A HSV Gene Transfer for Neuropathy and Pain

In some sense, the most natural target for the therapeutic use of HSV-mediated gene transfer is the PNS. Recombinant rep lication-defective vectors, such as wild-type HSV, target with high efficiency to dorsal root ganglion (DRG) neurons following subcutaneous inoculation, a process that benefits from high-level expression of the HveC HSV receptor on sensory nerve terminals in the skin (160) and specific interactions between capsid and tegument proteins and dynein to mediate retrograde axonal transport along microtubules (161), followed by injection of the DNA through a nuclear pore into the nucleus. We have used HSV-mediated gene transfer to the PNS in rodent models of peripheral neuropathy and in the treatment of pain. The underlying rationale for using HSVmediated gene transfer in these 2 conditions is similar. In both cases, peptides of proven efficacy have been identified. Peripheral neuropathy can be prevented in animal models by treatment with neurotrophic factors, and pain can be substantially ameliorated by the delivery of opioid peptides. However, it is difficult to deliver these short-lived peptide factors in adequate doses to achieve therapeutic effects without causing intolerable side effects that result from the widespread expression of the cognate receptors throughout the nervous system and in nonnervous structures in the body. Targeted gene delivery using HSV vectors can be used to overcome these limitations.

1. Neuropathy

We used subcutaneous inoculation of HSV-based vectors in the foot to deliver and express neurotrophic factors in DRG neurons in order to protect against peripheral nerve degeneration in models of neuropathy. We constructed vectors to express neurotrophin 3 (NT3) under the control of a fusion LAP2 HCMV promoter (vector QL2HNT3) and NGF under the control of the HCMV or LAP2 promoters (vectors SHN and SLN) (111). In the pyridoxine (PDX)-intoxication model of large-fiber sensory neuropathy, rats transduced by subcutaneous inoculation into the foot with QL2HNT3 and then intoxicated with pyridoxine PDX demonstrated a dramatic preservation of sensory nerve function, measured by sensory nerve recording (foot sensory amplitude and velocity), h-wave reflex, and behavioral assessment of proprioceptive function [Figs. 8A, B, and C from (162)]. Morphometric examination of the sciatic nerve demonstrated a substantial and statistically significant loss of predominantly large myelinated fibers in control vector-treated PDX-intoxicated animals, which was prevented by the NT-3 expressing vector QL2HNT3 (162). The NGF-ex-pressing vector SHN provided a similar protective effect in the same model.

Mice rendered diabetic by the injection of streptozotocin develop a pure sensory neuropathy, manifest by reduction in the evoked sensory amplitude. Injection of either vector SHN or vector SLN 2 weeks after the onset of diabetes protected the animals from the development of neuropathy, measured by the foot sensory amplitude at 6 weeks of diabetes (163). This protective effect persists through 6 months of diabetes (Goss and Fink, unpublished data, 2002). In addition, expression of neuropeptide (calcitonin gene-related peptide and substance P) genes in the in the DRG was preserved in the vector-inoculated animals, demonstrating protection of the neuro-

Gastrocnemius Muscle Transfer

Figure 8 Neuroprotective effects of HSV vector-mediated NT-3 expression. (A) Examples of individual M wave and H reflex (arrows) recorded from the gastrocnemius muscle after stimulation at the sciatic notch. Control is naive animal, PDX is an animal treated with pyroxidine, lacZ is treated with PDX and the control lacZ vector QOZHG, and NT-3 is treated with PDX and the NT-3 expression vector. (B) The mean H-wave reflex amplitude from the measurements in A. (C) Behaviorial analysis of propriceptive function. Rats were trained and tested on a 3-cm beam with the number of slips from the beam measured during a 3-minute session. The PDX-treated animals have great difficulty traversing the beam; however, treatment with the NT-3 expression vector prevents this deficit. (From Ref. 162.)

