A Novel Oncolytic Herpes Simplex Virus Design based on the Common Overexpression of microRNA-21 in Tumors.

BACKGROUND: Recognition sequences for microRNAs (miRs) that are down-regulated in tumor cells have recently been used to render lytic viruses tumor-specific. Since different tumor types down-regulate different miRs, this strategy requires virus customization to the target tumor. We have explored a feature that is shared by many tumor types, the up-regulation of miR-21, as a means to generate an oncolytic herpes simplex virus (HSV) that is applicable to a broad range of cancers. METHODS: We assembled an expression construct for a dominant-negative (dn) form of the essential HSV replication factor UL9 and inserted tandem copies of the miR-21 recognition sequence (T21) in the 3' untranslated region. Bacterial Artificial Chromosome (BAC) recombineering was used to introduce the dnUL9 construct with or without T21 into the HSV genome. Virus was produced by transfection and replication was assessed in different tumor and control cell lines. RESULTS: Virus production was conditional on the presence of the T21 sequence. The dnUL9-T21 virus replicated efficiently in tumor cell lines, less efficiently in cells that contained reduced miR-21 activity, and not at all in the absence of miR-21. CONCLUSION: miR-21-sensitive expression of a dominant-negative inhibitor of HSV replication allows preferential destruction of tumor cells in vitro. This observation provides a basis for further development of a widely applicable oncolytic HSV.

Effects of herpes simplex virus vectors encoding poreless TRPV1 or protein phosphatase 1α in a rat cystitis model induced by hydrogen peroxide.

Enhanced afferent excitability is considered to be an important pathophysiological basis of interstitial cystitis/bladder pain syndrome (IC/BPS). In addition, transient receptor potential vanilloid-1 (TRPV1) receptors are known to be involved in afferent sensitization. Animals with hydrogen peroxide (HP)-induced cystitis have been used as a model exhibiting pathologic characteristics of chronic inflammatory condition of the bladder. This study investigated the effect of gene therapy with replication-defective herpes simplex virus (HSV) vectors encoding poreless TRPV1 (PL) or protein phosphatase 1 α (PP1α), a negative regulator of TRPV1, using a HP-induced rat model of cystitis. HSV vectors encoding green fluorescent protein, PL or PP1α were inoculated into the bladder wall of female rats. After 1 week, 1% HP or normal saline was administered into the bladder, and the evaluations were performed 2 weeks after viral inoculation. In HP-induced cystitis rats, gene delivery of PL or PP1α decreased pain behavior as well as a reduction in the intercontraction interval. Also, both treatments reduced nerve growth factor expression in the bladder mucosa, reduced bladder inflammation characterized by infiltration of inflammatory cells and increased bladder weight. Taken together, HSV-mediated gene therapy targeting TRPV1 receptors could be effective for the treatment of IC/BPS.

Development of an oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-to-cell spread.

Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers.

Neuroblastomas vary widely in their sensitivities to herpes simplex virotherapy unrelated to virus receptors and susceptibility.

Although most high-risk neuroblastomas are responsive to chemotherapy, relapse is common and long-term survival is < 40%, underscoring the need for more effective treatments. We evaluated the responsiveness of 12 neuroblastoma cell lines to the Δγ134.5 attenuated oncolytic herpes simplex virus (oHSV), Seprehvir (HSV1716), which is currently used in pediatric phase I trials. We found that entry of Seprehvir in neuroblastoma cells is independent of the expression of nectin-1 and the sum of all four known major HSV entry receptors. We observed varying levels of sensitivity and permissivity to Seprehvir, suggesting that the cellular anti-viral response, not virus entry, is the key determinant of efficacy with this virus. In vivo, we found significant anti-tumor efficacy following Seprehvir treatment, which ranged from 6/10 complete responses in the CHP-134 model to a mild prolonged median survival in the SK-N-AS model. Taken together, these data suggest that anti-tumor efficacy cannot be solely predicted based on in vitro response. Whether or not this discordance holds true for other viruses or tumor types is unknown. Our results also suggest that profiling the expression of known viral entry receptors on neuroblastoma cells may not be entirely predictive of their susceptibility to Seprehvir therapy.

Relief of pain induced by varicella-zoster virus in a rat model of post-herpetic neuralgia using a herpes simplex virus vector expressing enkephalin.

