Recombinant adeno-associated virus for treating retinitis pigmentosa and uses thereof

Abstract

A recombinant adeno-associated virus for delivering a polynucleotide encoding a PRPF31 protein and its use in the treatment of retinitis pigmentosa caused by mutations in the PRPF31 gene.

Description

Technical Field

[0001] This invention relates to recombinant adeno-associated virus (AAV) for the treatment of retinitis pigmentosa, particularly retinitis pigmentosa caused by insufficient expression of the PRPF31 protein. The invention further relates to pharmaceutical compositions comprising the recombinant AAV, plasmids for producing the recombinant AAV, and the therapeutic and pharmaceutical uses of the recombinant AAV. The invention also relates to methods for establishing animal models of retinitis pigmentosa caused by the absence or insufficient expression of the PRPF31 protein, and related animal models. Furthermore, the invention relates to methods for screening candidate drugs or validating efficacy using these animal models. Background Technology

[0002] Retinitis Pigmentosa (RP) is a group of inherited fundus diseases characterized by the progressive degeneration of retinal photoreceptor cells (including rods and cones). Patients typically experience night blindness, narrowed visual field, and decreased vision, eventually leading to blindness. RP has diverse inheritance patterns, including autosomal recessive (50-60%), autosomal dominant (ad) (30-40%), and X-linked inheritance (5-15%). Mutations in the PRPF31 gene are one of the main causes of autosomal dominant RP (adRP). Statistics show that PRPF31 gene mutations account for approximately 9.4%-11.1% of adRP cases in my country, making it the second leading cause of adRP. Currently, there is no effective treatment for RP caused by PRPF31 gene mutations (PRPF31-RP), making it an incurable blinding disease.

[0003] The PRPF31 gene encodes a nuclear protein (U4 / U6 small nuclear ribonucleoprotein Prp31) involved in precursor mRNA splicing. This protein plays a crucial role in the normal function of retinal cells, and it is generally believed that PRPF31-RP disease is primarily related to RPE cells and / or photoreceptor cells. The vast majority of pathogenic PRPF31 mutations identified so far ultimately lead to RP due to insufficient normal PRPF31 haplotype dosage. Specifically, after a heterozygous PRPF31 mutation, the expression level of the remaining wild-type PRPF31 allele is insufficient to maintain normal physiological function, resulting in RP symptoms. Studies of PRPF31-RP patient families have found that asymptomatic mutation carriers express twice the amount of wild-type PRPF31 as symptomatic patients. This difference in wild-type gene expression dosage is the reason why PRPF31 gene mutation carriers do not develop the RP phenotype.

[0004] In recent years, gene therapy has shown great potential as an emerging treatment strategy in the field of hereditary diseases. Adeno-Associated Virus (AAV) has become the preferred vector for gene therapy due to its low immunogenicity, high transduction efficiency, and long-term expression characteristics. AAV vectors can deliver therapeutic genes to target cells and stably express them within the cells, thereby repairing or replacing functional defects caused by gene mutations. However, there are currently no gene therapy drugs for RP caused by PRPF31 gene mutations. Furthermore, the imperfections of animal disease models also hinder the development of PRPF31-RP gene therapy drugs.

[0005] Therefore, developing a gene therapy drug targeting PRPF31-RP based on an AAV vector has significant clinical implications, application value, and good feasibility. This invention aims to address one or more of the aforementioned technical problems and clinical needs, providing a gene therapy drug for treating retinitis pigmentosa, particularly fundus diseases caused by PRPF31 gene mutations. Summary of the Invention

[0006] One aspect of the present invention provides a recombinant adeno-associated virus (cceAAV) comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a PRPF31 protein, and a polyA, respectively; and wherein the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double helix.

[0007] Another aspect of the present invention provides a recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV) comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, said nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding the PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR; wherein the nucleotide sequence of said polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO: 2 or 5.

[0008] Another aspect of the present invention provides a recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV) comprising: (a) an rAAV8 capsid; and (b) a nucleic acid packaged within the rAAV capsid, said nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding a PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR.

[0009] Another aspect of the present invention provides a recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV) comprising: (a) an rAAV capsid; and (b) nucleic acid packaged within the rAAV capsid, said nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding a PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR; wherein said promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO: 11, 15, 17 or 18.

[0010] Another aspect of the present invention provides a pharmaceutical composition comprising the recombinant adeno-associated virus described herein, and a pharmaceutically acceptable vector.

[0011] Another aspect of the present invention provides a plasmid vector comprising a promoter, a polynucleotide encoding a PRPF31 protein, and a polyA, wherein: the promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO:11, 15, 17, or 18; the polynucleotide encoding the PRPF31 protein has a nucleotide sequence as shown in SEQ ID NO:2 or 5; and the polyA is a late SV40 polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:12.

[0012] Another aspect of the present invention provides a method for establishing an animal model of retinitis pigmentosa caused by the absence or insufficient expression of PRPF31 protein, the method comprising: inserting recombinase recognition sites flanking the PRPF31 gene in the genome of the animal to obtain a homozygous animal; preparing a recombinase-recombinant adeno-associated virus, the recombinase-recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding the recombinase, wherein the recombinase is capable of recognizing the recombinase recognition site; and introducing the recombinase-recombinant adeno-associated virus into the eye of the homozygous animal, the introduction causing the recombinase to recognize the recombinase recognition site and knock out the PRPF31 gene, thereby obtaining the retinitis pigmentosa animal model after 5-8 weeks.

[0013] Another aspect of the present invention provides a method for establishing an animal model of retinitis pigmentosa caused by the absence or insufficient expression of PRPF31 protein, the method comprising: inserting two recombinase recognition sites within or on one side (5' side (upstream) or 3' side (downstream)) of the PRPF31 gene in the genome of the animal, wherein the excision of the DNA sequence between the two recognition sites will affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein (e.g., inserting recombinase recognition sites into intron 3 and intron 5 respectively, and after excision of the DNA sequence between them by recombinase, exons 4 and 5 of PRPF31 will be cleaved). In addition, frameshift mutations occur, ultimately leading to abnormal PRPF31 protein expression (e.g., loss of expression, insufficient expression, or impaired function relative to wild-type PRPF31 protein), resulting in homozygous animals; recombinase-based recombinant adeno-associated virus is prepared, the recombinase-based ...

[0014] Another aspect of the present invention provides an animal model of retinitis pigmentosa, wherein the PRPF31 gene in the retina of the animal is knocked out while the PRPF31 gene in other organs of the animal is retained, and wherein the phenotype of the model includes one or more of the following features: osteocyte-like pigmentation, retinal vascular thinning, loss of the intra- and extra-segmental (IS / OS) junction, thinning or loss of the outer nuclear layer (ONL) of the retina, and reduced amplitude of a-wave and b-wave in ERG detection.

[0015] Another aspect of the present invention provides a method for screening candidate drugs for treating retinitis pigmentosa, comprising: preparing a recombinase-based recombinant adeno-associated virus, the recombinase-based recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding the recombinase; introducing the recombinase-based recombinant adeno-associated virus, alone and together with the candidate drug, into the eye of an animal, wherein a recombinase recognition site is inserted flanking a PRPF31 gene in the animal's genome, and the recombinase is capable of recognizing the recombinase recognition site; evaluating changes in the retinal phenotype of the animal after introduction together with the candidate drug versus introduction alone, wherein the candidate drug is identified as effective if the retinal phenotype is improved or prevented when introduced together with the candidate drug compared to when introduced alone.

[0016] Another aspect of the present invention provides a method for screening candidate drugs for treating retinitis pigmentosa, comprising: preparing a recombinase-based recombinant adeno-associated virus, said recombinase-based recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding said recombinase; introducing said recombinase-based recombinant adeno-associated virus, alone and together with said candidate drug, into the eye of an animal, wherein two recombinase recognition sites are inserted within or to one side (5' side (upstream) or 3' side (downstream)) of the PRPF31 gene in the animal's genome, and the DNA sequence between the two recognition sites is excised in a way that would affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein. The recombinase can (e.g., inserting recombinase recognition sites into introns 3 and 5 respectively, and after the recombinase removes the DNA sequence between them, it will result in the excision of exons 4 and 5 of PRPF31 and a frameshift mutation, ultimately leading to abnormal PRPF31 protein expression, such as loss of expression, insufficient expression, or impaired function relative to wild-type PRPF31 protein), and the recombinase can recognize the recombinase recognition site; evaluate the changes in retinal phenotype of the animal after administration with the candidate drug compared to administration alone, and if the retinal phenotype is improved or prevented when administration with the candidate drug compared to administration alone, the candidate drug is identified as effective.

[0017] Another aspect of the present invention provides a method for screening candidate drugs for treating retinitis pigmentosa, comprising: obtaining an animal model of retinitis pigmentosa provided by the present invention; introducing the candidate drug into the eye of the animal model; evaluating changes in the retinal phenotype of the animal after the introduction of the candidate drug relative to before the introduction, and identifying the candidate drug as effective when retinal phenotype improvement or phenotype deterioration is prevented; preferably, the candidate drug is a PRPF31 gene drug, such as a recombinant adeno-associated virus containing a polynucleotide encoding the PRPF31 protein.

[0018] Another aspect of the present invention provides a method for treating retinitis pigmentosa, the method comprising administering a therapeutically effective amount of the recombinant adeno-associated virus or pharmaceutical composition provided by the present invention to a subject in need.

[0019] Other aspects and advantages of the invention will become apparent from the following more detailed description. Attached Figure Description

[0020] Figure 1 The expression of the target gene in retinal cells after administration of AAV8 and AAV2(7m8) vectors was detected by IHC and RNAScope methods.

[0021] Figure 2 The expression of GFP protein in APRE-19 cells transfected with three GOI plasmids was observed under a fluorescence microscope.

[0022] Figure 3 Flow cytometry was used to detect the expression of GFP protein in APRE-19 cells transfected with three GOI plasmids.

[0023] Figure 4 Map of the .rAAV-PRPF31 GOI circular plasmid.

[0024] Figure 5 The RNAScope method was used to detect the expression levels and distribution of target genes for rAAV corresponding to different GOI coding sequences.

[0025] Figure 6 The expression levels of the target gene in rAAV-seq.2.0 and rAAV-seq.5.0 were detected by qPCR.

[0026] Figure 7 qPCR was used to detect and compare the expression levels of the target genes cceAAV, scAAV, and ssAAV.

[0027] Figure 8 Schematic diagram of the .rAAV-PRPF31 recombinant adeno-associated virus vector.

[0028] Figure 9 The expression of drug protein molecules at the cellular level was detected using Western blotting (WB).

[0029] Figure 10 The expression and distribution of Cre enzyme in the retina were detected using IHC and BaseScope assays.

[0030] Figure 11. (A) Analysis of animal model construction using OCT and fundus photography. (B and C) show the animal model construction at week 5 and week 8, respectively, using ERG detection analysis.

