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.


Introduction 1.Ovarian Cancer as a Significant Health Concern
Ovarian cancer is one of the most common and lethal types of gynecological cancers with an overall survival rate of 50 percent that has not changed significantly for several decades [1][2][3].Although it can occur at any age, it primarily affects women who have gone through menopause.The estimated rate of new cases and death in 2023 are 19,710 and 13,720, respectively [4].Due to the absence of noticeable symptoms, ovarian cancer often goes undetected in its early stages and hence is referred to as the "silent killer" [5][6][7][8].As the disease progresses, symptoms include abdominal bloating or swelling, difficulty eating or feeling full quickly, pelvic pain and fatigue.These symptoms can be indicative of other conditions as well, which makes early detection and accurate diagnosis challenging.As a result, patients are frequently diagnosed at an advanced stage when tumors have already spread beyond the ovaries.Thus, metastasis presents the greatest therapeutic challenge, restricting successful treatment of patients and dramatically reducing the overall survival rate [9,10].
There are different types of ovarian cancer, with epithelial ovarian cancer being the most common form, accounting for about 90% of cases [9,[11][12][13][14][15]. Highly aggressive highgrade serous ovarian carcinoma (HGSOC) is the most common epithelial subtype with a tendency to develop early chemotherapy resistance.HGSOC presents with various molecular abnormalities, especially TP53 mutations observed in the majority of tumors [16].Other less common types include germ cell tumors and stromal tumors, which develop in the cells that produce eggs and hormones within the ovary.The exact cause of ovarian cancer is not well understood, but certain factors have been identified that may increase a woman's risk of developing the disease.These risk factors include a family history of ovarian or breast cancer, certain inherited gene mutations (such as TP53, BRCA1, and BRCA2), increasing age, obesity, and certain hormonal factors [12,13,17,18].

Current Treatment Challenges and Limitations
Current treatment strategies typically involve a combination of surgery, systemic chemotherapy, and sometimes radiation therapy.Surgery, which is often the first line of treatment, aims to remove as much tumor tissue as possible (referred to as "debulking" [19,20]), which includes removal of one or both ovaries, fallopian tubes, uterus, and other affected tissues such as colon tissue.Adjuvant chemotherapy uses drugs to kill cancer cells that may have spread to other parts of the body [20,21].These procedures and their limitations are a major burden for patients.Clinical trials and new therapies that explore innovative approaches, including targeted therapies and immunotherapy, are much needed to improve outcomes for women with this disease.Additionally, ongoing efforts to raise awareness about the symptoms and risk factors of ovarian cancer are crucial in promoting early detection and better overall survival rates.

HGSOC Tumor Microenvironment Presents Obstacles to Treatment
Current immunotherapeutic approaches have not been as promising as for other cancers due to the unique tumor microenvironment (TME) of ovarian cancer that facilitates efficient metastasis and dramatically impairs immune surveillance.Several comprehensive reviews discuss the composition and effects of the ascites TME on ovarian cancer progression and treatment [22][23][24][25][26][27].
HGSOC cells mainly metastasize within the peritoneal cavity [28].The transition of epithelial ovarian cancer cells (EOCs) to a mesenchymal phenotype (EMT) involves aberrant expression of adhesion molecules [29,30] resulting in the loss of tight junctions and a more invasive behavior [31].Cells that exfoliate from the primary tumor survive detachment by forming spheroids composed of tumor and non-tumor cells that can adhere to the mesothelium, omentum, and organs within the peritoneal cavity [32].
Peritoneal membrane permeability and angiogenesis, induced by EOC overexpression of vascular endothelial growth factor (VEGF), contributes to the accumulation of ascites fluid [33].More than 90% of patients with stage III and IV ovarian cancer develop ascites fluid, the components of which constitute the TME [34].The ascites TME contains diverse cell types including mesothelial, endothelial cells, immune cells, adipocytes, and fibroblasts.Interaction between tumor cells and the TME can occur through direct cell contact, soluble molecules, or exosome vesicles released by cells.The crosstalk among TME components shapes cellular phenotypes ultimately determining tumor progression or suppression.

Anti-Tumor Immune Components in the TME
The migration of immune cells to the EOC tumor is orchestrated by various cytokines and chemokines (reviewed in [35][36][37]).Chemokine CCL5, constitutively expressed by EOC cells, induces the infiltration of CD8 T cells [38].Interaction with peptides presented by MHC I on tumor cells induces CD8 T cells to produce cytolytic factors, granzyme and perforin, and cytokines IL-12, IL-2 and IFNγ that act to kill tumor cells.This immunogenic cell death along with genome instability [39] releases danger signals (DAMPS) [40] that attract antigen presenting (APC) and innate natural killer (NK) cells.In this environment, tumor-associated antigens (TAAs) can be presented to activate and amplify cytolytic T cell and B cell anti-tumor responses.Prevalent TAAs in HGSOC include NY-ESO-1 [41], MAGE [42], p53 mutation, and WT1 [43,44].
Interferons play an important role in the TME by regulating the gene expression of tumor infiltrating lymphocytes (TILs) [37].Plasmacytoid dendritic cells, CD8 T cells, NK cells and T cell helper 1 (Th1) CD4 T cells are major sources of IFNγ.Anti-tumor M1 macrophages are induced by IFNγ and lipopolysaccharides [45].IFNγ induces myeloid cell CXCL9 secretion, which cooperates with CCL5 to enhance lymphocyte recruitment [38].IFN I induces CXCL13 expression in tumors, which correlates with the generation of tertiary lymphoid structures and infiltration of CD4, CD8 T cells and CD20 B cells [46].Oncolytic viruses represent an evolving field of research for the treatment of ovarian cancer due to their ability to target and directly kill cancer cells while synergistically stimulating the body's own anti-tumor immune response that also protects from tumor recurrence.Oncolytic viruses can also carry therapeutic genes to enhance their anti-tumor effects.These vectors are called "armed" oncolytic viruses.The expression of these genes is fully dependent on the replication of the virus [93,94].Unlike gene therapy and gene editing, which needs to be customized for each patient, oncolytic viruses offer a targeted, yet multifaceted approach that could potentially improve outcomes for patients with this challenging disease.
Different types of oncolytic viruses have been investigated in preclinical and clinical studies for ovarian cancer treatment [95].These viral vectors can be modified to target and kill ovarian cancer cells while stimulating immune responses.Table 3 lists studies of recent oncolytic virus-mediated therapies.The genomes of oncolytic viruses can consist of double-stranded DNA, single-stranded, negative sense RNA or single-stranded, positive sense RNA.Table 4 shows current clinical trials.Sindbis virus (SV), an enveloped, single-stranded, positive sense RNA virus, is a member of the alphavirus genus, Togavirus family [116].In depth reviews of alphavirus structure, expression, infection, replication, and evolution have been published [116][117][118].
Alphaviruses have many attributes that render them advantageous for gene expression vectors: (1) they exhibit a broad host range for mammalian cell infection [119]; (2) RNA genomes, which mimic mRNA, quickly form replication complexes in the cytoplasm of infected cells; (3) the lack of DNA intermediates avoids the risk of insertional chromosome mutagenesis [120][121][122]; (4) approximately 10 6 copies of viral RNA, coupled with a strong subgenomic promoter, enable very high expression levels of recombinant protein [120,123]; and (5) vectors are easy to manipulate and can accommodate at least 8000 bp of heterologous mRNA [124].

Vector Safety
In nature, SV has the safest profile among alphaviruses with mostly asymptomatic infections causing mild fever, rash or arthralgia [145][146][147].In addition, for added safety SV-derived vectors can be produced in a manner that prevents the amplification of virus particles (Figure 1).The cDNA sequence of the RNA genome is split into two segments and cloned into separate plasmids.A replicon DNA plasmid encodes the non-structural replicase proteins and contains a strong subgenomic promoter to express heterologous "genes of interest".A helper DNA plasmid, encoding the viral capsid and envelope proteins, lacks a packaging signal so that vector particles only contain the replicase and heterologous genes and can undergo only one round of infection [120,123,148].

Vector Safety
In nature, SV has the safest profile among alphaviruses with mostly asymptomatic infections causing mild fever, rash or arthralgia [145][146][147].In addition, for added safety SV-derived vectors can be produced in a manner that prevents the amplification of virus particles (Figure 1).The cDNA sequence of the RNA genome is split into two segments and cloned into separate plasmids.A replicon DNA plasmid encodes the non-structural replicase proteins and contains a strong subgenomic promoter to express heterologous "genes of interest".A helper DNA plasmid, encoding the viral capsid and envelope proteins, lacks a packaging signal so that vector particles only contain the replicase and heterologous genes and can undergo only one round of infection [120,123,148].

Sindbis Virus Selectively Targets and Replicates within Ovarian Cancer Cells While Sparing Healthy Cells
Immunohistochemical and bioluminescent in vivo imaging studies using SV vectors that express reporter genes indicate the specific targeting of tumor and metastatic cells in both murine xenotropic and syngeneic ovarian cancer models [125,149].Figure 2 shows that SV vector expressing Firefly luciferase (SV/Fluc) localizes to tumors and metastases in a syngeneic C57BL/6 MOSEC ovarian cancer model [150] while non-tumor bearing control mice show no significant bioluminescent signals.Similar results were observed in a nude mice human ovarian cancer model systemically treated with replication-positive SV [115].
Sindbis virus has been shown to bind with the 67 KDa high affinity laminin receptor (LAMR) protein [151,152].LAMR has a high affinity for laminin, a major component of cell basement membranes that plays an important role in cellular adhesion, morphology, differentiation, and migration (reviewed in [153]).The presence and conservation of LAMR among many distantly related species is consistent with the broad host range of the Sindbis virus.The overexpression of LAMR by cDNA transfection into Baby hamster kidney (BHK) cells increased the level of SV infection [152] while LAMR antisense RNA [152], silencing siRNA [125] or short hairpin shRNA [154] decreased infection.
both murine xenotropic and syngeneic ovarian cancer models [125,149].Figure 2 shows that SV vector expressing Firefly luciferase (SV/Fluc) localizes to tumors and metastases in a syngeneic C57BL/6 MOSEC ovarian cancer model [150] while non-tumor bearing control mice show no significant bioluminescent signals.Similar results were observed in a nude mice human ovarian cancer model systemically treated with replication-positive SV [115].Sindbis virus has been shown to bind with the 67 KDa high affinity laminin receptor (LAMR) protein [151,152].LAMR has a high affinity for laminin, a major component of cell basement membranes that plays an important role in cellular adhesion, morphology, differentiation, and migration (reviewed in [153]).The presence and conservation of LAMR among many distantly related species is consistent with the broad host range of the Sindbis virus.The overexpression of LAMR by cDNA transfection into Baby hamster kidney (BHK) cells increased the level of SV infection [152] while LAMR antisense RNA [152], silencing siRNA [125] or short hairpin shRNA [154] decreased infection.
Although the exact mechanism of SV infection via LAMR has not been elucidated (reviewed in [155]), it has been determined that the glutamic acid residue at amino acid position 70 of the envelope E2 protein is critical for in vivo tumor targeting [156].The overexpression of the 67 KDa LAMR occurs on the surface of many human cancer cells [157][158][159][160][161][162] including those of ovarian origin [162,163].LAMR monoclonal antibody binding studies suggest that tumor cells have varying degrees of unoccupied cell-surface LAMRs that conceivably result from the increased biosynthesis of LAMR or the Although the exact mechanism of SV infection via LAMR has not been elucidated (reviewed in [155]), it has been determined that the glutamic acid residue at amino acid position 70 of the envelope E2 protein is critical for in vivo tumor targeting [156].The overexpression of the 67 KDa LAMR occurs on the surface of many human cancer cells [157][158][159][160][161][162] including those of ovarian origin [162,163].LAMR monoclonal antibody binding studies suggest that tumor cells have varying degrees of unoccupied cell-surface LAMRs that conceivably result from the increased biosynthesis of LAMR or the dissolution of the surrounding basement membrane and extracellular matrix by tumor cells [164][165][166][167].It is thus plausible that SV uses unoccupied LAMRs as fortuitous binding sites on tumor cells.Other SV receptors have been more recently discovered.The natural resistance-associated macrophage protein (NRAMP), a divalent metal transport protein, has been identified as an SV receptor in Drosophila and the vertebrate homolog, NRAMP2, as an SV receptor in mammalian cells [168].VLDLR and ApoER2, members of the low-density lipoprotein family, were found to be receptors for certain alphaviruses including SV [169].Affinity purification followed by mass spectrometry identified the CD147 membrane protein as a receptor for SV and other alphaviruses in human cells [170].Notably, CD147, which has been found to be overexpressed on many cancer cells and cells within the tumor microenvironment, plays a role in tumor proliferation and the inhibition of apoptosis [171].The protumor effects of CD147 may potentially be obviated by SV binding, infection, and cell lysis.

Mechanisms of Action against Ovarian Cancer Cells
SV vector cancer therapy can involve several processes.SV replication complexes produce abundant RNA molecules with double stranded RNA intermediates triggering "danger signals" [40].The cytotoxic effects of SV infection result from the inhibition of cellular protein translation, activation of a stress response, and ultimately an apoptotic cascade [126].The expression of tumor-associated antigens (TAAs) or immunomodulatory agents can augment anti-tumor effects.

Immunotherapeutic Effects of SV Vectors
The ability of SV vector treatment to stimulate a rapid influx of activated innate natural killer cells (NK) was demonstrated using a xenotropic ES-2 human ovarian cancer [172] model in SCID (severe combined immunodeficiency) mice, which lack T and B cells [133].Significant inhibition of tumor growth and increased survival were observed.The incorporation of IL-12 into the SV vector enhanced the therapeutic effect by inducing IFNγ secretion from NK cells, which was shown to upregulate the expression of MHC class II on peritoneal macrophages promoting an M1 anti-tumor effect.
The potential of SV vectors to promote an adaptive immune response against tumors was studied in a syngeneic, immunocompetent BALB/c CT26 colon carcinoma tumor model [129].As CT26 cells are not susceptible to SV infection, this model separated SV oncolytic activity from potential immunogenic effects.A bioluminescent signal was first observed in the mediastinal lymph nodes that drain the peritoneum 3 h after SV/Firefly luciferase i.p. injection.When CT26 TAAs were expressed by SV vectors, an influx of activated CD8 T cells to the peritoneum occurred within one week.Effector and memory CD8 T cells were generated correlating with long-term survival.The cytolytic activity of immune cells released endogenous CT26 TAAs that were shown to be engaged by CD8 T cell specific tetramers revealing that SV vectors can be therapeutic via epitope spreading without oncolytic tumor targeting.
The observation that IL-12 increases the presence of OX40 (CD134) on the surface of CD4 T cells [181,183] prompted the study of a combined anti-tumor capacity.IL-12 activates T cells, stimulates the production of IFNγ and increases the expression of OX40 on effector CD4 T cells [183].The combination of SV.IL-12 with an agonistic antibody to OX40 exhibits strong therapeutic efficacy in CT26, colon, and MyC-CaP, prostate carcinoma, models [181].The transcriptome and metabolic reprogramming of T cells drove the development of activated effector T cells with enhanced tumor infiltration and anti-tumor capacity within the TME.
OX40 is a member of the tumor necrosis family that is expressed on activated T cells [184].OX40 promotes the clonal expansion, differentiation, and survival of CD4 Th1 helper cells, which produce IFNγ and IL-2 cytokines [185][186][187][188][189] that sustain the survival of primed CD8 T cells [184,190].The co-expression of OX40 with ICOS on follicular T helper cells (Tfh) facilitates the differentiation of antibody-producing B cells and long-lived plasma cells from germinal center B cells [191].In addition, OX40 signaling represses regulatory T cells (Treg) by downregulating the expression of Foxp3 [192].
Treatment with SV.IL-12, αOX40 and SV.IgGOX40 increased the migration of immune T cells into MOSEC.Fluc.p11tumors within 7 days, as shown by the overlapping of multiplex immunofluorescence staining of CD8 and CD4 T cells with Ki67 and granzyme B markers for proliferating cytotoxic T cells [114].Significantly more lysis of tumor tissue was observed when αOX40 was delivered by SV.αOX40 vector presumably as SV infection of MOSEC cells produce high levels of and αOX40 to stimulate a cytotoxic immune response and SV vector induced apoptosis.
The immune function of T cells require a high metabolic state.The ascites TME, characterized by hypoxia, acidosis, and low nutrient levels, impairs the metabolism and function of T cells (reviewed in [50,[193][194][195]).The metabolic profiles of splenic T cells were measured by Agilent Seahorse technology.Only SV.IL-12 combined with αOX40 provided T cells with a spare respiratory capacity and a higher basal energetic state [114].
To examine sustained protection from MOSEC growth, mice were treated with either SV.IL-12 or SV.IgGOX40.IL-12 and at 140 days, surviving mice were rechallenged with MOSEC cells (Figure 3).Naive control mice, inoculated with tumor cells at the same time as the rechallenged mice, succumbed after 35 days while both vectors provided long-term tumor suppression.The ability of SV.IL-12 to stimulate OX40 expression on T cells may account for the efficacy of both SV.IgGOX40.IL-12 and SV.IL-2 in this cancer model [114].
Treatment with SV.IL-12, ⍺OX40 and SV.IgGOX40 increased the migration of immune T cells into MOSEC.Fluc.p11tumors within 7 days, as shown by the overlapping of multiplex immunofluorescence staining of CD8 and CD4 T cells with Ki67 and granzyme B markers for proliferating cytotoxic T cells [114].Significantly more lysis of tumor tissue was observed when ⍺OX40 was delivered by SV.⍺OX40 vector presumably as SV infection of MOSEC cells produce high levels of and ⍺OX40 to stimulate a cytotoxic immune response and SV vector induced apoptosis.
The immune function of T cells require a high metabolic state.The ascites TME, characterized by hypoxia, acidosis, and low nutrient levels, impairs the metabolism and function of T cells (reviewed in [50,[193][194][195]).The metabolic profiles of splenic T cells were measured by Agilent Seahorse technology.Only SV.IL-12 combined with ⍺OX40 provided T cells with a spare respiratory capacity and a higher basal energetic state [114].
To examine sustained protection from MOSEC growth, mice were treated with either SV.IL-12 or SV.IgGOX40.IL-12 and at 140 days, surviving mice were rechallenged with MOSEC cells (Figure 3).Naive control mice, inoculated with tumor cells at the same time as the rechallenged mice, succumbed after 35 days while both vectors provided long-term tumor suppression.The ability of SV.IL-12 to stimulate OX40 expression on T cells may account for the efficacy of both SV.IgGOX40.IL-12 and SV.IL-2 in this cancer model [114].In vivo antibody depletion studies indicated that while both CD4 and CD8 T cells are important for SV.IgGOX40.IL-12 efficacy, CD4 T cells appear to play a more pivotal role [114].This observation coincides with previous results showing that SV.IL-12 and αOX40 elevate and sustain cytotoxic CD4 T cells [181].Effector CD4 T cell have been increasingly recognized for anti-tumor activity, independent of their helper function [196,197].
Transcriptome analysis, performed for untreated MOSEC tumors vs. tumors treated with SV empty vector, SV.IL-12, SV.αOX40 or SV.IgGOX40.IL-12 showed distinct gene expression profiles for all treatment groups.Empty SV vector showed the lowest number of differentially expressed genes implying that the "armed" vectors were deployed at tumor sites.Pathway and network analysis showed the downregulation of genes involved with DNA replication, transcription, and cell division correlating with the elimination of tumor cells.Upregulated genes were predominantly related to immune response pathways most likely correlating with infiltrated lymphocytes.
The modulation of anti-tumor immune responses is illustrated in Figure 4.The ability of SV.IgGOX40.IL-12 to systemically target metastatic tumors while altering the transcriptome signature and metabolic program of T cells, increasing their capacity to infiltrate the repressive TME, renders these vectors a promising therapy for ovarian and other types of cancers.
ways most likely correlating with infiltrated lymphocytes.
The modulation of anti-tumor immune responses is illustrated in Figure 4.The ability of SV.IgGOX40.IL-12 to systemically target metastatic tumors while altering the transcriptome signature and metabolic program of T cells, increasing their capacity to infiltrate the repressive TME, renders these vectors a promising therapy for ovarian and other types of cancers.

Summary
The unique TME of HGSOC complicates treatment strategies.More than 90% of stage III/IV patients develop malignant ascites fluid.Crosstalk among the myriad of the components of the ascites TME modulates the phenotypes of tumor and immune response cells.The goals of oncolytic virus-mediated therapies must include tumor targeting and killing, as well as, the skewing of the TME toward a strong immune response.Several recent studies and clinical trials have been presented.
A SV vector platform has been developed combining the expression of IL-12 and an agonistic antibody targeting the co-stimulatory OX40 receptor.The SV.IgGOX40.IL-12 vector decreases the tumor burden in ovarian cancer mouse models, increases survival and provides protection against tumor rechallenge.A notable influx of immune cells into the tumor microenvironment occurs.Treatment efficacy is associated with the

Summary
The unique TME of HGSOC complicates treatment strategies.More than 90% of stage III/IV patients develop malignant ascites fluid.Crosstalk among the myriad of the components of the ascites TME modulates the phenotypes of tumor and immune response cells.The goals of oncolytic virus-mediated therapies must include tumor targeting and killing, as well as, the skewing of the TME toward a strong immune response.Several recent studies and clinical trials have been presented.
A SV vector platform has been developed combining the expression of IL-12 and an agonistic antibody targeting the co-stimulatory OX40 receptor.The SV.IgGOX40.IL-12 vector decreases the tumor burden in ovarian cancer mouse models, increases survival and provides protection against tumor rechallenge.A notable influx of immune cells into the tumor microenvironment occurs.Treatment efficacy is associated with the transcriptional reprogramming of T cells leading to the expression of immune response genes and metabolic alterations that result in higher energy states.
The lack of clinical trials involving SV vectors, thus far, makes it difficult to evaluate their transition from animal models to human patients.Translation to clinical applications is feasible as SV vectors can be produced and purified under Good Manufacturing Practice (GMP) standards, ensuring high titers (10 11 transducing units per milliliter (TU/mL)) to compensate for dilution in the bloodstream or TME.Vectors might be administered directly to patients or after the debulking of tumors and ascites where they may have a greater chance of preventing a suppressive TME.Treatment efficacy may require multiple doses.While alphaviruses are not highly immunogenic, the requirement for many doses could pose challenges and may lead to patient reluctance to undergo treatment.The ease of SV vector modifications, however, will allow continued optimization.

Future Perspectives
The lytic activity of oncolytic viruses, which selectively replicate in tumor cells, initiated their role as tools for cancer treatment.Ultimately, lytic effects were observed to reshape "cold" into "hot" tumors that are more responsive to immunotherapies.Tables 3 and 4 present several families of oncolytic viruses that have been studied in combination with chemotherapy, checkpoint inhibitors, and other immunomodulatory agents for the treatment of ovarian cancer.Several oncolytic viruses have also been genetically modified to express therapeutic cargos.
Future studies should identify and optimize interactions between oncolytic viruses and the immune system within the TME.Epigenetic changes to cancer cells and effects on viral infection and replication should also be considered (reviewed in [198,199]).Epigenetic modifications of the genome, which include DNA methylation and histone acetylation, are often deregulated in tumors leading to cell proliferation.The inhibition of these modifiers have been shown to attenuate cellular anti-viral response, promote cancer cell cycle arrest and apoptosis and potentiate the immune response.The inhibition of histone deacetylase (HDACi) has been shown to augment adenovirus oncolysis in cisplatin-resistant ovarian cancer cells [200].
The considerable heterogeneity within patients with ovarian cancer involving genetic, epigenetic, and immunological systems presents a challenge to therapy.Oncolytic viruses combined with the modulators of the immune system, epigenome, and chemical drugs can provide powerful weapons in the arsenal against ovarian cancer.Funding: Funding was provided by NIH 5R44CA250627 and through a Research and Licensing Agreement between Cynvec and NYU Langone, which licenses the Sindbis technology to Cynvec.This work was also supported, in part, by The Experimental Pathology Research Laboratory at NYU Langone that is partially supported by the Cancer Center Support Grant P30CA016087.The Vectra3 multispectral imaging system was purchased through Shared Instrumentation Grant S10 OD021747.
Institutional Review Board Statement: All experiments were performed in accordance with the Institutional Animal Care and Use Committee of New York University Health.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequencing data that support the findings of this study will be.deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through the GEO Series accession number that will be provided and including all other relevant data included in the article, and further inquiries can be directed to the corresponding authors.

Acknowledgments:
We wish to thank all the postdoctoral fellows, graduate students and technicians who contributed to the studies described in this review.We appreciate the Funding was provided by NIH 5R44CA250627.We would like to thank the NYU High Throughput Biology Laboratory for Seahorse usage, the NYU Genome Technology Center for RNA sequencing service and the Experimental Pathology Research Laboratory at NYU Langone, which also supported this work, in part, by the Experimental Pathology Research Laboratory at NYU Langone, which is partially supported by the Cancer Center Support Grant P30CA016087.The Vectra3 multispectral imaging system was purchased through Shared Instrumentation Grant S10 OD021747.

Figure 1 .
Figure 1.Preparation of SV vector.Plasmids are linearized, transcribed by T7 polymerase and capped in vitro.Transcripts are electroporated into BHK cells and viral vectors harvested from media [148].T7, transcription promoter; Psg, Sindbis subgenomic promoter; GOI, gene of interest; AAA poly A tail; BHK, baby hamster kidney cells.Created with Biorender.

Figure 4 .
Figure 4. Summary of synergistic immune stimulating anti-tumor mechanism of armed SVs via (1) direct tumor oncolysis and (2) tumor influx of activated immune cells that enhance the anti-tumor response in the tumor microenvironment (TME) [114].

Figure 4 .
Figure 4. Summary of synergistic immune stimulating anti-tumor mechanism of armed SVs via (1) direct tumor oncolysis and (2) tumor influx of activated immune cells that enhance the anti-tumor response in the tumor microenvironment (TME) [114].
Author Contributions: D.M., C.P., A.H. and S.O.conceptualized the review; original papers, all authors; writing, C.P. and S.O.; editing, D.M. and A.H., preparation of figures, A.H., S.O. and C.P.; supervision, project administration, funding acquisition, D.M.All authors have read and agreed to the published version of the manuscript.
Interest: All authors are employed by NYU Langone School of Medicine and have no employment relationship or consultancy agreement with Cynvec, a biotechnology company that supports some studies under a Research and Licensing agreement with NYU.S.O., A.H., C.P., and D.M. are inventors on one or several issued patents and/or patent applications held by NYU that cover the Sindbis treatment of neoplasia and COVID-19.As part of the Research and Licensing agreement, the authors who are inventors on patents are entitled to a portion of the royalties that NYU Langone would receive, should Sindbis vectors be approved by the FDA for therapeutic or vaccination use.Data and materials availability: Correspondence should be addressed to D.M.

Table 2 .
Pro-Tumor signaling factors and pathways.

Table 3 .
Studies of Oncolytic Virus-mediated Treatment of Ovarian Cancer.

Table 4 .
Clinical trials of oncolytic virus mediated treatment of ovarian cancer.

. Sindbis Virus as a Novel Approach to Ovarian Cancer Treatment 2
.1.Sindbis Is a Prototypic Alphavirus