IP Library › Granted Patent US 12,564,647
Granted Patent B2
US 12,564,647 · App. 17/811,549 · Granted Mar 3, 2026

Optimized expression cassettes for gene therapy

Inventors: Huanyu Zhou (South San Francisco, CA); Christopher A. Reid (South San Francisco, CA)
Assignee: Tenaya Therapeutics, Inc.
A61K48/0058C07K14/47C07K14/4702C07K14/705C12N15/86C12N2750/14143C12N2750/14171C12N2830/008C12N2830/42C12N2830/48C12N2830/50
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Quick Facts
Patent No.
US 12,564,647
App. No.
17/811,549
Granted
Mar 3, 2026
Kind
B2
Abstract

In some aspects, cardiac-specific expression cassettes are provided herein. In some aspects, provided herein is an expression cassette comprising a polynucleotide sequence encoding a gene product for therapy of a heart disease, wherein the polynucleotide sequence is operably linked to promoter (e.g., a cardiac-specific promoter), and optionally an enhancer (e.g., a cardiac-specific enhancer). In some aspects, the disclosure provides recombinant adeno-associated virus (rAAV) virions, comprising a capsid protein and a viral genome comprising an expression cassette comprising a polynucleotide sequence encoding a therapeutic gene product, e.g., dwarf open reading frame (DWORF) polypeptide, operably linked to a promoter, the expression cassette flanked by inverted terminal repeats. The disclosure further provides pharmaceutical compositions and methods of treating or preventing heart disease.

Claims (48)

1 . An expression cassette comprising a polynucleotide sequence comprising, from 5′ to 3′:

an actin, alpha cardiac muscle 1 (ACTC1) cardiac enhancer;

an alpha-myosin heavy chain (αMHC) enhancer;

a cardiac troponin T (cTnT) promoter;

an intron; and

one or more copies of a transgene encoding a polypeptide for treating a heart disease or alleviating symptoms associated with a heart disease,

wherein the transgene encodes a dwarf open reading frame (DWORF) polypeptide.

2 . The expression cassette of claim 1 , comprising two copies of the transgene.

3 . The expression cassette of claim 2 , wherein one copy of the transgene is codon-optimized, and wherein one copy of the transgene is not codon-optimized.

4 . The expression cassette of claim 1 , wherein the polynucleotide sequence further comprises, 3′ of the transgene:

i) a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE); and

ii) a polyadenylation sequence (p(A)).

5 . The expression cassette of claim 4 , wherein the WPRE sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.

6 . The expression cassette of claim 4 , wherein the WPRE sequence comprises SEQ ID NO: 26.

7 . The expression cassette of claim 4 , wherein the polyadenylation sequence is selected from a BGH polyadenylation sequence and a SV40 polyadenylation sequence.

8 . The expression cassette of claim 7 , wherein the BGH polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27.

9 . The expression cassette of claim 7 , wherein the BGH polyadenylation sequence comprises SEQ ID NO: 27.

10 . The expression cassette of claim 7 , wherein the SV40 polyadenylation sequence shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28.

11 . The expression cassette of claim 7 , wherein the SV40 polyadenylation sequence comprises SEQ ID NO: 28.

12 . The expression cassette of claim 4 , comprising 5′-ACTC1 enhancer-αMHC enhancer-human TnT promoter-CMV intron-transgene-WPRE-bGHpA-3′.

13 . The expression cassette of claim 1 , wherein the cTnT promoter is a human cTnT promoter.

14 . The expression cassette of claim 13 , wherein the human cTnT promoter shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12 or SEQ ID NO: 13.

15 . The expression cassette of claim 13 , wherein the human cTnT promoter comprises SEQ ID NO: 12 or SEQ ID NO: 13.

16 . The expression cassette of claim 1 , wherein the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 78.

17 . The expression cassette of claim 1 , wherein the ACTC1 cardiac enhancer comprises SEQ ID NO: 78.

18 . The expression cassette of claim 1 , wherein the αMHC enhancer shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 79.

19 . The expression cassette of claim 1 , wherein the αMHC enhancer comprises SEQ ID NO: 79.

20 . The expression cassette of claim 1 , wherein the intron is selected from a CMV intron and a chimeric intron.

21 . The expression cassette of claim 20 , wherein the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 80.

22 . The expression cassette of claim 20 , wherein the CMV intron comprises SEQ ID NO: 80.

23 . The expression cassette of claim 20 , wherein the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 81.

24 . The expression cassette of claim 20 , wherein the chimeric intron comprises SEQ ID NO: 81.

25 . The expression cassette of claim 1 , wherein the expression cassette is flanked by ITRs.

26 . The expression cassette of claim 25 , wherein the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity to one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

27 . The expression cassette of claim 25 , wherein the ITRs comprise one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

28 . The expression cassette of claim 1 , wherein the transgene shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:33, SEQ ID NO:44, SEQ ID NO:76, or SEQ ID NO:77.

29 . The expression cassette of claim 1 , wherein the polypeptide shares at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:32, or SEQ ID NO:43.

30 . A recombinant vector comprising the expression cassette of claim 1 .

31 . A recombinant adeno-associated virus (rAAV) virion, comprising a capsid protein and a viral genome comprising the expression cassette of claim 1 , wherein the expression cassette is flanked by inverted terminal repeats (ITRs).

32 . The rAAV virion of claim 31 , wherein the ITRs share at least 90%, 95%, 96%, 97%, 98%, or 99% identity to one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

33 . The rAAV virion of claim 31 , wherein the ITRs comprise one or more of SEQ ID NO: 14 and SEQ ID NO: 15.

34 . The rAAV virion of claim 31 , wherein the capsid protein shares at least 98%, at least 99%, or 100% identity to an AAV9 capsid protein (SEQ ID NO: 143).

35 . The rAAV virion of claim 31 , wherein the capsid protein shares at least 98%, at least 99%, or 100% identity to an AAV5 capsid protein (SEQ ID NO: 144).

36 . The rAAV virion of claim 31 , wherein the capsid protein is a chimeric capsid protein.

37 . The rAAV virion of claim 31 , wherein the capsid protein is an AAV5/AAV9 chimeric capsid protein.

38 . The rAAV virion of claim 31 , wherein the capsid protein is selected from any one of SEQ ID NOs: 145-200.

39 . A pharmaceutical composition comprising the vector of claim 30 , or the rAAV virion of claim 31 , and a pharmaceutically acceptable carrier.

40 . A kit comprising the pharmaceutical composition of claim 39 .

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 26, 2022
From: ZHOU, HUANYU; REID, CHRISTOPHER A.
To: TENAYA THERAPEUTICS, INC.
Reel/Frame 061216/0036 →
Continuity (3)
Continuation PCTUS2021012708 · Jan 8, 2021
Provisional Application 62958430 · Jan 8, 2020
Related Publication 20230133924A1 · May 4, 2023
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