[0031] Figure 12 Expression of Cre enzyme in WT mice when the modeling vector is injected alone or simultaneously with the modeling vector and the therapeutic drug.

[0032] Figure 13 The efficacy verification was analyzed using OCT (A), fundus color photography (A), HE staining (B), and ERG detection (C).

[0033] Figure 14 Comparison of AAVs with different genomic structures: cceAAV, ssAAV, scAAV. Detailed Implementation

[0034] definition

[0035] PRPF31 protein, officially named U4 / U6 intranuclear small ribonucleoprotein Prp31 or pre-mRNA-processing factor 31, has the following examples of mature human PRPF31 sequences: UniProtKB / Swiss-Prot:Q8WWY3-1 or NCBI Reference Sequence:NP_056444.3 (as shown in SEQ ID NO:6); mouse PRPF31 sequences: GenBank:AAK77987.1; rat PRPF31 sequences: NCBI Reference Sequence:NP_001099689.1; and non-human primate PRPF31 sequences: NCBI Reference Sequence:NP_001253032.1. The protein sequences are relatively conserved across species.

[0036] The PRPF31 gene, Pre-mRNA-processing factor 31 (NCBI Gene ID: 26121), encodes the PRPF31 protein and is located on chromosome 19, spanning 16.3 kb. The association of genetic variation in this gene with retinitis pigmentosa (ADRP) was first discovered in 2001. It encodes six different protein-coding transcripts, with the most frequently expressed transcript consisting of 14 exons (13 coding and 1 non-coding). This is the largest transcript, producing a protein of 499 amino acids with a molecular weight of 55 kDa. The functional domains of PRPF31 include a Nop domain, a flexible loop, a coiled-coil domain, and a apex. The Nop domain has U4 binding specificity, and the flexible loop protects the RNA from free radical attack. Numerous genetic variations (>100) have been identified in PRPF31, covering 5%–10% of ADRP cases. The most common variations are associated with exons 6–10. The disease mechanism underlying these variants is haploid deficiency, which is also the most prominent feature leading to incomplete penetration of the disease phenotype. Reduction in PRPF31 protein was observed to affect splicing selection in genes including RHO, ROM1, FSCN2, and GNAT1.

[0037] The terms “adeno-associated virus vector,” “AAV vector,” “adeno-associated virus,” “AAV virus,” “AAV virus particle,” “AAV virus particle,” and “AAV particle” are used interchangeably herein and refer to a viral particle consisting of at least one AAV capsid protein (preferably composed of all capsid proteins of a specific AAV serotype) and a packaged recombinant viral genome. The particle contains a recombinant viral genome having heteropolynucleotides (encoding the PRPF31 protein of this invention) and a transcriptional regulatory region, which at least contains a promoter. The transcriptional regulatory region may also contain polyA.

[0038] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell in which a foreign nucleic acid and / or recombinant vector has been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to the specific test cell but also to its progeny. Due to mutations or environmental influences, certain modifications may occur in the progeny, and thus these progeny may actually differ from the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0039] The term "recombinant viral genome" refers to a viral genome or a portion thereof in which at least one expression cassette is inserted. As used herein, the term "AAV recombinant viral genome" refers to an AAV genome containing a polynucleotide with at least one expression cassette inserted. The minimal "genome" of the AAV genome according to the invention typically comprises cis-acting 5′ and 3′ inverted terminal repeats (ITRs) and an expression cassette.

[0040] As used herein, the term "operable link" refers to the functional relationship and position of a promoter sequence relative to the polynucleotide of interest (e.g., if a promoter or enhancer affects the transcription of a sequence, then it is operably linked to the coding sequence). Typically, an operably linked promoter is adjacent to the sequence of interest. However, an enhancer need not be adjacent to the sequence of interest to control its expression. In another embodiment, the promoter and the polynucleotide encoding the PRPF31 protein of the present invention are adjacent. In yet another embodiment, the promoter is adjacent to the polynucleotide encoding the PRPF31 protein of the present invention via an SV40 intron sequence.

[0041] The term "therapeuticly effective amount" refers to a non-toxic but sufficient amount of a viral vector encoding the PRPF31 protein of the present invention to provide the desired biological outcome. This outcome may be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired alteration of a biological system. For example, a therapeutically effective amount of the AAV vector according to the present invention is an amount sufficient to produce the desired biological outcome.

[0042] As used herein, the term "Cap protein" refers to a polypeptide possessing at least one functional activity of a native AAV Cap protein (e.g., VP1, VP2, VP3). Examples of functional activities of Cap proteins include the ability to induce capsid formation, promote single-stranded DNA accumulation, promote AAV DNA packaging into the capsid, bind to cell receptors, and facilitate viral particle entry into host cells. In principle, any Cap protein can be used in the context of this invention. As used herein, the term "capsid" refers to the packaging structure of the viral genome. The capsid consists of several oligomeric subunits composed of proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins, VP1, VP2, and VP3.

[0043] As used herein, the term "Rep protein" refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g., Rep 40, 52, 68, 78). "Functional activity" of a Rep protein refers to any activity associated with the protein's physiological function. Other functions include regulating transcription of AAV (or other heterologous) promoters and site-specific integration of AAV DNA into the host chromosome. In a particular embodiment, the AAV rep gene is derived from serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and their hybrids, as well as capsid protein mutants modified with the aforementioned capsid proteins as a backbone.

[0044] As used herein, "viral protein upon which AAV replication depends" refers to the polypeptide (i.e., "auxiliary functional polypeptide") upon which AAV replication depends. Auxiliary functions include those functions required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based auxiliary functions are derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus. Auxiliary functions include, but are not limited to, adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, ULB, UL52, and UL29, and herpesvirus polymerase. In another embodiment, the viral protein upon which AAV replication depends is derived from adenovirus.

[0045] As used herein, the terms "ITR," "adeno-associated virus ITRs," or "AAV ITRs" are used interchangeably and refer to inverted terminal repeat sequences present at both ends of the DNA strand of the adeno-associated virus genome. ITR sequences are essential for the efficient replication of the AAV genome. Another characteristic of these sequences is their ability to form hairpins. This characteristic facilitates self-initiation, thereby allowing the independent synthesis of a second DNA strand. Procedures for modifying these ITR sequences are known in the art.

[0046] As used herein, the term “polyadenylation signal” or “polyA” refers to a nucleic acid sequence that mediates the attachment of a polyadenylated chain to the 3′ end of mRNA. Suitable polyA signals include, but are not limited to, SV40 early polyA signal, SV40 late polyA signal, rabbit globin polyA, bGH polyA, and HSV thymidine kinase (TK) polyA signal.

[0047] Introns can be natural, synthetic, or artificial (e.g., heterologous). Examples of synthetic introns include intron sequences derived from SV-40 (referred to as SV-40T intron sequences) and intron sequences derived from the chicken β-actin gene. In some embodiments, the transgene described in this disclosure comprises one or more (1, 2, 3, 4, 5, or more) artificial introns. In some embodiments, one or more artificial introns are located between the promoter and the nucleic acid sequence encoding human PRPF31 (or the transgene).

[0048] The term “nucleotide or nucleic acid sequence” is used interchangeably with “polynucleotide” in this document and refers to any polymeric form of nucleotides of any length.

[0049] As used herein, the term "subject" or "object" refers to an individual mammal, such as a human, a non-human primate (such as a chimpanzee and other ape and monkey species), a farm animal (such as birds, fish, cattle, sheep, pigs, goats, and horses), a domesticated mammal (such as dogs and cats), or a laboratory animal (such as rodents, such as mice, rats, and guinea pigs). The term includes subjects of any age or sex. In another embodiment, the subject is a mammal, preferably a human.

[0050] As used in this article, single-stranded AAV (ssAAV) refers to rAAV that has the coding sequence of the transgene expression cassette on a separate strand and is packaged into the viral capsid. It requires the process of converting from single-stranded to double-stranded. The synthesis of the second strand of viral DNA has been shown to be the rate-limiting step in viral gene expression.

[0051] As used in this article, self-complementary AAV (scAAV) involves the deletion of a D sequence (packaging signal) in the right-hand ITR of the ssAAV genome and a terminal unwinding site mutation (Δtrs). This mutation prevents Rep protein modification from regulating the unwinding cleavage, thus enhancing the packaging of self-complementary double-stranded DNA. After entering the cell, the double-stranded AAV virus does not require a process of converting from single-stranded to double-stranded DNA; it can be expressed directly at a relatively high level.

[0052] As used herein, covalently closed-end AAV (cceAAV) is a newly emerging rAAV system formed by covalently linking one end of the complementary double-stranded DNA of the AAV genome through a single-stranded covalently closed-end (SS-CCE) domain. An example of cceAAV is the cceAAV system described in WO2020 / 092904, which is incorporated herein by reference in its entirety.

[0053] cceAAV contains a polynucleotide encoding the PRPF31 protein.

[0054] A first aspect of the present invention provides a recombinant adeno-associated virus comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a PRPF31 protein, and a polyA, respectively; and wherein the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double helix.

[0055] In some embodiments, the recombinant adeno-associated virus (AAV) capsid may comprise capsid proteins selected from the following serotypes of AAV: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof, as well as capsid protein mutants modified with the above capsid proteins as a backbone. In a preferred embodiment, the recombinant AAV capsid may comprise or be composed of capsid proteins selected from or composed of AAV1, AAV2, AAV5, AAV7, AAV8, and hybrids thereof. In a more preferred embodiment, the recombinant AAV capsid may comprise or be composed of the AAV8 capsid protein.

[0056] Examples of genomic sequences of different AAV serotypes can be found in literature or public databases such as GenBank. For example, GenBank accession numbers are NC_001401.2 (AAV2), NC_001829.1 (AAV4), NC / 006152.1 (AAV5), AF028704.1 (AAV6), NC-006260.1 (AAV7), NC.006261.1 (AAV8), AX753250.1 (AAV9), and AX753362.1 (AAV10). In some embodiments, the adeno-associated virus vector according to the invention comprises a capsid derived from a serotype selected from AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrh10 serotypes. In another embodiment, the AAV serotype is AAV8. If a viral vector contains a sequence encoding a capsid protein, it can be modified to include a foreign sequence, thereby directing AAV to a specific cell type or one cell type, improving the efficiency of targeted vector delivery to cells, facilitating the purification or detection of AAV, or reducing host response.

[0057] In some embodiments, the PRPF31 protein is the human PRPF31 protein. Therefore, the nucleic acid contains a polynucleotide encoding the human PRPF31 protein. An example of the amino acid sequence of the natural human PRPF31 protein is shown in SEQ ID NO:6. It is understood that the PRPF31 protein may also be a functional fragment of the natural human PRPF31 protein.

[0058] In some embodiments, the 5'-ITR and / or 3'-ITR are selected from the corresponding 5'-ITR and 3'-ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In a preferred embodiment, the 5'-ITR and / or 3'-ITR are ITRs of AAV2 or AAV8. In one embodiment, the 5'-ITR and 3'-ITR have the nucleotide sequences shown in SEQ ID NO:9 and 10, respectively.

