Prospects of chimeric RNA-DNA oligonucleotides in gene therapy

Abstract

A strategy called targeted gene repair was developed to facilitate the process of gene therapy using a chimeric RNA-DNA oligonucleotide. Experiments demonstrated the feasibility of using the chimeric oligonucleotide to introduce point conversion in genes in vitro and in vivo. However, barriers exist in the low and/or inconstant frequency of gene repair. To overcome this difficulty, three main aspects should be considered. One is designing a more effective structure of the oligonucleotide. Trials have included lengthening the homologous region, displacing the mismatch on the chimeric strand and inventing a novel thioate-modified single-stranded DNA, which was demonstrated to be more active than the primary chimera in cell-free extracts. The second aspect is optimizing the delivery system. Producing synthetic carriers for efficient and specific transfection is demanding, especially for treatment in vivo where targeting is difficult. The third and most important aspect lies in the elucidation of the mechanism of the strategy. Investigation of the mechanism of strand exchange between the oligonucleotide molecule and double-stranded DNA in prokaryotes may greatly help to understand the mechanism of gene repair in eukaryotes. The development of this strategy holds great potential for the treatment of genetic defects and other purposes.

Commentary: Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

“…Last but not least, the technique seems to offer opportunities for the future in the field of human gene therapy to correct point mutations, for instance in monogenic inherited diseases and cancer (Christensen et al, 2006;de Semir and Aran, 2006;Kmiec, 2003;Wu et al, 2001). The therapy using oligonucleotides or RNA/DNA chimeras can result in a fraction of cells in which the wild-type gene can be restored.…”

Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques

“…Chimeraplasts or chimeric RNA/DNA oligonucleotides are designed to target a homologous genomic sequence and induce a site-specific base change (Kmiec, 1999;Igoucheva and Yoon, 2000;Wu et al, 2001). The chimera is a single-stranded molecule, usually 70-80 bases in length, with sequence complementation so that it folds into a double-hairpin configuration (Fig.…”

Sequence-specific modification of genomic DNA by small DNA fragments

“…With the exception of an adeno-associated virus-based approach (9-11), most gene-targeting strategies are oligonucleotide based (12)(13)(14)(15)(16)(17)(18). The oligonucleotidebased gene-targeting strategies include those that use triplex-forming oligonucleotides (19)(20)(21)(22), RNA/DNA hybrid oligonucleotides (23)(24)(25)(26), and small DNA fragments (SDFs) (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). This Perspective will focus on one of these oligonucleotide strategies: small-fragment homologous replacement (SFHR) (12,13,32,36).…”