A novel AAV variant

Abstract

Provided is a variant AAV capsid protein, comprising one or more modifications, the modification comprises an insertion of 7 amino acids at a position between the amino acid position 587 and 588 of the capsid protein of AAV, and the polynucleotide, host cells, vectors, AAV virion or the pharmaceutical composition thereof. Further provided is a method of treating disease, the method comprising administering to a subject in need thereof an effective amount of the recombinant AAV virion.

Description

Title of Invention: A NOVEL AAV VARIANT PRIORITY

[0001] This application claims the benefit of, and priority to, PCT Application No. PCT / CN2024 / 086887, filed April 9, 2024, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD

[0002] The present invention relates to a variant AAV capsid protein, especially refers to a AAV2 Capsid Variants for Intravitreal Gene Therapy. BACKGROUND OF THE INVENTION

[0003] Adeno-associated virus (AAV) belongs to the Parvoviridae family and Dependovirus genus, whose members require co-infection with a helper virus such as adenovirus to promote replication, and AAV establishes a latent infection in the absence of a helper. Virions are composed of a 25 nm icosahedral capsid encompassing a 4.7 kb single-stranded DNA genome with two open reading frames: rep and cap. The non-structural rep gene encodes four regulatory proteins essential for viral replication, whereas cap encodes three structural proteins (VP1-3) that assemble into a 60-mer capsid shell. This viral capsid mediates the ability of AAV vectors to overcome many of the biological barriers of viral transduction-including cell surface receptor binding, endocytosis, intracellular trafficking, and unpackaging in the nucleus. AAV-based gene therapies have the potential to become promising treatments for many diseases. However, low transduction efficiency in certain tissues is a technical hurdle to the clinical use of AAV-based gene therapy in many situations.

[0004] To date, there remains a need for developing AAV-mediated gene delivery systems that have enhanced tissue transduction. SUMMARY

[0005] The present invention provides a variant AAV capsid protein comprising one or more modifications, the modification comprises an inserted sequence shown as formular I at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, YX1X2X3X4X5X5X77 (formular I), wherein X is any amino acid, Y is absent, LA (n) , or A (n), Z is A (n), n is any integer from 1 to 5.

[0006] In one embodiment, the variant AAV capsid protein comprising an inserted sequence shown as formular II at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, X1X2X3X4X5X6X7 (formular II) , wherein X is any amino acid.

[0007] In one embodiment, the variant AAV capsid protein comprising an inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, the inserted sequence is shown as XiX2X3X4X5X6X7 (formular II), X, 7 is any amino acid; preferably, the inserted sequence is shown as XiX2X3PX5RN, X5 is D or E, Xb3 is any amino acid, preferably, the inserted sequence is shown as XiX2X3X4R-PX7, X14 is any amino acid, X7 is E, Q, G, or S, preferably, the inserted sequence is shown as XiX2DX4RPQ, Xb2 is any amino acid, X4 is H, or T, preferably, the inserted sequence is shown as XiTEX4RP X7> Xi or X4 is any amino acid, X7 is Q, or S, or preferably, the inserted sequence is shown as XiX2X3X4KPE, X[ 4 is any amino acid.

[0008] In one embodiment, the variant AAV capsid protein comprising an inserted sequence is selected from the group consisting of SEQ ID NOs: 1-467; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-467.

[0009] In one embodiment, the variant AAV capsid protein comprising an inserted sequence is selected from the group consisting of SEQ ID NOs: 524-558; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 524-558.

[0010] In one embodiment, the variant AAV capsid protein comprising an inserted sequence selected from the group consisting of SEQ ID NOs: 23, 33, 36, 45, 57-67; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 33, 36, 45, 57-67.

[0011] In one embodiment, the AAV is selected from the group consisting of: serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, as well as any one of the AAV serotype isolated from human and nonhuman mammalians or variant thereof.

[0012] In one embodiment, the capsid protein further comprises a modification of any one of flanking substitutions and point mutations.

[0013] In one embodiment, the capsid protein comprises a combination of modification comprising flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, the flanking substitution is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGT, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG.

[0014] In one embodiment, the capsid protein comprises a combination of modification comprising flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, the flanking substitution is selected from the substitution of GPR, QQN, KAA, GIP, RTP, RDP, WAP, TAA, GPS, LAP, QQK, RDN, GRP, RSP, SGN, RGY, QQT, QAP, or SSD.

[0015] In one embodiment, the capsid protein comprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, the inserted sequence is SEQ ID NO: 33.

[0016] In one embodiment, the capsid protein comprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, RGG, the inserted sequence is SEQ ID NO: 23.

[0017] In one embodiment, the capsid protein comprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, RGG, the inserted sequence is SEQ ID NO: 36.

[0018] In one embodiment, the capsid protein comprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of KGG, the inserted sequence is selected from SEQ ID NO: 57-67.

[0019] In one embodiment, the capsid protein comprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RCN, the inserted sequence is selected from SEQ ID NO: 57-67.

[0020] In one embodiment, the capsid protein further comprises point mutations, wherein the point mutation comprises one or more amino acid substitutions corresponding to the amino acid sequence of parental AAV, wherein the one or more amino acid substitutions is selected from Y737F, Y444F, Y500F, K556E, R595L, R595M, A597V, A597S, and Q596H.

[0021] In one embodiment, the point mutation comprises one of the following substitution or substitution combinations: (i) Y737F; (ii) Y444F, Y500F, Y737F; (iii) K556E.

[0022] In one embodiment, the capsid protein comprises a combination of modification comprising flanking substitutions of RAA at the amino acid position 585 to 587, an amino acid substitution Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 33 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0023] In one embodiment, the capsid protein comprises a combination of modification comprising flanking substitutions of RAA at the amino acid position 585 to 587, amino acid substitutions Y444F, Y500F and Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 33 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0024] In one embodiment, the capsid protein comprises a combination of modification comprising flanking substitutions of RAA at the amino acid position 585 to 587, an amino acid substitution K556E, and an insertion of the amino acid sequences as shown in SEQ ID NO: 33 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0025] In one embodiment, the capsid protein comprises a combination of modification comprising an amino acid substitution Y737F and an insertion of the amino acid sequences as shown in SEQ ID NO: 23 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0026] In one embodiment, the capsid protein comprises a combination of modification comprising amino acid substitution Y444F, Y500F, Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 23 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0027] In one embodiment, the capsid protein comprises a combination of modification comprising an amino acid substitutions Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 36 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0028] In one embodiment, the capsid protein comprises a combination of modification comprising amino acid substitution Y444F, Y500F, Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 36 between the amino acid position 587 and 588 of the parental AAV capsid protein.

[0029] The present invention provides an isolated polynucleotide comprising a nucleotide sequence that encodes above variant AAV capsid protein or vectors comprising the above polynucleotides.

[0030] The present invention provides an isolated, genetically modified host cell comprising the above polynucleotide.

[0031] The present invention provides a recombinant AAV virion comprising above variant AAV capsid protein.

[0032] In one specific embodiment, the present invention provides a pharmaceutical composition comprising: a) a recombinant adeno-associated virus virion disclosed in the present invention; and b) a pharmaceutically acceptable excipient.

[0033] In another aspect, the present invention provides a recombinant AAV vector, comprising polynucleotide encoding a variant AAV capsid protein of invention, and an AAV 5’ inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional gene product, a regulatory sequence which directs expression of the gene product in a target cell, and an AAV 3’ ITR.

[0034] In another aspect, the present invention also provides a method of delivering a nucleic acid vector encoding a functional gene product to cells and / or tissues with a recombinant AAV virion or recombinant AAV vector of the present invention.

[0035] In one specific embodiment, the present invention also provides the use of a recombinant AAV virion or recombinant AAV vector of the present invention in the preparation of product for delivering a nucleic acid vector encoding a functional gene product to cells and / or tissues.

[0036] In another aspect, the present invention also provides a method of treating disease, the method comprising administering to a subject in need thereof an effective amount of a recombinant AAV virion of present invention, the recombinant AAV virion comprises functional gene product.

[0037] In one specific embodiment, the present invention also provides the use of a recombinant AAV virion or recombinant AAV vector of the present invention in the preparation of product for treating disease to a subject in need thereof; preferably, the disease is selected from the group consisting of liver disease, central nervous system diseases, and other diseases.

[0038] In one specific embodiment, the gene product is a polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows AAV2 library screening workflow.

[0040] Figure 2 shows dissection of the eyes of the cynomolgus macaque. The retina was split into 5 parts with 3 layers. Materials for NGS are SI, S2, Nl, N2, II, 12, Tl, T2, Ml, M2, RPE. Materials for 2nd round library packaging are RPE, PR. S: superior; N: Nasal; I: inferior; T: temporal; M: macula.

[0041] Figure 3 shows motifs with high retina tropism and penetration ability.

[0042] Figure 4 shows AVT225 achieve panretinal transduction in mice retina. Vector mediated GFP expression after intravitreal injection of novel capsid variants compared with controls in 6-week-old mice. A: AVT225; B: AAV7m8; C: AAVGL; D: AAV2.

[0043] Figure 5 shows first round of NHP validation design.

[0044] Figure 6 shows in vivo fundus GFP fluorescence following intravitreal delivery of rAAV. Sc-CB-GFP vector. In vivo fundus autofluorescence examinations of the NHP retina at 3 weeks post-intravitreal injection of 4E10 total vg for each capsid packaged with sc-CB-GFP.

[0045] Figure 7 shows Immunofluorescence of retinal cross sections and flat mount after 3 WPI.

[0046] Figure 8 shows comparation of AVT capsids with AAV2 and AV2.7m8 in cynomolgus. DETAILED DESCRIPTION

[0047] The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

[0048] The articles “a” , “an” , and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0049] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or l%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.

[0050] Throughout this disclosure, unless the context requires otherwise, the words “comprise” , “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0051] As used herein, the term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as cynomolgus, nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms "patient" or "subject" are used interchangeably.

[0052] The terms "treatment" and "therapeutic method" refer to both therapeutic treatment and prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.

[0053] "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term “AAV'includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7) , AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), AAV type 12 (AAV-12), AAV type 13 (AAV-13) avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. See, e.g., Mori et al. (2004) Virology 330: 375. The term “AAV” also includes chimeric AAV. “Primate AAV" refers to AAV isolated from a primate, "non-primate AAV" refers to AAV isolated from a non-primate mammal, "bovine AAV" refers to AAV isolated from a bovine mammal (e.g., a cow), etc.

