WO2025212745A1 - Engineered adeno-associated virus (aav) capsid protein and uses thereof - Google Patents

Abstract

The present disclosure, at least in part, relates to AAV capsid proteins grafted with a CPP between amino acid residues 587 and 588 of a wild-type AAV2 capsid protein. In some embodiments, a rAAV comprising a capsid protein described here shows enhanced transduction efficiency to retina cells (e.g., photoreceptors or RPE cells). In some embodiments, the present disclosure provides compositions and methods for treating retina diseases such as IRDs).

Description

ENGINEERED ADENO-ASSOCIATED VIRUS (AAV) CAPSID PROTEIN AND USES THEREOF

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of United States Provisional Application Serial Number 63/574,445, filed April 4, 2024, and entitled “ENGINEERED ADENO-ASSOCIATED VIRUS (AAV) CAPSID PROTEIN AND USES THEREOF,” the entire contents of which are herein incorporated by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The entire contents of the electronic sequence listing (U012070205WO00-SEQ- KZM.xml; Size: 255,072 bytes; and Date of Creation: March 28, 2025) are herein incorporated by reference.

BACKGROUND

Recombinant adeno-associated virus (rAAV) has emerged as the preferred delivery vector for both experimental and clinical gene therapies, particularly for treating inherited retinal diseases (IRDs). However, concerns have arisen regarding the low transduction efficacy and rAAV-induced immunogenicity of rAAV.

SUMMARY

The disclosure, at least in part, relates to the discovery that grafting certain cell penetrating peptides (CPP) (e.g., CPPs described in Table 1) into an AAV2 capsid protein produces capsid proteins that have enhanced transduction efficiency in retinal cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells). In some embodiments, capsid proteins described herein a useful in treating retinal diseases (e.g., AMD, diabetic retinopathy, Leber congenital amaurosis, or other inherited retinal diseases (IRDs)). In some embodiments, an AAV capsid protein described herein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, and comprises one or more amino acid insertion between position 587 and 588 of SEQ ID NO: 1. In some embodiments, a recombinant adeno-associated virus (rAAV) comprising a capsid protein described herein transduces retinal cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells) via a less invasive route (e.g., intravitreal injection (IVT)) relative to other more invasive route (e.g., subretinal injections). In some embodiments, injection (e.g., intravitreal injection) of an rAAV comprising an AAV capsid protein described herein and a transgene (e.g., a transgene encoding a therapeutic protein) to a subject results in enhanced pan-retinal transduction (e.g., relative to rAAV comprising wildtype AAV2 capsid or an AAV2.7m8 capsid protein). In some embodiments, a rAAV comprising a capsid protein described herein is less immunogenic (e.g., relative to rAAV comprising wildtype AAV2 capsid or an AAV2.7m8 capsid protein).

In some aspects, the present disclosure provides an adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, and an insertion of a cell penetrating peptide that comprises 5 to 18 amino acids between amino acid residues 587 and 588 of SEQ ID NO:1.

In some embodiments, the CPP is selected from a CPP set forth in Table 1. In some embodiments, the CPP is selected from any one of SEQ ID NOs: 2-39. In some embodiments, the CPP comprises the amino acid sequence of KLGVM (SEQ ID NO: 2).

In some embodiments, the AAV capsid protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 40-77. In some embodiments, the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 40.

In some embodiments, the AAV capsid protein has tropism for retinal cells. In some embodiments, the AAV capsid protein has tropism for photoreceptors or retinal pigment epithelium (RPE) cells.

In some embodiments, the capsid protein has reduced immunogenicity relative to wildtype AAV2 capsid protein or an AAV2.7m8 capsid protein.

In some aspects, the disclosure provides a nucleic acid encoding any one of the AAV capsid proteins described herein. In some embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs: 80-117.

In some aspects, the disclosure provides a vector comprising the nucleic acid encoding any one of the AAV capsid proteins described herein. In some embodiments, the vector is a plasmid.

In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein described herein; and (ii) an isolated nucleic acid comprising a transgene encoding a gene product (e.g., a therapeutic gene product, such as a therapeutic protein or interfering nucleic acid), flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, the therapeutic transgene is associated with a retinal disease.

In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein comprising the amino acid sequence of SEQ ID NO: 40; and (ii) an isolated nucleic acid comprising a transgene encoding a gene product (e.g., a therapeutic gene product), flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, the transgene further comprises a promoter operably linked the nucleic acid sequence encoding the gene product. In some embodiments, the promotor is a CB6 promoter or a GRK1 promoter.

In some aspects, the disclosure provides a host cell comprising an isolated nucleic acid, vector, or rAAV as described herein.

In some aspects, the disclosure provides a composition comprising an isolated nucleic acid, vector, rAAV, or host cell as described herein.

In some aspects, the disclosure provides a method of delivering a transgene to a retinal cell, the method comprising contacting the cell with an rAAV or composition as described herein. In some embodiments, the retinal cells comprise photoreceptor cells.

In some aspects, the disclosure provides a method of delivering a transgene to the retinal cells in a subject, the method comprising administering an rAAV or composition as described herein to a subject. In some embodiments, the retinal cells comprise photoreceptor cells.

In some aspects, the disclosure provides a method for treating a retinal disease, the method comprising administering an rAAV or composition as described herein to a subject.

In some embodiments, an rAAV or composition is administered via injection. In some embodiments, the injection is intravitreal injection.

In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the retinal disease is age-related macular degeneration (AMD), Leber congenital amaurosis, or diabetic retinopathy.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an illustration of library screening workflow through successive rounds of selection.

FIGs. 2A-2B show the identification of leading CPP-derived capsids in the mouse retina via intravitreal injection. FIG. 2A shows that the number of CPPs were gradually enriched in the mouse retina after two rounds of in vivo screening. FIG. 2B shows enrichment analysis of CPPs identified in the mouse retina after second round screening. Enrichment score of the individual CPP indicates the relative RNA abundance of each CPP normalized to its corresponding DNA abundance in the virus stock. The x-axis represents the CPP sequences.

FIGs. 3A-3D show the retinal transduction profile of CPP1 capsid in adult mice. FIG. 3A shows representative fundus images of adult C57BL/6 mice 4 weeks post-IVT injection of low (2.0X10⁸ vgs/eye) or high (2.0X10⁹ vgs/eye) dose of single- stranded (ss) AAVs via IVT injection. FIG. 3B shows relative mRNA expression of GFP in the mouse retina 4 weeks post-IVT injection of low or high dose of ssAAVs. FIG. 3C shows representative fundus images of adult C57BL/6 mice 4 weeks post-IVT injection of low dose of self-complementary (sc) AAVs. FIG. 3D shows relative mRNA expression of GFP in the mouse retina 4 weeks post-IVT injection of low dose of scAAV s.

FIGs. 4A-4D show ssAAV2.CPPl achieves pan-retinal and enhanced photoreceptor transduction with less AAV-induced immune responses via IVT injection. FIG. 4A shows immunolabelled cross sections of mouse retina 4 weeks post-IVT injection of high dose of ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP and ssAAV2.CPPl.CB6.GFP, respectively. White triangles indicate outer segments of transduced photoreceptors. FIG. 4B shows quantitative analysis of transduced photoreceptors in mouse retina receiving different viral vectors. FIG. 4C shows quantitative analysis of Ibal positive cells in different retinal layers in mice. FIG. 4D shows immunolabelled cross sections of mouse retina 4 weeks post-IVT injection of high dose of ssAAV2.GRKl.H2BGFP, ssAAV2.7m8.GRKl.H2BGFP and ssAAV2.CPPl.GRKl.H2BGFP, respectively. The dots indicate nuclear GFP expression. PNA indicates peanut agglutinin. ONE indicates outer nuclear layer - photoreceptor nuclei.

FIG. 5 shows the relative mRNA levels of TNF-a, IE-ip, IFN-y and IE-6 by ddPCR in the retinas of adult mice 4 weeks post-IVT injection of indicated AAV vectors at the dose of 2.0X10⁹ vgs/eye.

FIGs. 6A-6F show the identification of AAV2 capsid variant enriched in the retina of mice. FIG. 6A shows the schematic representation of the backbone plasmid construct containing the cellpenetrating peptide (CPP) library insert. FIG. 6B shows the workflow of the screening process in mice, involving two rounds of selection. FIG. 6C shows representative RT-PCR results of recovered RNA of capsid variants from pooled retina/RPE tissues 28 days after injection of the GRK1 -driven AAV library. FIG. 6D The reduction and enrichment of CPP variants during each round of selection. FIG. 6E shows the identification of AAV2.CPP1 as the leading capsid variant after the second round of selection based on enrichment and yield scores. FIG. 6F shows the sequence of the peptide insert on the AAV2.CPP1 capsid. CPP, cell-penetrating peptides; RT, reverse transcriptase. FIGs. 7A-7F show retinal transduction profile of the single-stranded AAV2.CPP1 vector in adult mice. FIG. 7A shows Representative fluorescence fundus images of adult C57BL/6 mice 4 weeks after intravitreal injection with a high dose (2.0 xlO⁹ vg/eye) of single-stranded AAVs. FIG. 7B shows the quantification of genomic DNA and mRNA expression levels of GFP in mouse retinas 4 weeks post-injection of high-dose ssAAVs. FIG. 7C Immunostaining of retinal cross sections 4 weeks after high-dose intravitreal injection of ssAAV2.CB6.GFP, ss7m8.CB6.GFP, and sAAV2.CPPl.CB6.GFP. The arrows show the GFP expression in the inner segments (ISs) or outer segments (OSs) of photoreceptors successfully transduced. The arrowheads indicate the transduced Muller cells. FIG. 7D shows quantitative analysis of the number of transduced photoreceptors in retinas treated with different viral vectors. FIG. 7E shows representative immunoassayed retinal cross sections showing nuclear GFP expression in photoreceptors 4 weeks after high-dose intravitreal injection of ssAAV2.GRKl.H2BGFP, ss7m8.GRKl.H2BGFP, and ssAAV2.CPPl.GRKl.H2BGFP. Peanut agglutinin (PNA) is a marker of photoreceptor OSs. ONL, outer nuclear layer. FIG. 7F shows quantitative analysis of transduced photoreceptors in retinas treated with the indicated vectors.

FIGs. 8A-8D show retinal transduction profile of self-complementary AAV2.CPP1 vector in adult mice. FIG. 8A shows representative fundus images showing retinal transduction 4 weeks after intravitreal (IVT) injection of a high dose (2.0x10⁹ vg/eye) of self-complementary (sc) AAVs in adult mice. FIG. 8B shows the quantification of genomic DNA and mRNA expression levels of GFP in the mouse retina 4 weeks post-injection of high-dose sc AAVs. FIG. 8C shows representative immunostained retinal cross sections from mice injected with high-dose scAAV2.CB6.GFP, sc7m8.CB6.GFP, and scAAV2.CPPl.CB6.GFP. FIG. 8D shows the quantitative analysis of transduced photoreceptors in retinas treated with different viral vectors.

FIGs. 9A-9D show that AAV2.CPP1 vector induces a minimal immune response comparted to AAV2.7m8. FIGs. 9A-9B show representative immunostaining of retinal microglia using the IBA1 marker in eyes treated with ssAAV2.CB6.GFP, ss7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP. FIG. 9C shows the quantification of IB Al -positive cells in different retinal layers following IVT injection of the indicated viral vectors. FIG. 9D shows relative mRNA expression levels of TNF-a, IL-ip, IFN-y, and IL-6, as a measure by ddPCR, in the retina 4 weeks after injection of 2.0xl0⁹ vg/eye of the specified AAV vectors.

FIGs. 10A-10F show heparan sulfate binding affinity of AAV2 and AAV2.7m8 vectors. FIG. 10A shows dot blot analysis showing that the AAV2 capsid strongly binds to heparan sulfate (HS), while the AAV2.7m8 capsid shows reduced HS binding. FIG. 10B shows a representative illustration of the Matrigel-HS -based Transwell model used to differentiate capsids with strong HS binding from those with weak HS binding. FIG. 10C shows representative images of the Transwell-based model distinguishes the HS binding affinities of AAV2, AAV2.7m8 and AAV2.CPP1 capsids. AAV9 capsids were added as a negative control. FIG. 10D shows the quantitative analysis of GFP expression indicating that the Matrigel-HS -based Transwell model can effectively distinguish between AAV2, AAV2.7m8, and AAV2.CPP1 capsids, based on their different affinities for HS binding. FIGs. 10E- 10F show representative molecular models of AAV2 containing the KLGVM (SEQ ID NO: 2) insertion (highlighted) at amino acid N587. The interaction between the inserted loop and other surface loops of the capsid may contribute to the novel properties observed with this vector.

