Sindbis Virus Vaccine Platform: A Promising Oncolytic Virus-Mediated Approach for Ovarian Cancer Treatment.

This review article provides a comprehensive overview of a novel Sindbis virus vaccine platform as potential immunotherapy for ovarian cancer patients. Ovarian cancer is the most lethal of all gynecological malignancies. The majority of high-grade serous ovarian cancer (HGSOC) patients are diagnosed with advanced disease. Current treatment options are very aggressive and limited, resulting in tumor recurrences and 50-60% patient mortality within 5 years. The unique properties of armed oncolytic Sindbis virus vectors (SV) in vivo have garnered significant interest in recent years to potently target and treat ovarian cancer. We discuss the molecular biology of Sindbis virus, its mechanisms of action against ovarian cancer cells, preclinical in vivo studies, and future perspectives. The potential of Sindbis virus-based therapies for ovarian cancer treatment holds great promise and warrants further investigation. Investigations using other oncolytic viruses in preclinical studies and clinical trials are also presented.

Channeling the Natural Properties of Sindbis Alphavirus for Targeted Tumor Therapy.

Sindbis alphavirus vectors offer a promising platform for cancer therapy, serving as valuable models for alphavirus-based treatment. This review emphasizes key studies that support the targeted delivery of Sindbis vectors to tumor cells, highlighting their effectiveness in expressing tumor-associated antigens and immunomodulating proteins. Among the various alphavirus vectors developed for cancer therapy, Sindbis-vector-based imaging studies have been particularly extensive. Imaging modalities that enable the in vivo localization of Sindbis vectors within lymph nodes and tumors are discussed. The correlation between laminin receptor expression, tumorigenesis, and Sindbis virus infection is examined. Additionally, we present alternative entry receptors for Sindbis and related alphaviruses, such as Semliki Forest virus and Venezuelan equine encephalitis virus. The review also discusses cancer treatments that are based on the alphavirus vector expression of anti-tumor agents, including tumor-associated antigens, cytokines, checkpoint inhibitors, and costimulatory immune molecules.

CCR7 signalling as an essential regulator of CNS infiltration in T-cell leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine-receptor interaction as a CNS 'entry' signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications.

Sindbis viral vectors transiently deliver tumor-associated antigens to lymph nodes and elicit diversified antitumor CD8+ T-cell immunity.

Tumors are theoretically capable of eliciting an antitumor immune response, but are often poorly immunogenic. Oncolytic viruses (OVs) have recently emerged as a promising strategy for the immunogenic delivery of tumor-associated antigens (TAAs) to cancer patients. However, safe and effective OV/TAA therapies have not yet been established. We have previously demonstrated that vectors based on Sindbis virus (SV) can inhibit tumor growth and activate the innate immune system in mice. Here, we demonstrate that SV vectors carrying a TAA generate a dramatically enhanced therapeutic effect in mice bearing subcutaneous, intraperitoneal, and lung cancers. Notably, SV/TAA efficacy was not dependent on tumor cell targeting, but was characterized by the transient expression of TAAs in lymph nodes draining the injection site. Early T-cell activation at this site was followed by a robust influx of NKG2D expressing antigen-specific cytotoxic CD8+ T cells into the tumor site, subsequently leading to the generation of long-lasting memory T cells which conferred protection against rechallenge with TAA-positive as well as TAA-negative tumor cells. By combining in vivo imaging, flow cytometry, cytotoxicity/cytokine assays, and tetramer analysis, we investigated the relationship between these events and propose a model for CD8+ T-cell activation during SV/TAA therapy.

The Transition of the 37-Kda Laminin Receptor (Rpsa) to Higher Molecular Weight Species: Sumoylation or Artifact?

The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.

Comprehensive proteomic analysis of nonintegrin laminin receptor interacting proteins.

Human nonintegrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. The laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion proteins, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from Mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through coimmunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/metabolism, protein processing, cytoskeleton/cell anchorage, DNA/chromatin, and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor and provides an essential stepping stone to a better mechanistic understanding of this protein's diverse functions.

Potent and Targeted Sindbis Virus Platform for Immunotherapy of Ovarian Cancer.

Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of ovarian and other types of cancers. In this study we show that SV.IL-12 combined with an agonistic OX40 antibody can eliminate ovarian cancer in a Mouse Ovarian Surface Epithelial Cell Line (MOSEC) model and further prevent tumors in mice rechallenged with tumor cells after approximately 5 months. Treatment efficacy is shown to be dependent upon T-cells that are transcriptionally and metabolically reprogramed. An influx of immune cells to the tumor microenvironment occurs. Combination of sequences encoding both IL-12 and anti-OX40 into a single SV vector, SV.IgGOX40.IL-12, facilitates the local delivery of immunoregulatory agents to tumors enhancing the anti-tumor response. We promote SV.IgGOX40.IL-12 as a safe and effective therapy for multiple types of cancer.