Acute and chronic pain (post-herpetic neuralgia or PHN) are encountered in patients with herpes zoster that is caused by reactivation of varicella-zoster virus (VZV) from a state of neuronal latency. PHN is often refractory to current treatments, and additional strategies for pain relief are needed. Here we exploited a rat footpad model of PHN to show that herpes simplex virus (HSV) vector-mediated gene delivery of human preproenkephalin (vHPPE) effectively reduced chronic VZV-induced nocifensive indicators of pain. VZV inoculated at the footpad induced prolonged mechanical allodynia and thermal hyperalgesia that did not develop in controls or with ultraviolet light-inactivated VZV. Subsequent footpad administration of vHPPE relieved VZV-induced pain behaviors in a dose-dependent manner for extended periods, and prophylactic vector administration prevented VZV-induced pain from developing. Short-term pain relief following low-dose vHPPE administration could be effectively prolonged by vector re-administration. HPPE transcripts were increased three- to fivefold in ipsilateral ganglia, but not in the contralateral dorsal root ganglia. VZV hypersensitivity and its relief by vHPPE were not affected by peripheral delivery of opioid receptor agonist or antagonist, suggesting that the efficacy was mediated at the ganglion and/or spinal cord level. These results support further development of ganglionic expression of enkephalin as a novel treatment for the pain associated with Zoster.

Effect of herpes simplex virus vector-mediated interleukin-4 gene therapy on bladder overactivity and nociception.

We investigated the effects of replication-defective herpes simplex virus (HSV) vector expression of interleukin-4 (IL-4) on bladder overactivity and nociception. HSV vector expressing murine interleukin-4 (S4IL4) or the control vector expressing ß-galactosidase (SHZ) were injected to the rat bladder wall. At 1 week after viral injection, in cystometry performed under urethane anesthesia, the S4IL4-treated group did not show the intercontraction intervals reduction during intravesical administration of 10 nM resiniferatoxin (RTx). At 2 weeks after viral injection, behavioral studies were performed on vector-injected animals in an awakened state. Freezing behavior induced by 3 µM RTx, administered for 1 min into the bladder, was significantly suppressed in the S4IL4 group compared with the SHZ group. Murine IL-4 levels examined by ELISA were significantly increased in bladder and bladder afferent dorsal root ganglia at 2 weeks after viral injection. The expression of IL-1ß and IL-2 and bladder inflammatory responses were significantly suppressed in the RTx-irritated bladder of S4IL4-injected rats. These results indicate that HSV vector-mediated interleukin-4 expression in the bladder and bladder afferent pathways reduces the inflammatory response, bladder overactivity and nociceptive behavior induced by bladder irritation in the rat model. Therefore, IL-4 gene therapy could be a new strategy for treating urinary frequency and/or bladder pain.

Expression of HSV-1 receptors in EBV-associated lymphoproliferative disease determines susceptibility to oncolytic HSV.

Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease (LPD) after hematopoietic stem cell or solid organ transplantation remains a life-threatening complication. Expression of the virus-encoded gene product, EBER, has been shown to prevent apoptosis via blockade of PKR activation. As PKR is a major cellular defense against Herpes simplex virus (HSV), and oncolytic HSV-1 (oHSV) mutants have shown promising antitumor efficacy in preclinical models, we sought to determine whether EBV-LPD cells are susceptible to infection by oHSVs. We tested three primary EBV-infected lymphocyte cell cultures from neuroblastoma (NB) patients as models of naturally acquired EBV-LPD. NB12 was the most susceptible, NB122R was intermediate and NB88R2 was essentially resistant. Despite EBER expression, PKR was activated by oHSV infection. Susceptibility to oHSV correlated with the expression of the HSV receptor, nectin-1. The resistance of NB88R2 was reversed by exogenous nectin-1 expression, whereas downregulation of nectin-1 on NB12 decreased viral entry. Xenografts derived from the EBV-LPDs exhibited only mild (NB12) or no (NB88R2) response to oHSV injection, compared with a NB cell line that showed a significant response. We conclude that EBV-LPDs are relatively resistant to oHSV virotherapy, in some cases, due to low virus receptor expression but also due to intact antiviral PKR signaling.

Ectopic matrix metalloproteinase-9 expression in human brain tumor cells enhances oncolytic HSV vector infection.

Oncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.

Herpes simplex virus vector-mediated gene delivery for the treatment of lower urinary tract pain.

Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a painful debilitating chronic visceral pain disorder of unknown etiology that affects an estimated 1 million people in the United States alone. It is characterized by inflammation of the bladder that results in chronic pelvic pain associated with bladder symptoms of urinary frequency and urgency. Regardless of the etiology, IC/PBS involves either increased and/or abnormal activity in afferent nociceptive sensory neurons. Pain-related symptoms in patients with IC/PBS are often very difficult to treat. Both medical and surgical therapies have had limited clinical utility in this debilitating disease and numerous drug treatments, such as heparin, dimethylsulfoxide and amitriptyline, have proven to be palliative at best, and in some IC/PBS patients provide no relief whatsoever. Although opiate narcotics have been employed to help alleviate IC/PBS pain, this strategy is fraught with problems as systemic narcotic administration causes multiple unwanted side effects including mental status change and constipation. Moreover, chronic systemic narcotic use leads to dependency and need for dose escalation due to tolerance; therefore, new therapies are desperately needed to treat refractory IC/PBS. This has led our group to develop a gene therapy strategy that could potentially alleviate chronic pelvic pain using the herpes simplex virus-directed delivery of analgesic proteins to the bladder.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord-injured rats.

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 kDa form of the glutamic acid decarboxylase (GAD(67)) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in rats with spinal cord injury (SCI). One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. Rats with SCI without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40-45% and 38-40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD(67) mRNA and protein levels were significantly increased in the L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group, while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD(67)-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO after SCI predominantly through the activation of spinal GABA(A) receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for treatment of neurogenic DO.

Myocarditis following adeno-associated viral gene expression of human soluble TNF receptor (TNFRII-Fc) in baboon hearts.

Sequestration of tumor necrosis factor-alpha (TNFalpha) by TNF-receptor immunoglobulin G (IgG)-Fc fusion proteins can limit heart failure progression in rodent models. In this study we directly injected an adeno-associated viruses (AAV)-2 construct encoding a human TNF receptor II IgG-Fc fusion protein (AAV-TNFRII-Fc) into healthy baboon hearts and assessed virally encoded gene expression and clinical response. Adult baboons received direct cardiac injections of AAV-TNFRII-Fc ( approximately 5 x 10(12) viral/genomes/baboon) or an equivalent dose of AAV-2 empty capsids, and were analyzed after 5 or 12 weeks. Viral genomes were restricted to the myocardium, and routine analyses (blood cell counts, clinical chemistries) remained unremarkable. Echocardiograms were unchanged but electrocardiograms revealed marked ST- and T-wave changes consistent with myocarditis only in baboons receiving AAV-TNFRII-Fc. TNFRII serum levels peaked at approximately 3 times the baseline levels at 1-2 weeks postinjection and subsequently declined to baseline levels. TNFRII-Fc protein and transcripts were detected in the heart at harvest. After AAV injection, anti-AAV-2 antibody levels increased in all baboons, while anti-TNFRII-Fc could not be detected. Baboons that received AAV-TNFRII-Fc developed myocardial infiltrates including CD8+ cells. Thus, a cellular immune response to cardiac delivery of AAV encoding foreign proteins may be an important consideration for AAV-based cardiac gene therapy.

Herpes simplex virus vector-mediated delivery of glial cell line-derived neurotrophic factor rescues erectile dysfunction following cavernous nerve injury.

Erectile dysfunction (ED) is frequently associated with injury to the cavernous nerve sustained during pelvic surgery. Functional recovery from cavernous nerve injury is generally incomplete and occurs over an extended time frame. We employed a therapeutic gene transfer approach with herpes simplex virus (HSV) vector expressing glial cell line-derived neurotrophic factor (GDNF). Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of purified vector stock was administered directly to and around the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein (GFP) and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before killing to assess nerve survival. Approximately 60% of major pelvic ganglion (MPG) cells were GFP positive after viral administration. At 4 weeks after nerve injury, rats treated with HSV-GDNF exhibited significant recovery of ICP/AP compared with control vector or untreated groups. The HSV-GDNF group also yielded more FG-positive MPG cells than the control vector group. HSV vector-mediated delivery of GDNF presents a viable approach for the treatment of ED following cavernous nerve injury.

Gene transfer to interfere with TNFalpha signaling in neuropathic pain.