FIGs. 11A-11C show corresponding DNA sequences of CPPs were confirmed by the next generation sequencing (NGS) at each round of screen. FIG. 11A shows the initial synthesized CPPs to make the plasmid library. FIG. 11B shows the CPPs identified in the retina from the first screen. FIG. 11C shows the CPPs identified in the retina from the second screen.

FIGs. 12A-12C show the identification of the leading AAV2 capsid variant enriched in the retina of mice using CMV/CB-AAV2-CPP library. FIG. 12A shows a representative schematic of the backbone plasmid construct containing the CPP library insert. FIG. 12B shows a representative RT- PCR results of recovered RNA capsid variants from pooled retina/RPE tissues 28 days after injection of the CMV/CBOdriven AAV library. FIG. 12C shows the reduction and enrichment of CPP variants during each round of selection and the identification of AAV2.CPP1 as the leading capsid variant after the second round of selection based on enrichment and yield scores.

FIGs. 13A-13C show engineered AAV2.CPP1 capsid can efficiently package AAV vectors with iodixanol gradient purification. FIG. 13A shows the titers of each AAV vectors packaged by AAV2, AAV2.7m8 and AAV2.CPPl capsids. FIG. 13B shows representative transmission electron microscopy images of scAAV2.CB6.GFP and scAAV2.CPPl.CB6.GFP. Light and black arrows indicate full virions and completely or partially empty virions, respectively. FIG. 13C shows the quantification analysis of full/empty capsid ratio of each vector.

FIGs. 14A-14B show the retinal transduction profile of the single-stranded AAV2.CPP1 vector in adult mice at low dose. FIG. 14A shows representative fluorescence funds images of adult C57BL/65 mice four weeks after intravitreal injection with a low dose (2.0xl0⁸ vg/eye) of singlestranded AAVs. FIG. 14B shows the quantification of genomic DNA levels of GFP in mouse retinas four weeks post-injection of low dose ssAAVs.

FIGs. 15A-15B show retinal transduction profile of the self-complementary AAV2.CPP1 vector in adult mice at low dose. FIG. 15A shows representative fluorescence fundus images of adult C57BL/6 mice four weeks after intravitreal injection with a low dose (2.0xl0⁸ vg/eye) of singlestranded AAVs. FIG. 15B shows quantification of genomic DNA levels of GFP in mouse retinas four weeks postinjection of low dose sc AAVs. DETAILED DESCRIPTION

Aspects of the disclosure, in part, relate to grafting of cell penetrating peptide (CPP) (e.g., CPPs described in Table 1) into AAV2 capsid protein to produce AAV capsid proteins that have enhanced transduction efficiency in retina cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells). In some embodiments, capsid proteins described herein a useful in treating retinal diseases (e.g., AMD, diabetic retinopathy, Leber congenital amaurosis, or other inherited retinal diseases (IRDs)). In some embodiments, an AAV capsid protein as described herein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, and comprises one or more amino acid insertion between position 587 and 588 of SEQ ID NO: 1. In some embodiments, a recombinant adeno-associated virus (rAAV) comprising an AAV capsid protein as described herein transduces retinal cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells) via a less invasive route (e.g., intravitreal injection (IVT)) relative to other more invasive route (e.g., subretinal injections). In some embodiments, injection (e.g., intravitreal injection) of a rAAV comprising a capsid protein described herein and a transgene to a subject results in enhanced pan-retinal transduction (e.g., relative to rAAV comprising wildtype AAV2 capsid or an AAV2.7m8 capsid protein). In some embodiments, a rAAV comprising a capsid protein described herein is less immunogenic (e.g., relative to (e.g., relative to rAAV comprising wild-type AAV2 capsid or an AAV2.7m8 capsid protein).

AAV Capsid Proteins

In some embodiments, the disclosure relates to AAV capsid proteins grafted with a cell penetrating peptide (CPP) (e.g., any one of the CPPs described in Table 1) in a wild-type capsid protein. “Graft” or “grafting” or “grafted, as used herein, refers to insertion of a peptide between two amino acid residues in an AAV capsid protein. In AAV, the cap gene encodes the three structural proteins of the AAV capsid. Differential splicing yields major and minor spliced products. The AAV VP1, VP2, and VP3 are translated from the same mRNA transcribed from the p40 promoter, and the three capsid proteins differ only in their N-terminal region. In some embodiments, VP3 capsid protein is the most abundant in an AAV capsid.

In some embodiments, a CPP is grafted into a wild type AAV2 capsid protein. In some embodiments, a wild type AAV2 VP1 protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, a wild type AAV2 VP2 protein comprises the amino acids 138- 735 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, a wild type AAV2

VP3 protein comprises amino acids 203-735 of the amino acid sequence of SEQ ID NO: 1.

In some embodiments, a CPP is grafted (i.e., inserted) into a wild type AAV2 capsid protein between amino acid residues 587 and 588 of an AAV2 VP1 protein (amino acid residues 450 and 451 in an AAV2 VP2 protein; amino acid residues 385 and 386 in an AAV2 VP3 protein). Amino acid residues 587 and 588 in an AAV2 VP1 protein is present at an overlapping region among AAV2 VP1, VP2, and VP3 proteins. Accordingly, inserting a CPP between amino acid residues 587 and 588 of an AAV2 VP1 protein results in VP1, VP2, and VP3 capsid proteins having the inserted CPP. A wild-type AAV2 VP1 capsid protein is set forth in SEQ ID NO: 1 (VP2 sequence underlined; VP3 sequence underlined and italicized; amino acid residues 587 and 588 bolded, underlined, and italicized).

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEP VNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEP L G L VE E P VKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPP AAP S GLGTNTMA TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTS TR TWALP TYNNHL YKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGS QAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNG RDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQY GSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNY NKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 1).

In some embodiments, a CPP is a peptide having a net positive charge, which enables it to penetrate cells (see., e.g., Herce et al., Cell Penetrating Peptides: How Do They Do It? J Biol Phys. 2007 Dec; 33(5-6): 345-356). In some embodiments, a CPP penetrates cells while carrying a cargo (e.g., proteins, oligonucleotide, or drugs). In some embodiments, grafting a CPP into an AAV capsid protein (e.g., AAV2 capsid protein) increases the transduction efficiency to a target cell and retinal pigment epithelium (RPE) cells). In some embodiments, a CPP described herein is between 3 and 42 amino acids in length (e.g., 3-42 amino acids, 3-40 amino acids, 3-35 amino acids, 3-30 amino acids, 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-10 amino acids, 3-5 amino acids, 5-42 amino acids, 5-40 amino acids, 5-35 amino acids, 5-30 amino acids, 5-25 amino acids, 5-20 amino acids, 5-18 amino acids, 5-15 amino acids, 5-12 amino acids, 5-10 amino acids, 5-8 amino acids, 8-18 amino acids, 8-15 amino acids, 8-12 amino acids, 8-10 amino acids, 10-18 amino acids, 10-16 amino acids, 10-15 amino acids, 10-12 amino acids, 12-18 amino acids, 12-16 amino acids, 12-15 amino acids, 15-18 amino acids, 15- 16 amino acids, 10-42 amino acids, 10-40 amino acids, 10-35 amino acids, 10-30 amino acids, 10-25 amino acids, 10-20 amino acids, 15-42 amino acids, 15-40 amino acids, 15-35 amino acids, 15-30 amino acids, 15-25 amino acids, 15-20 amino acids, 20-42 amino acids, 20-40 amino acids, 20-35 amino acids, 20-30 amino acids, 20-25 amino acids, 25-42 amino acids, 25- 40 amino acids, 25-35 amino acids, 25-30 amino acids, 30-42 amino acids, 30-40 amino acids, 30-35 amino acids, 35-42 amino acids, 35-40 amino acids, or 40-42 amino acids). In some embodiments, a CPP has an overall net positive charge, which can be calculated by suitable methods (e.g., by determining the total number of photos and the total number of electrons from all amino acids in the CPP, and subtracting the total number of electrons from the total number of photons to get the net charge).

In some embodiments, a CPP comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of the CPPs set forth in Table 1. In some embodiments, a CPP comprises the amino acid sequence of any one of the CPPs set forth in Table 1. In some embodiments, a CPP comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2-39. In some embodiments, a CPP comprises the amino acid sequence of any one of the CPPs set forth in Table 1.

Table 1. Amino Acid Sequences of Exemplary CPPs and the Capsid proteins comprising the CPPs

In some embodiments, a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a wild type AAV2 capsid protein (e.g., VP1 protein, VP2 protein, or VP3 protein) and comprises an insertion of a CPP between amino acid residue 587 and 588 of SEQ ID NO: 1. In some embodiments, a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a wild type AAV2 capsid protein (e.g., VP1 protein, VP2 protein, or VP3 protein) and comprises an insertion of a CPP comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2-39 between amino acid residue 587 and 588 of SEQ ID NO: 1. In some embodiments, a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a wild type AAV2 capsid protein (e.g., VP1 protein, VP2 protein, or VP3 protein) and comprises an insertion of a CPP consists of the amino acid sequence of any one of SEQ ID NOs: 2-39 between amino acid residue 587 and 588 of SEQ ID NO: 1. In some embodiments, a capsid protein described herein comprises an insertion of a CPP comprises or consists of KLGVM (SEQ ID NO: 2) between amino acid residue 587 and 588 of SEQ ID NO: 1.

In some embodiments, a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the AAV capsid protein (e.g., VP1, VP2, or VP3) in Table 1. In some embodiments, a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 40-77. In some embodiments, a capsid protein described herein comprises the amino acid sequence of SEQ ID NO: 40.

In some embodiments, a capsid protein described herein is not a capsid protein described in WO2018160686 (e.g., AAV2.7m8). An AAV2.7m8 capsid protein has an insertion of a peptide consists of amino acids LALGETTRPA (SEQ ID NO: 134) between amino acids residues 587 and 588 of a wild-type AAV2 VP1 protein as set forth in SEQ ID NO: 1 (see, e.g., AAV2.7m8 capsid protein as described in WO2018160686). For example, an AAV2.7m8 capsid VP1 protein is set forth in SEQ ID NO: 78. In some embodiments, a capsid protein described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 78. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEP VNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEP LGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPP AAP S GLGTNTMA TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTS TR TWALP TYNNHL YKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGS QAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNG RDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQY GSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL ( SEQ ID NO 78 ) .

In some embodiments, a capsid protein described herein can be produced by inserting the coding sequence for the CPP to between the codon encoding the asparagine at amino acid 587 (e.g., codon AAC as shown below in SEQ ID NO: 73) and the arginine at amino acid 588 (e.g., codon AGA as shown below in SEQ ID NO: 73) in a nucleic acid sequence encoding a wild type AAV2 protein. In some embodiments, a capsid protein described herein can be produced by inserting the coding sequence for the CPP to between nucleotides 1761 and 1762 of SEQ ID NO: 79. A nucleic acid sequence encoding the wild type AAV2 protein is set forth in SEQ ID NO: 79 (Codon AAC encoding N587 and codon AGA encoding R588 underlined).

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGT GGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAG GGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCG GTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCG GAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGA TACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCT CTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTC CTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATT GAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCA GCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACA ATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGAT GGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTC TACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCC CTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACT CATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTC AAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGG TGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCC GCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGT CAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCG GAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAG CCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACA AACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACA TTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGAC ATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGC AGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTT TTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGA AAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTAT GGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACAC AAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGC AAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAA CACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCA GTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGA GTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTAC AACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCA TTGGCACCAGATACCTGACTCGTAATCTGTAA ( SEQ ID NO : 79 )

In some embodiments, a capsid protein described herein is encoded by a nucleic acid sequence set forth in Table 2.

Table 2 Nucleic acid sequence encoding the AAV Capsid proteins. -Z1 -

In some embodiments, the disclosure provides isolated nucleic acids for encoding a capsid protein described herein. It is understood that all VP1, VP2, VP3 capsid proteins of a particular AAV capsid can be encoded by the same coding sequence. In some embodiments, a nucleic acid encoding a capsid protein described herein comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 80-117. In some embodiments, a nucleic acid encoding a capsid protein comprising the amino acid sequence of SEQ ID NO: 1 comprises the nucleic acid sequence of SEQ ID NO: 80.

