# US 2025/0144243 A1 · Aav capsids for improved heart transduction and detargeting of liver

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- Number: US 2025/0144243 A1
- Document ID: US20250144243A1
- Status: Published application
- Kind: A1
- Application number: 18/730,384
- Filed: 2023-01-24
- Published: 2025-05-08
- Earliest claimed: 2022-01-25
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: Jacob Hoffman; Joshua Joyner Sims
- Web page: https://patentgrep.com/patent/US20250144243A1

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- C07K 14/005: from viruses
- A61K 38/1719: {Muscle proteins, e.g. myosin or actin}
- 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 2750/14152: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are novel AAV capsids and recombinant AAV vectors comprising the same. In certain embodiment, vectors comprising a novel AAV capsid show improved transduction of cardiac tissue and/or reduced targeting of liver tissue compared to a prior art AAV capsid.

## Claims

### Claim 1

A recombinant adeno-associated virus (rAAV) having an AAV clade F capsid comprising a mutant galactose binding site,
wherein the mutant galactose binding site comprises (a) any amino acid residue (X) at position 446 (Y446X), (b) an arginine at position 470 (N470R), and (c) a histidine at position 503 (W503H) when residue positions are determined using the residue numbers of SEQ ID NO: 10 as a reference, and
a vector genome comprising a 5′ inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, regulatory sequences operably linked to the coding sequence that direct expression of the gene product, and a 3′ ITR sequence.

### Claim 2 (depends on 1)

The rAAV according to claim 1, wherein the mutant capsid comprises:
(a) H at position 446 (HRH);
(b) S at position 446 (SRH);
(c) V at position 446 (VRH);
(d) G at position 446 (GRH);
(e) F at position 446 (FRH);
(f) T at position 446 (TRH); or
(g) S at position 446 (SRH).

### Claim 3 (depends on 1)

The rAAV according to claim 1, wherein the parental AAV clade F capsid is an AAV9, AAVhu68, AAVhu31, AAVhu32, AAVhu95, or AAVhu96 capsid, or a mutant or variant of any of the aforementioned.

### Claim 4 (depends on 1)

The rAAV according to claim 1, wherein the mutant clade F capsid further comprises an insertional mutation in the HVR8 locus between position 588 and 589, based on the numbering of the amino acid sequence of SEQ ID NO: 10.

### Claim 5 (depends on 4)

The rAAV according to claim 4, wherein the insertional mutation comprises:
(a) the motif N- x- (T/I/V/A)-(K/R) (SEQ ID NO: 19), optionally wherein motif is flanked by two to seven amino acids at its carboxy- and/or amino terminus;
(b) an exogenous targeting peptide having the sequence: optional N-terminal linker- Y-G/A/R/K-Y/H-GNPA-T/R/H-RYFD-V/K (SEQ ID NO: 13)—optional C-terminal linker; inserted between amino acids 588 and 589 of an AAV9 capsid protein, based on the numbering of amino acid sequence: SEQ ID NO: 10; or
(c) a peptide comprising an RGD motif.

### Claim 6 (depends on 5)

The rAAV according to claim 5, wherein the capsid further comprises the motif of (a) which is NTVK, optionally selected from:
(SEQ ID NO: 14)
(a) SSNTVKLTSGH;
(SEQ ID NO: 12)
(b) EFSSNTVKLTS;
(SEQ ID NO: 18)
(c) GGVLTNIARGEYMRGG;
(SEQ ID NO: 17)
(d) GGIEINATRAGTNLGG;
(SEQ ID NO: 13)
(e) GGSSNTVKLTSGHGG;
(SEQ ID NO: 16)
(f) IEINATRAGTNL;
or
(SEQ ID NO: 15)
(g) SANFIKPTSY.

### Claim 7 (depends on 5)

The rAAV according to claim 5, wherein the capsid further comprises the motif of (b) selected from:
(SEQ ID NO: 28)
(a) Y-G/A/R/K-Y/H-GNPA-T/R/H-RYFD-V/K;
(SEQ ID NO: 20)
(b) YGYGNPATRYFDV;
(SEQ ID NO: 25)
(c) YGYGNPARRYFDV;
(SEQ ID NO: 26)
(d) YGYGNPAHRYFDV;
(SEQ ID NO: 27)
(e) YGYGNPATRYFDK;
(SEQ ID NO: 21)
(f) YAYGNPATRYFDV;
(SEQ ID NO: 22)
(g) YKYGNPATRYFDV;
(SEQ ID NO: 23)
(h) YRYGNPATRYFDV;
or
(SEQ ID NO: 24)
(i) YGHGNPATRYFDV.

### Claim 8 (depends on 1)

The rAAV according to claim 1, wherein the regulatory sequences comprise a constitutive promoter.

### Claim 9 (depends on 1)

The rAAV according to claim 1, wherein the regulatory sequences comprise a tissue-specific promoter.

### Claim 10 (depends on 9)

The rAAV according to claim 9, wherein the tissue-specific promoter is a cardiac promoter.

### Claim 11

A method for generating an a rAAV comprising a mutant capsid derived from a parental AAV capsid having liver specificity, the method comprising culturing a packaging host cell comprising:
(a) a nucleic acid sequence encoding a mutant AAV Clade F capsid operably linked to regulatory control sequences that direct its expression in the packaging host cell, wherein the encoded capsid protein comprises (i) any amino acid residue (X) at position 446 (Y446X), (ii) an arginine at position 470 (N470R), and (iii) a histidine at position 503 (W503H), where the amino acid residue positions are determined using the residue numbers of SEQ ID NO: 10 as a reference,
(b) a nucleic acid molecule comprising a vector genome comprising a 5′ inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, regulatory sequences operably linked to the coding sequence that direct expression of the gene product, and a 3′ ITR sequence; and
(c) helper functions necessary for packaging the vector genome of (b) into the mutant Clade F capsid.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the parental AAV capsid is an AAV9, AAVhu68, AAVhu31, AAVhu32, AAVhu95, or AAVhu96 capsid, or a mutant or variant of any of the aforementioned.

### Claim 13 (depends on 11)

An rAAV produced according to the method of claim 11.

### Claim 14 (depends on 1)

A method for reducing liver toxicity associated with delivery of an rAAV vector, the method comprising delivering to a subject the rAAV according to claim 1.

### Claim 15 (depends on 1)

A method for improved delivery of a gene product to cardiac cells or tissue, the method comprising administering to a subject the rAAV according to claim 1.

### Claim 16 (depends on 14)

The method according to claim 14, wherein the coding sequence comprises a LaminA gene, Kv11.1 voltage-gated potassium channel (ERG) gene, a tafazzin (TAZ gene, potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene, or a cardiac myosin binding protein C (MYBPC3) gene.

### Claim 17

A nucleic acid comprising a sequence encoding a mutant AAV Clade F VP1 protein having a mutant galactose binding pocket which comprises (a) any amino acid residue (X) at position 446 (Y446X), (b) arginine at position 470 (N470R), and (c) histidine at position 503 (W503H), where the amino acid residue positions are determined using the residue numbers of SEQ ID NO: 10 as a reference, the VP1 protein coding sequence being operably linked to expression control sequences which direct its expression in a packaging host cell.

### Claim 18 (depends on 17)

The nucleic acid according to claim 17, wherein the nucleic acid is a plasmid.

### Claim 19 (depends on 17)

A host cell comprising the nucleic acid according to claim 17.