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Adeno-associated virus vectors encoding modified g6pc and uses 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.

Active Publication Date: 2018-01-26
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although previous gene therapy studies using recombinant adeno-associated viruses (AAV) carrying G6Pase-α have been performed in animal models of GSD-Ia, none were able to completely correct hepatic G6Pase-α deficiency

Method used

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  • Adeno-associated virus vectors encoding modified g6pc and uses thereof
  • Adeno-associated virus vectors encoding modified g6pc and uses thereof
  • Adeno-associated virus vectors encoding modified g6pc and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0164] Example 1: Construction and characterization of human G6PC (G6Pase-α) mutants for AAV-mediated gene therapy

[0165] This example describes the generation of 18 human G6PC mutants and the identification of specific G6Pase-α mutants with increased phosphohydrolase activity.

[0166] Construction of G6PC mutant

[0167] To construct the human G6PC mutant, the pSVL vector containing nucleotides 1 to 1074 of human G6PC cDNA (the entire coding region, where the start codon ATG is at nucleotides 1-3; SEQ ID NO: 11) was used as a template . For PCR-directed mutagenesis, two external PCR primers are used to amplify the template. The external primers match nucleotides 1 to 20 (sense) and 1055 to 1074 (antisense), located in a 20-nucleotide long sense and The outer side of the antisense mutant primer (flanked), where the codon to be mutated is in the middle of the mutant primer (see Figure 4 And Table 1) below. The template of the hG6PC-S298C / A301V double mutant is the pSVL-hG6PC-S2...

Embodiment 2

[0189] Example 2: Evaluation of the minimum carrier dose required to correct liver G6Pase-α deficiency

[0190] This example describes a study to determine the minimum dose required to restore G6Pase-α activity to a level that prevents the development of HCA / HCC and maintains glucose homeostasis.

[0191] GSD-Ia is characterized by impaired glucose homeostasis and long-term complications 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 express ≥3% of normal liver G6Pase-α activity (which is equivalent to ≥5 units of G6Pase-α activity; 1 nmol / min / mg It is defined as a unit of G6Pase-α activity), which maintains glucose homeostasis to P70-P90 weeks of age and does not develop HCA (Lee et al., Hepatology 56: 1719-1729, 2012; PCT Publication No. WO 2015 / 081101, It is incorporated herein by reference).

[0192] This study was performed using purified rAAV8 vector (supplied...

Embodiment 3

[0200] Example 3: Treatment of human GSD-Ia using AAV-based gene therapy

[0201] This example describes an exemplary method of clinically using AAV vectors encoding modified G6PC to treat GSD-Ia.

[0202] Select patients diagnosed with GSD-Ia for treatment. Usually the patient is at least 18 years old and may or may not be pre-exposed to immunomodulation. The patient is administered a therapeutically effective amount of a recombinant AAV expressing modified G6PC, such as rAAV comprising SEQ ID NO: 4 or SEQ ID NO: 5, as described herein. Recombinant AAV can be administered intravenously. The appropriate therapeutic dose can be selected by the physician. In some cases, the therapeutically effective dose is 1×10 10 To 1×10 14 Virus particles (vp) / kg, such as about 1×10 11 Or 1×10 12 vp / kg. In most cases, a single dose is administered to the patient. In the absence of immune regulation, patients may only tolerate a single infusion of rAAV. If the subject is previously exposed to...

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Abstract

Modified G6PC (glucose-6-phosphatase, catalytic subunit) nucleic acids and glucose-6-phosphatase-alpha (G6Pase-alpha) enzymes with increased phosphohydrolase activity are described. Also described arevectors, such as adeno-associated virus (AAV) vectors, and recombinant AAV expressing modified G6Pase-alpha. 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 la (GSD-Ia), and complications thereof.

Description

[0001] Cross references to related applications [0002] This application claims the benefits of U.S. Provisional Application No. 62 / 096,400 filed on December 23, 2014, which is incorporated herein by reference in its entirety. Technical field [0003] The present disclosure relates to gene therapy vectors encoding modified glucose-6-phosphatase-α (G6PC) enzymes with enhanced activity and uses thereof, such as for the treatment of glycogen storage diseases (GSD) and complications related to GSD disease. Background of the invention [0004] Type Ia glycogen storage disease (GSD-Ia or von Gierke disease, MIM232200) is caused by a defect in glucose-6-phosphatase-α (G6Pase-α or G6PC), glucose- 6-phosphatase-α is an enzyme mainly expressed in the liver, kidney and intestine (Chou et al., Nat Rev Endocrinol 6:676-688, 2010). G6Pase-α is encoded by the G6PC gene and is a hydrophobic protein anchored in the endoplasmic reticulum (ER) through nine transmembrane helices (Chou et al., Nat Re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16C12N15/864C12N7/01A61K38/46A61P3/08A61P35/00A61P13/12
CPCA61K38/00C12N9/16C12Y301/03009A61K38/47C12Y302/00A61P1/16A61P13/12A61P3/00A61P35/00A61P3/08C12N15/85C12N15/8645A61K48/00A61K45/06C12N15/66
Inventor J·J·周
Owner UNITED STATES OF AMERICA
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