Combination of vinblastine and oncolytic herpes simplex virus vector expressing IL-12 therapy increases antitumor and antiangiogenic effects in prostate cancer models.

Oncolytic herpes simplex virus (oHSV)-1-based vectors selectively replicate in tumor cells causing direct killing, that is, oncolysis, while sparing normal cells. The oHSVs are promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We hypothesized that combining the direct oncolytic and antiangiogenic activities of the interleukin (IL)-12-secreting NV1042 oHSV with microtubule disrupting agents (MDAs) would be an effective means to enhance antitumor efficacy. Vinblastine (VB) was identified among several MDAs screened, which displayed consistent and potent cytotoxic killing of both prostate cancer and endothelial cell lines. In matrigel tube-forming assays, VB was found to be highly effective at inhibiting tube formation of human umbilical vein endothelial cells. The combination of VB with NV1023 (the parental virus lacking IL-12) or NV1042 showed additive or synergistic activity against prostate cancer cell lines, and was not due to increased oHSV replication by VB. In athymic mice bearing CWR22 prostate tumors, VB in combination with NV1042 was superior to the combination of VB plus NV1023 in reducing tumor burden, appeared to be nontoxic and resulted in a statistically significant diminution in the number of CD31(+) cells as compared with other treatment groups. In human organotypic cultures using surgical samples from radical prostatectomies, both NV1023 and NV1042 were localized specifically to the epithelial cells of prostatic glands but not to the surrounding stroma. These data highlight the therapeutic advantage of combining the dual-acting antitumor and antiangiogenic activities of oHSVs and MDAs.

Treatment of breast cancer stem cells with oncolytic herpes simplex virus.

Cancer stem cells have recently been isolated from several different solid tumors. In breast cancer, the CD44(+)CD24(-/low) population is considered to comprise stem-like cells. The identification of cancer stem cells has provided new targets for the development of therapeutics. Oncolytic herpes simplex viruses (oHSVs) are an effective strategy for killing breast cancer cells and treating breast tumors in preclinical models. Here, we examined the efficacy of the oHSV G47Δ in killing breast cancer stem cells. Human breast cancer cell line SK-BR-3 and human primary breast cancer cells were cultured in suspension under conditions conducive to the growth of stem cells. They generated mammospheres, which had cancer stem cell properties. The proportion of CD44(+)CD24(-/low) cells in these mammospheres exceeded 95%, as determined by flow cytometry. The mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. G47Δ contains the LacZ gene, and X-gal staining of infected cells in vitro and in vivo showed the replication and spread of the virus. G47Δ was found to be highly cytotoxic to the CD44(+)CD24(-/low) population in vitro, even when injected at low multiplicities of infection, and G47Δ treatment in vivo significantly inhibited tumor growth compared with mock treatment. This study demonstrates that oHSV is effective against breast cancer stem cells and could be a beneficial strategy for treating breast cancer patients.

Oncolytic herpes simplex virus armed with xenogeneic homologue of prostatic acid phosphatase enhances antitumor efficacy in prostate cancer.

Prostate cancer is one of the most prevalent cancers in men. Replication-competent oncolytic herpes simplex virus (oHSV) vectors are a powerful antitumor therapy that can exert at least two effects: direct cytocidal activity that selectively kills cancer cells and induction of antitumor immunity. In addition, oHSV vectors can also function as a platform to deliver transgenes of interest. In these studies, we have examined the expression of a xenogeneic homologue of the prostate cancer antigen, prostatic acid phosphatase (PAP), with the goal of enhancing virotherapy against PAP-expressing tumors. PAP has already been used for cancer vaccination in patients with prostate cancer. Here we show that treatment with oHSV bPDelta6 expressing xenogeneic human PAP (hPAP) significantly reduces tumor growth and increases survival of C57/BL6 mice bearing mouse TRAMP-C2 prostate tumors, whereas expression of syngeneic mouse PAP (mPAP) from the same oHSV vector did not enhance antitumor activity. Treatment of mice bearing metastatic TRAMP-C2 lung tumors with oHSV-expressing hPAP resulted in fewer tumor nodules. To our knowledge, this is the first report of oncolytic viruses being used to express xenoantigens. These data lend support to the concept of combining oncolytic and immunogenic therapies as a way to improve therapy of metastatic prostate cancer.

Analysis of genetically engineered oncolytic herpes simplex viruses in human prostate cancer organotypic cultures.

