title: High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones.
abstract: 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.
authors.affiliation: Department of Microbiology,University of California, David Geffen School of Medicine, Los Angeles, CA 90095, USA.
authors.forename: Sanggu
authors.initials: S
authors.lastname: Kim
authors.forename: Namshin
authors.initials: N
authors.lastname: Kim
authors.forename: Angela P
authors.initials: AP
authors.lastname: Presson
authors.forename: Dong Sung
authors.initials: DS
authors.lastname: An
authors.forename: Si Hua
authors.initials: SH
authors.lastname: Mao
authors.forename: Aylin C
authors.initials: AC
authors.lastname: Bonifacino
authors.forename: Robert E
authors.initials: RE
authors.lastname: Donahue
authors.forename: Samson A
authors.initials: SA
authors.lastname: Chow
authors.forename: Irvin S Y
authors.initials: IS
authors.lastname: Chen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000821:virology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D008253:Macaca mulatta; D017422:Sequence Analysis, DNA / Q000379:methods*; D016662:Virus Integration