Recombinant A A Vs (rAAVs)

Adeno-associated virus (AAV) is a small (~26 nm) replication-defective, non-enveloped virus that generally depends on the presence of a second virus, such as adenovirus or herpes virus, for its growth in cells. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. The disclosure, in some aspects, provides rAAVs comprising a capsid protein described that show enhanced tissue targeting (e.g., to retinal cells such as photoreceptors and RPE cells) capabilities for gene therapy (e.g., for treating IRDs) and research applications.

In some embodiments, an rAAV comprises a capsid protein described herein (e.g., any one of the AAV capsid protein described in Table 1 or a variant thereof) and an isolated nucleic acid encoding a transgene (e.g., any one of the transgenes associated with a retinal disease such as an IRD).

In some embodiments, an rAAV comprises a capsid protein comprising the amino acid sequence of any one of SEQ ID NOs: 40-77, and an isolated nucleic acid encoding a transgene (e.g., a therapeutic transgene, for example a transgene encoding a gene product associated with a retinal disease such as an IRD).

In some embodiments, an rAAV described herein has enhanced cell penetration capability (e.g., enhanced cell penetration by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) relative to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid. In some embodiments, an rAAV described herein has enhanced cell penetration capability (e.g., enhanced cell penetration by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) when administered to a subject via a less invasive route (e.g., intravitreal injection as opposed to subretinal injection) relative to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid. In some embodiments, an rAAV described herein has enhanced transduction efficiency (e.g., enhanced transduction efficiency by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) relative to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid. In some embodiments, an rAAV described herein has enhanced transduction efficiency (e.g., enhanced transduction efficiency by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) when administered to a subject via a less invasive route (e.g., intravitreal injection as opposed to subretinal injection) relative to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid.

In some embodiments, an rAAV described herein has reduced immunogenicity (e.g., reduced immunogenicity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) administered to a subject as compared to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid.

In some aspects, the disclosure provides isolated AAVs. As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially obtained or produced. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”. Recombinant AAVs (rAAVs) preferably have tissue- specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s) (e.g., retinal cells such as photoreceptors and/or RPE cells). The AAV capsid is an important element in determining these tissue-specific targeting abilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.

Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772), the contents of which are incorporated herein by reference in their entirety). Typically the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein (e.g., a nucleic acid encoding a capsid protein having a sequence as set forth in any one of SEQ ID NOs: 80-117) or fragment thereof; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by a cap gene of an AAV. In some embodiments, AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which may be expressed from a single cap gene. Accordingly, in some embodiments, the VP1, VP2 and VP3 proteins share a common core sequence. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the protein shell is primarily comprised of a VP3 capsid protein. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner. In some embodiments, VP1 and/or VP2 capsid proteins may contribute to the tissue tropism of the packaged AAV. In some embodiments, the tissue tropism of the packaged AAV is determined by the VP3 capsid protein. In some embodiments, the tissue tropism of an AAV is enhanced or changed by modification (e.g., insertion of a CPP as described herein) occurring in the capsid proteins.

In some embodiments, AAV variants described herein may be useful for delivering gene therapy to tissue or cells of the eye (e.g., retinal cells). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of disorders affecting the eye (e.g. retinal disorder). A disorder of the eye (e.g., retinal disorder) may affect the retina. A disorder of the eye (e.g., retinal disorder) may be of a genetic origin, either inherited or acquired through a somatic mutation. Non-limiting examples of disorders and diseases affecting the eye (e.g., retina) include, but are not limited to: age-related macular degeneration, diabetic retinopathy, Leber congenital amaurosis (LCA), X-linked retinitis pigmentosa (RP), achromatopsia, Stargardt disease, or cone-rod dystrophy (CRD).

The components to be cultured in the host cell to package a rAAV may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). In some embodiments, a single nucleic acid encoding all three capsid proteins (e.g., VP1, VP2 and VP3) is delivered into the packaging host cell in a single vector. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al, J. Virol., 70:520- 532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, rAAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (e.g., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell (e.g., across the cell membrane). A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV vector, an accessory function vector (e.g., rep/cap vector), or other transfer DNA associated with the production of rAAVs. The term includes the progeny of the original cell that has been transfected. Thus, a “host cell” as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

Cells may also be transfected with a vector (e.g., helper vector) that provides helper functions to the AAV. The vector providing helper functions may provide adenovirus functions, including, e.g., Ela, Elb, E2a, and E4ORF6. The sequences of adenovirus gene providing these functions may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 8, 9, 12 and 40, and further including any of the presently identified human types known in the art. Thus, in some embodiments, the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and/or AAV packaging.

As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment (e.g., nucleic acid sequence) to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from a transcribed gene.

In some cases, an isolated capsid gene can be used to construct and package recombinant AAVs, using methods well known in the art, to determine functional characteristics associated with the capsid protein encoded by the gene. For example, isolated capsid genes can be used to construct and package a recombinant AAV (rAAV) comprising a reporter gene (e.g., B- Galactosidase, GFP, Luciferase, etc.). The rAAV can then be delivered to an animal (e.g., mouse) and the tissue targeting properties of the novel isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g., heart, liver, kidneys) of the animal. Other methods for characterizing the novel isolated capsid genes are disclosed herein and still others are well known in the art.

The foregoing methods for packaging recombinant vectors in desired AAV capsids to produce the rAAVs of the disclosure are not meant to be limiting and other suitable methods will be apparent to the skilled artisan.

In some embodiments, a rAAV described herein also comprises an isolated nucleic acid encoding a transgene. In some embodiments, a recombinant AAV vector comprising the isolated nucleic acid encoding the transgene can be used to packaging the rAAV for transgene expression in a cell. “Recombinant AAV (rAAV) vectors” of the disclosure are typically composed of, at a minimum, a transgene (e.g., a transgene encoding one or more gene products) and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner that permits transgene transcription, translation, and/or expression in a cell of a target tissue.

In some embodiments, the isolated nucleic acid comprises inverted terminal repeats. The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and/or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene, and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR. In some embodiments, an isolated nucleic acid encoding a transgene is flanked by AAV ITRs (e.g., in the orientation 5’-ITR-transgene-ITR-3’). In some embodiments, the AAV ITRs are selected from the group consisting of AAV 1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR. In some embodiments, the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS). The term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR, or AITR). Without wishing to be bound by any particular theory, a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648- 1656. In some embodiments, vectors described herein comprise one or more AAV ITRs, and at least one ITR is an ITR variant of a known AAV serotype ITR. In some embodiments, the AAV ITR variant is a synthetic AAV ITR (e.g., AAV ITRs that do not occur naturally). In some embodiments, the AAV ITR variant is a hybrid ITR (e.g., a hybrid ITR comprises sequences derived from ITRs of two or more different AAV serotypes).

In some embodiments, the rAAVs of the present disclosure are pseudotyped rAAVs. Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle. With this method, the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles. In some aspects, a pseudotyped rAAV comprises nucleic acids from two or more different AAVs, wherein the nucleic acid from one AAV encodes a capsid protein and the nucleic acid of at least one other AAV encodes other viral proteins and/or the viral genome. In some embodiments, a pseudotyped rAAV refers to an AAV comprising an inverted terminal repeat (ITR) of one AAV serotype and a capsid protein of a different AAV serotype. For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX/Y (e.g., AAV2/1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.

In addition to the major elements identified above for the recombinant AAV vector, the vector also includes conventional control elements necessary which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters that are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory sequences are said to be “operably” linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5’ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame. In some embodiments, operably linked coding sequences yield a fusion protein. In some embodiments, operably linked coding sequences yield a functional RNA e.g., shRNA, miRNA, miRNA inhibitor).

For nucleic acids encoding proteins, a poly adenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence. A rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein that contains more than one polypeptide chains. Selection of these and other common vector elements are conventional, and many such sequences are available.

The precise nature of the regulatory sequences needed for gene expression in host cells may vary between species, tissues, or cell types, but shall in general include, as necessary, 5’ non-transcribed and 5’ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like. Especially, such 5’ non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the disclosure may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline -repressible system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al, J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue- specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

In some embodiments, a nucleic acid described herein utilizes a tissue-specific promoter (e.g., to promote expression of a transgene in a tissue-specific manner). In some embodiments, a tissue-specific promoter is retinal specific promoter. Examples of retinal specific promoters include rhodopsin, CB6, rhodopsin kinase (GRK1), cGMP phosphodiesterase (PDE), GRM6, RP1, calcium binding protein 5 (CABP5), Nefh, IRBP/GNAT2, PDE6B, RPB, red cone opsin, BEST1, ProA7, ProC29, cellular retinaldehyde binding protein (CRALBP), macular dystrophy 2 gene (VMD2), Rpr65, or human cone arrestin (hCAR) promoter.

In some embodiments, the regulatory sequences impart tissue- specific gene expression capabilities. In some cases, the tissue- specific regulatory sequences bind tissue- specific transcription factors that induce transcription in a tissue specific manner. Such tissue- specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, a gastrointestinal-specific mucin-2 promoter, an eye-specific retinoschisin promoter, an eye-specific K12 promoter, a respiratory tissue-specific CC10 promoter, a respiratory tissue-specific surfactant protein C (SP-C) promoter, a breast tissue- specific PRC1 promoter, a breast tissue-specific RRM2 promoter, a urinary tract tissue-specific uroplakin 2 (UPII) promoter, a uterine tissue-specific lactoferrin promoter, or a cardiac Troponin T (cTnT) promoter. Other exemplary promoters include Betaactin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron- specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron- specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others which will be apparent to the skilled artisan.

In some embodiments, one or more bindings sites for one or more of miRNAs are incorporated in a transgene of a rAAV vector, to inhibit the expression of the transgene in one or more tissues of a subject harboring the transgene. The skilled artisan will appreciate that binding sites may be selected to control the expression of a transgene in a tissue specific manner. For example, binding sites for the liver- specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. The target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region. Typically, the target site is in the 3’ UTR of the mRNA. Furthermore, the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may result in the cooperative action of multiple RISCs and provide highly efficient inhibition of expression. The target site sequence may comprise a total of 5-100, 10-60, or more nucleotides. The target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site.

The composition of the transgene sequence of the rAAV vector will depend upon the use to which the resulting vector will be put. For example, one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal. In another example, the transgene encodes a therapeutic protein or therapeutic functional RNA. In another example, the transgene encodes a protein or functional RNA that is intended to be used for research purposes, e.g., to create a somatic transgenic animal model harboring the transgene, e.g., to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of disease. Appropriate transgene coding sequences will be apparent to the skilled artisan.

Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding P-lactamase, P -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP, EGFP), chloramphenicol acetyltransferase (CAT), luciferase (e.g., Firefly luciferase), and others well known in the art. When associated with regulatory elements which drive their expression, the reporter sequences, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for P-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer. Such reporters can, for example, be useful in verifying the tissue- specific targeting capabilities and tissue specific promoter regulatory activity of an rAAV.

In some aspects, the disclosure provides rAAV for use in methods of preventing or treating one or more genetic deficiencies or dysfunctions in a mammal, such as for example, a polypeptide deficiency or polypeptide excess in a mammal, and particularly for treating or reducing the severity or extent of deficiency in a human manifesting one or more of the disorders linked to a deficiency in such polypeptides in cells and tissues (e.g., polypeptides associated with retinal diseases such as IRDs). The method involves administration of an rAAV that encodes one or more gene products (e.g., therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, etc.) in a pharmaceutically-acceptable carrier to the subject in an amount and for a period of time sufficient to treat the deficiency or disorder in the subject suffering from such a disorder.

Thus, the disclosure embraces the delivery of rAAV encoding one or more peptides, polypeptides, or proteins, which are useful for the treatment or prevention of disease states in a mammalian subject. Exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors.

The rAAV vectors may comprise a gene to be transferred to a subject to treat a disease associated with reduced expression, lack of expression or dysfunction of the gene. The rAAV vectors may comprise a gene to be transferred to a subject to treat a retinal disease associated with reduced expression, lack of expression or dysfunction of the gene. Exemplary genes and associated retinal disease states include, but are not limited to: PRPh2, MERTK, retinal- pigmented epithelium 65 (RPE65), retinoschisis 1 (RSI), cyclic nucleotide-gated cation channel alpha-3 (CNGA3), CNGB3, CHM, Rab escort protein 1 (REP-1), ATP binding cassette subfamily A member 4 (ABCA4), myosin VIIA (MY07A), VEGF, RGX 314, ADVM 022, phosphatidylinositol glycan anchor biosynthesis class F protein (PIGF), aflibercept protein, CEP290 (LCA10), cone-specific arrestin-3 (ARR3), Retinitis Pigmentosa GTPase Regulator (RPGR), Phosphodiesterase 6 (PDE6), Retinaldehyde-binding protein 1 (RLBP1), LCA2, or ND4.

