A chimeric oligonucleotide composed of DNA and modified RNA residues was used to direct correction of the mutation in the hemoglobin betaS allele. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the betaS mutation, there was a detectable level of gene conversion of the mutant allele to the normal sequence. The efficient and specific conversion directed by chimeric molecules may hold promise as a therapeutic method for the treatment of genetic diseases.

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 RNA/DNA 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 liver/bone/kidney 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 RNADNA 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 RNA-DNA hybrids and would feature double-hairpin capped ends avoiding destabilization or de-The publicatio...

Mutations in thevacuolar protein sorting 35 ortholog(VPS35) gene represent a cause of late-onset, autosomal dominant familial Parkinson’s disease (PD). A single missense mutation, D620N, is considered pathogenic based upon its segregation with disease in multiple families with PD. At present, the mechanism(s) by which familialVPS35mutations precipitate neurodegeneration in PD are poorly understood. Here, we employ a germlineD620N VPS35knockin (KI) mouse model of PD to formally establish the age-related pathogenic effects of the D620N mutation at physiological expression levels. Our data demonstrate that a heterozygous or homozygous D620N mutation is sufficient to reproduce key neuropathological hallmarks of PD as indicated by the progressive degeneration of nigrostriatal pathway dopaminergic neurons and widespread axonal pathology. Unexpectedly, endogenous D620N VPS35 expression induces robust tau-positive somatodendritic pathology throughout the brain as indicated by abnormal hyperphosphorylated and conformation-specific tau, which may represent an important and early feature of mutant VPS35-induced neurodegeneration in PD. In contrast, we find no evidence for α-synuclein–positive neuropathology in agedVPS35KI mice, a hallmark of Lewy body pathology in PD. D620N VPS35 expression also fails to modify the lethal neurodegenerative phenotype of human A53T-α-synuclein transgenic mice. Finally, by crossingVPS35KI and null mice, our data demonstrate that a singleD620N VPS35allele is sufficient for survival and early maintenance of dopaminergic neurons, indicating that the D620N VPS35 protein is fully functional. Our data raise the tantalizing possibility of a pathogenic interplay between mutant VPS35 and tau for inducing neurodegeneration in PD.

Chimeric oligonucleotides consisting of RNA and DNA residues have been shown to catalyze site-directed genetic alteration in mammalian cells both in vitro and in vivo. Since the frequency of these events appears to be logs higher than the rates of gene targeting, a process involving homologous recombination, we developed a system to study the mechanisms of chimera-directed gene conversion. Using a mammalian cell-free extract and a genetic readout in Escherichia coli, we find that point mutations and single base deletions can be corrected at frequencies of approximately 0.1% and 0.005%, respectively. The reaction depends on an accurately designed chimera and the presence of functional hMSH2 protein. The results of genetic and biochemical studies reported herein suggest that the process of mismatch repair functions in site-directed gene correction.