Adeno-associated virus vectors encoding modified G6PC and uses thereof
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10415044-B2
Publication Date
2019-09-17
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
The invention concerns modified glucose-6-phosphatase-α (G6Pase-α) enzymes with increased phosphohydrolase activity and nucleic acids encoding these modified enzymes. It describes vectors, particularly adeno-associated virus (AAV) vectors, and recombinant AAV particles expressing the modified G6Pase-α. These vectors are suitable for gene therapy applications aimed at treating glycogen storage disease (GSD), specifically glycogen storage disease type Ia (GSD-Ia), and associated complications.
The problem addressed is the inability of existing gene therapies using recombinant AAV encoding G6Pase-α to completely correct hepatic deficiency of G6Pase-α in GSD-Ia patients. GSD-Ia is caused by a deficiency in functional G6Pase-α, a hydrophobic endoplasmic reticulum enzyme crucial for glucose homeostasis. Patients suffer from fasting hypoglycemia and have high risks of developing hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC). Current dietary and gene therapy treatments fail to address the long-term complications of GSD-Ia effectively.
The invention identifies that the canine G6Pase-α enzyme has greater activity than the human enzyme and uses site-directed mutagenesis to create human G6Pase-α mutants containing canine amino acids at specific positions, particularly a serine to cysteine substitution at amino acid 298 (S298C). These modified enzymes exhibit significantly increased phosphohydrolase activity. The nucleic acids encoding these modified enzymes are incorporated into AAV vectors with specific promoter/enhancer elements and intronic and stuffer sequences to enable enhanced hepatic expression. This approach aims to increase therapeutic efficacy in gene therapy for GSD-Ia by achieving more complete correction of the enzyme deficiency and thereby improving glucose homeostasis and preventing complications such as HCA and HCC.
Claims Coverage
The claims cover six main inventive features focusing on nucleic acid molecules encoding modified G6Pase-α, vectors including AAV vectors encoding the modified enzyme, and isolated host cells.
Modified G6Pase-α nucleic acid molecule encoding serine to cysteine substitution at amino acid 298
An isolated nucleic acid molecule encoding a modified human glucose-6-phosphatase-α enzyme containing a serine to cysteine substitution at amino acid 298 of the human G6Pase-α sequence (SEQ ID NO: 2).
Specific amino acid and nucleotide sequences for the modified enzyme
The modified G6Pase-α amino acid sequence comprises or consists of SEQ ID NO: 8; the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 encoding the modified enzyme with the S298C substitution, optionally including codon optimization.
Vector comprising nucleic acid molecule encoding the modified G6Pase-α
A vector comprising the nucleic acid molecule encoding the modified G6Pase-α. The nucleic acid is operably linked to a promoter, including a G6PC promoter that comprises nucleotides 182-3045 of SEQ ID NO: 4 or SEQ ID NO: 5. The vector may include additional nucleotides 182-4441 of SEQ ID NO: 4 or 5.
AAV vector comprising the modified G6Pase-α nucleic acid
The vector is an adeno-associated virus (AAV) vector, including AAV serotype 8 (AAV8). The AAV vector may comprise nucleotides 17-4819 of SEQ ID NO: 4 or 5, inclusive of inverted terminal repeats (ITRs) flanking the expression cassette.
Isolated host cell comprising the nucleic acid molecule
An isolated host cell comprising the isolated nucleic acid molecule encoding the modified G6Pase-α, suitable for propagation or production of recombinant AAV particles.
The claims collectively cover isolated nucleic acids encoding human G6Pase-α modified at amino acid 298, vectors incorporating these nucleic acids operably linked to specific G6PC promoters/enhancers, recombinant AAV vectors of various serotypes including AAV8, and host cells containing these nucleic acids or vectors, all directed towards enhanced gene therapy for glycogen storage disease type Ia.
Stated Advantages
The modified human G6Pase-α enzymes exhibit increased phosphohydrolase activity compared to wild-type human G6Pase-α, potentially improving therapeutic outcomes.
Use of AAV vectors containing the modified G6Pase-α nucleic acid sequences achieves more efficient and stable hepatic transgene expression than previous vectors.
The incorporation of G6PC promoter/enhancer elements, introns, and stuffer sequences in the vectors enhances liver transduction and gene expression.
Codon optimization of the G6PC coding sequence increases translation efficiency, leading to higher enzyme expression in vivo.
Gene therapy with vectors encoding modified G6Pase-α at optimized doses can restore hepatic G6Pase-α to levels sufficient to maintain glucose homeostasis and prevent hepatocellular adenoma and carcinoma development.
Documented Applications
Gene therapy treatment of glycogen storage disease type Ia (GSD-Ia) in subjects with a deficiency in glucose-6-phosphatase-α.
Methods of promoting glucose homeostasis and inhibiting hypoglycemia in subjects with GSD-Ia via administration of recombinant AAV vectors encoding modified G6Pase-α.
Inhibiting or preventing the development of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) associated with GSD-Ia by gene therapy using the disclosed vectors.
Treating other complications of GSD-Ia including renal dysfunction or failure, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, and pulmonary hypertension by administering the disclosed recombinant AAV vectors.
Production of recombinant adeno-associated virus vectors and isolated host cells suitable for use in gene therapy and research applications related to GSD.
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