Abnormal Expression of Only the CD34 Part of a Transgenic CD34/Herpes Simplex Virus-Thymidine Kinase Fusion Protein Is Associated with Ganciclovir Resistance

Abstract

Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.

[ 18 F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility

“…Alternatively, the CD34-TK75 transgene may no longer have been expressed in the TdT cells, thus rendering them insensitive to the prodrug, GCV. 16 The latter is a well-described event after transduction of target cells with oncoretroviral vectors in contrast to lentiviral vectors. [27][28][29] Finally we cannot rule out alternative splicing (of the TK active site) as an alternative mechanism of GCV resistance.…”

“…Due to the limited sample size, the possibilities that CD34-TK75 was no longer expressed in patient cells or that the HSV-TK75 portion of the fusion protein was no longer functional were not investigated. 16 …”

Contributions of Gene Marking to Cell and Gene Therapies

“…Although the use of a fusion gene comprising the selectable marker and the gene of interest offers the advantage of equimolar production of a protein responsible for both functions, unexpected consequences can occur. In a recent paper Bennour et al (2008) reported an unusual loss of functionality of the fusion protein tCD34/cHSVtk arose from deletions in HSVtk that yielded ganciclovir-resistant transduced cells but left intact expression of the tCD34 þ surface marker. A truncated, nonsignaling CD19 (DCD19) surface marker was also tested recently for in vitro selection and enrichment of donor-positive T cells transduced with the human inducible caspase 9 (iCasp-9) suicide gene (Tey et al, 2007).…”

Clinical Impact of Suicide Gene Therapy in Allogeneic Hematopoietic Stem Cell Transplantation

“…A fusion HSV-TK=CD34 gene has been developed to ensure the suicide phenotype in selected cells (Fehse et al, 2002). However, posttranslational breakage of the fusion protein, leading to a loss of the suicide phenotype in transferred cells, was shown (Bennour et al, 2008).…”