Enhanced aav vectors and uses thereof

Abstract

The present invention provides enhanced AAV vectors and uses thereof. In particular, the present invention provides adenovirus-associated virus capsid protein variants, recombinant AAV virus particles containing the capsid proteins, methods of making the same, and uses in the treatment and prevention of disease.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to enhanced AAV vectors and their applications. Background Technology

[0002] Significant progress has been made in gene therapy using viruses to deliver therapeutic genetic material. Adeno-associated virus (AAV) has attracted considerable attention as a highly efficient viral vector for gene therapy due to its low immunogenicity and ability to efficiently transduce non-dividing cells. AAV can infect a variety of cell and tissue types, and its viral systems have made significant strides in the past decade, making them suitable for human gene therapy.

[0003] The existing technology (CN 107012171 A) uses the AAV2 variant AAV2.7m8 for ocular gene therapy. AAV2.7m8 is a modified adeno-associated virus (AAV) vector that enhances its infectivity to retinal cells by inserting a 10-peptide amino acid between amino acids 587 and 588 in the GH ring of the AAV2 capsid protein. Although AAV2.7m8 has shown enhanced retinal cell infectivity during directed evolution, there are still areas for improvement. First, although AAV2.7m8 penetrates the internal limiting membrane better than wild-type AAV2 after intravitreal injection, the viral load reaching the inner and outer nuclear layers remains limited. This uneven distribution caused by the physical barrier may directly affect gene delivery efficiency, especially for diseases involving multiple levels of retinal cells, where the penetration of the existing capsid is insufficient for clinical needs. Further optimization of the inserted peptide structure or the combination of penetration-enhancing technologies may be necessary directions.

[0004] Secondly, the cross-species activity differences in targeted screening warrant attention. Existing patented technologies use macaque models to assess transduction efficiency, but primates and humans differ significantly in retinal anatomy (such as internal limiting membrane thickness) and cellular receptor expression profiles, potentially leading to discrepancies between clinical efficacy and expectations. For example, the biochemical microenvironment or immune clearance mechanisms of the human vitreous cavity may further reduce the actual transduction efficiency of modified AAV2 variants. This potential disconnect between preclinical and clinical data can be verified through human organoid models or transgenic animal experiments.

[0005] Therefore, there is an urgent need in this field to develop AAV carrier subtypes that can improve retinal transduction efficiency, effectively solving the problems of insufficient transduction efficiency or insufficient expression intensity in intravitreal injection during intraocular treatment using existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide an AAV variant that can improve retinal transduction efficiency, effectively solving the problems of insufficient transduction efficiency or insufficient expression intensity in intravitreal injection during intraocular treatment in existing technologies.

[0007] The first aspect of the present invention provides an adeno-associated virus (AAV) capsid protein variant, wherein the VP1 of the AAV capsid protein variant has an insert peptide relative to the amino acid sequence of the parental AAV capsid protein VP1, the insert peptide being 9 amino acids in length, and its amino acid sequence differing from any of the sequences shown in SEQ ID NO:29-32 by no more than 3, 2, or 1 amino acids.

[0008] In another preferred embodiment, the amino acid sequence of the inserted peptide differs from any of the sequences shown in SEQ ID NO:29-32 by no more than one amino acid.

[0009] In another preferred embodiment, the VP1 of the AAV capsid protein variant has an insert peptide between positions 588 and 589 relative to the amino acid sequence of the parental AAV2 capsid protein VP1, the insert peptide being 9 amino acids in length, and its amino acid sequence differing from any of the sequences shown in SEQ ID NO:29-32 by no more than 3, 2, or 1 amino acids.

[0010] In another preferred embodiment, the insert peptide is an insert peptide of formula I:

[0011] X1X2X3X4X5X6X7X8X9 Formula I

[0012] in:

[0013] X1 is selected from Asp (D), Glu (E) and Met (M);

[0014] X2 is selected from Pro (P), Thr (T), Gly (G), and Asp (D);

[0015] X3 is selected from Pro (P) and Gln (Q);

[0016] X4 is selected from Glu (E), Asp (D), Pro (P) and Asn (N);

[0017] X5 is selected from Gln (Q), Thr (T), Pro (P), and Arg (R);

[0018] X6 is selected from Arg (R) and Ser (S);

[0019] X7 is selected from Pro (P), Gln (Q) and Arg (R);

[0020] X8 is selected from Ala (A), Ser (S) and Glu (E);

[0021] X9 is selected from Arg (R) and Val (V).

[0022] In another preferred embodiment, the inserted peptide is an inserted peptide of formula II:

[0023] X1X2PX4X5RPX8R Formula II

[0024] in:

[0025] X1 is selected from Asp (D), Glu (E) and Met (M);

[0026] X2 is selected from Pro (P), Thr (T), Gly (G), and Asp (D);

[0027] X4 is selected from Glu (E), Asp (D), Pro (P) and Asn (N);

[0028] X5 is selected from Gln (Q), Thr (T), Pro (P), and Arg (R);

[0029] X8 is selected from Ala (A), Ser (S) and Glu (E).

[0030] In another preferred embodiment, the inserted peptide is an inserted peptide of formula III:

[0031] X1X2X3X4X5X6X7AR Type III

[0032] in:

[0033] X1 is selected from Asp (D), Glu (E) and Met (M);

[0034] X2 is selected from Pro (P), Thr (T), Gly (G), and Asp (D);

[0035] X3 is selected from Pro (P) and Gln (Q);

[0036] X4 is selected from Glu (E), Asp (D), Pro (P) and Asn (N);

[0037] X5 is selected from Gln (Q), Thr (T), Pro (P), and Arg (R);

[0038] X6 is selected from Arg (R) and Ser (S);

[0039] X7 is selected from Pro (P), Gln (Q) and Arg (R).

[0040] In another preferred embodiment, the amino acid sequence of the inserted peptide is shown in any one of SEQ ID NO:29-32.

[0041] In another preferred embodiment, the amino acid sequence of the inserted peptide is shown in any one of SEQ ID NO: 29, 30 or 31.

[0042] In another preferred embodiment, the amino acid sequence of the inserted peptide is as shown in SEQ ID NO: 29 or 30.

[0043] In another preferred embodiment, the insert peptide is inserted between any two adjacent amino acid residues at positions 586-591 of the corresponding parental AAV capsid protein VP1 amino acid sequence.

[0044] In another preferred embodiment, the functional peptide is inserted between positions 586 and 587, 587 and 588, 588 and 589, 589 and 590, or 590 and 591 of the corresponding parental AAV capsid protein VP1 amino acid sequence.

[0045] In another preferred embodiment, the parental AAV capsid protein is AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, or AAV13 capsid protein.

[0046] In another preferred embodiment, the parental AAV capsid protein is AAV2 capsid protein.

[0047] In another preferred embodiment, the amino acid sequence of the AAV capsid protein VP1 includes the sequence shown in SEQ ID NO: 6.

[0048] In another preferred embodiment, the amino acid sequence of the AAV2 capsid protein VP1 is shown in SEQ ID NO: 6.

[0049] In another preferred embodiment, the amino acid sequence of the AAV capsid protein variant comprises the sequence shown in any of SEQ ID NO: 1-4.

[0050] In another preferred embodiment, the amino acid sequence of the AAV capsid protein variant is selected from the group consisting of:

[0051] (i) A sequence as shown in any of SEQ ID NO: 1-4;

[0052] (ii) A sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with any of the amino acid sequences shown in SEQ ID NO: 1-4.

