A Novel Method of Combined Preconditioning and In Vivo Chemoselection Using 6-Thioguanine Alone Results in Highly Efficient Reconstitution of Normal Hematopoiesis with HPRT-Deficient Bone Marrow

Abstract

Introduction: Hematopoietic stem cell transplantation (HSCT) is a mainstay of treatment for hereditary disorders and lymphohematopoietic malignancies, and is the most commonly employed modality for ex vivo gene therapy. However, this procedure often entails myeloablative conditioning by irradiation and/or cytotoxic drugs which incur significant morbidity, and low engraftment rates frequently limit efficacy in the absence of a selective advantage that amplifies donor cells. Methods & Results: Here we tested a novel strategy for combined conditioning and chemoselection based on the fact that 6-thioguanine (6TG) myelotoxicity is dependent on its conversion to thioguanine nucleotide by hypoxanthine-guanine phosphoribosyltransferase (HPRT), and that HPRT-deficiency per se does not impair hematopoietic cell development or function. In a murine model of bone marrow (BM) transplantation, our initial results demonstrated that, at appropriate doses, 6TG induces selective myeloablation in HPRT-wild type (wt) recipients, without detectable adverse effects on extra-hematopoietic tissues. In contrast, HPRT-deficient donor mice were highly resistant to its cytotoxic effects, and transplantation of HPRT-deficient BM achieved rescue of HPRT-wt recipients from lethal hematopoietic failure. Using an optimized protocol, we tested 6TG as a single agent, both for myeloablative conditioning of 6TG-sensitive HPRT-wt recipients, and for in vivo chemoselection of 6TG-resistant HPRT-deficient donor BM after transplantation. Combined 6TG conditioning and chemoselection achieved highly efficient engraftment (˜95%) without significant toxicity and in the absence of any other cytotoxic conditioning regimen. Longterm reconstitution of histologically and immunophenotypically normal BM was achieved in primary and secondary recipients, indicating that 6TG selection amplified the self-renewing, pluripotent HSC population from HPRT-deficient donor BM. In current studies, we are testing lentiviral vector-mediated shRNA expression for knockdown of HPRT expression, in order to apply this combined 6TG conditioning and chemoselection procedure to transplantation of HPRT-wt donor-derived BM after genetic modification. Conclusion: Our results suggest that this protocol for highly efficient conditioning and in vivo chemoselection may be generally applicable as a strategy to improve HSCT engraftment efficiency and confer a selective advantage to genetically modified cells after ex vivo gene therapy.