Abstract
AbstractIn our previous phase 1/2 study aimed at controlling graft-versus-host disease, 12 patients received Herpes simplex virus thymidine kinase (HSV-tk+)/neomycin phosphotransferase (NeoR+)–expressing donor gene-modified T cells (GMCs) and underwent an HLA-identical sibling T-cell–depleted bone marrow transplantation (BMT). This study's objective was to follow up, to quantify, and to characterize persistently circulating GMCs more than 10 years after BMT. Circulating GMCs remain detectable in all 4 evaluable patients. However, NeoR- and HSV-tk–polymerase chain reaction (PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. Further experiments, including a novel “transgene walking” PCR method, confirmed the presence of deletions. The deletions were unique, patient-specific, present in most circulating GMCs expressing NeoR, and shown to occur at time of GMC production. Unique patient-specific retroviral insertion sites (ISs) were found in all GMCs capable of in vitro expansion/cloning as well. These findings suggest a rare initial gene deletion event and an in vivo survival advantage of rare GMC clones resulting from an anti–HSV-tk immune response and/or ganciclovir treatment. In conclusion, we show that donor mature T cells infused with a T-cell–depleted graft persist in vivo for more than a decade. These cells, containing transgene deletions and subjected to significant in vivo selection, represent a small fraction of T cells infused at transplantation.
Highlights
(PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene
We showed the impairment of gene-modified T cells (GMCs)’ anti–Epstein-Barr virus (EBV) competence and demonstrated that it is linked to activation-induced cell death and toxicity of the neomycin phosphotransferase II (NeoR)-based selection process.[12,13]
The presence of persistently circulating GMCs was quantified by QPCR of the NeoR gene in posttransplantation peripheral blood mononuclear cells (PBMCs) samples from the 4 patients
Summary
(PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. In the field of ex vivo gene therapy, recent experimental studies using murine animal models[16,17,18] and clinical trials[19,20] have reported significant complications regarding retroviral-mediated genemodified hematopoietic stem cells, namely, the induction of target cell transformations by gene transfer.[21,22,23] mounting of immune responses against cells expressing HSV-tk has been reported.[24,25,26,27] These phenomena should be considered in the design of future gene therapy trials In this regard, molecular characterization of persistently circulating GMCs would help elucidate. The PCR reactions and amplification conditions for the 9 assays were identical: 35 cycles of denaturation for 1 minute at 95°C, annealing for 1 minute, and extension for 1 minute at 72°C
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