Oncolytic herpes simplex viruses type 1 (oHSVs) such as G47Delta and G207 are genetically engineered for selective replication competence in cancer cells. Several factors can influence the overall effectiveness of oHSV tropism, including HSV-1 receptor expression, extracellular matrix milieu and cellular permissiveness. We have taken advantage of human prostate organ cultures derived from radical prostatectomies to investigate oHSV tropism. In this study, we show that both G47Delta and G207 specifically replicate in epithelial cells of the prostatic glands but not in the surrounding stroma. In contrast, both the epithelial and stromal cell compartments were readily infected by wild-type HSV-1. Analysis of oHSV replication in prostate surgical specimens 3 days post infection showed that G47Delta generated approximately 30-fold more viral progeny than did G207. This correlated with the enhanced expression of G47Delta-derived glycoprotein gB protein levels as compared with G207. In benign prostate tissues, G207 and G47Delta titers were notably reduced, whereas strain F titers were maintained at similar levels compared with prostate cancer specimens. Overall, our results show that these oncolytic herpes vectors show both target specificity and replication competence in human prostate cancer specimens and point to the utility of using human prostate organ cultures in assessing oHSV tropism and cellular specificity.

Herpes simplex virus delivery to orthotopic rectal carcinoma results in an efficient and selective antitumor effect.

Cancer of the rectum poses a complex therapeutic challenge because of its proximity to adjacent organs and anal sphincters. The addition of radiotherapy before surgical resection has been shown to confer good survival rates while preserving sphincter function. Nevertheless, radiation is associated with significant side effects. On the basis of our previous work showing that herpes simplex virus type-1 (HSV-1) preferentially infects human colon cancer, we set out to examine the oncolytic effect of HSV-1 on orthotopic rectal tumors in mice. Two vectors were compared for oncolytic activity, HSV-1(Gbeta) with wild-type replication and an attenuated HSV-1 vector (HSV-G47Delta). Intratumoral injection of HSV-1(Gbeta) and HSV-G47Delta resulted in a significant reduction or disappearance of the tumors and increased survival of mice. Although the use of HSV-1(Gbeta) was associated with systemic toxicity, HSV-G47Delta appears to possess a selective oncolytic activity. Moreover, infection with HSV-G47Delta resulted in the activation of the double-stranded RNA-dependent protein kinase (PKR) pathway. A significant improvement in viral replication and the antitumor effect was observed when the PKR inhibitor 2-aminopurine was coadministered with HSV-G47Delta to the tumor. In conclusion, the efficacy of local delivery of HSV-G47Delta combined with a specific chemical inhibitor of antiviral activity points to a novel therapeutic modality for rectal cancer and other solid tumors.

Oncolytic herpes simplex virus vectors and taxanes synergize to promote killing of prostate cancer cells.

Genetically engineered oncolytic herpes simplex virus-1 (HSV-1) vectors selectively replicate in tumor cells causing direct killing whereas sparing normal cells. One clinical limitation of using oncolytic HSV vectors is their attenuated growth. We hypothesized that the appropriately chosen chemotherapeutic agent combined with an oncolytic HSV could be an effective means to promote augmented prostate cancer cell killing both in vitro and in vivo. Here we have identified that G47Delta synergizes with the microtubule-stabilizing taxane agents docetaxel and paclitaxel to enhance the in vitro killing of prostate cancer cells. In vivo efficacy studies show that when combined with docetaxel, G47Delta could be reduced at least 10-fold. Immunoblot analysis revealed that docetaxel-induced accumulation of the phospho-specific mitotic markers op18/stathmin or histone-H3 was markedly reduced by G47Delta, which correlated with enhanced apoptosis and required active viral replication. Furthermore, cell-cycle analysis demonstrated that in the presence of G47Delta, the majority of 4N cells arrested in mitosis were MPM-2-negative, indicative of cells exiting mitosis prematurely. These findings suggest that G47Delta may act in part, on mitotically blocked cells to enhance cell death, which may account for the enhanced antitumor efficacy observed in vivo.

Enhanced therapeutic efficacy of IL-12, but not GM-CSF, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers.