[0059] In some embodiments, the promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron. In a preferred embodiment, the promoter is a combination of a CMV promoter and an SV40 intron. In a preferred embodiment, the promoter has the nucleotide sequence shown in SEQ ID NO:11. In another embodiment, the promoter is a combination of an EF1α promoter and an SV40 intron. In a preferred embodiment, the promoter has the nucleotide sequence shown in SEQ ID NO:17. In other embodiments, the promoter is a CMV promoter having the nucleotide sequence shown in SEQ ID NO:18. In other embodiments, the promoter is an EF1α promoter having the nucleotide sequence shown in SEQ ID NO:15.

[0060] In some embodiments, the polyA is late SV40 polyA. Other polyAs are also feasible in this invention, including but not limited to early SV40 polyA, rabbit β-globin polyA, bGH polyA, and HSV TK polyA. In a preferred embodiment, the polyA has a nucleotide sequence as shown in SEQ ID NO:12.

[0061] In some embodiments, the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in any one of SEQ ID NO:1 to 5. In a preferred embodiment, the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:2 or 5. In a preferred embodiment, the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:2. In a preferred embodiment, the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:5.

[0062] In some embodiments, the covalently closed end domain comprises 10 to 50 nucleotide sequences (e.g., 15, 20, 25, 30, 35, 40, or 45), such as sequences from TelL or TelR. For example, in some embodiments, the covalently closed end domain comprises a nucleotide sequence as shown in SEQ ID NO:7 or 8. In a preferred embodiment, the covalently closed end domain comprises a nucleotide sequence as shown in SEQ ID NO:7.

[0063] In a preferred embodiment, the second polynucleotide chain comprises, in the 5' to 3' direction, a promoter, a polynucleotide encoding the PRPF31 protein, and a polyA, wherein the polyA is closer to the 3'-ITR than the promoter.

[0064] In a preferred embodiment, the nucleic acid has a cceAAV genome and contains a polynucleotide sequence as shown in SEQ ID NO:13.

[0065] Therefore, in some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA, respectively; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand.

[0066] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA, respectively; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand, the human PRPF31 protein having the amino acid sequence shown in SEQ ID NO:6.

[0067] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain comprising a nucleotide sequence as shown in SEQ ID NO: 7 or 8; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA, respectively; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand.

[0068] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV2 capsid or an rAAV8 capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain comprising a nucleotide sequence as shown in SEQ ID NO: 7 or 8; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA, respectively; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand.

[0069] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV2 capsid or an rAAV8 capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain comprising a nucleotide sequence as shown in SEQ ID NO:7 or 8; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA, in sequence; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand, wherein the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO:2 or 5.

[0070] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV2 capsid or an rAAV8 capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain comprising a nucleotide sequence as shown in SEQ ID NO:7 or 8; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA, in sequence; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand, wherein the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO:2 or 5, and the promoter has a nucleotide sequence as shown in SEQ ID NO:11.

[0071] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV2 capsid or an rAAV8 capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain comprising a nucleotide sequence as shown in SEQ ID NO:7 or 8; (iv) a second polynucleotide chain; and (v) a 3'-ITR; wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5' to 3' directions, a promoter, a polynucleotide encoding a human PRPF31 protein, and a polyA; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand, wherein the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO:2 or 5, the promoter has a nucleotide sequence as shown in SEQ ID NO:11, and the polyA has a nucleotide sequence as shown in SEQ ID NO:12.

[0072] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV2 capsid or an rAAV8 capsid; and a nucleic acid packaged within the rAAV capsid, the nucleic acid having a cceAAV genome, and the nucleic acid comprising a polynucleotide sequence as shown in SEQ ID NO:13.

[0073] In some embodiments, the first polynucleotide chain is 80 to 100% complementary to the second polynucleotide chain to form a complementary double helix, for example, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary.

[0074] In some embodiments, the recombinant adeno-associated virus provided by the present invention can significantly enhance the transcription and expression of polynucleotides encoding PRPF31 protein, thereby exhibiting significant therapeutic effects in a mouse model of retinitis pigmentosa.

[0075] As demonstrated in the embodiments of the present invention, recombinant adeno-associated virus with the cceAAV genome configuration has a higher PRPF31 gene expression level than recombinant adeno-associated virus with the ssAAV or scAAV genome configuration, and therefore has a better therapeutic effect.

[0076] Single-stranded recombinant adeno-associated virus (ssAAV) or self-complementary recombinant adeno-associated virus (scAAV)

[0077] Another aspect of the present invention provides a recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV) comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, said nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding the PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR; wherein the nucleotide sequence of said polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO: 2 or 5.

[0078] In some embodiments, the recombinant adeno-associated virus (AAV) capsid may comprise capsid proteins selected from the following serotypes of AAV: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof, as well as capsid protein mutants modified with the above capsid proteins as a backbone. In a preferred embodiment, the recombinant AAV capsid may comprise or be composed of capsid proteins selected from or composed of AAV1, AAV2, AAV5, AAV7, AAV8, and hybrids thereof. In a more preferred embodiment, the recombinant AAV capsid may comprise or be composed of the AAV8 capsid protein.

[0079] In some embodiments, the PRPF31 protein is the human PRPF31 protein. Therefore, the nucleic acid contains a polynucleotide encoding the human PRPF31 protein. An example of the amino acid sequence of the natural human PRPF31 protein is shown in SEQ ID NO:6. It is understood that the PRPF31 protein may also be a functional fragment of the natural human PRPF31 protein.

[0080] In some embodiments, the 5'-ITR and / or 3'-ITR are selected from the corresponding 5' or 3'-ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In a preferred embodiment, the 5'-ITR and / or 3'-ITR are ITRs of AAV2 or AAV8. In one embodiment, the 5'-ITR and 3'-ITR have the nucleotide sequences shown in SEQ ID NO:9 and 10, respectively.

[0081] In some embodiments, the promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron. In a preferred embodiment, the promoter is a combination of a CMV promoter and an SV40 intron. In a preferred embodiment, the promoter has the nucleotide sequence shown in SEQ ID NO:11. In another embodiment, the promoter is a combination of an EF1α promoter and an SV40 intron. In a preferred embodiment, the promoter has the nucleotide sequence shown in SEQ ID NO:17. In other embodiments, the promoter is a CMV promoter having the nucleotide sequence shown in SEQ ID NO:18. In other embodiments, the promoter is an EF1α promoter having the nucleotide sequence shown in SEQ ID NO:15.

[0082] In some embodiments, the polyA is late SV40 polyA. Other polyAs are also feasible in this invention, including but not limited to early SV40 polyA, rabbit β-globin polyA, bGH polyA, and HSV TK polyA. In a preferred embodiment, the polyA has a nucleotide sequence as shown in SEQ ID NO:12.

[0083] Another aspect of the present invention provides a recombinant adeno-associated virus comprising: an rAAV2 capsid or an rAAV8 capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding a PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR.

[0084] In some embodiments, the PRPF31 protein is the human PRPF31 protein. Therefore, the nucleic acid contains a polynucleotide encoding the human PRPF31 protein. An example of the amino acid sequence of the natural human PRPF31 protein is shown in SEQ ID NO:6. It is understood that the PRPF31 protein may also be a functional fragment of the natural human PRPF31 protein.

[0085] In some embodiments, the 5'-ITR and / or 3'-ITR are selected from the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In a preferred embodiment, the 5'-ITR and / or 3'-ITR are the ITRs of AAV2 or AAV8. In one embodiment, the 5'-ITR and 3'-ITR have the nucleotide sequences shown in SEQ ID NO:9 and 10, respectively.

[0086] In some embodiments, the promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron. In a preferred embodiment, the promoter is a combination of a CMV promoter and an SV40 intron. In a preferred embodiment, the promoter has the nucleotide sequence shown in SEQ ID NO:11. In another embodiment, the promoter is a combination of an EF1α promoter and an SV40 intron. In a preferred embodiment, the promoter has the nucleotide sequence shown in SEQ ID NO:17. In other embodiments, the promoter is a CMV promoter having the nucleotide sequence shown in SEQ ID NO:18. In other embodiments, the promoter is an EF1α promoter having the nucleotide sequence shown in SEQ ID NO:15.

[0087] In some embodiments, the polyA is late SV40 polyA. Other polyAs are also feasible in this invention, including but not limited to early SV40 polyA, rabbit β-globin polyA, bGH polyA, and HSV TK polyA. In a preferred embodiment, the polyA has a nucleotide sequence as shown in SEQ ID NO:12.

[0088] Another aspect of the present invention provides a recombinant adeno-associated virus comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding a PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR; wherein the promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron; preferably, the promoter is a combination of a CMV promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO:11.

[0089] In some embodiments, the PRPF31 protein is the human PRPF31 protein. Therefore, the nucleic acid contains a polynucleotide encoding the human PRPF31 protein. An example of the amino acid sequence of the natural human PRPF31 protein is shown in SEQ ID NO:6. It is understood that the PRPF31 protein may also be a functional fragment of the natural human PRPF31 protein.

[0090] In some embodiments, the polyA is late SV40 polyA. Other polyAs are also feasible in this invention, including but not limited to early SV40 polyA, rabbit β-globin polyA, bGH polyA, and HSV TK polyA. In a preferred embodiment, the polyA has a nucleotide sequence as shown in SEQ ID NO:12.

[0091] In some embodiments, the present invention provides a single-stranded recombinant adeno-associated virus (ssAAV) comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising, in sequence in the 5' to 3' direction: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding the PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR, wherein the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO:2 or 5.

[0092] In some embodiments, the present invention provides a self-complementary recombinant adeno-associated virus (scAAV) comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising, in the 5' to 3' direction,: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a mutant ITR; (iii) a second polynucleotide chain; and (v) a 3'-ITR, wherein the first polynucleotide chain or the second polynucleotide chain comprises, in the 5'-3' direction, a promoter, a polynucleotide encoding a PRPF31 protein, and polyA; the first polynucleotide chain and the second polynucleotide chain are complementary to form a complementary double strand; and the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO: 2 or 5. In some embodiments, the mutant ITR comprises the deletion of the packaging sequence (D) and the terminal unwinding site (TRS) of the wild-type ITR. In some embodiments, the sequence of the mutant ITR (mITR) is as shown in SEQ ID NO: 20 or 21. In some embodiments, the optional and preferred configurations of the components of the ssAAV and scAAV (including the rAAV capsid serotype, 5'-ITR and 3'-ITR, promoter, polyA) are the same as those described with respect to cceAAV.

[0093] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: an rAAV capsid; and nucleic acid packaged within the rAAV capsid, the nucleic acid comprising a polynucleotide encoding a PRPF31 protein, wherein the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO:2 or 5. In some embodiments, the optional and preferred configurations of the elements of the recombinant adeno-associated virus (including the rAAV capsid serotype, 5'-ITR and 3'-ITR, promoter, and polyA) are the same as those described with respect to cceAAV.

