Analysis of self-inactivating lentiviral vector integration sites and flanking gene expression in human peripheral blood progenitor cells after alkylator chemotherapy

Hum Gene Ther. 2010 Aug;21(8):943-56. doi: 10.1089/hum.2009.116.


Abstract Hematotoxicity is a major and frequently dose-limiting side effect of chemotherapy. Retroviral methylguanine-DNA-methyltransferase (MGMT; EC gene transfer to primitive hematopoietic progenitor cells (CD34(+) cells) might allow the application of high-dose alkylator chemotherapy with almost mild to absent myelosuppression. Because gammaretroviral vector integration was found in association with malignant or increased proliferation, novel lentiviral vectors with self-inactivating (SIN) capacity might display a safer option for future gene transfer studies. We assessed the influence of chemoselection on integration patterns in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-treated and untreated human CD34(+) cells transduced with an SIN lentiviral vector carrying the MGMT(P140K) transgene, using ligation-mediated PCR (LM-PCR) and next-generation sequencing. In addition, for the first time, the local influence of the lentiviral provirus on the expression of hit and flanking genes in human CD34(+) cells was analyzed at a clonal level. For each colony, the integration site was detected (LM-PCR) and analyzed (QuickMap), and the expression of hit and flanking genes was measured (quantitative RT-PCR). Analyses of both treated and untreated CD34(+) cells revealed preferential integration into genes. Integration patterns in BCNU-treated cells showed mild, but not significant, differences compared with those found in untreated CD34(+) cells. Most importantly, when analyzing the local influence of the provirus, we saw no significant deregulation of the integration-flanking genes. These findings demonstrate that SIN vector-mediated gene transfer might display a feasible and possibly safe option for MGMT(P140K)-mediated chemoprotection of CD34(+) cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34 / genetics
  • Antigens, CD34 / metabolism
  • Antineoplastic Agents, Alkylating / therapeutic use*
  • Blood Cells / chemistry
  • Blood Cells / metabolism
  • Gene Expression*
  • Genetic Therapy
  • Genetic Vectors
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Lentivirus / genetics*
  • Lentivirus / metabolism
  • Polymerase Chain Reaction
  • Terminal Repeat Sequences*
  • Transduction, Genetic
  • Transgenes


  • Antigens, CD34
  • Antineoplastic Agents, Alkylating