# US 2025/0084385 A1 · Adeno-associated virus with engineered capsid

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- Number: US 2025/0084385 A1
- Document ID: US20250084385A1
- Status: Published application
- Kind: A1
- Application number: 18/912,105
- Filed: 2024-10-10
- Published: 2025-03-13
- Earliest claimed: 2022-04-11
- Applicants: Tenaya Therapeutics, Inc.
- Inventors: Ze CHENG; Timothy C. HOEY; Christopher A. REID
- Web page: https://patentgrep.com/patent/US20250084385A1

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 9/00: Drugs for disorders of the cardiovascular system
- C07K 14/005: from viruses
- C12N 15/86: Viral vectors
- C12N 2750/14122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14145: Special targeting system for viral vectors
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present disclosure provides engineered capsid proteins and recombinant adeno-associated virus (rAAV) virions with an engineered capsid protein. In particular, the disclosure provides AAV9 virions with engineered AAV9 capsid, AAV5/9 chimeric capsid, or combinatory capsid that achieves increased transduction efficiency in heart, increased heart-to-liver ratio, and/or other desirable properties.

## Claims

### Claim 1

A recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein shares, or comprises a sequence sharing, at least 80% amino acid sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
an amino acid insertion at position 584, or between positions 583 and 584, comprising one or more of an asparagine (N), a threonine (T), a tyrosine (Y), phenylalanine (F), and an alanine (A);
an amino acid insertion at position 585, or between positions 584 and 585, comprising one or more of a histidine (H) and a methionine (M);
an amino acid insertion at position 586, or between positions 585 and 586, comprising one or more of a histidine (H), a tyrosine (Y), a valine (V), a threonine (T), an alanine (A), an isoleucine (I), a tryptophan (W), a methionine (M), and a leucine (L);
an amino acid insertion at position 587, or between positions 586 and 587, comprising one or more of an isoleucine (I) and a proline (P);
an amino acid insertion at position 588, or between positions 587 and 588, comprising one or more of an isoleucine (I), a threonine (T), and a proline (P);
an amino acid insertion at position 589, or between positions 588 and 589, comprising one or more of a glycine (G) and a glutamine (Q);
one or more amino acid substitutions selected from the group consisting of N452K, N452A, N452V, N452I, G453A, G453N, S454T, S454D, G455N, Q456L, Q456K, N457L, N457V, Q458I, and Q458H; and/or
one or more amino acid substitutions selected from the group consisting of T582D, T582L, T582E, T582A, T582F, T582R, T582P, N583V, N583T, H584R, H584Q, H584K, H584V, H584Y, H584M, H584T, H584W, H584E, H584D, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R, A587F, A587S, A587T, A587N, A587L, A587P, A587V, A587K, A587I, A587R, A587H, A587G, A587M, A587D, A587W, Q588L, Q588S, Q588F, Q588N, Q588G, Q588R, Q588I, Q588V, Q588T, Q588Y, Q588H, Q588M, Q588K, Q588D, A589R, A589I, A589N, A589S, A589V, A589Q, A589F, A589T, A589K, A589H, A589E, A589W, A589L, A589Y, A589M, Q590I, Q590S, Q590N, Q590G, Q590D, Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, Q590L, A591I, G594Q, and G594D.

### Claim 2 (depends on 1)

The capsid protein of claim 1, wherein the capsid protein comprises one, two, three, four or more substitutions or insertions in the VR-VIII site.

### Claim 3 (depends on 2)

The capsid protein of claim 2, wherein the capsid protein comprises, relative to reference SEQ ID NO:1, one, two, three, four or more substitutions or insertions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four or more substitutions or insertions at positions from 585 to 590 in the VR-VIII site.

