Journal of Molecular Neuroscience

, Volume 25, Issue 1, pp 95–103 | Cite as

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

  • Aristides D. Tagalakis
  • J. George Dickson
  • James S. Owen
  • J. Paul Simons
Original Article An Article Related To The 2004 Chemistry Nobel Prize

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 oligonucleotides (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 ɛ4 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.

Index Entries

Alzheimer’s disease apoE genotypes gene repair synthetic oligonucleotides 

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Copyright information

© Humana Press Inc 2005

Authors and Affiliations

  • Aristides D. Tagalakis
    • 1
  • J. George Dickson
    • 3
  • James S. Owen
    • 2
  • J. Paul Simons
    • 1
  1. 1.Department of Anatomy and Developmental BiologyRoyal Free and University College Medical SchoolLondonUK
  2. 2.Department of MedicineRoyal Free and University College Medical SchoolLondonUK
  3. 3.Centre for Biomedical Research, School of Biological SciencesRoyal Holloway University of LondonEghamUK

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