The skilled artisan will also realize that in the case of transgenes encoding proteins or polypeptides, mutations that result in conservative amino acid substitutions may be made in a transgene to provide functionally equivalent variants or homologs of a protein or polypeptide. In some aspects the disclosure embraces sequence alterations that result in conservative amino acid substitution of a transgene. In some embodiments, the transgene comprises a gene having a dominant negative mutation. For example, a transgene may express a mutant protein that interacts with the same elements as a wild-type protein, and thereby blocks some aspect of the function of the wild-type protein. Useful transgene products also include miRNAs. miRNAs and other small interfering nucleic acids regulate gene expression via target RNA transcript cleavage/degradation or translational repression of the target messenger RNA (mRNA). miRNAs are natively expressed, typically as final 19-25 non-translated RNA products. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated regions (UTR) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors that are subsequently processed into a miRNA duplex, and further into a “mature” single stranded miRNA molecule. This mature miRNA guides a multiprotein complex, miRISC, which identifies target sites, e.g., in the 3' UTR regions, of target mRNAs based upon their complementarity to the mature miRNA. miRNA binds to and leads to degradation of the mRNAs it targets and, as a result, inhibits expression of the polypeptides encoded by the mRNAs. Thus, blocking (partially or totally) the presence of the miRNA (e.g., silencing the miRNA) can effectively induce, or restore, expression of a polypeptide whose expression is inhibited (derepress the polypeptide). In one embodiment, derepression of polypeptides encoded by mRNA targets of a miRNA is accomplished by inhibiting the miRNA activity in cells through any one of a variety of methods. For example, blocking the activity of a miRNA can be accomplished by hybridization with a small interfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge, TuD RNA) that is complementary, or substantially complementary to, the miRNA, thereby blocking interaction of the miRNA with its target mRNA. As used herein, a small interfering nucleic acid that is substantially complementary to a miRNA is one that is capable of hybridizing with a miRNA, and blocking the miRNA’ s activity. In some embodiments, a small interfering nucleic acid that is substantially complementary to a miRNA is a small interfering nucleic acid that is complementary to the miRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases. In some embodiments, a small interfering nucleic acid sequence that is substantially complementary to a miRNA, or is a small interfering nucleic acid sequence that is complementary to the miRNA with at least one base.

A “miRNA Inhibitor” is an agent that blocks miRNA function, expression and/or processing. For instance, these molecules include, but are not limited to, microRNA specific antisense oligonucleotides, microRNA sponges, tough decoy RNAs (TuD RNAs), and microRNA oligonucleotides (double-stranded, hairpin, short oligonucleotides) that inhibit miRNA interaction with a Drosha complex. MicroRNA inhibitors can be expressed in cells from a transgene of a rAAV vector, as discussed above. MicroRNA sponges specifically inhibit miRNAs through a complementary heptameric seed sequence. Other methods for silencing miRNA function (derepression of miRNA targets) in cells will be apparent to one of ordinary skill in the art.

In some embodiments, the cloning capacity of the recombinant RNA vector may limit a desired coding sequence and may require the complete replacement of the virus's 4.8 kilobase genome. Large genes may, therefore, not be suitable for use in a standard recombinant AAV vector, in some cases. The skilled artisan will appreciate that options are available in the art for overcoming a limited coding capacity. For example, the AAV ITRs of two genomes can anneal to form head to tail concatemers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript. Other options for overcoming a limited cloning capacity will be apparent to the skilled artisan.

Methods

In some aspects, the disclosure provides compositions (e.g., rAAVs comprising a capsid protein described herein) and methods for treating retinal diseases. In some embodiments, the retinal disease is an inherited retinal disease (IRDs). Inherited retinal diseases (IRDs) are a heterogeneous group (e.g., clinically and genetically) of disorders characterized by photoreceptor degeneration or dysfunction. In some embodiments, subjects with IRDs present with severe vision loss (see, e.g., Georgiou, Inherited retinal diseases: Therapeutics, clinical trials, and end points — A review, Clinical & Experimental Ophthalmology, Volume 49, Issue 3 p. 270-288). In some embodiments, IRD are divided into two sub-types: stationary and progressive. In some embodiments, a stationary IRD (e.g., cone and rod dysfunction syndromes) is congenital or early-infantile onset and give rise to predominantly cone or rod dysfunction. In some embodiments, a progressive IRD (e.g., progressive cone dystrophy (COD), cone -rod dystrophy (CORD), and rod-cone dystrophy (RCD)) is usually of later-onset. In other embodiments, a progressive IRD is congenital onset (e.g., Leber congenital amaurosis/early onset severe retinal dystrophy (LCA/EOSRD)). In some embodiments, a subject having LCA/EOSRD is blind from birth or early infancy. Non-limiting examples of IRD include macular dystrophies (e.g., Stargardt disease (STGD), Best disease (BD), X-linked retinoschisis (XLRS), Pattern dystrophy (PD), Sorsby fundus dystrophy (SFD), etc.), cone dysfunction syndromes (e.g., Achromatopsia, Blue cone monochromatism (BCM), Bornholm eye disease (BED), etc.), cone and cone-rod dystrophies (e.g., AD GUCAlA-associated COD/CORD, AD GUCY2D-associated COD/CORD, Autosomal dominant PRPH2-associated CORD, Autosomal recessive ABCA4-associated COD/CORD, X-linked RPGR-associated COD/CORD, etc.), ROD dysfunction syndromes (e.g., Complete and incomplete congenital stationary night blindness (cCSNB/iCSNB), Fundus Albipunctatus (FA), etc.), rod-cone dystrophies (e.g., retinitis pigmentosa (RCD), MERTK-RCD, MYO7A-RCD, USH2A-RCD, PDE6B-RCD, RLBP1-RCD, RHO-RCD, RPGR-RCD, RP2-RCD, Enhanced S-cone syndrome (ESCS), Bietti crystalline comeoretinal dystrophy (BCD), etc.), leber congenital amaurosis (lea) and early-onset severe retinal dystrophy (EOSRD) (e.g., GUCY2D - LCA/EOSRD, CEP290 - LCA/EOSRD, RPE65 - LCA/EOSRD, AIPL1 - LCA, etc.), chorioretinal dystrophies (e.g., Choroideremia (CHM)).

In some embodiments, a retinal disease is not an IRD. A non-IRD regina disease include but are not limited to Age-related macular degeneration (AMD), or diabetic retinopathy. In some embodiments, the disclosure provides methods for treating IRD (e.g., any of the IRD described herein), AMD or diabetic retinopathy, the method comprising administering the subject an effective amount of a rAAV or a composition comprising a rAAV described herein.

The rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. The rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments a host animal does not include a human.

Delivery of the rAAVs to a mammalian subject may be by, for example, intravitreal, subretinal, intramuscular injection or by administration into the bloodstream of the mammalian subject such as intravenous injection. In some embodiments the rAAV is delivered to the eye. In some embodiments, the rAAV is delivered to the eye via intravitreal injection. In some embodiments, the rAAV is delivered to the eye via subretinal injection. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Pharmaceutical Compositions

The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g.. a second rAAV encoding having one or more different transgenes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes).

Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ e.g., retina), intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.

The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.

An effective amount of an rAAV is an amount sufficient to target infect an animal or target a desired tissue (e.g., retinal cells such as photoreceptors or RPE cells). In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary between animals or tissues. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 10⁷ to 10¹⁶ genome copies. In some embodiments the rAAV is administered at a dose of 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per subject. In some embodiments the rAAV is administered at a dose of 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per kg. In some cases, a dosage between about 10¹¹ to 10¹² rAAV genome copies is appropriate. In some embodiments the rAAV is administered to the retinal cells (e.g., photoreceptors or RPE cells) e.g., by intravitreal injection at a dose of about 10⁹ to 10¹⁶ genome copies per subject. In some embodiments, the rAAV is administered to the retina (e.g., by intravitreal injection) at a dose of about 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or 10¹⁶ genome copies per subject. In some embodiments, the rAAV is administered to the retina (e.g., by intravitreal injection) at a dose of IxlO⁹ genome copies per subject. In some embodiments, the rAAV is administered to the retina (e.g., by intravitreal injection) at a dose of 3.6xl0⁹ genome copies per subject. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~10¹³ gc/mL or more). Methods for reducing aggregation of rAAVs are well-known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)

Formulation of pharmaceutically acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, , intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAVs. In some embodiments, a preferred mode of administration is by portal vein injection. In some embodiments, a preferred mode of administration is by facial vein injection.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500. ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Sonophoresis (z.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899).

EXAMPLES

Example 1: Engineering adeno-associated virus 2 (AAV2) capsid proteins with cell-penetrating peptides for enhanced retinal transduction via intravitreal injection

Recombinant adeno-associated virus (rAAV) has emerged as a key delivery vector for both experimental and clinical gene therapies, particularly for treating inherited retinal diseases (IRDs), due to its favorable safety profile. Following the approval by the US Food and Drug Administration (FDA) of Luxturna, the first rAAV-based gene therapy product for Leber congenital amaurosis type 2 (LCA2), many rAAV-based clinical trials for other IRDs are currently underway. IRDs primarily cause degeneration of two outer retinal cell types including photoreceptors and retinal pigment epithelium (RPE) cells due to various genetic mutations. Both cells play crucial roles in phototransduction and retina metabolism. Current rAAV-based gene therapy mainly targets these two cell types through subretinal injection.

However, concerns have arisen regarding the low transduction efficacy and vector-induced immunogenicity of rAAV. Besides, subretinal injection, while effective, is invasive and only transduces a limited number of photoreceptors and RPEs, significantly reducing therapeutic efficacy. Consequently, efforts are being directed towards engineering novel capsids capable of photoreceptor and RPE transduction via intravitreal (IVT) injection, a non-invasive and easily administered route with access to the entire retina. The major limitation for IVT injection of rAAV2-based vectors is its inability to transduce photoreceptors and RPEs due to strong binding of enriched heparan sulfate proteoglycans (HSPGs) in the inner limiting membrane (ILM), a major structural barrier for rAAV2- based ocular gene therapy through IVT route. One promising development is the engineered AAV2 capsid 7m8 which has demonstrated the capability to transduce photoreceptors via ITV route in mice. 7m8 was discovered through direct evolution-based screening of a seven-amino-acid library inserted at the 3-fold spike region of AAV2, specifically N587 of VP1. However, its robust photoreceptor transduction observed in mice was not replicated in non-human primates (NHPs), where transduction was limited to the macula and peripheral retina. This was likely due to the absence of or significantly thinner ILM in these regions compared to the central retina.

Cell-penetrating peptides (CPPs) are short amino acid sequences, often rich in positively charged residues like arginine, which enable them to interact with and penetrate negatively charged cell membranes. Originally inspired by viral proteins like TAT, CPPs have been synthetically engineered to enhance the delivery of therapeutic molecules such as drugs and genes. Cationic CPPs like polyarginine enter cells through mechanisms such as micropinocytosis or direct membrane interaction, while amphiphilic CPPs leverage both hydrophilic and hydrophobic regions to exploit the amphipathic nature of cell membranes for cargo delivery. These peptides can be optimized for enhanced uptake, stability, and reduced toxicity. In ocular drug delivery, CPPs present a promising non-invasive alternative as eye drops to repeated intravitreal injections for conditions such as age- related macular degeneration (AMD). A study has shown that CPP-conjugated anti- vascular endothelial growth factor drugs delivered topically can be as effective as intravitreal injections in AMD animal models. A similar approach in designing AAV9 variants with CPPs resulted in two variants, AAV.CPP.16 and AAV.CPP.21, demonstrating improved transduction of CNS cells in both mice and macaques, showing promise for delivering therapeutic payloads such as in glioblastoma models. However, whether modifying AAV2, a commonly used vector in retinal gene therapy, with CPPs can enhance retinal cell transduction via intravitreal injection remains unclear.