[0054] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes. "

[0055] The term "inserted sequence" is used below to describe a peptide that is inserted into a parental AAV capsid. In some cases, the insertion peptide has a length of from 7 amino acids to 20 amino acids. In some cases, the insertion peptide has a length of from 7 amino acids to 16 amino acids. In some cases, the insertion peptide has a length of from 7 amino acids to 10 amino acids. In some cases, the insertion peptide has a length of from 7 amino acids to 12 amino acids. The insertion peptide has a length of 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids. In some cases, the insertion peptide has a length of 7 amino acids. In some cases, the insertion peptide has a length of 8 amino acids. In some cases, the insertion peptide has a length of 9 amino acids. In some cases, the insertion peptide has a length of 10 amino acids. In some cases, the insertion peptide has a length of 11 amino acids. In some cases, the insertion peptide has a length of 12 amino acids. In some cases, the insertion peptide has a length of 13 amino acids. In some cases, the insertion peptide has a length of 14 amino acids. In some cases, the insertion peptide has a length of 15 amino acids. In some cases, the insertion peptide has a length of 16 amino acids. EXAMPLES

[0056] The equipments and regents

[0057] Table 1. The equipment used in the invention Equipment Product number Supplier microcentrifuge 5425R Eppendorf Tissue Lyser II QIAGEN Biosafety cabinet HF Safe 1800LC Heal Force Shaker MAXQ 436 HP Thermo Ultra centrifuge XPN-80 Beckman Centrifuge Auanti J-15R Beckman Vortex MVM-2500 Titan High speed centrifuge LYNX6000 Thermo CO2 incubator 371 Thermo Electron microscope T2 Nikon PCR Pro FlexTM Base Applied Biosystems QX200 Droplet Digital PCR QX200 Droplet Digital Bio-Rad PCR plat sealer PX1 Bio-Rad qPCR CFX96 Bio-Rad Nanodrop One 840-317400- Thermo ChemiDoc MP 12003154 Bio-Rad

[0058] Table 2. The regents and supplies used in the study Reagents Supplies Product Number Cell culture 0.25%Tiypsin-EDTA Gibco 25200-072 293T National Collection of Authenticated Cell SCSP-502 FBS Transgen FS301 DMEM Yeasen 41401ES76 AAV library generation PEI Yeasen 40816ES02 NaCl solution, 5 M in H2O, Bio-Reagent Sigma S5150-1L Ultra-Pure™ IM Tris-HCI, pH 8.0 Invitrogen 15568-025 Benzonase>90% (SDS-PAGE) Sigma E1014-25KU Opti-PrepTfvI Density Gradient Medium. Sigma D1556-250ML Sodium deoxycholate Sigma 30970-25G MgCJ2, 1M Sigma 63069-100ML KCI Sigma 12636-500g Phenol Red solution Sigma P0290-100ML Pluronic™ F-68 Non-ionic Surfactant (100x) Thermo 24040032 Opti-MEM Gibco 2193178 Construction of AAV plasmid Phanta Max Super-Fidelity DNA Polymerase vazyme P5O5 ClonExpress MultiS One Step Cloning vazyme C113 Kit DNA Ladder Generay DL2502 Wizard® SV Gel and PCR Clean-Up System Promega A9281 FastDigest Smil Thermo FD1244 FastDigest Pfl23II Thermo FD0854 FastDigest Eco311 Thermo FD0293 FastPure EndoFree Plasmid Mini Kit vazyme DC203-01 2T1 GeneCppoeia CC007 TaKaRa MidiBEST Endo-free Plasmid Purification Kit Takara 9783 Tissue extraction and testing DNeasy Blood & Tissue Kit QIAGEN 69506 TransZol Up plus RNAkit Transgen ER501-01 HiScript IV 1st Strand cDNA Synthesis Kit C+gDNA wiper) Vazyme R412 Premix Ex TaqTM (Probe qPCR) TAKARA RR390B ddPCR Super mix for Probes (No dUTP) Bio-Rad 1863025 TaqMan™ Copy Number Reference Assay, human, RNase P TaqMan 4403328 Copy Number Reference Assay, mice, TFRC Applied Biosystems 4458367 DistilledWater Invitrogen 10977-015 10% PluRoNic F-68 Gibco 24040-032 Anti-VSV-G tag Antibody abeam abl874 Anti-AUl Epitope Tag Antibody Bio legend 903101 Anti-Avi-Tag Antibody Cell Signaling 10643S Anti-Flag -Tag Antibody Sigma-Aldrich F3165-.2MG Anti-HA-Tag (C29F4) Rabbit mAh CST 3724S Anti-Myc-tag Antibody proteintech 60003-2-Ig mCherry (E5D8F) Rabbit mAb Cell Signaling. 43590S GFP Monoclonal Antibody (GF28R) Thermo MA5-15256 IM Tris PH 8.0 Invitrogen AM9865 SSS DNA Invitrogen AM9680

[0059] Table 3. The various oligos used in the study AAV library generation SEQ ID NO Cap2PCR-P Forward GAGACTCTCTGGTGAATCCGGGCCCGGC 468 Reverse AACGCCTTGTGlGTTGACATCTGCGGTAGCrGCTTGTCTMNNMNNMNNMN NMNNMNNMNNGTTGCCTCTCTGGAGGTTGGTAGATAC 469 Flanking substitution AVT225 Forward GAGACTCTCTGGTGAATCCGGGCCCGGC 468 Reverse ITGACATCTGCGGTAGCTGCTTGTCTATTCTTCGAAGCATTATCAACMNNM NNMNNCTGGAGGTTGGTAGATACAG 470 AVT235; Forward GAGACTCTCTGGTGAATCCGGGCCCGGC 468 Reverse ITGACATCTGCGGTAGCTGCTTGTCTACCAGGACGCATATCCTGCTGMNNM NNMNNCTGGAGGTTGGTAGATACAG 471 AVT236 Forward GAGACTCTCTGGTGAATCCGGGCCCGGC 468 Reverse TTGACATCTGCGGTAGCTGCTTGTCTCTGCTTCTGAGGATCAGTAGAMNNM NNMNNCTGGAGGTTGGTAGATACAG 472 AVT237 Forward GAGACTCTCTGGTGAATCCGGGCCt GGC 468 Reverse TTGACATCTGCGGTAGCTGCTTGTCTATTCCTATCAGGCTGAATCTGMNNM NNMNNCTGGAGGTTGGTAGATACAG 473 AVT238 Forward GAGACTCTCTGGTGAATCCGGCjCCCGGC 468 Reverse TTGACATCTGCGGTAGCTGCTTGTCTATTCCTCTCAACAATCATAAGMNNM NNMNNCTGGAGGTTGGTAGATACAG 474 AVT239 Forward GAGACTCTCTGGTGAAR ( < it iGC X CGGC 468 Reverse TTGACATCTGCGGTAGCTGCTTGTCTCTCAGGCTTATGAATATTAATMNNMN NMNNCTGGAGGTTGGTAGATACAG 475 Construction of AAV plasmids AAV2 AAV2F ACTGACTCGGAGTACCAGCTCCC 476 AAV2R ATTGATTAACAAGCAAACTAGT 477 pAVT225 Overlap F AGAGGCAACGTTGATAATGCTTCGAAGAATAGACAAGCAGCTACCGCAGA TG 478 Overlap R TTGTCTATTCTTCGAAGCATTATCAACGTTGCCTCTCTGGAGGTTGGTAGAT ACAG 479 pAVT235 Overlap F AGAGGCAACCAGCAGGATATGCGTCCTGGTAGACAAGCAGCTACCGCAGA TG 480 Overlap R TTGTCTACCAGGACGCATATCCTGCTGGTTGCCTCTCTGGAGGTTGGTAGAT ACAG 481 pAVT239 Overlap F agaggcaacattaatattcataagcctgagagacaagcagctaccgcagat G 482 Overlap R ttgtctctcaggcttatgaatattaatgttgcctctctggaggttggtagat ACAG 483 pAVT242 Overlap F agaggcaacaagcagggtgttactcaggctagacaagcagctaccgcaga TG 484 Overlap R ttgtctagcctgagtaacaccctgcttgttgcctctctggaggttggtagat ACAG 485 pAVT248 Overlap F agaggcaacattattcagcctgataggaatagacaagcagctaccgcagat G 486 Overlap R ttgtctattcctatcaggctgaataatgttgcctctctggaggttggtagat ACAG 487 pAVT250 Overlap F agaggcaacactacgaatttggctaaggatagacaagcagctaccgcaga TG 488 Overlap R ttgtctatccttagccaaattcgtagtgttgcctctctggaggttggtagat ACAG 489 pAVT260 Overlap F cgggctgcgcagcaggatatgcgtcctggtagacaagcagctaccgcaga TG 490 Overlap R ttgtctaccaggacgcatatcctgcitgcgcagcccgctggaggttggtaga tacag 491 pAVT261 Overlap F AAGCtGGGGTCTGCAGGATATGCGTCCTGGTAGACAAGCAGCTACCGCAGA tgt 492 Overlap R ttgtctaccaggacgcatatcctgcagaccccccttctggaggttggtaga TACAG 493 pAVT262 Overlap F agaggcaacctggccatcgtggtgggtaccaagatggccagacaagcagc taccgcagatg 494 Overlap R ttgtctggccatcttggtacccaccacgatggccaggttgcctctctggag gttggtagatacag 495 pAVT263 Overlap F agaggcaacgctattcagtctgatcggaatagacaagcagctaccgcagat G 496 Overlap R ttgtctattccgatcagactgaatagcgttgcctctctggaggttggtagat ACAG 497 pAVT264 Overlap F AGAGGCAACGCAGCTGCAAATTTGATGCCTGGGACAAGGGCTGCTAGACA AGCAGCTACCGCAGATG 498 Overlap R TTGTCTAGCAGCCCTTGTCCCAGGCATCAAATTTGCAGCTGCGTTGCCTCT CTGGAGGTTGGTAGATACAG 499 pAVT265 Overlap F AGGGCGCCGCAGCAGGATATGCGTCCTGGTAGACAAGCAGCTACCGCAGA TG 500 Overlap R TTGTCTACCAGGACGCATATCCTGCTGCGGCGCCCTCTGGAGGTTGGTAGA TACAG 501 Titering and Mixing of AAV Capsid Library ITR Forward GGA ACC CCT AGT GAT GGA GTT 502 Reverse CGG CCT CAG TGA GCG A 503 Probe / 5 ’FAM / CACTC CCT C / ZEN / T CTG CGC GCT CG / 3TBFQ / 504 Note: M represents A or C; N represents A, C, G, or T; K represents G or T. Example 1: Variants generation 1.1 AAV library generation

[0060] The DNA fragment libraries were generated by PCR with AAV2 Rep-Cap plasmid DNA template. The primer is Cap2PCR.

[0061] The purified PCR products were assembled into the inverted terminal repeat (ITR) -containing library backbone with GRK1 promoter, p41, AAV2 (Cap2) VP1 and bovine growth hormone (bGH) poly A. AAV2 (Cap2) VP1 Amino acids sequence (SEQ ID NO: 508) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERIIKDDSRGLVLPGYKYLGPFNGLDK GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVKQA KKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNW GFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQS LDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTS ADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIEGKQGSEKTNVDI EKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQ GPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVS VEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

[0062] The p41 promoter was used to drive capsid gene expression for AAV library production only in the presence of Ad5 helper gene products. The GRK1 promoter was aimed to recover Cap2 mRNA expressed in retina tissues. The assembled library plasmid DNA was transformed to the competent cells to amplify. The amplified cap2 library plasmids were used for AAV library production by co-transfecting pHelper and pRep2-AAP in HEK293 cells as follows:

[0063] HEK293T cells were purchased from National Collection of Authenticated Cell (SCSP-502). HEK293 was thawed rapidly by gentle agitation in a 37°C water bath for 2 minutes. Thawed cells were added to pre-warmed media, centrifuged at low speed to remove freeze media and subsequently resuspended in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) , supplemented with Fetal Bovine Serum (FBS) to a final concentration of 10%. Then the cells were grown in a humidified incubator at 37°C, supplemented with 5%CO2, growth media should be replaced every 2-3 days. For cell transfection: a) Inoculating cells: 24 hours before transfection, the cell plating was performed, and the cell state was observed before transfection. When the cell density was 70%-80%and in good condition without pollution, it could be transfected; b) Configuration plasmid-PEI transfection complex: plasmid: PEI=1: 1, the Helper plasmid, Rep-cap plasmid and GOI plasmid were added to each 150 dish at 58 qg of the total amount of plasmid, and the transfection complex was prepared and left for 15 min; c) Transfection was performed with transfection complex: medium with serum = 1: 10, and transfection was completed by dropping the transfection complex into 150 mm dish; d) cultured for 72 hours, virus was harvested.