[0053] In another preferred embodiment, the amino acid sequence of the AAV capsid protein variant is as shown in any of SEQ ID NO: 1-4.

[0054] In another preferred embodiment, the amino acid sequence of the AAV capsid protein variant is as shown in any of SEQ ID NO: 1-3.

[0055] In another preferred embodiment, the amino acid sequence of the AAV capsid protein variant is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0056] In a second aspect of the invention, a separated polynucleotide is provided, the polynucleotide encoding a variant of the AAV capsid protein described in the first aspect of the invention.

[0057] In another preferred embodiment, the polynucleotide sequence is as shown in any one of SEQ ID NO: 7-10.

[0058] In a third aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the second aspect of the invention.

[0059] In another preferred embodiment, the vector is a plasmid.

[0060] In a fourth aspect of the invention, a host cell is provided, the host cell containing a vector as described in the third aspect of the invention, or having a genome integrated with polynucleotides as described in the second aspect of the invention.

[0061] In another preferred embodiment, the host cell further contains an auxiliary plasmid containing the target nucleic acid.

[0062] In another preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

[0063] In another preferred embodiment, the host cell is a plant cell, an insect cell, or an animal cell, preferably a mammalian cell.

[0064] In another preferred embodiment, the host cell is a HEK-293T cell.

[0065] In a fifth aspect of the invention, a recombinant adeno-associated virus (rAAV) particle is provided, the rAAV particle comprising:

[0066] (i) AAV capsid protein variants as described in the first aspect of the invention.

[0067] (ii) The target nucleic acid packaged within the AAV capsid.

[0068] In another preferred embodiment, the target nucleic acid is a nucleic acid encoding an ophthalmic disease-related gene or a protein used to treat ophthalmic diseases.

[0069] In another preferred embodiment, the ratio of the transduction efficiency E1 of the rAAV particles to the target cells to the transduction efficiency E0 of the AAV particles containing the parental AAV capsid protein is E1 / E0≥2, preferably E1 / E0≥5, and more preferably E1 / E0≥10.

[0070] In a sixth aspect of the present invention, a method for preparing rAAV particles as described in the fifth aspect of the present invention is provided, comprising the steps of: culturing host cells as described in the fourth aspect of the present invention under suitable conditions to obtain rAAV particles.

[0071] In another preferred embodiment, the method further includes the step of isolating and / or purifying the rAAV particles from the culture.

[0072] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0073] (a) rAAV particles as described in the fifth aspect of the invention; and

[0074] (b) Pharmaceutically acceptable carriers.

[0075] In an eighth aspect of the invention, a pharmaceutical combination is provided, the pharmaceutical combination comprising:

[0076] (a) First active ingredient: rAAV particles as described in the fifth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention; and

[0077] (b) Second active ingredient.

[0078] In another preferred embodiment, the second active ingredient is an immunomodulator, such as an immunosuppressant.

[0079] In another preferred embodiment, the second active ingredient is an ophthalmic therapeutic agent.

[0080] In a ninth aspect of the invention, the use of rAAV particles as described in the fifth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention, or a pharmaceutical combination as described in the eighth aspect of the invention, or a combination thereof, in the preparation of a medicament for treating a disease is provided.

[0081] In another preferred embodiment, the disease is an eye disease.

[0082] In another preferred embodiment, the ocular disease is selected from the group consisting of: dry age-related macular degeneration (dAMD), geographic atrophy, crystalline retinal degeneration (BCD), wet age-related macular degeneration (wAMD), retinitis pigmentosa (RP), Fabry disease, choroidal dysplasia, Leber hereditary optic neuropathy (LHON), Stargardt disease, X-linked retinoschisis and X-linked retinitis pigmentosa, Leber congenital amaurosis, and hereditary retinal degeneration (IRD).

[0083] In another preferred embodiment, the medicament further comprises an active ingredient for treating diseases selected from the group consisting of: hemophilia, Canavan disease, Alzheimer's disease, lysosomal storage disease, adrenal medullary neuropathy, Parkinson's disease, amyotrophic lateral sclerosis (ALS), hereditary cardiomyopathy, familial hypercholesterolemia, Wilson's disease, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), epidermolysis bullosa (EB), hereditary deafness, and type 1 diabetes.

[0084] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0085] Figure 1 The figures shown are the fluorescence intensity detection results (a) and QPCR detection results (b) of different AAV variant viruses on day 14 after infection with the optic cup in this embodiment of the invention. The MOI is 1E9 and the mScarlet primer is used to detect the transcription intensity of the target viral RNA.

[0086] Figure 2 The figures shown are the efficacy test results of different AAV vectors injected into the intravitreal (IVT) of mice in the embodiments of the present invention. Among them, a is the result of in vivo autofluorescence (AF) examination at the 4th and 6th weeks after administration, b and c are the results of the average fluorescence intensity and total fluorescence intensity of AF in both eyes of mice after 6 weeks of administration, respectively, and d is the result of qPCR quantitative analysis of retinal RNA extracted from both eyes of mice after 6 weeks of administration.

[0087] Figure 3 The image shows the fluorescence staining results of frozen sections of the retinas of mice that had been administered different AAV carriers for 6 weeks, as described in this embodiment of the invention. Here, IVT represents intravitreal (IVT) injection of different AAV carriers, and 1E9vg represents a dose of 1 × 10⁻⁶ per eye. 9Viral genome (vg); green fluorescent labeling for rhodopsin (R), blue fluorescent labeling for cell nuclei with DAPI, and red fluorescent labeling for red fluorescent protein (S) produced by mScarlet expression carried by AAV. The leftmost column shows a complete retinal slice, and the remaining columns show partial retinal staining images with a scale bar of 75 μm, respectively showing the retinal ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL).

[0088] Figure 4 The graph shown is a result of the inhibition efficiency of IVIG against different viruses in the embodiments of the present invention. IVIG is an intravenous immunoglobulin, and RLUs represent relative luminescent units. Detailed Implementation

[0089] Through in-depth research and extensive screening, the inventors unexpectedly discovered a 9-amino acid (9mer) short peptide inserted between amino acids 588 and 589 of the AAV2 capsid protein (VP1). This led to the successful screening of a series of novel AAV variants with higher penetration and transduction efficiency in the retina, effectively addressing the problems of insufficient transduction efficiency or expression intensity encountered in intravitreal injection during intraocular treatment using existing technologies. The invention also validated the effectiveness in cross-species models, further verifying the transduction efficiency of these novel AAV variants in human retinal organoids. This overcomes the limitation of existing technologies, which can only be validated in primate models, providing more direct and reliable experimental evidence for the application of AAV vectors in the treatment of ophthalmic diseases. Based on this, the invention was completed.

[0090] the term

[0091] For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural, and vice versa, where appropriate. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0092] The term "about" or "approximately" includes a range of values ​​that are statistically significant. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% or 1%. The permissible variation covered by the term "about" or "approximately" depends on the specific system being studied and can be readily understood by those skilled in the art.

[0093] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0094] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the stated elements, integers, or steps constitute the whole. For example, when referring to a polypeptide that “comprising” a specific sequence, it is also intended to cover polypeptides composed of that specific sequence.

[0095] As described in this article, adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus *Dependent Virus* of the family Parvoviridae. It is currently the simplest single-stranded DNA-deficient virus discovered, requiring a helper virus (usually adenovirus) to participate in replication. It encodes the VP1 and rep genes located between two terminal inverted repeat sequences (ITRs). The ITRs play a crucial role in viral replication and packaging. The VP1 gene encodes the viral capsid protein, and the rep gene participates in viral replication and integration. AAV can infect various cell types. Due to its smaller size compared to other viral vectors, non-pathogenicity, and ability to transfect both dividing and non-dividing cells, gene therapy based on AAV vectors targeting ocular diseases, particularly hereditary retinal degeneration, has received widespread attention.

