Transduction of human pancreatic tumor cells with vesicular stomatitis virus G-pseudotyped retroviral vectors containing a herpes simplex virus thymidine kinase mutant gene enhances bystander effects and sensitivity to ganciclovir

We examined the suitability of Moloney murine leukemia virus (MLV) 4070A-, cat endogenous virus (CEV) RD114-, or vesicular stomatitis virus G (VSV-G)-pseudotyped retroviruses containing the humanized enhanced green fluorescent protein (hEGFP) or one of two herpes simplex virus thymidine kinase (HSV-TK) genes to transduce and provide gene expression in human pancreatic tumor cells. Fluorescence-activated cell sorter analysis demonstrated that VSV-G-pseudotyped hEGFP vector infected a greater percentage of cells and generated more robust gene expression than MLV 4070A- or CEV RD114-pseudotyped vectors. Dot blot and Southern blot analysis of genomic DNA revealed up to 10-fold more gene copies in G418-selected VSV-G hEGFP vector-transduced cells compared with genomic DNA from cells transduced with MLV 4070A or CEV RD114 pseudotypes. Cells transduced with VSV-G pseudotypes of HSV-TK(WT) or the HSV-TK30 vectors were 5- to 10-fold more sensitive to ganciclovir (GCV) than other pseudotype-transduced cells. A 40- to 61-fold difference in sensitivity to GCV was observed between cells transduced with VSV-G HSV-TK30 vector and cells transduced with MLV 4070A HSV-TK(WT) vector in vitro. A 13-fold reduction in tumor volume was observed in severe combined immunodeficient mice inoculated with PancTuITK30 cells compared with mice inoculated with PancTuITK(WT) cells during GCV treatment. We conclude that the choice of glycoprotein envelope and the potency of a particular suicide gene were therapeutically additive and increased the number of HSV-TK-positive cells and sensitivity toward GCV in human pancreatic tumors cells for prodrug gene therapy.     

In vivo Adenovirus-mediated Prodrug Gene Therapy for Carcinoembryonic Antigen-producing Pancreatic Cancer

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)-ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus-carrying HSVtk gene under the control of a carci-noembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA-producing Su.86.86, BxPC-3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA-nonproducing HeLa, were used for analysis of GCV sensitivity induced by adeno-viral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA-positive pancreatic cancer in vivo , a subcutaneously implanted tumor-bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA-production. In contrast, when the cells were transduced with a CEA promoter, the cell-killing effect of GCV was increased in only CEA-producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus-mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA-producing pancreatic cancer cells in vitro and in vivo . This strategy may provide a useful tool for treating pancreatic cancer, especially CEA-producing tumor cells.     

Ganciclovir-mediated cell killing and bystander effect is enhanced in cells with two copies of the herpes simplex virus thymidine kinase gene

Delivery and expression of the herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir is currently being evaluated for the treatment of many types of cancer. After initial phosphorylation by HSVtk, cellular kinases generate the toxic triphosphate form of ganciclovir (GCV). To further define the role of GCV metabolism in cells expressing HSVtk, two human tumor cell lines, UMSCC29 and T98G, were transduced with HSVtk and screened for insertion of one or two copies of the viral transgene by Southern blot analysis. Both the relative capacities for incorporating labeled GCV and the levels of GCV metabolites were determined for each of the parental cell lines and their derivatives containing either one or two copies of the HSVtk gene. The efficiency of GCV killing and the magnitude of the bystander effect were compared for the single- and double-copy HSVtk cell lines. Consistently, cells that expressed two copies of HSVtk metabolized GCV more efficiently, were more sensitive to GCV, and demonstrated improved bystander killing relative to single-copy HSVtk cells. The implications of these results for future and current therapies employing HSVtk and GCV are discussed.     

