Extending the transposable payload limit of Sleeping Beauty (SB) using the Herpes Simplex Virus (HSV)/SB amplicon-vector platform.

The ability of a viral vector to safely deliver and stably integrate large transgene units (transgenons), which not only include one or several therapeutic genes, but also requisite native transcriptional regulatory elements, would be of significant benefit for diseases presently refractory to available technologies. The herpes simplex virus type-1 (HSV-1) amplicon vector has the largest known payload capacity of approximately 130 kb, but its episomal maintenance within the transduced cell nucleus and induction of host cell silencing mechanisms limits the duration of the delivered therapeutic gene(s). Our laboratory developed an integration-competent version of the HSV-1 amplicon by adaptation of the Sleeping Beauty (SB) transposon system, which significantly extends transgene expression in vivo. The maximum size limit of the amplicon-vectored transposable element remains unknown, but previously published plasmid-centric studies have established that DNA segments longer than 6-kb are inefficiently transposed. Here, we compared the transposition efficiency of SB transposase in the context of both the HSV amplicon vector as well as the HSV amplicon plasmid harboring 7 and 12-kb transposable reporter transgene units. Our results indicate that the transposition efficiency of the 12-kb transposable unit via SB transposase was significantly reduced as compared with the 7-kb transposable unit when the plasmid version of the HSV amplicon was used. However, the packaged HSV amplicon vector form provided a more amenable platform from which the 12-kb transposable unit was mobilized at efficiency similar to that of the 7-kb transposable unit via the SB transposase. Overall, our results indicate that SB is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery of the platform via HSV amplicons.

A rough morphology of the adsorbed fibronectin layer favors adhesion of neuronal cells.

A range of substrates made of polystyrene (PS) and poly(methyl methacrylate)-poly(methacrylic acid) copolymer (PMMA-PMAA) containing 98 and 80% PMMA (PA98, PA80) and presenting a homogeneous or a patterned surface were used to study fibronectin adsorption and neuronal cell behavior. Fibronectin adsorption showed weak differences regarding the adsorbed amount (evaluated by XPS), but large differences in adsorbed layer morphology as observed by AFM. A fine granular morphology, with dimensions up to 8 nm height and 50-150 nm width, was observed on top of a thin adsorbed layer in the case of PS, PA98, and of a surface made of nanoscale inclusions of the latter in PS. In contrast, the layer adsorbed on PA80, which carries more ionizable groups, showed a higher roughness on the PA80 zones with differences in height up to 30 nm and characteristic lateral dimensions of 400 nm. On substrates of the former category, the cells formed large clusters, revealing poor interactions with the substrate. On PA80, the cells formed large networks with only a few small clusters. The adsorbed layer roughness, resulting from aggregation of fibronectin upon adsorption and from the substrate surface chemical composition, is responsible for neuronal cell spreading and growth. Its effect is not prevented by the presence of inclusions (< 30% of the surface) responsible for smoother areas of adsorbed fibronectin and for protrusions below 40 nm.

Isoform-specific effects of ApoE on HSV immediate early gene expression and establishment of latency.

Alzheimer's disease (AD) is a common and devastating neurodegenerative disease in which most cases are of unknown, sporadic origin. In addition to age, the most prevalent known risk factor for developing AD is carriage of the epsilon4 allele of Apolipoprotein E (ApoE). Carriage of the epsilon2 or epsilon3 allele of ApoE confers protection or no change in risk for AD, respectively. Latent herpes simplex virus type 1 (HSV-1) infection in the brain concurrent with ApoE4 carriage exacerbates risk for AD, suggesting that these two factors interact to promote neuronal dysfunction and degeneration in selective brain areas. Indeed, HSV-1 DNA has been found in regions primarily affected by AD, such as the temporal lobes, hippocampus, and neocortex. We hypothesize that HSV-1 infection in the background of ApoE4, but not ApoE2 or ApoE3, promotes an environment more conducive to neuronal degeneration. To investigate this idea, we have utilized transgenic mice that express human ApoE2, 3, or 4 alleles from astrocytes in a murine ApoE -/- background. We find that carriage of the different ApoE alleles dramatically affects HSV-1 immediate early gene expression as well as the establishment of latency. Both of these factors are poised to impact neuronal viability, inflammation, and viral spread. Our data support the concept that HSV-1 and ApoE4 interact to provide an environment conducive to the development and/or spread of AD.