[0096] Recombinant adeno-associated virus vectors (rAAVs) are derived from non-pathogenic wild-type adeno-associated viruses. Due to their good safety profile, broad host cell range (dividing and non-dividing cells), low immunogenicity, and long in vivo expression time of exogenous genes, they are considered one of the most promising gene transfer vectors and are widely used in gene therapy and vaccine research worldwide. In medical research, rAAVs are used for gene therapy research on various diseases (including in vivo and in vitro experiments). Simultaneously, as a distinctive gene transfer vector, they are also widely used in gene function research, disease model construction, and gene knockout mouse creation.

[0097] The term "capsid protein" refers to a protein that forms part of the viral capsid. For adeno-associated virus (AAV), the capsid proteins are generally referred to as VP1, VP2, and / or VP3, and each is encoded by a single VP1 gene. For AAV, these three AAV capsid proteins are generated by overlapping capsid open reading frames (ORFs) via alternating mRNA splicing and / or alternating translation start codons. All three proteins use a common stop codon (Warrington et al. (2004) J.Virol. 78:6595). The amino acid sequences of the AAV capsid proteins are well known in the art and are generally conserved. Accordingly, although the amino acid positions provided herein can be provided relative to the AAV2 capsid protein VP1, and unless otherwise specified, the amino acid positions provided herein are determined with reference to the amino acid positions of the AAV2 capsid protein shown in SEQ ID NO: 6, those skilled in the art can readily and independently determine the corresponding positions of the same amino acids in different AAV serotypes. The “capsid proteins” mentioned in this article include capsid proteins of existing serotypes such as type 1 AAV (AAV1), type 2 AAV (AAV2), type 3 AAV (AAV3), type 4 AAV (AAV4), type 5 AAV (AAV5), type 6 AAV (AAV6), type 7 AAV (AAV7), type 8 AAV (AAV8), and type 9 AAV (AAV9).

[0098] The term "rAAV" used in this article refers to recombinant adeno-associated virus, also known as recombinant adeno-associated virus particles or recombinant AAV.

[0099] The term "retinal cell" in this document may refer to any cell type, including the retina, such as retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells and photoreceptor cells (including rod cells and cone cells), Müller's glial cells and retinal pigment epithelial cells.

[0100] As used herein, the phrase "operable connection" includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other, or otherwise coordinate with each other to participate in biological events, such juxtaposition achieving or allowing such interactions and / or coordination. In some embodiments, "operable connection" involves the covalent connection of related components or elements with each other. However, those skilled in the art will understand that in some embodiments, covalent connections are not required to achieve an effective operable connection.

[0101] The term "capsid protein variant" includes a capsid protein that has at least one mutation (e.g., substitution, deletion, or insertion) compared to the corresponding capsid protein that is the parent.

[0102] In this document, amino acid mutations can be amino acid substitutions, deletions, or insertions. Any combination of substitutions, deletions, or insertions can be performed to obtain optimized variants with desired properties. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. In some embodiments, amino acid mutations are amino acid substitutions, such as single amino acid substitutions, or combinations of several amino acid substitutions. In some embodiments, amino acid mutations are insertions, such as the insertion of several amino acid segments. The inserted amino acid can simply be inserted between two given amino acids of the capsid protein. Amino acid insertions can also be performed in conjunction with the deletion of a given amino acid of the capsid protein at the insertion site.

[0103] In this paper, when referring to the amino acid position of the capsid protein to be mutated, it is determined by referring to the amino acid sequence shown in SEQ ID NO: 6. The corresponding amino acid position on a hybrid protein or polypeptide with other amino acid sequences can be identified by comparing the amino acid sequence with SEQ ID NO: 6.

[0104] The terms "transduction" or "infection" refer to the introduction of nucleic acids into target cells via a viral vector. The term "transduction efficiency" refers to the percentage (e.g., fraction) of cells expressing the target nucleotide after incubation with a predetermined number of viral vectors containing the target nucleotide. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting using fluorescent reporter gene transduction and PCR for target nucleotide expression.

[0105] The “identity” or “percentage of identity” of an amino acid sequence or nucleic acid sequence refers to the percentage of amino acid residues / nucleotides in the candidate sequence that are identical to the specific sequence shown in this specification after comparing the candidate sequence with the specific sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention includes variants of the protein, polypeptide, or nucleic acid of the invention that have a considerable degree of identity with respect to the polypeptide, protein, or nucleic acid specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. The variants may contain conserved changes.

[0106] The terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual is a human.

[0107] The term "treatment" includes the administration of a composition or hybrid polypeptide to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, to alleviate symptoms, or to stop or inhibit the further development of a disease, symptom, or condition. The term "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0108] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0109] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0110] The term "effective amount" refers to such an amount or dose of the rAAV or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient requiring treatment or prevention.

[0111] The term "therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of rAAV or the composition or combination thereof are less than the beneficial therapeutic effects. Relative to an untreated subject, the "therapeutic effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, and more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0112] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. As used herein, the term polynucleotide may refer alternately to both double-stranded and single-stranded molecules.

[0113] The term "target nucleic acid" refers to the nucleic acid to be transduced by recombinant AAV viral particles, which encodes, for example, preventive or therapeutic proteins, especially proteins used to prevent or treat ophthalmic diseases, such as AIPL1, PROM1, RS1, RPE65, macromolecular antibodies and antibody analogs.

[0114] AAV capsid protein variant

[0115] In some embodiments, the present invention relates to a novel AAV capsid protein variant having a 9aa insert peptide relative to the parental AAV2 capsid protein.

[0116] In some embodiments, the amino acid sequence of the insert peptide has ≥60%, ≥70%, ≥80%, or ≥90% sequence identity with any of the sequences shown in SEQ ID NO:29-32. In some embodiments, the amino acid sequence of the insert peptide may differ from any of the sequences shown in SEQ ID NO:29-32 by 3, 2, or 1 amino acids, and such difference does not change or substantially does not change the retinal transduction efficiency of AAV viral particles containing the capsid protein variant of the present invention. In some embodiments, the amino acid sequence of the insert peptide is as shown in Formula I, Formula II, or Formula III. In some embodiments, the amino acid sequence of the insert peptide may be as shown in any one of SEQ ID NO:29-32. In some embodiments, the amino acid sequence of the insert peptide is as shown in any one of SEQ ID NO:29, 30, or 31. In another preferred embodiment, the amino acid sequence of the insert peptide is as shown in SEQ ID NO:29 or 30.

[0117] The present invention also relates to a plasmid comprising nucleic acid encoding a variant of the capsid protein of the present invention.

[0118] Purpose nucleic acid

[0119] The capsid protein of this invention can package the target nucleic acid to form viral particles.

[0120] The target nucleic acid encoded by the viral particles of this invention is any nucleic acid encoding a therapeutic or preventative protein, particularly a nucleic acid encoding a protein for the prevention or treatment of ophthalmic diseases, such as ophthalmology-related genes, such as RPE65, AIPL1, PROM1, RS1, etc. In some embodiments, the protein for the prevention or treatment of ophthalmic diseases includes, but is not limited to, RPE65, AIPL1, PROM1, RS1, or antibody analogs.