Figure 8 Neuroprotective effects of HSV vector-mediated NT-3 expression. (A) Examples of individual M wave and H reflex (arrows) recorded from the gastrocnemius muscle after stimulation at the sciatic notch. Control is naive animal, PDX is an animal treated with pyroxidine, lacZ is treated with PDX and the control lacZ vector QOZHG, and NT-3 is treated with PDX and the NT-3 expression vector. (B) The mean H-wave reflex amplitude from the measurements in A. (C) Behaviorial analysis of propriceptive function. Rats were trained and tested on a 3-cm beam with the number of slips from the beam measured during a 3-minute session. The PDX-treated animals have great difficulty traversing the beam; however, treatment with the NT-3 expression vector prevents this deficit. (From Ref. 162.)

chemical phenotype (163). We have observed similar protective effects against the development of neuropathy using the same vectors (QL2HNT3, SHN, and SLN) in the rodent model of neuropathy resulting from administration of the chemothera-peutic drug cisplatin (164).

In each model tested, the HSV vector coding for a neuro-trophic factor has been inoculated prior to the onset of neuropathy. We have not examined whether the vectors might be used to enhance recovery from neuropathy, but achieving prevention alone would be a major advance in the treatment of neuropathy. Most sensory neuropathies develop gradually over a period of months to years; patients with diabetes, for example, characteristically present with loss of sensation in their toes, but there is no available therapy that will prevent progression of neuropathy. Gene transfer at that stage, if it prevented the development of a severe sensory neuropathy, would represent a substantial advance (165). Neuropathy caused by chemotherapeutic drugs represents one model in which the onset of neuropathy is temporally defined and pre-treatment is possible. This type of neuropathy may serve as a convenient model for the first human trials of such therapy.

2. Pain

The treatment of pain uses the same approach, subcutaneous inoculation to transduce DRG neurons, to achieve local expression of peptides in sensory neurons. Wilson first constructed a replication-competent HSV-based vector to express proenkephalin (117). Subcutaneous injection of that vector into the dorsum of the foot in mice produced an antihyperalge-sic effect that could be demonstrated by an increased latency to withdraw the foot from noxious heat after sensitization of C fibers by application of capsaicin, or sensitization of A8 fibers by application of dimethyl sulfoxide (117). Although no antinociceptive effect was demonstrated in the absence of prior sensitization using the experimental paradigms reported, substantial expression of methionine-enkephalin could be demonstrated by immunocytochemistry (117). A replication-competent HSV-based vector expressing proenkephalin reduced pain in a rodent model of arthritis (166).

We extended these results by constructing a replication-incompetent HSV vector with the human PE gene (coding for 6 copies of met-enkephalin and 1 copy of leu-enkephalin) in the tk locus of the ICP4~ recombinant d120 (designated vector SHPE). Inoculation of the vector into the foot transduced DRG neurons, and expression of the human proenkephalin gene and RNA was confirmed in DRG by PCR and RT-PCR. Expression of the peptide could be demonstrated in DRG neurons transduced in vitro by immunocytochemistry (167). We examined the biological activity of enkephalin (ENK) expression of SHPE transduction in the formalin model of inflammatory pain. Rats injected with SHPE 1 week prior to formalin testing showed a significant reduction in nocisponsive behaviors during the delayed phase (30-60 min after formalin injection). The antinociceptive effect was reversed by intra-peritoneal naloxone (50 mg/kg i.p.) and by intrathecal administration of naltrexone. The effect of SHPE transduction waned over time. Animals tested 2 weeks after SHPE trans-duction showed a smaller reduction in nocisponsive behavior than those tested 1 week after transduction, and by 4 weeks after transduction there was no longer any significant antinoci-sponsive effect. Reinoculation of SHPE at 4 weeks reestablished the antinociceptive effect, measured 1 week later (5 weeks after the initial inoculation) by formalin test. These results suggest that the loss of effect was not a result of tolerance and demonstrate that prior inoculation with the nonrepli-cating vector does not prevent the reapplication of therapeutic gene transfer with the HSV vector.

To determine whether the effect of vector-mediated ENK release in DH is restricted to the distribution of transduced neurons projecting to DH, we repeated the experiment, but compared the pain response in animals injected with formalin ipsilateral to vector footpad inoculation or contralateral to vector inoculation. There was a significant reduction in nocifen-sive behavior in the delayed phase of the formalin test when formalin was injected ipsilateral to SHPE inoculation 1 week after vector inoculation. There was no reduction in nocifensive behavior observed in animals injected with formalin into foot contralateral to the SHPE inoculation. SHZ injection had no effect on nocifensive behavior observed in either foot. In conjunction with the evidence that intrathecal naltrexone blocks the vector-mediated effect, these results suggest that vector-mediated ENK released from the central terminal of transduced primary afferent neurons to act locally in DH in the distribution of the central projection of those neurons.