Treatment of rat experimental brain tumors by herpes simplex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir

Transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, followed by administration of ganciclovir (GCV), generates the "bystander effect," in which HSVtk-negative wild-type cells are killed by GCV, as are HSVtk-expressing cells. Our previous study demonstrated that intracranial 9L gliomas could be efficiently treated due to this bystander effect by injecting the 9L glioma cells transduced with the HSVtk gene in the vicinity of the preimplanted wild-type 9L glioma and then administering GCV. For a possible clinical application of the bystander effect-mediated cell killing, we tested HSVtkgene-transduced allogeneic C6 glioma cells (C6tk) instead of syngeneic 9L glioma cells transduced with the HSVtk gene. Fisher rats were implanted intracranially with wild-type 9L glioma cells, subsequently injected with C6tk cells at the same brain coordinate, and thereafter treated with GCV or saline. When the rats were treated with GCV, a significant retardation of tumor growth was observed by serial magnetic resonance imaging, although this growth retardation was less prominent than that observed with 9L glioma cells transduced with the HSVtk gene; consequently, survival was prolonged (P < .01). Tumors that received C6tk cells contained almost no HSVtk-positive cells after treatment with GCV. Rejection of allogeneic tumor cells, although possibly incomplete in the brain, can also contribute to the safety of this therapeutic strategy.     

Bystander-mediated regression of osteosarcoma via retroviral transfer of the herpes simplex virus thymidine kinase and human interleukin-2 genes

Current treatment of osteosarcoma produces disappointing outcomes, and innovative therapies must be investigated. We have used retroviral vectors to transfer the herpes simplex virus thymidine kinase (HSVtk) and interleukin-2 genes to human osteosarcoma cells. Each gene was stably transduced and expressed; the HSVtk gene effectively conferred ganciclovir (GCV) susceptibility to transduced cells. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to GCV killing. Human osteosarcoma cells were used to develop a series of experiments in athymic nude mice to treat experimental osteosarcoma. Subcutaneously implanted mixtures of tumor cells and HSVtk vector producer cells developed into tumors that completely regressed upon administration of GCV. Subcutaneously implanted mixtures of transduced and wild-type cells showed a potent bystander effect upon administration of GCV, with complete tumor ablation when as little as 10% of the cells were HSVtk+. A significant (P < .05) antitumoral response was seen against primary tumors composed of unmodified cells when a secondary tumor of transduced cells was implanted at a distance of 1 cm, suggesting a diffusible bystander factor. The presence of interleukin-2-transduced cells improved the efficacy of treatment. A significant (P < .03) antitumoral response was seen in the treatment of established osteosarcomas by the injection of HSVtk vector producer cells.     

Herpes simplex virus thymidine kinase and granulocyte macrophage colony-stimulating factor combination gene therapy in a murine CT26 cell colon cancer model

We evaluated the antitumor effects of combination gene therapy on CT26 mouse colon cancer cells, using the genes for herpes simplex virus thymidine kinase gene HSV-TK combined with granulocyte macrophage colony-stimulating factor (GM-CSF) compared with HSV-TK alone. Cells, unmodified or retrovirally transduced with HSV-TK or GM-CSF, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSV-TK and GM-CSF were also delivered using different routes (in separate cells vs doubly transfected single cells). Both HSV-TK (with i.p. ganciclovir - GCV - treatment) and GM-CSF genes had independent antitumor effects, and given together they caused significant reduction in tumor volumes compared with the HSV-TK gene alone (P < 0.001). Following GCV treatment, however, the treated/control ratios for tumor volumes were not different between tumors containing either HSV-TK alone or both genes (0.27 vs 0.25, respectively). Thus, the presence of GM-CSF did not increase the bystander effect of HSV-TK. Tumors receiving genes transferred in separate cells tended to be more consistently suppressed after GCV treatment than when both genes were transferred in the same cells, although this was not statistically significant. Thus, combination GM-CSF and HSV-TK gene therapy produced greater therapeutic efficacy than HSV-TK alone, but the bystander effect was not enhanced by GM-CSF.     