[0121] The target nucleic acid can be contained in an expression cassette and packaged within an AAV capsid.

[0122] In some implementations, the expression cassette contains at least one ITR sequence, thereby enabling the vector genome to be successfully assembled by the capsid. The expression cassette can be single-stranded DNA, double-stranded DNA, or single-stranded RNA or double-stranded RNA.

[0123] In some embodiments, the expression cassette may include one or more regulatory sequences to guide the expression of a target nucleic acid coding sequence in target cells (e.g., retinal target cells, such as photoreceptor cells or optic nerve cells). The regulatory sequences may be selected from transcription initiation sequences, termination sequences, promoter and / or enhancer sequences operatively linked to the coding sequence; effective RNA processing signals such as splicing and polyadenylation (polyA) regions, including human growth hormone polyadenylation regions; inverted repeat sequences (e.g., L-ITR or R-ITR); selective markers or reporter genes, such as resistance genes; microRNAs; posttranscriptional regulatory sequences, such as WPRE (posttranscriptional regulatory sequences for marmot hepatitis virus); sequences stabilizing cytoplasmic mRNA; nucleic acid restriction sites; homologous recombination sequences; sequences enhancing translation efficiency (e.g., Kozak sequences); sequences enhancing protein stability; and sequences enhancing the secretion of the encoded product when desired.

[0124] In some preferred embodiments, the regulation sequence is located in the 5' UTR or the 3' UTR. In some preferred embodiments, the regulation sequence is selected from one or more of the following:

[0125] Promoters, inverted repeat sequences, introns, enhancers, posttranscriptional regulatory sequences, polyadenylated regions, selective markers or reporter genes.

[0126] Examples of promoters suitable for use in this invention include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including apes and humans). Promoters can be constitutive or inducible. Constitutive promoters initiate RNA synthesis independently of regulatory influences.

[0127] The expression cassette of the present invention may also include a selective marker or reporter gene, for example, to determine the expression of the vector in a growth system (e.g., bacterial cells) or in target cells. The “selective marker” or “reporter gene” of the present invention may be selected from those known in the art. Suitable reporter genes include, but are not limited to, enhanced green fluorescent protein, red fluorescent protein, luciferase, and secreted embryonic alkaline phosphatase (seAP), which may include sequences encoding resistance to hygromycin, puromycin, or other antibiotics, etc. Such selective markers or reporter genes (which may or may not be located outside the viral genome to be packaged into viral particles) can be used to signal the presence of plasmids in bacterial cells, such as antibiotic resistance marker genes, for example, ampicillin or tetracycline resistance or kanamycin resistance.

[0128] The expression cassette or expression vector of the present invention may also include a polyadenylated region, such as hGHpA (human growth hormone polyadenylated region).

[0129] The expression cassette or expression vector of the present invention may also contain introns, such as introns of chimeric or natural genes.

[0130] Virus particles

[0131] This invention relates to a recombinant AAV virus (rAAV) particle, which comprises

[0132] (i) the AAV capsid protein variant of the present invention; and

[0133] (ii) The target nucleic acid, such as the nucleic acid encoding an ophthalmic disease-related gene or a protein used to treat an ophthalmic disease, packaged within the AAV capsid.

[0134] Preparation method

[0135] This invention relates to a method for preparing recombinant AAV viral particles (rAAV). Many methods are known in the art for packaging and producing rAAV. Currently, commonly used rAAV packaging systems mainly include three-plasmid co-transfection systems, systems using adenovirus as a helper virus, packaging systems using herpes simplex virus type 1 (HSV1) as a helper virus, and baculovirus-based packaging systems. Each packaging system has its own characteristics, and those skilled in the art can make appropriate selections according to their needs.

[0136] rAAV production cultures used to produce rAAV viral particles require: 1) suitable host cells, including, for example, human cell lines such as HEK-293T cells, or insect cell lines (in the case of baculovirus production systems); 2) suitable helper molecules provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide helper functions; 3) AAV rep and VP1 genes and gene products; 4) the target gene / target nucleic acid flanked by at least one fully functional AAV ITR sequence and preferably driven by an operable promoter; and 5) a suitable culture system to support rAAV production.

[0137] In some embodiments, the present invention relates to a method for producing recombinant AAV virus particles, comprising culturing packaging cells under conditions sufficient to produce recombinant AAV virus particles, wherein the packaging cells comprise a plasmid containing a nucleic acid encoding a capsid protein variant according to the present invention or a nucleic acid encoded by a capsid protein variant according to the present invention.

[0138] In some embodiments, the packaging cells further include helper plasmids and / or transfer plasmids containing the target nucleic acid.

[0139] In some embodiments, the method further includes separating recombinant AAV virus particles from the culture supernatant.

[0140] In some embodiments, the method further includes lysing the packaging cells and separating recombinant AAV virus particles from the cell lysis products.

[0141] In some implementations, the method further includes one or more of the following steps:

[0142] a. Remove cell debris,

[0143] b. Treat the supernatant containing recombinant AAV virus particles with a totipotent nuclease.

[0144] c. Concentrated recombinant AAV virus particles,

[0145] d. Purify the recombinant AAV virus particles.

[0146] Therefore, the present invention also relates to a packaging cell for producing recombinant AAV virus particles, said packaging cell comprising a plasmid containing a nucleic acid encoding a variant of the capsid protein described in the present invention, or a capsid protein encoding nucleic acid of the present invention, or an expression cassette of the present invention.

[0147] Composition, drug or preparation

[0148] The present invention provides a formulation, composition, or drug comprising (a) the rAAV described herein, and (b) pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0149] As used herein, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. For information on the use and applications of pharmaceutical excipients, see also The Pharmaceutical Excipients Handbook (8th Edition), RCRowe, PJ Sheskey, and SC Owen, Pharmaceutical Publishing House, London and Chicago.

[0150] In some embodiments, pharmaceutical excipients include, but are not limited to, one or more compatible solid or liquid fillers or gelling substances suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Suitable pharmaceutical excipients will be known to those skilled in the art. Examples of pharmaceutically acceptable carrier portions include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0151] The formulations, compositions, or drugs of the present invention can be liquids or solids, such as powders, gels, or pastes. Preferably, the formulations, compositions, or drugs of the present invention are liquids, and more preferably, injectable liquids. Preferably, the injectable liquids are provided as capsules or in syringes.

[0152] The rAAV of the present invention, or formulations, compositions, or drugs comprising thereof, can be administered intravenously, intramuscularly, subcutaneously, orally, via mucosal contact, intraperitoneally, and intralesionally, preferably topically to the eye, for example, via intraretinal or intravitreal administration, such as intravitreal injection, subretinal injection, or suprachoroidal injection. In some embodiments, the rAAV of the present invention, or formulations, compositions, or drugs comprising thereof, can be administered intraretinically or intravitreally, such as via intravitreal or subretinal administration, such as intravitreal administration (IVT). In any mode of administration, preferably, the formulations, compositions, or drugs of the present invention are provided as injectable liquids.

[0153] The composition, formulation, or drug may comprise physiologically acceptable sterile aqueous or anhydrous water, dispersion, suspension, or emulsion, and sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0154] The compositions of the present invention, such as pharmaceutical compositions or pharmaceutical preparations, may also contain other active ingredients, such as one or more other therapeutic agents, such as immunomodulators (e.g., immunosuppressants).

[0155] Combination products

[0156] The present invention also provides combination products (e.g., pharmaceutical combination products) comprising the rAAV of the present invention, and one or more other therapeutic agents. The combination products of the present invention can be used in the treatment methods of the present invention.

