Development and Safety Assessment of Lentiviral Vector Gene Therapy for SCID-X1

This thesis work focuses on exploring ways to improve hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency disease (SCID-X1) using lentiviral vectors and a mouse model of the disease. This involved 3-part approach: modifying some components of the vector to improve efficacy and safety, improving the transplant conditions of the stem cell recipient mice, and bioinformatics analysis of vector integration sites to create a safety profile of the vectors under investigation. A series of experiments in SCID-X1 mice were undertaken to demonstrate the effectiveness and safety of the new vectors in vivo. The results of these experiments showed that self-inactivating lentiviral vectors using a codon optimized version of the therapeutic IL2RG gene and eukaryotic promoter elements were able to restore full immune system functionality to SCID-X1 mice while having a low risk of triggering adverse events. This research also underlined that pre-transplant conditioning of hematopoietic stem cell (HSC) recipients is vital to improve engraftment of gene therapy treated donor HSCs and maximize lymphocyte reconstitution. Over 100,000 vector integration sites were sequenced and analyzed, which required the creation of a new bioinformatics pipeline to efficiently annotate and analyze the resulting data. This led to a collaboration with the Erasmus MC department of Bioinformatics, resulting in a new web-based integration site analysis tool for public use.