Optimization of hematopoietic stem cell gene therapy culture conditions and engraftment

RAG2 severe combined immunodeficiency (SCID) is a rare, inherited primary immunodeficiency (PID), characterized by a T-/B-/NK+ phenotype and is treated with hematopoietic stem cell (HSC) transplantation. When no suitable donor is available, these patients have no other treatment options. Therefore, it is important to develop gene therapy (GT) strategies, where patient-derived autologous cells can be used. For SCID patients, usually reduced conditioning regimens (RIC) are used to support homing and long-term engraftment of the HSCs in the bone marrow (BM). In order to prevent RIC -related toxicity, alternative conditioning regimen protocols should be developed. In this thesis, we aimed to 1) optimize ex vivo mouse HSC culture conditions, 2) compared the effects of the harmless granulocyte-colony stimulating factor (G-CSF) and very late antigen 4 inhibitor (VLA-4I) on HSC engraftment, with Busulfan (BU), and 3) transplanted RAG2 mice with HSCs genetically modified with LV-SF-RAG2co or LV-UCOE RAG2co. Here we showed that during short-term ex vivo HSC cultures, addition of mTpo alone is sufficient, and therefore we used these conditions during LV transductions. The methylation-resistant UCOE promoter displayed better results than the stronger SF promoter at 6 months after GT in transplanted RAG2 mice in terms of peripheral blood and spleen CD3 and CD19 numbers. Although the engraftment kinetics after HSC transplantation and HSC GT of mice treated with G-CSF or VLA-4I were different from the mice treated with BU, use of both G-CSF and VLA-4I was at least as effective as BU in terms of immune recovery at 6 months after transplantation. With this thesis, we developed a new conditioning and treatment protocol that can be applied to not only RAG2 SCID, but also to other SCIDs and can serve as the basis for future clinical research and treatment/support of SCID patients.