We next examined the antinociceptive effect of the PE-expressing vector in the spinal nerve ligation (SNL) model of neuropathic pain, established according to the method described by Kim and Chung (168). Under chloral hydrate anesthesia, the left transverse process of L6 was removed, and the L5 spinal nerve was gently isolated and ligated tightly with 6.0 black silk suture distal to DRG but proximal to the formation of the sciatic nerve. Tactile threshold was determined using calibrated von Frey filaments (169,170). Subcutaneous inoculation of SHPE 1 week after SNL caused a significant reduction in tactile allodynia in the injected foot (171). The antiallodynic effect was maximal 2.5 weeks after vector inoculation and declined over the subsequent 4 weeks. Reinoculation of the vector reestablished the analgesic effect (171), which then persisted for approximately 6 weeks. The antinoci-ceptive effect of SHPE was reversed by intraperitoneal naloxone. These results are consistent with published evidence, suggesting that mechanical allodynia in SNL models of neuropathic pain results from aberrant electrical activity in undamaged fibers from DRG, that project along with degenerating fibers from the ligated sensory root into the same peripheral nerve (172). The time course of the biological effect of transgene expression from this vector, in which the HCMV IEp is used to drive transgene expression suggests that at least 6 weeks of expression may be achieved in transduced DRG neurons.

Vector transduction enhanced the effect of morphine. Un-inoculated animals with neuropathic pain responded to intra-peritoneal morphine with an ED50 of 1.8 mg/kg (CI95 1.1-2.8 mg/kg). Animals inoculated with SHPE 1 week after SNL and tested with morphine 1-2 weeks later demonstrated a reduction in the ED50 of morphine to 0.15 mg/kg (CI95 0.06-0.34 mg/kg, P < 0.05) (171). Inoculation with SHZ had no effect on the response to morphine. In addition, twice daily administration of morphine resulted in a graduate decrease in the antiallodynic effect in the neuropathic rats, with no detectable effect by day 7. Animals that had been inoculated with SHPE 1 week after SNL showed a significantly greater response to morphine at days 1 and 2, consistent with the interaction of SHPE with morphine, and continued to exhibit an antiallodynic effect at day 7, when morphine alone was no longer effective (171).

Pain resulting from cancer metastatic to bone has features of both inflammatory and neuropathic pain. To evaluate the potential therapeutic effect of HSV-mediated gene transfer and expression of PE in pain due to cancer, we tested the vector in the osteogenic sarcoma model in the mouse (173). Tumor-injected mice demonstrated spontaneous pain, increasing to 2 weeks after tumor inoculation and remaining at the same level up to 4 weeks postinoculation. Subcutaneous inoculation of SHPE 1 week after tumor implantation resulted in a substantial and significant reduction in spontaneous behavior recorded 2 and 3 weeks after tumor implantation (1 and 2 weeks after vector inoculation) (174). The analgesic effect of the vector was reversed by intrathecal naltrexone.

Taken together, the results of studies demonstrate that a nonreplicating genomic HSV vector expressing PE is antino-ciceptive in models of neuropathic pain, inflammatory pain, and pain resulting from cancer in rodent models. A proposal for the first human trial employing these vectors in the treatment of intractable pain resulting from cancer metastatic to a vertebral body was presented to the Recombinant DNA Advisory Committee at the National Institutes of Health in June 2002. In the near future, we should be able to determine if this approach will be as successful in treating the human disease as it has been in the animal models.

B. Cancer

Cancer gene therapy may offer a treatment modality to patients who have exhausted all other standard treatment regimens such as surgery, chemotherapy, and radiation therapy. There are a number of considerations in applying gene therapy to the treatment of cancer that include the selection of the appropriate therapeutic gene(s), the specific effect or mechanism, target tissue, and method of gene delivery. The overriding problem is that cancer is generally a systemic disease, and thus, even if gene transfer is effective in destroying a tumor locally, metastases will promote continued disease.