[0157] The present invention also provides a complete set of medicine boxes comprising the combined products, for example, the complete set of medicine boxes comprising, within the same package:

[0158] A first container containing the rAAV of the present invention or a drug comprising the present invention;

[0159] A second container for a pharmaceutical composition containing one or more other therapeutic agents (e.g., immunomodulators).

[0160] In some implementations, other therapeutic agents are immunomodulators, such as immunosuppressants, for example, used to reduce the immune response, such as the immune inflammatory response, generated by rAAV particles.

[0161] Treatment

[0162] In one embodiment, the rAAV, formulation, composition, or drug of the present invention is used to treat eye diseases.

[0163] In some implementations, eye diseases include, but are not limited to, the following: dry age-related macular degeneration (dAMD), geographic atrophy, crystalline retinal degeneration (BCD), wet age-related macular degeneration (wAMD), retinitis pigmentosa (RP), Fabry disease, choroidal agenesis, Leber hereditary optic neuropathy (LHON), Stargardt disease, X-linked retinoschisis and X-linked retinitis pigmentosa, Leber congenital amaurosis, hereditary retinal degeneration (IRD), congenital cataracts, glaucoma, congenital retinal, iris or choroidal defects, retinoblastoma, pathological myopia, congenital optic neuropathy, strabismus, keratoconus, etc.

[0164] In one embodiment, the rAAV, formulation, composition, or drug of the present invention is administered intraocularly, such as via intraretinal administration or intravitreal administration, such as via subretinal or intravitreal administration. In one embodiment, the administration is by injection.

[0165] In one embodiment, the invention also relates to the use of recombinant AAV virus particles, formulations or compositions comprising them, or combination products in the preparation of a medicament for treating the ocular disease of the present invention.

[0166] AAV variants with enhanced retinal cell transduction

[0167] To explore the role of peptides in enhancing the retinal penetration ability of AAV vectors, this invention synthesized a series of DNA sequences corresponding to 9mer short peptides and precisely inserted these 9mer sequences between 588 and 589 of the VP1 protein of wild-type AAV2 using polymerase chain reaction (PCR) and Gibson ligation technology. Subsequently, these capsid variants containing 9mers were packaged to construct a 9mer-AAV library.

[0168] To screen for viral variants with high retinal penetration capabilities, this invention performed tissue-specific screening by injecting a 9mer-AAV library into a mouse model of rod cells specifically expressing GFP. One week after injection, the mouse eyes were dissected, and the retina was processed and dissociated into a single-cell suspension. Subsequently, GFP-labeled photoreceptor cells were isolated using flow cytometry, and the amplified viral gene fragments were analyzed using next-generation sequencing. Sequencing results showed that the most enriched viral variants contained the following 9mer amino acid sequences: “DPPEQRPAR”, “ETPDTRPSR”, “MGQPPSQAR”, and “EDPNRRREV”. These variants exhibited significant advantages in retinal penetration capabilities, providing important reference for the development of novel AAV vectors.

[0169] The technical solution of the present invention has the following main advantages:

[0170] (1) The novel AAV variants RC-V63 and RC-V68 provided by the present invention are significantly superior to the existing AAV2.7m8 variant in terms of transduction efficiency and penetration in the eyes of live animals.

[0171] (2) When injected intravitreally into the mouse eye, the novel AAV variants RC-V63 and RC-V68 provided by this invention showed a wider distribution in fundus photography and section examination compared to the existing AAV2.7m8 variant. Specifically, RC-V63 and RC-V68 were able to carry the reporter gene to the outermost intraocular regions, especially the nuclear and outer nuclear layers, indicating that they have higher penetration and transduction efficiency in the retina.

[0172] (3) The transduction efficiency of the novel AAV variants RC-V53, RC-V62, RC-V63, and RC-V68 provided by this invention in human retinal organoids has been fully verified. The transduction effects of RC-V53 and RC-V62 in human retinal cells are significantly higher than those of AAV2.7m8 (the expression levels of RC-V53 and RC-V63 are 4.5 times and 6.8 times that of AAV2.7m8, respectively), while RC-V63 and RC-V68 can also reach similar levels to AAV2.7m8. This finding indicates that these variants, especially RC-V53 and RC-V62, have significant advantages in the treatment of human eye diseases and can effectively overcome the problem of low transduction efficiency of existing AAV variants in the treatment of eye diseases.

[0173] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0174] Example 1: Screening for AAV variants with enhanced retinal cell transduction

[0175] (1) Experimental methods and principles:

[0176] AAV insertion and display peptides can enhance gene delivery capabilities, primarily through the following mechanisms: First, the displayed peptides can specifically bind to novel receptors or high-abundance molecules on the target cell surface, enhancing initial viral attachment and cell entry capabilities. Second, certain peptides (such as pH-sensitive peptides or membrane-penetrating peptides) can promote endosome escape or direct penetration of the cell membrane, improving viral delivery efficiency. Furthermore, displayed peptides can mask capsid antigenic epitopes, reducing the recognition by neutralizing antibodies and prolonging the virus's duration of action in vivo. Finally, some peptides can activate non-classical endocytosis pathways, preventing viral degradation by lysosomes. These modifications collectively optimize AAV targeting, internalization efficiency, and intracellular transport, thereby significantly improving gene delivery performance.

[0177] (2) Experimental Procedure: In this embodiment of the invention, a series of 9mer sequences were synthesized, and these fragments were precisely inserted between the R588 and Q589 residues of the AAV2 capsid using molecular cloning technology. The plasmids containing the 9mer sequences were then packaged into a viral library in 293T cells. After the viral library passed quality assessment, it was injected into the vitreous cavity of a mouse model whose rod cells specifically express GFP. One week after injection, the mouse eyes were dissected, the retina was separated, and it was dissociated into a single-cell suspension. Next, photoreceptor cells labeled with GFP were isolated using flow cytometry (FACS), and viral gene fragments were recovered by PCR. The recovered target fragments were then subjected to second-generation sequencing analysis. Finally, bioinformatics analysis of the sequencing data was performed to statistically analyze the enrichment of different 9mer sequences and to screen for dominant sequences with high transduction capacity in photoreceptor cells.

[0178] The results show that this invention successfully identified a series of novel capsid variants with varying degrees of enhanced retinal penetration, including RC-V53, RC-V62, RC-V63, and RC-V68. The VP1 amino acid sequence of RC-V53 is shown in SEQ ID NO.1; the VP1 amino acid sequence of RC-V62 is shown in SEQ ID NO.2; the VP1 amino acid sequence of RC-V63 is shown in SEQ ID NO.3; and the VP1 amino acid sequence of RC-V68 is shown in SEQ ID NO.4.

[0179] To more comprehensively evaluate the performance of these variants, this invention not only validated them one by one in mouse models, but also introduced a human retinal organoid model to further characterize the infectivity of each variant.

[0180] Example 2: Preparation of AAV variants

[0181] Variants RC-V53, RC-V62, RC-V63, and RC-V68 were designed, with short peptides "DPPEQRPAR (SEQ ID NO. 29)", "ETPDTRPSR (SEQ ID NO. 30)", "MGQPPSQAR (SEQ ID NO. 31)", and "EDPNRRREV (SEQ ID NO. 32)" inserted after position 588 of VP1 in AAV2. The VP1 amino acid sequences of RC-V53, RC-V62, RC-V63, and RC-V68 are shown in SEQ ID NO. 1-4, respectively; the VP1 amino acid sequence of the AAV variant AAV2.7m8 disclosed in the prior art is shown in SEQ ID NO. 5; and the VP1 amino acid sequence of wild-type AAV2 is shown in SEQ ID NO. 6.