[0064] Table 4. sequence information Name Nucleotide SEQ ID NO GRK1 promoter gggccccagaagcctggtggttgtttgtccttctcaggggaaaagtgaggcggccccttggaggaaggggccgggcagaatgatctaa tcggattccaagcagctcaggggattgtctttttctagcaccttcttgccactcctaagcgtcctccgtgaccccggctgggatttagcctgg tgctgtgtcagccccgggctcccaggggcttcccagtggtccccaggaaccctcgacagggccagggcgtctctctcgtccagcaagg gcagggacgggcsacaggccaagggc 505 p41 promoter tgttcaaatttgaactgactaagcggcteccgccagattttggcaagattactaagcaggaagtcaaggacttttttgcttgggcaaaggtc aatcaggtgccggtgactcaegagtttaaagttccCagggaattggcgggaactaaaggggcggagaaatctctaaaacgcccactgg gtgacgtcaccaatactagctataaaagtctggagaagcgggccaggctctcagttgttcccgagacgcctcgcagttcagacgtgact gttgatcccgctcctctgcgaccgct 506 AAV2 (Cap2) VP1-NT atggctgccgatggttatcttccagattggctcgaggacactctctctgaaggaataagacagtggtggaagctcaaacctggcccacca ccaccaaagcccgcagagcggcataaggacgacagcaggggtcttgtgcttcctgggtacaagtacctcggacccttcaacggactcg acaagggagagccggtcaacgaggcagacgccgcggccctcgagcacgacaaagcctacgaccggcagctcgacagcggagac aacccgtacctcaagtacaaccacgccgacgcggagtttcaggagcgccttaaagaagatacgtcttttgggggcaacctcggacgag cagtcttccaggcgaaaaagagggttcttgaacctctgggcctggttgaggaacctgttaagacggctccgggaaaaaagaggccggt agagcactctcctgtggagccagactcctcctcgggaaecggaaaggcgggccagcagcctgcaagaaaaagattgaattttggtcag 507 actggagacgcagactcagtacctgacccccagcctctcggacagccaccagcagccccctctggtctgggaactaatacgatggcta caggcagtggcgcaccaatggcagacaataacgagggcgccgacggagtgggtaattcctcgggaaattggcattgcgattccacat ggatgggcgacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaacaaccacctctacaaacaaatttccagcca atcaggagcctcgaacgacaatcactactttggctacagcaccccttgggggtattttgacttcaacagattccactgccacttttcau’cac gtgaclggcaaagactcatcaacaacaactggggattccgacccaagagai:tcaactt(:aagctctttaacattcaagtcaaagagglca cgcagaatgacggtacgacgafgattgfcaataaccttaccagcacggtti-aggtgmactgactfggagtacragcK:ccgtacgtci: tfggcfcggcgc-atcaaggatgcctCL’cgccgttcccagc-agacgtcttcalggtgccacagtatggatacctcaccctgaacaaeggg agtcaggcagtaggacgftcttcattttactgcctggagtactttcfttctcagatgctgcgtaccggaaacaactttaocttc’agctacactt ttgaggacgttcctttccacagcagctacgctcacagccagagtctggaccgtctcatgaatcctctcatcgaccagtaccrgtattacttga gcagaacaaacactccaagtggaaccaccacgcagtcaaggcttcagtttrctcaggccggagcgagtgacattcgggaccagtctag gaactggcttcctggaccctgttaccgccagcagcgagtatcaaagacatctgcggataacaacaacagtgaatactcgtggactggag ctaccaagtaccacctcaatggcagagactctctggtgaatccgggcccggccatggcaagccaeaaggacgatgaagaaaagtttttt cctcagagcggggttctcatetttgggaagcaaggctcagagaaaacaaatgtggacattgaaaaggtcatgattacagacgaagagga aatcaggacaaccaatcccgtggctacggagcagtatggttctgtatctaccaacctccagagaggcaacagacaagcagctaccgca gatgteaacacacaaggcgttcttccaggcatggtctggcaggacagagatgtgtaccttcaggggcccatctgggcaaagattccaea cacggacggacattttcacccctctcccctcatgggtggattcggacttaaacaccctcctccacagattctcatcaagaacaccccggta cctgcgaatccttcgaccaccttcagtgcggcaaagtttgcttccttcatcacacagtactccacgggacaggtcagcgtggagatcgagt gggagctgcagaaggaaaacagcaaacgctggaatcccgaaatteagtacacttccaactacaacaagtctgttaatgtggactttactg tggacactaatggcgtgtattcagagcctcgccccattggcaccagatacctgactcgtaatctgtaa AAV2 (Cap2) VP1-AA MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERIIKDDSRGLVLPGYKYLGPF NGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARK RLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSS GNWHCDSTWMGDRV1TTSTRTWALPTYNNHLYKQ1SSQSGASNDNHYFGYSTPWGY FDFNRFHCHFSPRDWQRL1NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTT1ANNLTST VQVFTDSEYQLPYYTGSAHQGCLPPFPADYTMATQYGYLTLNNGSQAVGRSSFYCLE YFPSQMLRTGNNFTFSYTFEDVPFHSSYAElSQSLDRLMNPLIDQYLYYLSRTNTPSGrr TQSRLQFSQAGASDIRIXJSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNG RDSLWPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPV ATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG HFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL* 508 bGH poly A ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataa aatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattggga agacaatagcaggcatgctgggga 509 pRep2-AAP ggcaaaaagtttagceatttttttccaga'tttgttcaacaggccaaccattacgttggtoatcaaaatcattcgcatcaaocaaaccgttattca: ttcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaa caCtgccagcgcatcaacaatatttteacctgaatcaggatattettctaatacctggaatgctgttHtccggggatcgcagtggtgagtaa ccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaa catcattggcaacgctacctttgccatgtttcagaaacaaetetggcgcatcgggettcccatacaagcgatagattgtcgcacctgattgc ccgacattategcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttccegttgaatatggctc atactcttcctttttcaatattattgaagcatttatcagggttattgtetcatgagcggatacatatttgaalgtatttagaaaaataaaeaaatag gggttcogcgcacatttocccgaaaagtgecacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattt tttaaCcaataggccgaaateggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaaga gtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaa tcaagttttttggggtegaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggc gaaogtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcaugctgcgcgraac caccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtg cgggcctcttegctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggtttteccagtcacg acgttgtaaaacgacggwagtgagcgcgcgtaatacgaftcaotatagggcgaattgggtaccgggcccfCcetcgatL’gaggtcga cggtategggggagctcgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaTgccggggtttTacgagattgtgatta aggtccccagcgaccttgacgagcatctgcccggcatttctgacagctttgtgaaclgggtggccgagaaggaatgggagttgccgcca gattctgacatggatctgaatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcgactttctgacggaatggcgccg tgtgagtaaggccccggaggcccttttctttgtgcaatttgagaagggagagagctacnccacatgcacgtgctcgtggaaaccaccgg ggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaactgattcagagaatttaccgcgggatcgagccgactttgcca 510 aactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattacttgctcc ccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtttgaatctcacggagcgtaaacggttgg tggcgcagcatctgacgcacgtgtcgeagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatc aaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccaggaggacca ggectcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgac taaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggta cgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaac taccgggaagaccaacatcgcggaggCcatagcccaQactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttca acgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagteggccaaagccattctcggag gaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtg cgccgtgattgacgggaactcaacgaccttqgaacaccagcagccgrtgcaagaccggatgttcaaatttgaactcacccgccgKitgg atcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattct acgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcg cagccatcgacgtcagacgcggaagctfcgatcaactacgcagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgat gctgtttccctgcagacaatgcgagagaatgaateagaattcaaatatctgcttcacteacggacagaaagactgtttagagtgctttcccg tgtcagaatctcaacccgtttctgtcgtcaaaaaggegtatcagaaactgtgctacattcatcatatcatgggaaaggtgccagacgcttgc actgcctgcgatctggtcaatgtggatttggatgaclgcatctttgaacaataaatgatttaaatcaggtatggctgccgatggttaacttcca gattgactcgaggacactetctctgaaggaataagacaglggtggaagctcaaacctggcccaccaccaccaaagcccgcagagcgg cataaggacgacagcaggggtettgtgcttcctgggracaagtacctcggacccttcaacggactcgacaagggagagccggtcaacg aggcagacgccgcggccctcgagcacgacaaagcctacgaccggcagctcgacagcggagacaacccgtacctcaagtacaacca cgccgacgcggagtttcaggagcgccttaaagaagatacgtcttttgggggcaaccteggacgagcagtcttccaggcgaaaaagag ggttcttgaacctctgggcctggttgaggaacctgttaagacggctccgggaaaatagaggccggtagagtagtctcctgtggagccag actcctcctcgggaaccggaaaggcgggccagcagcctgcaagaaaaagattgaattttggtcagactggagacgcagactcagtacc tgacccccagcctetcggacagccaccagcagccccdctggtctgggaactaatacgatggctacaggcagtggcgcaccaatggc agacaataactagggcgccgacggagtgggtaattcctcgggaaattggcattgcgattccacatggatgggcgacagagtcatcacc accagcacccgaacctgggccctgcccacctacaacaaccacctctacaaacaaatttccagccaatcaggagcctcgaacgacaatc actactttggctacagcaccccttgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcaaagactcatcaaca acaaCtggggattccgacecaagagactcaacttcaagctctttaaCattcaagtcaaagaggtcacgcagaatgaCggtacgacgacg attgccaataaccttaccagcacggtteaggtgtttactgactcggagtaccagcteccgtacgtectcggcfcggcgcateaaggatgcc tcccgccgttcccagcagacgtcttcatggtgccacagtatggatacctcaccctgaacaacgggagtcaggcagtaggacgctcttcat tttactgcctggagtactttccttctcagatgctgcgtaccggaaacaactttaccttcagctacacttttgaggacgttcctttccacagcagc tacgcteacagccagagtctggaccgtcteatgaatoctctcatcgaccagtacctgtattacttgagcagaacaaacgcagatgteaaca cacaaggcgttcttccaggcatggtctggcaggacagagatgtgtaccttcaggggcccatetgggcaaagattccacacacggacgg acattttcaeccctctcccctcatgggtggattcggacttaaacaccctcctecacagattctcatcaagaacaccccggtacctgcgaatc cttcgawacctteagtgcggcaaagtttgcttccttcaTcai.'acagtactccacgggacaggt'.'agcgtggagatcgagtgggagctgt' agaaggaaaacagcaaacgctggaatcccgaaattcagtacacttccaactacaacaagtctgttaaigtggactttactgtggacactaa tggcgtgtattcagagcctegccvcattggcacx'agatacctgactcgtaatetgtaaacvggttgattcgtttcagttgaactttggtctetg cgaagggc'gaattcgtttaaacctgi.'aaggaaccartagtgatggagttggccacbxctctetgi.'gcgctcgctogcteactgaggctg caggactagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggteacgctgggtatttaagcccgagtga gcacgcagggtctctattttgaagcgggaggtttgaaugcgcagvcgttaagccgaattctgcagatatccatcaccactggcggccg ctcgactagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcata gctgtttectgtgtgaaattgttatecgctcacaattecacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatga gtgagctaaclcacattaattgcgttgcgcteactgcctgctttocagtcgggaaacctgtcgtgccagctgcattaatgaateggccaai; gcgCggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgCtgcgctcggtegtteggctgcggcgagcg gratcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaa aaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcateacaaaaatcgacgcteaa gtcagaggtggcgaaacccgacaggactataaagataccaggcgtttecccctggaagctecctcgtgcgctetcctgttccgaccctg ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtc gttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccg gtaagacacgacttategccactggcagcagCcactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttettgaa gtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagc tcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaa gatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatettca cctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttagaaaaactcaicgagcarc aaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagt tccataggatggcaagatcetggtatcggtctgCgattccgactcgtccaacateaatacaacctattaatttcccctcgtcaaaaataaggt tatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccag ccattacgctcgtcatcaaaatcactcgcatcaaccaa CMV enhancer cgttacataacttaGggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgaegtatgttcccat-agta acgccaatagggactttccattgacg^tcaatgggtggagtatttacggtaaaCtgcccacttggcagtaCatcaagtgtatcatatgccaag tacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtaca tctac 511 CB promoter cfacgttctgcttcactctcccfatctcccccccctccfcacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggfggg ggggBggggggggcgcgcgcraggcggggcggggcggggcgaggggcggggcggggcgaggcggagJggtgcggcggca gecaatcagagcggLgcgctccgaaagtttecmtatggcgaggcggi:ggcggcggcggccctataaaaagcgaagcgcgi.ggcg ggcgggag 512 Sv40 intron gtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttcta g 513 FXN atgtggactttcggacgccgcgcagttgccggcctcctggcgtccccgagcccggcccaggcccagaccctcacccgggccccgcg gctggcagagttggcccagctctgcagccgccggggcctgcgcaccggcatcaatgcgacctgcacaacccaccacaccagttcgaa cctccgtggcctcaaccagattcggaatgtcaaaaggcagagtgtctacttgatgaatttgaggaaatcgggaactttgggccacccagg ctctctagatgacaccacctatgaaagactagcagaggaaacgctggactctttagcagagttttttgaagaccttgcagacaagccatac acctttgaggactatgatgtttcctttgggagtggtgtcttaactgttaaactgggtggagatctaggaacctacgtgateaacaageagac gccaaacaagcaaatctggttatcttctccatccagtggacccaagegttatgactggactgggaaaaactgggtgtattcccacgacgg cgtttccctccatgagctgctgggcgcagagctcactaaagccttaaaaaccaaactggacttgtcttccttggcctattccggaaaagac get 514 VSV tag tatacagacatagagatgaaccgacttggaaag 515 A VT tag ggcctgaacgacatcttcgaggctcagaaaatcgaatggcacgaa 516 AU tag gatacctatcgttatatt 517 HA tag tacccatacgatgttccagattacgct 518 Flag tag gactacaaagacgatgacgacaag 519 Myo tag gaacaaaaactcatctcagaagaggatctg 520 GFP atgagcaagggcgaggaactgttcactggcgtggtcccaattctcgtggaactggatggcgatgtgaatgggcacaaattttctgtcagc ggagagggtgaaggtgatgccacatacggaaagctcaccetgaaattcatctgcafcactggaaagctecctgtgccatggecaacact ggteactaccctgacctatggcgtgcagtgcttttccagatacccagaceatatgaagcagcatgactttttcaagagcgccatgcccgag ggctatgtgcaggagagaaccatetttttcaaagatgacgggaactacaagacccgcgctgaagtcaagttcgaaggtgacaccctggt gaatagaatcgagetgaagggcattgactttaaggaggatggaaacattctcggccacaagctggaatacaactataactcecacaatgt gtacateatggccgacaagcaaaagaatggcatcaaggtcaacttcaagatcagacacaacattgaggatggatccgtgcagctggcc gaccattatcaacagaacactccaatcggcgacggccctgtgctcctcccagacaaccattacctgtccacccagtctgccctgtctaaa gatcccaacgaaaagagagaccacatggtcctgctggagtttgtgaccgctgctgggatcacacatggcatggacgagctgtacaagt ga 521 mCherry atggtgagcaagggegaggaggataaeatggceatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggc cacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtg;gc cccctgcccttcgcctgggacatcctgtccccteagttcatgtacggctccaaggcctacgtgaagcaccecgccgacatccccgactac ttgaagctgtccttccccgagggctteaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcct ccctgcaggacggcgagttcatetacaaggtgaagctgcgcggcaccaacttcccctccgaqggccccgtaatgcagaagaagacca tgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaagga cggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagetgcccggcgcctacaacgtcaacatca agttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactceaccggcggcatg gacgagctgtacaagtag 522 RPE65 promoter attctctccaagatccaacaaaagtgattataccccccaaaatatgatggtagtatcttatactaccatcattttataggcatagggctcttagc tgcaaataatggaactaactetaataaagcagaacgcaaatattgtaaatattagagagctaacaatctetgggatggctaaaggatggag cttggaggctafccagccagtaacaatattccgggctecaetgttgaatggagacactacaaftgecttggatgggcagagatattatgg atgctaagccccaggtgctaccattaggacttctaccactgtectaacgggtggagcccatcacatgcctatgccctcaetgtaaggaaat gaagctactgttgtatatcttgggaagcacttggattaattgttatacagttttgttgaagaagaccectagggtaagtagccataactgcaca ctaaatttaaaattgttaatgagtttctcaaaaaaaatgttaaggttgttagctggtatagtatatatcttgcctgttttccaaggacttctttgggc agtaecttgtctgtgctggcaagcaactgagacttaatgaaagagtattggagatatgaatgaattgatgctgtatactctcagagtgccaa aCatataCcaatggacaagaaggtgaggCagagagcagacaggcattagtgacaagcaaagatatgeagaatttcattetcagcaaatc aaaagtcctcaacctggttggaagaatattggcactgaatggtatcaataaggttgctagagagggttagaggtgcacaatgtgcttccat aacattttatacttctccaatcttagcactaatcaaacatggttgaatactttgtttactataactcttaeagagttataagatctgtgaagacagg 523 gacagggacaatacccatctctgtctggttcataggtggtatgtaatagatatttttaaaaataagtgagttaatgaatgagggtgagaatga aggcacagaggtattagggggaggtgggccccagagaatggtgccaaggtccagtggggtgactgggatcagcteaggcctgacgc tggccactCccacctagctcctttctttctaatCtgttctcattGtCGttgggaaggattgaggtetctggaaaacagcGaaacaa^ ggaacagcaagcccaaataaagccaagcatcagggggatctgagagctgaaagcaacttctgttccccctccctcagctgaaggggtg gggaagggctcccaaagccataactccttttaagggatttagaaggcataaaaaggcccctggGtgagaacttccttcttcattctgcagtt ggtaatc 1.2 Screening in vivo