Functional expression of thymidine kinase in human leukaemic and colorectal cells, delivered as EGFP fusion protein by herpesvirus saimiri-based vector

Herpesvirus saimiri (HVS) has the capacity to incorporate large amounts of heterologous DNA and can infect many different human cell types. To develop its potential as a gene therapy vector, we cloned herpes simplex virus thymidine kinase (TK) gene into the HVS genome in the form of an enhanced green fluorescent protein (EGFP) fusion protein, using a cosmid-based approach. At multiplicity of infection = 100 over 90% of human leukemic K562 and Jurkat cells were transduced with HVS/EGFP-TK. Conditions of no selective pressure expression were maintained at > 92% per cell division. Expression of the EGFP-TK fusion protein rendered transfected leukaemic cells sensitive to cytotoxic treatment with the prodrugs ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2'deoxyuridine (BVDU) at concentrations as low as 10 ng/ml. The viral vector was also screened against a panel of colorectal and pancreatic carcinoma cell lines. All cell lines were transduced but showed a range of sensitivity to infection. Three of the most easily transduced cell lines: Mia PaCa, HCT116 and SW948 transduced with HVS/EGFP-TK were effectively ablated by subsequent treatment with GCV or BVDU. Our results show that in its current form HVS/EGFP-TK could be utilized as an antitumour agent, or it could be developed further by inclusion of a therapeutic gene, with TK presence ensuring a mechanism of controlled removal of modified cells when no longer necessary. These results suggest that HVS/EGFP-TK has a great potential for a number of gene therapy applications.     

Enhanced anti-tumor effects of herpes simplex virus thymidine kinase/ganciclovir system by codelivering monocyte chemoattractant protein-1 in hepatocellular carcinoma.

The therapeutic efficacy of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system in many types of tumors is unsatisfactory due to the insufficient spread of gene transfer and insufficient cell killing. In the current study, we investigated whether adenovirally delivered monocyte chemoattractant protein (MCP)-1 potentiates the antitumor effects of the HSV-tk/GCV system in hepatocellular carcinoma (HCC) cells. Subcutaneous tumor foci of the human HCC cell line, HuH7, established in athymic mice were directly transduced with a recombinant adenovirus (rAd) harboring an HSV-tk gene driven by a human alpha-fetoprotein promoter, followed by GCV administration. Subsequently, another rAd expressing MCP-1 under the universal CAG promoter was injected. The growth of tumors was markedly suppressed by codelivering HSV-tk and MCP-1 genes compared to that by either HSV-tk/GCV or MCP-1 delivery. In the tumor tissues, monocyte/macrophage infiltration was detected immunohistochemically. The antitumor effects of the rAd expressing MCP-1 were markedly reduced by the administration of carrageenan, a compound known to inactivate macrophage. These results indicate that adenovirally delivered MCP-1 enhanced the antitumor effects of the HSV-tk/GCV system synergistically by recruitment/activation of macrophages in tumor tissues, suggesting an effective immunotherapy for HCC and other lineages of tumors when used adjuvantly with a suicide gene.     

Suicide gene therapy for human oral squamous cell carcinoma cell lines with adeno-associated virus vector

The purpose of this study was to test the possibility of gene transfer as a new therapy for oral cancer. Adeno-associated virus (AAV) has already been used in the fields of cystic fibrosis and Parkinson's disease as a potential vector for gene therapy because of its wide host range, high transduction efficiency, and lack of cytopathogenicity. Four human oral squamous cell carcinoma cell lines were transduced with an AAV vector containing the beta-galactosidase gene (AAVlacZ) in vitro. Gene transduction efficiency was from 20 to 50% at a multiplicity of infection (MOI; for the purposes of this study the number of vector genomes per target cell) of 1x10(3), and nearly 100% of each cell line were transduced at an MOI of 1x10(4). Next, four cell lines were transduced with an AAV vector containing the herpes simplex virus thymidine kinase (HSVtk) gene, which sensitizes transduced cells to ganciclovir (GCV). Subsequent administration of GCV resulted in nearly 100% tumor cell killing at an MOI of 1x10(4) and from 70 to 80% tumor cell killing at an MOI of 1x10(3). These results suggest that AAV-mediated gene transfer of HSVtk and administration of GCV has potential as a new therapy for oral squamous cell carcinoma.     