Displaying random 7-mer peptides on the capsid is a standard technique in AAV capsid engineering via directed evolution. This method has led to the development of several tissue-specific, high-performing capsids, such as AAV.PHP.B and AAV. PHP. eB for CNS transduction and AAV2.7m8 for retinal applications. To date, peptides of varying lengths have not generally been used for capsid bio-panning in vivo, likely due to the inherent limitations in variant diversity and the challenges related to the efficient synthesis and cloning of DNA libraries and the tolerability of longer peptide insertions within the capsid. In this study, a combined rational design and semi-directed evolution approach was utilized, efficiently constructing a library of AAV2 capsid variants featuring a wide range (3- to 42-mer) of known CPPs with varying lengths inserted at HSPG-binding regions. The library underwent two rounds of screening via intravitreal injection in mice, leading to the identification of AAV2.CPP1 (a 5-mer insertion), a variant with enhanced pan-retinal transduction and reduced immune response compared to AAV2.7m8. Notably, AAV2.CPP1 demonstrated superior photoreceptor transduction. The present disclosure demonstrates that the CPP1 insertion reduces HS binding, likely facilitating greater vector penetration through the ILM and more effective retinal cell transduction. These results indicate that AAV2.CPP1 holds promise for AAV based retinal gene therapies via intravitreal injection. This example involves engineering the AAV2 capsid to enable photoreceptor transduction via IVT injection by inserting a library of cell-penetrating peptide (CPP) between amino acid residues 587 and 588 of a wild type AAV2 VP1 protein set forth in SEQ ID NO:1 (FIG. 1). CPPs are positively charged amino acid sequences that penetrate cells effectively, transporting diverse cargoes such as proteins, oligonucleotides, and drugs. Through two rounds of screening, 38 capsid variants with different CPP insertions enriched in the mouse retina at the transcriptional level were identified. Among these variants, the capsid with insertion of KLGVM (SEQ ID NO: 2), termed CPP1, demonstrated pan-retinal and enhanced photoreceptor transduction with reduced immunogenicity via IVT injection compared to the AAV2 and 7m8.

A CPP library (comprising 1090 CPPs) was synthesized as the DNA fragments and the library was cloned into the backbone encoding AAV2 Cap gene at between amino acid residues 587 and 588 of AAV2 (SEQ ID NO:1) under the regulation of the CB6 promoter (FIG. 1 and FIG. 2A). The resulting plasmid library 1 (comprising 725 CPPs) was then packaged into AAV library 1 (comprising 565 CPPs) which was intravitreally injected into the eyes of adult C57BL/6 mice. CPPs were retrieved from the retina at mRNA level one-week post-injection and cloned into the plasmid backbone, resulting in plasmid library 2 (comprising 174 CPPs) and subsequent AAV library 2 (comprising 180 CPPs). This AAV library 2 was again intravitreally injected into mouse eyes, and the CPPs (comprising 38 CPPs) were retrieved at mRNA level one-week post-injection and subjected to Next-Generation Sequencing (NGS). Enrichment analysis was performed to rank these CPPs based on the enrichment score (FIG. 2B). Among these CPPs, KLGVM (SEQ ID NO: 2) (CPP1) was selected for further validation due to its higher enrichment score (ranked #2 out of 38 CPPs) and mRNA abundance (-15.7% of entire reads) in the retina after second round screening.

To assess its transduction efficiency, rAAV encoding GFP gene controlled by either the CB6 promoter or the photoreceptor-specific promoter GRK1, in both single-stranded (ss) and self- complementary (sc) genome formats, was packaged with the CPP1 capsid as well as with AAV2 and 7m8 capsid. The titer of the resulting AAVs indicated that CPP1 can be more efficiently packaged compared to AAV2 and 7m8, indicating the positive effects of CPP1 insertion on rAAV production. IVT injection of single-stranded rAAV s at low (2.0X10⁸ vgs/eye) or high (2.0X10⁹ vgs/eye) dose demonstrated homogenous retinal transduction by ssAAV2.CPPl.CB6.GFP, assessed by fluorescent fundus images (FIG. 3A). Quantitative analysis (FIG. 3B) revealed that ssAAV2.CPPl.CB6.GFP achieved ~2-fold increase in transduction in mice receiving a high dose of viral vectors compared to other vectors. Similarly, GFP expression was uniform in the mouse eyes receiving low dose of scAAV2.CPPl.CB6.GFP (FIG. 3C), with no significant changes in GFP mRNA expression observed among groups. Table 3. Summary of titers of various AAVs packaged by AAV2, 7m8 and AAV2.CPP1 capsids.

The immunostaining results revealed that when injected at high dose, ssAAV2.CPPl.CB6.GFP uniformly transduced nearly all retinal cells across the entire retina by the fourth week following IVT injection, resulting in robust GFP expression. In contrast, retinas receiving ssAAV2.CB6.GFP and ss7m8.CB6.GFP exhibited weaker and patchier GFP expression patterns (FIG. 4A). This expression pattern aligns with the observations from fundus images, indicating that ssAAV2.CPPl.CB6.GFP achieved pan-retinal transduction. It was found that ssAAV2.CPPl.CB6.GFP transduced ~2.5-fold more photoreceptors compared to ss7m8.CB6.GFP (FIG. 4B), a well-established viral vector known for its efficacy in transducing outer retinal cell via IVT injection. Furthermore, it was found that the percentage of resident immune cells in the retina, microglia (labelled with anti-Ibal antibody), was significantly reduced in the inner plexiform layer of the retina receiving ssAAV2.CPPl.CB6.GFP compared to those receiving ss7m8.CB6.GFP (FIG. 4C), indicating a lower level of immunogenicity induced by CPP1 capsid. To specifically evaluate the CPP1 capsid's potential for photoreceptor transduction via IVT injection, viral vectors packaged with AAV2 capsid, 7m8 capsid, and CPP1 capsid, individually, all controlled by the photoreceptorspecific promoter, rhodopsin kinase (GRK1), and encoding nuclear-expressing GFP (H2BGFP) were utilized. These vectors, including ssAAV2.GRKl.H2BGFP, ss7m8.GRKl.H2BGFP, and ssAAV2.CPPl.GRKl.H2BGFP, were administered via IVT injection in mice at a dose of 2.0X10⁹ vgs/eye and retinas were collected 4 weeks post-injection. Immunostaining results revealed markedly enhanced nuclear GFP expression in the photoreceptor layers (outer nuclear layer, ONL) of eyes receiving ssAAV2.CPPl.GRKl.H2BGFP compared to those receiving ss7m8.GRKl.H2BGFP. Conversely, minimal nuclear GFP expression was observed in mice receiving ssAAV2.GRKl.H2BGFP (FIG. 4D). These findings further support the observations depicted in FIG. 4A.

The mRNA expression levels of a set of inflammatory cytokines, including tumor necrosis factor alpha (TNF-a), interleukin- 1 beta (IL-1 |3), interferon gamma (IFN-y), and interleukin-6 (IL-6) in the retina were further evaluated by the fourth week following IVT injection of different viral vectors encoding GFR Among these inflammatory cytokines, the expression level of IL-6 was increased in the retina receiving ss7m8.CB6.GFP compared to both uninjected retinas and those injected with ssAAV2.CB6.GFP (FIG. 5), indicating a robust immune response induced by ss7m8.CB6.GFP. Increased expression level of IL-6 in the retina receiving ssAAV2.CPPl.CB6.GFP was not observed (FIG. 5), indicating a lower degree of immunogenicity associated with this vector.

Altogether, the results indicated that AAV vectors packaged with CPP1 capsids enable pan- retinal and robust transduction with the capability to transduce photoreceptors via IVT injection. Compared to benchmark 7m8 vector, the CPP1 vector demonstrated the capacity to transduce nearly the entire retina and enhance photoreceptor transduction by ~2.5-fold, while significantly reducing immunogenicity. This example presents a strategy for capsid engineering, offering a novel modality to facilitate and improve transduction of outer retinal cells via IVT injection.

Example 2: Development and in vivo evaluation ofAAV2 capsid libraries featuring CPPs and photoreceptor-targeted vectors

CPPs were grafted onto AAV2 capsids to determine if they could enhance retinal cell transduction following ITV injection. First, a CPP library was curated by electing peptides with unique amino acid sequences from CPPsite2.0 database (Agrawal et al., CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides. Nucleic Acids Res. 44, D1098-D1103.) which contains approximately 1,700 unique CPPs.

The corresponding DNA sequences were synthesized, generating a library of 1 ,090 CPPs, ranging from 3-mer to 42-mer, confirmed by next-generation sequencing (NGS) and ready for cloning (Table 4).

Table 4: Primers

Following a method previously described, a diverse capsid library was constructed by grafting the CPP sequences between residues 587 and 588 in the hypervariable surface loop VIII of AAV2 capsid genes by Gibson assembly, which was combined with AAV2 ITRs and a photoreceptor- specific G-protein-coupled receptor kinase 1 (GRK1) promoter (FIG. 6A). This site permits genetic modifications without disrupting capsid assembly or genome packaging. The resulting plasmid library retained 66.4% (724/1,090) of the CPPs from the initial library. Rep proteins were provided via a separate plasmid during virus production. Viral libraries were generated in HEK293T cells under low-DNA-input conditions to minimize capsid mosaicism and cross -packaging. The viral libraries were intravitreally (IVT) injected into both eyes of adult C57BL/6 mice (n = 5). After 28 days, the retina and RPE complex were harvested and processed, and capsid library sequences were recovered by RT-PCR (FIG. 6B). Amplicon pools were recloned into GRK1 AAV vectors for a second round of selection. A limited number of capsid amplicons were detected in retinal samples from mice injected with the AAV libraries, as evidenced by the relatively weak intensity of the expected band on the gel (FIG. 6C). In contrast, saline-injected controls showed no detectable amplicons.

NGS analysis was conducted after each round of in vivo selection to assess variant diversity and enrichment in the retina. The first round reduced the number of unique sequences from 724 (plasmid library) to 84 (retinal tissue), eliminating 88% of the variants. In the second round, 76% of the remaining variants were removed, reducing the pool to 20 unique sequences in the retinal tissue (FIGs. 6D and 11A-11C; Table 5). Interestingly, many CPP sequences were absent following AAV packaging, indicating that certain CPPs with variable length and sequences may interfere with VP3 protein structure and function, rendering them non- packageable. AAV enrichment and packaging efficiency was analyzed after the second round of screening, using an enrichment score threshold of >2.0 and a yield score >0.5. The results identified a distinct CPP sequence, KLGVM (SEQ ID NO: 2), which met the selection criteria and was designated AAV2.CPP1 (FIG. 6E). To validate these findings, the original CPP library was cloned into an AAV genome driven by the CB6 promoter (CMV enhancer/CB promoter) and two rounds of in vivo selection in adult C57BL/6mice via intravitreal injection was performed. AAV2.CPP1 consistently emerged as the top candidate, meeting the selection criteria (FIGs. 6F and 12A-12C).

Table 5: The CPP sequences identified in the mouse retina after the second-round screen

Example 3: Enhanced photoreceptor transduction by single-stranded AAV2.CPP1 in mice via intravitreal injection

Several batches of rAAVs were packaged using AAV2, AAV2.7m8, and AAV2.CPP1 capsids. These carried either single-stranded (ss) genomes encoding cytoplasmic green fluorescent protein (GFP) driven by the CB6 promoter (ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6. GFP) or histone H2B-tagged GFP (nuclear- localized H2BGFP) under the control of the GRK1 promoter (ssAAV2.GRKl.H2BGFP, ssAAV2.7m8.GRKl.H2BGFP, and ssAAV2.CPPl.GRKl.H2BGFP). In addition, self- complementary(sc) AAVs encoding cytoplasmic GFP (scAAV2.CB6.GFP, scAAV2.7m8.CB6. GFP, and scAAV2.CPPl.CB6. GFP) were packaged. Vectors packaged with the CPP1 capsid showed an approximately 4-fold increase in viral titers compared to AAV2, regardless of whether the genome was single stranded or self-complementary (FIGs. 13A-13C). This indicates that the insertion of the CPP1 peptide does not interfere with AAV production. To evaluate the integrity of rAAV particles purified by iodixanol gradient purification, high- resolution transmission electron microscopy (TEM) was utilized to examine the morphology of the virions (FIG. 13B). A semi-quantitative assessment was performed by counting empty/partially full and fully packaged virions across six representative fields. The analysis revealed that the full-to-empty ratio for scAAV2.CPPl.CB6. GFP (-60%) was comparable to that of scAAV2.CB6.GFP (-70%) (FIG. 13C).

