Adeno-associated virus vectors for treatment of glycogen storage disease
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10113183-B2
Publication Date
2018-10-30
Expiration Date
2034-11-25
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Abstract
The present disclosure describes improved adeno-associated virus (AAV) vectors for gene therapy applications in the treatment of glycogen storage disease, particularly glycogen storage disease type Ia (GSD-Ia). Described are recombinant nucleic acid molecules, vectors and recombinant AAV that include a G6PC promoter/enhancer, a synthetic intron, a G6PC coding sequence (such as a wild-type or codon-optimized G6PC coding sequence), and stuffer nucleic acid sequence situated between the G6PC promoter/enhancer and the intron, as well as between the intron and the G6PC coding sequence. The recombinant AAVs disclosed herein exhibit highly efficient liver transduction and are capable of correcting metabolic abnormalities in an animal model of GSD-Ia.
Core Innovation
The invention provides recombinant nucleic acid molecules, adeno-associated virus (AAV) vectors, and recombinant AAV useful for gene therapy of glycogen storage disease type Ia (GSD-Ia). The recombinant nucleic acid molecules include a G6PC promoter/enhancer, a synthetic intron flanked by stuffer nucleic acid sequences, and a G6PC coding sequence that may be wild-type or codon-optimized. These sequences are arranged such that the stuffer sequences are situated between the promoter/enhancer and the intron, and also between the intron and the coding sequence. The AAV vectors further include inverted terminal repeat (ITR) sequences and comprise any suitable AAV serotype, including AAV8 vectors.
GSD-Ia is caused by a deficiency in the enzyme glucose-6-phosphatase-α (G6Pase-α) encoded by the G6PC gene, which is expressed mainly in liver, kidney, and intestine. This deficiency impairs glucose homeostasis with symptoms including fasting hypoglycemia and hepatomegaly. Current treatments such as dietary therapies manage hypoglycemia but do not correct chronic complications such as hepatocellular adenoma and carcinoma. Previous gene therapy approaches using recombinant AAV with alternative promoters did not fully correct hepatic G6Pase-α deficiency.
The disclosed recombinant AAV vectors with the G6PC promoter/enhancer (AAV-GPE) demonstrate significantly improved hepatic transgene expression and correction of metabolic abnormalities as evidenced in animal models. The inclusion of stuffer sequences flanking the intron notably enhances liver transduction and expression. Moreover, the upstream enhancer elements within the G6PC promoter are critical for optimal expression. Codon-optimization of the G6PC coding sequence further increases translation efficiency, yielding higher G6Pase-α expression. Gene therapy with the recombinant AAV-GPE vector was efficacious in GSD-Ia mice for at least 70-90 weeks, normalizing biochemical and pathological parameters.
Claims Coverage
The patent contains one set of independent claims focusing on recombinant nucleic acid molecules and related compositions and methods for treating GSD-Ia.
Recombinant nucleic acid molecule with modified G6PC coding region
A recombinant nucleic acid molecule comprising a G6PC promoter/enhancer, a synthetic intron, and a G6PC coding region with at least 95% sequence identity to nucleotides 182-4441 of SEQ ID NO: 1, which includes at least one nucleotide substitution resulting in a coding change at specified residues of the human G6PC protein.
Inclusion of inverted terminal repeat (ITR) sequences
Recombinant nucleic acid molecules further comprising 5′ and 3′ inverted terminal repeat sequences with at least 95% identity to nucleotides 17-4819 of SEQ ID NO: 1.
Vectors comprising the recombinant nucleic acid molecule
Vectors that include the recombinant nucleic acid molecule; vectors are specifically adeno-associated virus (AAV) vectors, particularly serotype 8 (AAV8) vectors.
Isolated host cells carrying the vector
Isolated host cells comprising the vector encoding the recombinant nucleic acid molecule.
Recombinant AAV particles encapsidating the recombinant nucleic acid
Recombinant AAV (rAAV) particles comprising the recombinant nucleic acid molecule, including rAAV8.
Pharmaceutical compositions with the recombinant AAV
Pharmaceutical compositions including the rAAV and a pharmaceutically acceptable carrier, formulated for intravenous administration.
Methods of treating GSD-Ia using the rAAV
Methods for treating subjects diagnosed with glycogen storage disease type Ia by administering a therapeutically effective amount of the recombinant AAV, including intravenous administration at specified dose ranges, as single or multiple doses.
The claims cover recombinant nucleic acid molecules comprising a G6PC promoter/enhancer, synthetic intron, and modified G6PC coding region with precise sequence identity and substitutions, vectors comprising these molecules including AAV8 vectors, recombinant AAV particles, pharmaceutical compositions thereof, isolated host cells, and methods of treatment for GSD-Ia using these compositions.
Stated Advantages
The recombinant AAV vectors with the G6PC promoter/enhancer direct significantly more efficient and persistent hepatic expression of G6Pase-α than vectors using alternative promoters.
Gene therapy using the disclosed recombinant AAV vectors achieves complete normalization of hepatic G6Pase deficiency and correction of metabolic abnormalities in animal models of GSD-Ia.
Inclusion of stuffer sequences flanking the intron enhances liver transduction and transgene expression efficiency.
Upstream enhancer elements of the G6PC promoter are critical for optimal transgene expression and therapeutic efficacy.
Codon-optimization of the G6PC coding sequence increases translation efficiency, yielding substantially greater hepatic G6Pase-α expression.
Long-term efficacy of gene therapy treatment is demonstrated, with normalization of blood metabolites, glucose tolerance, hepatic fat storage, and absence of hepatic abnormalities such as hepatocellular adenoma.
Documented Applications
Gene therapy treatment of glycogen storage disease type Ia (GSD-Ia) in animal models using recombinant AAV vectors expressing G6Pase-α.
Administration of recombinant AAV expressing G6Pase-α, including codon-optimized sequences, for the correction of metabolic and pathological abnormalities associated with GSD-Ia.
Intravenous administration of recombinant AAV vectors to subjects diagnosed with GSD-Ia to achieve therapeutic hepatic transgene expression.
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