[0182] The following describes the construction of plasmids and the packaging of a series of AAV viruses with mScarlet transgenes, including RC-V53, RC-V62, RC-V63, RC-V68, AAV2, and AAV2.7m8, for subsequent in vitro cell and mouse in vivo transduction experiments. The nucleic acid sequences of VP1 for RC-V53, RC-V62, RC-V63, and RC-V68 are shown in SEQ ID NO. 7-10, respectively; the nucleic acid sequence of VP1 for the AAV variant AAV2.7m8 disclosed in the prior art is shown in SEQ ID NO. 11; and the nucleic acid sequence of VP1 for wild-type AAV2 is shown in SEQ ID NO. 12.

[0183] (1) Construction of RC-V53 plasmid

[0184] The AAV2 plasmid's cap gene and downstream poly sequence were completely digested with Swa I at 2003 bp and Sma I at 4348 bp to obtain a linearized vector. The AAV2 cap gene and downstream poly sequence were removed, and a 2372 bp fragment containing the RC-V53 cap and downstream poly sequence was used to replace them via homologous recombination. This fragment was amplified by polymerase chain reaction (PCR). Using the AAV2 plasmid as a template, PCR yielded two amplification products.

[0185] (a) Upstream of the 589-597aa mutation region:

[0186] The 5' end amplification primer is RC-V53-F1: AACAATAAATGATTTAAATCAGGTATGG (SEQ ID NO.13), and the 3' end amplification primer is RC-V53-R1: GGTCTCTGTTCCGGAGGATCTCTGTTGCCTCTCTGGAGGT (SEQ ID NO.14).

[0187] (b) Downstream of the 589-597aa mutation region:

[0188] The 5' end amplification primer was RC-V53-F2: ATCCTCCGGAACAGAGACCGGCTAGACAAGCAGCTACCGCAGATGTC (SEQ ID NO.15), and the 3' end amplification primer was RC-V53-R2: CGCTGTTTAAACGCCCGGGCTGTAG (SEQ ID NO.16). The two amplification products were overlapped to obtain a 2372bp fragment containing RC-V53cap and a downstream poly sequence.

[0189] (2) Construction of RC-V62 plasmid

[0190] Similar to the above construction method, the RC-V62 vector was constructed by homologous recombination of a fragment containing the RC-V62 cap with a linearized vector. The fragment containing the RC-V62 cap was obtained by PCR amplification. Using the AAV2 plasmid as a template, two amplification products were obtained: (a) upstream of the 589-597aa mutation region, the 5' amplification primer was RC-V62-F1: AACAATAAATGATTTAAATCAGGTATGG (SEQ ID NO.17), and the 3' amplification primer was RC-V62-R1: CTAGGCCTGGTGTCGGGTGTTTCTCTGTTGCCTCTCTGGAGGT (SEQ ID NO.18); (b) downstream of the 589-597aa mutation region: the 5' amplification primer was RC-V62-F2: ACACCCGACACCAGGCCTAGTAGACAAGCAGCTACCGCAGATGT (SEQ ID NO.19), and the 3' amplification primer was RC-V62-R2: CGCTGTTTAAACGCCCGGGCTGTAG (SEQ ID NO.19). NO.20), the two amplification products were overlapped to obtain a 2372bp fragment containing RC-V62 cap and downstream poly sequence.

[0191] (3) Construction of RC-V63 plasmid

[0192] Similar to the above construction method, the RC-V63 vector was constructed by homologous recombination of a fragment containing the RC-V63 cap with a linearized vector. The fragment containing the RC-V63 cap was obtained by PCR amplification. Using the AAV2 plasmid as a template, two amplification products were obtained: (a) Upstream of the 589-597aa mutation region: 5' end amplification primer was RC-V63-F1: AACAATAAATGATTTAAATCAGGTATGG (SEQ ID NO.21), and 3' end amplification primer was RC-V63-R1: CTTGGCTAGGTGGCTGACCCATTCTGTTGCCTCTCTGGAGGT (SEQ ID NO.22); (b) Downstream of the 589-597aa mutation region: 5' end amplification primer was RC-V63-F2: GGGTCAGCCACCTAGCCAAGCTAGGCAAGCAGCTACCGCAGATGT (SEQ ID NO.23), and 3' end amplification primer was RC-V63-R2: CGCTGTTTAAACGCCCGGGCTGTAG (SEQ ID NO.23). NO.24), the two amplification products above were overlapped to obtain a 2372bp fragment containing RC-V63 cap and downstream poly sequence.

[0193] (3) Construction of RC-V68 plasmid

[0194] Similar to the above construction method, the RC-V68 vector was constructed by homologous recombination of a fragment containing the RC-V68 cap with a linearized vector. The fragment containing the RC-V68 cap was obtained by PCR amplification. Using the AAV2 plasmid as a template, two amplification products were obtained: (a) Upstream of the 589-597aa mutation region: 5' end amplification primer was RC-V68-F1: AACAATAAATGATTTAAATCAGGTATGG (SEQ ID NO.25), 3' end amplification primer was RC-V68-R2: CGGCGGCGGTTGGGGTCCTCTCTGTTGCCTCTCTGGAGGTT (SEQ ID NO.26); (b) Downstream of the 589-597aa mutation region: 5' end amplification primer was RC-V68-F2: AGGACCCCAACCGCCGCCGCGAGGTCCAAGCAGCTACCGCAGATGT (SEQ ID NO.27), 3' end amplification primer was RC-V68-R2: CGCTGTTTAAACGCCCGGGCTGTAG (SEQ ID NO.26). NO.28), the two amplification products were overlapped to obtain a 2372bp fragment containing the RC-V68 cap and a downstream poly sequence.

[0195] (2) Packaging and purification of AAV

[0196] Dilute seed cells to 1E+6 / ml using 300ml of suspension medium (SMM 293-CD1, Sinocare), and culture at 37℃, 120rpm, 5% CO2 on a shaker. Add 300ug of plasmid to 15ml of antibiotic-free and GlutaMax-free SMM 293-TII, with a molar ratio of 1:1:1 for the three plasmid packages: LX-GOI-E10 (mScarlet reporter genome plasmid), RC-V53 / RC-V62 / RC-V63 / RC-V68 (Rep-Cap plasmid), and pHelper (helper packaging plasmid). Add 300ul of Fecto VIR-AAV to the dilution, vortex, and incubate at room temperature for 30min. Add the transfection mixture dropwise to the suspension cells and culture at 37℃, 120rpm, 5% CO2 on a shaker. After 24 and 48 hours of packaging, cells were fed with 35 ml / L of SMS 293-SUPI (Stimulin) per liter of cell culture. After 72 hours, cells were collected by centrifugation at 1500 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in PBS. Totipotency enzyme was added at 50 U / ml of sample, and the cells were digested on a shaker at 37°C for 1 hour. After centrifugation at 4000 rpm for 15 minutes, the supernatant was collected and subjected to iodixanol ultracentrifugation. The 40% iodixanol layer was collected and replaced with PBS using a 50 ml Millipore 100KD ultrafiltration tube. The virus was stored at -80°C.

