Gene correction of the apolipoprotein (Apo) E2 phenotype to wild-type ApoE3 by in situ chimeraplasty

A D Tagalakis, I R Graham, D R Riddell, J G Dickson, J S Owen

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

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 --> 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 epsilon2 allele. We conclude that chimeraplasty has the potential to convert the apoE2 mutation in patients with Type III hyperlipidemia to apoE3.

Original languageEnglish
Pages (from-to)13226-30
Number of pages5
JournalJournal of Biological Chemistry
Volume276
Issue number16
DOIs
Publication statusPublished - 20 Apr 2001

Keywords

  • Amino Acid Substitution
  • Animals
  • Apolipoprotein E2
  • Apolipoprotein E3
  • Apolipoproteins E/chemistry
  • Base Sequence
  • CHO Cells
  • Cricetinae
  • Crosses, Genetic
  • Female
  • Genetic Therapy
  • Genomic Library
  • Genotype
  • Humans
  • Lymphocytes/cytology
  • Male
  • Mice
  • Mice, Transgenic
  • Oligodeoxyribonucleotides
  • Oligoribonucleotides
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length
  • RNA, Messenger/genetics
  • Recombinant Fusion Proteins/biosynthesis
  • Transfection

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