[0094] In some embodiments, the present invention provides a polynucleotide encoding a PRPF31 protein, wherein the nucleotide sequence of said polynucleotide is as shown in SEQ ID NO:2 or 5.

[0095] In some embodiments, the present invention provides a vector, such as a plasmid, comprising a polynucleotide encoding a PRPF31 protein, wherein the nucleotide sequence of said polynucleotide is as shown in SEQ ID NO:2 or 5.

[0096] Production of recombinant AAV and GOI plasmid

[0097] Recombinant AAV can be generated using a triple transfection (triple plasmid transfection) method. Typically, recombinant AAV is generated by transfecting host cells with an AAV vector (containing a transgene flanked by ITR elements) to be packaged into AAV particles, an AAV helper vector, and an accessory function vector. The AAV helper vector encodes “AAV helper function” sequences (e.g., rep and cap), which trans-provide the functional proteins required for AAV replication and encapsulation. The accessory function vector encodes nucleotide sequences for non-AAV-derived viral and / or cellular functions that AAV depends on for replication (e.g., “accessory functions”). Accessory functions include those functions required for AAV replication, including but not limited to those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0098] In some embodiments, the present invention provides a vector, such as a plasmid, containing the polynucleotide. In some aspects, an antibiotic resistance gene is introduced into the plasmid. Antibiotic resistance markers can be used to identify positive transgenic cells in recombinant virus generation. In some aspects, the antibiotic marker contains a sequence encoding an antibiotic resistance gene. For example, markers conferring resistance may include, but are not limited to, kanamycin, gentamicin, ampicillin, chloramphenicol, tetracycline, doxycycline, or hygromycin. In some aspects, the antibiotic resistance gene is a non-beta-lactam antibiotic resistance gene, such as kanamycin. In some embodiments, the vector or plasmid is constructed to form ssAAV, scAAV, or cceAAV. Figure 14 The comparison of these three different genome structures / configurations is shown.

[0099] The cells used to produce recombinant AAV can be HEK293 cells. Other cells are also feasible, such as CHO cells. In some embodiments, the cells are transfected with the polynucleotide-containing vector. Furthermore, the cells are simultaneously transfected with helper plasmids and accessory plasmids, such as those described above, wherein the helper plasmid provides, for example, the rep and cap genes for AAV, and the accessory plasmid provides, for example, genes such as E4, E2a, and / or VA for AAV replication.

[0100] In some embodiments, the present invention provides a plasmid vector comprising a promoter, a polynucleotide encoding the PRPF31 protein, and a polyA, wherein: the promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO:11, 15, 17, or 18; the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:2 or 5; and the polyA is a late SV40 polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:12.

[0101] In some embodiments, the plasmid vector further comprises polynucleotides encoding an ITR sequence, such as the 5'-ITR and 3'-ITR of AAV2 or AAV8. In some embodiments, in the case of cceAAV or scAAV, the ITR has a nucleotide sequence as shown in SEQ ID NO: 9 or 10. In some embodiments, in the case of ssAAV, the ITR has a nucleotide sequence as shown in SEQ ID NO: 9 and 10. In some embodiments, in the case of ssAAV, the ITR has a nucleotide sequence as shown in SEQ ID NO: 9 or 10.

[0102] In some embodiments, the plasmid vector further comprises TelL and TelR sequences. In some embodiments, the TelL and TelR sequences comprise nucleotide sequences as shown in SEQ ID NO:7 and 8, respectively.

[0103] Animal models of retinitis pigmentosa and their applications

[0104] Another aspect of the present invention provides a method for establishing an animal model of retinitis pigmentosa caused by the absence or insufficient expression of PRPF31 protein, the method comprising: (a) inserting a recombinase recognition site flanking the PRPF31 gene in the genome of the animal to obtain a homozygous animal; (b) preparing a recombinase-recombinant adeno-associated virus, the recombinase-recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding the recombinase, wherein the recombinase is capable of recognizing the recombinase recognition site; and (c) introducing the recombinase-recombinant adeno-associated virus into the eye of the homozygous animal, the introduction causing the recombinase to recognize the recombinase recognition site and knock out the PRPF31 gene, thereby obtaining the retinitis pigmentosa animal model after 5-8 weeks.

[0105] Another aspect of the present invention provides a method for establishing an animal model of retinitis pigmentosa caused by the absence or insufficient expression of PRPF31 protein, the method comprising: (a) inserting two recombinase recognition sites into the PRPF31 gene in the genome of the animal, or into the gene and on one side of the gene (5' side (upstream) or 3' side (downstream)), wherein the excision of the DNA sequence between the two recognition sites will affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein (e.g., inserting recombinase recognition sites into intron 3 and intron 5 respectively, and after the recombinase excises the DNA sequence between them, it will result in the excision of exons 4 and 5 of PRPF31 being cleaved). (a) Obtaining homozygous animals by: (b) preparing a recombinase-based recombinant adeno-associated virus (RAV) comprising a retinal-specific promoter and a polynucleotide encoding the recombinase, wherein the recombinase is capable of recognizing the recombinase recognition site; and (c) introducing the recombinase-based RAV into the eye of the homozygous animal, wherein the introduction causes the recombinase to recognize the recombinase recognition site and knock out the PRPF31 gene, and obtaining the retinitis pigmentosa animal model after 5-8 weeks.

[0106] In some embodiments, the recombinase and recombinase recognition site are a Cre / LoxP system or a Flp / Frt system. In a preferred embodiment, the recombinase and recombinase recognition site are a Cre / LoxP system. Therefore, in step (a), the method inserts LoxPs flanking the PRPF31 gene in the animal's genome, and in step (b), prepares a recombinase Cre recombinant adeno-associated virus, which includes a retina-specific promoter and a polynucleotide encoding the recombinase Cre.

[0107] In some embodiments, in step (a), the method inserts two LoxPs into the PRPF31 gene of the animal genome, or into the gene and on one side of the gene (5' side (upstream) or 3' side (downstream)). Removal of the DNA sequence between the two LoxP sites will affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein (e.g., inserting LoxP sites into introns 3 and 5, respectively, and then removing the DNA sequence between them using the recombinase Cre, will result in the removal of exons 4 and 5 of PRPF31 and a frameshift mutation, ultimately leading to abnormal PRPF31 protein expression, such as loss of expression, insufficient expression, or impaired function relative to the wild-type PRPF31 protein). In step (b), a recombinase Cre recombinant adeno-associated virus is prepared, comprising a retina-specific promoter and a polynucleotide encoding the recombinase Cre.

[0108] In some embodiments, the retina-specific promoter is a photoreceptor-specific promoter, preferably the hGRK1 promoter. In some embodiments, the retina-specific promoter is an RPE cell-specific promoter, preferably the RPE65 promoter or the BEST1 (VMD2) promoter. In a preferred embodiment, the retina-specific promoter is the hGRK1 promoter. In a preferred embodiment, the hGRK1 promoter has the nucleotide sequence shown in SEQ ID NO:16. Other examples of retina-specific promoters contemplated for use in this invention include the CRX promoter (photoreceptor-specific), the Rho promoter (rod-specific), the Nrl promoter (photoreceptor-specific), the IRBP promoter (photoreceptor-specific), the PDE6B promoter (rod-specific), the Opn1mw and Opn1sw promoters (both cone-specific), the Gnat1 promoter (rod-specific), the PR2.1 promoter (cone-specific), and the cone arrestin promoter (cone-specific).

[0109] In some embodiments, the intraocular space is the subretinal space, so in step (c) the recombinase-recombinant adeno-associated virus is introduced into the subretinal space of the homozygous animal.

[0110] In some embodiments, the introduction is by injection, thus the recombinase-recombined adeno-associated virus is formulated into an injectable.

[0111] In some embodiments, in step (c), the model is established using at least two of the following methods: electroretinography (ERG), optical coherence tomography (OCT), fundus photography, and pathological sections. For example, in step (c), model establishment is determined using ERG, OCT, and fundus photography. For example, in step (c), the model is considered established when its phenotype includes one or more of the following features: osteocyte-like pigmentation, retinal vessel narrowing, loss of the intra- and extra-segmental (IS / OS) junction, thinning or disappearance of the outer nuclear layer (ONL), and decreased amplitude of the a and b waves as detected by ERG. In a preferred embodiment, the model exhibits all of the following features: osteocyte-like pigmentation, retinal vessel narrowing, loss of the intra- and extra-segmental (IS / OS) junction, thinning or disappearance of the outer nuclear layer (ONL), and decreased amplitude of the a and b waves as detected by ERG.

[0112] In some embodiments, the animal is selected from non-human primates, rodents, and ungulates. In a preferred embodiment, the animal is a rodent, preferably a rat or mouse, and more preferably a mouse.

[0113] Therefore, in some embodiments, the present invention provides a method for establishing a mouse model of retinitis pigmentosa caused by the absence or insufficient expression of PRPF31 protein, the method comprising: (a) inserting LoxPs flanking the PRPF31 gene in the genome of the animal to obtain a homozygous animal; (b) preparing a recombinase Cre recombinant adeno-associated virus, the recombinase Cre recombinant adeno-associated virus comprising a retinal-specific promoter hGRK1 and a polynucleotide encoding the recombinase Cre, wherein the recombinase Cre is capable of recognizing the recombinase recognition site LoxP; and (c) injecting the recombinase Cre recombinant adeno-associated virus into the subretinal space of the homozygous animal, the introduction causing the recombinase Cre to recognize the recombinase recognition site LoxP and knock out the PRPF31 gene, and obtaining the retinitis pigmentosa animal model after 5-8 weeks.

[0114] Therefore, in some embodiments, the present invention provides a method for establishing a mouse model of retinitis pigmentosa caused by the absence or insufficient expression of PRPF31 protein, the method comprising: (a) inserting two LoxPs into the PRPF31 gene within the genome of the animal, or into the gene and on one side of the gene (5' side (upstream) or 3' side (downstream)). The removal of the DNA sequence between the two LoxP sites will affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein (e.g., inserting LoxP sites into intron 3 and intron 5 respectively, and removing the DNA sequence between them using the recombinase Cre, will result in the removal of exons 4 and 5 of PRPF31 and a frameshift mutation, ultimately leading to...). (a) Obtaining homozygous animals by aberrant expression of PRPF31 protein (e.g., loss of expression, insufficient expression, or impaired function relative to wild-type PRPF31 protein); (b) preparing recombinase Cre recombinant adeno-associated virus, the recombinase Cre recombinant adeno-associated virus comprising a retinal-specific promoter hGRK1 and a polynucleotide encoding the recombinase Cre, wherein the recombinase Cre is capable of recognizing the recombinase recognition site LoxP; and (c) injecting the recombinase Cre recombinant adeno-associated virus into the subretinal space of the homozygous animals, the introduction causing the recombinase Cre to recognize the recombinase recognition site LoxP and knock out the PRPF31 gene, obtaining the retinitis pigmentosa animal model after 5-8 weeks.