[0197] Example 3: Retinal Organoid Experiment

[0198] Retinal organoids (optic cups) differentiate from human iPSC cells and can form three-dimensional structures containing all retinal cell types. Optic cups are highly similar in structure to the human retina, allowing for better simulation of the effect of AAV infection on the human retina. When the optic cups differentiate to the photoreceptor cell maturation stage (approximately 150-200 days), this invention examined the transduction activity of RC-V52, RC-V62, RC-V63, AAV2, and AAV2.7m8 viral vectors in human retinal organoids.

[0199] (1) Visual cup differentiation

[0200] Wild-type H9 cells were used for retinal organoid differentiation. On Day 0, human embryonic stem cell line H9 cells were mildly digested at 37°C and embryoid bodies (EBs) were established in 6-well plates with ultra-low adsorption. On Days 1-5, NIM medium (DMEM / F12 + 1xN2 + MEM-NEAA + Heparin) was changed every 2 days. On Days 7-15, EBs were transferred to 6-well plates with Matrigel Coat using Pasteur pipettes, and NIM medium was partially changed on Days 9, 12, and 15. On Days 16-25, 3:1 medium (DMEM / F12 + 1xB27 + MEM-NEAA) was used and the medium was changed every 2 days. The optic cups were then separated. Cells were scraped off using a pipette tip cross-section and transferred to low-absorption 6-well plates via Pasteur pipette. After isolating the optic cups, the culture medium was changed to 3D-RDM (DMEM / F12 + 10% FBS + MEM-NEAA + 1x B27 + 100uM Taurine + CDLS). On Days 30-40, well-defined optic cups were picked under a stereomicroscope for long-term culture. The first stage of optic cup differentiation occurs no later than week 6 after isolation, characterized by the appearance of a black core. 17-24 weeks after optic cup isolation, most iPSC-derived optic cups develop into the second stage, characterized by the reappearance of prominent surface hair-like appendages and a thin outer edge. At the onset of the third stage of differentiation (later than 148-196 days), the optic cups reach a higher stage of photoreceptor development, including the formation of inner and outer segments, the outer nuclear layer, and the outer plexiform layer, making them suitable for AAV infection testing.

[0201] (2) Vision cup infection

[0202] AAV2, AAV2.7m8, RC-V53, RC-V62, and RC-V63 viruses were uniformly diluted to 1E11 vg / ul. Differentiated mature retinal organoids were infected at 1E9 vg, with the 3D-RDM medium changed every three days. Fluorescence photography was performed on Day 14 after infection.

[0203] (3) Tissue RNA extraction and identification

[0204] The retinal organoid culture medium was removed, and the sample was washed twice with PBS. RNA was then extracted using a DNA / RNA co-extraction kit (Tiangen, DP422), and finally dissolved in 50 μL of DEPC water. 5 μL of RNA was reverse transcribed into cDNA using the PrimeScript™ Genomic DNA Clearance Reverse Transcription Kit (Takara, RR047A). Quantitative PCR was performed using TB Green® Premix Ex Taq™ (TliRNaseH Plus (Takara, RR420A)). The mScarlet primer was used to detect the transcription intensity of the target viral RNA, and the β-actin primer was used for normalization.

[0205] The results are as follows Figure 1 As shown in the fluorescence imaging results, at MOI=1E9, after Day 14 of infection with each virus, the fluorescence intensity of RC-V53 and RC-V62 was significantly higher than that of AAV2 and AAV2.7m8, while the fluorescence intensity of RC-V63 and RC-V68 was higher than that of AAV2 and close to that of AAV2.7m8. Figure 1 (a) Tissue RNA extraction and qPCR quantification results showed that at MOI=1E9, after Day 14 of viral infection, the mScarlet mRNA expression levels of RC-V53 and RC-V62 were higher than those of AAV2 and AAV2.7m8 (expression levels were 8.7-13 times higher than AAV2, while AAV2.7m8 was only 1.9 times higher than AAV2); the mScarlet mRNA expression level of RC-V63 was 4.1 times higher than AAV2, although not as high as RC-V53 and RC-V62, it was still higher than AAV2.7m8; while the mScarlet mRNA expression level of RC-V68 was 1.6 times higher than AAV2, slightly lower than AAV2.7m8. Figure 1 (b)

[0206] Example 4: In vivo intravitreal (IVT) injection efficacy test in animals

[0207] RC-V63 and RC-V68 were packaged into vectors containing CAG-mScarlet transgenes to enable their transduction properties to be expressed.

[0208] To evaluate the transduction efficiency of different AAV vectors in the retina, three 6- to 8-week-old C57 wild-type (WT) mice were used. Four different AAV vectors were used in the experiment: RC-V63-mScarlet, RC-V68-mScarlet, AAV2.7m8-mScarlet, and AAV2-mScarlet. These vectors were administered via intravitreal (IVT) injection at a dose of 1 × 10⁻⁶ per eye. 9 Viral genome (vg). In vivo autofluorescence (AF) and retinal section examinations were performed on mice at weeks 4 and 6 post-drug administration to assess in vivo transduction efficiency.

[0209] The results showed that the fluorescence intensity of RC-V63 and RC-V68 was significantly higher than that of AAV2.7m8 and AAV2 ( ) in vivo autofluorescence (AF) examination. Figure 2 (a)

[0210] Next, ImageJ software was used to statistically analyze the AF fluorescence intensity in both eyes of mice after 6 weeks of drug administration. The statistical results are shown in Table 1 and... Figure 2As shown in (bc), the average fluorescence intensity and total fluorescence intensity of RC-V63 and RC-V68 are significantly higher than those of AAV2.7m8 and AAV2.

[0211] Table 1

[0212]

[0213] To more accurately analyze the differences in transcriptional levels among variants, the eyes of mice treated for 6 weeks were dissected, retinal tissue was isolated, and retinal RNA was extracted and quantitatively analyzed by qPCR (extraction and identification methods were the same as for retinal organoids). Statistical results are as follows: Figure 2 As shown in Figure d, the delivery capacity of RC-V63 and RC-V68 in the mouse retina is higher than that of AAV2.7m8 and AAV2, respectively, being 22.4 times and 8.4 times that of AAV2, while AAV2.7m8 is only 2.3 times that of AAV2.

[0214] The above results indicate that, in in vivo experiments in mice, RC-V63 and RC-V68 exhibit significantly higher transduction efficiency in the retina than AAV2.7m8 and AAV2.

[0215] The inventors of this application further cryosectioned the retinas of mice that had been drugged for 6 weeks, labeled rhodopsin with green fluorescence, and labeled the cell nuclei with DAPI. The experimental results are as follows. Figure 3 As shown, RC-V63 and RC-V68 exhibited significantly enhanced expression levels and tissue penetration capabilities. These two vectors were not only highly expressed in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL), but also achieved efficient expression in the outer nuclear layer (ONL) nuclei. In contrast, the AAV2 vector could only be delivered to the GCL, and the target gene expression was weak; no efficient transduction in the INL and ONL layers was observed. While AAV2.7m8 possessed some penetration ability, its expression intensity was significantly lower than that of RC-V63 and RC-V68. These in vivo experimental data fully demonstrate that RC-V63 and RC-V68 are significantly superior to AAV2 in terms of transduction efficiency and penetration depth in retinal tissue.

[0216] AV2.7m8 and AAV2 exhibited stronger target protein expression levels and broader cell type coverage.

