Correction/mutation of acid α-D-glucosidase gene by modified single-stranded oligonucleotides: in vitro and in vivo studies
- 22 September 2003
- journal article
- Published by Springer Nature in Gene Therapy
- Vol. 10 (22) , 1910-1916
- https://doi.org/10.1038/sj.gt.3302096
Abstract
Deficiency in acid -D-glucosidase results in Pompe's disease. Modified single-stranded oligonucleotide (ODN) was designed to correct the acid -D-glucosidase gene with a C1935 A (Asp Glu) point mutation which causes a complete loss of enzymatic activity for glycogen digestion in the lysosome. The ODN vectors contained a stretch of normal oligonucleotide flanked by phosphorothioated sequences. The 25mer and 35mer ODNs were homologous to the target sequence, except for a mismatched base in the middle. The ODNs caused permanent and inheritable restoration of acid -D-glucosidase activity in skin fibroblast cells carrying this mutation derived from a Pompe's disease patient. Gene correction was confirmed by amplification refractory mutation system-PCR (ARMS-PCR), restriction fragment length polymorphism (RFLP) and direct DNA cloning and sequencing. The increased acid -D-glucosidase activity was detected using 4-MUG as the artificial substrate. The correction efficiency, ranging from 0.5 to 4%, was dependent on the length and polarity of the MSSOV used, the optimal design being a sense-strand 35mer ODNs. Repeated treatment of the mutant fibroblast cells with the ODNs substantially increased correction. We also constructed ODN vectors to trigger specific and in vivo nonsense mutation in the mouse acid -D-glucosidase gene. The ODNs were in complex with YEEE-K18, an asialoglycoprotein-receptor ligand tagged with polylysine and targeted to hepatocytes and renal cells in vivo through intravenous injection. The mutated genotype was detected in the liver and the kidney by ARMS-PCR and glycogen accumulation in the lysosome of the liver cells. The studies demonstrate the utility of single-stranded ODN to direct targeted gene correction or mutation in a human hereditary disease and in an animal model. Our data open the possibility of developing ODN vector as a therapeutic approach for treatment of human hereditary diseases caused by point mutation.Keywords
This publication has 17 references indexed in Scilit:
- DNA oligonucleotide treatment corrects the age‐associated decline in DNA repair capacityThe FASEB Journal, 2002
- In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotidesNucleic Acids Research, 2001
- Targeted gene correction by small single-stranded oligonucleotides in mammalian cellsGene Therapy, 2001
- Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-α-glucosidaseProceedings of the National Academy of Sciences, 1999
- Persistence of an [E1-, Polymerase-] Adenovirus Vector Despite Transduction of a Neoantigen into Immune-Competent MiceHuman Gene Therapy, 1999
- Adenovirus-Mediated Transfer of the Acid -Glucosidase Gene into Fibroblasts, Myoblasts and Myotubes from Patients with Glycogen Storage Disease Type II Leads to High Level Expression of Enzyme and Corrects Glycogen AccumulationHuman Molecular Genetics, 1998
- Cell-Type Specific and Ligand Specific Enhancement of Cellular Uptake of Oligodeoxynucleoside Methylphosphonates Covalently Linked with a Neoglycopeptide, YEE(ah-GalNAc)3Bioconjugate Chemistry, 1995
- Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligandBioconjugate Chemistry, 1992
- Transformation of yeast with synthetic oligonucleotides.Proceedings of the National Academy of Sciences, 1988
- Identification of heterozygotes for glycogenosis 2 (Acid maltase deficiency)Clinical Genetics, 1981