Multiple functions of the 37/67-kd laminin receptor make it a suitable target for novel cancer gene therapy.

The 37/67-kd laminin receptor, LAMR, is a multifunctional protein that associates with the 40S ribosomal subunit and also localizes to the cell membrane to interact with the extracellular matrix. LAMR is overexpressed in many types of cancer, playing important roles in tumor-cell migration and invasion. Here, we show that LAMR is also vital for tumor-cell proliferation, survival, and protein translation. Small-interfering RNA (siRNA)-mediated reduction in expression of LAMR leads to G1 phase cell-cycle arrest in vitro by altering cyclins A2/B1, cyclin-dependent kinases (CDKs) 1/2, Survivin, and p21 expression levels. In vivo, reduction in LAMR expression results in inhibition of HT1080 cells to develop tumors. We also found that LAMR's ribosomal functions are critical for translation as reduction in LAMR expression leads to a dramatic decrease in newly synthesized proteins. Further, cells with lower expression of LAMR have fewer 40S subunits and 80S monosomes, causing an increase in free 60S ribosomal subunits. These results indicate that LAMR is able to regulate tumor development in many ways; further enhancing its potential as a target for gene therapy. To test this, we developed a novel Sindbis/Lenti pseudotype vector carrying short-hairpin RNA (shRNA) designed against lamr. This pseudotype vector effectively reduces LAMR expression and specifically targets tumors in vivo. Treatment of tumor-bearing severe combine immunodeficient (SCID) mice with this pseudotype vector significantly inhibits tumor growth. Thus, we show that LAMR can be used as a target in novel therapy for tumor reduction and elimination.

Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

Sindbis viral vector induced apoptosis requires translational inhibition and signaling through Mcl-1 and Bak.

BACKGROUND: Sindbis viral vectors are able to efficiently target and kill tumor cells in vivo, as shown using pancreatic and ovarian cancer models. Infection results in apoptosis both in vitro and in vivo. Sindbis vector uptake is mediated by the LAMR, which is upregulated on a number of different tumor types, thus conferring specificity of the vector to a wide range of cancers. In this study we elucidate the mechanism of apoptosis in two tumor cell lines, MOSEC, derived from the ovarian epithelium and Pan02, derived from a pancreatic adenocarcinoma. A comprehensive understanding of the mechanism of apoptosis would facilitate the design of more effective vectors for cancer therapy. RESULTS: The initial phase of Sindbis vector induced apoptosis in MOSEC and Pan02 models reconfirms that viral infection is sensed by PKR due to double-stranded RNA intermediates associated with genomic replication. PKR activation results in translation inhibition through eIF2alpha phosphorylation and initiation of the stress response. Our studies indicate that the roles of two proteins, Mcl-1 and JNK, intimately link Sindbis induced translational arrest and cellular stress. Translational arrest inhibits the synthesis of anti-apoptotic Bcl-2 protein, Mcl-1. JNK activation triggers the release of Bad from 14-3-3, which ultimately results in apoptosis. These signals from translational arrest and cellular stress are propagated to the mitochondria where Bad and Bik bind to Bcl-xl and Mcl-1 respectively. Formation of these heterodimers displaces Bak, which results in caspase 9 cleavage and signaling through the mitochondrial pathway of apoptosis. CONCLUSION: The host cell response to Sindbis is triggered through PKR activation. Our studies demonstrate that PKR activation and subsequent translational arrest is linked to both cellular stress and apoptosis. We have also found the linkage point between translational arrest and apoptosis to be Mcl-1, a protein whose constant translation is required for inhibition of apoptosis. With this information vectors can be designed, which express or repress proteins implicated in this study, to enhance their therapeutic potential.

Sindbis Virus with Anti-OX40 Overcomes the Immunosuppressive Tumor Microenvironment of Low-Immunogenic Tumors.

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors.

Molecular and metabolic pathways mediating curative treatment of a non-Hodgkin B cell lymphoma by Sindbis viral vectors and anti-4-1BB monoclonal antibody.

