Michael C. Rice

Correction of the Mutation Responsible for Sickle Cell Anemia by an RNA-DNA Oligonucleotide

A chimeric oligonucleotide composed of DNA and modified RNA residues was used to direct correction of the mutation in the hemoglobin βS allele. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the βS 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.

Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

ABSTRACT Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.

Genetic Correction for Gene Therapy

The development of gene targeting systems has been enabled by the great advances in molecular genetics and cell culture technology. The success of producing genetic knock-outs in mice through the use of embryonic stem cells allowed the conception of efficient targeting in mammalian cells to become a distinct possibility. The availability of cloned genes and DNA sequences, combined with the ability to transfer and express genes in mammalian cells, forms the basis of gene targeting strategies. The challenges of gene targeting in mammalian cells are enormous, however, and they fall into three general categories.