A phase I/II study of herpes simplex virus type 1 thymidine kinase "suicide" gene therapy for recurrent glioblastoma. Study Group on Gene Therapy for Glioblastoma

Despite extensive surgery for glioblastoma, residual tumor cells always lead to relapse. Gene therapy based on retrovirus-mediated gene transfer of herpes simplex virus type 1 thymidine kinase (HSV-1 TK), which specifically sensitizes dividing cells to ganciclovir (GCV) toxicity, may help eradicate such cells. During glioblastoma surgery, HSV-1 TK retroviral vector-producing cells (M11) were injected into the surgical cavity margins after tumor debulking. After a 7-day transduction period, GCV was administered for 14 days. Safety was assessed by clinical and laboratory evaluations, and efficacy was assessed by MRI-based relapse-free survival at month 4 and by overall survival. Twelve patients with recurrent glioblastoma were treated without serious adverse events related to M11 cell administration or GCV. Quality of life was not negatively influenced by this treatment. Overall median survival was 206 days, with 25% of the patients surviving longer than 12 months. At 4 months after treatment, 4 of 12 patients had no recurrence; their median overall survival was 528 days, compared with 194 days for patients with recurrence (p=0.03 by the log rank test). One patient is still free of detectable recurrence, steroid free and independent, 2.8 years after treatment. Thus, brain injections of M11 retroviral vector-producing cells for glioblastoma HSV-1 TK gene therapy were well tolerated and associated with significant therapeutic responses. These results warrant further development of this therapeutic strategy in brain tumor, including recurrent glioblastoma.     

Suicide gene therapy for human uterine adenocarcinoma cells using herpes simplex virus thymidine kinase

We report the first large-scale screening of mitochondrial (mt) DNA in 77 Caucasian patients with relapsing-remitting or secondary progressive form of multiple sclerosis (MS) and in 84 Caucasian controls by using the method of restriction site polymorphism and haplotype analysis. No pathogenic mtDNA mutation was found in association with MS. However, mtDNA haplotypes K* and J* defined by the simultaneous presence of Ddel restriction sites at nucleotides 10,394 and 14,798 of the mtDNA in haplogroups K and J showed association with MS at a P-value of 0.001. A relative increase of MS patients compared to controls either with the J* or with the K* haplotype (+10,394Ddel/+14,798Ddel in haplogroup J or K) also was detected (each with a P<0.05). No distinct phenotypic characteristics of MS were observed when clinical data of patients with haplotypes K* or J* were analyzed. In addition to previous complete sequencing in several MS patients, the population screening of mtDNA presented here suggests that mtDNA point mutations are not likely to be involved in the pathogenesis of typical forms of MS. However, the mitochondrial genetic background (haplotype K* and J*) may moderately contribute to MS susceptibility. The reported association between MS and Leber's hereditary optic nerve atrophy, a disease caused by mtDNA point mutations preferentially occurring in haplogroup J, may be at least in part related to the overlapping mitochondrial genetic background of the two diseases.     

Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors

A number of packaging materials are being used not only to contain food during distribution but also to serve as the cooking container. The higher temperatures that these materials reach led the US Food and Drug Administration (FDA) to issue an intent to publish new regulations in 1989. The food and packaging industries responded by conducting extensive research and submitting the results to FDA. The methods used and results obtained are discussed. Most of the data were focused on microwave susceptors and the volatile compounds generated. One project showed that for a specific product, popcorn, there was no transfer into the food. Work is continuing to validate methods to test for non-volatile compounds. In addition to susceptors, various paper and plastic materials are used in dual ovenable (microwave and conventional ovens) applications. Most of the research on these materials has investigated the food contact temperatures on testing for migrants. An update on the current regulatory status of packaging materials intended for high temperature use in the US is discussed.     

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.     

