Engineered nucleic acid regulatory elements and methods and uses thereof
AssigneeREGENXBIO Inc.
InventorsRandolph Qian, Chunping Qiao, Devin McDougald
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Abstract
The present invention relates to nucleic acid expression cassettes that are engineered to enhance gene expression. Vectors and methods employing the expression cassettes containing novel chimeric regulatory elements are provided. The invention is particularly useful for delivery of transgenes to target cells and confers desirable properties for liver-directed and muscle-gene therapy. Moreover, the invention relates to gene therapy methods of delivery of therapeutics for treating various disorders.
Claims35
A recombinant expression cassette comprising a composite nucleic acid regulatory element comprising a) Muscle cis regulatory element (CRE) and b) at least two promoters arranged in tandem wherein at least one promoter is a muscle specific promoter and one promoter is hAAT, wherein the hAAT is start-codon deleted (ΔATG) (SEQ ID NO:
- 4), operably linked to a transgene.
A recombinant expression cassette comprising a composite nucleic acid regulatory element comprising a) Muscle cis regulatory element (CRE) and b) a muscle specific promoter, operably linked to a transgene.
A method for enhancing expression of a transgene in a subject, said method comprising delivery of viral vectors comprising a nucleic acid expression cassette having a 5′ to 3′ arrangement of a) a Mus022 sequence, b) at least one muscle-specific promoter and at least one liver-specific promoter, wherein the 3′ promoter comprises a deleted start codon, c) a transgene;
- and d) a polyadenylation signal sequence.
A pharmaceutical composition for use in treating a disease or disorder in a subject in need thereof comprising recombinant AAV particles comprising an expression cassette having a synthetic ApoE enhancer sequence (SEQ ID NO:
- 7) and/or a Mus022 enhancer sequence (SEQ ID NO:
- 8) upstream of a muscle-specific promoter and a liver-specific promoter, wherein at least the muscle specific promoter or the liver-specific promoter comprises a deleted start codon (ΔATG), operably linked to a transgene.
A method of producing recombinant AAVs comprising:
- (a) culturing a host cell containing:
- (i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a composite nucleic acid regulatory element comprising a) a synthetic ApoE Enhancer and a Mus022 enhancer, and b) at least two promoters arranged in tandem wherein at least one promoter is a CK promoter and at least one promoter is a hAAT, wherein the hAAT is start-codon deleted (ΔATG), operably linked to a transgene coding for one or more RNA or protein products;
- (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans;
- (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein;
- and
- (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.
A host cell comprising (i) a plasmid comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a composite nucleic acid regulatory element comprising a) a synthetic ApoE Enhancer and a Mus022 enhancer, and b) at least two promoters arranged in tandem wherein at least one promoter is a CK promoter and at least one promoter is a hAAT, wherein the hAAT is start-codon deleted (ΔATG), operably linked to a transgene coding for one or more RNA or protein products.
Dependencies are inferred from the claim text.