[0065] AAV preparations are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate for in vivo studies. Preparations are then aliquoted and stored at -80 °C. AAV particles are titered by droplet digital PCR (ddPCR) using primers and probes targeting the ITR elements and an internal control of known titer (protocol modified from Lock et al., 2014.) The endotoxin levels are determined using a chromogenic endotoxin detection assay based on the amebocyte lysate method. Then the purified AAV viral libraries (First round random library and 2nd-3rd round recovery library) were intravitreal injected to 3-6 years old Cynomolgus macaque at 3Ellvg / eye. Twenty-one days after injection, monkeys were euthanized and eyeballs were collected for separating retinal sublayers after necropsy to evaluate the level of transgene product. The separation followed the steps below:

[0066] Washed a sterile petri dish with chilled physiological saline twice, then placed on the wet ice. Placed the eyeball on the chilled glass petri. Removed the anterior pole of eye from the position of ciliary body, then cut the optic cup into several parts (superior, nasal, inferior, temporal and macula), immersed the retina with sclera in a trypsin solution PH 7.2 (0.1%trypsin (1: 250) and 10 mM HEPES in a Ca27Mg2+ -free Hank’s solution) for 10 minutes, at 37°C in a CO2 incubator (95%air / 5%CO2). After the immersion, the trypsin solution was removed quickly with a Pasteur pipette and the dish was wiped dry with sterilized filter paper.

[0067] The procedures of separation retina were followed: 1. The surface of the flatted retina was covered gently with sterilized Millipore filter. During this step, prevented air from entering between the tissue and Millipore filter. 2. Removed the entire retina including the Millipore covering from choroid with RPE and sclera, the retina with retina with Millipore filter and choroid with RPE and sclera was placed in two petri dishes. 3. Turned over the retina with Millipore filter so that the outer nuclear layer faced upwards, covered the outer nuclear layer with filter paper, then separated the outer nuclear layer by gently removing the filter paper. The outer nuclear layer and remaining retinal tissue attached with the filter paper was transferred into the labeled tubes and frozen in dry ice, then stored in a freezer set to maintain <-60°C until shipped. 4. Covered the choroid with RPE and sclera with Millipore filter, then separated RPE by gently removing the filter paper. RPE with Millipore filter was transferred into the labeled tubes and frozen in dry ice, then stored in a freezer set to maintain <-60°C until shipped. The total RNA was isolated and treated with Dnase I. The cap2 specific mRNA was reverse transcribed and amplified by PCR (RT-PCR) for next-generation sequencing (NGS). The enriched candidates were selected by customized enrichment score analysis. A total of 2035 candidates including variants predicted by artificial intelligence modeling were synthesized for the second round of screening. Meanwhile, 58 top variants were individually packaged for in vitro and in vivo characterization. These top variants were selected by NSG enrichment score. The top variant candidates selected for validation are shown in Table 5.

[0068] Table 5. Top variant candidates selected for validation. Capsid name Amino acids sequence of an insertion between the amino acid position 587 and position 588 SEQ ID NO pAVT202 KNPSIND 1 pAVT203 VNDSQRN 1 pAVT204 TSNPDRN 3 pAVT205 YDNTRTQ 4 pAVT206 PDNYTKQ 5 pAVT207 RNTTRDE 6 pAVT208 SYDTTRA 7 pAVT209 ENTPNRN 8 pAVT210 ENNVTKN 9 pAVT211 KNHSIND 10 pAVT212 KNPSINA 11 pAVT213 TDNPNRN 12 pAVT214 ITQMKHE 13 pAVT215 QIQPDRN 14 pAVT216 PNETKPP 15 pAVT217 PNDQSRT 16 pAVT218 PNDQSRT 16 pAVT219 SYDANKN 17 pAVT220 SHSNATN 18 pAVT221 NPLTNRD 19 pAVT222 HVHNDRG 20 pAVT223 HDTDRPQ 21 pAVT224 DSKHNQN 22 pAVT225 VDNASKN 23 pAVT226 ENPSIND 24 pAVT227 TTNTHHQ 25 pAVT228 LAVDNASKNA 26 pAVT229 LAENPSINDA 27 pAVT230 LATTNTHHQA 28 pAVT231 SVDTTRN 29 pAVT232 LASVDTTRNA 30 pAVT233 AAASVDTTRNAA 31 pAVT234 LALSNNTRAA 32 pAVT235 QQDMRPG 33 pAVT236 STDPQKQ 34 pAVT237 QIQPDRN 14 pAVT238 LMIVERN 35 pAVT239 INIHKPE 36 pAVT240 SDNSRVG 37 pAVT241 NVDHTKQ 38 pAVT242 KQGVTQA 39 pAVT243 IPNPERN 40 pAVT244 QINVARD 41 pAVT245 MIQPDRN 42 pAVT246 PNQPDRN 43 pAVT247 LPNPERN 44 pAVT248 IIQPDRN 45 PAVT249 ATQQERN 46 pAVT250 TTNLAKD 47 pAVT251 LTNPDRN 48 pAVT252 INPHRTD 49 pAVT253 VSTPERN 50 pAVT254 AVMQDRN 51 pAVT255 RSHDRNE 52 pAVT256 RPYDNKD 53 pAVT257 KEHRPND 54 pAVT258 LYDTNKN 55 pAVT259 SMDVSRN 56 1.3 Flanking sequence optimization and point mutations

[0069] Six candidates were selected for flanking optimization at insertion position (R585G586N587). Random primers were used for substitution of RGN to random amino acids.

[0070] Table 6. Random primers were used for substitution of RGN to random amino acids. Capsid name Amino acids sequence of an insertion between the amino acid position 587 and position 588 SEQ ID NO AVT225 VDNASKN 23 AVT235 QQDMRPG 33 AVT236 STDPQKQ 34 AVT237 QIQPDRN 14 AVT238 LMIVERN 35 AVT239 INIHKPE 36

[0071] Library assemble, in vivo selection and NGS was the same as descripted before. After NGS, another 16 top variants were selected by NSG enrichment score and individually packaged for in vitro and in vivo characterization. Meanwhile, point mutations were performed on AVT225, AVT239 and AVT260.

[0072] Table 7. Candidates selected form flanking optimization library and point mutations. Capsid name Flanking optimization (position 585-587) Amino acids sequence of an insertion between the amino acid position 587 and position 588 SEQ ID NO Other mutations pAVT260 RAA QQDMRPG 33 / pAVT261 KGG LQDMRPG 57 / pAVT262 / LAIVVGTKMA 58 i pAVT263 / AIQSDRN 59 / pAVT264 / AAANLMPGTRAA 60 / pAVT265 RAP QQDMRPG 33 / pAVT266 RAA QQDMRPG 33 Y737F pAVT267 RAA QQDMRPG 33 Y444F, Y500F, Y737F pAVT268 RAA QQDMRPG 33 K556E pAVT269 / VDNASKN 23 Y737F pAVT270 / VDNASKN 23 Y444F, Y5O0F, Y737F pAVT271 / fNIHKPE 36 Y737F pAVT272 . / INIHKPE 36 Y444F, Y500F, Y737F pAVT273 RGM INIHKPE 36 / ■ pAVT274 RGP INIHKPE 36 / pAVT275 RGM INIHKPE 36 / pAVT276 RGP INIHKPE 36 / pAVT277 KAG QQDMRPG 33 / pAVT278 / ASVHKPE 61 / ■ pAVT279 / QFQPDRN 62 i pAVT280 / TQDTRPQ 63 / pAVT281 '1 ATEARPQ 64 / pAVT282 RCN TYNPDRN 65 / pAVT283 7 KTEFRPE 66 i pAVT284 i TSNDRPQ 67 / Note:    means no substitutions or mutations are at the corresponding positions of AAV variant.