To evaluate the retinal transduction efficiency, ssAAV2.CB6.GFP, ssAAV2.7m8.CB6. GFP, and ssAAV2.CPPl.CB6. GFP were intravitreally injected (2.0xl0⁹ viral genomes [vg]/eye) into 2-month-old male C57BL/6mice (n = 5-11). Four weeks post-injection, in vivo fluorescence fundus imaging was performed to assess GFP expression. ssAAV2.CPPl.CB6. GFP exhibited robust, homogeneous GFP expression throughout the retina, in contrast to the more localized, blood-vessel-centric GFP expression observed with ssAAV2.7m8.CB6. GFP and the weaker GFP signal seen with ssAAV2.CB6.GFP (FIG. 7A). Following imaging, retinal tissues were harvested for DNA biodistribution, RNA expression analysis, and immunohistology. ssAAV2.CPPl.CB6.GFP displayed DNA biodistribution similar to that of ssAAV2.CB6.GFP and ssAAV2.7m8.CB6.GFP. However, it resulted in significantly higher GFP expression at RNA levels, increasing by 3.5- and 5.8-fold, respectively. This divergence between DNA and RNA levels indicates that the improved transgene expression by ssAAV2.CPPl.CB6. GFP was primarily driven by enhanced transcription rather than biodistribution. The elevated RNA/DNA ratios further support this finding (FIG. 7B). At a lower dose of 2.0xl0⁸ vg/eye, moderate increases in GFP expression were observed in mice receiving ssAAV2.CPPl.CB6. GFP via fluorescence fundus imaging; however, no significant differences at RNA levels were detected between the vectors (FIGs. 14A-14B).

Immunohistological analysis of GFP expression in retinal cross sections confirmed that ssAAV2.CPPl.CB6. GFP achieved uniform transduction across most retinal cell types and exhibited stronger GFP expression compared to ssAAV2.CB6.GFP and ssAAV2.7m8.CB6. GFP, which showed weaker and more patchy expression patterns (FIG. 7C). These results align with the fundus imaging findings, confirming that ssAAV2.CPPl.CB6. GFP delivers pan-retinal transduction. ssAAV2.CPPl.CB6. GFP transduced approximately 2.5 times more photoreceptors, as indicated by GFP expression in their inner or outer segments, compared to ssAAV2.7m8.CB6. GFP (FIG. 7D), a well-known vector for effective outer retinal cell transduction via intravitreal injection, while ssAAV2 rarely transduced photoreceptors. A significantly higher number of Muller cells transduced by ssAAV2.CPPl.CB6. GFP compared to other vectors was also observed, as indicated by its extended radial processes spanning the retina from the ganglion cell layer to the outer nuclear layer (FIG. 7C).

Given the challenges in assessing cytoplasmic GFP expression in photoreceptors by quantifying the number of outer segments expressing GFP, a new AAV genome encoding nuclear-localized H2BGFP was developed, driven by the GRK1 promoter. It was packaged into AAV2, AAV2.7m8, and AAV2.CPP1 capsids (ssAAV2.GRKl.H2BGFP, ssAAV2.7m8.GRKl.H2BGFP, and ssAAV2.CPPl.GRKl.H2BGFP). This approach allows for clearer and more straightforward assessment of H2BGFP expression specifically in photoreceptor nuclei (FIG. 7E). Upon intravitreal injection, H2BGFP was homogeneously expressed in the photoreceptor nuclei of eyes treated with ssAAV2.7m8.GRKl.H2BGFP and ssAAV2.CPPl.GRKl.H2BGFP, but not in those treated with ssAAV2.GRKl.H2BGFP. Quantitative analysis showed that ssAAV2.CPPl.GRKl.H2BGFP increased photoreceptor transduction nearly 4-fold compared to ssAAV2.7m8.GRKl.H2BGFP.

Example 4: Enhanced photoreceptor transduction by scAAV2.CPPl in mice via intravitreal injection

To evaluate the transduction capability of the scAAV2.CPPl, 2.0xl0⁹ vg/eye of scAAV2.CB6.GFP, scAAV2.7m8.CB6.GFP, and scAAV2.CPPl.CB6.GFP was administered intravitreally (ITV) in mice. Fundus imaging revealed a similar expression pattern in scAAV2.CPPl-treated mice, but with significantly higher GFP expression compared to the single- stranded vectors (FIG. 8A). Molecular analysis of transgene expression corroborated the findings from the single- stranded vectors. While all three vectors exhibited similar DNA biodistributions in the retina (FIG. 8B), scAAV2.CPPl.CB6. GFP showed a 5- and 2.9-fold increase in GFP expression at the RNA level compared to scAAV2.CB6.GFP and scAAV2.7m8.CB6. GFP, respectively. The increase in RNA/DNA ratios of scAAV2.CPPl.CB6. GFP was consistent with the results observed for the single-stranded vectors.

Immunohistological analysis showed that GFP expression was more uniform and pronounced with scAAV2.CPPl.CB6. GFP than with the other vectors (FIG. 8C). Photoreceptor transduction was notable in eyes receiving scAAV2.7m8.CB6. GFP, but scAAV2.CPPl.CB6. GFP led to even more extensive transduction. Quantitative analysis confirmed that scAAV2.CPPl.CB6.GFP transduced twice as many photoreceptors as scAAV2.7m8.CB6. GFP (FIG. 8D). Similar to FIG. 7C, it is observed enhanced transduction of Muller cells by scAAV2.CPPl.CB6. GFP (FIG. 8C). GFP expression was also assessed at a lower dose (2.0xl0⁸ vg/eye). Although fundus imaging showed an increase in GFP expression with both scAAV2.7m8.CB6. GFP and scAAV2.CPPl.CB6. GFP compared to that with scAAV2.CB6.GFP, no significant differences at the RNA level were observed among the vectors (FIGs. 15A-15B).

Example 5: Decreased immune responses by AAV2.CPP1 vector

Intravitreal injections of AAV vectors typically elicit stronger immune responses compared to subretinal injections. Engineered vectors, such as AAV2.7m8, appear to cause increased immune responses compared to their wild-type parent, AAV2, likely due to the higher degree of heterogeneity in vector genomes packaged by AAV2.7m8. To assess whether AAV2.CPP1 induces immune responses in the retina, ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP at a dose of 2.0xl0⁹ vg/eye were injected ITV in mice. Four weeks post-injection, retinal cross sections were analyzed using immunohistochemistry for IBA1, a marker of microglial cells. Increased IB Al staining intensity indicates microglial activation, which is predominantly seen in the inner retinal layers. A significantly higher number of IBA1 -positive microglia in the retinas of mice treated with ssAAV2.7m8.CB6.GFP compared to those treated with ssAAV2.CPPl.CB6.GFP or ssAAV2.CB6.GFP was observed (FIGs. 9A-9B). Further quantitative analysis revealed that the inner plexiform layer (IPL) of retinas treated with ssAAV2.7m8.CB6.GFP had a substantial increase in IBA1 -positive microglia, indicating that microglial cells had migrated into the IPL. In contrast, this increased immune response was not observed in retinas treated with ssAAV2.CPPl.CB6.GFP (FIG. 9B), indicating a likely reduced immunogenicity of the AAV2.CPP1 vector.

Activated microglia, along with other cell types, may upregulate proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin- 1 beta (IL- lb), interferongamma (IFN-g), and interleukin-6 (IL-6). To explore this possibility, RNA levels for these cytokines using droplet digital PCR (ddPCR) were examined in retinas 4 weeks post-injection of different GFP-encoding vectors. Among these cytokines, IL-6 expression was notably higher in retinas injected with ssAAV2.7m8.CB6.GFP compared to both uninjected controls and retinas injected with ssAAV2.CB6.GFP and ssAAV2.CPPl.CB6.GFP (FIG. 9C). This indicates that ssAAV2.7m8.CB6.GFP likely induces a robust immune response even 4 weeks post-injection. Interestingly, retinas injected with ssAAV2.CPPl.CB6.GFP did not show elevated IL-6 levels, indicating a lower degree of immunogenicity associated with the AAV2.CPP1 vector.

Example 5: Reduced. HS binding affinity ofAAV2.CPPl

AAV2 is known to utilize HSPG as its primary receptor for binding to host cells, with subsequent cell entry facilitated by proteinaceous co-receptors. Studies have shown that modifying the AAV2 capsid can enhance photoreceptor transduction via intravitreal injection by reducing its HS binding affinity, thereby enabling more vectors to cross the ILM, as demonstrated by AAV2.7m8. To investigate the mechanism behind the enhanced retinal transduction of AAV2.CPP1, the HS binding affinity of ssAAV2. CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP was assessed. Dot blot analysis using anti-HS antibody to detect vector-HS interactions revealed that AAV2 exhibited strong HS binding, while both AAV2.7m8 and AAV2.CPP1 showed significantly reduced binding to HS (FIG. 10A). This reduced HS binding in AAV2.CPP1 indicates that CPP1 may facilitate greater vector penetration across the ILM, leading to enhanced retinal cell transduction.

To further test this, a Matrigel-HS-based Transwell model was developed to partially mimic ILM by coating culture Transwells with a mixture of Matrigel and HS and seeding HeLa cells, a cell line rich in HSPG, underneath the Transwells. It is proposed that AAVs with strong HS binding would be unable to penetrate this in vitro model, thus failing to transduce the HeLa cells. Conversely, AAVs with reduced HS binding would pass through the HS-enriched Transwells and transduce the cells, resulting in detectable GFP expression (FIG. 10B). Using this system, the transduction profile of ssAAV2.CB6.GFP, ssAAV2.7m8.CB6. GFP, ssAAV2.CPPl.CB6. GFP, and ssAAV9.CB6.GFP was compared. After 48 h of infection, it was observed that in the absence of in vitro model, GFP expression was comparable among ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP, except for cells transduced by ssAAV9.CB6.GFP, which is known for poor transduction with cells enriched with HSPG. In the presence of the Matrigel-HS-based Transwell model, GFP expression in ssAAV2.CB6.GFPtreated cells was significantly reduced, whereas cells treated with ssAAV2.7m8.CB6. GFP or ssAAV2.CPPl.CB6. GFP maintained GFP expression (FIG. 10C). Quantitative analysis (FIG. 10D) confirmed these observations, showing a clear distinction in GFP expression levels among the vectors in this in vitro model. Importantly, these findings indicate that the CPP1 vector, like 7m8, exhibits reduced HS binding affinity.

The AAV2.CPP1 capsid structure was modeled by superimposing it onto the AAV2 capsid (FIGs. 10E and 10F). Notably, the KLGVM (SEQ ID NO: 2) peptide insertion disrupted the VR-VIII region, which forms the top of the second highest of the three protrusions at the three-fold axis of symmetry. This alteration potentially affects the spacing between R585 and R588 and effectively interrupts the HSPG binding motif.

Example 7: Methods

Vector construction

Library backbone vectors containing an AAV capsid expression cassette driven by either the GRK1 or the CB6 promoter were constructed using the Gibson assembly method. For the first-round screening, the backbone plasmid pAAV-Cap2-scl included inverted terminal repeats (ITRs) and a partial Rep2 C-terminal sequence, which is crucial for capsid gene splicing and protein assembly. A full-length cap2 gene was inserted between residues N587 and R588 with a unique Aflll restriction site for CPP library cloning. For the second-round screening, the backbone plasmid pAAV-Cap2-sc2 also contained ITRs and the partial Rep2 C-terminal sequence, but with a cap2 fragment that featured a unique Aflll site for cloning the CPP sequences recovered from the retina. A plasmid, pRep2-3stop, was constructed to exclusively express the Rep protein by introducing three stop codons in the Cap2 gene, thereby preventing the expression of the VP 1/2/3 proteins.

Plasmid library, AAV library construction, and virus production

A double- stranded CPP library was synthesized by GenScript, containing overlapping sequences of the Cap2 gene at the 5’ (5’-CCAACCTCCAGAGAGGCAAC-3’) (SEQ ID NO: 128) and 3’ (3’-TCTGCGGTAGCTGCTTGTCT-5’) (SEQ ID NO: 129) ends for Gibson assembly, to create the plasmid library for the first round of screening. The library was cloned into the backbone plasmid pAAV-Cap2-scl using the standard Gibson assembly method. The pAAV-Cap2-scl plasmid (2 mg) was digested with Aflll enzyme and purified using a DNA and gel purification kit (D4008, Zymo Research, USA). In a 20-mL Gibson assembly reaction, the purified linear pAAV-Cap2-scl was combined with the synthesized double- stranded CPP library inserts (backbone/inserts = 1/5 ratio) in the Gibson Assembly Master Mix (10 mL) and water. The resulting plasmid library was precipitated using isopropanol and concentrated in 5 mL of water. The library (<100 ng/mL in 1 mL) was electroporated into ElectroMAX DH10B cells (18290015, Invitrogen, USA), following the manufacturer’s instructions. Electroporated cells were pooled and cultured in 500 mL of TB medium overnight. The resulting plasmid library was extracted and purified. For the second round of screening, pAAV-Cap2-sc2 was digested with Aflll and purified in the same manner. The recovered CPP sequences from the retina (after the first round) were cloned into the pAAVCap2-sc2 using Gibson assembly to generate the second plasmid library.

