Adeno-associated virus vector encoding modified g6pc and use thereof
A carrier and encoding technology, applied in the direction of viruses/phages, medical preparations containing active ingredients, and the use of vectors to introduce foreign genetic materials, etc.
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Embodiment 1
[0166] Example 1: Construction and characterization of human G6PC (G6Pase-α) mutants for AAV-mediated gene therapy
[0167] This example describes the generation of 18 human G6PC mutants and the identification of specific G6Pase-alpha mutants with increased phosphohydrolase activity.
[0168] Construction of G6PC mutants
[0169] For the construction of human G6PC mutants, the pSVL vector comprising nucleotides 1 to 1074 of the human G6PC cDNA (the entire coding region with the start codon ATG at nucleotides 1-3; SEQ ID NO: 11) was used as a template . For PCR-directed mutagenesis, the template was amplified using two outer PCR primers matching nucleotides 1 to 20 (sense) and 1055 to 1074 (antisense), located between the sense and The outer side (flanked) of the antisense mutation primer, wherein the codon to be mutated is in the middle of the mutation primer (see Figure 4 and table 1 below). The template for the hG6PC-S298C / A301V double mutant was the pSVL-hG6PC-S298C mu...
Embodiment 2
[0191]Example 2: Evaluation of the minimum vector dose required to correct hepatic G6Pase-α deficiency
[0192] This example describes the studies used to determine the minimum dose required to restore G6Pase-[alpha] activity to a level that prevents HCA / HCC development and maintains glucose homeostasis.
[0193] GSD-Ia is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma (HCA) (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). The inventors have previously demonstrated that G6pc- / - mice treated with rAAV8-G6PC expressed ≥3% of normal hepatic G6Pase-α activity (which is equivalent to ≥5 units of G6Pase-α activity; 1 nmol / min / mg defined as one unit of G6Pase-α activity), maintain glucose homeostasis to P70-P90 weeks of age and do not develop HCA (Lee et al., Hepatology 56:1719-1729, 2012; PCT Publication No. WO2015 / 081101, which incorporated herein by reference).
[0194] This study was performed using purified rAAV8 vectors o...
Embodiment 3
[0202] Example 3: Treatment of human GSD-Ia using AAV-based gene therapy
[0203] This example describes an exemplary method for the clinical use of an AAV vector encoding a modified G6PC to treat GSD-Ia.
[0204] Patients diagnosed with GSD-Ia are selected for treatment. Typically patients are at least 18 years of age and may or may not have been pre-exposed to immunomodulation. The patient is administered a therapeutically effective amount of a recombinant AAV expressing a modified G6PC, such as an rAAV comprising SEQ ID NO:4 or SEQ ID NO:5, as described herein. Recombinant AAV can be administered intravenously. Appropriate therapeutic dosages can be selected by a physician. In some cases, the therapeutically effective dose is between 1 x 10 10 to 1×10 14 In the range of virus particles (vp) / kg, such as about 1×10 11 or 1×10 12 vp / kg. In most cases, patients are administered a single dose. In the absence of immune modulation, patients may only tolerate a single infu...
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