Integrated homology modelling and X-ray study of herpes simplex virus I thymidine kinase: a case study

Knowledge-based homology modelling together with site-directed mutagenesis, epitope and conformational mapping is an approach to predict the structures of proteins and for the rational design of new drugs. In this study we present how this procedure has been applied to model the structure of herpes simplex virus type 1 thymidine kinase (HSV1 TK, HSV1 ATP-thymidine-5'-phosphotransferase, EC 2.7.1.21). We have used, and evaluated, several secondary structure prediction methods, such as the classical one based on Chou and Fastman algorithm, neural networks using the Kabsch and Sander classification, and the PRISM method. We have validated the algorithms by applying them to the porcine adenylate kinase (ADK), whose three-dimensional structure is known and that has been used for the alignment of the TKs as well. The resulting first model of HSV1-TK consisted of the first beta-strand connected to the phosphate binding loop and its subsequent alpha-helix, the fourth beta-strand connected to the conserved FDRH sequence and two alpha-helix with basic amino acids. The 3D structure was built using the X-ray structure of ADK as template and following the general procedure for homology modelling. We extended the model by means of COMPOSER, an automatic process for protein modelling. Site-directed mutagenesis was used to experimentally verify the predicted active-site model of HSV1-TK. The data measured in our lab and by others support the suggestion that the FDRH motif is part of the active site and plays an important role in the phosphorylation of substrates. The structure of HSV1 TK, recently solved in collaboration with Prof. G. Schulz at 2.7 A resolution, includes 284 of 343 residues of the N-terminal truncated TK. The secondary structures could be clearly assigned and fitted to the density. The comparison between crystallographically determined structure and the model shows that nearly 70% of the HSV1 TK structure has been correctly modelled by the described integrated approach to knowledge based ligand protein complex structure prediction. This indicate that computer assisted methods, combined with "manual" correction both for alignment and 3D construction are useful and can be successful.     

Monitoring gene therapy with herpes simplex virus thymidine kinase in hepatoma cells: uptake of specific substrates

The uptake as well as the export of citric acid by Aspergillus niger occur by active, deltapH-driven, H(+)-symport dependent systems. They are inhibited by nonmetabolizable tricarboxylic acid analogues and phthalic acid, and by several other mono-, di- and tribasic organic acids. However, citrate export could only be demonstrated in a mycelium cultivated under manganese-deficient growth conditions, whereas the uptake of citrate from the medium was only detectable upon precultivation of A. niger in a medium supplemented with Mn2+ ions. In addition, the uptake of citrate was dependent on the presence of Mn2+ ions in the assay, and inhibited by EDTA. This requirement for Mn2+ could also be partially fulfilled by Mg2+, Fe2+ or Zn2+, whereas Cu2+ ions inhibited citrate transport. The observed divergent effects of manganese ions on citrate uptake and citrate export may be a major reason for the well documented requirement for manganese deficiency of citric acid accumulation.     

Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy

Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) 'prodrug' system. Since retinoids have been reported to increase GJIC by induction of connexin expression, we hypothesized that these compounds could be used to augment the HSVtk/GCV bystander effect. Addition of all-trans retinoic acid increased GJIC in tumor cell lines, augmented expression of connexin 43, and was associated with more efficient GCV-induced in vitro bystander killing in cells transduced with HSVtk via either retrovirus or adenovirus vectors. This augmentation of bystander effect could also be seen in vivo. HSVtk-transduced tumors in mice treated with the combination of GCV and retinoids were significantly smaller than those treated with GCV or retinoids alone. These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effects and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk/GCV system to treat tumors or vascular restenosis.     

Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy

Gene therapy via the herpes simplex virus thymidine kinase (tk) gene and ganciclovir (GCV) treatment eliminates experimental tumors. In this approach, cells expressing the tk gene (tk+) and neighboring tumor cells which do not express the gene are killed. We have demonstrated this bystander effect is enhanced in vitro by gap junctional intercellular communication (GJIC). In order to extend our in vitro results into in vivo situations, we injected into nude mice different ratios of tk+/tk- HeLa cells, either lacking or transfected with connexin43 (Cx43), a gene coding for a gap junction protein. When GCV was administered before tumors were palpable, fewer animals developed tumors, even after a longer period, if the injected cells were mixtures of Cx43(+)-tk+ and Cx43(+)-tk- while tumor growth was not prevented with mixtures of HeLa cells not expressing Cx43, i.e. Cx43(+)-tk+/Cx43(-)-tk-. When GCV was given after the appearance of tumors, the size of the tumors from Cx43- cells was 30% reduced for 3 weeks if 50% of the injected cells were tk+. However, for cells expressing Cx43, the tumor size was 66% reduced if 10% of the cells were tk+. Such a reduction demonstrates a long-term bystander effect which is dependent on Cx43 expression.     

The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK- cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK+ phenotype on 143B TK- cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK- cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N-, a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2'-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.     

