Oligonucleotide-mediated gene targeting in human hepatocytes: implications of mismatch repair.
Gene therapy using viral vectors for liver diseases, particularly congenital disorders, is besought with difficulties, particularly immunologic reactions to viral antigens. As a result, nonviral methods for gene transfer in hepatocytes have also been explored. Gene repair by small synthetic single-stranded oligodeoxynucleotides (ODNs) produces targeted alterations in the genome of mammalian cells and represents a great potential for nonviral gene therapy. To test the feasibility of ODN-mediated gene repair within chromosomal DNA in human hepatocytes, two new cell lines with stably integrated mutant reporter genes, namely neomycin and enhanced green fluorescent protein were established. Targeting theses cells with ODNs specifically designed for repair resulted in site-directed and permanent gene conversion of the single-point mutation of the reporter genes. Moreover, the frequency of gene alteration was highly dependent on the mitotic activity of the cells, indicating that the proliferative status is an important factor for successful targeting in human hepatocytes. cDNA array expression profiling of DNA repair genes under different cell culture conditions combined with RNA interference assay showed that mismatch repair (MMR) in actively growing hepatocytes imposes a strong barrier to efficient gene repair mediated by ODNs. Suppression of MSH2 activity in hepatocytes transduced with short hairpin RNAs (shRNAs) targeted to MSH2 mRNA resulted in 25- to 30-fold increase in gene repair rate, suggesting a negative effect of MMR on ODN-mediated gene repair. Taken together, these data suggest that under appropriate conditions nonviral chromosomal targeting may represent a feasible approach to gene therapy in liver disease.
Igoucheva
Olga
O
Department of Dermatology and Cutaneous Biology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA19107, USA. Olga.Igoucheva@jefferson.edu
Alexeev
Vitali
V
Anni
Helen
H
Rubin
Emanuel
E
D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D053843:DNA Mismatch Repair; D005240:Feasibility Studies; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D008113:Liver Neoplasms / Q000378:metabolism; D009841:Oligonucleotides / Q000008:administration & dosage / Q000235:genetics*; D017354:Point Mutation; D014162:Transfection
D005456:Fluorescent Dyes; D008081:Liposomes; D009841:Oligonucleotides; D016650:Fluorescein-5-isothiocyanate