Reconstitution of T-lineage differentiation potential of induced pluripotent stem cells derived from severe combined immunodeficiency patients by retroviral gene transfer.

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Severe combined immunodeficiency (SCID) is a heterogenous disease caused by different monogenic mutations that result in a complete block of T-cell differentiation. Immune reconstitution can be achieved either by hematopoietic stem cell transplantation, limited by donor availability and potential severe side-effects, or gene transfer into the patients¿ hematopoietic cells, which is currently only available for ADA- and XSCID. As several cases of insertional mutagenesis occured, a self-inactivating vector lacking viral enhancer sequences was created to improve safety issues for ongoing trials. The aim of this project is to test, if T-cell reconstitution can be achieved by gene transfer also in other forms of SCID. Therefore, the same vector backbone modified by replacement of the appropriate transgene will be used for transduction of patient-derived induced pluripotent stem cells (iPSC) carrying mutations in ADA, RAG2, ILR2G, JAK3, LIG4, DCLRE1C, and AK2. The read-out will be the assessment of T-cell differentiation in vitro using the OP9-DL4 co-culture system. IPSCs, functioning as a disease model, will be generated from patient’s fibroblast lines by retroviral transfer of the four reprogramming factors OCT4, KLF4, SOX2 and c-MYC with enzymatically excisable viruses. It is of great significance to know, wether the retroviral gene transfer using a safe vector system is able to restore T-cell differentiation potential in these other SCID diseases, as this will be the basis for future gene therapy trials.