Gene Correction by RNA‐DNA Oligonucleotides

Abstract

of several hairs and DOPA staining of hair follicles in the An oligonucleotide composed of a contiguous stretch of RNA and DNA residues has been developed to facilitate the correctreated skin of albino mice. Such RDOs might hold a promise tion of single-base mutations of episomal and chromosomal as a therapeutic method for the treatment of skin diseases. targets in mammalian cells. The design of the oligonucleotide However, the frequency of gene correction varies among different cells, indicating that cellular activities, such as reexploited the highly recombinogenic RNA-DNA hybrids and featured hairpin capped ends avoiding destruction by cellular combination and repair, may be important for gene conversion helicases or exonucleases. The RNA-DNA oligonucleotide by RDOs. As this technology becomes more widely utilized in the scientific community, it will be important to understand (RDO) was designed to correct a point mutation in the the mechanism and to optimize the design of RDOs to imtyrosinase gene and caused a permanent gene correction in mouse albino melanocytes, determined by clonal analysis at prove their efficiency and general applicability. the level of genomic sequence, protein and phenotypic change. Recently, we demonstrated correction of the tyrosinase gene

Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides

“…Our laboratory and others have shown that various forms of oligonucleotides, RNA-DNA oligonucleotide (RDO) and single-stranded oligodeoxynucleotide (ODN), are capable of chromosomal gene alterations in mammalian cells. 27 One of the major limitations of gene targeting is the low frequency of homologous recombination (10 À5 -10…”

Targeted gene correction by small single-stranded oligonucleotides in mammalian cells

“…These approaches use oligonucleotide technology related to triple-helix-forming oligonucleotide [1][2][3][4] and chimeric RNA-DNA oligonucleotide [5][6][7][8][9][10][11][12][13][14][15][16] for recognition and repair of the DNA sequence, and antisense oligonucleotides and ribozymes for editing RNA sequence. [17][18][19] Unlike gene replacement, where a therapeutic gene is transferred to the cell or affected organ, usually by virus-mediated gene transfer, this approach intends to produce predefined alterations in the DNA or RNA of eukaryotic cells.…”

“…The RNA-DNA oligonucleotide was shown to cause a site-specific chromosomal correction or mutation in tissue culture cells and in vivo. [6][7][8][9][10][11][12][13][14][15][16] A permanent and stable gene correction by the RNA-DNA oligonucleotide was demonstrated by clonal analysis at the level of genomic sequence, protein and phenotypic change. 15 The RNA-DNA oligonucleotide might hold promise as a therapeutic method for the treatment of genetic diseases.…”

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides

“…Recently, the chimeric RNA/DNA oligonucleotides (COs), a molecule consisting of RNA and DNA bases configured into a double-stranded, double hairpin conformation (1,2), has been used successfully to accomplish single-base exchange. These vectors have been used to repair point and frameshift mutations in mammalian cell culture (3)(4)(5)(6)(7)(8)(9), cell-free extracts (10)(11)(12) and animal models (13)(14)(15). In addition, gene repair using COs has been carried out successfully in plants ( [16][17][18] and plant cell-free extracts (19).…”