Nace, Rebecca A.

Abstract VSV-IFNβ-NIS (VV1) is a Vesicular Stomatitis Virus (VSV) that encodes the interferon beta (IFNβ) and the iodide symporter (NIS) gene. The IFNβ gene confers tumor selectivity and enhances host antitumor immunity while the NIS gene acts as an imaging biomarker, allowing noninvasive imaging of viral infection sites using PET/SPECT. Multiple myeloma (MM) is a cancer of plasma cells and poses a challenge for treatment due to its biological heterogeneity. As MM usually relapses and is refractory, current therapies face the issue of drug resistance and patients may have to turn to novel therapies. Therefore, we set out to understand the activity of VSV which could be a new approach for patients with relapsed refractory MM (RRMM). Our study utilized two unique bortezomib resistant Vk*MYC transplantable models, Vk12598 and Vk12653, with distinct genomic features. Upon re-transplantation of splenocytes from these mice, flow cytometry revealed that Vk12598 and Vk12653 mice had high MM populations in the spleen and bone marrow. To assess VV1 efficacy in tumor bearing mice, we performed a VV1 dose escalation study, showing efficacy in delaying MM development and prolonging survival in both mice models, with Vk12598 being the more responsive model. Later studies also showed that VV1 induces tumor remission in the Vk12598 model when treatment was initiated at low tumor burden (gamma/albumin ratio ∼0.2). IFNβ levels increased significantly post 24-72h of VV1 treatment, indicating targeted infection and replication of the virus in MM cells. However, high doses of VV1 led to toxicity, as observed from early mortality and weight loss. To determine the sites of MM metastasis and VV1 infection, timepoint harvests were carried out. Hematoxylin and eosin and immunostainings showed the onset of MM of Vk*MYC in the bone marrow and spleen first, then later in the liver. Immunofluorescent staining confirmed that the higher the tumor burden observed, the more VV1 was present in the tissues. To enhance VV1 efficacy, we performed in vivo combination therapies of VV1 with anti-PD-1, an inhibitor of PD-1 which is overexpressed on MM patient T cells; and bortezomib, which inhibits NF-κB which in turn prevents IFN pathway protein transcription, potentially enhancing the replication of VV1 virus. In Vk12598 mice, VV1 combined with both drugs induced tumor regression and doubled the median survival time compared to treatment with VV1 only or VV1 coupled with either of the drugs individually. Addition of VV1 to either anti-PD-1 or bortezomib alone also increased survival time significantly. Studies are still ongoing to understand the mechanisms of VSV and its combination treatments in Vk*MYC models. This translational study will allow us to gain the knowledge needed to refine VSV treatments and provide novel therapy combinations to improve MM patient survival. Citation Format: Yi Ying Ma, Lianwen Zhang, Rebecca A. Nace, Marta Chesi, Stephen J. Russell, Kah Whye Peng. Harnessing oncolytic virus VSV and combination therapy to advance treatments for relapsed refractory multiple myeloma using Vk*MYC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 940.

Background The varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a potential association between VZV infection and cancer remission. Methods Here, we constructed oncolytic VZV candidates based on the vaccine strain vOka and the laboratory strain Ellen. These newly engineered viruses were subsequently assessed for their oncolytic properties in the human MeWo melanoma xenograft model and the mouse B16-F10-nectin1 melanoma syngeneic model. Results In the MeWo xenograft model, both vOka and Ellen exhibited potent antitumor efficacy. However, it was observed that introducing a hyperfusogenic mutation into glycoprotein B led to a reduction in VZV’s effectiveness. Notably, the deletion of ORF8 (encodes viral deoxyuridine triphosphatase) attenuated the replication of VZV both in vitro and in vivo, but it did not compromise VZV’s oncolytic potency. We further armed the VZV Ellen-ΔORF8 vector with a tet-off controlled mouse single-chain IL12 (scIL12) gene cassette. This augmented virus was validated for its oncolytic activity and triggered systemic antitumor immune responses in the immunocompetent B16-F10-nectin1 model. Conclusions These findings highlight the potential of using Ellen-ΔORF8-tet-off-scIL12 as a novel VZV-based oncolytic virotherapy.