Improved HSV-1 amplicon packaging using virion host shutoff mutants lacking mRNAse activity.

Given their generous transgene capacity and inherent neurotropism, herpes simplex virus (HSV-1)-based viral vectors are promising tools for gene delivery to the central nervous system. Despite their widespread pre-clinical use, vector toxicity remains a concern with regard to the use of herpes vectors in humans. One potential source of toxicity stems from the tegument-associated virion host shutoff protein (vhs), which induces translational arrest in the host cell through non-specific mRNAse activity. In the current study we utilized a series of HSV-1 viruses containing a deletion in the U(L)41 open reading frame to investigate: (1) the requirement of intact vhs function in amplicon packaging and (2) whether vhs influences the post-transduction survival of dissociated cortical neurons. Our results demonstrate that while amplicon yield was reduced an order of magnitude, U(L)41 deletion was associated with reduced vector toxicity. Furthermore, partial reconstitution of vhs function using mRNAse-inactive point mutants improved amplicon titers without imparting the toxicity observed with wild-type controls. These findings offer a novel approach to improving the titer and toxicity profiles of HSV-based viral vectors.

Synapsin I Cre transgene expression in male mice produces germline recombination in progeny.

The cre/LoxP system can produce conditional loss of gene function in specific cell types such as neurons. A transgenic mouse line, utilized by multiple studies, used the Synapsin I promoter to drive expression of cre (SynCre) to achieve neuronal-specific cre expression. Herein we describe that cre expression can also be observed in SynCre mice within the testes after being bred into a floxed transgenic mouse line. Cre transcript was expressed in testes resulting in recombination of the floxed substrate in testes. In the majority of cases, progeny of male SynCre mice inherited a germline recombined floxed allele, while this was never observed in progeny from female mice carrying the SynCre allele. This observation should alert investigators to a potential confound using these mice and enables male germ cell "deletor" strategies.

Novel gene therapeutic strategies for neurodegenerative diseases.

The convergent pathobiologic model of Parkinson's disease stipulates that disparate insults initiate a disease process that obligately share a common pathway leading to cell death. A combinatorial treatment which targets various steps in this pathway is likely to be the most successful therapeutic strategy. As advances are made in the field of neuroimaging and pharmacogenomics, early detection of sporadic PD will become a reality. Early intervention will likely spare more dopaminergic neurons and extend the quality of life for the patient. Continued advancements in the fields of pharmacology, neurosurgery, and gene therapy will strengthen the armamentarium available for the treatment of PD patients.

Temporal evolution of mouse striatal gene expression following MPTP injury.

The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (SN) causes parkinsonism characterized by slow, halting movements, rigidity, and resting tremor when neuronal loss exceeds a threshold of approximately 80%. It is estimated that there is extensive compensation for several years prior to symptom onset, during which vulnerable neurons asynchronously die. Recent evidence would argue that much of the compensatory response of the nigrostriatal system is multimodal including both pre-synaptic and striatal mechanisms. Although parkinsonism may have multiple causes, the classic syndrome, Parkinson's disease (PD), is frequently modeled in small animals by repeated administration of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Because the MPTP model of PD recapitulates many of the known behavioral and pathological features of human PD, we asked whether the striatal cells of mice treated with MPTP in a semi-chronic paradigm enact a transcriptional program that would help elucidate the response to dopamine denervation. Our findings reveal a time-dependent dysregulation in the striatum of a set of genes whose products may impact both the viability and ability to communicate of dopamine neurons in the SN.

Redefining neuroprotective gene therapeutic strategies: Lessons learned from caloric restriction and NAD(+) metabolism.

Herein a case is made for the development of novel cytoprotective approaches based upon molecular mechanisms thought to underlie the caloric restriction phenomenon. This analysis leads to the prediction that molecular genetic perturbations affecting the metabolism of nuclear NAD(+) and metabolites will be neuroprotective.

HSV amplicon delivery of glial cell line-derived neurotrophic factor is neuroprotective against ischemic injury.

Direct intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) is neuroprotective against ischemia-induced cerebral injury. Utilizing viral vectors to deliver and express therapeutic genes presents an opportunity to produce GDNF within localized regions of an evolving infarct. We investigated whether a herpes simplex virus (HSV) amplicon-based vector encoding GDNF (HSVgdnf) would protect neurons against ischemic injury. In primary cortical cultures HSVgdnf reduced oxidant-induced injury compared to the control vector HSVlac. To test protective effects in vivo, HSVgdnf or HSVlac was injected into the cerebral cortex 4 days prior to, or 3 days, after a 60-min unilateral occlusion of the middle cerebral artery. Control stroke animals developed bradykinesia and motor asymmetry; pretreatment with HSVgdnf significantly reduced such motor deficits. Animals receiving HSVlac or HSVgdnf after the ischemic insult did not exhibit any behavioral improvement. Histological analyses performed 1 month after stroke revealed a reduction in ischemic tissue loss in rats pretreated with HSVgdnf. Similarly, these animals exhibited less immunostaining for glial fibrillary acidic protein and the apoptotic marker caspase-3. Taken together, our data indicate that HSVgdnf pretreatment provides protection against cerebral ischemia and supports the utilization of the HSV amplicon for therapeutic delivery of trophic factors to the CNS.

Immune responses to replication-defective HSV-1 type vectors within the CNS: implications for gene therapy.

Herpes simplex virus (HSV) is a naturally occurring double-stranded DNA virus that has been adapted into an efficient vector for in vivo gene transfer. HSV-based vectors exhibit wide tropism, large transgene size capacity, and moderately prolonged transgene expression profiles. Clinical implementation of HSV vector-based gene therapy for prevention and/or amelioration of human diseases eventually will be realized, but inherently this goal presents a series of significant challenges, one of which relates to issues of immune system involvement. Few experimental reports have detailed HSV vector-engendered immune responses and subsequent resolution events primarily within the confines of the central nervous system. Herein, we describe the immunobiology of HSV and its derived vector platforms, thus providing an initiation point from where to propose requisite experimental investigation and potential approaches to prevent and/or counter adverse antivector immune responses.

Neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing IL-12 effectively treats the parent tumor and protects against recurrence-after resection.