Strategies to treat cancer by gene therapy can be considered in 3 categories: (1) tumor cell destruction using conditionally replicating viruses that selectively replicate in and kill tumor cells (175,176) compared with the surrounding normal tissue, (2) tumor cell destruction by expression of transgenes whose products induce cell death, or sensitize the cells to chemo-(177) or radiation therapy (178), and (3) tumor vaccination through expression of transgenes whose products recruit, activate, or costimulate immunity or provide tumor antigens. The latter approach is more likely to be effective in treating meta-static disease. Because these strategies are complementary, it has also been suggested that they can be used in combination. Examples of these various approaches include the use of (1) prodrug-activating genes such as thymidine kinase (TK) or cytosine deaminase (179); (2) cytokines such as TNF-a, 7IFN and various interleukins (180); (3) MHC products such as costimulatory molecules (B7.1) (181-183); (4) allotypic class I or class II molecules (181-184); and (5) tumor antigens (185,186), which together may assist in the recruitment and activation of nonspecific inflammatory responses (187) or the induction of tumor-specific immunity.

The first strategy involves the use of vectors that are replication competent, but depend on attributes unique to the tumor cell to support viral growth. For example, E1b-deficient adenoviral vectors can replicate in tumor cells mutant for p53 but generally not in normal cells (175,188-190). Thus, the intent of this strategy is to provide a mechanism for virus spread locally in the tumor to increase the number of infected tumor cells. However, this treatment is limited to p53-defec-tive tumors. Similarly, HSV vectors have been engineered that replicate in dividing cells, such as tumor cells, but not in normal neurons. The use of conditional replication-competent viruses could in theory allow for spread in tumor tissue without damaging normal brain, thereby increasing the specificity and effectiveness compared with nonreplicating vectors that express transgenes that augment tumor cell killing. HSV vectors of this type include mutants lacking the 34.5 gene, which is required for growth specifically in neurons (191 -193). Deletion of this gene alone (1716), or in combination with the UL39 ribonucleotide reductase (RR) large subunit (G207), creates viruses that are highly compromised for their ability to replicate in and kill neuronal cells, yet that retain the ability to replicate in and kill dividing tumor cells.

Although these vectors were originally used to treat animal models of malignant glioma (194-198), they have now been employed to treat breast (199,200), lung (201,202), head and neck (203), melanoma (204,205), colorectal (206-209), prostate (210-213), ovarian (214,215), peritoneal (216-218), bladder (211,219), renal (220), cervical (221), and gallbladder (222) tumors in various animal models, demonstrating their utility. Moreover, in addition to their application for direct tumor cell killing, they have also been employed in tumor vaccination models (201,206,209,223,224). They have also been employed to augment the host immune response to the tumor by expressing either cytokines such as IL-12 (225) or immunomodulatory molecules such as B7-1 (226). In addition, they have also been used in conjunction with suicide gene therapy (227), low-dose ionizing radiotherapy (208,221), and chemotherapeutic agents like cisplatinum (202,228).

The safety and biodistribution of these vectors following intracranial administration has been examined in both rodents and nonhuman primates. The G207 vector deleted for 34.5 and UL39 (RR) displayed a very good safety profile (229-232) in both mice and nonhuman primates (rhesus macaques and Aotus nancymae), and vector remained locally surrounding the injection site. In addition, injection of vector was unable to reactivate endogenous resident wild-type HSV (230,233), a major issue concerning the use of these vectors in a human population that may already possess latent virus, again demonstrating the safety of HSV as a therapeutic vector. Safety studies using the 1716 vector have shown that deletion of 34.5 alone does not necessarily render the vector nontoxic. Vector injection into the ventricles of immune-deficient nude mice resulted in animal fatalities (234), or persistent ependymal cell loss in immune-competent mice (235), with virus spread to distal sites being observed in immune- competent rats (236). Another major question that needs to be addressed with the use of HSV vectors in a human population that is 60% to

90% seropositive for the virus, concerns whether preexisting immunity to the virus will prevent effective therapeutic treatment following vector administration. Although prior immunized animals displayed both humoral and cell-mediated responses to the virus, these responses did not significantly alter the ability of the vector to kill tumor cells and increase animal survival in preimmune vs. naive animals (218,228,237).

