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

Cole-Strauss, Allyson; Yoon, Kyung-Chul; Xiang, Yufei; Byrne, Bruce C.; Rice, Michael C.; Gryn, Jeffrey; Holloman, William K.; Kmiec, Eric B.

doi:10.1126/science.273.5280.1386

Cited by 303 publications

(177 citation statements)

References 15 publications

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“…1,2 Since then, oligonucleotide-mediated modification of chromosomally and episomally located genes has been extensively studied in Escherichia coli, S. cerevisiae and mammalian cells using a variety of different oligonucleotide designs including single-stranded DNA oligonucleotides, doublestranded RNA-DNA hybrids and single-stranded DNA oligonucleotides that contain phosphorothioate linkages or 2 0 -O-methyluracil residues to improve their intracellular stability. [3][4][5][6] These studies have firmly established the feasibility of oligonucleotide-mediated gene modification and identified a number of parameters affecting its efficacy. In yeast and mammalian cells, the efficiency of chemically modified DNA oligonucleotides was enhanced by transcriptional activity of the target gene and antisense oligonucleotides (complementary to the non-transcribed strand) appeared to be more effective than sense oligonucleotides.…”

Section: Introductionmentioning

confidence: 99%

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

et al. 2006

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We have previously demonstrated that site-specific insertion, deletion or substitution of one or two nucleotides in mouse embryonic stem cells (ES cells) by single-stranded deoxyribo-oligonucleotides is several hundred-fold suppressed by DNA mismatch repair (MMR) activity. Here, we have investigated whether compound mismatches and larger insertions escape detection by the MMR machinery and can be effectively introduced in MMR-proficient cells. We identified several compound mismatches that escaped detection by the MMR machinery to some extent, but could not define general rules predicting the efficacy of complex base-pair substitutions. In contrast, we found that fournucleotide insertions were largely subject to suppression by the MSH2/MSH3 branch of MMR and could be effectively introduced in Msh3-deficient cells. As these cells have no overt mutator phenotype and Msh3-deficient mice do not develop cancer, Msh3-deficient ES cells can be used for oligonucleotide-mediated gene disruption. As an example, we present disruption of the Fanconi anemia gene Fancf. Gene Therapy (2006) 13, 686-694.

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Section: Introductionmentioning

confidence: 99%

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

et al. 2006

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“…Early reports claimed mutation rates up to 30% with chimeric RNA/DNA constructs. [7][8][9][10] However, when we performed several experiments microinjecting a chimeric RNA/DNA construct into mouse embryonic pronuclei, we were unable to detect any mutation events (data not shown). Morpholinos, having the advantages of minimal embryonic toxicity and complete resistance to nucleases, were also tested as a mediator of targeted mutagenesis in our experiments.…”

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confidence: 97%

Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition

Morozov

Wawrousek

2007

Gene Ther

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Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the aB-crystallin gene in onecelled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported. Gene Therapy (2008) Keywords: targeted mutagenesis; animal models; gene correction/modification; oligonucleotide-based therapies Site-specific gene alteration by precise gene repair mechanisms is the ultimate goal in somatic gene therapy and in generating animal models of human genetic diseases via knockout/knock-in technology. Targeted genome alteration in the one-celled embryo would be ideal, however, attempts to accomplish this have not yet been successful. 1 Currently, the production of knockout and knock-in animals utilizes gene recombination techniques in mouse embryonic stem cells, followed by transferring selected stems cells into blastocysts to create a chimeric animal. Several laboratories have studied gene correction in mouse embryonic stems cells. 2,3 Even if the mutation efficiency can be increased sufficiently, the selection and screening of embryonic stem cell clones and the microinjection of selected stem cells into blastocysts are still required. Gene alteration directly in one-celled embryos will eliminate many costly and timeconsuming steps in this process. However, early mouse embryos have never been demonstrated to potentiate targeted gene alteration. This is a unique, rapidly developing and changing system distant from any other described for genomic DNA alteration. There is limited data for mouse ova and early embryos suggesting a rapid change in double-strand DNA (dsDNA) break repair pathways, 4 which could greatly affect gene correction efficiency.In other model systems, a variety of synthetic oligonucleotide constructs has been successfully used: triple helix-forming oligonucleotides (TFO), 5,6 chimeric RNA/DNA constructs, 7-10 peptide nucleic acids, 11 short DNA fragments, 12 and single-strand DNA (ssDNA). [13][14][15] However, the necessity of having a specific sequence of targeted DNA, and restrictions for triplex formation under physiological conditions, 16 impose serious limitations on the universality of TFOs as mutation mediators. Moreover, the previously reported efficiency of gene alteration mediated by chimeric RNA/DNA constructs cannot be reproduced by many e...

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“…In such cases, an independent HR event is expected at each end of the homologously aligned transgene: such events would not necessarily be identical in nature. Indeed, it is also possible that HR may occur at one transgene end and nonhomologous in 1996 [10,52], COs have been used to correct various point mutations in a range of mammalian cells. However, reported rates of gene repair have been highly variable (ranging from zero to ca.…”

Section: Chimeric Oligonucleotides and Hrmentioning

confidence: 99%

Theoretical mechanisms in targeted and random integration of transgene DNA

Smith

2001

Reprod. Nutr. Dev.

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

Cited by 303 publications

References 15 publications

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition

Theoretical mechanisms in targeted and random integration of transgene DNA

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