Search

Here, we describe the construction and testing of a novel herpes simplex virus type 1 (HSV-1) derived oncolytic virus (OV): 34.5ENVE (viral ICP34.5 Expressed by Nestin promotor and Vstat120 Expressing), for the treatment of cancer. This virus showed significant glioma-specific killing and antiangiogenic effects in vitro and in vivo. Treatment of subcutaneous and intracranial glioma-bearing mice with 34.5ENVE showed a significant increase in median survival of mice in four different glioma models. Histology and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) revealed reduced microvessel density (MVD) and increased tumoral necrosis in 34.5ENVE-treated tumor tissue compared to control OV-treated tumor tissue. Collectively, these results describe the construction, efficacy, and impact on tumor microenvironment of a transcriptionally driven OV armed with Vstat120 gene expression. These preclinical results will facilitate future clinical testing of 34.5ENVE.

The microtubule-associated protein tau (MAPT) and alpha-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the human MAPT and SNCA transgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kb MAPT or 135 kb SNCA locus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPT vector carrying the complete MAPT locus. On transduction into neuronal cultures, multiple MAPT transcripts were expressed from iBAC-MAPT under strict developmental and cell type-specific control. In primary neurons from Mapt(-/-) mice, the iBAC-MAPT vector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity of Mapt(-/-) neurons to Abeta peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes.

Oncolytic herpes simplex virus (oHSV) mutants are under development as anticancer therapeutics. One such vector, rRp450, is ICP6-deleted and expresses a prodrug enzyme for cyclophosphamide (CPA) (rat CYP2B1). Little is known about rRp450's toxicity profile, especially in combination with CPA. We tested rRp450/CPA for antitumor efficacy in an aggressive human xenograft sarcoma model, measured virus production in primary cells, and tested rRp450/CPA for safety in immunocompetent mice. CPA enhanced the antitumor efficacy of rRp450. Relative to wild-type HSV-1, rRp450 replication was attenuated approximately 10,000-fold in human primary hepatocytes, differentiated primary foreskin keratinocytes, and primary Schwann cells. In vivo, intravenous and intracranial (IC) rRp450 injection at the strength of 10(8) plaque-forming units (pfu) alone or followed 24 hours later by intraperitoneal (IP) CPA was well tolerated and had no significant effect clinically on blood counts or chemistries. By contrast, intravenous KOS was found to be uniformly neurotoxic at 10(5) and fatal at 10(6) pfu, and IC virus was fatal in most mice at 10(4) pfu. Low levels of virus DNA were detected in some organs following intravenous and IC virus injection, but were not significantly altered by CPA. HSV replication was not detected in reactivation studies of isolated organs. Our findings suggest rRp450/CPA is safe and warrants further study as a potential combination in anticancer therapeutics.

The herpes simplex virus (HSV) amplicon vector is a powerful gene delivery vehicle that can accommodate up to 150 kilobase of exogenous DNA. However, amplicon-mediated transgene expression is often transient outside the nervous system. In order to define the role of host immune responses in silencing amplicon-encoded transgenes, we evaluated the kinetics of cytokine-/chemokine-expression after tail-vein injection of a luciferase-encoding amplicon into mice. Type I interferons (IFNs) were induced earliest, within an hour after injection, and several other cytokines/chemokines were subsequently upregulated in the livers of wild-type (WT) mice. When the same experiment was performed in signal transducers and activators of transcription 1 (STAT1)-knockout (KO) mice, the levels of type I IFN expression were significantly lower and chemokine induction was almost non-existent. Importantly, STAT1-KO mice exhibited significantly higher and more sustained luciferase activity than did the WT mice, which is attributable to increased transcriptional activity rather than increased copy numbers of luciferase-encoding vector DNA. Further studies using primary cultured fibroblasts derived from WT and STAT1-KO mice revealed the significance of STAT1 signaling in transcriptional silencing of the amplicon-encoded transgene in vitro. Our results indicate that type I IFNs induced by systemic delivery of HSV amplicon vectors initiate a cascade of immune responses and suppress transgene expression at the transcriptional level by activation of STAT1.