The objective of the study was to evaluate the utility of NV1042, a replication competent, oncolytic herpes simplex virus (HSV) containing the interleukin-12 (IL-12) gene, as primary treatment for hepatic tumors and to further assess its ability to reduce tumor recurrence following resection. Resection is the most effective therapy for hepatic malignancies, but is not possible in the majority of the patients. Furthermore, recurrence is common after resection, most often in the remnant liver and likely because of microscopic residual disease in the setting of postoperative host cellular immune dysfunction. We hypothesize that, unlike other gene transfer approaches, direct injection of liver tumors with replication competent, oncolytic HSV expressing IL-12 will not only provide effective control of the parent tumor, but will also elicit an immune response directed at residual tumor cells, thus decreasing the risk of cancer recurrence after resection. Solitary Morris hepatomas, established in Buffalo rat livers, were injected directly with 10(7) particles of NV1042, NV1023, an oncolytic HSV identical to NV1042 but without the IL-12 gene, or with saline. Following tumor injection, the parent tumors were resected and measured and the animals were challenged with an intraportal injection of 10(5) tumor cells, recreating the clinical scenario of residual microscopic cancer. In vitro cytotoxicity against Morris hepatoma cells was similar for both viruses at a multiplicity of infection of 1 (MOI, ratio of viral particles to target cells), with >90% tumor cell kill by day 6. NV1042 induced high-level expression of IL-12 in vitro, peaking after 4 days in culture. Furthermore, a single intratumoral injection of NV1042, but not NV1023, induced marked IL-12 and interferon-gamma (IFN-gamma) expression. Both viruses induced a significant local immune response as evidenced by an increase in the number of intratumoral CD4(+) and CD8(+) lymphocytes, although the peak of CD8(+) infiltration was later with NV1042 compared with NV1023. NV1042 and NV1023 reduced parent tumor volume by 74% (P<.003) and 52% (P<.03), respectively, compared to control animals. Treatment of established tumors with NV1042, but not with NV1023, significantly reduced the number of hepatic tumors after resection of the parent tumor and rechallenge (16.8+/-11 (median=4) vs. 65.9+/-15 (median=66) in control animals, P<.025). In conclusion, oncolytic HSV therapy combined with local immune stimulation with IL-12 offers effective control of parent hepatic tumors and also protects against microscopic residual disease after resection. The ease of use of this combined modality approach, which appears to be superior to either approach alone, suggests that it may have clinical relevance, both as primary treatment for patients with unresectable tumors and also as a neoadjuvant strategy for reducing recurrence after resection.

HSV vector-mediated gene delivery to the central nervous system.

The efficient and targeted transfer of genes is the goal of gene therapy. In the central nervous system (CNS), this is challenging due in part to the exquisite anatomy of the brain. Herpes simplex virus (HSV) vectors are particularly amenable to CNS therapies as they are capable of transducing a variety of cells, have a large transgene capacity and can exist as either oncolytic or non-immunogenic vectors. The versatility of this vector platform and its potential molecular therapeutic use in two CNS disorders, Alzheimer's disease and malignant brain tumors, will be discussed.

Combination vascular delivery of herpes simplex oncolytic viruses and amplicon mediated cytokine gene transfer is effective therapy for experimental liver cancer.

BACKGROUND: Herpes simplex type I (HSV)-based vectors have been used experimentally for suicide gene therapy, immunomodulatory gene delivery, and direct oncolytic therapy. The current study utilizes the novel concept of regional delivery of an oncolytic virus in combination with or serving as the helper virus for packaging herpes-based amplicon vectors carrying a cytokine transgene, with the goal of identifying if this combination is more efficacious than either modality alone. MATERIALS AND METHODS: A replication competent oncolytic HSV (G207) and a replication incompetent HSV amplicon carrying the gene for the immunomodulatory cytokine IL-2 (HSV-IL2) were tested in murine syngeneic colorectal carcinoma and in rat hepatocellular carcinoma models. Liver tumors were treated with vascular delivery of (1) phosphate-buffered saline (PBS), (2) G207, (3) HSV-IL2, (4) G207 and HSV-IL2 mixed in combination (mG207/HSV- IL2), and (5) G207 as the helper virus for packaging the construct HSV-IL2 (pG207/HSV-IL2). RESULTS: Tumor burden was significantly reduced in all treatment groups in both rats and mice treated with high-dose G207, HSV-IL2, or both (p < 0.02). When a low dose of virus was used in mice, anti-tumor efficacy was improved by use of G207 and HSV-IL2 in combination or with HSV-IL2 packaged by G207 (p < 0.001). This improvement was abolished when CD4(+) and CD8(+) lymphocytes were depleted, implying that the enhanced anti-tumor response to low-dose combined therapy is immune mediated. CONCLUSIONS: Vascular regional delivery of oncolytic and amplicon HSV vectors can be used to induce improved anti-tumor efficacy by combining oncolytic and immunostimulatory strategies.

Dendritic cells transduced with HSV-1 amplicons expressing prostate-specific antigen generate antitumor immunity in mice.

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.

