Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNAzDNA oligonucleotide

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNAyDNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liveryboneykidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents. Targeted correction of disease-related mutations or sitedirected inactivation of viral genes by homologous recombination would be a effective strategy for gene therapy. Unfortunately, homologous recombination in mammalian cells between a target gene and an exogenous DNA vector takes place at relatively low frequencies and is complicated by interference from an illegitimate recombination pathway that does not depend on sequence homology (1–5). An alternative approach involves targeted mutagenesis facilitated by triple-helixforming oligonucleotides coupled to cross-linking agents (6, 7). Such oligonucleotides have been used previously to change DNA sequences thereby altering gene expression but these approaches have been limited by the sequence restriction of the target that must consist of homopurine or homopyrimidine stretches (8, 9). Moreover, the generation of a specific type of mutation or correction has been difficult to achieve (6, 7). We have developed an experimental strategy to enable correction of single-base mutations of episomal sequences by using a chimeric oligonucleotide of unique design. This strategy evolved from in vitro studies on homologous recombination conducted with the RecA and Rec2 proteins (10–12). Analysis of the homologous pairing reaction promoted by the Rec2 protein of Ustilago maydis revealed that RNAzDNA hybrids were more active in homologous pairing reactions than corresponding DNA duplexes (10). Since pairing would appear to be the rate-limiting step during the gene targeting process (13), the overall frequency of recombination should be elevated if the number of pairing events is elevated. It was also discovered that joint molecule formation proceeded efficiently even when the ends of the hybrid were capped with double hairpin structures. These observations led us to a strategy for gene targeting in which vector design would exploit the natural recombinogenicity of RNAzDNA hybrids and would feature double-hairpin capped ends avoiding destabilization or destruction by cellular helicases or exonucleases. A chimeric oligonucleotide can be designed so that it aligns in perfect register with a specified genomic target or in imperfect register such that a single base pair is different between the oligonucleotide and a specified targeted nucleotide. In the latter case, structural distortion created by the mismatched base pair should be recognized by the endogenous repair systems (14, 15) and a change in sequence on either chimeric oligonucleotide or the target sequence would ensue. An additional feature in the design of chimeric oligonucleotides was modification of the RNA residues by 29-O-methylation of the ribose sugar (16) to render the oligonucleotide resistant to the RNase H activity present in mammalian cells. To test the feasibility of chimeric oligonucleotide-based targeting in mammalian cells, we chose an episomal target utilizing human liveryboneykidney alkaline phosphatase cDNA, whose gene product is important in skeletal mineralization. Mutations in the structural gene give rise to hypophosphatasia, a metabolic bone disease with variable clinical severity ranging from still birth with almost no mineralized bone to pathological fractures in adults (17). A wellcharacterized mutant form has a missense mutation (G3 A) at position 711 of the cDNA that results in the loss of enzymatic activity (18). This particular gene was chosen for study since direct biochemical and histochemical assays are available to monitor its activity. With the use of an appropriate chromogenic substrate dye, enzymatic activity can be detected by deposition of pigment on the cells since the enzyme localizes to the cell surface (19). Chinese hamster ovary (CHO) cells were chosen as hosts since there is little detectable endogenous alkaline phosphatase expressed (19, 20). A gene correction event mediated by chimeric oligonucleotides would restore enzymatic activity and be visualized by pigment development. Thus, targeted gene correction can be detected directly without selection or screening for the rare successful targeting events. In this paper, we demonstrate that targeted mutagenesis by chimeric oligonucleotide occurs at a high frequency in a sequence-specific manner. MATERIALS AND METHODS Synthesis and Purification of Oligonucleotides. The chimeric oligonucleotides were synthesized on a 0.2-mmol scale by using the 1000-Å-wide-pore CPG on the ABI 394 DNAyRNA synthesizer. The exocyclic amine groups of DNA phosphoramidites (Applied Biosystems) are protected with benzoyl for adenosine and cytidine and isobutyryl for guanosine. The 29-O-methyl RNA phosphoramidites (Glen Research, Sterling, VA) are protected with a phenoxyacetyl group for adenosine, dimethylformamide for guanosine and an isobutyryl group for cytidine. After the synthesis was complete, the base-protecting groups were removed by heating in ethanolyconcentrated ammonium hydroxide, 1:3 (volyvol), for 20 h at 55°C. The crude oligonucleotides were purified by polyacrylamide gel The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: FBS, fetal calf serum; SV40, simian virus 40. *To whom reprint requests should be addressed.