Based on these preclinical studies, two phase I clinical trials have now been reported using conditionally replicating HSV vectors. These studies have demonstrated the feasibility of inoculating recombinant HSV viruses into glioblastomas. No evidence of viral encephalitis or reactivation of wild-type virus was seen in either study. Furthermore, no adverse events could be unequivocally attributed to the vectors. One study, which used the G207 virus deleted for the 34.5 neurovirulence gene and the UL39 ribonucleotide reductase gene, showed safety up to a dose of 3 X 109 pfu (176). A second study using a 34.5 mutant with a complementing mutation in the US11 gene, which restores the growth characteristics of wildtype virus without reversing the attenuated neurovirulence phenotype of the 34.5 mutant, showed safety to a final dose of 105 pfu (238). A total of 30 patients were treated in the 2 trials. These trials were not designed to test whether the vectors were efficacious in the treatment of glioblastoma (GBM); phase II efficacy studies are ongoing.

The second approach to treat cancer involves suicide gene therapy (SGT) for the treatment of cancer in experimental animals and in phase I human clinical protocols (177,239-243). This strategy uses the bystander destruction of tumor cells mediated by a variety of mechanisms other than virus spread, including the recruitment of natural killer (NK) cells by expressing the appropriate cytokines, the activation of anticancer drugs at the tumor site that kill multiple tumor cells in addition to those transduced by the vector, and the use of antigens and cytokine-expressing genes to elicit specific antitumor immunity. Transfer of the HSV gene TK into tumor cells results in tumor cell death when combined with the antiviral drug ganciclovir (GCV). TK has been shown to convert the prodrug into a toxic nucleoside analog that, upon incorporation into nascent DNA, results in the interruption of DNA replication by chain termination. A uniquely powerful characteristic of the TK-GCV approach is that only a small fraction of the tumor cells need to be transduced with the suicide gene to result in significant antitumor activity, an activity known as the ''bystander effect'' (239,240,243-245). It has been demonstrated that cell-to-cell transfer of activated GCV via gap junctions between transduced tumor cells and untrans-duced neighboring cells is a major mechanism of the bystander effect (246-249). A variation on this strategy is to introduce cell lines into the tumor site that produce TK-expressing re-troviruses that then infect tumor cells locally. Although setting up virus production factories in this manner is logical, the practice of this strategy has been disappointing because the xenogeneic producer cell lines induce rapid inflammatory processes that lead to brain swelling and little detectable gene transfer (250). Although these TK-GCV SGT strategies suffer from limitations, approaches of this nature are under evalua tion for efficacy in phase I—II clinical trials for patients with brain tumors; however, they have met with limited success.

In our initial experiments using replication-defective HSV vectors expressing HSV-TK for SGT, we employed both vectors deleted for single (ICP4 " ) and multiple (ICP4 "/ICP27 "/ ICP22~) essential IE genes that expressed the TK gene from an HSV IE promoter to ensure its expression from the replication-defective virus backbone (Fig. 9A). We tested the ability of these TK overexpressing replication-defective HSV vectors to act as a treatment for established tumors in rodent glioma models and demonstrated significant increases in survival following administration of the HSV-TK vector and GCV (114,251). However, the magnitude of the bystander effect was inversely proportional to the overall toxicity of the vector. Thus, more cytotoxic vectors like the ICP4 deletion mutant resulted in the cytotoxic death of the transduced tumor cells before they were able to produce and release significant levels of modified GCV, thereby dampening the overall bystander-mediated killing (251). We have now seen similar results using vectors that express CD alone or in combination with HSV-TK (252), suggesting that further modifications will be required to achieve more effective tumor cell killing.