### Claim 4 (depends on 1, 2, 3)

The capsid protein of any one of claims 1-3, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
(i) one or more amino acid substitutions selected from the group consisting of T582D, T582E, N583V, H584Q, S586K, A587P, A587S, Q588G, Q588M, A589S, A591I, G594Q, and G594D;
(ii) one or more amino acid substitutions selected from the group consisting of T582L, T582A, T582F, T582R, T582P, H584R, H584K, H584V, H584Y, H584M, H584Q, H584W, H584E, H584D, Q585T, Q585N, Q585M, Q585E, Q585V, Q585H, S586T, S586G, S586Q, S586I, S586L, S586F, S586D, S586R, S586M, A587F, A587I, A587H, A587M, A587N, A587W, Q588Y, Q588S, Q588T, and Q588R;
(iii) one or more amino acid substitutions selected from the group consisting of Q585C, Q585S, S586I, A587V and A587G; or
(iv) one or more amino acid substitutions selected from the group consisting of Q585V, Q585T, Q585L, Q585C, Q585N, Q585S, Q585M, Q585E, Q585P, Q585A, Q585G, Q585H, Q585I, S586D, S586G, S586T, S586M, S586N, S586L, S586R, S586I, S586K, A587S, A587T, A587N, A587L, A587V, A587K, A587I, A587F, A587P, A587R, A587D, Q588L, Q588S, Q588F, Q588N, Q588R, Q588I, Q588V, Q588T, Q588H, Q588Y, Q588M, Q588K, Q588D, Q588G, A589R, A589I, A589N, A589S, A589V, A589Q, A589F, A589T, A589K, A589H, A589E, A589W, A589L, A589Y, A589M, Q590I, Q590S, Q590N, Q590G, Q590D, Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, and Q590L.

### Claim 5 (depends on 1, 2, 3, 4)

The capsid protein of any one of claims 1-4, wherein the capsid protein: (i) is cardiotrophic, (ii) exhibits increased transduction efficiency in cardiac cells compared to the parental sequence, (iii) exhibits decreased transduction efficiency in liver cells compared to the parental sequence, and/or (iv) exhibits increased selectivity for the cardiac cells over liver cells compared to the parental sequence.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, one or more amino acid substitutions selected from the group consisting of N452K, N452A, N452V, N452I, G453A, G453N, S454T, S454D, G455N, Q456L, Q456K, N457L, N457V, Q458I, and Q458H.

### Claim 7 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, at position 452 an amino acid selected from the group consisting of: K and N.

### Claim 8 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, an amino acid substitution N452K.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 584 an amino acid selected from the group consisting of: R and H;
at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, L and Q;
at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, I and S;
at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, P and A;
at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, G and Q;
at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, Y and A; and/or
at position 590 an amino acid selected from the group consisting of: G, R, S, I, H, N, Y, L, M and Q.

### Claim 10 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N;
at position 584 an amino acid selected from the group consisting of: R and H;
at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, L and Q;
at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, I and S;
at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, P and A;
at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, G and Q;
at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, Y and A; and
at position 590 an amino acid selected from the group consisting of: G, R, S, I, H, N, Y, L, M and Q.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 584 amino acid R;
at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H and, L;
at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, and I;
at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, and P;
at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, and G;
at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, and Y; and/or
at position 590 an amino acid selected from the group consisting of: G, R, S, I, H, N, Y, L, and M.

### Claim 12 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, at least two, three, four, five, six, seven or all eight of any of the following:
(i) at position 452 amino acid K;
(ii) at position 584 amino acid R;
(iii) at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, and L;
(iv) at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, and I;
(v) at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, and P;
(vi) at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, and G;
(vii) at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, and Y; and
(viii) at position 590 an amino acid selected from the group consisting of: G, R, S, I, H, N, Y, L, and M

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V, T and Q;
at position 586 an amino acid selected from the group consisting of: N, T, M, G, D, and S;
at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N, V and A;
at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I, R and Q;
at position 589 an amino acid selected from the group consisting of: S, N, L, T, I, R and A; and/or
at position 590 an amino acid selected from the group consisting of: I, S, G, H, R and Q.

### Claim 14 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N;
at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V, T and Q;
at position 586 an amino acid selected from the group consisting of: N, T, M, G, D, and S;
at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N, V and A;
at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I, R and Q;
at position 589 an amino acid selected from the group consisting of: S, N, L, T, I, R and A; and
at position 590 an amino acid selected from the group consisting of: I, S, G, H, R and Q.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V and T;
at position 586 an amino acid selected from the group consisting of: N, T, M, G, and D;
at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N and V;
at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I and R;
at position 589 an amino acid selected from the group consisting of: S, N, L, T, I and R; and/or
at position 590 an amino acid selected from the group consisting of: I, S, G, H and R.

### Claim 16 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, at least two, three, four, five, six or all seven of any of the following:
(i) at position 452 amino acid K;
(ii) at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V and T;
(iii) at position 586 an amino acid selected from the group consisting of: N, T, M, G, and D;
(iv) at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N and V;
(v) at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I and R;
(vi) at position 589 an amino acid selected from the group consisting of: S, N, L, T, I and R; and
(vii) at position 590 an amino acid selected from the group consisting of: I, S, G, H and R.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 585 an amino acid selected from the group consisting of: E, N, M, C, and Q;
at position 586 an amino acid selected from the group consisting of: A, M, G, D, N and S;
at position 587 an amino acid selected from the group consisting of: T, N, V and A;
at position 588 an amino acid selected from the group consisting of: V, Y, T, S, I and Q;
at position 589 an amino acid selected from the group consisting of: S, G, L, I, R and A; and/or
at position 590 an amino acid selected from the group consisting of: I, S, G, R and Q.

### Claim 18 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N;
at position 585 an amino acid selected from the group consisting of: E, N, M, C, and Q;
at position 586 an amino acid selected from the group consisting of: A, M, G, D, N and S;
at position 587 an amino acid selected from the group consisting of: T, N, V and A;
at position 588 an amino acid selected from the group consisting of: V, Y, T, S, I and Q;
at position 589 an amino acid selected from the group consisting of: S, G, L, I, R and A; and
at position 590 an amino acid selected from the group consisting of: I, S, G, R and Q.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 585 an amino acid selected from the group consisting of: E, N, M, and C;
at position 586 an amino acid selected from the group consisting of: A, M, G, D, and N;
at position 587 an amino acid selected from the group consisting of: T, N, and V;
at position 588 an amino acid selected from the group consisting of: V, Y, T, S, and I;
at position 589 an amino acid selected from the group consisting of: S, G, L, I and R; and/or
at position 590 an amino acid selected from the group consisting of: I, S, G, and R.

### Claim 20 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, at least two, three, four, five, six or all seven of any of the following:
(i) at position 452 amino acid K;
(ii) at position 585 an amino acid selected from the group consisting of: E, N, M, and C;
(iii) at position 586 an amino acid selected from the group consisting of: A, M, G, D, and N;
(iv) at position 587 an amino acid selected from the group consisting of: T, N, and V;
(v) at position 588 an amino acid selected from the group consisting of: V, Y, T, S, and I;
(vi) at position 589 an amino acid selected from the group consisting of: S, G, L, I and R; and
(vii) at position 590 an amino acid selected from the group consisting of: I, S, G, and R.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The capsid protein of any one of claims 1-20, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N; and
at position 587 amino acid substitution A587T; and optionally comprises amino acid N or R at one, two or more positions selected from the group consisting of: 584, 585, 586, 588, 589, and 590.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The capsid protein of any one of claims 1-21, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N; and
amino acid N or R at one, two or more positions selected from the group consisting of: 584, 585, 586, 588, 589, and 590.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The capsid protein of any one of claims 1-22, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N; and
amino acid S at two or more positions selected from the group consisting of: 585, 586, 587, 588, 589 and 590.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The capsid protein of any one of claims 1-23, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at position 452 an amino acid selected from the group consisting of: K and N; and
at three, four, five or six positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, R and I.

### Claim 25 (depends on 24)

The capsid protein of claim 24, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
at three, four, five or six positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, and R.

### Claim 26 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586N, A587T, Q588V, A589S, Q590I, and N452K.

### Claim 27 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions S586T, A587L, Q588F, A589N, Q590S, and N452K.

### Claim 28 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585N, A587T, Q588Y, A589L, Q590G, and N452K.

### Claim 29 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585G, A587I, Q588L, A589T, Q590H, and N452K.

### Claim 30 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585M, S586M, A587T, Q588T, and Q590R; and amino acid N at position 452.

### Claim 31 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585N, A587T, Q588Y, A589L, and Q590G; and amino acid N at position 452.

### Claim 32 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585C, A587T, Q588S, A589I, and Q590R; and amino acid N at position 452.

### Claim 33 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, and Q590S; and amino acid N at position 452.

### Claim 34 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, Q590S, and N452K.

### Claim 35 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585N, S586N, A587V, Q588I, A589S, Q590G, and N452K.

### Claim 36 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions S586G and Q588Y; and amino acid N at position 452.

### Claim 37 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions S586A, A587N, Q588Y, A589G, and N452K.

### Claim 38 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The capsid protein of any one of claims 1-37, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acids ATN at positions 581-583, and amino acids AQTG at positions 591-594.

### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The capsid protein of any one of claims 1-37, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO:1, amino acids ATNH at positions 581-584, and amino acids AQTG at positions 591-594.

