TARGETED NUCLEOTIDE EXCHANGE IN THE ALKALINE-PHOSPHATASE GENE OF HUH-7 CELLS MEDIATED BY A CHIMERIC RNA DNA OLIGONUCLEOTIDE/

Although a variety of methods has been devised for modification of hep atic genes, none has been effective for long-term correction of geneti c disorders. In this study, we employed a recently described novel exp erimental strategy for site-directed nucleotide exchange in genomic DN A of HuH-7 human hepatoma cells. A chimeric 2'-O-methylated-RNA/DNA ol igonueleotide containing sequences complementary to 25 bases of the al kaline phosphatase gene was constructed as a duplex containing a G to A substitution at nucleotide 935. Cells were transfected with oligonuc leotides for 48 hours, then harvested for DNA isolation and polymerase chain reaction (PCR) amplification of exon 6 of the alkaline phosphat ase gene. Colony lifts were hybridized to 17 mer P-32-labeled oligonuc leotide probes specific to the 935-G and 935-A sequences. Hybridizing colonies were grown, plasmid DNA isolated, and sequenced. Transfection efficiency was determined at 24 hours by nuclear uptake of fluorescei n-12-dUTP-labeled chimeric oligonucleotides, Colonies hybridizing with the 935-A probe were identified only from cells transfected with the specific chimeric oligonucleotide; and there was no evidence of cross- hybridization. Conversion of G to A at nucleotide 935 occurred at an o verall frequency of up to 11.9% and when corrected for transfection ef ficiency approached 43%. No other alterations were detected in the seq uence of exon 6 with the targeted nucleotide exchange. These results s how that a single base pair alteration in the alkaline phosphatase gen e of HuH-7 cells can be introduced at a relatively high frequency foll owing transfection with chimeric RNA/DNA oligonucleotides. This techni que offers a novel and potentially powerful strategy for site-directed hepatic gene alteration without the use of viral-based vectors.