An Accelerated Incidence of T-Cell Malignancies in a Mouse Model of XSCID Gene Therapy Is Associated with an Expanded Progenitor Cell Pool in the Bone Marrow.
Authors: Shou Yan , Stepanova Lilia , Sorrentino Brian P. 
Published: October 2019

Three patients in the French X-SCID gene therapy trial have developed T-cell lymphomas associated with integration of a γ c-expressing oncoretroviral vector into the LMO2 locus. These occurrences have raised important questions regarding the safety of gene therapy for hematopoietic diseases: 1) are there unique risk factors for XSCID gene therapy that increase the risk of insertional mutagenesis; 2) does deregulated expression of the vector-encoded γ c gene contribute to transformation; 3) what other genetic lesions may contribute to transformation; 4) can safer vectors be designed that result in lower levels of cellular gene activation? To address these questions, we generated a mouse model of XSCID gene therapy in which both the Arf tumor suppressor gene and the γ c gene were ablated. Bone marrow cells from these Arf −/−, γ c−/− mice were transduced with a MSCV-γ c-ires-GFP vector or with a control MSCV-GFP vector. These transduced cells were then transplanted into lethally-irradiated, CD45.1+ wild-type mice. After 1 year of follow-up, 13 out of 15 mice in γ c-transplanted group developed lymphomas in which 12 were T-cell lymphomas and 1 was a B-cell lymphoma. All of these lymphomas except one were highly positive for GFP expression and were derived from transplanted donor cells. In contrast, there were only 3 lymphomas in the MSCV-GFP control group, all of which lacked GFP expression and two of which were derived from recipient hematopoietic cells. Southern blot analysis of lymphoma cells in the γ c-group demonstrated that the lymphomas were clonally-derived. Ligation-mediated PCR analysis showed integrations near or within established proto-oncogenes in 8 cases demonstrating that T-cell transformation was associated with potential insertional oncogene activation. To examine whether the X-SCID background was essential to the increased transformation rate, a second transplant experiment was performed in which bone marrow cells from ARF−/−, γ c+/+ mice were transduced with a MFG-γ c vector. Only 4 out of 18 mice developed lymphomas at 54 weeks indicating that the XSCID background was required for accelerated transformation and that there was no direct effect from deregulated γ c expression per se. We hypothesized that γ c gene deletion could lead to an increase in the number of early progenitors with a resultant increase in target cells susceptible to insertional mutagenesis. Flow cytometry analysis indeed revealed that XSCID mice had a 5-fold increase in the number of Lin−, c-kit+, Sca1+ progenitor cells in the bone marrow relative to wild-type mice, suggesting potential expansion of common lymphocyte progenitors was present. Our results show that unique risk factors exist for gene therapy-induced transformation in XSCID suggesting that the risk for gene therapy in other hematopoietic disorders may be significantly less. One unique risk factor is likely to be an expansion of early progenitors resulting from loss of γ c gene function. Loss of tumor suppressor gene function is likely to be a required secondary event; a hypothesis that is consistent with the relatively long latency for tumor development in patients. Lastly, our animal model should now allow us to test vector safety modifications such as the use of self-inactivation vectors and chromatin insulators and could define a new γ c vector suitable for use in XSCID gene therapy.