The ability to deliver efficiently a complete genomic DNA locus to human and rodent cells will likely find widespread application in functional genomic studies and novel gene therapy protocols. In contrast to a cDNA expression cassette, the use of a complete genomic DNA locus delivers a transgene intact with its native promoter, the exons, all the intervening introns, and the regulatory regions. The presence of flanking, noncoding genomic DNA sequences could prove critical for prolonged and appropriate gene expression. We have recently developed a technology for the rapid conversion of bacterial artificial chromosome (BAC) clones into high-capacity herpes simplex virus-based amplicon vectors. Here, we express the human low-density lipoprotein receptor (LDLR), mutated in familial hypercholesterolemia (FH), from a 135-kb BAC insert. The infectious LDLR genomic locus vectors were shown to express at physiologically appropriate levels in three contexts. First, the LDLR locus was expressed appropriately in the ldl−/−a7 Chinese hamster ovary (CHO) cell line immediately following infectious delivery; second, the locus was maintained within a replicating episomal vector and expressed at broadly physiological levels in CHO cells for 3 months following infectious delivery; and third, the locus was efficiently expressed in human fibroblasts derived from FH patients. Finally, we show that the infectious LDLR locus retains classical expression regulation by sterol levels in human cells. This long-term expression and physiological regulation of LDLR prepares the way for in vivo functional studies of infectious delivery of BAC inserts.

A variety of oncolytic viruses (OVs) are being tested in clinical trials for different human cancers. Although the innate immune response is critical as the first line of defense in thwarting viral infection of mammalian cells, little is known of this response in the context of OV therapy of tumors. Investigations of activities against a herpes simplex OV demonstrated that HSV-seronegative sera from rats, mice, and humans efficiently neutralize this OV in vitro. Although this neutralization is due to complement, activation of this innate host defense differs in its pathways among species routinely used in preclinical tumor trials. In rats, both natural immunoglobulins and mannan-binding lectin (MBL) activate complement against the OV, while in mice only MBL is relevant to this activation. However, in humans only natural immunoglobulins play a role in complement activity. Quantitative analyses confirm that in vivo complement depletion facilitates the initial infection of tumors by systemic OVs. Therefore, complement activation against oncolytic HSV vectors proceeds through different pathways in different species. These findings are relevant to preclinical rodent studies of OV therapy and their application to human clinical trials.

Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (

Neuroblastoma, a heterogeneous tumor that arises from the neural crest, is the most common extracranial solid tumor in children. Fewer than half of patients with high-risk features survive long-term, despite multi-modal therapy with surgery, chemotherapy, antibody therapy, retinoic acid, high-dose chemotherapy, and autologous hematopoietic progenitor cell transplant. We previously reported neuroblastoma xenograft models were responsive to direct intratumoral injection of oncolytic Herpes Simplex Viruses (Parikh et al., Ped Blood Can 44:469, 2005; Mahller et al., Can Res 68:1170, 2008). We have confirmed these findings using an oncolytic HSV, Seprehvir (HSV1716), which is in clinical trials that include pediatric patients (NCT00931931, NCT02031965). The models showed wide variability, however, with CHP134 being highly responsive (4/8 complete responses (CR), 4/8 maintained complete responses (MCR)), CHLA-20 intermediately responsive (4/7 partial responses (PR), 3/7 MCR), SKNBE(2) somewhat responsive (6/7 PR, 1/7 MCR) and SK-N-AS minimally responsive (3/7 progressive disease (PD), 4/7 delayed progression (PD1), 0/7 PR). Because DNA viruses are highly dependent on normal nuclear machinery for DNA synthesis/replication and virus packaging/assembly, we postulated that increased transit time in mitosis may be beneficial to an oncolytic DNA virus. Aurora A Kinase is an enzyme critical for formation of centrioles and mitotic spindles during mitosis and is often dysregulated in cancer. The selective AAK inhibitor, Alisertib (MLN8237), arrests cells in G2/M and is highly effective in neuroblastoma models, making it an attractive candidate for combination studies with Seprehvir. We administered repeated 7-day cycles of combination therapy (10 mg/kg Alisertib once daily by gavage, days 1-5; 10e7 pfu Seprehvir day 5) to mice bearing SK-N-AS xenografts and found significant antitumor synergy (2/10 PD1, 4/10 SD, 2/10 PR, 2/10 CR). Our preliminary data assessing kinetics of virus infection suggest that Alisertib pretreatment enhances virus uptake and spread, accounting for the combinatorial effects. Overall, our findings support clinical testing of Alisertib combined with Seprehvir in patients with refractory neuroblastoma.

