723. In Vivo Expansion of MDS1/EVI1, PRDM16 and SETBP1 Integration Clones in Successful Chronic Granulomatous Disease (CGD) Gene Therapy Trial.

The potential of gene therapy to cure inherited immunodeficiencies of the lymphoid compartment has been demonstrated in clinical trials of ADA-SCID and SCID-X1. To circumvent the missing in vivo selection advantage of the corrected cells in myeloid diseases the two patients in the recent successful chronic granulomatous disease (CGD) gene therapy trial received nonmyeloablative conditioning before transplantation of the corrected cells. 3 months after transplantation 20% and 10% of the granulocytes in the peripheral blood expressed gp91phox in patient 1 and patient 2, respectively. 5 to 9 months after therapy a 3 to 4 fold expansion of marked granulocytes in the peripheral blood appeared in both patients. The proportion of gene-corrected granulocytes in both patients was then stable until 524 days post transplantation in patient 1 and 343 days post transplantation in patient 2 which were the last time points analyzed. To characterize the clonality of the hematopoietic repopulation, the long term activity of individual clones and the distribution of the SFFV vector integration sites we analyzed peripheral blood leucocytes and sorted cells derived from different time points post transplantation with linear amplification mediated PCR (LAM-PCR) and accomplished high throughput sequencing and mapping of the insertion sites. For patient 1 we could identify 435 exactly mappable insertion sites. For patient 2 330 unique integrations could be assigned to the human genome. We detected 3 common integrations sites (CIS) in or near the loci of the zinc finger transcription factor homologous MDS1/EVI1 and PRDM16 in both patients and SETBP1 as a third CIS gene in patient 1. RNA analysis demonstrated an activating influence of the retroviral vector on the CIS genes in both patients. LAM-PCR analysis of CFU-GM and BFU-E derived colonies from both patients revealed that 1 to 4 integrations are present per cell and confirmed the increasing dominance of one MDS1/EVI1 clone in patient 1 seen in the peripheral blood LAM-PCR pattern. Our data show that the upregulation of specific human genes as a side effect of retroviral integration may lead to an unprecedented in vivo expansion of the concerned cell clones and have therefore great impact on future gene therapy strategies.Molecular Therapy (2006) 13, S279–S279; doi: 10.1016/j.ymthe.2006.08.803 [ABSTRACT FROM AUTHOR]