Functional Assessment of the Engraftment Potential of Gammaretrovirus-Modified CD34 + Cells,Using a Short Serum-Free Transduction Protocol

Abstract

The successful transduction and engraftment of human mobilized peripheral blood (MBP) CD34⁺ cells are determined to a large extent by the ex vivo cell-processing conditions. In preparation for upcoming clinical trials, we investigated essential culture parameters and devised a short and efficient gammaretroviral transduction protocol entailing minimal manipulation of MBP CD34⁺ cells. The engraftment potential and in vivo transgene expression in the progeny of repopulating CD34⁺ cells were measured to assess the functionality of CD34⁺ cells transduced under these conditions. Using a competitivein vivo repopulation assay in nonobese diabetic/severe combined immunodeficient mice, we demonstrate equivalent engraftment of CD34⁺ cells transduced under serum-free conditions as compared with CD34⁺ cells cultured with serum. We also took advantage of this in vivo model to demonstrate that ex vivo manipulation of CD34⁺ cells can be shortened to 60 hr, using 36 hr of prestimulation and two cycles of transduction 12 hr apart. These minimally manipulated CD34⁺ cells engraft in a manner similar to cells transduced under longer protocols and the vector-encoded transgene is expressed at the same frequency in cells derived from repopulating CD34⁺ cells in vivo. We have thus developed a short and efficient human MBP CD34⁺ transduction protocol under serum-free conditions that is suitable and broadly applicable for phase I clinical trials.