[0073] Table 8. The NGS data and enrichment scores. Capsid name Flanking optimization (position 585-587) Amino acids sequence of an insertion between the amino acid position 587 and position 588 SEQ ID NO Other mutations (position 595-597) Enrichment score RGM INIHKPE 36 / 0.621522255 RGP INIHKPE 36 ■ / ■ 0.545065429 / TYNPDRN 65 J 0.522940154 / KTEFRPE 66 I 0.510433927 / TQDTRPQ 63 / 0.533319447 AVT248 ! IIQPDRN 45 . / 0.531580864 RGT INIHKPE 36 / 0,533798277 . / TNHPDRN 68 / 0.475236147 / VIQPDRN 69 0.496587426 r QFQPDRN 62 / 0.485640635 KAG QQDMRPG 33 / 0.425898894 AVT239 / INIHKPE 36 7 0.543805295 / ISMTRPE 70 / 0 583454089 AVT245 ! MIQPDRN 42 / 0.466030318 / TSNDRPQ 67 0.45356099 RGV INIHKPE 36 / 0.539095191 RGL INIHKPE 36 / 0.55956905 i TSVTRPD 71 0.476153309 , / MQTSRPD 72 / 0.419602332 / IMVTRPE 73 0.546705888 . / YNQERPA 74 / 0.463333313 LSDTRPQ 75 . / 0.422857415 I QYAQRPE 76 / 0.496623268 / [ QYAQRPE 76 / 0.367168689 I KDMMRPE 77 / 0.443475923 , / REDMRPA 78 z 0.384540507 KGS QQDMRPG 33 / 0.372978534 / MAQPERN 79 / 0.338403658 i AHADRPQ 80 / 0.412647776 / TVTMRPE 81 7 0.402092361 . / QNDNRPA 82 / 0.413107547 / TSDYRPG 83 ■ / 0.468558197 / MTNADRN 84 / ■ 0 328704796 . / WSESRPQ 85 : / 0.36785046 7 REPQRPD 86 7 0.375390639 i MTMPERN 87 / 0.31097903 / MQQHRPD 88 z 0,344367787 / VNQPDRN 89 / 0.340060815 , / MNSERPA 90 / 0.419989585 1 AQQHKPE 91 / : 0.372605287 I ASVIIKPE 61 0.356772833 . / MVQPDRN 92 . / 0.332594616 / TSNPERN 93 , / 0,32293406 / QSASRPE 94 7 0.445014795 / MTVHRPD 95 / 0.365113404 . / MVHADRN 96 z 0.342589032 AVT260 RAA QQDMRPG 33' z 0.386773712 / RNPGREE 97 / 0.340168186 . / - VSNNRNE 98 / 0.343457168 , / QFISRPE 99 0.416472021 KGA QQDMRPG 33 / 0.336423432 . / ATEARPQ 64 / 0.310255183 / TQNVRPE 100 / 0.35510681 / NTESRPQ 101 . / 0.298658827 . / ITVTRPE 102 .. / 0.399481752 / ITENRPM 103 / 0.316620196 i ANPQIKD 104 / 0.340387017 / ENPRIQQ 105 / 0,294798971 , / INMPDRN 106 / 0.286022074 / TNDSRPA 107 / 0.333252657 7 VAILRPE 108 0.500935431 AVT265 RAP QQDMRPG 33 / 0.377296582 / LHTDRPN 109 0.301295979 / FNTERPP 110 / 0.332157285 TVQQRPE 111 . / 0,41119035 / YTETRPQ 112 / 0.301540922 . / QQIISKPE 113 z 0.289717268 / REHMRPE 114 0:321450085 , / INAERPT 115 / 0.330081814 / TlDHRPQ 116 / 0.325855055 I TFETRPS 117 / 0.281203922 RAN QQDMRPG 33 / 0.298676473 7 QMNIRPE 118 7 0.355953543 , / IFVSRPE 119 . / 0.484500304 i LSESRPA 120 ■ / 0.308494573 RAT QQDMRPG 33 7 0.303640428 / MNAQRPE 121 / 0,363873816 ! TTEARPS 122 / 0.308043171 / TSMHKAE 123 0.255886559 / TGETRPP 124 / 0:.281274631 / IMESRPQ 125 / 0.274883662 KSG QQDMRPG 33 0.377798498 ■ / SNPQIKD 126 / 0.323821755 / VMVSRPE 127 0.452900495 . / MVHMRPE 128 .. / 0.300774084 MTHERPG 129 . / 0.23520231 I TLISRPE 130 / 0.41047152 / ATEQRPS 131 / 0.281622578 I- TTEHRPT 132 / 0.269636028 , / SYVQERN 133 z 0.267095985 / FNEQRPA 134 / 0.265112097 / INDRKNE 135 / 0.220068512 i SMNQRPD 136 / 0.202610446 / NHQYKPE '137 7 0.235074749 . / LRDVRPE 138 j 0.212293134 / RSMPERD 139 ■ / 0.210328168 / MSIHRPE 140 / 0:264179456 , / ASNTERN 141 : / 0.194903345 7 AYDQRHN 142 7 0.307011065 AVT246 i PNQPDRN 43 / 0:.189925434 / THDNRPS 143 / z 0 234046609 / SQETRPQ 144 0.192057273 KAG QQDMRPV 145 / 0.8:66916441 1 KEYPGRD 146 / 0.278196489 I ATQQERN 46 0.179600221 1. QIDHRPG 447 . / 0.198123184 RDM INIHKPE 36 , / 0.770583192 / MPDTRPG 148 7 0.203867122 / ASNTDRN 149 / 0.197976512' . / IMPQRPE 150 z 0.434259669 / ISLHKPE 151 z 0.219204506 / TEIIAKNP 152 / 0.174247054 . / - TREQRPE 153 / 0.233885111 , / FNQQRPE 154 , / 0.25175271 ! TQHNKAE 155 / 0.207036827 / AMEQRPQ 156 / 0.176158985 / ADIVAKN 157 / 0 2404110^1 / ANPN1NQ 158 / 0 228279973 . / SMQNRPE 159 .. / 0 29705409 / IFNTRPD 160 / 0 231369083 i TMELRPG 161 / 0 228293396 / ISMTRPD 162 I 0 436024836 KAG QHDMRPG 163 1 0.787756406 / MNPNRND 164 / 0.143688144 7 SQQHKPE 165 0.647467199 , / TGINRPD 166 / 0.191510424 / TDCNRPQ 167 0.735632753 / ALNIRPE 168 / 0.193910675 FIQPDRN 169 . / 0.658886993 / LNENRPT 170 / 0.165358929 . / TMESRPG 171 z 0.175799783 / QLNQERN 172 0.149474418 RGI INIHKPE 36 / 0.275189915 AVT243 / IPNPERN 40 / 0.19944513 I SINQRPD 173 / 0.718911996 i FQETRPQ 174 / 0.162436219 7 VSTPERN 50 7 0.248459339 ,z ENHRIQQ 175 . / 0,64383 / VINVRPE 176 , / 0.173397286 / MTIPERN 177 7 0.652829264 / IQVSRPE 178 / 0,315989097 ! MSEIRPQ 179 / 0.424915185 . / SIDHRPN 180 t 0566429745 / STEQRPS 181 0:.589475096 / IQTSRPD 182 / 0.576659151 J WSEHKQP 183 0.169292308 , / ENPSIQQ 184 / 0.564904178 KAV QQDMRPG 33 0.660641573 TAG QQDMRPG 33 . / 0.458550363 RAG QQDMRPG 33 .. / 0,201133333 RSF QQDMRPG 33 / 0.173869564 / SSNTERN 185 / 0.502977268 . / YNQERPD 186 / 0.55825152 , / FNQPDRN 187 z 0.541088758 / SSNTDRN 188 1 0.596951245 / TLETRPA 189 I 0.169919742 , / RTYDRPE 190 / 0.233304376 / TDVNRPQ 191 7 0.546051217 I TYNPERN 192 j 0.346157596 / KEHRPND 54 , / 0.214024183 / ADQPNRN 193 / ■ 0.133941723 ,. / VNHPDRN 194 : / 0,544492168 7 SMEQRPQ 195 7 0,517678048 i INIPDRN 196 / 0.585363152 / PGTSRPE 197 z 0,188278174 .. / AQQHKPD 198 / 0,602096923 , / LIQPDRN 199 / 0.561943869 .. / THDTRPQ 200 / : 0.417028633 KAG QQDIRPG 201 0.592099518 I FNPTRPE 202 . / 0.216557062 I TQVHRPE 203 , / 0.193234549 KVG QQDMRPG 33 7 0.474714362 / TNHPYRN 204 / 0.611653924 . / SVDTNRN 205 z 0.146092573 t SYVHERN 206 z 0.593843112 / TTAIIRPD 207 / 0.159356302 QTEQRPA 208 / 0.238254248 , / STQQERN 209 , / 0.478609237 ! ADNINRN 210 / 0,130758538 . / MTAERPG 211 / 0,170878586 / ANHPDRN 212 / 0.443212583: / SNTERPQ 213 / 0.469812813 .. / ATVARPE 214 .. / 0.251938847 / MTMPESN 215 / 0,515220551 KAG QQDMRPG 33 R595L 0,526640369 IAG QQDMRPG 33: I 0,529584322 , / IIQPERN 216 1 0.47:9814686 / MNLETRPA 217 / 0.34795618 7 SNHPDRN 218 0.556591124 , / MAQPERH 219 / 0.540511433 / TI-INPDRM 220 0.551267511 .. / REDMRPD 221 / 0.549393997 PNHPDRN 222 . / 0.382005207 I QIQPDRN 14 / 0.153040982 )■ ADI1PNRN 223 z 0.564878772 RCM INIHKPE 36 0.497450431 , / IVQPDRN 224 / 0.498713321 RGT INIHKPE 36 A597V 0.49606015 I SDTNRPN 225 / 0.119913596 , / MIIQIIRPD 226 / 0.554465911 7 SDNINRN 227 7 0,520314318 , / IATTRPE 228 . / 0.279691069 i SDQPNRN 229 , / 0.381248784 / ISNPDRM 230 7 0.11564709 KAG QQDMRPC 231 / 0.539029148 ! QHILRPD 232 / 0.253599348 / SYNPDRN 233 0480001418 / QTEFRPE: 234 / 0.49593239 / PATIIRPD 235 / 0.189088764 J TTNADRN 236 0.520489961 KAG QQDMRPG 33 A597S 0.535749237 KAG HQDMRPG 237 0.447713641 . / NHQYKPD 238 / 0.612414067 ITMPERN 239 . / 0.515875835 I TYNPYRN 240 / 0.540838051 / SQDTRPQ 241 / 0.411171693 I- PNQPDSN 242 / 0.499454497 , / MSQPERN 243 z 0.511560679 / TYHPDRN 244 / 0.39629465 RAG QQDMRPG 33 A597V 0.524600425 i STNPERN 245 / 0.518834727 / ATDQRPS 246 7 0.506874997 . / QHNPDRT 247 / 0.507769761 / DNPRIQQ 248 ■ / 0.484688699 RGC QQDMRPG 33 / ■ 0.511533666 . / TSVTRPE 249 7 0.373839412 7 MTNADSN 250 7 0.474960797 i PNQPDMN 251 / 0.482881999 / MNHNRND 252 z 0.407251897 / SIETRPS 253 / 0.415771526 , / RDSMRAE 254 / 0.201065043 1 QQHNKPD 255 / : 0.106438056 I TSIIIKAE 256 0.4274453 . / REDMCPA 257 . / 0.424399079 RAD QQDMRPG 33 , / 0.406297833 / AHQHKPE 258 7 0.418961947 / LTHQKPE 259 / 0.134440964 . / MFQPDRN 260 z 0.405707227 / THHPDRN 261 z 0.444898655 / TYNPDRM 262 / 0.440720798 KAG QLDMRPG 263 / 0.413264892 KAG QQDMRPG 33 A597V 0.451475238- ! SPSHKPE 264 / 0,169561324 / INPNRND 265 ■ / 0.33419542 / ASNPERN 266 / 0375503284 / KNYLRNE 267 . / 0:442651323 . / IMEYRPQ 268 . / 0.450447586 / KTEFRPD 269 I 0,359070977 i AFEMRPQ 270 i 0.100360928 / ANPHIKD 271 / 0:,46156816 , / SVMQDRN 272 1 0.384640613 / VNIIIKPE 273 / 0.402702291 7 NTEYRPQ 274 0.415855966 , / NHQYTPE 275 / 0.426542038 / ATHQERN 276 0.401812184 / KNEFRPE 277 / 0.384085602 LVQPDRN 278 . / 0.400089563 / MNEANRN 279 / 0.087560346 . / NTESRPE 280 z 0.326329328 / AQHHKPE 281 0.382133607 , / MNPNRNY 282 I 0.392078741 / TVATRPE 283 / 0.242408424 I MQQHRPE 284 / 0.228593707 , / LTMPERN 285 / 0.337068296 RSG QQDMRPG 33 7 0.103475569 RGM FNIHTPE 286 . / 0.400249994 i TNIHKPE 287 ■i 0.113104846 / YAQTRPD 28.8 7 0.114138683 / REDMSPA 289 / 0.416637008 ! ATESRPQ 290 / 0.363807733 NAG QQDMRPG 33: 0.345999604 / ANPQ1ND 291 / 0:.425587944 / TEIIDKNP 292 / 0.344838776' J TYNPDRT 293 0.362673707 , / MNSERPQ 294 / 0.405590326 / TINQRAE 295 0.388275253 . / LNDNRPT 296 .. / 0.40048665 IAQPERN 297 . / 0.363651426 I FSHNKME 298 / 0.386986153 RAG QIIDMRPG 163 / 0.390071781 I- FNQLRPE 299 / 0.23957731 , / IIESRPQ 300 z 0.290362861 RGG QQDMRPG 33 / 0:.177412222 / DSVHKPE 301 / 0.308411199 i FTEQRPG 302 / 0.112777534 / 1TEFRPE 303 7 0.334293262 KAG LQDMRPG 57 j 0.366814574 / SNDNRHN 304 ■ / 0.272638807 / i:\adrx 305 / 0'33:8622755 . / DTEQRPS 306 : / 0.379821183 7 AAQHRPD 307 7 0.146903366 i NYNPDRN 308 / 0:.324334548 / SEHMRPE 309 z 0.352441167 / ITESRPQ 310 / 0.5:34327651 , / SEDMRPA 311 / 0.310712676 1 RDDMRPA 312 / 0.29679194 I KTEFMPE 313 0.348117617 . / KTEFSPE 314 . / 0.363222409 / QFQPDRN 62 Q596H 0.342615326 / NNHPDRN 315 7 0.322650907 RPS QQDMRPG 33 / 0.137602864 . / TNHPERN 316 z 0.260914805 / MVQPERN 317 z 0.29645778 KAG QQDMSPG 318 / 0.323671991 , / AIQPDRN 319 / 0.360944988 , / SMNQRPY 320 , / 0.342574477 I FNATRPE 321 / 0.14741885 / QYEMRPQ 322 / 0.093013:416 / TIETRPD 323 / 0222113384 / FNENRAQ 324 / 0.331986238 . / LSESRPQ 325 .. / 0.234162993 / YTESRPQ 326 / 0,310249074 KGS QHDMRPG 163 / 0.291185345 / MEDMRPA 327 / 0290734515 , / REDMRPS 328 / 0.356008365 / IQILRPE 329 / 0.192686235 7 MENPARD 330 0.258582194 , / QDNVSKN 331 / 0.104042067 / AQQHTPE 332 0.357334945 / TETHKTA 333 / 0.503218836 IGTVRPE 334 . / 0.321737452 / VNPNINQ 335 / 0.33893754 . / SDGNRPQ 336 z 0.286831163 / MTEFRPE 337 0260937807 , / QMTHRPD 338 / 0.163445681 / QMNQERN 339 / 0.344759225 . / MAQPDRN 340 / 0242978773 , / TNMPDRN 341 / 0.444948081 7 VNDSRPQ 342 7 0.108030954 , / MAQPERY 343 . / 0.352307957 I TNHPDRN 68 Q596H 0.482516117 / TFETRPG 344 7 0.476343442 / MTHERPC 345 / 0.316382301 ! TSVTRPA 346 / 0.44503882 / TDHAKNP 347 0214835622 / VSTQRPE 348 / 0:.203462962 / TSGTRPD 349 / 0.23160963 RVN TNHPDRN 68 0269006419 , / SHETRPQ 350 / 0.235640208 / FHTDRPP 351 0.167188383 . / LQTSRPD 352 / 0.29502774 DSNTDRN 353 .. / 0261840878: I SQDINRN 354 / 0.361091807 / TDIIPDRN 355 / 0.331555532 I- PNQTDRN 356 / 0.322053046 , / LNENRPN 357 z 0.328710365 / VHNQKPD 358 / 0.075837429 / AIEQRPQ 359 / 0.23484195 i QQDARPS 360 / 0.179211552 / AGHGKNE 361 / 0.129424337 / TYKPDRN 362 / 0.263369224 / INSHKPE 363 ■ / 0.308900371 / YTEHKPS 364 / ■ 0.104001121 RGM INIHNPE 365 : / 0282873563 I IVHADRN 366 7 0285996303 i QNDNRPD 367 / 0:.3193:67977 / TMEQRPE 368 z 0.319910934 . / MTYADRN 369 / 0275382135 , / FNEQRPD 370 / 0.30065556 1 MTNSDRN 371 / : 0.237809294 I TEIISKNP 372 0.280075707 / TRVTRPD 373 . / 0290050738 I TDNPNRT 374 , / 0.109361762 / DNPQIKD 375 / 0.290358076 RCN IIQPDRN 45 / 0.284512597 . / VTQPDRN 376 z 0.231184609 / SYNPDKi 377 z 0.309632191 / MFMTRPE 378 / 0.161868923 . / - TSKDRPQ 379 / 0.285092097 , / MTNAERN 380 , / 0.238666454 ! MSIHRPD 381 / 0.402327166 / MNNADRN 382 / 0.282267889 / ENPRIQQ 105 A597S 0213917182 / HQNPDRT 383 / 0.251641016 . / TSEQRPE 384 .. / ■ 0251283811 / AVIQDRN 385 1 0274529483 i MAQPERN 79 R595M 0.311530052 / QQNPDRT 386 / 0.094079548 , / SEPQRPD 387 1 0.276:117608: / INLPDRN 388 / 0 234120454 / MTMPERY 389 0 269429237 , / TYMHKAE 390 / 0.257562965 RCN TQDTRPQ 63 0284575626 RCN KTEFRPE 66 / ■ 0.261969115 RVN IIQPDRN 45 .. / 0258661911 I DSNTERN 391 / 0.18609641 )■ TLETRPD 392 z 0 285716952 / VDRPDRI 393 0277311592 , / AYNTERN 394 I 0.244045889 / QHHSKPE 395 1. 0 229133651 I NNDLRNE 396 1 0.223022785 , / MIQPDRN 42 Q596II 0237481677 / ATQHERN 397 / 0209920269 ,z PDNPRPP 398 . / 0.057574085 RPA QQDMRPG 33 , / 0.142070799 / MEHPSTA 399 7 0.239588291 RON TNHPDRN 68 / 0.24219975 ! LQQHRPD 400 / 0.261604469 / AYQPNRN 401 0.250588641 / T1ESRPG 402 / 0.240526742 / MNEDNRN 403 / 0.231432287 J MADYNKN 404 0.150030829 RCN TYNPDRN 65 / 0.222841598 / YNHERPA 405 0.304783448 . / KNPTRDE 406 / 0.135818184 TSVTRPD 71 Q596H 0.259170948 I TNHPDRM 407 / 0.279314403 / MQTSRPQ 408 / 0.288453341 I- QFQPF.RN 409 / 0.223055512 KAG QQDMRPG 33 Q596H 0.230004715 / TENQRAP 410 0.124870883: / TYNPDRN 65 Q596H 0.252767112 7 LNENRPS 411 / 0.255373577 RCN MQTSRPD 72 / 0.251226806 I TYNPNRT 412 i 0.253725925 / FNENRNA 413 7 0.262736154 / LTHERPG 414 / 0:230020679 . / THHNKAE 415 7 0.237873741 RVN TSNDRPQ 67 7 0.24236329 i INHPDRN 416 / 0.233598239 / IIQPDRN 45 Q596H 0 230716053 / TQNPDRL 417 / 0.261709381 , / IQQHRPD 418 / 0.214992266 1 TGEQRPE 419 / 0.245456375: I QQDIIRPA 420 0.252708475 . / KNDLRND 421 7 0.249020651 .. / ISNPDRN 422 7 0.103810922 / TNDSRPD 423 / 0.235499117 RVN MQTSRPD 72 / 0.236915981 . / MIQPERN 424 / 0.217701318 / RTEDRHE 425 / 0.221883408 / RSMPESD 426 / 0.229937402 . / - ITQPDRN 427 / 0.252261187 , / D.MEQRPQ 428 , / 0.20725592: ! THVHRPE 429 / 0.253447615 . / YNPQIKD 430 7 0.313396671 / DTQQERN 431 / 0.176656253 KCS QQDMRPG 33 7 0.216522017 .. / YTTSRPE 432 7 0.137407316 RGT INLHKPE 433 / 0.26782554 i AMEQSPQ 434 / 0.281364874 . / IYVQERN 435 / 0.227159884 , / ADQPNSN 436 1 0.23376883 / ADQIINRN 437 / 0.208261563 / DDQPNRN 438 0.218842961 7 REQPNRN 439 / 0.447853805 RGG HQDMRPG 237 0.271425166 .. / RNDLRNE 440 / ■ 0.273672077 RVN LSDTRPQ 75 .. / 0.216596572 / INDSKNE 441 / 0.2135947 . / TREARPE 442 / 0.122172146 / AFDTNRD 443 0.208802542 , / MEHMRPE 444 I 0.200884685 / MENPSRA 445 / 0.22283801 RCN QFQPDRN 62 / 0.190990768 , / MIIVARPE 446 / 0.159146595 / QLNQERH 447 / 0.174374499 .. / HLNQERN 228 7 0.21352946 . / TYNPDRK 448 ■i 0.210772264 / IHHLKPE 449 / 0.254341363 / ■ QIIDARPS 450 / 0.204048104 RVN YNQERPA 74 / 0.295966674 RCN TDGNRPQ 451 / 0.224703502 I TQNPDRM 452 / 0.115136189 / SHHLKPE 453 / 0.131793664 I ENTRIQQ 454 / 0.230880618 / VSHNTME 455 / 0.202453954 / QHEDRPS 456 / 0.196154541 / QTEQRPD 457 / 0.285099293 i ACHGKNE 458 / 0.216484826 / SSNDRPQ 459 / 0.188258827 / KTEFRPE 66 Q596H 0.212547923 ■ / KTEFRPQ 460 / 0.222863034 ■ / IMNQRPD 461 ! 0.142164289 RVN V1QPDRN 69 / 0.196296741 RVN TQDTRPQ 63 j 0.193905891 . / TNANRME 462 / 0.077502622 / SQEHNKN 463 f 0.197746876 RAN QHDMRPG 163 / 0.206297973 / FHETRPQ 464 / 0.166745199 RVN TYNPDRN 65 / 0.169768106 / AYNPDRN 465 / 0.209006017 RVN QFQPDRN 62 / 0.175118071 . / KTEFRPA 466 ! 0.30467348 7m8 / LALGETTRPA 467 / 0.132734248 WT (RGNRQA) / / / / -1.362183587 Note:    means no substitutions or mutations are at the corresponding positions of AAV variant.