[0217] Example 5: Detection of anti-IVIG (human immunoglobulin) neutralizing capacity

[0218] In the cell preparation stage, 293T cells in the logarithmic growth phase were harvested, digested, and then adjusted to a cell density of 1.2 × 10⁻⁶ cells using high-glucose DMEM medium containing 10% FBS. 5 Cells / mL, add 500 μL of cell suspension (corresponding to 6 × 10⁶ cells / mL) to each well of a 48-well plate.4 Cells / well were pre-cultured at 37°C in a 5% CO2 incubator for 18 hours to achieve 30-40% confluence. Subsequently, an IVIG (Taibang Biotechnology) gradient dilution system was constructed. The IVIG to be tested was serially diluted with serum-free high-glucose DMEM at a 1:3 (v / v) ratio (100 μL of each dilution was added to 200 μL of culture medium) to generate seven gradient dilutions: 3×, 9×, 27×, 81×, 243×, 729×, and 2187×. A blank control without IVIG was also included. For virus treatment, 50 μL of serum-free high-glucose DMEM was added to each AAV virus required for MOI=2000, mixed thoroughly, and then 50 μL of each gradient IVIG dilution was added. After vortexing and mixing, the cells were incubated at 37°C for 1 hour (during which the EP tube was gently tapped every 15 minutes to promote neutralization). After neutralization, the original culture medium in the cell culture plate was aspirated, and 100 μL of virus-IVIG mixture was added to each well. A positive control (IVIG-free pure virus group) and a negative control (uninfected cell group) were simultaneously set up. After infection at 37℃ and 5% CO2 for 24 h, 500 μL of high-glucose DMEM containing 10% fetal bovine serum (FBS) was added to each well. Flow cytometry was performed 72 h after infection. Different capsid viruses are uniformly packaged as CAG-mScarlet, so the fluorescence intensity of the mScarlet can be used to represent viral infectivity. After normalization using the IVIG-free pure virus group, the inhibitory efficiency of IVIG against different viruses can be analyzed. Neutralization effect is represented by ID... 50 This indicates the concentration of neutralizing antibodies corresponding to a 50% reduction in viral activity, ID 50 The lower the value, the higher the concentration of neutralizing antibodies required, meaning a stronger ability to neutralize the virus. The test results are shown in Table 2 and... Figure 4 As shown, the results of the anti-IVIG neutralization ability are as follows: the inhibitory effect of IVIG from largest to smallest is: RC-V62, AAV2.7m8, RC-V68, AAV2, RC-V63, RC-V53, that is, the anti-IVIG neutralization ability of each variant is: RC-V53>RC-V63>AAV2>RC-V68>AAV2.7m8>RC-V62.

[0219] Table 2

[0220]

[0221] Note: ID 50 This refers to a 50% infection dose.

[0222] Furthermore, the inventors packaged AAV2, AAV2.7m8, and RC-V53 / V62 / V63 / V68 separately, with each virus's genome carrying a specific DNA barcode for subsequent analysis. The six viruses were mixed at the same titer and injected into the eyes of cynomolgus monkeys at 1.1E11 vg / eye. Twenty-three days after injection, retinal samples were collected, and RNA was recovered, reverse transcribed, and then sequenced. The DNA barcode content of different variants was compared, allowing for a side-by-side comparison to obtain the corresponding percentages and rankings. The results are shown in Table 3 below.

[0223] Table 3

[0224]

[0225] In cynomolgus monkeys, the percentage of DNA barcode content of different variants, from largest to smallest, is: V53>V63>V62>AAV2.7m8>AAV2>V68. That is, the transcription level of the target gene of the RC-V53, RC-V62, and RC-V63 variants is much higher than that of AAV2, and higher than that of AAV2.7m8. This indicates that the AAV2 variant provided by this invention has the following three advantages: (1) It has a high efficiency in gene delivery: Compared with AAV2 or AAV2.7m8, it significantly improves the transcription level of the target gene in the primate retina (which is closer to the physiological structure of humans), and can achieve more efficient and stable gene expression. (2) Excellent animal adaptability and clinical application prospects: Non-human primates, represented by cynomolgus monkeys, are advanced animal models for the development of retinal-related diseases and gene therapy vectors. The high transduction performance of the neocapsids of RC-V53, RC-V62, and RC-V63 indicates that their potential for treating human retinal diseases in future clinical translation is superior to that of existing mainstream vectors. (3) Reduced risk of dose-related side effects: Improved expression efficiency means that the dose of AAV viral vector required to achieve the same therapeutic effect can be significantly reduced, which helps to reduce potential immune responses and toxic side effects and improve the safety margin of treatment.

[0226] The following is some of the sequence information involved in this invention:

[0227] SEQ ID NO.1: (VP1 amino acid sequence of RC-V53)

[0228] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRDPPEQRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0229] SEQ ID NO.2: (Amino acid sequence of VP1 of RC-V62)

[0230] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRETPDTRPSRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0231] SEQ ID NO.3: (Amino acid sequence of VP1 of RC-V63)

[0232] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRMGQPPSQARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0233] SEQ ID NO.4: (Amino acid sequence of VP1 of RC-V68)

[0234] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNREDPNRRREVQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0235] SEQ ID NO.5: (Amino acid sequence of VP1 of AAV2.7m8)

[0236] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASH

[0237] SEQ ID NO.6: (Amino acid sequence of VP1 of AAV2)

[0238] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0239] SEQ ID NO.7: (VP1 nucleic acid sequence of RC-V53)

[0240]

[0241] SEQ ID NO.8: (VP1 nucleic acid sequence of RC-V62)

[0242]

[0243] SEQ ID NO.9: (VP1 nucleic acid sequence of RC-V63)

[0244]

[0245] SEQ ID NO.10: (VP1 nucleic acid sequence of RC-V68)

[0246]

[0247] SEQ ID NO.11: (AAV2.7m8 nucleic acid sequence)

[0248]

[0249] SEQ ID NO.12: (VP1 nucleic acid sequence of AAV2)

[0250]

[0251] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An adeno-associated virus (AAV) capsid protein variant, characterized in that, the VP1 of the AAV capsid protein variant has an insertion peptide relative to the VP1 amino acid sequence of the parent AAV capsid protein, the insertion peptide has a length of 9 amino acids, and its amino acid sequence differs from the sequence shown in any one of SEQ ID NOs: 29-32 by no more than 1 amino acid; and the amino acid sequence of the AAV capsid protein variant is as shown in any one of SEQ ID NOs: 1-4.

2. The AAV capsid protein variant of claim 1, wherein, the amino acid sequence of the AAV capsid protein variant is as shown in any one of SEQ ID NOs: 1-3.

3. The AAV capsid protein variant of claim 1, wherein, the amino acid sequence of the AAV capsid protein variant is as shown in SEQ ID NO: 1 or 2.

4. An isolated polynucleotide, comprising, the polynucleotide encodes the AAV capsid protein variant of claim 1.

5. A vector, characterized in that, the vector contains the polynucleotide of claim 4.

6. A host cell, characterized in that, the host cell contains the vector of claim 5, or the polynucleotide of claim 4 is integrated into the genome of the host cell.

7. A recombinant adeno-associated virus (rAAV) particle, characterized in that, the rAAV particle comprises: (i) the AAV capsid protein variant of claim 1; (ii) a nucleic acid of interest packaged within the AAV capsid.

8. A method of making the rAAV particle of claim 7, wherein, comprising the step of culturing the host cell of claim 6 under suitable conditions, thereby obtaining the rAAV particle.

9. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises: (a) the rAAV particle of claim 7; and (b) a pharmaceutically acceptable carrier.

10. Use of the rAAV particle of claim 7, or the pharmaceutical composition of claim 9, or a combination thereof, in the manufacture of a medicament for treating a disease; the disease being an ocular disease.

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