To augment the cell killing seen in SGT we have taken two approaches. In the first approach, we attempted to augment the bystander effect by altering the makeup of tumor-cell gap junction complexes. Connexins are the components of gap junctions (253) that play a major role in intercellular communication to control homeostasis and cell proliferation. Reduced intercellular communication through gap junctions has been regularly observed in transformed cells (254-256) and may be due to reduced connexin expression. Retrovirus-mediated introduction of the connexin 43 (Cx43) gene limited the growth of transformed cells that shared the characteristic of reduced gap junctional activity (257), and other studies have reported similar findings supporting the suggestion that con-nexins alone have tumor-suppressor activity (258-261). Gap junctions also play a critical role in the HSV-TK/GCV bystander effect by enabling the transfer of activated GCV from TK-positive to neighboring TK-negative cells (246,262-265). Tumor cells having reduced gap junction formation are less susceptible to the bystander effect (265), suggesting that transfer of connexin genes into these cells to restore or augment intercellular communication would improve the effectiveness of HSV-TK/GCV therapy. Indeed, it has been demonstrated both in vitro and in vivo that the bystander effect can be potentiated by the expression of connexin (264,266,267). The bystander effect requires connexin expression not only by the TK-positive GCV-activating cell, but also by the TK-negative recipient cell (265). This suggests that gene therapy approaches aimed at codelivery of connexin with HSV-TK to mediate an enhanced bystander effect may not be extremely effective against tumors that express no connexin at all. However, it may from a study of 17 cell lines measuring bystander effects and gap junctional activity (263); this will be an extremely small group. Furthermore, overexpression of con-nexin in the transduced cells will increase transfer of activated

Tranfers Diagram Therapy

Figure 9 HSV-vector mediated tumor cell killing. Diagrams of replication-defective HSV-1 expression vectors for expressing (A) HSV-TK (SGT), (B) HSV-TK and connexin 43 (SGT/ Cx43), (C) HSV-TK and TNF-a (SGT/TNF), and (D) HSV-TK, connexin 43, and TNF-a (SGT/TNF/Cx43). All vectors express ICP0 from both copies of the inverted repeat element flanking the unique long segment (UL) of the HSV genome, and TK as an IE gene from a copy of the ICP4 IE promoter replacing the native TK promoter in the UL23 gene locus. All vectors have the ICP4 and ICP27 genes deleted, and all but the SGT/TNF/Cx43 vector have inactivating deletions in ICP22. This vector has the IE genes ICP22 and ICP47 turned into early genes by mutation of the TAATGARAT sequences in these promoters converting these to early (P) genes. The SGT/ Cx43 vector contains the Cx43 gene driven by the ICP0 IE promoter inserted into the UL41 locus. The SGT/TNF vector has TNF inserted into the ICP22 gene locus under control of the HCMV IE promoter. The SGT/TNF/Cx43 vector has both the ICP0p-Cx43 and HCMV IEp-TNF expression cassettes inserted into the UL41 locus. The ability of these vectors to destroy tumors and effect animal survival in the immunocompe-tent rat 9L tumor model was evaluated by intratumoral vector injection 3 days following implantation of the 9L tumor cells into the frontal lobe of Fisher rats. Animals received i.p. injections of GCV at the time of vector injections for 10 consecutive days and gamma-knife radiosurgery (GKR) at 2 days post vector injection.

GCV, providing even limited connexin expression in neighboring cells.

We tested the potential benefit of coexpressing connexin with HSV-TK for the treatment of glioblastoma (268). Human U-87MG tumor cells express detectable amounts of Cx43 and are sensitive to bystander killing in vitro, but this effect is not sufficient to control tumor formation with TK-transduced cells and GCV treatment alone (254). Replication-defective vectors (ICP4 "/ICP27 "/ICP22 " ) that we engineered to express Cx43 (Fig. 9B) were able to enhance TK-GCV tumor cell killing in cultures of U-87MG tumor cells that express Cx43 (268). Thus, although U-87MG cells already showed a good bystander effect, the expression of additional Cx43 in a fraction of the population further enhanced the effectiveness of GCV treatment. We also were able to demonstrate a pronounced bystander effect with L929 cells (268) that did not show a bystander effect when infected with a TK-expressing vector, in agreement with recent findings by others (265). This indicated that vector-directed connexin expression enabled bystander killing among these otherwise bystander-resistant cells. Together, these results suggested that vector-directed Cx43 expression should be beneficial regardless of whether the target cells express significant levels of connexin.

These in vitro assays were extended to animals using an ex vivo flank tumor model and in vivo using animals bearing U-87MG tumors in the CNS (268). Dramatically, all animals implanted with Cx43 vector-infected cells and treated with GCV in were tumor free 1 week after cessation of GCV administration, whereas no other animal was tumor free at this time, including animals treated with Cx43 vector alone or the TK-expressing vector plus GCV. Moreover, all animals in this treatment group were alive at the end of the observation period (72d), whereas no animals in any of the other groups survived past day 41. Encouraged by these promising results, experiments were initiated to test the effectiveness of combined Cx43/tk gene delivery in vivo because the ex vivo approach enables one to infect every tumor cell that does not readily mimic the actual situation in human patients with glioblas-toma. In the in vivo experiments, virus was injected directly into the tumor mass 3 days following tumor cell implantation and GCV was administered for 10 days after vector inoculation. In these studies, all animals in every treatment group died by day 50, whereas 50% of the Cx43-expression vector-treated animals survived past 50 days and one-third were still alive at the end of the study (70d), indicating a beneficial effect of connexin/tk gene codelivery in vivo.