Replication competent oncolytic herpes simplex viruses (HSV) with broad-spectrum activity against various cancers, including prostate cancer, exert a dual effect by their direct cytocidal action and by eliciting tumor-specific immunity. These viruses can deliver immunoregulatory molecules to tumors so as to enhance the cumulative antitumor response. This is particularly desirable for prostate cancers, which are usually poorly immunogenic. Initial studies described herein comparing the efficacy of three different oncolytic HSVs (G207, G47Delta, and NV1023) to inhibit the growth of the poorly immunogenic TRAMP-C2 mouse prostate tumors demonstrated that NV1023 was most effective in treating established tumors. The expression of IL-12 on an NV1023 background (NV1042), but not the expression of GM-CSF (NV1034), further enhanced the efficacy of NV1023 in two murine prostate cancer models with highly variable MHC class I levels, Pr14-2 with 91% and TRAMP-C2 with 2% of cells staining. NV1042 also inhibited the growth of distant noninoculated tumors in both prostate cancer models. NV1042 treated tumors exhibited increased immune cell infiltration and decreased levels of angiogenesis. Thus, an IL-12 expressing oncolytic herpes virus, which is capable of direct cytotoxicity and can modulate the otherwise suboptimal immune response through concomitant expression of the cytokine at the site of tumor destruction, could serve as a valuable clinical agent to seek out both overt and occult prostate cancers.

Intracarotid delivery of oncolytic HSV vector G47Delta to metastatic breast cancer in the brain.

Delivery of viral vectors to tumors in the brain is a challenge, especially via systemic administration, which is key to targeting the invasive margins of malignant glioma and the multiple foci of metastatic disease. Like for other cancer therapeutics, the blood-brain barrier or even the blood-tumor barrier significantly limits delivery and efficacy. Blood-brain barrier disruption (BBBD) is one strategy for transiting the cerebrovasculature. G47Delta is a third-generation oncolytic replication-competent herpes simplex virus (HSV) vector, containing deletions of the gamma34.5 and alpha47 genes and an inactivating LacZ insertion in UL39 (ICP6). Intracarotid artery delivery of G47Delta after BBBD with 25% mannitol significantly extended the life of nude mice bearing intracerebral human MDA-MB-435 breast tumors, whereas, G47Delta injection contralateral to the tumor, in the absence of mannitol or mannitol alone had no effect on survival. G47Delta replication was extensive after BBBD, as visualized by X-gal staining. Staining of peripheral organs, lung and liver, was minimal and not altered by BBBD. This is the first demonstration of intracarotid arterial delivery of oncolytic HSV vectors and antitumor efficacy in a mouse model and opens the door to the use of mouse syngenic tumor models and transgenic/knockout animals.

Ionizing radiation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous tumor models of human and murine prostate cancer.

Viral gene therapy against malignant tumors holds great promise for tumors that are susceptible to the oncolytic activity of viruses. One advantage of oncolytic viral therapy is that it can potentially be combined with other therapies, such as radiotherapy, to obtain an enhanced tumor response. In the case of prostate cancer, herpes simplex virus-mediated therapies have been shown to be highly effective in animal models; however, studies of the efficacy of combined viral and radiation therapy have not yet been reported. In this study, we have combined G207, a multimutated HSV type 1 vector, with external beam radiation therapy of prostate tumors grown subcutaneously in mice. We examined both the human LNCaP tumor in athymic mice and the mouse transgenic TRAMP tumor in either athymic mice or its syngeneic host, C57BL/6 mice. Virus was delivered either intravenously, in the case of LNCaP, or intratumorally, in the case of TRAMP. We found that individually, either G207 or radiation was effective in delaying tumor growth in these models. However, delivering the treatments simultaneously did not produce an enhanced effect.

Preclinical safety evaluation of G207, a replication-competent herpes simplex virus type 1, inoculated intraprostatically in mice and nonhuman primates.

G207, a replication-competent herpes simplex virus type 1 (HSV-1) virus, has been previously shown to be effective against human prostate cancer xenografts in mice. This study assesses its safety in the prostate of two animal models known for their sensitivity to HSV-1. BALB/c mice were injected intraprostatically with either HSV-1 G207 or strain F and observed for 5 months. None of the G207-injected animals exhibited any clinical signs of disease or died. However, 50% of strain F-injected mice displayed sluggish, hunched behavior and died by day 13. Histopathologically, the G207-injected prostates were normal whereas strain F-injected prostates showed epithelial flattening, sloughing, and stromal edema. Four Aotus nancymae monkeys were also injected with G207 intraprostatically and observed short term (up to 21 days) and long term (56 days). Safety was assessed on the basis of clinical observations, viral biodistribution, virus shedding, and histopathology. None of the injected monkeys displayed evidence of clinical disease, shedding of infectious virus, or spread of the virus into other organs. Except for minor histological changes unrelated to the study, no significant abnormalities were observed. These results demonstrate that G207 can be safely inoculated into the prostate and should be considered for human trials for the treatment of prostate cancer.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing.

