240. Stable and Clinically Benign Clonal Dominance in an ADA-SCID Patient Treated With Retroviral Gene Therapy

To date, adverse events involving genotoxicity and oncogenic transformation have occurred in several gene therapy trials using oncoretroviral vectors. In striking and surprising contrast, insertional oncogenesis has not been observed in multiple trials treating SCID caused by adenosine deaminase deficiency (ADA-SCID), despite the use of very similar oncoretroviral vectors. In our trial for ADA-SCID, we employed non-restrictive LAM-PCR to monitor clonality and observed that one patient developed a marked clonal dominance that was stable over multiple years. Despite this, the patient has been clinically well up to present. The dominant clone was confirmed using an integration site-specific digital PCR assay, and it accounted for up to 85% of vector integrations in peripheral blood mononuclear cell (PBMC) DNA. The vector integration in the dominant clone is in a gene-poor region of chromosome 21, which is also a common insertion site observed in our cohort of patients. RNA-seq expression analysis of cells of this clone did not detect any dysregulation of nearby genes. Importantly, this analysis would have also detected unannotated non-coding RNAs or novel transcripts being produced from the vector, and we observed neither. The majority of vector copies detected in PBMC DNA were contributed by the NK lineage, and the clone was most dominant in NK cells. Compared to healthy donor NK cells, the patient's NK cells more highly expressed the NK receptor NKG2C. The beginning of the clonal expansion coincides with the onset of Epstein-Barr virus (EBV) viremia in the patient, which also correlates with the expansion of a potential EBV-reactive T-cell clone, as indicated by flow cytometric TCR beta family analysis. We hypothesize that NK cells expressing NKG2C and bearing the chr21 IS expanded in response to the EBV infection, which was not fully controlled by the patient's adaptive immune response.