We thank Drs Lemaire and Halperin for their comments on our article in Molecular Therapy.1,2 They raise important concerns about the effects of rapid cell lysis on the physiology of nontumor tissues; these effects have been recognized as tumor lysis syndrome and are of particular concern in cancers with high cell turnover rate, such as lymphomas and leukemias. However, we think that the time course of cell death (hours to days) will spread these effects over a period sufficiently long that normal homeostatic mechanisms will buffer the ionic effects of tumor cell death.

The prognosis for patients with glioblastoma remains poor despite numerous phase I–II clinical trials on systemic chemotherapies such as the vascular endothelial growth factor–neutralizing antibody bevacizumab.1 Indeed, only two chemotherapies—implantable carmustine-containing Gliadel wafers and temozolomide2—have been approved for glioblastoma by the Food and Drug Administration, because these two agents showed benefit in phase III clinical trials by prolonging median survival by approximately 8 weeks. Therefore, there is a continued need to explore new treatments or to further refine and optimize older treatments for this type of brain cancer.

Top of pageAbstract Stem cell-based replacement therapy has great potential in treating neurological disorders, including brain and spinal cord injuries. Although several studies have demonstrated neuronal and glial differentiation of transplanted neural stem cells (NSCs) in vivo, the efficacy of the transplanted NSCs to generate neurons and of the newly generated neurons to form new synaptic connections with the remaining host neurons still needs clarification. To address these issues, we have constructed an HSV amplicon-based indicator vector that allows visualization of neuronal differentiation and neurite projection. Synapsin I (SYN) is a well characterized gene that expresses selectively in neurons. Thiel and colleagues found that a 400-bp 5' untranslated promoter region of SYN directed prominent expression of a reporter gene in various neuronal cell lines, but that no expression was detected in cell lines derived from non-neuronal tissue (Thiel G et al., PNAS 1991). On the other hand, microtubule- associated protein tau-reporter fusion protein has been used as a genetic reporter to visualize neuronal processes in vivo (Callahan CA et al., PNAS 1994). We, therefore, combined the two genetic elements in the context of HSV amplicon vectors, producing an indicator herpes simplex virus (HSV) amplicon vector, HSV-SYN-tauR, that carries tau fused with a monomeric red fluorescent protein (mRFP, Campbell et al., PNAS 2002) under the control of the 400- bp SYN promoter. When a mixed culture of mouse cortical neurons and glia was transduced with the HSV-SYN-tauR amplicon vector, RFP reporter was expressed only in neurons along their neurites, allowing visualization of every axon, dendrite, and spine. Time-lapse imaging of mixed cortical cultures transduced with the HSV-SYN-tauR amplicon vector allowed noninvasive real-time monitoring of extension and retraction of the neurites of live neurons. This indicator HSV amplicon vector could prove useful in studying neuronal differentiation, neurite projections, and regeneration of neuronal networks in vitro and in vivo.