AAV library production was conducted in HEK293 cells. Calcium phosphate transfection was performed using 1.5 mg of pAd-DeltaF6 (containing adenovirus helper genes), 1.0 mg of pRep2-3stop, and 100 mg of either the first or the second plasmid library in 10 roller bottles. Cells and culture medium were harvested 72 h post-transfection by scraping, and the cells were pelleted via low-speed centrifugation. The cells were lysed by undergoing three freeze-thaw cycles, and the supernatant was precipitated on ice using 1:0.3 volume of 40% polyethylene glycol solution, followed by centrifugation. The lysate and supernatant were pooled and fractionated through four rounds of iodixanol gradient purification. Buffer exchange was performed on Amicon-100 columns (Millipore) using phosphate-buffered saline (PBS). Final viral preparations were analyzed by ddPCR using a poly(A)-specific primer/probe set (Table 3), and the samples were tested via silver staining of PAGE gels. Full sequences of all the plasmids used in AAV library production can be found in the supplemental information.

Characterization of AAV particles by high-resolution TEM

The morphology of negatively stained AAV virions was analyzed using TEM at the Core Electron Microscopy Facility of the University of Massachusetts Chan Medical School, following established protocols (Gao et al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Mol. Ther. Methods Clin. Dev. 2014, 1, 20139.). Briefly, 5 mL of the AAV preparation was applied to a Formvar-coated grid and allowed to adhere for 30 s. Excess liquid was removed, and the sample was negatively stained by sequentially flowing six drops of 1% uranyl acetate over the grid, enhancing contrast and fixing the virus particles. After drying in a controlled humidity chamber, the grid was examined using TEM, and images were recorded. The resulting visualization prominently displayed AAV empty particles as characteristic donut-like shapes, formed by the accumulation of uranyl acetate stain in the capsid dimples. To assess the full/empty capsid ratio, six fields of images were analyzed, and the numbers of full and completely or partially empty virions were counted. The average counts from these fields were used to calculate the full/empty capsid ratio.

Animals

Adult male C57BL/6J mice (5-8 weeks of age) were purchased from The Jackson Laboratory and received intravitreal injections. The mice were maintained on a 12-h light/dark cycle at a temperature of 70F-74F and a humidity level of 35%-46%. They were provided with a standard chow diet (ISO-pro 300 irradiated diet, #5P76). All animal procedures in this study were approved by the UMass Chan Medical School Animal Care and Use Committee. Library screening by in vivo selection

AAV libraries were intravitreally injected into the eyes of C57BL/6J mice (n = 10) at a dose of 2.0xl0⁹ vg/eye. Four weeks post-injection, the animals were euthanized, and the retina/RPE complexes were harvested, pooled, snap-frozen in liquid nitrogen, and stored at -80C. Total retinal RNA was extracted using the DNA/RNA extraction kit (SKU 47700, Norgen Biotek). mRNA was further purified from the total RNA using the Dynabeads mRNA Purification Kit (#61006, Invitrogen). Reverse transcription was performed using 400 ng of purified mRNA and the gene-specific Cap2-RT primer (Table SI) with the SuperScript IV First- Strand Synthesis Kit (Life Technologies). To amplify the Cap sequence with CPP insertions, a nested PCR approach was used. In the first round of PCR, the target sequence was amplified using the initial set of primers (Nes-IF and Nes-IR) in multiple reactions containing 2 mL of cDNA, 25 mL of Q5 HotStart High-Eidelity 2x Master Mix (NEB), and a total reaction volume of 50 mL for 10 cycles. In the second round, amplification was performed with the second set of primers (Nes-2E and Nes-2R) in multiple reactions using 1 mL of amplicon purified and concentrated from the first-round PCR, 12.5 mL of Q5 2x Master Mix, and a total reaction volume of 25 mL for 20 cycles. The resulting amplicons were purified and concentrated for NGS preparation, using 100 ng of input amplicons in 15 cycles of amplification. The final purified amplicon was ready for NGS sequencing.

AAV vector production and intravitreal administration in mice

The leading CPP variant enriched in the retina was identified through NGS and subsequently cloned into the Rep/Cap2 plasmid at position N587. Plasmids encoding the Rep and AAV2 (Rep/Cap2), 7m8 (Rep/7m8), and AAV2-CPP (Rep/CPP) capsids were used to produce single- stranded or self-complementary AAV vectors encoding GPP or H2BGPP under the control of the CB6 or GRK1 promoters. This was achieved using the same method as for AAV library production, involving the co-transfection of HEK293 cells. A total of 1.5 mg of pAd-DeltaP6; 150 mg of pssAAV-CB6-GPP, pssAAV-GRKl-H2BGPP (a generous gift of Dr. Claudio Punzo, UMass Chan), or pscAAV-CB6-GPP; and 1.5 mg of Rep/Cap, Rep/7m8, or Rep/CPP was transfected into cells in 10 roller bottles. Seventy-two hours post-transfection, the cells were harvested, lysed, and subjected to iodixanol gradient purification and buffer exchange, following the same protocol used for the AAV libraries. The final titer was confirmed by ddPCR using a GPP probe. For in vivo experiments, viral vectors were intravitreally injected into the eyes of mice at different doses. The injections were administered using glass micropipettes (Clunbury Scientific LLC), introduced through the corneal limbus, with fast green dye added at a concentration of 0.1% to visualize the injection site. Each mouse received 1 mL of vector. Twenty-eight days post-injection, mice were anesthetized for fluorescence fundus imaging and subsequently euthanized for retinal harvesting for immunohistochemistry and molecular analysis.

NGS and bioinformatic analysis

Amplicon libraries for NGS were prepared by performing 15 cycles of PCR amplification on 100 ng of the second round of nested PCR amplicons, using Q5 High-Fidelity DNA polymerase (NEB) with the NGS- IF and NGS-1R primers. The amplicon libraries were then purified using the Zymo Gel DNA Recovery Kit (Zymo Research). The final libraries were quantified with the Qubit dsDNA HS kit (Life Technology) before being submitted for sequencing at the Massachusetts General Hospital (MGH) DNA core facility. FASTQ files generated from the sequencing were processed using Geneious Prime software, which enabled quantitative and qualitative assessment of the peptide sequence inserts.

Fluorescence fundus imaging and immunohistochemistry

To perform fundus imaging, mice were first administered one drop each of phenylephrine and tropicamide to dilate the pupils. The mice were then anesthetized via intraperitoneal injection of a ketamine/xylazine mixture (100 and 10 mg/kg). A mouse was positioned on a fundus scope platform, and a heating pad was used to maintain lens clarity by keeping the mouse warm. The platform was adjusted to position the camera directly over the eye. Appropriate wavelengths were selected to capture focused images of the retina, and images were acquired with the Micron IV system from the Phoenix Technology Group (Lakewood, CO, USA).

The eye cups, after removing the cornea, were fixed overnight at 4°C in 4% paraformaldehyde, followed by dehydration in 30% sucrose at 4°C overnight before embedding. Cryosections, 12 mm thick, were prepared as previously described (Cheng et al., Altered photoreceptor metabolism in mouse causes late-stage age-related macular degeneration-like pathologies. Proc. Natl. Acad. Sci. USA. 2020, 117, 13094-13104.). The sections were analyzed using a Leica DM6 Thunder microscope equipped with a 16-bit monochrome camera. After post-fixation and rinsing in PBS, the sections were blocked and incubated with antibodies overnight at 4C. The following antibodies were used: chicken anti-EGFP (abl3970; 1:1,000; Abeam), rabbit anti-IBAl (019-19741; 1:300; Wako), and fluorescein peanut agglutinin lectin (PNA) (FL1071; 1:1,000; Vector Laboratories). All images were visualized with a Leica DM6 Thunder microscope with a 16-bit monochrome camera. Images were processed by LAS X Life Science Microscope Software.

Quantification of photoreceptor transduction

Quantification of photoreceptor transduction in retinas treated with vectors encoding cytosolic GFP under the CB6 promoter was performed by manually counting GFP-positive photoreceptor segments. For each group of injected mice, four rectangular regions of equal area surrounding the optic nerve head were selected from a single retinal section per eye. GFP- positive segments within these selected areas were manually counted. In addition, the total number of photoreceptor nuclei in the outer nuclear layer within the same areas was determined using the Imaris software package (8.2)(Petit et al., Rod Outer Segment Development Influences AAV-Mediated Photoreceptor Transduction After Subretinal Injection. Hum. Gene Ther. 28, 464-481) Photoreceptor transduction efficiency was then calculated as the average per eye and per group by determining the ratio of GFP-positive photoreceptors to the total number of photoreceptors. For retinas treated with vectors encoding nuclear GFP under the GRK1 promoter, GFP-positive photoreceptor nuclei were manually counted across the entire retinal section. The total number of photoreceptors and the photoreceptor transduction efficiency were calculated as described previously.

Quantitative analysis of viral genome and transcripts

Snap-frozen retinas were processed for total DNA and RNA extraction using the DNA/RNA Extraction Kit (SKU 47700, Norgen Biotek). RNA was reverse transcribed into cDNA using the cDNA Reverse Transcription Kit (#4374966; Thermo Fisher Scientific). Vector DNA and cDNA were quantified using duplex TaqMan ddPCR assays targeting EGFP (assay ID Mr00660654_cn; Thermo Fisher Scientific) and Tfrc genomic sequences (#4458367; Thermo Fisher Scientific) or EGFP and Gusb cDNA (#4448490; Thermo Fisher Scientific). The ddPCR was performed on a QX200 system (Bio-Rad), and data analysis was conducted using QuantaSoft software (Bio-Rad). HS binding affinity assay and dot-plot analysis

For the HS affinity assay, 500 mL of diluted AAV vector containing l.OxlO¹⁰ vg was incubated with or without 2 mL of HS solution (100 mg/mL) for 1 h at room temperature, with shaking at 500 rpm. After incubation, 100 mL of the mixture was loaded onto a nitrocellulose membrane using a blot filtration system. The membrane was quickly rinsed once with 0.5% PBST and then blocked with blocking solution for 1 h at room temperature. HS antibody (clone F58-10E4; 1:200; Amsbio) was used for incubation overnight at 4°C. The membrane was washed three times with 0.5% PBST for 5 min each, followed by incubation with the secondary antibody (IRDye 680; 1:5,000; LLCOR). A final quick rinse with 0.5% PBST was performed before imaging.

The Matrigel-HS-based Transwell model

A Matrigel-HS-based Transwell model was established to assess the HS binding affinity of AAV2 variants. HeLa cells were seeded at a density of l.OxlO⁵ cells per well in a 24- well plate the day before AAV infection. The culture inserts (CLS3470; 6.5-mm Transwell with 0.4- mm pore polyester membrane insert; Corning) were coated with Matrigel (5 mg/cm2; CLS356231; Coming) mixed with HS solution (300 mg HS in 70 mL of Matrigel solution; S5992; Selleck Chemicals) and incubated at 37C for 5 h. After incubation, the remaining solution in the inserts was removed, and the inserts were mounted into the wells containing the seeded cells. AAV vectors encoding the GFP gene were then added to the inserts at a multiplicity of infection (MOI) of 100,000 in 100 mL of culture medium mixed with Ad5 helper vims (MOI of 100).44 GFP expression was assessed through fluorescence microscopy to evaluate transduction efficiency 2 days post-infection.

Molecular Modeling

Themonomeric stmcture of the VP1 capsid (amino acid residues 219-740) was predicted using AlphaFold 3.0 (alphafoldserver.com). The prediction was based on the AAV2 VP1 reference model (PDB:6IH9) with a resolution of 2.8 A. Default automated settings were applied to build the VP3 monomer. The predicted VP3 monomer was then aligned with the wild-type AAV2 VP3 monomer using PyMOL software. Capsid reconstruction was carried out using the Viper server (viperdb.org/Oligomer_Generator.php). Statistical analysis

Data were analyzed using GraphPad Prism version 10 (GraphPad Software) and are reported as means ± standard deviation (SD). Depending on the experimental setup, statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was defined as an adjusted p value of less than 0.05.