[0115] A related aspect of the present invention provides an animal model of retinitis pigmentosa, wherein the PRPF31 gene in the retina of the animal is knocked out while the PRPF31 gene in other organs of the animal is retained, and wherein the phenotype of the model includes one or more of the following features: osteocyte-like pigmentation, retinal vascular thinning, loss of the intra- and extra-segmental (IS / OS) junction, thinning or loss of the outer nuclear layer (ONL) of the retina, and reduced amplitude of a-wave and b-wave in ERG detection.

[0116] In some embodiments, the PRPF31 gene in the retina includes the PRPF31 gene in rod cells and cone cells.

[0117] In some embodiments, the expression level of PRPF31 protein in the retina of the animal is reduced compared to the wild type. In some embodiments, the expression level of PRPF31 protein in the rod and cone cells of the animal is reduced compared to the wild type.

[0118] In some embodiments, the animal is selected from non-human primates, rodents, and ungulates. In a preferred embodiment, the animal is a rodent, preferably a rat or mouse, and more preferably a mouse.

[0119] Therefore, in some embodiments, the present invention provides a mouse model of retinitis pigmentosa, wherein the PRPF31 gene in the rod and cone cells of the animal is knocked out, while the PRPF31 gene in other organs of the animal is retained, and wherein the phenotype of the model includes one or more of the following features: osteocyte-like pigmentation, retinal vascular thinning, loss of the inner-outer segment / outer-segment (IS / OS) junction, thinning or loss of the outer nuclear layer (ONL) of the retina, and reduced amplitude of a-wave and b-wave in ERG detection, and the expression level of PRPF31 protein in the rod and cone cells of the animal is reduced relative to the wild type.

[0120] A related aspect of the present invention provides a method for screening candidate drugs for treating retinitis pigmentosa, comprising: (a) preparing a recombinase-based recombinant adeno-associated virus, the recombinase-based recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding the recombinase; (b) introducing the recombinase-based recombinant adeno-associated virus, alone and together with the candidate drug, into the eye of an animal, wherein a recombinase recognition site is inserted flanking a PRPF31 gene in the animal's genome, and the recombinase is capable of recognizing the recombinase recognition site; and (c) evaluating changes in the retinal phenotype of the animal after introduction together with the candidate drug versus introduction alone, wherein the candidate drug is identified as effective if the retinal phenotype is improved or prevented when introduced together with the candidate drug compared to when introduced alone.

[0121] A related aspect of the present invention provides a method for screening candidate drugs for treating retinitis pigmentosa, comprising: (a) preparing a recombinase-based recombinant adeno-associated virus, said recombinase-based recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding said recombinase; and (b) introducing said recombinase-based recombinant adeno-associated virus, alone and together with said candidate drug, into the eye of an animal, wherein two recombinase recognition sites are inserted within or to one side (5' side (upstream) or 3' side (downstream) of the PRPF31 gene in the animal's genome, wherein excision of the DNA sequence between the two recognition sites would affect the normal expression of the PRPF31 gene or the normal expression of the PRPF31 protein. Function (e.g., inserting recombinase recognition sites into introns 3 and 5, respectively, and after the recombinase removes the DNA sequence between them, exons 4 and 5 of PRPF31 are removed and frameshift mutations occur, ultimately leading to abnormal PRPF31 protein expression, such as loss of expression, insufficient expression, or impaired function relative to wild-type PRPF31 protein), and the recombinase is capable of recognizing the recombinase recognition site; and (c) evaluating the changes in retinal phenotype of the animal after administration with the candidate drug compared to administration alone, wherein the candidate drug is identified as effective if the retinal phenotype is improved or prevented when administration with the candidate drug compared to administration alone.

[0122] In some embodiments, the candidate drug is a PRPF31 gene drug, such as a recombinant adeno-associated virus containing a polynucleotide encoding the PRPF31 protein. In some embodiments, the candidate drugs containing the recombinant adeno-associated virus encoding the polynucleotide encoding the PRPF31 protein differ from each other in one or more of the following aspects: capsid serotype, genomic conformation, promoter, polynucleotide encoding the PRPF31 protein, polyA, and the presence or absence of one or more regulatory elements.

[0123] In some embodiments, the recombinase and recombinase recognition site are a Cre / LoxP system or an Flp / Frt system. In a preferred embodiment, the recombinase and recombinase recognition site are a Cre / LoxP system.

[0124] In some embodiments, the retina-specific promoter is a photoreceptor-specific promoter, preferably the hGRK1 promoter. In some embodiments, the retina-specific promoter is an RPE cell-specific promoter, preferably the RPE65 promoter or the BEST1 (VMD2) promoter. In a preferred embodiment, the retina-specific promoter is the hGRK1 promoter. In a preferred embodiment, the hGRK1 promoter has the nucleotide sequence shown in SEQ ID NO:16. Other examples of retina-specific promoters contemplated for use in this invention include the CRX promoter (photoreceptor-specific), the Rho promoter (rod-specific), the Nrl promoter (photoreceptor-specific), the IRBP promoter (photoreceptor-specific), the PDE6B promoter (rod-specific), the Opn1mw and Opn1sw promoters (both cone-specific), the Gnat1 promoter (rod-specific), the PR2.1 promoter (cone-specific), and the cone arrestin promoter (cone-specific).

[0125] In some embodiments, the intraocular space is the subretinal space, so in step (c) the recombinase-recombinant adeno-associated virus is introduced into the subretinal space of the homozygous animal.

[0126] In some embodiments, the animal is selected from non-human primates, rodents, and ungulates. In a preferred embodiment, the animal is a rodent, preferably a rat or mouse, and more preferably a mouse.

[0127] A related aspect of the present invention provides a method for screening candidate drugs for treating retinitis pigmentosa, comprising: (a) obtaining an animal model of retinitis pigmentosa provided by the present invention; (b) introducing the candidate drug into the eye of the animal model; and (c) evaluating changes in the retinal phenotype of the animal after the introduction of the candidate drug relative to before the introduction, identifying the candidate drug as effective when retinal phenotype improvement or phenotype deterioration is prevented.

[0128] In some embodiments, the candidate drug is a PRPF31 gene drug, such as a recombinant adeno-associated virus containing a polynucleotide encoding the PRPF31 protein. In some embodiments, the candidate drugs containing the recombinant adeno-associated virus encoding the polynucleotide encoding the PRPF31 protein differ from each other in one or more of the following aspects: capsid serotype, genomic conformation, promoter, polynucleotide encoding the PRPF31 protein, polyA, and the presence or absence of one or more regulatory elements.

[0129] In some embodiments, the intraocular space is the subretinal space, so in step (c) the recombinase-recombinant adeno-associated virus is introduced into the subretinal space of the homozygous animal.

[0130] In some embodiments, the introduction is an injection, thus the recombinant adeno-associated virus is formulated into an injectable.

[0131] In some embodiments, the animal is selected from non-human primates, rodents, and ungulates. In a preferred embodiment, the animal is a rodent, preferably a rat or mouse, and more preferably a mouse.

[0132] Drug Compositions, Therapies and Indications

[0133] Another aspect of the present invention provides a pharmaceutical composition comprising any of the recombinant adeno-associated virus (rAAV) described herein, and pharmaceutically acceptable excipients.

[0134] In some embodiments, the rAAV composition is formulated to reduce the aggregation of AAV particles in the composition, especially in the presence of high rAAV concentrations (e.g., about 10). 13 In cases where GC / mL or higher is used. Methods for reducing rAAV aggregation are well known in the art and include, for example, adding surfactants, adjusting pH, adjusting salt concentration, etc. The formulation of pharmaceutically acceptable excipient and excipient solutions, and the development of suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens, are well known to those skilled in the art.

[0135] In certain circumstances, it is desirable to deliver the rAAV-based therapeutic construct in a suitably formulated pharmaceutical composition disclosed herein via one of the following methods: intravitreal, intraocular, subretinal, subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, oral, intraperitoneal, or inhalation. In some embodiments, a preferred administration method is intraocular injection, such as subretinal injection.

[0136] Suitable forms of pharmaceuticals for injection include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In many cases, the form is sterile and has a flowability sufficient for easy injection. It must remain stable under manufacturing and storage conditions, and its preservation must be protected against contamination by microorganisms such as bacteria and fungi. Excipients can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Suitable flowability can be maintained, for example, by using coating agents such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Prolonged absorption of injectable compositions can be caused by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0137] For example, for the administration of injectable aqueous solutions, the solution may be appropriately buffered if necessary, and the liquid diluent may first be isotonic with sufficient saline or glucose. Sterile aqueous media that can be used in this regard are known to those skilled in the art.

[0138] A sterile injectable solution is prepared by incorporating the desired amount of active rAAV with various other ingredients listed herein (as needed) into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, which produce a powder from its previously sterile filtered solution containing the active ingredient plus any other desired ingredients.

[0139] The rAAV compositions disclosed herein can also be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (forming with the free amino group of a protein), which form with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Through formulation, the solution will be administered in a dosage form compatible with the dosage formulation and at a therapeutically effective amount. The formulation is readily applicable in various dosage forms, such as injectable solutions, drug-release capsules, etc.

[0140] As used herein, “excipients” include any and all solvents, dispersion media, carriers, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption-retarding agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Supplemental active ingredients may also be incorporated into the composition. The phrase “pharmaceuticalally acceptable” refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to a host.

[0141] Delivery media such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles can be used to introduce the compositions of this disclosure into suitable host cells. In particular, transgenes delivered via rAAV vectors can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles. Such formulations are preferably used for pharmaceutically acceptable formulations that introduce the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Currently, liposomes with improved serum stability and circulating half-life have been developed.

[0142] In some cases, targeting ocular (e.g., corneal) tissue via intrastromal or subcutaneous administration may require a different (e.g., higher or lower) dose than that achieved by another method (e.g., systemic or topical application). Therefore, in some embodiments, the injection is an intrastromal injection (IS). In some embodiments, the injection is a topical application (e.g., topical application to the eye). In some cases, multiple doses of rAAV are administered.

[0143] In some implementations, as described herein, administration of rAAV results in the delivery of the transgene to ocular tissues. rAAV can be delivered to the ocular tissues of a mammalian subject by, for example, intraocular injection, subretinal injection, topical application (e.g., eye drops), or by injection into the eye of a mammalian subject (e.g., intravitreal injection). As used herein, “ocular tissues” means any tissue derived from or contained within the eye. Non-limiting examples of ocular tissues include neurons, the retina (e.g., photoreceptor cells), sclera, choroid, vitreous body, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea (e.g., keratinocytes, corneal endothelial cells, corneal basal cells, corneal pterygoid cells, and corneal squamous cells), conjunctival ciliary body, and optic nerve. The retina is located at the back of the eye and contains photoreceptor cells. These photoreceptor cells (e.g., rod cells, cone cells) contribute visual acuity by distinguishing colors, as well as contrast in the visual field.