[0074] Base on the NGS data and enrichment scores, our AI team found many motifs with high retina tropism and penetration, these motifs and form different groups and families (Fig 3). Example 2: Validation 2.1 Construction and Purification of AAV plasmids

[0075] Rep-cap plasmids construction for variants were performed by overlap PCR as follows: a, AAV2 Rep-cap plasmid was used as template, and AAV2F, overlap R was used as primers to amplify fragment 1; AAV2 Rep-cap plasmid was used as template and amplified with overlap F, AAV2R to obtain fragment 2. b, Fragmentl and fragment 2 were used as template, AAV2 F and AAV2 R were used as primers to amplify fragment 3. c, The AAV2 repcap plasmid and fragments were digested with BsiwI and Spel, and fragment 3 was inserted between BsiwI and Spel of AAV2 Repcap plasmid to complete the construction. After sanger sequencing, the plasmids were used for AAV production.

[0076] Three variants (AVT225, AVT226 and AVT228) plus controls (AAV2, AAV7m8 and AAVGL) were individually packaged with green fluorescent protein (GFP) transgene driven by Chicken [3-actin (CB) promoter. All 6 AAVs were produced in HEK293 cells by triple plasmid transfection with PEIpro (Polyplus). Three days after transfection, the cells were harvested for lysis, Benzonase (Sigma, E1014-25KU) treatment and proceeded to iodixanol gradient ultracentrifugation purification method. After buffer exchange using PBS with 0.001%F68, the AAV was filtered through 0.22 pm. The titer was measured by ddPCR using ITR primer and probes. The purity of AAV was assessed by SDS-PAGE.

[0077] Table 9. The titer of AAV. Variants Titer (vg / ml) Packaging seal AVT225 5E+12 vg / ml 6*150 nun dish AVT226 5 F. 12 vg / ml 6*150 mm dish AVT228 5E+12y g / ml 6*150 mm dish 2.2 Validation of novel capsids in C57BL / 6J mice.

[0078] Six-week-old C57BL / 6J mice were intravitreally injected with those 6 AAVs (1E9 vg / eye). Animals were anesthetized by intraperitoneal injection of Zoletil (60 mg / kg) and xylazine (10 mg / kg). The pupil was dilated with tropicamide eye drops (National Medicine Permit No. J20180051). The injection system was a Nanofil microliter injection system equipped with a 36G bevel needle (World Precision Instrument, Nanofil-10 pL). The whole field operation was performed under a stereomicroscope (OLYMPUS, SZ61). For injection, the needle was beveled downward and inserted approximately 2 mm from the limbus side against the sclera, while penetrating the sclera, choroid, and retina to reach the vitreous. After microscopically confirming the correct position of the needle in the vitreous, a slow bolus of 1 pL AAV solution was administered. Special care should be taken to avoid damaging the lens during the injection.

[0079] Three weeks post injection (wpi), animals were anesthetized, systemic blood was replaced with 4°C precooled saline by means of cardiac perfusion. The eyes were removed and fixed in FAS fixative solution for 4 hours, then after removal of cornea and lens under stereomicroscope, transferred to 30%sucrose / PBS solution for dehydration overnight, and finally embedded in OCT embedding agent.

[0080] Before sectioning, the embedded tissue block was equilibrated in a cryostat incubator at -20°C for at least 30 minutes. The embedded block was then fixed to the chuck using additional OCT, and sagittal sections of the eyeball with a thickness of 10 to 20 pm were cut, and sections were collected with a positively charged adhesive slide. The slides were finally dried at room temperature for 1 h and stored at -20°C.

[0081] For staining, slides were first removed from -20°C and dried for 20 min at room temperature. The sections were outlined on the slide using an IHC pen to create a hydrophobic ring. The slides were then washed three times with 500 pl PBS for 5 min each. The cells were incubated for 1 h with PBS blocking solution containing 0.5%Triton X-100 and 10%goat serum. After removal of the blocking solution, the primary antibody incubation solution was added and incubated overnight at 4°C. Slides were then washed five times for 5 min each with PBS solution containing 0.5%Triton X-100. After the washing solution was removed, the secondary antibody incubation solution was added and incubated for 1 h at room temperature. Finally, the plates were washed 5 times with PBS for 5 min each time and sealed with a drop of capping agent containing DAPI dye.

[0082] Image acquisition was performed with the use of a light microscope (Nikon, TS2R) with 4x, lOx, and 20x objectives. For brightfield, DAPI, GFP, and Cy3 signaling pathways, images were taken using brightfield, DAPI, GFP, and Alexa Fluor 555 channels, respectively. For images that needed to be stitched into large maps, serial multichannel image shots with approximately 10%overlap were performed.

[0083] The results indicate that AVT225 showed very extensive infection and good tissue penetration compared to controls. No signal was detected for AVT226 and AVT228 (Fig 4) . This may be due to differences between species, as candidates were screened from monkeys, whereas validation was performed in mice. Example 3: Intravitreal injection of NHP

[0084] Six variants (AVT225, AVT235, AVT239, AVT248, AVT260, AVT264) plus controls (AAV2, AAV7m8 and R100) were individually packaged with green fluorescent protein (GFP) transgene or mCherry or FXN-HA tag driven by Chicken [3-actin (CB) promoter. All AAVs were produced in HEK293 cells by triple plasmid transfection with PEIpro (Polyplus).

[0085] Seven adult cynomolgus macaque (4-5 years old, ^3 kg) was intravitreally injected with 8E10 vg / eye (4E10 / eye / capsid), each eye was injected with two capsid packaging with different marker gene as follows:

[0086] Table 10. First round of NHP validation design. Animal Number Animal ID OD; OS 1101 19324 AV2-m, 7m8-G; 260-m, 260-G 1201 Hl 909082 260-m, 225-G; 248-m, 7m8-G 1301 Hl 908050 7m8-m, 248-G; 225-m, 264-G 1401 H1901001 248-m, 260-G; 7m8-m, 225-G 1501 H1907069 225-m, 7m8-G; 264-m, 248-G 1601 19306 225-m, 235-G; RIOO-m, 239-G 1701 H1906022 7m8-m, 235-G; 260-m, 239-G Example 4: Terminal, fundus autofluorescence and tissue collection

[0087] Animals were anesthetized by intramuscular injection of Zoletil (50 mg / kg) and xylazine (10 mg / kg), and topical application of proparacaine hydrochloride and tropicamide was used to relieve pain or mydriatic the pupil. The injection system used a 31g insulin syringe, which was inserted into the sclera (approximately 3mm behind the limbus), and 50qL intravitreal injection was performed in two separate doses to each eye, with a total injection volume of 100 pl / eye. The needle was beveled down, and the position of the needle was confirmed under the operating microscope. The AAV solution was delivered to the middle vitreous body by slow injection. After completion of the bolus, the needle was kept stable for at least 2 min to minimize backflow of the injection solution. In addition, 40 to 80 mg of methylprednisolone was administered weekly after the injection.

[0088] Ophthalmic examination and ocular imaging were performed on all animals by a board-certified veterinary ophthalmologist before and at various time points after administration. Ophthalmic examinations included indirect ophthalmoscopy and slit-lamp biomicroscopy. Fundus imaging was performed with the use of a confocal scanning laser fundus imaging system (Spectralis HRA + OCT, Heidelberg Engineering). The images recorded included infrared reflection (IR), blue autofluorescence (BAF) and optical coherence tomography (OCT). During BAF imaging, multiple regions of the central and peripheral retina were recorded to reflect the distribution of GFP expression, and the sensitivity was set to 100. OCT imaging is performed by scanning multi-line horizontal, high-resolution images from the optic nerve head (ONH) to the fovea.

[0089] Novel capsids AVT225, AVT239, AVT248, and AVT260 produced very strong and broadly distributed GFP signals in the NHP retina after 3 WPI. This signal of AVT225, AVT248, and AVT260 was significantly stronger than the positive control AAV7m8 (Fig 6) . Example 5: Retinal sections, flat mount and immunofluorescence

[0090] For staining, slides were first removed from -20°C and dried for 20 min at room temperature. The sections were outlined on the slide using an IHC pen to create a hydrophobic ring. The slides were then washed three times with 500 pl PBS for 5 min each. The cells were incubated for 1 h with PBS blocking solution containing 0.5%Triton X-100 and 10%goat serum. After removal of the blocking solution, the primary antibody incubation solution was added and incubated overnight at 4°C. Slides were then washed five times for 5 min each with PBS solution containing 0.5%Triton X-100. After the washing solution was removed, the secondary antibody incubation solution was added and incubated for 1 h at room temperature. Finally, the plates were washed 5 times with PBS for 5 min each time and sealed with a drop of capping agent containing DAPI dye.

[0091] Retinal flat mount: after completion of the cryosections, the remaining embedded blocks were thawed at room temperature. The retina was carefully peeled off under a microscope, and the retina was washed five times repeatedly with PBS to remove residual OCT embedding agent. Retinas were then blocked in PBS solution containing 2%Triton X-100, 10%normal bovine serum for 1 h, followed by incubation with rabbit anti-mCherry primary antibody or anti-HA primary antibody at a dilution ratio of 1: 100 overnight. Retinas were washed five times and incubated with secondary antibody solution for 2h at room temperature. After washing, the samples were prepared into planar slides and sealed with mounting agent containing DAPI dye droppers.

[0092] Image acquisition was performed with the use of a light microscope (Nikon, TS2R) with 4x, lOx, and 20x objectives. For brightfield, DAPI, GFP, and Cy3 signaling pathways, images were taken using brightfield, DAPI, GFP, and Alexa Fluor 555 channels, respectively. For images that needed to be stitched into large maps, serial multichannel image shots with approximately 10%overlap were performed.

[0093] Immunolabelled retinal cross sections were stained for GFP and DAPI, in fovea area AAV2 has no infection. AVT264 has weak infection which is better than AAV2 but weaker than AAV7m8, well AVT 260, AVT225 and AVT248 shows stronger infection than the positive control AAV7m8. Fovea is the thinnest part of retina, out of fovea is much harder for AAV to infect and penetrate the retina layers. AAV2 shows no infection out of fovea, well AVT260, AVT225 and AVT248 shows better infection and penetration than AAV7m8. Retina flat mount shows AVT260, AVT225 and AVT248 has wide spread infection of NPH retina (Fig 7).

[0094] Adult cynomolus monkeys were intravitreally injected with AAV-CB-GFP at a dose of 1E12 vg / eye. Three weeks after the injection, in vivo fundus autofluorescence examinations were conducted and Immunolabelled retinal cross sections were stained for GFP and DAPI. AAV2 has no infection, AVT 260, AVT277 and AVT279 shows stronger infection than the positive control AAV7m8 (Fig 8). Example 6: The AAV variants enhanced the ocular tropism of AAV8

[0095] The peptides selected in AAV2 library were synthesized on a chip. Subsequently, the Q588 to P593 of AAV8 were substituted by RGNRQA, and the AAV2 library were inserted after RGN to generate a AAV8 library. Then, the AAV8 library was injected into the eyes of non -human primates (NHPs) at a dose of 1E12 vg / eye. After three weeks, DNA and RNA were extracted and subjected to next-generation sequencing (NGS) . The top -ranked variant candidates enhanced the ocular tropism of both AAV2 and AAV8.