Although the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

Abstract Oncolytic virotherapy aims to activate host antitumor immunity, but the effectiveness of current intratumoral herpes-simplex virus (HSV) based monotherapy is limited. Here, to boost the potency of cancer cell targeting α-herpesviruses derived from HSV or Varicella-Zoster Virus (VZV), we coinjected them with myeloid cell-tropic β-herpesviruses derived from cytomegalovirus (CMV) which can powerfully modulate immune responses. Co-administered CMV was shown to promote intratumoral replication of HSV, increase local release of pro-inflammatory cytokines, inhibit adaptive antiviral immune responses, enhance systemic antitumor responses, and lead to superior control of both injected and contralateral tumors. CMV-encoded myeloid-tropic genes, NK evasins (i.e. the vMHC-I homolog), and the m02-m16 region (involved in downregulating classical MHC-I) were implicated in enhancing antitumor activities. CMV is a promising adjuvant to reshape and enhance the antitumor immune responses following α-herpesvirus therapy.

The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR “fingerprinting” approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.

The infectivity of certain retroviruses is limited to dividing cells, which makes them attractive tools for targeting cancer cell proliferation. Previously developed replication-competent gammaretroviral vectors spread efficiently in rapidly dividing cancer cells, but not in cancer cells that divide more slowly.

Abstract Measles viruses (MV) are rapidly inactivated by anti-measles neutralizing antibodies, which has limited their clinical performance as oncolytic agents. Here, by substituting the H and F surface glycoproteins of MV with those from the homologous canine distemper virus (CDV) and engineering the CDV H attachment protein to target EGFR or CD38, we generated a fully retargeted MV capable of resisting neutralization by measles-immune human serum. The resultant recombinant MVs encoding retargeted CDV envelope glycoproteins had similar growth kinetics as the control MV, showed the expected engineered receptor specificities for cell entry, intercellular fusion, and target cell killing, and were blind to native CDV receptors. In contrast to the control MV, recombinant MVs incorporating CDV F and H glycoproteins retained full infectivity when exposed to high concentrations of pooled measles-immune human serum. Comparing viruses bearing MV or CDV glycoproteins in the SKOV3ip.1 model, only the virus bearing an EGFR-retargeted CDV envelope glycoprotein complex was capable of limiting tumor growth and extending the survival in measles immune mice. MV, “stealthed” and retargeted using engineered CDV surface glycoproteins, may be a promising platform to advance for systemic cancer therapy in measles immune patients.

Abstract Abstract 378 The need for new and more effective long-term treatments for Multiple Myeloma (MM) has led to the utilization and engineering of replication competent (oncolytic) viruses as potential therapies. Vesicular stomatitis virus (VSV) is a potent oncolytic agent with several features that make it a favorable choice as a potential myeloma therapy. Specifically, (i) VSV replicates rapidly and can be grown to high titers (for effective clinical use) (ii) VSV undergoes transcription and replication exclusively in the cytoplasm avoiding host genome integration (iii) there is low/absent pre-existing immunity against VSV among the general population, (iv) naturally occurring human VSV infections are generally asymptomatic or result in minimal flu-like symptoms, (v) VSV is not easily transmitted between individuals (natural transmission is by hematophagous insects). Previously we showed weak oncolytic efficacy with an attenuated strain of VSV coding for the sodium iodide symporter (NIS) gene, VSV-D51-NIS, in the immune competent 5TGM1 syngeneic MM mouse model (C57Bl/KalwRijHsd) (Goel at. al. Blood. 2007 Oct 1;110(7):2342-50). Since VSV replication is strongly inhibited by IFN-induced innate immune responses in normal cells, but not in myeloma cells and IFN has known anti-myeloma activity, we hypothesized that the IFN-coding VSVs would be safer and more potent than previously tested VSV recombinants. We therefore constructed and tested the efficacy of VSVs coding for b-Interferon (VSV-IFN) or b-IFN and NIS (VSV-IFN-NIS). Interestingly, all of the newly constructed viruses, including VSV-IFN-NIS, showed greatly superior replication kinetics compared to the previously reported VSV-D51-NIS virus. Furthermore, compared to VSV-D51-NIS, VSV-IFN-NIS vectors induced higher NIS expression in vitro. In vivo therapy studies showed that a single intravenous dose of each of the IFN-coding VSVs promoted tumor regression and significantly prolonged survival of immunocompetent mice bearing subcutaneous or orthotopic 5TGM1 myeloma tumors. Tc-99m imaging studies conducted in mice treated with VSV-IFN-NIS, showed tumor specific virus mediated NIS expression and radio-isotope uptake that increased concurrently with intratumoral viral spread. Most importantly, there was no evidence of neurotoxicity following treatment with the IFN-coding VSVs. These studies indicate that VSV-IFN-NIS has potential as a novel therapeutic agent for multiple myeloma that can be combined with radio-isotopes for both non-invasive imaging of viral biodistribution and radiovirotherapy. A phase I clinical study is currently planned. Disclosures: No relevant conflicts of interest to declare.