Sequences:

Nucleotide sequence of CPP1 (KLGVM (SEQ ID NO: 2)): AAGCTGGGCGTGATG (SEQ ID NO: 130) pAAV-Cap2-scl (6418bp)

Features:

11-140: 5’ ITR

214-505: GRK1

523-731: AAV2 Rep C-terminal sequence

1027-3225: Cap2 fragment sequence

2788-2792: Inserted digestion site of Aflll

3263-3389: Rabbit globin polyA

3607-3478: 3’ ITR

4370-5227: Ampicillin resistance

CTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCC GGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATG CTACTTATCTACCAGGGTAATGGGACGCGTGATCCTCTAGAACTATAGGGGCCCCA GAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGG AGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGAT TGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCT GGGATTTAGCCTGGTGCTGTGTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGG TCCCCAGGAACCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGA CGGGCCACAGGCCAAGGGCCGGGAGCAAGCTGCTAGCGGTCACCAAGCAGGAA GTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGA

ATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAG

ATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCA

GACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCG

TCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGA

ATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCT

TTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAAC

TGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGC

GATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAA

AACAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAG

GAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGG

CATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAAC

GGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACA

AAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCC

GACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACG

AGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGT

TAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCT

CCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAG

ACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCC

CTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA

CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGA

TGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAAC

CACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCT

ACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGA

CTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCT

CTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAA

CCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGG

CTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGT

ATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCC

TGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTT

GAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAAT

CCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCA CGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGG

AACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAAC

AACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCT

CTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCT

CAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAA

AAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCA

GTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACTTAAGAGACAAGCAGCTACCGCA

GATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTT

CAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTC

ATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTA

CCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACT

CCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACG

CTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACT

GTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACACGA

AATCTGTAATAAGCGGCCGCCTCGAGTGATCCGATCT77TTCCCTCTGCCAAAAA7TAT

GGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCA

TTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGCAATTCGAAGATCAGAA

TTTCGACCACCCATAATACCCATTACCCTGGTAGATAAGTAGCATGGCGGGTTAATC

ATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGC

TCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGC

GGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCG

TTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAG

CACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTT

CCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTA

AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCT

AGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCC

CGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC

CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTG

ATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTG

TTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGG

ATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAAC

GCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATG TGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT

GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA

TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTT

GCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA

GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCG

AAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC

CCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGA

CTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA

GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA

CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATG

TAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGC

GTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCG

AACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGT

TGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT

GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG

CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA

AATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGAkkCAGACkGkC

CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGA

TCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT

CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT

TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG

TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCA

GAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA

AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG

CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCG

GATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA

GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA

CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAAC

AGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG

TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC

GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT

GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTAT TACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGC GCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCT

TTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTT CACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGC (SEQ ID NO: 131) pAAV-Cap2-sc2 (6373bp)

Features:

11-140: 5’ ITR

214-505: GRK1

523-731: AAV2 Rep C-terminal sequence

1027-3180: Cap2 fragment sequence

2770-2774: Inserted digestion site of Aflll

3218-3344: Rabbit globin polyA

3562-3433: 3’ ITR

4325-5182: Ampicillin resistance

CTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCC

GGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATG CTACTTATCTACCAGGGTAATGGGACGCGTGATCCTCTAGAACTATAGGGGCCCCA

GAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGG

AGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGAT

TGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCT

GGGATTTAGCCTGGTGCTGTGTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGG

TCCCCAGGAACCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGA

CGGGCCACAGGCCAAGGGCCGGGAGCAAGCTGCTAGCGGTCACCAAGCAGGAA

GTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGA

ATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAG

ATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCA GACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCG

TCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGA

ATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCT

TTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAAC

TGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGC

GATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAA

AACA AATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAG

GAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGG

CATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAAC

GGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACA

AAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCC

GACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACG

AGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGT

TAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCT

CCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAG

ACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCC

CTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAA

CGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGA

TGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAAC

CACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCT

ACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGA

CTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCT

CTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAA

CCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGG

CTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGT

ATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCC

TGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTT

GAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAAT

CCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCA

CGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGG

AACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAAC

AACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCT CTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCT

CAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAA

AAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCA

GTATGGTTCTGTATCTACCTTAAGACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGAC

AGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTT

CACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCA

AGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCT

TCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAG

GAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTA

ATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCA

GATACCTGACTCGTAATCTGTAATAAGCGGCCGCCTCGAGTGATCCGATC777TTCCCT

CTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAG

GAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAA A

CGTTGATCTGAATTTCGACCACCCATAATACCCATTACCCTGGTAGATAAGTAGCAT

GGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCT

CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG

CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATT

CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTA

ATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGC

ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTG

TAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACAC

TTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT

CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAG

TGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGG

GCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAAT

AGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTT

GATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAA

CAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTT

TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA

TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGG

AAGAGAATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG

CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG TTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAG

AGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG

GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACT

ATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGG

CATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCC

AACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACA

TGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC

CAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAAC

TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA

GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATT

GCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGG

CCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACT

ATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGA

AACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTA

ATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA

ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTT

CTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC

TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA

CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAG

GCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT

TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA

CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTAT

GAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG

CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT

CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCT

CGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC

CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT

GGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGA

CCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACC

GCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA

CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCG

GATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAA

GGC (SEQ ID NO: 132) pRep2-3stop

Features:

151-750: stuffer

775-2717: Rcp2 fragment

2178-4468: Cap2 fragment

3144-3146, 3144-3146, 3363-3365: stop codon

5797-6654: Ampicillin resistance

CTCTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGAC

ACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTG

AAGCGGGAGGTTTGAACGCGCAGCCGCCAAGCCGAATTCTGCAGATATCCCCGAG

TCCTTCAATGCTATCATTCCCTTTGATATTGGACCATATGCATAGTACCGAGAA

ACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTAGATATCCCCGAGTCCTTCA

ATGCTATCATTCTCTTTGATATTGGACCATATGCATAGTACCGAGAAACTAGTG

CGAAGTAGTGATCAGGTATTGCTGTTAGATATCCCCGAGTCCTTCAATGCTATC

ATTCCCTTTGATATTGGACCATATGCATAGTACCGAGAAACTAGTGCGAAGTA

GTGATCAGGTATTGCTGTTAGATATCCCCGAGTCCTTCAATGCTATCATTCTCT

TTGATATTGGACCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCA

GGTATTGCTGTTAGATATCCCCGAGTCCTTCAATGCTATCATTCCCTTTGATAT

TGGACCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTG

CTGTTAGATATCCCCGAGTCCTTCAATGCTATCATTTCCTTTGATATTGGATCA

TATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTAA

GGATCCATCACACTGGCGGCCGCTCGAGGGGAGCTCGCAGGGTCTCCATTTTGAAG

CGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGG

TCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGG

TGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATT

GAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATG

GCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGAAGGGAG AGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTT

TTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGG

GATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCG

GAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAA

ACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTG

TTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGC

AGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGAT

CAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGG

GGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTC

AATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAA

GATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGG

AGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCC

CAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAA

CACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCA

TAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCT

TCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCC

AAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACC

AGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAAC

ACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCC

GTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGG

GAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGG

TTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGC

CCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGC

CATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAA

TGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGA

ATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG

CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACT

GTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCT

GGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTA

TGGCTGCCGATGGTTAGCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGAC

AGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGAC

GACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGA CAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTAC

GACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGG

AGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCT

TCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACG

GCTCCGGGAAAA AGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGG

GAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGA

GACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGG

TCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAAC AGG

GCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGC

GACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCT

CTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGC

ACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGC

AAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAA

CATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTAC

CAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGG

CGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGAT

ACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGG

AGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTT

TTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCA

TGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTG

GAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGG

GACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAA

GACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACC

ACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGGTCAGCG

TGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAAT

TCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAA

TGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTA

ATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGT

ATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGCGGCCGCCACCGCGGTGGAGCTCC

AGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAG

CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA

AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGC GTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG

AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC

GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTC

AAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG

TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT

TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG

AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTC

CCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC

CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG

TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC

TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG

CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGC

TACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG

GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT

CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA

CGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC

GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG

GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT

A A GAGAAAACAAGGACAGACAGAAACCAATGCTTAATCAGTGAGGCACCTATCTCAG

CGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGAT

ACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCAC

CGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT

CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTA

GTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCAC

GCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT

GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAA

GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGT

CATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA

TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC

ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA

AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTT

CAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT

ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTA

TTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCT

AAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTC ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGA

CCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC

GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACG

TGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCG

GAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTG GCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGT

GTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACA

GGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGC

GGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTA

AGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGA GCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGG

TCGACGGTATGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGGCTTTGTAGT

TAATGATTAACCCGGCATGCTACTTATCTACGTAGCCATG (SEQ ID NO: 133)

Claims

CLAIMS What is claimed is:

1. An adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, and an insertion of a cell penetrating peptide (CPP) that comprises 5 to 18 amino acids between amino acid residues 587 and 588 of SEQ ID NO:1.

2. The AAV capsid protein of claim 1, wherein the CPP is selected from a CPP set forth in Table 1.

3. The AAV capsid protein of claim 1 or 2, wherein the CPP is selected from any one of SEQ ID NOs: 2-39.

4. The AAV capsid protein of any one of claims 1 to 3, wherein the CPP comprises the amino acid sequence KLGVM (SEQ ID NO: 2).

5. The AAV capsid protein of any one of claims 1 to 4, wherein the capsid protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 40-77.

6. The AAV capsid protein of any one of claims 1 to 5, wherein the capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 40.

7. The AAV capsid protein of any one of claims 1 to 6, wherein the capsid protein has tropism for retinal cells.

8. The AAV capsid protein of any one of claims 1 to 7, wherein the capsid protein has tropism for photoreceptor cells or retinal pigment epithelium (RPE) cells.

9. The AAV capsid protein of any one of claims 1 to 8, wherein the capsid protein has reduced immunogenicity in a subject relative to a wildtype AAV2 capsid protein or an AAV2.7m8 capsid protein.

10. An isolated adeno-associated virus (AAV) capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO: 40-77.

11. An isolated adeno-associated virus (AAV) capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 40.

12. An isolated nucleic acid encoding the AAV capsid protein of any one of claims 1 to 11.

13. The isolated nucleic acid of claim 12, wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 80-117.

14. A vector comprising the isolated nucleic acid of claim 12 or 13.

15. The vector of claim 14, wherein the vector is a plasmid.

16. A recombinant adeno-associated virus (rAAV) comprising:

(i) the AAV capsid protein of any one of claims 1 to 10; and

(ii) an isolated nucleic acid comprising a transgene encoding a therapeutic gene product, flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).

17. A recombinant adeno-associated virus (rAAV) comprising:

(i) an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 40; and

(ii) an isolated nucleic acid comprising a transgene encoding a gene product, flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).

18. The rAAV of claim 16 or 17, wherein the transgene further comprises a promoter operably linked the nucleic acid sequence encoding the gene product.

19. The rAAV of any one of claims 16 to 18, wherein the promotor is a CB6 promoter or a GRK1 promoter.

20. A host cell comprising the isolated nucleic acid of claim 12 or 13, the vector of claim 14 or 15, or the rAAV of any one of claims 16 to 19.

21. A composition comprising the isolated nucleic acid of claim 12 or 13, the vector of claim 14 or 15, the rAAV of any one of claims 16 to 19, or the host cell of claim 20.

22. A method of delivering a transgene to a retinal cell, the method comprising contacting the retinal cell with the rAAV of any one of claims 16 to 19, or the composition of claim 21.

23. A method of delivering a transgene to the retinal cells in a subject, the method comprising administering the rAAV of any one of claims 16 to 19, or the composition of claim 21 to a subject.

24. A method for treating a retinal disease, the method comprises administering the rAAV of any one of claim claims 16 to 19, or the composition of claim 21 to a subject.

25. The method of claim 23 or 24, wherein the rAAV or composition is administered via injection

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

27. The method of any one of claims 23 to 25, wherein the subject is a mammal.

28. The method of any one of claims 23 to 27, wherein the subject is human.

29. The method any one of claims 24 to 28, wherein the retinal disease is age-related macular degeneration (AMD), Leber congenital amaurosis, or diabetic retinopathy.

30. The method of any one of claims 23 to 29, wherein the retinal cells are photoreceptor cells.