Adeno-associated virus vectors for treatment of glycogen storage disease
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-11060110-B2
Publication Date
2021-07-13
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 in gene therapy applications for the treatment of glycogen storage disease, especially glycogen storage disease type Ia (GSD-Ia). The recombinant nucleic acid molecules include a G6PC promoter/enhancer, a synthetic intron, the G6PC coding region (which can be codon-optimized), and stuffer nucleic acid sequences situated between the G6PC promoter/enhancer and the intron, as well as between the intron and the G6PC coding sequence. Recombinant AAVs harboring these nucleic acids demonstrate highly efficient liver transduction and ability to correct metabolic abnormalities in animal models of GSD-Ia.
The problem addressed arises from the deficiency in glucose-6-phosphatase-α (G6Pase-α), due to mutations in the G6PC gene, which is essential to glucose homeostasis. Patients with GSD-Ia experience severe hypoglycemia, growth retardation, hepatomegaly, nephromegaly, and metabolic abnormalities without current cure. Prior gene therapy attempts using recombinant AAV vectors driven by various promoters including CBA promoter/CMV enhancer or canine/human G6PC promoters achieved some correction but failed to completely restore hepatic G6Pase-α deficiency and metabolic normalization.
This invention improves on prior art by identifying critical features of an optimized AAV vector that efficiently drives persistent, high-level hepatic G6Pase-α expression. Important elements include the use of the full G6PC promoter/enhancer sequence (nucleotides −2684 to −1), the inclusion of a synthetic intron flanked by stuffer sequences, and optional codon optimization of the G6PC coding region. These features collectively enhance transduction efficiency, maintain expression over the long term, and correct metabolic abnormalities and pathological manifestations of GSD-Ia, including prevention of hepatocellular adenoma in animal models.
Claims Coverage
The patent includes multiple independent claims covering recombinant nucleic acid molecules, vectors, recombinant AAV, compositions, and methods of treatment. The main inventive features focus on the structure of the nucleic acid molecule, vector composition, and therapeutic use.
Recombinant nucleic acid molecule with G6PC promoter/enhancer, synthetic intron, and modified coding region
A recombinant nucleic acid molecule comprising a G6PC promoter/enhancer, a synthetic intron, and a G6PC coding region, wherein the nucleotide sequence is at least 90% identical to nucleotides 182-4441 of SEQ ID NO: 1. The nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 4 but with residue changes at specified positions (including positions 3, 54, 139, 196, 199, 242, 247, 292, 298, 301, 318, 324, 332, 347, 349, 350, or 353) of the human G6PC protein.
Inclusion of inverted terminal repeat sequences in the recombinant nucleic acid molecule
The recombinant nucleic acid molecule further comprises 5′ and 3′ inverted terminal repeat (ITR) sequences, with nucleotide identity at least 90% to nucleotides 17-4819 of SEQ ID NO: 1, forming a complete cassette suitable for packaging into AAV vectors.
Vector comprising the recombinant nucleic acid molecule
Vectors comprising the above recombinant nucleic acid molecule, specifically adeno-associated virus (AAV) vectors such as AAV8 serotype vectors, for efficient gene delivery.
Isolated host cells comprising the vector
Isolated host cells containing the recombinant nucleic acid molecule or AAV vectors, useful for production of recombinant AAV particles.
Recombinant AAV particles encoding the recombinant nucleic acid
Recombinant AAV particles (e.g., rAAV8) packaged with the recombinant nucleic acid molecules comprising the modified G6PC elements for gene therapy applications.
Pharmaceutical compositions and administration routes
Pharmaceutical compositions containing the recombinant AAV particles in pharmaceutically acceptable carriers, formulated for intravenous administration.
Method of treating glycogen storage disease type Ia
A method of treating a subject diagnosed with GSD-Ia by administering a therapeutically effective amount of the recombinant AAV, intravenously or by multiple dosings within a dose range about 1×10¹¹ to 1×10¹⁴ viral particles per kilogram body weight, optionally as a single or multiple dose treatment.
The independent claims broadly cover recombinant nucleic acid molecules with specified G6PC promoter/enhancer and coding modifications, vectors and recombinant AAV derived therefrom (notably AAV8), pharmaceutical compositions, and clinical methods for treating glycogen storage disease type Ia by administering these AAV-based gene therapy agents.
Stated Advantages
The recombinant AAV vectors exhibit significantly improved hepatic transduction efficiency and persistent gene expression compared to vectors using alternative promoters like the chicken β-actin promoter/CMV enhancer.
Gene therapy mediated by the disclosed AAV vectors achieves complete normalization of hepatic G6Pase-α deficiency and correction of metabolic abnormalities in animal models of GSD-Ia.
The inclusion of the full G6PC promoter/enhancer, synthetic intron flanked by stuffer sequences, and codon optimization of the G6PC coding sequence collectively enhance expression efficiency and durability.
Treatment with the recombinant AAV vectors prevents pathological manifestations such as hepatocellular adenoma and hepatomegaly in animal models while maintaining normal blood metabolite and glucose tolerance profiles.
Documented Applications
Gene therapy treatment of glycogen storage disease type Ia (GSD-Ia) in subjects by administration of recombinant AAV vectors encoding G6Pase-α.
Use of AAV vectors for liver-targeted gene therapy to correct metabolic and pathological abnormalities associated with GSD-Ia.
Production of recombinant AAV vectors and host cell lines for therapeutic applications in GSD-Ia.
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