We examined the role of spinal tumor necrosis factor-alpha (TNFalpha) in neuropathic pain of peripheral nerve origin. Two weeks after selective L5 spinal nerve ligation (SNL), rats exhibiting mechanical allodynia and thermal hyperalgesia showed a marked increase in full-length membrane-associated TNFalpha (mTNFalpha) in the dorsal horn of spinal cord, in the absence of detectable soluble TNFalpha peptide. Local release of the soluble p55 TNF receptor, achieved by herpes simplex virus vector-based gene transfer to dorsal root ganglion, resulted in a reduction of mTNFalpha and concomitant reductions in interleukin-1beta and phosphorylated p38 MAP kinase. Subcutaneous inoculation of soluble p55 TNF receptor expressing HSV vector into the plantar surface of the hind foot ipsilateral to the ligation 1 week before SNL delayed the development of both mechanical allodynia and thermal hyperalgesia; subcutaneous inoculation into the hind foot ipsilateral to the ligation 1 week after SNL resulted in a statistically significant reduction in mechanical allodynia and thermal hyperalgesia that was apparent 1 week after inoculation. These results suggest a novel 'reverse signaling' through glial mTNFalpha, which may be exploited to downregulate the neuroimmune reaction in spinal cord to reduce chronic neuropathic pain.

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-beta peptide in vivo.

Accumulation of insoluble aggregates of amyloid-beta peptide (Abeta), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer's disease (AD). Consequently, downregulation of APP, or enhanced clearance of Abeta, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Abeta accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Abeta (HSV-neprilysin), substantially inhibited accumulation of Abeta. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APP(Sw)), resulted in rapid Abeta accumulation. Co-inoculation of LV-APP(Sw) with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Abeta accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Abeta accumulation in the brain, thus providing useful tools to clarify the role of Abeta in AD that may facilitate the development of novel therapies for this important disease.

I kappa B-mediated apoptotic gene therapy against acute myelogenous leukemia using replication-defective HSV-1 vector expressing TK and mutant I kappa B alpha.

Overexpression of NF-kappa B reportedly plays anti-apoptotic roles in the growth of AML cells. Control of AML cell growth was attempted using a replication-defective herpes simplex virus-1 vector, T0I kappa B alpha, overexpressing mutant I kappa B alpha to inhibit NF-kappa B in vitro. T0I kappa B alpha displays defective ICP4/ICP22/ICP27, isogenic thymidine kinase, and mutant I kappa B alpha. T0Z.1 expressing lacZ instead of I kappa B was used for controls. Infection of T0I kappa B alpha at 15 multiplicity of infection (MOI) with cells of AML lines, HL60, K562, and NB4 displaying >90% infection efficiency and tumor killing in vitro. Use of 10 microM of Ara-C alone was clinically equivalent to high-dose Ara-C, displaying 11% tumor killing. Neither ganciclovir (GCV) nor Ara-C enhanced T0I kappa B- alpha mediated tumor killing. Attenuation of NF-kappa B by T0I kappa B alpha was confirmed by EMSA. T0I kappa B alpha induced caspase-3 activity, with subsequent apoptosis confirmed by colorimetric and TUNEL assays. Fresh AML cells from 8 patients were infected with T0I kappa B alpha at 3 MOI, with or without GCV or 10 microM of Ara-C in vitro. Infection efficiency was 10%. T0I kappa B alpha displayed 8-15% tumor killing, superior to Ara-C in 6 of the 8 patients. Administration of Ara-C enhanced tumor killing in 5 of these 6 cases. Our results suggest that T0I kappa B alpha-mediated gene therapy induces apoptosis of AML cells in vitro.

HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy.

We examined the utility of herpes simplex virus (HSV) vector-mediated gene transfer of vascular endothelial growth factor (VEGF) in a mouse model of diabetic neuropathy. A replication-incompetent HSV vector with VEGF under the control of the HSV ICP0 promoter (vector T0VEGF) was constructed. T0VEGF expressed and released VEGF from primary dorsal root ganglion (DRG) neurons in vitro, and following subcutaneous inoculation in the foot, expressed VEGF in DRG and nerve in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of T0VEGF prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibers in the skin and reduction of neuropeptide calcitonin gene-related peptide and substance P in DRG neurons of the diabetic mice. HSV-mediated transfer of VEGF to DRG may prove useful in treatment of diabetic neuropathy.

Safety and biodistribution studies of an HSV multigene vector following intracranial delivery to non-human primates.

