# US 2025/0084129 A1 · Aav5 capsid variants

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- Number: US 2025/0084129 A1
- Document ID: US20250084129A1
- Status: Published application
- Kind: A1
- Application number: 18/727,527
- Filed: 2023-01-10
- Published: 2025-03-13
- Earliest claimed: 2022-01-10
- Assignees: University of Florida Research Foundation, Inc.
- Applicants: University of Florida Research Foundation, Incorporated
- Inventors: Oleksandr Kondratov; Anatolii Mamchur; Maria Ukhanova; Sergei Zolotukhin
- Web page: https://patentgrep.com/patent/US20250084129A1

## CPC

- C07K 14/005: from viruses
- A61K 48/0041: {the non-active part being polymeric}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- 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
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are recombinant AAV variant (e.g., variant serotype 5 (AAV5)) capsid proteins and variant capsid protein-containing viral particles with enhanced ability to transduce adult stem cells and neurons. Viral particles containing these capsid variants are capable of enhanced transduction of mammalian mesenchymal stem cells and neurons. The recombinant AAV5 variant proteins and viral particles disclosed herein were identified from a variant AAV5 capsid library that was engineered by making substitutions in variable regions of the capsid. Compositions of these variant AAV5 particles are provided that are useful for transducing and delivering therapeutic transgenes to cells, such as mesenchymal stem cells, and thus treat diseases and disorders pertaining to these cells.

## Claims

### Claim 1

A variant recombinant adeno-associated virus (rAAV) serotype 5 (AAV5) capsid protein comprising mutations in each of amino acid residues 377 and 378 in the wild-type AAV5 VP1 sequence of SEQ ID NO: 1.

### Claim 2 (depends on 1)

The capsid protein of claim 1, wherein the protein comprises an N378K substitution.

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

The capsid protein of claim 1 or 2, wherein the protein comprises an E377D substitution.

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

The capsid protein of claim 1 or 2, wherein the protein comprises an E377G substitution.

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

The capsid protein of any one of claims 1-3, wherein the protein comprises the amino acid sequence of SEQ ID NO: 2 (AAV5-DK).

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

The capsid protein of any one of claims 1, 2 and 4, wherein the protein comprises the amino acid sequence of SEQ ID NO: 3 (AAV5-GK).

### Claim 7

A variant recombinant adeno-associated virus (rAAV) serotype 5 (AAV5) capsid protein comprising either of the following sequences:
(a) LFRFVSTDATGNLKF (SEQ ID NO: 6) in variable region (VR) IV, or
(b) LSAGGNRNYLSAKA (SEQ ID NO: 7) in VR V, of AAV5 VP1 protein.

### Claim 8

A variant recombinant adeno-associated virus (rAAV) serotype 5 (AAV5) capsid protein comprising mutations in at least two, at least three, at least four, at least five, or at least six of amino acid residues 436, 442, 443, 446, 447, and 448 in the wild-type AAV5 VP1 sequence of SEQ ID NO: 1.

### Claim 9 (depends on 8)

The capsid protein of claim 8, wherein the protein comprises mutations at each of amino acid residues 436, 442, 443, 446, 447, and 448.

### Claim 10 (depends on 8)

The capsid protein of claim 8, wherein the protein comprises at least two, at least three, at least four, at least five, or at least six of the following substitutions: Y436F, N442D, N443A, G446N, V447L, and Q448K.

### Claim 11 (depends on 8, 9, 10)

The capsid protein of any one of claims 8-10, wherein the protein comprises each of the following substitutions: Y436F, N442D, N443A, G446N, V447L, and Q448K.

### Claim 12 (depends on 8, 9, 10, 11)

The capsid protein of any one of claims 8-11, wherein the protein comprises the amino acid sequence of SEQ ID NO: 4 (AAV5-FDA).

### Claim 13

A variant recombinant adeno-associated virus (rAAV) serotype 5 (AAV5) capsid protein comprising mutations in at least two, at least three, at least four, at least five, at least six, or at least seven of amino acid residues 478, 479, 481, 484, 485, 486 and 489 in the wild-type AAV5 VP1 sequence of SEQ ID NO: 1.

### Claim 14 (depends on 13)

The capsid protein of claim 13, wherein the protein comprises mutations at each of amino acid residues 478, 479, 481, 484, 485, 486 and 489.

