Targeted gene repair

Kmiec, Eric B.

doi:10.1038/sj.gt.3300789

Cited by 50 publications

(26 citation statements)

References 11 publications

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“…Several gene-targeting approaches have been developed for correction of mutations associated with genetic disease. These include RNA/DNA chimeric oligos, [4][5][6][7][8] triplex forming oligonucleotides, 9,10 and small fragment homologous replacement (SFHR).…”

mentioning

confidence: 99%

Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

et al. 2001

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confidence: 99%

Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

et al. 2001

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“…One area that has seen significant progress has been gene targeting via oligonucleotides. [13][14][15] These advances include the use small DNA fragments (SDF), [16][17][18][19] chimeric DNA/RNA oligonucleotides (RDOs) 20,21 or triplex-forming oligonucleotides (TFOs) 22,23 to effect sequence-specific modification of genomic DNA. The strengths of the individual gene targeting strategies, when combined, have the potential to develop therapeutic approaches that overcome the limitations of individual strategies into an efficaciously synergistic therapy.…”

Section: Recent Advances In Genome Medicinementioning

confidence: 99%

Genome medicine: gene therapy for the millennium, 30 September–3 October 2001, Rome, Italy

et al. 2002

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“…As the technique has also been used in bacteria (29) and plants (30), there appears no obvious restriction to DNA sequences that can be targeted.…”

Section: Fig 3 Modifying the Chimeraplast Designmentioning

confidence: 99%

Gene Correction of the Apolipoprotein (Apo) E2 Phenotype to Wild-type ApoE3 by in Situ Chimeraplasty

Tagalakis

Graham

Riddell

et al. 2001

Journal of Biological Chemistry

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Apolipoprotein (apo) E is a polymorphic plasma protein, synthesized mainly by liver. Here, we evaluate whether synthetic DNA-RNA oligonucleotides (chimeraplasts) can convert a dysfunctional isoform, apoE2 (C 3 T, R158C), which causes Type III hyperlipidemia and premature atherosclerosis, into apoE3. First, we treated recombinant Chinese hamster ovary cells stably secreting apoE2 with a 68-mer apoE2 to apoE3 chimeraplast. About one-third of apoE2 was converted to apoE3, and the repair was stable through 12 passages. Subcloning treated cells produced both apoE2 and apoE3 clones. Direct sequencing and reverse transcription polymerase chain reaction confirmed the genotype, whereas phenotypic change was verified by isoelectric focusing and immunoblotting of secreted proteins. Second, we established that the APOE2 gene can be targeted both in vivo, using transgenic mice overexpressing human apoE2, and in chromosomal context, using cultured lymphocytes from a patient homozygous for the ⑀2 allele. We conclude that chimeraplasty has the potential to convert the apoE2 mutation in patients with Type III hyperlipidemia to apoE3. Apolipoprotein (apo)1 E is a 34-kDa polymorphic protein that has anti-atherogenic actions by clearing remnant lipoproteins and promoting cholesterol efflux from cells (1-4). Low apoE is a risk factor for coronary artery disease (5), and apoE deficiency results in severe hyperlipidemia and atherosclerosis (6, 7). By contrast, infusing apoE into hyperlipidemic rabbits regresses atherosclerotic lesions (8), and apoE transgenic mice resist diabetic or dietary hyperlipidemia (9, 10). The three common isoforms of apoE differ in two amino acid positions; apoE2 (Cys-112 and Cys-158), apoE3 (Cys-112 and Arg-158), the most prevalent or wild-type protein, and apoE4 (Arg-112 and Arg-158). The rarest variant, apoE2, is the primary molecular cause of Type III hyperlipidemia (11), which is characterized by accumulation of remnant lipoproteins and premature heart disease.Approaches to treat or prevent atherosclerosis include systemic gene therapy, using viral or non-viral delivery strategies, to overexpress proteins that inhibit atherogenesis or stabilize lesions. Indeed, adenoviral-mediated apoE gene transfer ameliorates experimental hyperlipidemia and atherosclerosis (12, 13). The recent development of targeted gene repair, using synthetic DNA-RNA oligonucleotides (chimeraplasts), offers another possibility (14 -16). Chimeraplasts contain short regions of correcting DNA bounded by long stretches of 2Ј-Omethyl RNA; hairpin loops and GC clamps are also incorporated to make the structure self-associating. These features promote strong, specific binding to the target genomic sequence and allow the DNA repair machinery of the cell to identify and correct the point mutation in situ (15).Here, we apply chimeraplasty to recombinant Chinese hamster ovary (CHO) cells secreting human apoE2 and show that the gene conversion of apoE2 to apoE3 is stable and permanent at the DNA, mRNA, and protein level. In addition, we demonst...

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Targeted gene repair

Cited by 50 publications

References 11 publications

Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

Genome medicine: gene therapy for the millennium, 30 September–3 October 2001, Rome, Italy

Gene Correction of the Apolipoprotein (Apo) E2 Phenotype to Wild-type ApoE3 by in Situ Chimeraplasty

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