[0096] Table 11. Top variant candidates validated in AAV8 Flanking optimization (position 585-587) Amino acids sequence of an insertion between the amino acid position 587 and position 588 SEQ ID NO Flanking optimization (position 595-597) Enrichment score Ranking score RAP QQDMRPG 33 i 1.209219606 42327.95324 i LALGETTRPA 467 / 1.12602565 35818.49906 / TTNLAKD 47 / 0.537641959 22898.3999 GPR RPEQRAGD 524 RGP 0,532875943 22289.53989 RAA QQDMRPG 33 / ■ 0.650830335 21366.26638 J TTTLAKD 525 / ' 1.181099482 21209.00255 QQN RPNSRPP 526 TAP 0.714944182 20976.61249 KAA MPSRPER 527 AS 0.59506866 18795.76698 QQN TRHPERN 528 TAP 0.59917512 17658.47183 / TTNRAKD 529 / 1.035350641 16827.2304 GIP SKDTRAA 530 RPP 0.523513297 14634.05117 i TPNLAKD 531 r 1.676508524 14032.27471 RAP QQDMRPG 33 RQP 1.009199553 13674.75505 RAP QQEMRPG 532: / 0.925607519 13368.95761 RAP QQDMCPG 533 / 1.444569365 13347.70896 J TTHLAKD 534 i 1.53409711 13255.70055 GPR RPDQRAGD 535 RGP 1.058211408 13058.7779 RTP QQDMRPG 33 I 1.769166733 12306.9215 GPR RPEPRAGD 536 RGP 1.020970725 12171.22227 i TTNLAKD 47 RHA 1,036845412 12010.23395 i VDNASKN 23 / 0.363423495 11999.44946 i TTNFAKD 537 / 1.849185721 11509.78805 I IIQPDRN 45 / 0.352678008 11391.50364 RDP QQDMRPG 33 / 1.672448799 11244.82328 / TTKLAKD 538 / 1.221741294 11240.73619 7 TTNLATD 539 I 1,359267345 11113.02678 RAP QQDMRPG 33 RQT 1.267456627 10934.78236 QQN LNKTRPD 540 PAP 1.079494664 10912.08995 RAP QQDMRPG 33 RHA 1.707127292 10441.89371 / QQDMRPG 33 7 0.362798787 10337.5841 / TTNLAKD 47 RPA 1.259139294 10114.73303 GIP SKDPRAA 541 RPP 1,04457441 10073.53038 WAP QQDMRPG 33 7 1.345102903 9821.359575 QQN RPKSRPP 542 TAP 1.283253717 9590.374987 TAA MPSRPER 527 AS 1.312898597 9547.1297 RAP QQDIRPG 201 I 1.361904454 9521.445014 / TTNLAKE 528 1.051305585 9331,027254 GPS RPEQRAGD 524 RGP 0.937691562 9264.822911 / ' PTNLAKD 529 7 1.222455765 9080,273618 RAP QQDMRPG 231 7 1.311015615 9042.502036 RAP Q1IDMRPG 163 1.281364716 9023,536866 GTP SNDTRAA 530 RPP 1,255858711 9015.100578 RAP QQDMRPV 145 7 1.426673876 8978.258749 LAP QQDMRPG 33 7 1.295114209 8818.101788 RAP QQDMRPG 33 RQS 1.30897854 8614.680308 QQK TRHPERN 528 TAP 1.154935479 8582.065493 RAP IIQDMRPG 237 7 1.19421902 8576.604834 RDN LALGETTRPA 467 7 1.345017611 8521.756948 / VDNASKN 23 RHA 0.909504647 8505,596337 RAP QQYMRPG 531 / 1.303614235 8424.704523 / VDTASKN 532 / 1.042103167 8340.677903 RAP QQDLRPG 533 / 1,46422371 8305.354432 GRP SKDTRAA 530 RPP 1.252530798 8303.828483 RSP QQDMRPG 33 / 1.334047776 8249.931186 / MIQPDRN 42 7 0.266480842 8194.73021 / TTNLAKD 534 7 1.293025834 8124.659563 RVN LALGETTRPA 467 7 1.229072593 8090.03021 / SPNPERN 535 / / 0.759999109 8034.558348 RAP QQDMSPG 318 7 1.382056718 7964.798952 RAP QLDMRPG 263 7 1.591926169 7954,519552 SGN TTNLAKD 47 7 0.849486882 7933.396803 RVN TTNLAKD 47 / 0.736568951 7818 462808 GPR RSEQRAGD 536 RGP 0.83030584 7779 066404 QQN INHTRPE 537 PAP 1.265224076 7700 ^27^72 RAT QQDMRPG 33 7 0.846238912 7622 497865 QQN RPTSRPP 538 TAP 1.3363341 7542 47106 GIP SKETRAA 539 RPP 0.922466706 7520 59+385 / : IPTPERN 540 / 1.069081458 7492 473738 / VDNASKN 23 RPA 0.868871572 7355 210492 RAP QQDMLPG 541 / 1.349171415 7344 941016 RGY LALGETT 542 RPARQA 1.310070937 7267348391 7 TTNLTKD 543 7 1,119258573 7196.884499 RAP QQDMRPG 33 RLA 1.330726714 7050.672466 7 TTNLAKD 47 SQA 0.874345276 7048.051483 RAP QQVMRPG 544 / 1.337988551 7001.397064 RGT TTNLAKD 47 7 1.187405591 6963,587898 GPR RPEQWAGD 545 RGP 0.746557722 6961.033036 RCN TTNLAKD 47 7 0.74758343 6886.832677 RAP QQDMRTG 546 7 1.2082127 6863.62354 QQN RPNSRPP 526 PAP 1.249391936 6850.797604 / QINVARD 41 / 0.242701434 6740.955503 GIP STDTRAA 547 RPP 1.111235102 6730.444313 / QITVARD 548 I 0.850076279 6717.15613 QQT TRHPERN 528 TAP 1.063126223 6707.163468 / TTNLAKD 47 RQV 0.920934989 6705.775975 QAP QQDMRPG 33 7 0,829254987 6664.196113 SSD QKSMKPPKP 549 TAA 0.315896692 6641.05879 / : LALGETT 542 RPARQA 1.20748111 6589.114363 / QINGARD 550 7 0.619594604 6544.031234 / STNLAKD 551 7 0.727231272 6461.990364 / TTNLAND 552 / 0.661029248 6460.742132 / QMNVARD 553 / 1.124508447 6414.761129 7 LALGETTRPA 554 / 1.398138733 6412.551537 / ISQPDRN 555 / 0.974891024 6382.569024 / TTNLAKY 556 / 0.665617146 6344.289263 / MSQPDRN 557 / 0.808218159 6327.645143 KAA MPRRPE 558 RAS 1.050533609 6272.535014 RGI TTNLAKD 47 / ' 0,843265964 6264.321297 RAA QQDMCPG 533 / 0.913469027 6140.465345 (WT) RGN / / (WT) RQA f /

[0097] The embodiments of the present invention have been described above, but the present invention is not limited thereto, and those skilled in the art can understand that modifications and changes can be made within the scope of the purport of the present invention. The manner of modifications and changes should fall within the scope of protection of the present invention.

Claims

Claims

1. A variant AAV capsid protein comprising one or more modifications, the modification comprises an inserted sequence shown as formulae I at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, YX1X2X3X4X5X6X7Z (formulae I), wheeein X is any amino acid, Y is absent, LA (n) , oe A (n), Z is A (n), n is any integee feom 1 to 5.

2. The variant AAV capsid protein of claim 1, wherein the capsid protein comprises an inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the inserted sequence is shown as X^X^^XsXgX-? (formulae II), X17 is any amino acid; preferably, the inserted sequence is shown as XiX2X3PX5RN, X5 is D or E, Xb3 is any amino acid, preferably, the inserted sequence is shown as XiX2X3X4RPX7, X[ 4 is any amino acid, X7 is E, Q, G, or S, preferably, the inserted sequence is shown as XiX2DX4RPQ, Xb2 is any amino acid, X4 is H, or T, preferably, the inserted sequence is shown as XiTEX4RP X7> Xi or X4 is any amino acid, X7 is Q, or S, or preferably, the inserted sequence is shown as XiX2X3X4KPE, Xb4 is any amino acid.

3. The variant AAV capsid protein of any one of claim 1 to 2, wherein the inserted sequence is selected from the group consisting of SEQ ID NOs: 1-467; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-467.

4. The variant AAV capsid protein of any one of claim 1 to 2, wherein the inserted sequence is selected from the group consisting of SEQ ID NOs: 524-558; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 524-558.

5. The variant AAV capsid protein of claim 3, wherein the insertion peptide is selected from the group consisting of SEQ ID NOs: 23, 33, 36, 45, 57-67, ; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 33, 36, 45, 57-67.

6. The variant AAV capsid protein of claim 1, wherein the AAV isselected from the group consisting of: serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, as well as any one of the AAV serotype isolated from human and nonhuman mammalians or variant thereof.

7. The variant AAV capsid protein of any one of claims 1 to 6, whereinthe capsid protein further comprises a modification of any one of flanking substitutions and point mutations.

8. The variant AAV capsid protein of claim 7, wherein the capsid proteincomprises a combination of modification comprising flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, the flanking substitution is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG.

9. The variant AAV capsid protein of claim 7, wherein the capsid proteincomprises a combination of modification comprising flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, the flanking substitution is selected from the substitution of GPR, QQN, KAA, GIP, RTP, RDP, WAP, TAA, GPS, LAP, QQK, RDN, GRP, RSP, SGN, RGY, QQT, QAP, or SSD.

10. The variant AAV capsid protein of claim 8, wherein the capsid proteincomprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, the inserted sequence is SEQ ID NO: 33.

11. The variant AAV capsid protein of claim 8, wherein the capsid proteincomprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, the inserted sequence is SEQ ID NO: 23

12. The variant AAV capsid protein of claim 8, wherein the capsid proteincomprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, the inserted sequence is SEQ ID NO: 36.

13. The variant AAV capsid protein of claim 8, wherein the capsid proteincomprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of KGG, the inserted sequence is selected from SEQ ID NO: 57-67.

14. The variant AAV capsid protein of claim 8, wherein the capsid proteincomprises flanking substitutions at the amino acid position 585 to 587 and the inserted sequence at a position between the amino acid position 587 and 588 of the parental AAV capsid protein, wherein the flanking substitutions is selected from the substitution of RCN, the inserted sequence is selected from SEQ ID NO: 57-67.

15. The variant AAV capsid protein of any one of claims 7-14, whereinthe point mutation comprises one or more amino acid substitutions corresponding to the amino acid sequence of parental AAV, wherein the one or more amino acid substitutions is selected from Y737F, Y444F, Y500F, K556E, R595L, R595M, A597V, A597S, and Q596H.

16. The variant AAV capsid protein of claim 15, wherein the pointmutation comprises one of the following substitution or substitution combinations:(i) Y737F; (ii) Y444F, Y500F, Y737F; (iii) K556E.

17. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising flanking sub-stitutions of RAA at the amino acid position 585 to 587, an amino acid substitution Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 33 between the amino acid position 587 and 588 of the parental AAV capsid protein.

18. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising flanking substitutions of RAA at the amino acid position 585 to 587, amino acid substitutions Y444F, Y500F and Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 33 between the amino acid position 587 and 588 of the parental AAV capsid protein.

19. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising flanking substitutions of RAA at the amino acid position 585 to 587, an amino acid substitution K556E, and an insertion of the amino acid sequences as shown in SEQ ID NO: 33 between the amino acid position 587 and 588 of the parental AAV capsid protein.

20. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising an amino acid substitution Y737F and an insertion of the amino acid sequences as shown in SEQ ID NO: 23 between the amino acid position 587 and 588 of the parental AAV capsid protein.

21. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising amino acid substitution Y444F, Y500F, Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 23 between the amino acid position 587 and 588 of the parental AAV capsid protein.

22. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising an amino acid substitutions Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 36 between the amino acid position 587 and 588 of the parental AAV capsid protein.

23. The variant AAV capsid protein of claim 16, wherein the capsid proteincomprises a combination of modification comprising amino acid substitution Y444F, Y500F, Y737F, and an insertion of the amino acid sequences as shown in SEQ ID NO: 36 between the amino acid position 587 and 588 of the parental AAV capsid protein.

24. An isolated polynucleotide comprising a nucleotide sequence thatencodes a variant AAV capsid protein of any one of claims 1 to 23.

25.

26. A vector comprising the isolated polynucleotide of claim 24. An isolated, genetically modified host cell comprising the polynucleotide of claim 24.

27. A recombinant AAV virion comprising a variant AAV capsid protein of any one of claims 1 to 23.

28. A pharmaceutical composition comprising: a) a recombinant adeno-associated virus virion of claim 27; and b) a pharmaceutically acceptable excipient.

29. A recombinant AAV vector, comprising polynucleotide encoding a variant AAV capsid protein of any one of claims 1 to 23, and an AAV 5’ inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional gene product, a regulatory sequence which directs expression of the gene product in a target cell, and an AAV 3’ ITR.

30. A method of delivering a nucleic acid vector encoding a functional gene product to cells and / or tissues with a recombinant AAV virion of claim 27 or recombinant AAV vector of claim 29.

31. A method of treating disease, the method comprising administering to a subject in need thereof an effective amount of a recombinant AAV virion of claim 27, the recombinant AAV virion comprises functional gene product.

32. The method of anyone of claim 30 or 31, wherein the gene product is a polypeptide.

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