In the second approach to augment SGT, we created vectors that express cytokines in the hope of stimulating a host response to the tumor. There has been a considerable amount of recent interest in using cytokine genes, costimulatory molecules, tumor antigens, and recruitment molecules to enhance the immune response to the tumor. Antitumor immunity should prove effective in treatment of metastatic cancer. The development of antitumor immunity could circumvent the need for replication-competent vectors because tumor-specific cytotoxic T lymphocytes constantly move through the brain parenchyma searching for target cells. A growing body of literature suggests that local expression of cytokines can enhance CTL activation at least in animal model systems and these bear testing in human brain cancer. HSV offers the potential for combinational gene therapy in this regard because multiple immunomodulatory genes can be recombined into the virus and comparatively tested (47).

Tumor necrosis factor-a (TNF-a) has been demonstrated to possess an array of antitumor activities, including potent cytotoxicity exerted directly on tumor cells (269), enhancement of the expression of HLA antigens (270) and ICAM-1 (271) on tumor cell surfaces, enhancement of interleukin-2 receptors on lymphocytes (272), and stimulation of such effector cells as NK cells, lymphokine-activated killer cells, and cytotoxic T lymphocytes (CTL) (272-276). However, despite this promising antitumor profile, the clinical use of TNF-a has been constrained by the toxicity of systemic TNF-a delivery (275,276). This problem could be minimized by local production of TNF-a at the site of tumor growth, which may allow for effective use of this cytokine as an antitumor agent. Furthermore, TNF-a has a radiosensitizing ability that could optimize its antitumor effects. In an effort to augment the effectiveness of HSV-TK-mediated suicide gene therapy, we created the replication-defective, triple IE gene-deleted HSV vector (ICP4-, ICP27", ICP22") that expresses HSV-TK and TNF-a (Fig. 9C).

In vitro studies demonstrated that high levels of TNF-a could be detected in the media of vector-infected cells during the first 24-h period, but this was followed by a precipitous decline in production on day 2 and subsequent days with the protein being no longer detectable on day 7 (114). The bioac-tivity of TNF-a produced in this experiment was tested by exposure of cultured TNF-a-sensitive L929 fibrosarcoma cells to medium collected after the first day of infection. A dramatic reduction in cell viability was observed for cells treated with medium from TNF vector-infected cells, and this reduction was essentially identical over time to that seen with unconditioned medium supplemented with 10 ng recombinant TNF-a protein (114). These results demonstrated that TNF-a produced by the TNF vector was biologically active and comparable in specific activity to recombinant TNF-a. We then determined that intracellular production of TNF-a could enhance HSV-TK/GCV-mediated cell killing of both the TNF-a-sensitive L929 cell line, as well as the TNF-a-resistant U-87MG cell line. Although the mechanism is unclear, this enhancement indicated that the combination of vector-directed TNF-a expression and HSV-TK expression with GCV treatment could be beneficial not only against TNF-a-sensitive tumor cells such as L929, but also against TNF-a-resistant tumors.

To determine if the increased effectiveness of combination gene treatment evident in vitro could also be observed in vivo, we first tested the effect of intratumoral vector injection followed by GCV treatment on established L929 tumors (TNF-a sensitive) in the flanks of immune-competent mice (114). Tumor treatment with the TNF vector plus GCV resulted in significantly greater growth inhibition and extended animal survival compared with all other treatments. These results demonstrated the promise of combination TNF/TK gene therapy for the treatment of TNF-sensitive tumors and added an incentive to test the same treatment against TNF-resistant tumors. However, the results in the U-87MG TNF-a-resistant intracerebral tumor model in immunodeficient mice were not as significant as those observed with the L929 model, although 2 of 14 animals in the TNF/GCV treatment group survived past 80 days (114). This reduced response in the U-87MG model may be due to the fact that the tumor cell killing mediated by TNF is only the result of the cytotoxic effects of TNF expression intracellularly and is not augmented by its ability immunomodulatory ability. Together, these results suggest that combined HSV-TK/TNF therapy is beneficial, but that a more effective strategy may be required.