Malignant glioma is a fatal human cancer in which surgery, chemo- and radiation therapies are ineffective. Therapeutic gene transfer used in combination with current treatment methods may augment their effectiveness with improved clinical outcome. We have shown that NUREL-C2, a replication-defective multigene HSV-based vector, is effective in treating animal models of glioma. Here, we report safety and biodistribution studies of NUREL-C2 using rhesus macaques as a model host. Increasing total doses (1 x 10(7) to 1 x 10(9) plaque forming units (PFU)) of NUREL-C2 were delivered into the cortex with concomitant delivery of ganciclovir (GCV). The animals were evaluated for changes in behavior, alterations in blood cell counts and chemistry. The results showed that animal behavior was generally unchanged, although the chronic intermediate dose animal became slightly ataxic on day 12 postinjection, a condition resolved by treatment with aspirin. The blood chemistries were unremarkable for all doses. At 4 days following vector injections, magnetic resonance imaging showed inflammatory changes at sites of vector injections concomitant with HSV-TK and TNFalpha expression. The inflammatory response was reduced at 14 days, resolving by 1 month postinjection, a time point when transgene expression also became undetectable. Immunohistochemical staining following animal killing showed the presence of a diffuse low-grade gliosis with infiltrating macrophages localized to the injection site, which also resolved by 1 month postinoculation. Viral antigens were not detected and injected animals did not develop HSV-neutralizing antibodies. Biodistribution studies revealed that vector genomes remained at the site of injection and were not detected in other tissues including contralateral brain. We concluded that intracranial delivery of 1 x 10(9) PFU NUREL-C2, the highest anticipated patient dose, was well tolerated and should be suitable for safety testing in humans.

Intra-articular adenoviral-mediated gene transfer of trail induces apoptosis of arthritic rabbit synovium.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that primarily affects joints. In rheumatoid joints there is extensive synovial proliferation with diseased synovium becoming highly aggressive, attaching to the articular cartilage and bone to form what is termed a pannus. The formation of active pannus is central to erosive disease and resulting joint destruction. In this study, we examined the ability to eliminate the hyperplastic synovium by adenoviral-mediated gene transfer of human TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family that is able to induce apoptosis through interaction with receptors containing death domains, DR4 and DR5. Infection of synovial cells derived from RA patients with Ad.TRAIL resulted in significant apoptosis in three out of five lines. Moreover, primary rabbit synovial fibroblasts were also sensitive to Ad.TRAIL-mediated gene transfer. In a rabbit model of arthritis, intra-articular gene transfer of TRAIL induced apoptosis in cells within the synovial lining, reduced leukocytic infiltration and stimulated new matrix synthesis by cartilage. These results demonstrate that TRAIL can affect the viability of the cells populating the activated synovium in arthritic joints and suggest that the delivery of TRAIL to arthritic joints may represent a non-invasive mechanism for inducing pannus regression.

Sustained release of low-dose ganciclovir from a silicone formulation prolonged the survival of rats with gliosarcomas under herpes simplex virus thymidine kinase suicide gene therapy.

A silicone formulation of ganciclovir (GCV-pellet) was developed to enhance the cytotoxic effects of herpes simplex virus thymidine kinase suicide gene therapy. The effectiveness of this drug delivery system was assessed in a rat 9L gliosarcoma model. The GCV-pellets (1 mm in length and in diameter) used in this experiment contained a total amount of 0.15 mg of GCV. In vitro experiments demonstrated that GCV was gradually released over a period of 7 days. Five days after stereotactic tumor inoculation into the right caudate nucleus, a herpes simplex virus type 1 (HSV-1) vector expressing herpes simplex virus thymidine kinase (HSV-tk) (T1, 2x10(6) pfu) was administered at the same location. The survival rate of the group treated with the GCV-pellet was compared with that of the T1 group injected intraperitoneally (IP) with GCV (30 mg/kg/day for 7 days). The GCV-pellet-treated group had a significantly prolonged survival (a median of more than 80 days) compared with the GCV IP group (a median of 65 days) and with control groups (P<0.05). The control groups (untreated or receiving only the virus vector) had a survival of 35-38 days. The survival rate of the GCV-pellet group over 80 days was 75%, and all the rats that survived more than 80 days and did not show tumors upon histological examination of the brain were deemed cured. No toxic effects or immunological reactions were observed histologically around the pellet in brain sections from the rats treated with the GCV-pellet. After GCV-pellet inoculation into the tumor, drug concentrations were kept at 1-10 microg/g tissue for 3-4 days. When the same dose of GCV (0.15 mg) in aqueous solution was injected into the tumor, GCV concentrations reached a peak of 0.5 mg/g tissue after 30 min and decreased below measurable level within 12 h. After IP injections of 3 mg GCV, GCV concentrations in the tumor reached a peak of 5.7 microg/g tissue after 30 min and also decreased below measurable level within 12 h. This sustained release of a low and effective GCV dose with the silicone formulation significantly prolonged survival in combinations with HSV-tk expression if compared to IP administration of GCV. Histological examination suggests that the treatment appears to be safe.