Transduction of CD34-enriched human peripheral and umbilical cord blood progenitors using a retroviral vector with the Fanconi anemia group C gene.

BACKGROUND

Fanconi anemia (FA) is an autosomal recessive inherited form of bone marrow failure. FA cells are characterized by their extreme sensitivity to DNA cross-linking agents that cause DNA instability and cell death. Four genetic complementation groups for FA have been identified and the gene for the complementation C group (FACC) has been cloned. Genetic transfer of the FACC gene should provide a growth advantage in transduced hematopoietic cells. We have previously demonstrated efficient retroviral-mediated gene transduction and correction of FA(C) cell lines and peripheral blood-derived CD34+ progenitors from patients carrying mutant FACC alleles. In this report we sought to define the optimal conditions for transduction of CD34+ progenitors from mobilized peripheral blood and umbilical cord blood.

METHODS

Peripheral blood hematopoietic progenitors were obtained by G-CSF mobilization followed by apheresis. Human fetal cord blood cells were obtained from full-term gestation deliveries. Cells were immunoselected for CD34 antigen expression and then incubated with recombinant retroviruses containing a selectable marker gene (neomycin). Recombinant colony stimulating factors were added to facilitate viral transduction. Cells were plated in methylcellulose and resulting hematopoietic colonies were isolated and analyzed by PCR.

RESULTS

Transduction efficiency of peripheral blood progenitors (from normal individuals) using a retrovirus encoding the FACC cDNA was comparable to that of the retroviral producer G1Na.40 currently being used in clinical gene therapy marking studies. We extended our standard transduction protocol to analyze CD34+ and CD34+ CD38-subpopulations of progenitors derived from umbilical cord blood (from normal pregnancies). In addition, we tested whether FACC cDNA transduction could be improved by vector infection supported by autologous stroma. For FA(C) hematopoietic cell infection, vector supernatant transduction in the presence of recombinant human IL-3, IL-6, and SCF was found to be superior to transduction supported by autologous FA(C) patient stroma.

CONCLUSIONS

We documented efficient retroviral transduction of umbilical cord blood and peripheral blood enriched for hematopoietic progenitor cells. These results suggest the feasibility of a clinical gene therapy protocol utilizing progenitor cells from both peripheral blood and umbilical cord blood.