[0144] In some embodiments, administration of rAAV as described herein leads to an increase in PRPF31 protein levels. In some embodiments, administration of rAAV as described herein leads to an increase in PRPF31 protein in ocular tissues. In some embodiments, administration of rAAV as described herein leads to improvement in retinitis pigmentosa in ocular tissues. In some embodiments, one or more of the following characteristics are improved in subjects receiving the rAAV: osteocyte-like pigmentation, retinal vascular thinning, loss of the intra- and extra-segmental (IS / OS) junction, thinning or disappearance of the outer nuclear layer (ONL), and decreased amplitude of a-waves and b-waves on ERG testing.

[0145] The compositions disclosed herein may comprise a single rAAV, or a combination of rAAVs with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the compositions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0146] An effective amount of rAAV or a composition is an amount sufficient to target and infect an animal and a target tissue (e.g., muscle tissue, eye tissue, etc.). In some embodiments, the effective amount will depend primarily on factors such as species, age, weight, the health of the subject, and the tissue to be targeted, and therefore can vary between animals and tissues. For example, an effective amount of rAAV is typically in the range of about 1 ml to about 100 ml of solution containing about 10 6 Up to 10 16 One genome copy (e.g., 1 x 10^15) 6 Up to 1x10 16 (including endpoints). In some implementations, the effective amount of rAAV ranges from 1x10. 9 Up to 1x10 14 Between 10 rAAV genome copies. In some cases, approximately 10 11 Up to 10 12 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 11 Up to 10 13 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 11 Up to 10 14 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 11 Up to 10 15 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 10 12 Up to 10 14A dose of one rAAV genome copy is appropriate. In some implementations, approximately 10 13 Up to 10 14 A dose of one rAAV genome copy is appropriate. In some implementations, approximately 1 x 10^ ... 12 Approximately 1.1 x 10 12 Approximately 1.2 x 10 12 Approximately 1.3 x 10 12 Approximately 1.4 x 10 12 Approximately 1.5 x 10 12 Approximately 1.6 x 10 12 Approximately 1.7 x 10 12 Approximately 1.8 x 10 12 Approximately 1.9 x 10 12 Approximately 1 x 10 13 Approximately 1.1 x 10 13 Approximately 1.2 x 10 13 Approximately 1.3 x 10 13 Approximately 1.4 x 10 13 Approximately 1.5 x 10 13 Approximately 1.6 x 10 13 Approximately 1.7 x 10 13 Approximately 1.8 x 10 13 Approximately 1.9 x 10 13 Or approximately 2.0 x 10 14 One vector genome (vg) copy per kilogram (kg) of body weight is suitable. In some implementations, approximately 4 x 10^6 copies per kilogram (kg) of body weight is suitable. 12 Up to 2x 10 13 The dosage between rAAV genome copies is appropriate. In some implementations, approximately 1.5 x 10^6 kiloliters are administered intravenously. 13 A dose of vg / kg is appropriate. In some embodiments, 10 12 -10 13 One rAAV genome copy is effective against target tissues (e.g., the eye). In some implementations, 10 13 -10 14 One copy of the rAAV genome is effective against target tissues (e.g., the eye).

[0147] In some embodiments, rAAV is injected into the subject. In other embodiments, rAAV is administered to the subject by topical application (e.g., eye drops). In some embodiments, an effective amount of rAAV is an amount sufficient to express an effective amount of the human PRPF31 protein in the subject's target tissue (e.g., the eye).

[0148] In a related aspect, the present invention provides the use of any of the recombinant adeno-associated virus or pharmaceutical compositions provided herein in the preparation of a medicament for the treatment of retinitis pigmentosa. In a related aspect, the present invention provides any of the recombinant adeno-associated virus or pharmaceutical compositions provided herein for the treatment of retinitis pigmentosa.

[0149] In some embodiments, the retinitis pigmentosa is at least partially caused by insufficient expression of the PRPF31 protein. In some embodiments, the retinitis pigmentosa is at least partially caused by a PRPF31 mutation. In some embodiments, the treatment includes a dose of 1 × 10 6 vg / eye to 1×10 13 The recombinant adeno-associated virus was administered at a dose of vg / eye.

[0150] In a related aspect, the present invention provides a method for treating retinitis pigmentosa, the method comprising administering to a subject in need a therapeutically effective amount of any of the recombinant adeno-associated virus or pharmaceutical composition described herein. In some embodiments, the retinitis pigmentosa is at least partially caused by insufficient expression of the PRPF31 protein. In some embodiments, the retinitis pigmentosa is at least partially caused by a PRPF31 mutation. In some embodiments, the treatment comprises administering 1 × 10 6 vg / eye to 1×10 13 The recombinant adeno-associated virus was administered at a dose of vg / eye.

[0151] sequence list

[0152] Example 1. Selection of capsid proteins

[0153] Based on the same GOI plasmid and cceAAV genome configuration (containing the same target gene expression cassette: CMV promoter + SV40 intron (SEQ ID NO:11), EGFP coding sequence, SV40 late PolyA (SEQ ID NO:12)), rAAV viral vectors were packaged using two serotypes, AAV2 (7m8) and AAV8, respectively, and then injected using the same subretinal injection method at the same dose (1x10). 9AAV8 and the same volume (1 μl) of virus were injected into the subretinal space of wild-type C57BL / 6J mice. Twenty-eight days post-injection, samples were collected, paraffin-embedded, and then subjected to IHC and BaseScope assays. The ability of the two different serotypes to deliver and express the target gene (EGFP) into target cells (e.g., RPE cells and photoreceptor cells) was compared at both protein and transcriptional levels. The results showed that AAV8 had better efficiency in delivering and expressing the target gene in RPE and photoreceptor cells (see...). Figure 1 ).

[0154] Example 2. Promoter Selection

[0155] Based on the same plasmid backbone (except for the promoter sequence driving the expression of the target gene, all other sequences are exactly the same, and the target gene expression cassette is: promoter + SV40 intron, EGFP coding sequence, SV40 late PolyA), three GOI circular plasmids were constructed using CMV promoter + SV40 intron (SEQ ID NO:11), EF-1α promoter + SV40 intron (SEQ ID NO:17), and GRK1 promoter + SV40 intron (SEQ ID NO:19), respectively. The following steps were used to transfect ARPE-19 cells, and the expression of EGFP protein in ARPE-19 cells was detected by fluorescence microscopy and flow cytometry: ① Wipe the safety cabinet from the inside out with alcohol and non-woven fabric; ② Before transfection, the cells were seeded into 24-well plates to achieve a confluence of 60-80% on the day of transfection; ③ Dilute Lipofec 3000 reagent and DNA to the required concentration and volume; ④ Mix Lipofec 3000 diluent and DNA diluent at a 1:1 ratio; ⑤ Incubate at room temperature for 10-15 min; ⑥ Add DNA-Lipofect mixture to each well; ⑦ 48 h after transfection, the expression of GFP in cells transfected with different plasmids was detected by fluorescence microscopy and flow cytometry.

[0156] The results showed that the CMV promoter + SV40 intron resulted in the highest expression efficiency of the target gene in ARPE-19 cells, followed by the EF-1α promoter + SV40 intron, and then the GRK1 promoter + SV40 intron (see...). Figure 2 and Figure 3 ).

[0157] Example 3. Optimization and selection of the target protein coding sequence

[0158] Based on the human U4 / U6 small nuclear ribonucleoprotein Prp31 protein (hPrp31) sequence, three optimized candidate PRPF31 gene coding sequences were obtained through different optimizations including codon frequency, mRNA secondary structure, GC content, RNase splicing sites, and repetitive sequences. These three coding sequences (SEQ ID NO:2, 3, 4) and the human PRPF31 gene wild-type cDNA sequence (SEQ ID NO:1) were packaged using the same serotype (AAV8), the same genome conformation (cceAAV), and the same expression regulatory frames (CMV promoter, SV40 intron, GOI coding sequence, and SV4 late PolyA, where the CMV promoter and SV40 intron sequences are the same as in Example 2, the SV40 late PolyA sequence is shown in SEQ ID NO:12, and the 5'-ITR and 3'-ITR use the AAV2 wild-type ITR) using the same process to obtain the corresponding rAAV vectors (the GOI circular plasmid used is shown in [link to GOI circular plasmid]). Figure 4 Then, using the same injection method (subretinal injection), and injecting the same dose and volume (1x10⁻¹²). 9 Vg / eye / 1μl) was injected into the subretinal space of wild-type C57BL / 6J mice. Twenty-eight days post-injection, samples were collected, paraffin-embedded, and then subjected to BaseScope assays. (The target protein is highly conserved across different species; human PRPF31 protein and mouse PRPF31 protein differ by only a few amino acids, and no antibody was found to distinguish them. Therefore, BaseScope technology was used to detect transcriptional levels, rather than IHC technology to detect protein levels.) The results showed that rAAV-Seq.2.0 exhibited the strongest expression in the target cells (see...). Figure 5 ).

[0159] Example 4. Further optimization of rAAV-Seq.2.0 sequences

[0160] The target gene coding sequence of rAAV-Seq.2.0 obtained in Example 3 was further optimized (SEQ ID NO:5) according to the requirements of viral packaging process. The optimized sequence was packaged into the corresponding rAAV vector (rAAV-Seq.5.0) using the same serotype (AAV8), the same genome conformation (cceAAV), and the same expression regulatory frame (CMV promoter, SV40 intron, GOI coding sequence, SV40 latePolyA) and the same process. Then, rAAV-Seq.2.0 and rAAV-Seq.5.0 were injected using the same injection method (subretinal injection) and the same dose and volume (1x10⁻¹²). 9vg / eye / 1μl) was injected into the subretinal space of wild-type C57BL / 6J mice. Samples were collected 14 days post-injection for qPCR detection to compare the target gene expression levels. The results showed that rAAV-Seq.5.0 had a higher target gene expression efficiency (see...). Figure 6 ).

[0161] Example 5. Selection of AAV genome conformation

[0162] Based on the same target gene expression cassette (same promoter, introns, PolyA and other regulatory elements, and the same hPRPF31 coding sequence) and the same serotype (AAV8), AAV viruses were packaged using three different genomic configurations: cceAAV, scAAV, and ssAAV. The three different types of rAAV vectors were then injected using the same injection method (subretinal injection) at the same dose and volume (1x10⁻¹²). 9 vg / eye / 1μl) was injected into the subretinal space of wild-type C57BL / 6J mice. Twenty-one days post-injection, tissue samples were collected for qPCR detection. The expression levels of the target gene were compared among the three. Results showed that cceAAV had a significantly higher target gene expression efficiency compared to ssAAV and scAAV (see...). Figure 7 ).

