819. Molecular Follow-Up of Patients Treated with Allogeneic Hematopoietic Stem Cell Transplantation and Donor Lymphocytes Transduced with a Retroviral Vector Expressing HSV-TK and ÄLNGFR

Abstract

Donor lymphocyte infusion (DLI) plays a crucial role in promoting immune reconstitution and anti-tumor activity in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). The efficacy of DLI is, however, limited by the occurrence of graft-versus-host disease (GvHD). We showed that the infusion of lymphocytes transduced by a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (|[Delta]|LNGFR) allows to control GvHD in 100% of the cases, while preserving anti-tumor and antiviral activities in 65% and 83% of the patients. Expansion (up to 40% of circulating cells) and long-term persistence (>10 years) of transduced cells was observed in >40 patients treated with HSV-TK+ DLI in the context of HLA-identical and haplo-identical HSCT. No acute or chronic adverse or toxic effect due to the gene transfer procedure was observed in these patients, who were treated with a total of >1011 cells generated by >60 independent transductions. Analysis of gene expression profiles showed that <1% of the 16,000 genes contained in an Affimetrix HG-U133A Gene chip|[reg]| were differentially expressed in |[Delta]|LNGFR+ (transduced) vs. |[Delta]|LNGFR- (untransduced) T-cells ex vivo, suggesting the substantial biological identity of T-cell populations generated by HSCT and those administered as DLI. Administration of NGF to |[Delta]|LNGFR+ T cells in culture caused no significant variation in their gene expression profile. Vector integration sites were analyzed by sequencing the vector-genome junctions amplified by LM-PCR from DNA of |[Delta]|LNGFR+ lymphocytes obtained up to 10 years after treatment from 4 different patients. Over 85% of proviral integrations occurred within transcription units, with a preference for first introns (27%) and regions upstream of transcription start sites (24%). Over 75% of intragenic integrations occurred in genes active in T cells at the time of transduction. Interestingly, distribution of integration sites and transcriptional orientation of integrated proviruses showed different patterns in T cells obtained ex vivo from transplanted patients compared to cultured T cells. Persistence of T-cell clones carrying specific vector integrations was followed in individual patients by quantitative PCR analysis of PBL-derived genomic DNA at different times after infusion. The effect of vector integration on the expression of hit genes was analyzed by microfluidic real-time PCR in RNA extracted from individual T-cell clones harboring proviral integrations in the first intron or <10 kb upstream of the transcription start site of one or more genes. This analysis indicated that only a minority of the selected, potentially |[ldquo]|dangerous|[rdquo]| integration events led to detectable perturbation of gene expression.