Stable Marker Gene Transfer into Human Bone Marrow Stromal Cells and Their Progenitors Using Novel Herpesvirus Saimiri-Based Vectors

Abstract

We have evaluated the ability of new herpesvirus saimiri (HVS)-based vectors to deliver a marker gene green fluorescent protein (GFP) into human bone marrow (BM) stromal cells and their progenitors. Stromal cells expanded from adherent layers of long-term BM cultures (LTC) were susceptible to HVS-based infection in a dose-dependent manner, and the efficiency of 94.8 +/- 2.0% was achieved using single exposure with HVS/EGFP vector at multiplicity of infection (moi) of approximately 50. Colony-forming unit-fibroblast (CFU-F) assay established the ability of HVS-based vectors to infect progenitors for bone marrow stroma fibroblasts and transfer the marker gene over multiple cell divisions in the absence of selective pressure. HVS was not toxic for stromal cells and progenitors and no viral replication was detected upon growth of modified stroma. On the basis these data, we believe that HVS-based constructs can offer a new opportunity for selective gene delivery into bone marrow stromal cells and progenitors. The ability of HVS to infect nondividing cells can be considered advantageous in the development of both ex vivo and in vivo strategies.

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

“…18 Alternatively, HSVbased vectors efficiently transduce MSCs but further development is required for the production of safe replication-deficient vectors. 19 All in all, viral vectors permit efficient gene delivery into MSCs but raise safety concerns that are critical when considering clinical applications.…”

Efficient infection and persistence of a herpesvirus saimiri-based gene delivery vector into human tumor xenografts and multicellular spheroid cultures

“…For example, the potential of HVS as a hematopoietic stem cell (HSC) vector has been highlighted by its ability to maintain heterologous gene expression throughout mouse embryonic stem cell differentiation in vitro. 13,14 HVS-based vectors are able to infect totipotent mouse embryonic stem cells with high efficiency. The presence of the HVS genome was maintained and had no apparent effect on cell/colony morphology of the transduced mouse ES cells and no virus replication or production was observed.…”

Rhadinovirus vector-derived human telomerase reverse transcriptase expression in primary T cells

“…25,37 The markerless release of the enhanced green fluorescent protein (EGFP) cassette and the reconstitution of the original virus sequence was achieved by en passant mutagenesis. 38,39 For constructing RT-hTERT BAC488, the expression cassette for hTERT was inserted into orf75 by homologous recombination.…”

“…22 Moreover, they retain most of the parental T-cell properties, such as surface phenotype, major histocompatibility complex-restricted antigen recognition, interleukin-2 (IL-2)-dependent growth and early signal transduction events, suggesting the possible use of HVS as T-cell vectors for heterologous gene expression. 14,[23][24][25] By rhadinoviral vector-mediated transduction of primary T cells, we achieved efficient long-term expression of the suicide gene thymidine kinase and the targeted elimination of transduced T cells upon low doses of ganciclovir. 23 Consistent with these results, the reinfusion of transduced autologous T cells in macaques was well tolerated without the occurrence of lymphoproliferative disease.…”

“…26 In addition to T-cell applications, HVS vectors have been suggested for various other applications. 25,[27][28][29][30] This led us to the conclusion that rhadinoviral vectors could be beneficial even for clinical usage, if the potential for oncogenic transformation was excluded.…”