### Claim 15 (depends on 13)

The capsid protein of claim 13, wherein the protein comprises at least two, at least three, at least four, at least five, or at least six of the following substitutions: G478S, S479A, V481G, A484N, S485Y, V486L, and F489K.

### Claim 16 (depends on 13, 14, 15)

The capsid protein of any one of claims 13-15, wherein the protein comprises each of the following substitutions: G478S, S479A, V481G, A484N, S485Y, V486L, and F489K.

### Claim 17 (depends on 13, 14, 15, 16)

The capsid protein of any one of claims 13-16, wherein the protein comprises the amino acid sequence of SEQ ID NO: 5 (AAV5-SAG).

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

A variant recombinant AAV5 particle comprising the recombinant AAV capsid protein of any one of claims 1-17.

### Claim 19 (depends on 18)

The variant recombinant AAV5 particle of claim 18, further comprising a nucleic acid comprising a transgene of interest.

### Claim 20 (depends on 18, 19)

The variant recombinant AAV5 particle of claim 18 or 19, wherein the nucleic acid is single stranded.

### Claim 21 (depends on 18, 19)

The variant recombinant AAV5 particle of claim 18 or 19, wherein the nucleic acid is self-complementary.

### Claim 22 (depends on 18, 19, 20, 21)

A composition comprising a plurality of the variant recombinant AAV5 particle of any one of claims 18-21.

### Claim 23 (depends on 22)

The composition of claim 22 further comprising a pharmaceutically acceptable carrier.

### Claim 24 (depends on 22, 23)

The composition of claim 22 or 23, wherein the plurality is in an amount of between 1×1011 vgs/ml and 2×1011 vgs/ml, or between 1×1012 and 4×1012 vgs/ml.

### Claim 25 (depends on 18, 19, 20, 21)

A cell comprising a plurality of the variant recombinant AAV5 particle of any one of claims 18-21.

### Claim 26 (depends on 25)

The cell of claim 25, wherein the cell is a mesenchymal stem cell.

### Claim 27 (depends on 18, 19, 20, 21, 22, 23, 24)

A method of transducing a mesenchymal stem cell with a transgene of interest, the method comprising providing to the cell the variant recombinant AAV particle of any one of claims 18-21 or the composition of any one of claims 22-24.

### Claim 28 (depends on 27)

The method of claim 27, wherein the method provides about a 15%, a 30%, a 50%, a 100%, a 200%, a 300%, a 400%, a 500%, a 750%, or a 1000% increase in transduction of the transgene of interest in the mesenchymal stem cell, relative to a wild-type recombinant AAV5 particle.

### Claim 29 (depends on 27, 28)

The method of claim 27 or 28, wherein the mesenchymal stem cell is derived from a mammalian subject.

### Claim 30 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26)

A method of treating a disease or disorder comprising administering the variant recombinant AAV particle of any one of claims 18-21, the composition of any one of claims 22-24, or the cell of claim 25 or 26, to a subject in need thereof.

### Claim 31 (depends on 30)

The method of claim 30, wherein the subject is a mammal.

### Claim 32 (depends on 29, 30, 31)

The method of any one of claims 29-31, wherein the subject is human.

### Claim 33 (depends on 30, 31, 32)

The method of any one of claims 30-32 further comprising re-administering the recombinant AAV particle, the composition, or the cell to the subject.

### Claim 34 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26)

A method of administering the variant recombinant AAV particle of any one of claims 18-21, the composition of any one of claims 22-24, or the cell of claim 25 or 26, to a subject in need thereof who has previously been administered the recombinant AAV particle, the composition, or the cell.

### Claim 35 (depends on 18, 19, 20, 21, 22, 23, 24)

A method of transducing a neuron or glial cell with a transgene of interest, the method comprising providing to the cell the variant recombinant AAV particle of any one of claims 18-21 or the composition of any one of claims 22-24.

### Claim 36 (depends on 27)

The method of claim 27, wherein the method provides about a 15%, a 30%, a 50%, a 100%, a 200%, a 300%, a 400%, a 500%, a 750%, or a 1000% increase in transduction of the transgene of interest in the neuron or glial cell, relative to a wild-type recombinant AAV5 particle.

### Claim 37 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26)

The variant recombinant AAV particle of any one of claims 18-21, the composition of any one of claims 22-24, or the cell of claim 25 or 26, for use as a medicament.

### Claim 38 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A polynucleotide encoding the capsid of any one of claims 1-17.

### Claim 39 (depends on 38)

A host cell comprising the polynucleotide of claim 38.