Optimized expression cassettes for gene therapy

Inventors

Zhou, Huanyu • REID, Christopher A.

Assignees

Tenaya Therapeutics Inc

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.

Core Innovation

The invention relates to expression cassettes for cardiac-specific gene therapy that drive expression of a dwarf open reading frame (DWORF) polypeptide. The cassette includes a polynucleotide sequence encoding a DWORF polypeptide operatively linked to a cardiac troponin T (cTnT) promoter, an actin, alpha cardiac muscle 1 (ACTC1) cardiac enhancer, and an alpha-myosin heavy chain (αMHC) enhancer. The disclosure also includes a polyadenylation sequence.

The ACTC1 cardiac enhancer comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 78. The disclosure further describes optional regulatory components such as introns, PTRE elements including WPRE, orientation-specific arrangements of elements, and selected polyadenylation sequences including BGH polyadenylation sequences or SV40 polyadenylation sequences.

The disclosure also provides recombinant AAV (rAAV) virions that include genomes bearing the expression cassette flanked by AAV inverted terminal repeats (ITRs). Capsid identity options include AAV9 and AAV5, as well as chimeric capsids including AAV5/AAV9, and the document describes therapeutic contexts in heart disease, including increasing DWORF expression in cardiac cells and treating cardiomyopathy and heart failure models.

Claims Coverage

The independent claims cover a cardiac expression cassette architecture for DWORF, defined by specific promoter and enhancer elements, a polyadenylation sequence, and a sequence-identity constraint on the ACTC1 cardiac enhancer. Dependent refinements add optional cassette components and constrain specific sequence identities, as well as rAAV capsid identity constraints.

Overall, the claim coverage centers on a DWORF-encoding cardiac expression cassette defined by operatively linked cTnT promoter plus ACTC1 and αMHC enhancers, with inclusion of a polyadenylation sequence and a defined identity threshold for ACTC1 relative to SEQ ID NO: 78. Dependent claims further narrow optional intron and PTRE elements and impose additional sequence-identity and capsid-identity constraints.

Stated Advantages

Documented Applications