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Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts)

Tagalakis, AD; Dickson, JG; Owen, JS; Simons, JP; (2005) Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts). J MOL NEUROSCI , 25 (1) 95 - 103. Green open access

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Abstract

Apolipoprotein E (apoE) is a multifunctional circulating 34-kDa protein, whose gene encodes single-nucleotide polymorphisms linked to several neurodegenerative diseases. Here, we evaluate whether synthetic RNA/DNA oligonucleoticles (chimeraplasts) can convert a dysfunctional gene, APOE4 (C -> T, Cys112Arg), a risk factor for Alzheimer's disease and other neurological disorders, into wild-type APOE3. In preliminary experiments, we treated recombinant Chinese hamster ovary (CHO) cells stably secreting apoE4 and lymphocytes from a patient homozygous for the epsilon 4 allele with a 68-mer apoE4-to-apoE3 chimeraplast, complexed to the cationic delivery reagent, polyethyleneimine. Genotypes were analyzed after 48 h by routine polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and by genomic sequencing. Clear conversions of APOE4 to APOE3 were detected using either technique, although high concentrations of chimeraplast were needed (>= 800 nM). Spiking experiments of PCR reactions or CHO-K1 cells with the chimeraplast confirmed that the repair was not artifactual. However, when treated recombinant CHO cells were passaged for 10 d and then subcloned, no conversion could be detected when > 90 clones were analyzed by locus-specific PCR-RFLP. We conclude that the apparent efficient repair of the APOE4 gene in CHO cells or lymphocytes 48 h post-treatment is unstable, possibly because the high levels of chimeraplast and polyethyleneimine that were needed to induce nucleotide substitution are cytotoxic.

Type: Article
Title: Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts)
Open access status: An open access version is available from UCL Discovery
Keywords: Alzheimer's disease, apoE genotypes, gene repair, synthetic oligonucleotides, RNA-DNA OLIGONUCLEOTIDE, SICKLE-CELL-ANEMIA, CEREBROSPINAL-FLUID, ALZHEIMERS-DISEASE, REPAIR STRATEGIES, VALIDATION, EFFICIENCY, CONVERSION, ASTROCYTES, MUTATIONS
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Inflammation
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept
URI: https://discovery.ucl.ac.uk/id/eprint/383
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