### Claim 40 (depends on 1, 2, 3, 4, 5)

The capsid protein of any one of claims 1-5, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO:1:
(i) amino acid sequence ATNHENTVSIAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(ii) amino acid sequence ATNHQTLFNSAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(iii) amino acid sequence ATNHNSTYLGAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(iv) amino acid sequence ATNHGSILTHAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(v) amino acid sequence ATNHMMTTARAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(vi) amino acid sequence ATNHNSTYLGAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(vii) amino acid sequence ATNHCSTSIRAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(viii) amino acid sequence ATNHEDNIRSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(ix) amino acid sequence ATNHEDNIRSAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(x) amino acid sequence ATNHNNVISGAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(xi) amino acid sequence ATNHQGAYAQAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xii) amino acid sequence ATNHQANYGQAQTG at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452;
(xiii) amino acid sequence ATNHNMNRVNAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xiv) amino acid sequence ATNHNNVISGAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xv) amino acid sequence ATNHSNSVQSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xvi) amino acid sequence ATNHSSTFQGAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xvii) amino acid sequence ATNHVSSFTSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xviii) amino acid sequence ATNHSTTNFRAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xix) amino acid sequence ATNHSSIFNSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xx) amino acid sequence ATNHAGNYNNAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxi) amino acid sequence ATNHTSVISIAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxii) amino acid sequence ATNHHSRVEIAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxiii) amino acid sequence ATNHSSIIYSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxiv) amino acid sequence ATNHSGRDSYAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxv) amino acid sequence ATNHSSSYNNAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxvi) amino acid sequence ATNHHNPSINAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxvii) amino acid sequence ATNHNRNGLLAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxviii) amino acid sequence ATNHESTSVRAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxix) amino acid sequence ATNHNIRTEMAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxx) amino acid sequence ATNHQTLFNSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxxi) amino acid sequence ATNHLSVSSIAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxxii) amino acid sequence ATNHEDIIRSAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452;
(xxxiii) amino acid sequence ATNRQTAQAQAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452; or
(xxxiv) amino acid sequence ATNRQIAQAQAQTG at the VR-VIII positions 581-594, and amino acid N at the VR-IV position 452.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The capsid protein of any one of claims 1-8, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
(i) an amino acid insertion at position 584 comprising one or more of an asparagine (N), a threonine (T), a tyrosine (Y), phenylalanine (F), and an alanine (A);
(ii) an amino acid insertion at position 585 comprising one or more of a histidine (H) and a methionine (M);
(iii) an amino acid insertion at position 586 comprising one or more of a histidine (H), a tyrosine (Y), a valine (V), a threonine (T), an alanine (A), an isoleucine (I), a tryptophan (W), a methionine (M), and a leucine;
(iv) an amino acid insertion at position 587 comprising one or more of an isoleucine (I) and a proline (P);
(v) an amino acid insertion at position 588 comprising one or more of an isoleucine (I), a threonine (T), and a proline (P); and/or
(vi) an amino acid insertion at position 589 comprising one or more of a glycine (G) and a glutamine (Q).

### Claim 42 (depends on 41)

The capsid protein of claim 41, wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1:
(i) an amino acid insertion at position 584 consisting of a TY, FN, or AT;
(ii) an amino acid insertion at position 585 consisting of MH;
(iii) an amino acid insertion at position 586 consisting of HY, VT, AI, WM, or ML;
(iv) an amino acid insertion at position 587 consisting of PI; and/or
(v) an amino acid insertion at position 588 consisting of IT or PT.

### Claim 43 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)

The capsid protein of any one of claims 1-42, wherein the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to an AAV9 VP3 sequence according to SEQ ID NO: 487, except for the specified modifications.

### Claim 44 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)

The capsid protein of any one of claims 1-43, wherein the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to an AAV9 VP2 sequence according to SEQ ID NO: 486, except for the specified modifications.

### Claim 45 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)

The capsid protein of any one of claims 1-44, wherein the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to an AAV9 VP1 sequence according to SEQ ID NO: 1, except for the specified modifications.

### Claim 46 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

The capsid protein of any one of claims 1-45, wherein the capsid protein comprises, consists essentially of, or consists of an amino acid sequence sharing at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from the group consisting of: SEQ ID NOs: 488, 499, 504, 505, 506, 510, 512, 513, 516, 518, 521, 522, 533, 536, 539, 558, 562, 566, 571, 576, 578, 579, 580, 581, 585, 588, 589, 705, 706, 707, 708, 710, 772, and 774, or a functional fragment thereof.