Suicide Gene Therapy for Chemically Induced Rat Bladder Tumor Entailing Instillation of Adenoviral Vectors

The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV-tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short-term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apopto-sis. Following a long-term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.     

Suicide gene therapy for chemically induced rat bladder tumor entailing instillation of adenoviral vectors.

The efficacy of an in vivo gene therapy protocol making use of an adenoviral vector in the treatment of bladder cancer was examined. Bladder tumors were induced in rats by oral administration of BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine). Histologically, such tumors resemble those seen in human bladder cancer, and the cells can be selectively transduced using adenoviral vectors. The therapeutic protocol thus entailed instillation of an adenoviral vector containing the HSV-tk suicide gene into rat bladder followed by a regimen of intraperitoneal ganciclovir (GCV) injections. Histological examination after a short-term GCV regimen (3 days) revealed marked vacuolization of the tumor cells. Moreover, TUNEL assays showed that the cytotoxic reaction was mediated by apoptosis. Following a long-term GCV regimen (14 days), tumor growth was significantly inhibited and glandular metaplasia was observed. This is the first report demonstrating the efficacy of in vivo suicide gene therapy in a chemically induced transitional cell carcinoma like that seen in most human bladder cancer. Intravesical instillation is already a well established clinical technique. Our findings indicate that now there is a strong potential for its incorporation into new and useful gene therapies aimed at the treatment of human bladder cancer.     

Combined suicide gene and immunostimulatory gene therapy using AAV-mediated gene transfer to HPV-16 transformed mouse cell: decrease of oncogenicity and induction of protection

To test the effects of combined transduction of a suicide gene and genes coding for various immunostimulatory factors on the oncogenicity and immunogenicity of TC-1 cells (HPV-16 transformed C57BL/6 mouse cells), several bicistronic recombinant adeno-associated viruses (rAAV) were constructed. Each of these constructs carried, and in infected cells expressed, the herpes simplex type 1 thymidine-kinase gene (HSV-TK) and the gene of one of the following immunostimulatory factors: human monocyte chemoattractant protein 1 (MCP-1), mouse B7.1 costimulatory molecule (B7.1), or mouse granulocyte-macrophage colony-stimulating factor (GM-CSF). For control purposes, an rAAV carrying the HSV-TK gene and neomycin resistance gene (neo) and an rAAV containing the lacZ gene were used. All of these constructs proved functional both in mouse TC-1 and human 293T cells. For experiments in mice, TC-1 cells were infected in vitro with the AAV recombinants at an input multiplicity of 50 particles/cell; these cells were then administered to 5-week-old mice. As from day 5, half of the animals were given ganciclovir (GCV) (2.5 mg/day) for 10 days. With a single exception, none of the mice inoculated with cells treated with rAAV expressing HSV-TK + B7.1 or HSV-TK + MCP-1 developed tumour irrespective of GCV treatment. The tumour suppressive effect was less marked in animals inoculated with TC-1 cells infected with rAAV expressing HSV-TK + GM-CSF, and among these it was somewhat more pronounced in GCV-untreated animals. A clear antitumour effect of GCV treatment was only observed in mice inoculated with TC-1 cells transduced with rAAV expressing HSV-TK but no immunostimulatory factor. Mice that remained tumour-free on day 54 were challenged with untreated TC-1 cells. The tumour resistance rates found were related not only to the immunostimulatory gene used for the transduction, but also to GCV treatment. The best protection was recorded in mice pre-inoculated with TC-1 cells transduced with either B7.1 or MCP-1-expressing rAAV and not given GCV.     

