Adeno-associated virus vectors encoding modified G6PC and uses thereof
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-11535870-B2
Publication Date
2022-12-27
Expiration Date
2035-12-22
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Abstract
Modified G6PC (glucose-6-phosphatase, catalytic subunit) nucleic acids and glucose-6-phosphatase-α (G6Pase-α) enzymes with increased phosphohydrolase activity are described. Also described are vectors, such as adeno-associated virus (AAV) vectors, and recombinant AAV expressing modified G6Pase-α. The disclosed AAV vectors and rAAV can be used for gene therapy applications in the treatment of glycogen storage disease, particularly glycogen storage disease type Ia (GSD-Ia), and complications thereof.
Core Innovation
This invention discloses modified glucose-6-phosphatase-α (G6Pase-α) enzymes and nucleic acids encoding them, characterized by increased phosphohydrolase activity compared to the wild-type human enzyme. Specifically, the invention identifies a serine to cysteine substitution at amino acid position 298 of human G6Pase-α, derived from comparison with the canine G6Pase-α enzyme which exhibits higher activity. The invention includes isolated nucleic acid molecules encoding such modified enzymes, vectors such as adeno-associated virus (AAV) vectors harboring these nucleic acids, recombinant AAV constructs, and host cells containing these materials for propagation or therapeutic use.
The underlying problem addressed by the invention, as described in the background, relates to glycogen storage disease type Ia (GSD-Ia), a metabolic disorder caused by deficiency of functional G6Pase-α. Current gene therapy methods using recombinant AAV vectors encoding wild-type human G6Pase-α have not achieved complete correction of hepatic G6Pase-α deficiency, leaving significant clinical complications such as hypoglycemia and the development of hepatocellular adenoma (HCA) or carcinoma (HCC) uncorrected. The invention aims to overcome these limitations by providing modified G6Pase-α enzymes with increased enzymatic activity to enhance therapeutic efficacy in gene therapy for GSD-Ia and associated complications.
Claims Coverage
The patent includes one independent claim directed to recombinant AAV compositions encoding modified G6Pase-α and their features.
Recombinant AAV encoding modified G6Pase-α with serine to cysteine substitution at position 298
The rAAV comprises a nucleic acid molecule encoding a modified human G6Pase-α characterized by a serine to cysteine substitution at amino acid 298. This nucleic acid is operably linked to a promoter to drive expression.
Specific amino acid sequence of modified G6Pase-α
The modified G6Pase-α encoded by the nucleic acid molecule comprises or consists of the amino acid sequence set forth as SEQ ID NO: 8, representing the S298C mutant.
Nucleic acid sequence encoding the modified G6Pase-α
The nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6 or the codon optimized sequence SEQ ID NO: 7, encoding the S298C mutation.
Use of a G6PC promoter in the vector
The promoter operably linked to the modified G6Pase-α nucleic acid comprises a G6PC promoter, specifically nucleotides 182-3045 of SEQ ID NO: 4 or SEQ ID NO: 5.
Vector nucleotide sequences
The nucleic acid molecule contained within the rAAV includes sequences comprising nucleotides 182-4441 or 17-4819 of SEQ ID NO: 4 or 5 covering promoter/enhancer, intron, stuffer, coding sequences, and terminal repeats.
AAV serotype specificity
The rAAV vector employed can be of serotype 8 (rAAV8) for enhanced gene delivery efficiency, though other serotypes are contemplated.
Pharmaceutical composition and formulation
The recombinant AAV can be formulated in a pharmaceutically acceptable carrier for intravenous administration as a composition suitable for therapeutic use.
The independent claim covers recombinant AAV vectors encoding a human G6Pase-α modified by a serine to cysteine substitution at amino acid 298, operably linked to a G6PC promoter, with specific nucleic acid sequences disclosed, formulated for therapeutic intravenous administration, primarily as an rAAV8 vector.
Stated Advantages
The modified G6Pase-α enzymes exhibit increased phosphohydrolase activity compared to wild-type human G6Pase-α, enhancing therapeutic efficacy.
Recombinant AAV vectors comprising the modified enzyme achieve more efficient and sustained hepatic expression of G6Pase-α, improving correction of hepatic deficiency in GSD-Ia.
Codon optimization of the nucleic acid sequences further increases translation efficiency and protein expression in the liver.
Use of G6PC promoter/enhancer elements including upstream enhancers and introns improves in vivo transgene expression levels.
The minimal effective dose of rAAV encoding modified G6Pase-α is reduced compared to wild-type vectors, potentially improving safety and clinical feasibility.
Documented Applications
Gene therapy treatment of subjects diagnosed with glycogen storage disease type Ia (GSD-Ia) by administration of recombinant AAV vectors encoding modified G6Pase-α.
Methods of promoting glucose homeostasis and inhibiting hypoglycemia in GSD-Ia subjects through AAV-mediated delivery of modified G6Pase-α.
Prevention or inhibition of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) development associated with GSD-Ia by gene therapy utilizing modified G6Pase-α expressing vectors.
Treatment or prevention of renal dysfunction or failure, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, pulmonary hypertension, or other complications associated with GSD-Ia.
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