High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones

J Virol. 2010 Nov;84(22):11771-80. doi: 10.1128/JVI.01355-10. Epub 2010 Sep 15.


Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5' and the 3' vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Cells, Cultured
  • Clone Cells
  • Genetic Therapy
  • Genetic Vectors / genetics
  • Genetic Vectors / physiology
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / virology*
  • High-Throughput Screening Assays / methods*
  • Lentivirus / genetics
  • Lentivirus / physiology*
  • Macaca mulatta
  • Sequence Analysis, DNA / methods*
  • Virus Integration*