Suppression of tumor growth and pulmonary metastasis in murine osteosarcoma using gene therapy

We evaluated the effect of gene therapy in the murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs. LM8 cells were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ). We investigated the cytotoxicity of LM8 cells bearing an HSV-tk gene after treatment with ganciclovir (GCV). LM8 cells bearing an HSV-tk gene were more sensitive than non-transduced cells. The remarkable inhibition of tumor growth and pulmonary metastases was confirmed in vivo. Our findings indicated that GCV kills tumor cells transduced with HSV-tk in vitro and in vivo.     

Effects of retroviral-mediated herpes simplex virus thymidine kinase gene transfer to murine neuroblastoma cell lines in vitro and in vivo

Selective introduction of genes conferring chemosensitivity on proliferating tumor cells can be used to treat cancer. We investigated the efficacy of retrovirus-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell lines (neuro-2a) in vitro and in vivo. Retrovirus-mediated HSV-TK gene transfer to the neuro-2a cells resulted in sensitivity to ganciclovir (GCV) in vitro. In A/J mice, tumors produced from HSV-TK transduced neuro-2a cells regressed after GCV treatment. Intratumoral injection of recombinant retrovirus expressing HSV-TK gene also inhibited growth of the tumor established in A/J mice. These results demonstrate that HSV-TK gene therapy might be a feasible approach for inhibiting the growth of neuroblastoma.     

HSV-tk、重组人IL-2、TNF-α融合基因对喉癌细胞Hep-2杀伤作用的体外研究

目的:观察自杀基因HSV-tk与不同细胞因子基因(IL-2、TNF-α)构建的不同融合基因表达产物对喉癌细胞系的体外杀伤作用。方法:分别构建不同融合基因逆转录表达载体PL(TI)SN,PL(N)SN,PL(TK)SN,在LipofectAMINETM2000介导下转染PA317包装细胞,G418筛选,直至出现抗性克隆,扩大培养,用NIH3T3测定病毒滴度,将各重组病毒分别感染人喉癌细胞系Hep-2,G418筛选出抗性克隆,分别命名为Hep/TI,Hep/TT,Hep/TK,以RT-PCR及Southern blot检测各融合基因的整合及表达。通过观察各基因修饰细胞生长状况及GCV杀伤实验鉴定融合基因的表达及杀伤效应。结果:RT-PCR及South-ern blot分析证明各重组外源基因在人喉癌细胞系Hep-2中均有整合及表达。Hep/TI,HeP/TK生长速度无明显差别,Hep/TT细胞增殖速度受到抑制。在GCV杀伤试验中,Hep/TI,Hep/TT,Hep/TK均显示出对GCV的高敏感性,其中GCV对Hep/TT的杀伤作用最为明显,各基因修饰细胞与不同比例亲本细胞混合,均显示出明显旁观者效应。结论:自杀基因与细胞因子基因共表达体系对喉癌细胞系Hep-2体外杀伤具有协同作用,并且有明显的旁观者效应,可望成为肿瘤基因治疗的新途径。     

Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1(-)E3(-) backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.     

Bone marrow-derived cells as carriers of recombinant immunomodulatory cytokine genes to lymphoid organs