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Delta and R47Delta, have been constructed by deleting the alpha47 gene and the promoter region of US11 from gamma34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the alpha47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Delta-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early alpha47 promoter, which suppresses the reduced growth properties of gamma34.5-deficient mutants. G47Delta and R47Delta showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Delta was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 x 10(6) plaque forming units, G47Delta was as safe as G207. These results suggest that G47Delta may have enhanced antitumor activity in humans.

Biological purging of breast cancer cells using an attenuated replication-competent herpes simplex virus in human hematopoietic stem cell transplantation.

Autologous hematopoietic stem cell transplantation after myelosuppressive chemotherapy is used for the treatment of high-risk breast cancer and other solid tumors. However, contamination of the autologous graft with tumor cells may adversely affect outcomes. Human hematopoietic bone marrow cells are resistant to herpes simplex virus type 1 (HSV-1) replication, whereas human breast cancer cells are sensitive to HSV-1 cytotoxicity. Therefore, we examined the utility of G207, a safe replication-competent multimutated HSV-1 vector, as a biological purging agent for breast cancer in the setting of stem cell transplantation. G207 infection of human bone marrow cells had no effect on the proportion or clonogenic capacity of CD34+ cells but did enhance the proliferation of bone marrow cells in culture and the proportion of CD14+ and CD38+ cells. On the other hand, G207 at a multiplicity of infection of 0.1 was able to purge bone marrow of contaminating human breast cancer cells. Because G207 also stimulates the proliferation of human hematopoietic cells, it overcomes a limitation of other purging methods that result in delayed reconstitution of hematopoiesis. The efficient infection of human bone marrow cells in the absence of detected toxicity suggests that HSV vectors may also prove useful for gene therapy to hematopoietic progenitor cells.

Combination suicide/cytokine gene therapy as adjuvants to a defective herpes simplex virus-based cancer vaccine.

We have used syngeneic, established bilateral subcutaneous tumor models to examine the antitumor activity of herpes simplex virus (HSV) vectors, including the induction of an immune response against non-inoculated distant tumors. In such a model with CT26 murine colon adenocarcinoma, unilateral intratumoral inoculation of replication-deficient HSV-1 tsK inhibited the growth of both the inoculated and noninoculated established tumors. To enhance this limited antitumor immune response, we generated a defective HSV vector, dvIL12-tk encoding both interleukin-12 (IL-12) and HSV thymidine kinase (TK), with tsK as the helper virus. In a 'suicide gene' strategy, ganciclovir (GCV) treatment after intratumoral inoculation of dvlacZ-tk/tsK, encoding E. coli lacZ instead of IL-12, resulted in enhanced antitumor activity. Antitumor activity was also enhanced by local expression of IL-12 from dvIL12-tk/tsK. The combination of IL-12 cytokine therapy with GCV treatment was the most efficacious approach, with significantly greater inhibition of tumor growth than IL-12 or TK + GCV alone. These results illustrate the power of combining different cancer therapy approaches; 'suicide gene' therapy, cytokine therapy, and HSV vector infection. HSV vectors are particularly well suited to this because they can accommodate the insertion of large and multiple gene sequences.

Therapeutic efficacy of G207 in a novel peripheral nerve sheath tumor model.

Nerve involvement poses a significant obstacle for the management of peripheral nervous system tumors, and nerve injury provides a frequent source of postoperative morbidity. The lack of suitable animal models for peripheral nerve tumors has impeded the development of alternative nerve-sparing therapies. To evaluate the effect of a multimutated replication-competent herpes simplex virus (G207) on the growth of peripheral nerve tumors and on nerve function, we developed a novel peripheral nerve sheath tumor model. Human neuroblastoma-derived cells injected into murine sciatic nerve consistently caused tumor development within the nerve sheath after 2 weeks followed by increasingly severe impairment of nerve function. Tumor treatment by a single intratumoral injection of G207 resulted in significant reduction of functional impairment, inhibition of tumor growth and prolonged survival. Direct injection of G207 viral particles into the healthy nerve sheath caused no obvious neurologic sequelae, whereas injections of wild-type virus resulted in uniform lethality. The results indicate that viral therapy might be considered as a safe alternative to surgical removal of tumors with peripheral nerve involvement.

