Gene Therapy for RAG-deficient Severe Combined Immunodeficiency

textabstractSevere combined immunodeficiency (SCID) is a rare class of primary, inherited, immunodeficiency causing infants to suffer from persistent diarrhea, opportunistic infections and a failure to thrive. RAG proteins play a crucial role in the initiation of V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) gene segments. Inactivating mutations in RAG1 or RAG2 lead to a developmental block at early checkpoints in B- and T-cell development. SCID can be cured by hematopoietic stem cell transplantation, but an HLA-matched donor is not always available. For two types of SCID, ADA- SCID and X-linked SCID, gammaretrovirus-based gene therapy has proven a successful treatment. For my thesis, several aspects of T-cell development and gene therapy were studied. A feature all forms of SCID have in common, is the occurrence of a block in T-cell development. Therefore, one of the studies in my thesis aimed at gaining more detailed knowledge of normal human T-cell development. Subsets corresponding to consecutive stages of human T- cell development were isolated and their gene expression profile was correlated to the T-cell receptor (TCR) gene rearrangement status. This analysis resulted in the identification of candidate factors involved in regulation of TCR gene rearrangements. In one of the X-linked SCID gene therapy trials using hematopoietic stem cells, lymphoproliferations were observed in four patients. In two cases, insertion of the therapeutic vector was found near the T- ALL oncogene LMO2. We found that gammaretrovirus-driven overexpression LMO2, but not the therapeutic IL2RG gene resulted in a block in human T-cell development. HIV-based vectors have been shown to have a more favorable integration pattern then gammaretroviruses, and self-inactivating (SIN) versions of this transfer system have been developed. We tested the feasibility of RAG gene therapy using HIV-based SIN vectors. We evaluated RAG1 and RAG2 gene transfer into hematopoietic stem cells using murine models for RAG-SCID. Correction of both T- and B-cell development was established when using a RAG2 transfer vector in Rag2-/- mice, showing proof-of-principle for the use of lentiviral SIN vectors for RAG gene transfer