The Effect of Recombinant Fibronectin Fragment (Retronectin) and Cytokines during deltaLNGFR Retroviral Transduction of CD34+ Cells

Lee, Seok; Min, Yoo Hong; Ko, Yun Woong

Abstract

BACKGROUND
Hematopoietic stem cells (HSC) remain one of the most promising target cells for gene therapeutic approaches, but is currently limited by a number of issues, including the low gene transfer efficiency.
METHODS
We evaluated the effect of recombinant fibronectin fragment (retronectin) and cytokines during retroviral transduction of CD34+ cells and analyzed the characteristics of transduced cells. To rapidly characterize and isolate transduced cells, we used a retroviral vector coding for the truncated form of the low-affinity nerve growth factor receptor (delta LNGFR).
RESULTS
The total number of CD34+ cells (1.5~6.3-fold) and the amount of cycling (S+G2/M) cell fractions (2~6-fold) were increased after cytokine prestimulation, especially using thrombopoietin (TPO)-based cytokines. The immunophenotype of CD34+ cells showed no significant differences according to the cytokines. However, CD34+AC133+ cells were more effectively maintained in the presence of TPO. The transduction efficiency of CD34+ cells was significantly increased in the presence of retronectin (6.7+/-2.3% vs 23.1+/-4.4%). Immunomagnetic selection of the transduced cells allowed us to enrich these effectively (6.7+/-2.3% --> 86.3+/-6.5%). Compared with control, delta LNGFR transduction did not influence on the immunophenotype and cloning efficiency of CD34+ cells. Among the delta LNGFR+/-derived colonies, 85.0% were shown to contain an integrated delta LNGFR gene.
CONCLUSION
These data show that retronectin and TPO-based cytokines can be used to facilitate retroviral transduction of CD34+ cells. Also, delta LNGFR transduced cells could be rapidly characterized by FACS analysis and effectively enriched by immunomagnetic selection method. Further improvement of transduction conditions for stimulation of HSC proliferation without differentiation and development of new vectors that obviate the requirement for cell division are likely to enhance transduction of primitive HSC.