BACKGROUND: Limitations to current therapies for treating non-Hodgkin B cell lymphoma include relapse, toxicity and high cost. Thus, there remains a need for novel therapies. Oncolytic viral (OV) therapy has become a promising cancer immunotherapy because of its potential effectiveness, specificity and long-lasting immunity. We describe and characterize a novel cancer immunotherapy combining Sindbis virus (SV) vectors and the agonistic monoclonal antibody (mAb) to the T cell costimulatory receptor, 4-1BB (CD137). METHODS: A20 lymphoma was transfected with luciferase and tumor cells were inoculated to BALB/c mice. Tumor growth was monitored by IVIS imaging. Tumor bearing mice were treated with Sindbis virus, α4-1BB Ab or SV plus α4-1BB Ab. On day 7 after treatment, splenocytes were harvested and surface markers, cytokines, and transcription factors were measured by flow cytometry or Elispot. Splenic T cells were isolated and RNA transcriptome analysis was performed. Tumor cured mice were rechallenged with tumor for testing immunological memory. RESULTS: SV vectors in combination with α4-1BB monoclonal antibody (mAb) completely eradicated a B-cell lymphoma in a preclinical mouse model, a result that could not be achieved with either treatment alone. Tumor elimination involves a synergistic effect of the combination that significantly boosts T cell cytotoxicity, IFNγ production, T cell proliferation, migration, and glycolysis. In addition, all mice that survived after treatment developed long lasting antitumor immunity, as shown by the rejection of A20 tumor rechallenge. We identified the molecular pathways, including upregulated cytokines, chemokines and metabolic pathways in T cells that are triggered by the combined therapy and help to achieve a highly effective anti-tumor response. CONCLUSIONS: Our study provides a novel, alternative method for B cell lymphoma treatment and describes a rationale to help translate SV vectors plus agonistic mAb into clinical applications.

Systemically Administered Sindbis Virus in Combination with Immune Checkpoint Blockade Induces Curative Anti-tumor Immunity.

Oncolytic viruses represent a promising form of cancer immunotherapy. We investigated the potential of Sindbis virus (SV) for the treatment of solid tumors expressing the human cancer testis antigen NYESO-1. NYESO-1 is an immunogenic antigen frequently expressed in numerous cancers, such as ovarian cancer. We show that SV expressing the tumor-associated antigen NYESO-1 (SV-NYESO1) acts as an immunostimulatory agent, inducing systemic and rapid lymphocyte activation, leading to a pro-inflammatory environment. SV-NYESO1 treatment combined with anti-programmed death 1 (anti-PD-1) markedly augmented the anti-tumor immunity in mice over the course of treatment, resulting in an avid systemic and intratumoral immune response. This response involved reduced presence of granulocytic myeloid-derived suppressor cells in tumors and an increase in the activation of splenic and tumor-infiltrating T cells. Combined therapy also induced enhanced cytotoxic activity of T cells against NYESO-1-expressing tumors. These results were in line with an observed inverse correlation between T cell activation and tumor growth. Finally, we show that combined therapy resulted in complete clearance of NYESO-1-expressing tumors in vivo and led to long-term protection against recurrences. These findings provide a rationale for clinical studies of SV-NYESO1 combined with immune checkpoint blockade anti-PD-1 to be used in the treatment of NYESO-1-expressing tumors.

Systemic tumor targeting and killing by Sindbis viral vectors.

Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects.

Tumor-specific in vivo transfection with HSV-1 thymidine kinase gene using a Sindbis viral vector as a basis for prodrug ganciclovir activation and PET.

UNLABELLED: One type of gene therapy of tumors, gene-directed enzyme-prodrug therapy (GDEPT), holds considerable promise, although practical considerations limit its clinical applicability. These include the lack of acceptable noninvasive methods that are adaptable to humans for selective tumor targeting of the therapeutic genetic material. Sindbis virus is an oncolytic, alpha-virus that selectively targets tumors through the 67-kDa laminin receptor (LAMR). In this report we describe a novel approach that permits tumor-selective tumor targeting and quantitative in vivo monitoring using PET of a commonly applied GDEPT, based on herpes simplex virus thymidine kinase type 1 (HSVtk) and ganciclovir (GCV). METHODS: Sindbis/tk vectors were harvested from the supernatant of in vitro cultures of a packaging cell produced by electroporation of both replicon RNA (SinRep5/tk) and helper RNA (DH-BB) into baby hamster kidney (BHK) cells. The therapeutic effect of GCV was determined by incubation of transfected tumor cells with increasing concentrations of GCV. BHK tumors growing as xenografts in severe combined immunodeficiency disease (SCID) mice were transfected by parenteral administration of the vector. Imaging was performed using small-animal PET at 2 h after injection of 18F fluoro-ethyl-arabinosyluridine (18F-FEAU) and 24 h after the final parenteral injection of Sindbis/tk viral vector. RESULTS: The vector efficiently expresses the HSVtk enzyme in infected tumor cells, both in vitro and in vivo. High levels of HSVtk expression ensure sufficient prodrug GCV conversion and activation for bystander effects that kill the surrounding untransduced tumor cells. Tumor localization of intravenously administered 18F-FEAU after 2 and 3 parenteral vector treatments of Sindbis/tk demonstrated uptake of 1.7 and 3.1 %ID/g (percentage injected dose per gram), respectively. CONCLUSION: The vector efficiently targets the HSVtk enzyme gene into Sindbis-infected tumor cells. High levels of HSVtk expression ensure sufficient prodrug GCV conversion and activation for bystander effects that killed many surrounding untransduced tumor cells. In addition, the HSVtk activities in tumors can be noninvasively monitored using PET after systemic Sindbis/tk treatments as a basis for determining the levels and tissue distribution of vector, noninvasively in living animals, and for optimizing in vivo transfection rates of tumor.