ABSTRACT Mengovirus, a member of the Picornaviridae family, has a broad cell tropism and can cause encephalitis and myocarditis in multiple mammalian species. Attenuation has been achieved by shortening the polycytidine tract in the 5′ noncoding region (NCR). A poly(C)-truncated strain of mengovirus, vMC 24 , resulted in significant tumor regression in immunocompetent BALB/c mice bearing syngeneic MPC-11 plasmacytomas, but the associated toxicities were unacceptable. To enhance its safety profile, microRNA target sequences complementary to miR-124 or miR-125 (enriched in nervous tissue), miR-133 and miR-208 (enriched in cardiac tissue), or miR-142 (control; enriched in hematopoietic tissues) were inserted into the vMC 24 NCRs. The microRNA-detargeted viruses showed reduced replication and cell killing specifically in cells expressing the cognate microRNAs, but certain insertions additionally were associated with nonspecific suppression of viral fitness in vivo. In vivo toxicity testing confirmed that miR-124 targets within the 5′ NCR suppressed virus replication in the central nervous system while miR-133 and miR-208 targets in the 3′ NCR suppressed viral replication in cardiac tissue. A dual-detargeted virus named vMC 24 -NC, with miR-124 targets in the 5′ NCR and miR-133 plus miR-208 targets in the 3′ NCR, showed the suppression of replication in both nervous and cardiac tissues but retained full oncolytic potency when administered by intratumoral (10 6 50% tissue culture infectious doses [TCID 50 ]) or intravenous (10 7 to 10 8 TCID 50 ) injection into BALB/c mice bearing MPC-11 plasmacytomas. Overall survival of vMC 24 -NC-treated tumor-bearing mice was significantly improved compared to that of nontreated mice. MicroRNA-detargeted mengoviruses offer a promising oncolytic virotherapy platform that merits further development for clinical translation. IMPORTANCE The clinical potential of oncolytic virotherapy for cancer treatment has been well demonstrated, justifying the continued development of novel oncolytic viruses with enhanced potency. Here, we introduce mengovirus as a novel oncolytic agent. Mengovirus is appealing as an oncolytic virotherapy platform because of its small size, simple genome structure, rapid replication cycle, and broad cell/species tropism. However, mengovirus can cause encephalomyelitis and myocarditis. It can be partially attenuated by shortening the poly(C) tract in the 5′ NCR but remains capable of damaging cardiac and nervous tissue. Here, we further enhanced the safety profile of a poly(C)-truncated mengovirus by incorporating muscle- and neuron-specific microRNA target sequences into the viral genome. This dual-detargeted virus has reduced pathogenesis but retained potent oncolytic activity. Our data show that microRNA targeting can be used to further increase the safety of an attenuated mengovirus, providing a basis for its development as an oncolytic platform.