Gene transfer into sympathetic preganglionic neurons in vivo using a non-replicating thymidine kinase-deficient herpes simplex virus type 1

The suitability of non-replicating thymidine kinase deficient herpes simplex virus type 1 expressing bacterial beta-galactosidase (tk-lacZ HSV-1) as a transfer vehicle into sympathetic preganglionic neurons in vivo was assessed. Many sympathoadrenal preganglionic neurons (451 +/- 105) with normal morphology were identified using beta-galactosidase histochemistry two days after inoculation of tk-lacZ HSV-1 into the adrenal gland of hamsters. Beta-galactosidase activity co-localized with nicotinamide adenine dinucleotide phosphate-diaphorase-positive sympathetic preganglionic neurons in the nucleus intermediolateralus, pars principalis. The maximal number of beta-galactosidase expressing neurons was found two days post-inoculation but this number dropped dramatically after this time. An inflammatory infiltrate was abundant around infected neurons and in the white matter at five days and infected neurons appeared morphologically abnormal. At 26 days, the infiltrate was still present but no infected sympathoadrenal preganglionic neurons were detected. Approximately 25% fewer nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the nucleus intermediolateralis, pars principalis were counted ipsilaterally than contralaterally in animals infected for 14, 21 or 26 days with tk-lacZ HSV-1, compared to the 3% difference in animals mock-infected for 26 days. Approximately 33% of the estimated number of sympathoadrenal preganglionic neurons infected with tk-lacZ HSV-1 at five days were apoptotic or necrotic. About 60% of neurons infected with tk-lacZ HSV-1 at two days no longer expressed nicotinamide adenine dinucleotide phosphate-diaphorase at 14-26 days. In conclusion, the non-replicating thymidine kinase deficient HSV-1 was efficiently retrogradely transported from the adrenal gland to infect sympathoadrenal preganglionic neurons. These gene transfer experiments using tk-lacZ HSV-1 suggest that foreign gene expression in sympathetic preganglionic neurons in vivo may be maximal two days after inoculation when beta-galactosidase was expressed in the greatest number of sympathetic preganglionic neurons. After two days, fewer neurons expressed beta-galactosidase and the presence of tk-lacZ HSV-1 appeared to be altering protein expression in sympathetic preganglionic neurons and/or leading to the demise of the infected neuron.     

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.     

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments.     

Some 6-aza-5-substituted-2'-deoxyuridines show potent and selective inhibition of herpes simplex virus type 1 thymidine kinase

The synthesis and X-ray crystal structures of a series of 5-substituted-6-aza-2'-deoxyuridines is reported. These nucleoside analogues inhibit the phosphorylation of thymidine by HSV-1 TK but have no effect on the corresponding human enzyme. Detailed examination of one analogue proves it to be a competitive inhibitor of thymidine with a Ki of 0.34 microM and is a very poor substrate. The analogues are not substrates for the enzyme and also do not inhibit the degradation of thymidine by thymidine phosphorylase. Molecular modelling showed that the inhibitors fit well in the active site of HSV-1 TK, provided the conformation of the sugar moiety is the same for thymidine in the complex.     

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.     

Histopathological responses in the CNS following inoculation with a non-neurovirulent mutant (1716) of herpes simplex virus type 1 (HSV 1): relevance for gene and cancer therapy

OBJECTIVE: To determine the efficacy of intranasally administered immunoglobulin in preventing symptoms of rhinitis in children. METHODS: Forty children ages 1 to 4 years who attended day-care centers in Turku, Finland, were enrolled in the double blind, placebo-controlled study. The children were randomly assigned to receive treatment with immunoglobulin, composed mainly of immunoglobulin A, or placebo, both administered as nasal sprays twice daily for 8 weeks. During this medication period and an additional 8-week follow-up period, the parents recorded the symptoms of the children daily in the diaries provided. One child who met an exclusion criterion was withdrawn from the study after a few days of medication. RESULTS: During the 8-week medication period the 19 children in the immunoglobulin group had 42% fewer days with rhinitis than the 20 children receiving placebo (mean, 10.8 vs. 18.7 days; P=0.004). The total numbers of episodes of rhinitis in the immunoglobulin and placebo groups were 33 and 51, respectively. No significant differences were observed between the groups during the postmedication follow-up period. CONCLUSIONS: Intranasal administration of immunoglobulin appears to be an effective method to prevent symptoms of rhinitis in children, and further studies of this approach are needed.