A series of letters to Nature Biotechnology have highlighted the controversy surrounding chimeric RNA/DNA oligonucleotide (RDO) technology1–3, including a failed attempt to modify the human, X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus that established specific criteria for validating gene-targeting experiments3. We believe an alternative gene-targeting approach based on single-stranded parvoviral vectors, such as adeno-associated virus (AAV), satisfies these criteria. In our laboratory, we have shown that AAV vectors can introduce specific mutations (including insertions, deletions, and substitutions) into homologous chromosomal sequences at multiple chromosomal positions4–7. With this method, up to 1% of normal human cells undergo accurate gene targeting at a single-copy chromosomal locus without preselection for reporter gene expression, which is 4–6 logs higher than the targeting rates obtained in normal human cells with conventional techniques based on electroporation or transfection of plasmid constructs8, 9. Although AAV-mediated gene targeting rates are lower than some of those reported for RDOs, the method is notable for its accuracy, flexibility, and success in human cells, so it should prove useful for both therapeutic and scientific applications requiring precise manipulation of the human genome. The four established validation criteria3 for AAV-mediated gene targeting are addressed below. First, the assay used must require conversion of a wild type to a rare mutant genotype to rule out spontaneous reversions. This has been shown for HPRTtargeting experiments in which AAV vectors introduced a 4 bp insertion or several different single-base substitutions into coding exons of wild-type alleles, at frequencies well above background mutation rates and in a dosedependent manner4, 7.

Second, the mutation should be absent in the cells used. This has been demonstrated both for HPRT gene targeting experiments4, 7 and for mutation correction experiments with reporter genes, such as neomycin phosphotransferase (neo) 4, 5 or alkaline phosphatase6. In the HPRT experiments, cell lines containing the mutated alleles did not exist in the laboratory until created by gene targeting, ruling out cell culture contamination artifacts. Third, gene targeting must be documented in