[0163] Example 6. Design of rAAV-PRPF31 drug molecule

[0164] Based on the data from the foregoing embodiments, rAAV-Seq.5.0 was identified as the final candidate rAAV drug (its hPRPF31 protein-coding DNA sequence is SEQ ID NO:5). In this embodiment, the main plasmid backbone adopts the cceAAV genome configuration, the promoter element adopts the CMV promoter sequence (SEQ ID NO:11), the PolyA element adopts the SV40 late PolyA sequence (SEQ ID NO:12), and the viral capsid adopts the AAV8 serotype (e.g., Figure 8 The single-stranded nucleotide sequence is shown in SEQ ID NO:13. The obtained rAAV is labeled as rAAV-PRPF31.

[0165] Example 7. Preparation of rAAV drug

[0166] AAV virus was packaged by co-transfecting HEK293 cells with three plasmids: the main plasmid (GOI plasmid), the R / C plasmid (helper plasmid), and the pHelper plasmid.

[0167] The GOI plasmid, based on the cceAAV genome configuration, was obtained through upstream fermentation and downstream purification in *E. coli* to form a circular GOI plasmid. This was then digested in vitro with TelN enzymes, endonucleases, and exonucleases, followed by ion chromatography and UF / DF purification to obtain a linearized GOI plasmid. Helper plasmids (containing gene fragments essential for AAV replication in adenovirus, such as E2A, E4, and VA) and R / C plasmids (expressing Rep and Cap proteins) were transfected into HEK293 cells at a mass ratio of 6.5%:23.5%:70% using HieffTrans PEI transfection reagent to package AAV virus containing the target gene. After packaging, the virus was lysed and harvested, and then purified through clarification filtration, affinity chromatography, anion exchange chromatography, and ultrafiltration concentration to obtain high-purity rAAV virus. Finally, relevant quality tests were performed on the rAAV virus, such as viral genome titer (Vg), capsid protein purity, viral particle number (Vp), host cell DNA residue (HCD), host cell protein residue (HCP), and bacterial endotoxin.

[0168] Example 8. In vitro expression experiment of the target protein

[0169] Cell lines with PRPF31 gene knockout were infected with rAAV-PRPF31 drugs at different MOIs (the target protein is conserved across species and widely expressed in various cells; therefore, the PRPF31 gene in the NIH / 3T3 cell line, derived from Cyagen Biosciences, was knocked out using CRISPR-Cas9 technology) to verify the expression capacity of the rAAV-PRPF31 drug protein.

[0170] Experimental Design: NIH / 3T3 cells were seeded into culture plates and cultured for a period of time to reach an appropriate cell density. Then, an appropriate amount of virus was added to the cells for infection. 72 hours after infection, cell samples were collected for Western blot analysis to detect the expression level of the target protein PRPF31. The experimental design is as follows:

[0171] Results: rAAV-PRPF31 drug protein was expressed, and the expression level showed a dose-related relationship (e.g., Figure 9 (As shown).

[0172] Example 9. Construction of a mouse model of PRPF31-RP disease

[0173] The modeling vector, namely the AAV8-GRK1-Cre vector (GRK1 is an adeno-associated virus vector with Cre enzyme expression driven by the promoter), was prepared using the same method as in Example 6. After successful vector preparation, the AAV8-GRK1-Cre vector (5*10) was injected subretinally. 8(vg / eye) was delivered into the eyes of C57BL / 6J wild-type (WT) mice. Eye samples were harvested and paraffin-embedded 28 days post-injection (D28). Immunohistochemistry (IHC) and Base Scope assays were used to detect Cre enzyme expression in the WT mouse retina at both protein and transcriptional levels. The results showed that the IHC and Base Scope results were consistent, indicating that Cre enzyme was specifically expressed in the outer nuclear layer (photoreceptor cells) at both the protein and transcriptional levels, as expected. Figure 10 (As shown).

[0174] Divided into three groups: ① KO group: AAV8-GRK1-Cre vector (5*10) was injected subretinally. 8 ① AAV solvent buffer was delivered intraocularly to the eyes of Prpf31(flox / flox) homozygous mice (Prpf31-flox mice were bred and provided by Cyagen Biosciences); ② Buffer group: 1 μl of AAV solvent buffer was delivered intraocularly to the eyes of Prpf31(flox / flox) homozygous mice via subretinal injection; ③ WT group: AAV8-GRK1-Cre vector (5*10) was delivered intraocularly to the eyes of Prpf31(flox / flox) homozygous mice via subretinal injection. 8 (vg / eye / 1μl) was delivered intraocularly to the eyes of C57BL / 6J wild-type mice (n≥3 in each group). Five to eight weeks after injection, the KO group developed a mouse disease model conforming to the clinical phenotype of PRPF31-RP, including: osteocyte-like pigmentation, retinal vascular thinning, loss of the intraocular / outer segment (IS / OS) junction, thinning or disappearance of the retinal retina (ONL), and decreased amplitude of a and b waves in ERG detection. Figure 11A , 11B (as shown in 11C).

[0175] The procedures for subretinal injection, electroretinography (ERG), optical coherence tomography (OCT), and other examinations are as follows:

[0176] retinal injection experimental procedure

[0177] Aspirate an appropriate amount of sample using a microsyringe. Place the anesthetized mouse on the operating table. Locally anesthetize the eye with lidocaine hydrochloride. Hold the head down with your left hand to partially protrude the eyeball. With your right hand, insert the microsyringe into the vitreous cavity 1 mm from the posterior limbus of the cornea. Insert the needle vertically first, then at an angle until it reaches the contralateral retina (via vitreous injection). Stop the needle insertion when resistance is encountered, and slowly inject the sample. Alternatively, use a syringe needle at a 15° angle to the axial length to create a channel in the sclera 1-2 mm from the exposed corneal limbus, piercing the sclera but not penetrating the RPE. Stop the needle insertion once the bevel is submerged (via scleral injection), and slowly inject. After injection, slowly withdraw the needle. The subretinal injection volume is 1 μl / eye.

[0178] Injection effect verification: An OCT examination is performed immediately after administration to confirm successful administration; photos are required.

[0179] Optical coherence tomography (OCT)

[0180] Mice were weighed and anesthetized by an intraperitoneal injection of an appropriate amount of 1% sodium pentobarbital solution. They were then placed on a specialized small animal table, with their limbs and upper body wrapped in tissue paper, exposing only their heads. One drop of compound tropicamide was administered to both eyes, causing pupil dilation within seconds. 1% sodium carboxymethyl cellulose was used to prevent corneal dryness and refractive media opacity. During fundus photography, the position of the mouse's eyes was adjusted, and the lens was slowly advanced towards the cornea, focusing until the fundus was clearly visible and the optic disc was approximately centered. Image acquisition was then initiated. After capturing the fundus color image, OCT scanning of the retina was performed along both the horizontal and vertical diameters of the optic disc. The scanning signal was adjusted until a clear tomographic image appeared, at which point the image was acquired.

[0181] ERG: Detection of retinal membrane potential

[0182] ① Mouse preparation: Weigh the mice to be tested one day in advance and keep them in a dark room for 12 hours overnight. The next day, anesthetize the mice by intraperitoneal injection according to their body weight, and then instill 1% tropicamide into the eyes to dilate the pupils and apply a topical anesthetic.

[0183] ②Electrode insertion: After the mouse is under stable anesthesia, apply gel coupling agent to the surface of the mouse cornea, place the mouse on the stage, and let the mouse cornea contact the objective lens electrode. Insert the ground electrode and reference electrode subcutaneously into the tail and head of the mouse, respectively.

[0184] ③ Detection: Electroretinograms (ERGs) were recorded using the Celeris ERG system. Dark-adapted ERG and light-adapted ERG measurements were performed using different flare stimulation intensities from low to high, with five recordings for each intensity. After the detection was completed, the data were saved, exported, and analyzed.

[0185] Example 10. Validation of the efficacy of rAAV-PRPF31

[0186] Based on the disease mouse model construction method of Example 8, and using the same subretinal intracavitary injection method, three groups were used for modeling and drug administration: ① WT group, AAV8-GRK1-Cre vector (5*10 8 ① AAV8-GRK1-Cre vector (5*10) was delivered intraocularly to the eyes of C57BL / 6J wild-type mice; ② In the KO group, ... 8(vg / eye) was delivered into the eyes of Prpf31(flox / flox) homozygous mice; ③ In the treatment group, the modeling vector and the therapeutic drug were administered simultaneously (data from simultaneous injection in WT mice showed that this administration method did not affect the expression of Cre enzyme in the modeling vector). Figure 12 As shown), the AAV8-GRK1-Cre vector (5*10) was used. 8 vg / eye) and rAAV-PRPF31 drug (1*10) 9 (vg / eye) was simultaneously delivered into the eyes of Prpf31(flox / flox) homozygous mice. Each group had n≥3, and the injection volume was 1 μL / eye.

[0187] The results showed that, compared with the KO group, the drug-treated group successfully prevented the appearance of the phenotype in mice, indicating that the rAAV-PRPF31 drug exhibited a significant therapeutic effect. Figure 13 (As shown).

Claims

1. A recombinant adeno-associated virus (cceAAV) comprising: (a)rAAV capsid; and (b) A nucleic acid packaged within an rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a first polynucleotide chain; (iii) a single-stranded covalently closed end (SS-CCE) domain; (iv) a second polynucleotide chain; and (v) a 3'-ITR; in, The first or second polynucleotide chain contains, in the 5' to 3' direction, a promoter, a polynucleotide encoding the PRPF31 protein, and a polyA, respectively. The first polynucleotide chain is complementary to the second polynucleotide chain to form a complementary double helix.

2. The recombinant adeno-associated virus according to claim 1, having one or more of the following characteristics: (a)rAAV capsid is AAV8 capsid; (b) The PRPF31 protein is the human PRPF31 protein (hPRPF31), preferably having the amino acid sequence shown in SEQ ID NO:6; (c) The 5'-ITR and 3'-ITR are wild-type 5'-ITR and 3'-ITR of AAV2 or AAV8; (d) The promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO: 11, 15, 17 or 18; (e)polyA is SV40 late polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:12; (f) The nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:2 or 5; (g) The single-stranded covalently closed end domain includes a nucleotide sequence as shown in SEQ ID NO:7 or 8; (h) The second polynucleotide chain contains, in the 5' to 3' directions, a promoter, a polynucleotide encoding the PRPF31 protein, and a polyA; (i) The nucleic acid contains a polynucleotide sequence as shown in SEQ ID NO:

13.

3. A recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV), comprising: (a)rAAV capsid; and (b) A nucleic acid packaged within an rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding the PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR; in, The nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:2 or 5.

4. The recombinant adeno-associated virus according to claim 3, having one or more of the following characteristics: (a)rAAV capsid is AAV8 capsid; (b) The PRPF31 protein is the human PRPF31 protein, preferably having the amino acid sequence shown in SEQ ID NO:6; (c) The 5'-ITR and 3'-ITR are wild-type 5'-ITR and 3'-ITR of AAV2 or AAV8; (d) The promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO: 11, 15, 17 or 18; (e)polyA is SV40 late polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:

12.

5. A recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV), comprising: (a)rAAV8 capsid; and (b) A nucleic acid packaged in an rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding the PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR.

