. A recombinant AAV virus particle produced according to the method of any one of claims 14 to 18.

20. A packaging cell for producing recombinant AAV virus particles, comprising a plasmid containing a nucleic acid encoding the capsid protein variant of any one of claims 1 to 3, or a nucleic acid of claim 4 or 5.

21. 20. A formulation or composition or medicament comprising the recombinant AAV viral particle of any one of claims 6 to 13 or 19, and optionally pharmaceutically acceptable auxiliary substances, such as pharmaceutically acceptable carriers, excipients such as buffers, known in the art.

22. A combination product comprising a recombinant AAV viral particle according to any one of claims 6 to 13 or 19, and one or more additional therapeutic agents, for example immunomodulatory agents such as immunosuppressants.

23. 22. A method of treating an eye disease in an individual, comprising administering to said individual a recombinant AAV viral particle according to any one of claims 6 to 13 or 19, or a formulation or composition or drug according to claim 21, or a combination product according to claim 22.

24. 24. The method of claim 23, wherein the eye disease is selected from the group consisting of retinopathy or chorioretinopathy, age-related macular degeneration, diabetic retinopathy, and other non-genetic eye diseases such as physically or chemically induced retinal damage, such as diseases caused by X-linked retinoschisis or retinitis pigmentosa.

25. 25. The method of claim 23 or 24, wherein the administration is via intraocular administration, such as intraretinal or intravitreal administration, such as subretinal or intravitreal administration (IVT).

26. 26. The method of claim 25, wherein the administration is by injection.

27. The recombinant AAV virus particles are ３ vg / eye / time, 10 ４ vg / eye / number of times, 10 ５ vg / eye / number of times, 10 ６ vg / eye / number of times, 10 ７ vg / eye / number of times, 10 ８ vg / eye / number of times, 10 ９ vg / eye / number of times, 10 １０ vg / eye / number of times, 10 １１ vg / eye / times, or 10 １２ vg / eye / time or more, preferably 10 １２ vg / eye / time or less, e.g., 2 x 10 ８ vg / eye / number of times, 3 x 10 ８ vg / eye / number of times, 4 x 10 ８ vg / eye / number of times or 5 x 10 ８ vg / eye / time or more, for example, about 10 to about 5×10 ８ vg / eye / number of times, 1 x 10 ７ ~Approx. 5×10 ８ vg / eye / time, e.g., about 2 x 10 ８ vg / eye / number of times, 3 x 10 ８ vg / eye / number of times, 4 x 10 ８ vg / eye / number of times or 5 x 10 ８ The method of any one of claims 23 to 26, wherein the dose is administered in a dose of vg / eye / time.

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