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

Abstract

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.