HSV mutants (e.g. G207 and MGH1), possessing deletions in both copies of the ICP34.5 gene and an insertional mutation in the ICP6 gene (ribonucleotide reductase), have proven safe through a number of animal experiments and clinical trials. Although these double mutants clearly demonstrate oncolytic effects as well as anti-tumor immunization, their oncolytic effects are markedly reduced. ICP34.5-null viruses fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. In HSV-infected cells, viral double-stranded RNA will activate PKR by autophosphorylation, and activated PKR phosphorylates eIF-2a, which inhibits initiation of protein translation within the cells and in turn inhibits viral replication. The ICP34.5 dephosphorylates eIF-2a, thus allowing continued protein synthesis and viral replication. In order to increase therapeutic efficacy and tumor selectivity, glioma selective expression of ICP34.5 in oncolytic HSV has been explored. As a candidate for glioma selective promoters, we examined a synthetic nestin promoter, which consists of the nestin enhancer and hsp68 minimum promoter. We first performed RT-PCR to examine nestin expression in various types of cultured cells. All the glioma cell lines tested (U251, U87dEGFR, Gli36d5, T98G, MGH238, U138) were confirmed to be positive for nestin expression. Astrocytes, on the other hand, were negative. We, therefore, constructed a novel HSV mutant, rQNestin34.5, which expresses ICP34.5 under control of the synthetic nestin promoter. In the experiments using cultured human glioma cell lines in vitro, rQNestin34.5 showed remarkable enhancements in both viral propagation and oncolysis compared to the ICP34.5-null virus (rHsvQ1). In four primary glioblastoma cells, rQNestin34.5 also showed remarkable enhancement in both viral propagation and oncolysis compared to control rHsvQ1. In normal human astrocytes, rQNestin34.5 showed equivalent toxicity to control rHsvQ1. We also performed Western blotting to examine the phosphorylation state of eIF2a. In glioma cell lines infected with rQNestin34.5, the level of phspho-eIF2a was lower than that of control rHsvQ1, confirming that the ICP34.5 is expressed selectively in glioma cells and resulted in a decrease of phospho-eIF2a level. This was responsible for its efficient propagation in glioma cells. We further characterized the anti-cancer efficacy of this new virus in vivo using an athymic mouse subcutaneous tumor model (U87dEGFR cells) and a brain tumor model (U87dEGFR cells). The rQNestin34.5 showed significantly more potent inhibition of tumor growth compared to control rHsvQ1. The mice with brain tumor treated by rQNestin34.5 survived longer than those treated by rHsvQ1.

Glioblastoma is among the most devastating malignancies in humans and often rapidly fatal despite aggressive modern treatments. Recent evidence suggests that neoplastic tumors are initiated and maintained exclusively by a rare fraction of cancer cells with stem cell properties (cancer stem cells). Several groups of investigators reported the presence of stem cell-like cells in human brain tumor specimens, and Dirks and his colleagues nicely demonstrated that only the CD133 positive fraction of human brain tumor cells has stem-like properties including ability of self-renewal, multipotency and potential to develop brain tumors in SCID mice.

Replication-competent, oncolytic herpes simplex virus type 1 (HSV-1) vectors are useful tools for cancer gene therapy. The virus genome is genetically engineered so that the vector selectively replicates in and destroys tumor cells while sparing the surrounding normal tissues. A marker gene is frequently used in order for replicating virus vectors to be easily detected. Green Fluorescent Protein (GFP) is now a popular marker, because it can be detected in living cells and also be used for selection. MGH-1 is a double-mutated HSV-1 with deletions in both copies of the γ34.5 gene and an insertion of the LacZ gene down-stream of the ICP6 promoter inactivating the ICP6 gene. Using this MGH-1 as a backbone and a bacterial artificial chromosome technology, we newly created multiple types of “armed” oncolytic HSV-1 vectors (vHSV series) in which the ICP6 and LacZ genes were replaced with the ICP6 promoter-driven GFP gene and a transgene cassette driven by the HSV IE4/5 promoter. Prior to testing “armed” oncolytic vectors, we compared the antitumor activity of vHSV-null, the control oncolytic vector expressing only the GFP marker, with that of MGH-1. The replication capability of vHSV-null in cultured Vero cells at a MOI of 0.01 was approximately 10 times lower than that of MGH-1. Despite decreased replication capability, vHSV-null showed slightly faster cytopathic effect than MGH-1 in Neuro2a murine neuroblastoma cells at MOI=0.1. A/J mice bearing bilateral subcutaneous Neuro2a tumors were used for in vivo evaluations. When vectors were inoculated directly into the left tumor at 2×106 plaque-forming units (days 0 and 3), vHSV-null was significantly more effective than MGH-1 for inhibiting the growth of inoculated tumors (p < 0.05). Contralateral non-inoculated tumors also tended to be smaller in vHSV-null-treated animals than MGH-1-treated animals although not statistically significant. Such difference in tumor growth inhibition was not observed in athymic mice, indicating that the greater antitumor activity of vHSV-null in vivo compared with MGH-1 is associated with T cell-mediated immune responses most likely due to expression of GFP. While this possible immunostimulatory effect by GFP may be beneficial to oncolytic virus therapy, a caution is needed to evaluate the antitumor activity of oncolytic HSV-1 vectors when the GFP gene is used as a marker for virus replication.