A novel approach to cancer therapy using an oncolytic herpes virus to package amplicons containing cytokine genes.

There are two promising herpes viral-based anticancer strategies: one involves replication-defective viruses to transfer therapeutic transgenes, and the other involves replication-conditional oncolytic viruses, which selectively infect and destroy cancer cells directly. This study examines a novel dual herpesvirus preparation, which combines the immunostimulatory effects of amplicon-mediated IL2 expression with direct viral-induced oncolysis. The oncolytic virus G207 was used as the helper virus to package a herpes simplex virus (HSV)-amplicon vector carrying the gene IL2 (HSV-IL2), yielding a single preparation with two complementary modes of action. In vivo comparison was carried out in a syngeneic squamous cell carcinoma flank tumor model. We directly injected established tumors with HSV-IL2, G207, G207 mixed with HSV-IL2, or G207-packaged HSV-amplicon carrying the IL2 transgene (G207[IL2]). Significant inhibition of tumor growth was seen at 2 weeks in the G207[IL2]-treated tumors relative to controls (0.57+/-0.44 cm(3) versus 39.45+/-5.13 cm(3), P<0.00001), HSV-IL2 (20.97+/-4.60 cm(3)), and the G207 group (7.71+/-2.10 cm(3)). This unique use of a replication-conditional, oncolytic virus to package a replication-incompetent amplicon vector demonstrates impressive efficacy in vitro and in vivo, and avoids the theoretical concerns of recombination with reversion to wild type.

Efflux of glutathione conjugate of monochlorobimane from striatal and cortical neurons.

Evidence for the presence of a novel transporter in primary cultures of rat striatal neurons and mouse cortical neurons similar in function to the multidrug resistance-associated protein (MRP1) is presented. Functional activity was assessed by efflux studies with the glutathione conjugate of monochlorobimane (B-SG). The glutathione transferase-catalyzed formation of B-SG in rat striatal neurons and mouse cortical neurons was inhibited by ethacrynic acid. The efflux of B-SG from rat striatal neurons and mouse cortical neurons was lower at 20 degrees C than at 37 degrees C and was lower in cells with reduced ATP concentrations compared with cells with constitutive ATP concentrations. In addition, the efflux of B-SG was inhibited by MK-571 in both rat striatal and mouse cortical neurons and by probenecid in rat striatal neurons, but not in mouse cortical neurons. Verapamil did not inhibit B-SG efflux in either rat striatal or mouse cortical neurons. Although functionally similar to MRP1, Western blot analysis with commercially available antibodies directed against human and mouse MRP1 failed to show MRP1-like protein in either whole-cell homogenates of rat striatal neurons or mouse cortical neurons, indicating that the described neuronal transporter differs in structure from human or mouse MRP1 or lacks epitopes in common with MRP1.

Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Positron emission tomography imaging for herpes virus infection: Implications for oncolytic viral treatments of cancer.

Molecular therapy using viruses would benefit greatly from a non-invasive modality for assessing dissemination of viruses. Here we investigated whether positron emission tomography (PET) scanning using [(124)I]-5-iodo-2'-fluoro-1-beta-d-arabinofuranosyl-uracil (FIAU) could image cells infected with herpes simplex viruses (HSV). Using replication-competent HSV-1 oncolytic viruses with thymidine kinase (TK) under control of different promoters, we demonstrate that viral infection, proliferation and promoter characteristics all interact to influence FIAU accumulation and imaging. In vivo, as few as 1 x 107 viral particles injected into a 0.5-cm human colorectal tumor can be detected by [(124)I]FIAU PET imaging. PET signal intensity is significantly greater at 48 hours compared with that at 8 hours after viral injection, demonstrating that PET scanning can detect changes in TK activity resulting from local viral proliferation. We also show the ability of FIAU-PET scanning to detect differences in viral infectivity at 0.5 log increments. Non-invasive imaging might be useful in assessing biologically relevant distribution of virus in therapies using replication-competent HSV.