Allyson Cole-Strauss

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.

Targeted gene conversion in a mammalian CD34+-enriched cell population using a chimeric RNA/DNA oligonucleotide.

Abstract Gene conversion of genetically inherited point mutations is a fundamental methodology for treating a variety of diseases. We tested the feasibility of a new approach using an RNA/DNA chimeric oligonucleotide. The β-globin gene was targeted at the point mutation causing sickle cell anemia. The chimera is designed to convert an A residue to a T after creating a mismatched basepair. In a CD34+-enriched population of normal cells a 5–11% conversion rate was measured using restriction enzyme polymorphism and direct DNA sequence analyses. The closely related δ-globin gene sequence appeared unchanged despite successful conversion at the β-globin locus.

Targeted gene correction: a new strategy for molecular medicine

Advances, over the past 20 years, in the genetic manipulation of mammalian cells form the scientific basis of gene therapy. A number of strategies are presently being used to replace or augment a dysfunctional gene with a correct copy of itself. Now, a novel approach to correct the dysfunctional gene in the chromosome is being developed. Data obtained from biochemical, cell-based and animal studies suggest that the era of gene repair is dawning. It is now conceivable that inherited and non-inherited disorders might be treated with a small molecular tool designed to fix the mutation directly. Here, the conceptualization of the technique and its barriers to success are discussed.

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.