The purpose of this study was to determine the feasibility of cytokine gene delivery to lymphatic tissue using transduced bone marrow-derived cells. MBAE and pBABE retroviral vectors carrying the genes for murine interleukin-4 and the selection marker neomycin phosphotransferase (neo) were used to transduce bone marrow-derived dendritic cells (DC) and hematopoietic stem cells (HSC). A transduction efficiency of 11-33% for HSC and 2-10% for DC was achieved. Transduced HSC and DC released 55-170 pg of recombinant interleukin-4 per 1 x 10(6) cells/mL in vitro. To study the migration of the cells in vivo, we introduced the transduced cells into syngenic mice. DC were injected subcutaneously into the front limbs of unconditioned mice and HSC were intravenously administered to irradiated mice. The distribution of the transduced cells was studied by quantitative polymerase chain reaction for the neo gene as a marker. After 3 days, DC migrated to the axillary lymph nodes in the drainage area of the injection site and were detectable up to 5 days. After intravenous administration of transduced HSC, the neo gene could be found in up to 100 copies/5 x 10(3) cells in mesenterial lymph node, spleen, bone marrow, thymus, and liver. The distribution of the transduced cells was heterogenous: in different mice, different organs showed high copies of the neo gene after 10 and 13 days. After 39 days, two of three mice were negative for neo in all organs analyzed. In conclusion, bone marrow-derived cells can be genetically engineered ex vivo to deliver recombinant cytokine genes to lymphoid organs in vivo. In particular, DC might be candidate cells for use in immunomodulatory gene therapy for autoimmune diseases and cancer.     

Cell‐specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant sindbis virus vector

Transfer of the herpes simplex virus type 1 thymidine kinase (HSV‐TK) gene into tumor cells using virus‐based vectors in conjunction with ganciclovir (GCV) exposure provides a potential gene therapy strategy for the treatment of cancer. The possibility of using a novel targetable Sindbis virus expression vector containing the HSV‐TK gene was examined. Baby hamster kidney (BHK) cells and several human tumor cells infected with a Sindbis virus containing the HSV‐TK gene showed strong expression of HSV‐TK protein. Cells transduced with the HSV‐TK gene exhibited increased TK activity, ranging from 3‐ to 20‐fold over an average baseline level. The human HeLa‐CD4⁺ cells infected with recombinant Sindbis virus containing the HSV‐TK gene were sensitive to low concentrations of GCV (0.1–1 μg/ml) and the 50% growth inhibitory concentration (IC₅₀) was 0.6 μg/ml. We also demonstrated applications of cell type‐specific Sindbis virus‐mediated antigen‐antibody targeting of the HSV‐TK/GCV system in vitro. Sindbis virus containing the HSV‐TK gene packaged in a helper virus displaying the IgG‐binding domain of protein A on its envelope could infect various tumor cell lines in the presence of specific antibodies that recognize antigens on their surfaces. HSV‐TK‐transduced tumor cell lines exhibited sensitivity to GCV. Our data suggest the potential for targeted gene therapy of the HSV‐TK/GCV system using a cell type‐specific recombinant Sindbis virus vector‐antibody system. Int. J. Cancer 80:110–118, 1999. © 1999 Wiley‐Liss, Inc.     

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (BM-hMSCs) are nonhematopoietic stem cells that have the potential to differentiate into adipocytes, osteocytes and chondrocytes. Because of its propensity to migrate to the sites of injury and the ability to expand them rapidly, BM-hMSCs have been exploited as potential gene transfer vehicles to deliver therapeutic genes. Herein, we evaluated the feasibility of employing herpes simplex virus type I (HSV-1) amplicon viral vector as a gene delivery vector to BM-hMSCs. High transduction efficiencies were consistently observed in different isolates of BM-hMSCs following infection with HSV-1 amplicon viral vectors. Furthermore, we demonstrated that transduction with HSV-1 amplicon viral vector did not alter the intrinsic properties of the BM-hMSCs. The morphology and cellular proliferation of the transduced BM-hMSCs were not altered. Chromosomal stability, as confirmed by karyotyping and soft agar colony assays, of the transduced BM-hMSCs was not affected. Similarly, transduction with HSV-1 amplicon viral vectors has no effect on the pluripotent differentiation potential and the tumor tropism of BM-hMSCs. Taken together, these results demonstrated that BM-hMSCs could be transduced efficiently by HSV-1 amplicon viral vector in an 'inert' manner and thus enable strategies to express potential therapeutic genes in BM-hMSCs.