Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides

Kenner, Oliver; Kneisel, Andrea Reinhilde; Klingler, J. A.; Bartelt, Britta; Speit, Günter; Vogel, Walther; Kaufmann, Dieter

doi:10.1016/s0006-291x(02)02749-3

Cited by 21 publications

(23 citation statements)

References 30 publications

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“…mutations was demonstrated by the RFLP analysis of both RT-PCR products and genomic DNA. While the data presented here indicate an effi ciency that ranges between 0.1% and 2% that is consistent with the effi ciencies observed by other groups studying SFHR (Gruenert et al, 2003), several studies evaluating SSOs or shorter DNA fragments in rodent cells (Kenner et al, 2002(Kenner et al, , 2004 or human lymphoblasts (Hunger-Bertling et al, 1990) have observed effi ciencies of 10 −4 to 10 −5 and 6 × 10 −6 , respectively. The higher frequencies observed in this study may refl ect differences in the cells used, the size of the correcting DNA fragments, and/or the methods of transfection.…”

Section: Discussionsupporting

confidence: 91%

“…This preliminary fi nding is several orders of magnitude higher than was observed previously using a similar gene targeting strategies (Hunger-Bertling et al, 1990;Kenner et al, 2002Kenner et al, , 2004 and may refl ect that apparently small differences can have a signifi cant effect on gene targeting effi ciencies. Since previous studies have indicated that homologous recombination is inhibited by wt p53 (Mekeel et al, 1997;Lu et al, 2003;Yoon et al, 2004;Saintigny et al, 2005;Gatz and Wiesmuller, 2006), the fact that these cells express wt p53 suggests that SFHR may be independent of the recombinatorial pathway, which is regulated by p53.…”

Section: Transfectioncontrasting

confidence: 53%

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Sequence-Specific Correction of Genomic Hypoxanthine-Guanine Phosphoribosyl Transferase Mutations in Lymphoblasts by Small Fragment Homologous Replacement

Bedayat

Abdolmohamadi

et al. 2010

Oligonucleotides

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Oligo/polynucleotide-based gene targeting strategies provide new options for achieving sequence-specifi c modifi cation of genomic DNA and have implications for the development of new therapies and transgenic animal models. One such gene modifi cation strategy, small fragment homologous replacement (SFHR), was evaluated qualitatively and quantitatively in human lymphoblasts that contain a single base substitution in the hypoxanthine-guanine phosphoribosyl transferase (HPRT1) gene. Because HPRT1 mutant cells are readily discernable from those expressing the wild type (wt) gene through growth in selective media, it was possible to identify and isolate cells that have been corrected by SFHR. Transfection of HPRT1 mutant cells with polynucleotide small DNA fragments (SDFs) comprising wild type HPRT1 (wtHPRT1) sequences resulted in clones of cells that grew in hypoxanthine-aminopterin-thymidine (HAT) medium. Initial studies quantifying the effi ciency of correction in 3 separate experiments indicate frequencies ranging from 0.1% to 2%. Sequence analysis of DNA and RNA showed correction of the HPRT1 mutation. Random integration was not indicated after transfection of the mutant cells with an SDF comprised of green fl uorescent protein (GFP) sequences that are not found in human genomic DNA. Random integration was also not detected following Southern blot hybridization analysis of an individual corrected cell clone.

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Section: Discussionsupporting

confidence: 91%

Section: Transfectioncontrasting

confidence: 53%

Sequence-Specific Correction of Genomic Hypoxanthine-Guanine Phosphoribosyl Transferase Mutations in Lymphoblasts by Small Fragment Homologous Replacement

Bedayat

Abdolmohamadi

et al. 2010

Oligonucleotides

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“…1). Oligonucleotides were chemically modified to contain 4 terminal phosphorothioate linkages at either end to increase nuclease resistance (Kenner et al, 2002).…”

Section: Fig 1)mentioning

confidence: 99%

Mutagenesis by an Antisense Oligonucleotide and Its Degradation Product

Reshat

Priestley

Gooderham

2012

Toxicological Sciences

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The European Medicines Agency (EMA) has expressed concern regarding (i) the potential for antisense oligonucleotide (ASO) therapeutics to induce sequence specific mutation at genomic DNA and (ii) the capability of ASO degradation products (nucleotide analogues) to incorporate into newly synthesised genomic DNA via DNA polymerase and cause mutation if base-pairing occurs with reduced fidelity. Treating human lymphoblastoid cells with a biologically active antisense molecule induced sequence specific mutation within genomic DNA over four fold, in a system where RAD51 protein expression was induced. This finding has implications for ASO therapeutics with individuals with an induced DNA damage response, such as cancer patients. Furthermore, a phosphorothioate nucleotide analogue potently induced mutation at genomic DNA two orders of magnitude above control. This study shows that a biologically active ASO molecule can induce heritable sequence alterations, and if degraded, its respective analogue may incorporate into genomic DNA with mutagenic consequences.

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“…22 Transition to single strands was prompted initially by in vitro studies on mechanism carried out by Gamper et al, and by results in mammalian cell culture (see Liu et al 2 and references therein). Important proof-of-concept experiments have been conducted in model systems where the repair of mutations in eGFP, 8,23,24 LacZ, 25,26 HPRT, 27,28 and retinoblastoma genes, 21 albeit at different frequencies, is effectively directed by a single-stranded vector. Partially duplexed oligonucleotides containing regions of singlestranded character have also been shown to be effective, but do not appear to be as active as the completely single-stranded molecule.…”

Section: Single-stranded Dna Catalyzes Gene Repair Most Effectivelymentioning

confidence: 99%

Gene therapy progress and prospects: targeted gene repair

et al. 2005

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The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models. Gene Therapy (2005) 12, 639-646.

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Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides

Cited by 21 publications

References 30 publications

Sequence-Specific Correction of Genomic Hypoxanthine-Guanine Phosphoribosyl Transferase Mutations in Lymphoblasts by Small Fragment Homologous Replacement

Sequence-Specific Correction of Genomic Hypoxanthine-Guanine Phosphoribosyl Transferase Mutations in Lymphoblasts by Small Fragment Homologous Replacement

Mutagenesis by an Antisense Oligonucleotide and Its Degradation Product

Gene therapy progress and prospects: targeted gene repair

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