278. Successful Correction of Rag1 Severe Combined Immunedefciency at Clinically Relevant Vector Copy Numbers
Authors: Karin Pike-Overzet, Miranda R.M. Baert, Axel Schambach, Michael Rothe, Fang Zhang, Arjen C. Lankester, Robbert G.M. Bredius, Adrian Thrasher, Bobby Gaspar, Frank J.T. Staal
Journal: Molecular Therapy (2015)

RAG1 -SCID patients lack B- and T lymphocytes due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment alternative for RAG1-SCID patients lacking an appropriate bone marrow donor. However, developing such therapy for RAG1 has proven difficult. As proof-of-principle, we have previously shown with an SFFV promoter element that sufficient RAG1 expression could be achieved for correction at clinically acceptable vector copy numbers (VCN). In our current study, we tested clinically relevant lentiviral SIN vectors with different internal promoter elements (UCOE, PGK, MND, and UCOE-MND in tandem) to deliver human RAG1 sequences. In vitro assessment of promoter strength was done on transduced mouse lineage-depleted cells. The highest RAG1 expression per VCN was reached with the MND and the UCOE-MND containing vectors, followed by UCOE. The PGK displayed the lowest promoter strength, 3 to 10 times weaker than the other elements. Next, we used Rag1-/- mice as a preclinical model for RAG-SCID to assess the capacity of the various vectors to correct the SCID phenotype at very low to moderate VCN (0.01-0.2), where the VCN positively correlating with the RAG1 expression levels. All gene therapy-treated mice reconstituted their peripheral T cell compartment, however in animals that received a transplant with low RAG1 expression (n=9) this effect was limited to peripheral blood. When assessing phenotypic data from thymus 17 weeks after transplantation, only mice that received a transplant with high RAG1 expression, showed a thymocyte subset composition comparable to mice receiving wild type cells. This included a high percentage and number of double positive (DP) cells, assuring an ongoing supply of peripheral T cells. Mice receiving a transplant with low RAG1 expression levels, were mostly lacking the DP population. Similarly, when looking at developing B cells in the bone marrow, animals that were transplanted with cells with low RAG1 expression did not have a substantial number of surface IgM+ cells. This in contrast to animals that received cells with high RAG1 expression levels in which developing B cell subsets were readily detectable. These findings were paralleled in the peripheral blood, where the fraction of B220+ cells positively correlated with RAG1 expression in the transplant material. In the low RAG1 expression groups, we found that a number of mice developed skin rashes, while animals in the high RAG1 expression group as well as animals that received wild type cells or uncorrected Rag1-/- cells did not display any health problems. We are currently determining the TCR repertoire, since a limited repertoire could point to autoreactive T cells causing the rashes. To conclude, the level of B- and T cell development in primary lymphoid organs is directly correlated to the RAG1 expression levels in the transplant and we were able to obtain sufficient RAG1 expression for full T and B cell reconstitution even at a moderate, but clinically relevant, VCN.