The Functional Effect of Repeated Cryopreservation on Transduced CD34+ Cells from Patients with Thalassemia.

Stable gene marking and effective engraftment of gene-modified CD34⁺ hematopoietic stem cells is a prerequisite for gene therapy success but may be challenged by the inevitable cryopreservation of the final product prior to extensive quality assurance testing. We investigated the β-globin gene transfer potency in fresh and cryopreserved CD34⁺ cells from mobilized patients with β-thalassemia, as well as the qualitative impact of repeated freeze/thaw cycles on the functionality of cultured and unmanipulated CD34⁺ cells in terms of engrafting capacity in a xenotransplantation model, under partial myeloablation. Cells transduced fresh or after one freeze-thaw cycle yielded similar clonogenic and gene transfer frequencies. Repeated cryopreservation cycles did not affect the transduction rates whereas either one or two freeze-thaw cycles of cultured-but not of unmanipulated-cells significantly reduced their clonogenicity. No differences in the engrafting potential of gene-corrected cells subjected to either none or up to two cryopreservation cycles, were encountered post xenotransplantation. Overall, we assessed the gene transfer efficiency, clonogenicity and engrafting capacity of cryopreserved CD34⁺ cells and the impact of repeated freeze/thaw cycles in their performance. These observations may prove essential in the design of gene therapy trials, considerably facilitating their logistics.