Herpes Simplex Virus Thymidine Kinase Gene Transfer for Controlled Graft-versus-Host Disease and Graft-versus-Leukemia: Clinical Follow-up and Improved New Vectors

Abstract

We previously demonstrated that severe graft-versus-host disease (GvHD), associated with the therapeutic infusion of donor lymphocytes after allogeneic marrow transplantation (BMT), can be efficiently controlled by the SFCMM-2-mediated expression of the herpes simplex virus thymidine kinase (HSV-tk) suicide gene into the allogeneic lymphocytes. This was achieved by selective elimination of transduced lymphocytes by ganciclovir (GCV) infusion. Despite the positive results of the pilot clinical trial, two vector-related limitations were observed. The induction of a strong immune response against genetically modified cells was observed in two patients. In addition, the only patient who developed chronic GvHD showed only partial response to ganciclovir treatment. In an attempt to overcome these limitations, we developed a new generation of vectors. The neomycin resistance (neo^R ) gene was removed from the SFCMM-3 and SFCMM-4 retroviral vectors. These vectors are less immunogenic and able to confer higher ganciclovir sensitivity to transduced human lymphocytes. All the vectors carry a modified form of the low-affinity nerve growth factor receptor cDNA, as cell surface selectable marker (ΔLNGFR). The vectors were compared in terms of gene transfer efficiency, and ability to confer high and specific sensitivity to ganciclovir. The SFCMM-3 vector, carrying the entire HSV-tk gene driven by the LTR promoter, allows efficient transduction of human T lymphocytes and confers the highest GCV sensitivity to transduced lymphocytes with a high and a low proliferation index. The expression of the ΔLNGFR marker allows an easier in vitro manipulation and a faster immune selection of transduced cells compared with neo^R selection. Finally, the elimination of the neo^R gene removes a potent immunogen from transduced lymphocytes.

Overview summary

Transfer of a suicide gene into donor lymphocytes could allow their in vivo selective elimination and abrogation of severe GvHD occurring after allogeneic BMT. In various experimental settings, different suicide genes have been proposed. The HSV-tk/GCV strategy seems the most effective and specific, as confirmed by several preclinical studies and by initial clinical applications. However, some limitations are associated with this specific suicide gene strategy. First, the combined expression of a viral protein (HSV-tk) and a bacterial protein (NeoR) results in a strong immunogenicity of transduced cells; second, the cell cycle dependence of ganciclovir activity reduces the possibility of eliminating transduced cells with a low proliferation index. In this regard, chronic GvHD seems to be more resistant than acute GvHD to this strategy. The possibility to rapidly eliminate also low-proliferating transduced cells present at the early phase of chronic GvHD may be crucial to circumvent this limitation. Here, we show that both of the above-described limitations can be addressed or reduced by new-generation vectors.