Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts).
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 oligonucleotides (chimeraplasts) can convert a dysfunctional gene, APOE4 (C, A and E, 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 (> or =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.
Tagalakis
Aristides D
AD
Department of Anatomy and Developmental Biology, Royal Free and University College Medical School, London, UK.
Dickson
J George
JG
Owen
James S
JS
Simons
J Paul
JP
D000818:Animals; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D015316:Genetic Therapy / Q000379:methods*; D005838:Genotype; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D008969:Molecular Sequence Data; D009422:Nervous System Diseases / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D012313:RNA
D053318:Apolipoprotein E3; D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D009841:Oligonucleotides; D012313:RNA; D004247:DNA