Gene therapy that safely targets and kills tumor cells throughout the body.

The authors studied the therapeutic value of Sindbis vectors for advanced metastatic cancer by using a variety of clinically accurate mouse models and demonstrated through imaging, histological, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in vivo, leading to significant suppression of tumor growth and enhanced survival. Use of two different bioluminescent genetic markers for the IVIS Imaging System (Xenogen Corp., Alameda, CA) permitted demonstration of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor, which is elevated in tumor versus normal cells, and downregulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. Laser capture microdissection of mouse tumor implants showed equivalent laminin receptor expression levels in the different tumor metastases in the peritoneal cavity. Incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer.

In vivo antitumor activity of Sindbis viral vectors.

BACKGROUND: Sindbis virus, a blood-borne virus transmitted by mosquitoes, has been used as a vector to efficiently express exogenous genes in vitro and in vivo and to induce apoptosis. Because Sindbis virus infects mammalian cells by interacting with the high-affinity laminin receptors, which are expressed at higher levels in several human cancers than in normal cells, we determined whether a Sindbis viral vector could be used to target cancers in vivo. METHODS: C.B-17-SCID mice with established xenografts were given daily intraperitoneal injections of the Sindbis viral vector SinRep/LacZ containing the bacterial beta-galactosidase gene. Control mice were untreated or received injections with phosphate-buffered saline. Tumor size was measured daily. Expression of beta-galactosidase and Factor VIII (a marker for endothelial cells) was determined by immunohistochemical staining of tumor sections. Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining. C.B-17-SCID beige mice, which lack natural killer (NK) cells, were used to assess the importance of NK cells in antitumor efficacy of Sindbis viral vectors. RESULTS: Tumors from mice treated with SinRep/LacZ were statistically significantly smaller than tumors from control mice. This effect was observed for tumor xenografts derived from BHK (kidney, hamster), LS174T (colon, human), HT29 (colon, human), and CFPAC (pancreas, human) cells. Expression of beta-galactosidase co-localized with that of Factor VIII in tumor sections. Tumors from SinRep/LacZ-treated mice contained more apoptotic cells than tumors from control mice. Complete tumor regression was observed in three of five C.B-17-SCID mice but in none of five C.B-17-SCID beige mice treated with SinRep/LacZ. CONCLUSION: Sindbis viral vectors efficiently targeted tumors in vivo, were apparently delivered through the circulation, and were more effective in the presence of NK cells.

Enhanced ubiquitinylation of heat shock protein 90 as a potential mechanism for mitotic cell death in cancer cells induced with hypericin.

A unique property of the photodynamic signal transduction inhibitor hypericin is functionality in the dark. We show in tumor cells that hypericin targets the heat shock protein (Hsp) 90 chaperone but not Hsp70 (Hsc70) to enhanced ubiquitinylation. As a consequence Hsp90 chaperone functionality is abrogated and the client proteins, mutant p53, Cdk4, Raf-1, and Plk, are displaced from complexes with Hsp90, destabilized, and degraded via a proteasome-independent pathway. Decline in Raf-1 prevents downstream activation of extracellular signal-regulated kinase 1/2 kinases, the Ras/Raf pathway is inhibited, and tumor cell proliferation is arrested. The cells exhibit multiple aberrations including retardation at G(2)-M, increased cell volume, and multinucleation, all of which are hallmarks of mitotic cell death. The studies demonstrate that ubiquitinylation of Hsp90 inactivates the chaperone, destabilizes the plethora of client proteins, and creates deficiencies in multiple unrelated cellular functions. This combination constitutes a mechanism by which hypericin generates mitotic cell death in cancer cells.

Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models.

We studied the therapeutic value of Sindbis vectors for advanced metastatic ovarian cancer by using two highly reproducible and clinically accurate mouse models: a SCID xenograft model, established by i.p. inoculation of human ES-2 ovarian cancer cells, and a syngenic C57BL/6 model, established by i.p. inoculation of mouse MOSEC ovarian cancer cells. We demonstrate through imaging, histologic, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in the peritoneal cavity, leading to significant suppression of the carcinomatosis in both animal models. Use of two different bioluminescent genetic markers for the IVIS Imaging System permitted demonstration, for the first time, of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis vector infection and growth suppression of murine MOSEC tumor cells indicate that Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor. Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. We show that incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer.