Multiple displacement amplification enables large-scale clonal analysis following retroviral gene therapy

J Virol. 2008 Mar;82(5):2448-55. doi: 10.1128/JVI.00584-07. Epub 2007 Dec 12.

Abstract

Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • Animals
  • Base Sequence
  • Cell Line
  • DNA Primers
  • Gene Amplification*
  • Genetic Therapy*
  • Humans
  • Mice
  • Mice, SCID
  • Retroviridae / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • DNA Primers