In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity.

In vivo delivery of immunomodulatory genes is a promising strategy for solid tumor vaccination. A drawback is that it necessitates induction of a large effect from transgene expression in a small percentage of tumor cells. Although the B7 family is known to be the most potent of the costimulatory molecules, gene transduction of B7 alone has not been effective in inducing antitumor immunity in nonimmunogenic tumors by ex vivo methods, much less in vivo. We have developed a novel approach where a gene encoding soluble B7-1, a fusion protein of the extracellular domain of murine B7-1 and the Fc portion of human IgG1, is delivered to tumor cells in vivo in the context of an oncolytic replication-competent herpes simplex virus, and the gene product is secreted by tumor cells rather than expressed on the cell surface. Defective herpes simplex virus vectors containing the B7-1-immunoglobulin (B7-1-Ig) fusion transgene (dvB7Ig) were generated using G207 as a helper virus and tested in the poorly immunogenic murine neuroblastoma, Neuro2a, in syngeneic A/J mice. Intraneoplastic inoculation of dvB7Ig/G207 at a low titer successfully inhibited the growth of established s.c. tumors, despite the expression of B7-1-Ig being detected in only 1% or fewer of tumor cells at the inoculation site, and prolonged the survival of mice bearing intracerebral tumors. Immunohistochemistry of dvB7Ig/G207-inoculated tumors revealed a significant increase in CD4+ and CD8+ T-cell infiltration compared with control tumors inoculated with defective vector expressing alkaline phosphatase (dvAP/G207). The antitumor effect of dvB7Ig/G207 was not manifested in athymic mice. In vivo depletion of immune cell subsets in A/J mice further revealed that CD8+ T cells, but not CD4+ T cells, were required. Animals cured of their tumors by dvB7Ig/G207 treatment were protected against rechallenge with a lethal dose of Neuro2a cells but not SaI/N cells. The results demonstrate that the use of soluble B7-1 for immune gene therapy is a potent and clinically applicable means of in situ cancer vaccination.

Viral shedding and biodistribution of G207, a multimutated, conditionally replicating herpes simplex virus type 1, after intracerebral inoculation in aotus.

G207 is a multimutated, conditionally replicating herpes simplex virus type 1 (HSV-1) that is currently in clinical trial for patients with malignant glioma. G207 exhibits an efficient oncolytic activity in tumor cells, yet minimal toxicity in normal tissue when injected into the brains of HSV-susceptible mice or nonhuman primates. In this study, we evaluated the shedding and biodistribution of clinical-grade G207 after intracerebral inoculation (3 x 10(7) pfu) in four New World owl monkeys (Aotus nancymae). Using PCR analyses and viral cultures, neither infectious virus nor viral DNA was detected from tear, saliva, or vaginal secretion samples at any time point up to 1 month postinoculation. Analyses of tissues obtained at necropsy at 1 month from two of the four monkeys, plus one monkey inoculated with laboratory-grade G207 (10(9) pfu) 2 years earlier, showed the distribution of G207 DNA restricted to the brain, although infectious virus was not isolated. Histopathology revealed normal brain tissues including the sites of inoculation. A measurable increase of serum anti-HSV antibody titer was observed in all monkeys, as early as 21 days postinoculation. The results ascertain the safety of G207 in the brain and indicate that strict biohazard management may not be required for G207-treated patients.

Tumor growth inhibition by intratumoral inoculation of defective herpes simplex virus vectors expressing granulocyte-macrophage colony-stimulating factor.

To evaluate the potential of defective herpes simplex virus (HSV) amplicon vectors as in vivo cytokine gene transfer vehicles for active immunotherapy, we generated a defective HSV vector that encodes the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, using a replication-defective HSV as helper virus. A variety of murine tumor cell lines were efficiently infected in vitro with the defective GM-CSF vector (dvGM), and this led to the synthesis and secretion of murine GM-CSF. In an established bilateral subcutaneous tumor model with Harding-Passey murine melanoma, unilateral intratumoral inoculation of dvGM significantly inhibited tumor growth of both the inoculated and noninoculated contralateral tumors. This tumor inhibition was dose-dependent and resulted in increased survival of the dvGM-treated mice. Inoculation of a lacZ-expressing defective vector had no effect on tumor growth. We conclude that this defective HSV vector system offers an effective method for cytokine gene delivery in vivo and that GM-CSF expression in tumors has antitumor activity.

CNS induced neurogenic cystitis is associated with bladder mast cell degranulation in the rat.

PURPOSE: To determine if bladder mast cell degranulation is involved in the genesis of neurogenic cystitis induced by pseudorabies virus (PRV) invasion of the central nervous system (CNS). MATERIALS AND METHODS: Rats received a total of 4 x 106 plaque forming units (pfu) of PRV-Bartha in the abductor caudalis dorsalis (ACD) muscle. Granulated bladder mast cells per mm2 of bladder tissue and urine histamine content were monitored as the cystitis developed over the next few days. In a subgroup of rats, intravesical resiniferatoxin was used to remove capsaicin-sensitive sensory bladder afferents, while another subgroup was pretreated with a mast cell degranulator. RESULTS: PRV injection into the ACD muscle leads to neurogenic cystitis. Histamine levels were elevated in the urine of virus injected rats before any behavioral or microscopical signs of cystitis were present. When the cystitis became clinically manifest, urine histamine returned to control levels, and the number of granulated mast cells dropped significantly. Rats in which capsaicin-sensitive afferents had been removed did not show any signs of cystitis, or increase in urine histamine, or change in the number of granulated mast cells. Pretreatment of animals with a mast cell degranulator completely prevented the appearance of cystitis without altering the CNS disease. CONCLUSION: These results provide further evidence that mast cells are involved in neurogenic cystitis induced by changes in CNS activity.

Evaluation of ganciclovir-mediated enhancement of the antitumoral effect in oncolytic, multimutated herpes simplex virus type 1 (G207) therapy of brain tumors.

G207 is a multimutated, conditionally replicating herpes simplex virus type 1 (HSV-1) that retains an intact viral thymidine kinase (HSV-tk) gene. The virus exhibits oncolytic activity in various tumors and is being evaluated in patients with recurrent malignant glioma. In view of the potential for ganciclovir (GCV) to either enhance or inhibit the antitumoral activity of HSV-tk-retaining HSV-1 vectors, we evaluated the effect of GCV administration on the antitumoral activity of G207. In culture, addition of GCV either had no effect or inhibited the cytocidal action of G207 at replication-permissive temperatures, while it significantly increased the cell killing in three of the four cell lines studied when virus replication was inhibited at nonpermissive temperatures. Using a G207-permissive immunocompetent mouse tumor model, subcutaneous N18 neuroblastoma in syngeneic A/J mice, we found that GCV treatment did not affect G207-mediated tumor growth inhibition at a variety of viral doses (10(5), 10(7), and 10(7) x 2 plaque-forming units). In A/J mice harboring intracerebral N18 tumors, GCV administration had no significant effect on the prolongation of survival by G207 inoculation. These findings suggest that GCV administration may not be beneficial to the efficacy of G207 tumor therapy under conditions that favor active viral replication, because the potential HSV-tk/GCV-mediated enhancement of G207 oncolytic activity may be balanced out by the inhibitory action of GCV on viral replication.

Clinical mutations in the L1 neural cell adhesion molecule affect cell-surface expression.

Mutations in the L1 neural cell adhesion molecule, a transmembrane glycoprotein, cause a spectrum of congenital neurological syndromes, ranging from hydrocephalus to mental retardation. Many of these mutations are single amino acid changes that are distributed throughout the various domains of the protein. Defective herpes simplex virus vectors were used to express L1 protein with the clinical missense mutations R184Q and D598N in the Ig2 and Ig6 extracellular domains, respectively, and S1194L in the cytoplasmic domain. All three mutant proteins were expressed at similar levels in infected cells. Neurite outgrowth of cerebellar granule cells was stimulated on astrocytes expressing wild-type or S1194L L1, whereas those expressing R184Q and D598N L1 failed to increase neurite length. Live cell immunofluorescent staining of L1 demonstrated that most defective vector-infected cells did not express R184Q or D598N L1 on their cell surface. This greatly diminished cell-surface expression occurred in astrocytes, neurons, and non-neural cells. In contrast to wild-type or S1194L L1, the R184Q and D598N L1 proteins had altered apparent molecular weights and remained completely endoglycosidase H (endoH)-sensitive, suggesting incomplete post-translational processing. We propose that some missense mutations in human L1 impede correct protein trafficking, with functional consequences independent of protein activity. This provides a rationale for how expressed, full-length proteins with single amino acid changes could cause clinical phenotypes similar in severity to knock-out mutants.