### Claim 47 (depends on 1)

The capsid protein of claim 1, wherein the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence selected from the group consisting of: SEQ ID NOs: 488, 499, 504, 505, 506, 510, 512, 513, 516, 518, 521, 522, 533, 536, 539, 558, 562, 566, 571, 576, 578, 579, 580, 581, 585, 588, 589, 705, 706, 707, 708, 710, 772, and 774.

### Claim 48 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)

A recombinant adeno-associated virus (rAAV) virion, comprising a capsid protein according to any one of claims 1-47 and a vector genome comprising a polynucleotide cassette flanked by inverted terminal repeats (ITRs).

### Claim 49 (depends on 48)

The rAAV virion of claim 48, wherein the rAAV virion transduces heart cells.

### Claim 50 (depends on 48, 49)

The rAAV virion of claim 48 or claim 49, wherein the rAAV virion transduces cardiomyocytes.

### Claim 51 (depends on 48, 49, 50)

The rAAV virion of any one of claims 48-50, wherein the rAAV virion traffics to at least one organ other than the liver.

### Claim 52 (depends on 48, 49, 50)

The rAAV virion of any one of claims 48-50, wherein the rAAV virion traffics to the heart.

### Claim 53 (depends on 48, 49, 50, 51, 52)

The rAAV virion of any one of claims 48-52, wherein the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1.

### Claim 54 (depends on 48, 49, 50, 51, 52, 53)

The rAAV virion of any one of claims 48-53, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

### Claim 55 (depends on 48, 49, 50, 51, 52, 53, 54)

The rAAV virion of any one of claims 48-54, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

### Claim 56 (depends on 48, 49, 50, 51, 52, 53, 54, 55)

The rAAV virion of any one of claims 48-55, wherein the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate.

### Claim 57 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56)

The rAAV virion of any one of claims 48-56, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

### Claim 58 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)

The rAAV virion of any one of claims 48-57, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

### Claim 59 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The rAAV virion of any one of claims 48-58, wherein the polynucleotide cassette comprises a polynucleotide sequence encoding MYBPC3, DWORF, PKP2, KCNH2, TRPM4, DSG2, TGFBR2, TGFBR1, EMD, KCNQ1, TAZ, COL3A1, JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAF1, SOS1, FBN1, LAMP2, FXN, RAF1, BAG3, KCNQ1, MYLK3, CRYAB, ALPK3, ACTN2, JPH2, PLN, ATP2A2, CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, MYH6, MYH7, Cas9, RBM20, MYOCD, ASCL1, GATA4, MEF2C, TBX5, miR-133, or MESP1.

### Claim 60 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)

The rAAV virion of any one of claims 48-59, wherein the polynucleotide cassette comprises a polynucleotide sequence which encodes a protein selected from the group consisting of: MYBPC3, DWORF, PKP2, LMNA, LAMP2, BAG3, CRYAB, JPH2, PLN, TTNI3, MYOCD, ASCL1, DSP, JUP, DSP, MYH6, MYH7, RBM20, and Cas9.

### Claim 61 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)

A pharmaceutical composition comprising an rAAV virion according to any one of claims 48-60 and a pharmaceutically acceptable carrier.

### Claim 62 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)

A polynucleotide encoding the capsid protein of any one of claims 1-47.

### Claim 63 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)

A method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion according to any one of claims 48-60, wherein the rAAV virion transduces the cardiac cell.

### Claim 64 (depends on 63)

The method of claim 63, wherein the cardiac cell is a cardiomyocyte.

### Claim 65 (depends on 63, 64)

The method of claim 63 or claim 64, wherein the rAAV virion exhibits higher transduction efficiency in the cell than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1.

### Claim 66 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)

A method of delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with an rAAV virion according to any one of claims 48-60.

### Claim 67 (depends on 66)

The method of claim 66, wherein the cardiac cell is a cardiomyocyte.

### Claim 68 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)

A method of treating a cardiac pathology in a subject in need thereof, comprising administering a therapeutically effective amount of an rAAV virion according to any one of claims 48-60 to the subject, wherein the rAAV virion transduces cardiac tissue.

### Claim 69 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)

A method of treating a heart disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of an rAAV virion according to any one of claims 48-60 to the subject.

### Claim 70 (depends on 61)

A kit comprising a pharmaceutical composition according to claim 61 and instructions for use.