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

Research output: Contribution to journalArticle

10 Citations (Scopus)

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, 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.

Original languageEnglish
Pages (from-to)95-103
Number of pages9
JournalJournal of Molecular Neuroscience
Volume25
Issue number1
DOIs
Publication statusPublished - 31 Jan 2005

Fingerprint

Apolipoprotein E4
Oligonucleotides
Cricetulus
RNA
Ovary
DNA
Polyethyleneimine
Genes
Apolipoproteins E
Restriction Fragment Length Polymorphisms
Polymerase Chain Reaction
Apolipoprotein E3
Apolipoproteins C
Lymphocytes
Nervous System Diseases
Neurodegenerative Diseases
Single Nucleotide Polymorphism
Alzheimer Disease
Nucleotides
Clone Cells

Keywords

  • Animals
  • Apolipoprotein E3
  • Apolipoprotein E4
  • Apolipoproteins E/genetics
  • Base Sequence
  • CHO Cells
  • Cricetinae
  • DNA
  • Genetic Therapy/methods
  • Genotype
  • Humans
  • Lymphocytes/cytology
  • Molecular Sequence Data
  • Nervous System Diseases/genetics
  • Oligonucleotides/chemistry
  • RNA

Cite this

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title = "Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts)",
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, 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.",
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Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts). / Tagalakis, Aristides D; Dickson, J George; Owen, James S; Simons, J Paul.

In: Journal of Molecular Neuroscience, Vol. 25, No. 1, 31.01.2005, p. 95-103.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts)

AU - Tagalakis, Aristides D

AU - Dickson, J George

AU - Owen, James S

AU - Simons, J Paul

PY - 2005/1/31

Y1 - 2005/1/31

N2 - 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.

AB - 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.

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KW - Base Sequence

KW - CHO Cells

KW - Cricetinae

KW - DNA

KW - Genetic Therapy/methods

KW - Genotype

KW - Humans

KW - Lymphocytes/cytology

KW - Molecular Sequence Data

KW - Nervous System Diseases/genetics

KW - Oligonucleotides/chemistry

KW - RNA

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DO - 10.1385/JMN:25:1:095

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VL - 25

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EP - 103

JO - Journal of Molecular Neuroscience

JF - Journal of Molecular Neuroscience

SN - 0895-8696

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