# US 2021/0340539 A1 · Modified aav constructs and uses thereof

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- Number: US 2021/0340539 A1
- Document ID: US20210340539A1
- Status: Published application
- Kind: A1
- Application number: 17/316,769
- Filed: 2021-05-11
- Published: 2021-11-04
- Earliest claimed: 2015-04-24
- Assignees: University of Massachusetts
- Applicants: University of Massachusetts
- Inventors: Guangping Gao; Jun Xie; Phillip D. Zamore
- Web page: https://patentgrep.com/patent/US20210340539A1

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/86: Viral vectors
- C12N 2310/11: Antisense
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2330/51: Specially adapted vectors
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14171: Demonstrated in vivo effect
- 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)

## Abstract

The present disclosure relates to the field of rAAV delivery of transgenes. In some aspects, the disclosure relates to RNAi. Provided herein are recombinant adeno-associated virus (rAAV) vectors comprising modified ITRs. In some embodiments, the modified ITRs comprise a sequence encoding a shRNA, miRNA, or AmiRNA.

## Claims

### Claim at position 1

1-18. (canceled)

### Claim 19

An isolated nucleic acid having one inverted terminal repeat at a first terminus and a promoter operably linked with a sequence encoding a hairpin-forming RNA at a second terminus, wherein the isolated nucleic acid is configured for forming a self-complementary AAV (scAAV) vector.

### Claim 20

(canceled)

### Claim 21 (depends on 19)

The isolated nucleic acid of claim 19, wherein the sequence encoding a hairpin-forming RNA is substituted at a position of the scAAV vector normally occupied by a mutant ITR, or wherein the sequence encoding a hairpin-forming RNA forms a shRNA, miRNA, or artificial microRNA (AmiRNA).

### Claim 22

(canceled)

### Claim 23 (depends on 21)

The isolated nucleic acid of claim 21, wherein the wherein the AmiRNA construct comprises:
(i) a nucleic acid sequence encoding a pri-miRNA scaffold;
(ii) a nucleic acid sequence encoding a guide strand; and,
(iii) a nucleic acid sequence encoding a passenger strand,
wherein, the pri-miRNA scaffold is derived from a naturally-occurring pri-miRNA and comprises at least one flanking sequence and a loop-forming sequence comprising at least 4 nucleotides.

### Claim 24 (depends on 23)

The isolated nucleic acid of claim 23, wherein
(i) the guide strand and/or the passenger strand share at least 50% complementarity to a target nucleic acid sequence but are not 100% complementary to one another; or
(ii) the nucleic acid sequence encoding the guide strand and the nucleic acid sequence encoding the passenger strand are inserted into the pri-miRNA scaffold between the flanking sequence and the loop-forming sequence, thereby forming a stem.

### Claim 25

(canceled)

### Claim 26 (depends on 23)

The isolated nucleic acid of claim 23, wherein the nucleic acid sequence encoding the guide strand and the nucleic acid sequence encoding the passenger strand have at least one base pair mismatch.

### Claim 27

(canceled)

### Claim 28 (depends on 24)

The isolated nucleic acid of claim 24, wherein the nucleic acid sequence encoding the guide strand and the nucleic acid sequence encoding the passenger strand have mismatches at no more than ten consecutive base pairs.

### Claim at position 12

29-30. (canceled)

### Claim 31 (depends on 23)

The isolated nucleic acid of claim 23, wherein the pri-miRNA scaffold is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451.

### Claim 32 (depends on 23)

The isolated nucleic acid of claim 23, wherein the guide strand targets a gene associated with a gain of function mutation disease, an oncogene, or a gene associated with a metabolic disorder.

### Claim 33 (depends on 32)

The isolated nucleic acid of claim 32, wherein the guide strand targets SOD1, Huntington gene, p53, HER2/neu, LDLR, or beta-glucosidase.

### Claim at position 16

34-46. (canceled)

### Claim 47

A preparation comprising a plurality of rAAVs, wherein at least 80% of the rAAVs comprise a non-truncated genome comprising a sequence encoding an artificial miRNA (AmiRNA).

### Claim 48 (depends on 47)

The preparation of claim 47, wherein the non-truncated genome comprises two ITRs flanking the sequence encoding an artificial miRNA (AmiRNA).

### Claim at position 19

49-51. (canceled)

### Claim 52

A self-complementary adeno-associated virus (scAAV) comprising:
(i) a viral genome comprising a nucleic acid sequence encoding at least one inverted terminal repeat and a promoter operably linked with a nucleic acid sequence encoding a hairpin-forming RNA; and
(ii) at least one AAV capsid protein serotype.

### Claim 53

(canceled)

### Claim 54 (depends on 52)

The scAAV of claim 52, wherein the wherein the sequence encoding a hairpin-forming RNA is substituted at a position of the scAAV normally occupied by a mutant ITR.

### Claim 55 (depends on 52)

The scAAV of claim 52, wherein
the sequence encoding a hairpin-forming RNA forms a shRNA, miRNA, or AmiRNA, optionally wherein the AmiRNA construct comprises:
(i) a nucleic acid sequence encoding a pri-miRNA scaffold;
(ii) a nucleic acid sequence encoding a guide strand; and,
(iii) a nucleic acid sequence encoding a passenger strand,
wherein, the pri-miRNA scaffold is derived from a naturally-occurring pri-miRNA and comprises at least one flanking sequence and a loop-forming sequence comprising at least 4 nucleotides.

### Claim at position 24

56-57. (canceled)

### Claim 58 (depends on 55)

The scAAV of claim 55, wherein the nucleic acid sequence encoding the guide strand and the nucleic acid sequence encoding the passenger strand are inserted into the pri-miRNA scaffold between the flanking sequence and the loop-forming sequence, thereby forming a stem.

### Claim at position 26

59-63. (canceled)

### Claim 64 (depends on 55)

The scAAV of any claim 55, wherein the pri-miRNA scaffold is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451.

### Claim 65 (depends on 55)

The scAAV of claim 55, wherein the guide strand targets a gene associated with a gain of function mutation disease, an oncogene, or a gene associated with a metabolic disorder.

### Claim 66 (depends on 65)

The isolated nucleic acid of claim 65, wherein the guide strand targets SOD1, Huntington gene, p53, HER2/neu, LDLR, or beta-glucosidase.

### Claim 67 (depends on 52)

The scAAV of claim 52, wherein the size of the viral genome is between about 150 bp and 5 kb.

### Claim 68

(canceled)

### Claim 69 (depends on 52)

The scAAV of claim 52, wherein the at least one capsid protein is an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 capsid protein.

### Claim at position 33

70-76. (canceled)