6. The recombinant adeno-associated virus according to claim 5, having one or more of the following characteristics: (a) The 5'-ITR and 3'-ITR are wild-type 5'-ITR and 3'-ITR of AAV2 or AAV8; (b) The PRPF31 protein is the human PRPF31 protein, preferably having the amino acid sequence shown in SEQ ID NO:6; (c) The promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO: 11, 15, 17 or 18; (d)polyA is SV40 late polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:

12.

7. A recombinant adeno-associated virus, said recombinant adeno-associated virus being a single-stranded recombinant adeno-associated virus (ssAAV) or a self-complementary recombinant adeno-associated virus (scAAV), comprising: (a)rAAV capsid; and (b) A nucleic acid packaged within an rAAV capsid, the nucleic acid comprising: (i) a 5'-ITR; (ii) a promoter; (iii) a polynucleotide encoding the PRPF31 protein; (iv) a polyA; and (v) a 3'-ITR; in, The promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO: 11, 15, 17 or 18.

8. The recombinant adeno-associated virus according to claim 7, having one or more of the following characteristics: (a) The PRPF31 protein is the human PRPF31 protein, preferably having the amino acid sequence shown in SEQ ID NO:6; (b) polyA is SV40 late polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:

12.

9. A pharmaceutical composition comprising the recombinant adeno-associated virus according to any one of claims 1 to 8; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is an injection; more preferably, the pharmaceutical composition is configured as an injection suitable for intraocular delivery.

10. Use of the recombinant adeno-associated virus according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating retinitis pigmentosa; preferably, the retinitis pigmentosa is at least partially caused by insufficient expression of PRPF31 protein; preferably, the retinitis pigmentosa is at least partially caused by PRPF31 mutation; preferably, the treatment comprises using 1 × 10 6 vg / eye to 1×10 13 The recombinant adeno-associated virus was administered at a dose of vg / eye.

11. A plasmid vector comprising a promoter, a polynucleotide encoding a PRPF31 protein, and polyA, wherein: (a) The promoter is a CMV promoter, an EF1α promoter, a combination of a CMV promoter and an SV40 intron, or a combination of an EF1α promoter and an SV40 intron, preferably having a nucleotide sequence as shown in SEQ ID NO: 11, 15, 17 or 18; (b) The nucleotide sequence of the polynucleotide encoding the PRPF31 protein is shown in SEQ ID NO:2 or 5; and (c) The polyA is SV40 late polyA, preferably having a nucleotide sequence as shown in SEQ ID NO:

12.

12. The plasmid vector of claim 11, wherein the plasmid vector further comprises polynucleotides encoding 5'-ITR and 3'-ITR of AAV2 or AAV8; preferably having nucleotide sequences as shown in SEQ ID NO:9 and 10, respectively.

13. A method for establishing an animal model of retinitis pigmentosa caused by the absence or inadequacy of PRPF31 protein expression, the method comprising: (a) Recombinase recognition sites were inserted on both sides of the PRPF31 gene in the genome of the animal to obtain homozygous animals; (b) Preparation of recombinase recombinant adeno-associated virus, the recombinase recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding the recombinase, wherein the recombinase is capable of recognizing the recombinase recognition site; and (c) The recombinase-recombinant adeno-associated virus is introduced into the eye of the homozygous animal, the introduction causing the recombinase to recognize the recombinase recognition site and knock out the PRPF31 gene, and the retinitis pigmentosa animal model is obtained after 5-8 weeks.

14. A method for establishing an animal model of retinitis pigmentosa caused by the absence or inadequacy of PRPF31 protein expression, the method comprising: (a) Inserting two recombinase recognition sites into or within the PRPF31 gene and on one side of the gene in the genome of the animal. After the DNA sequence between the two recognition sites is removed, it will affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein, thus obtaining a homozygous animal; preferably, inserting recombinase recognition sites into intron 3 and intron 5 respectively. After the DNA sequence between the two is removed by the recombinase, exons 4 and 5 of PRPF31 will be removed and frameshift mutations will occur, ultimately leading to abnormal expression of PRPF31 protein; (b) Preparation of recombinase recombinant adeno-associated virus, the recombinase recombinant adeno-associated virus comprising a retinal-specific promoter and a polynucleotide encoding the recombinase, wherein the recombinase is capable of recognizing the recombinase recognition site; and (c) The recombinase-recombinant adeno-associated virus is introduced into the eye of the homozygous animal, the introduction causing the recombinase to recognize the recombinase recognition site and knock out the PRPF31 gene, and the retinitis pigmentosa animal model is obtained after 5-8 weeks.

15. The method according to claim 13 or claim 14, comprising one or more of the following features: (a) The recombinase and the recombinase recognition site are either the Cre / LoxP system or the Flp / Frt system, preferably the Cre / LoxP system; (b) The retina-specific promoter is a photoreceptor cell-specific promoter, preferably the hGRK1 promoter; or the retina-specific promoter is an RPE cell-specific promoter, preferably the RPE65 promoter or the BEST1 (VMD2) promoter; more preferably, the retina-specific promoter is the hGRK1 promoter, preferably having the nucleotide sequence shown in SEQ ID NO:16; (c) The intraocular space is the subretinal space; (d) The importation is an injection; (e) The model established in step (c) is determined by at least two of the following: electroretinography (ERG), optical coherence tomography (OCT) fundus photography, and pathological sections; (f) The phenotype of the model includes one or more of the following features: osteocyte-like pigmentation, retinal vessel thinning, loss of the intra- and extra-segmental (IS / OS) junction, thinning or loss of the outer nuclear layer (ONL) of the retina, and reduced amplitude of a and b waves as detected by ERG. (g) The animal is selected from non-human primates, rodents and ungulates, preferably rats or mice.

16. An animal model of retinitis pigmentosa, wherein the PRPF31 gene in the retina of the animal is knocked out while the PRPF31 gene in other organs of the animal is retained, and wherein the phenotype of the model includes one or more of the following features: osteocyte-like pigmentation, retinal vascular thinning, loss of the intra- and extra-segmental (IS / OS) junction, thinning or loss of the outer nuclear layer (ONL) of the retina, and reduced amplitude of a-wave and b-wave as detected by ERG.

17. The animal model of retinitis pigmentosa according to claim 15, having one or more of the following characteristics: (a) The PRPF31 gene in the retina includes the PRPF31 gene in rod cells and cone cells; (b) The expression level of PRPF31 protein in the retina of the animals was reduced compared with that of the wild type; (c) The animal is selected from non-human primates, rodents and ungulates, preferably rats or mice.

18. A method for screening candidate drugs for the treatment of retinitis pigmentosa, comprising: (a) Preparation of recombinase recombinant adeno-associated virus, wherein the recombinase recombinant adeno-associated virus comprises a retinal-specific promoter and a polynucleotide encoding the recombinase; (b) The recombinase-recombinant adeno-associated virus is introduced into the eye of an animal, alone and together with the candidate drug, wherein the recombinase recognition site is inserted flanking the PRPF31 gene of the animal's genome, and the recombinase is capable of recognizing the recombinase recognition site. as well as (c) Evaluate the changes in retinal phenotype in the animals after administration with the candidate drug versus administration alone. If the retinal phenotype is improved or prevented when the candidate drug is administered with the animal compared to administration alone, the candidate drug is identified as effective.

19. A method for screening candidate drugs for the treatment of retinitis pigmentosa, comprising: (a) Preparation of recombinase recombinant adeno-associated virus, wherein the recombinase recombinant adeno-associated virus comprises a retinal-specific promoter and a polynucleotide encoding the recombinase; (b) The recombinase-recombinant adeno-associated virus is introduced into the eye of an animal, either alone or together with the candidate drug, wherein two recombinase recognition sites are inserted into or to one side of the PRPF31 gene in the animal's genome. Removal of the DNA sequence between the two recognition sites will affect the normal expression of the PRPF31 gene or the normal function of the PRPF31 protein, and the recombinase is capable of recognizing the recombinase recognition sites. Preferably, the recombinase recognition sites are inserted into introns 3 and 5, respectively. Removal of the DNA sequence between these two sites by the recombinase will result in the removal of exons 4 and 5 of PRPF31 and a frameshift mutation, ultimately leading to abnormal PRPF31 protein expression. (c) Evaluate the changes in retinal phenotype in the animals after administration with the candidate drug versus administration alone. If the retinal phenotype is improved or prevented when the candidate drug is administered with the animal compared to administration alone, the candidate drug is identified as effective.

20. The method according to claim 18 or claim 19, comprising one or more of the following features: (a) The candidate drug is a PRPF31 gene drug, such as a recombinant adeno-associated virus containing a polynucleotide encoding the PRPF31 protein; (b) The recombinase and recombinase recognition site are Cre / LoxP system or Flp / Frt system, preferably Cre / LoxP system; (c) The retina-specific promoter is a photoreceptor cell-specific promoter, preferably the hGRK1 promoter; or the retina-specific promoter is an RPE cell-specific promoter, preferably the RPE65 promoter or the BEST1 (VMD2) promoter; preferably, the retina-specific promoter is the hGRK1 promoter, preferably having the nucleotide sequence shown in SEQ ID NO:16; (d) The intraocular space described is the subretinal space; (e) The animal is selected from non-human primates, rodents and ungulates, preferably rats or mice.

21. A method for screening candidate drugs for the treatment of retinitis pigmentosa, comprising: (a) Obtaining an animal model of retinitis pigmentosa as described in claim 16 or 17; (b) Introducing the candidate drug into the eye of the animal model; (c) Evaluate the changes in retinal phenotype in the animal model after the introduction of the candidate drug relative to before the introduction, and identify the candidate drug as effective when retinal phenotype improvement or phenotype deterioration is prevented; preferably, the candidate drug is a PRPF31 gene drug, such as a recombinant adeno-associated virus containing a polynucleotide encoding the PRPF31 protein.

22. A method for treating retinitis pigmentosa, the method comprising administering to a subject in need a therapeutically effective amount of the recombinant adeno-associated virus of any one of claims 1 to 8 or the pharmaceutical composition of claim 9; preferably, the retinitis pigmentosa is at least partially caused by insufficient expression of the PRPF31 protein; preferably, the retinitis pigmentosa is at least partially caused by a PRPF31 mutation; preferably, the treatment comprises administering 1 × 10 6 vg / eye to 1×10 13 The recombinant adeno-associated virus was administered at a dose of vg / eye.

23. A recombinant adeno-associated virus, comprising: (a)rAAV capsid; and (b) A nucleic acid packaged in an rAAV capsid, the nucleic acid containing a polynucleotide encoding a PRPF31 protein, wherein the nucleotide sequence of the polynucleotide encoding the PRPF31 protein is as shown in SEQ ID NO:2 or 5.

24. A polynucleotide encoding a PRPF31 protein, wherein the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO:2 or 5.

25. A vector comprising the polynucleotide of claim 22; preferably, the vector is a plasmid.

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