Insertional Activation of MDS1/EVI1, PRDM16 and SETBP1 in a Successful Chronic Granulomatous Disease (CGD) Gene Therapy Trial.

The potential of gene therapy to correct genetic diseases of the lymphoid compartment has been demonstrated in ADA-SCID and X-linked SCID clinical gene therapy trials. The first successful correction of the myeloid compartment could be achieved in the latest chronic granulomatous disease (CGD) gene therapy trial. CD34+ bone marrow derived cells of 2 patients were transduced using a SFFV based retroviral vector encoding the therapeutic transgene gp91phox. After non-myeloablative conditioning the autologous cells were reinfused. 3 months post therapy the proportion of marked granulocytes was 20% in patient 1 and 10% in patient 2. 5 to 9 months after treatment the proportion of gp91phox expressing granulocytes expanded 4-fold in both patients. Until the latest time points analyzed, (P1: d820; P2: d560) the marking efficiency persisted at that level. In order to define the clonality of the corrected hematopoietic repopulation we accomplished linear amplification mediated PCR (LAM-PCR) on peripheral blood and bone marrow samples as well as sorted lymphoid and myeloid fractions derived from successive time points after therapy. To characterize the retroviral insertion site distribution, we carried out high throughput sequencing and mapping of the vector genome junctions. The hematopoietic repopulation in patient 1 was polyclonal up to day 542 after therapy. Subsequently the number of corrected cell clones and the activity of a predominant clone decreased up to 820 days post transplantation, when the patient succumbed to infectious complications. In this time frame, a different predominant clone appeared. The repopulation in patient 2 has been polyclonal until the latest time point analyzed. Identification of 435 integration sites from patient 1 and 330 insertion sites from patient 2 revealed the gene coding region of the zinc finger transcription factor homologues MDS1/EVI1 and PRDM16 as common integration sites (CIS) in both patients and the SETBP1 locus as a third CIS in patient 1. RT-PCR analysis demonstrated an activating influence of vector LTR on individual CIS genes. Our data show that prospectively studying insertions and stem cell contributions is feasible and that retroviral vector insertion may lead to an upregulation of genes causing an in vivo expansion of the affected cell clones, which can augment gene-corrected hematopoietic repopulation as an unexpected, thus far non-adverse side effect.