Fractionated radiotherapy has been shown to confer a small but significant survival benefit to patients with glioblastoma. Unfortunately, the dose of radiotherapy that may be tolerated by the brain (about 60 Gy) is inadequate for tumor eradication. To circumvent inherent toxicity problems, techniques have been developed that allow focusing of radiation to the tumor bed, allowing a higher dose to be delivered (radiosurgery). This enables eradication of the central portion of the tumor, but does not allow delivery of an augmented radiation dose to the tumor periphery. Gamma-knife radiosurgery (GKR) allows for precise delivery of a single high dose of radiation to brain tumors without opening the skull. In this technique, tumors are targeted by the application of a tightly focused high-energy radiation field, which results in minimal collateral damage to the surrounding normal tissue. Radiosurgery has been used for boost irradiation of patients with malignant glial tumors, in addition to conventional wide-margin fractionated radiotherapy (277,278). Unfortunately, glioma cells are often seen invading the normal tissue surrounding the tumor, often migrating along normal white matter tracts (279,280). This feature of glioma is largely responsible for the inability to effect a surgical cure by resection and the correspondingly poor prognosis. We have therefore examined ways in which the response to radiotherapy may be enhanced by gene delivery. One such approach involves selectively sensitizing tumor cells to radiotherapy. This would confer a major advantage, in that the sensitized cells could be effectively killed by a low dose of radiotherapy that is not toxic to surrounding brain tissue. In this strategy, we combine GKR with the injection of replication-defective HSV vectors expressing HSV-TK, Cx43, and TNF.

In the first series of experiments, we used GKR in conjunction with vector-mediated TNF expression because TNF has previously been shown to have a synergistic effect with ionizing radiation when delivered as a recombinant protein (281-287), by plasmid-based delivery (288-292) or by using an adenoviral vector (293-297). Moreover, the TNF-a approach has proven safe and shown some efficacy in human phase I clinical trials (297,298). Experiments were carried out to determine whether HSV-TK/TNF gene transfer along with GCV treatment was more effective in the presence of low-dose gamma-knife radiation. Both the TNF/TK vector and gamma-knife radiation alone were effective in protecting a proportion of animals from tumor growth and animal death when used in the U-87MG model of glioblastoma in nude mice (299). The results using the TNF/TK vector with SGT and GKR demonstrated that the combination of TNF, GCV, and GKR was superior to other treatments, such that 89% of the animals in that treatment group surviving for the length of the study (75d) and 67% of the animals were found to be tumor free at 75 days (299).

We then extended these results to an immunocompetent tumor model that may more closely mimic the human disease by carrying out survival studies comparing the efficiency of HSV-TK, TNF, and Cx43 in combination with GCV, with and without radiosurgery using the 9L gliosarcoma model in immunocompetent Fisher 344 rats (Niranjan and Glorioso, unpublished data, 2002). In this study, GKR was found to enhance the survival of 9L intracranial tumor-bearing rats compared with the control (Fig. 9E); however, treatment resulted in an overall survival of 15%, similar to what has been reported in human patients (277,300,301). Combining the TNF and connexin-43 genes in a HSV-TK/ICP0 vector (Fig. 9D) further improved animal survival (Fig. 9E). Eleven of 15 animals treated with the HSV-TK/TNF/Cx43 vector, GCV, GKR survived for more than 150 days compared with 7 of 15 treated with TNF, GCV, and GKR. These results demonstrate that our most effective current strategy for the treatment of animal brain tumors involves the multigene vector, which simultaneously expresses TNF-a Cx43, HSV-TK, and HSV-ICP0, combined with radiosurgery and GCV treatment. This combination approach employs genes whose products that are tumorcidal (HSV-TK, TNF), augment this process by increasing the bystander effect (Cx43), sensitize tumor cells to radiation (TNF), and stimulate the host immune response to the tumor (TNF). This combination approach may also prove to be effective against metastatic disease, but remains to be tested in these animal models.

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