In vivo selection with lentiviral expression of Bcl2(T69A)(/)(S70A)(/)(S87A) mutant in hematopoietic stem cell-transplanted mice

摘要

Current in vivo selections for hematopoietic stem cell (HSC)-based gene therapy are drug dependent and not without risk of cytotoxicity or tumorigenesis. We developed a new in vivo selection system with the non-phosphorylatable Bcl2 mutant Bcl2(T69A/S70A/S87A) (BCl2(AAA)), which makes in vivo selection drug independent and without risk of cytotoxicity or tumorigenesis. We demonstrated in HSC-transplanted mice that Bcl2(AAA) facilitated efficient in vivo selection in the absence of any exogenously applied drugs under both myeloablative and non-myeloablative conditioning. In mice transplanted with retrovirally transduced sca-1-positive bone marrow cells, the marked cell level increased from 26.38% of input transduced cells to 92.61 +/- 0.95% of peripheral blood cells for myeloablative transplantation or to 37.82 +/- 6.35% for non-myeloablative transplantation 6 months after transplantation. Bcl2(AAA) did not induce tumorigenesis and does not influence hematopoiesis and the function of the reconstituted blood system. However, the high-level constitutive expression of Bcl2(AAA) mediated by retroviral vector induced exhaustion of the marked cells after tertiary transplantation. Fortunately, low-level constitutive expression of Bcl2(AAA) driven by an internal promoter in lentiviral vector could both maintain the marked cell level (24.13 +/- 5.27%, 27.17 +/- 5.51%, 24.33 +/- 5.08%, and 22.07 +/- 4.44% for primary, secondary, tertiary, and quaternary recipients) and avoid the exhaustion of the marked cells even in quaternary recipients. Importantly, the low-level constitutive expression of Bcl2(AAA) did not induce tumorigenesis. Thus, the in vivo selection employing the low-level constitutive expression of Bcl2(AAA) provides a general platform which is relevant for widespread applications of gene therapy.