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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.
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Herpesvirus Humano 3 , Melanoma Experimental , Humanos , Animais , Camundongos , Herpesvirus Humano 3/genética , Interleucina-12RESUMO
Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough 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.
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COVID-19 , Sarampo , Humanos , Animais , Camundongos , Vacinas contra COVID-19 , Anticorpos Neutralizantes , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/prevenção & controle , Vacina contra Sarampo/genética , Vírus do Sarampo/genética , Anticorpos Antivirais , Glicoproteínas de MembranaRESUMO
Oncolytic virotherapy aims to activate host antitumor immunity. In responsive tumors, intratumorally injected herpes simplex viruses (HSVs) have been shown to lyse tumor cells, resulting in local inflammation, enhanced tumor antigen presentation, and boosting of antitumor cytotoxic lymphocytes. In contrast to HSV, cytomegalovirus (CMV) is nonlytic and reprograms infected myeloid cells, limiting their antigen-presenting functions and protecting them from recognition by natural killer (NK) cells. Here, we show that when co-injected into mouse tumors with an oncolytic HSV, mouse CMV (mCMV) preferentially targeted tumor-associated myeloid cells, promoted the local release of proinflammatory cytokines, and enhanced systemic antitumor immune responses, leading to superior control of both injected and distant contralateral tumors. Deletion of mCMV genes m06, which degrades major histocompatibility complex class I (MHC class I), or m144, a viral MHC class I homolog that inhibits NK activation, was shown to diminish the antitumor activity of the HSV/mCMV combination. However, an mCMV recombinant lacking the m04 gene, which escorts MHC class I to the cell surface, showed superior HSV adjuvanticity. CMV is a potentially promising agent with which to reshape and enhance antitumor immune responses following oncolytic HSV therapy.
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Infecções por Citomegalovirus , Herpesvirus Humano 1 , Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Camundongos , Herpesvirus Humano 1/genética , Citomegalovirus , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Apresentação de Antígeno , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismoRESUMO
Mengovirus is an oncolytic picornavirus whose broad host range allows for testing in immunocompetent cancer models. Two pathogenicity-ablating approaches, polycytidine (polyC) tract truncation and microRNA (miRNA) targets insertion, eliminated the risk of encephalomyocarditis. To investigate whether a polyC truncated, miRNA-detargeted oncolytic Mengovirus might be boosted, we partially or fully rebuilt the polyC tract into the 5' noncoding region (NCR) of polyC-deleted (MC0) oncolytic constructs (NC) carrying miRNA target (miRT) insertions to eliminate cardiac/muscular (miR-133b and miR-208a) and neuronal (miR-124) tropisms. PolyC-reconstituted viruses (MC24-NC and MC37-NC) replicated in vitro and showed the expected tropism restrictions, but reduced cytotoxicity and miRT deletions were frequently observed. In the MPC-11 immune competent mouse plasmacytoma model, both intratumoral and systemic administration of MC0-NC led to faster tumor responses than MC24-NC or MC37-NC, with combined durable complete response rates of 75%, 0.5%, and 30%, respectively. Secondary viremia was higher following MC0-NC versus MC24-NC or MC37-NC therapy. Sequence analysis of virus progeny from treated mice revealed a high prevalence of miRT sequences loss among MC24- and MC37- viral genomes, but not in MC0-NC. Overall, MC0-NC was capable of stably retaining miRT sites and provided a more effective treatment and is therefore our lead Mengovirus candidate for clinical translation.
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Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.
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Foamy Viruses are cell cycle-dependent retroviruses capable of persisting unintegrated in quiescent cells until cell division occurs. This unique ability allows them to target slowly dividing human tumor cells which remains an unmet need in oncolytic virotherapy. We have previously reported the generation of oncolytic Foamy Virus (oFV) vector system and demonstrated its superiority over oncolytic Murine Leukemia Virus vectors in infecting slowly dividing cancer cells. In the present study we evaluated (i) the ability of oFV to carry foreign transgenes and (ii) the genetic stability of these vectors upon serial passage. The thymidine kinase (TK) and inducible caspase 9 (iCasp9) cDNAs could be detected in the oFV backbone for up to 3 in vitro passages. In vivo, GFP-, TK- and iCasp9- carrying oFV vectors propagated efficiently in subcutaneous xenograft glioblastoma tumors and drove transgene expression for up to 66 days. However, in vivo oFV vector spread eventually resulted in complete loss of the iCasp9 cDNA, minor loss of the TK cDNA and negligible loss of the GFP. Our results suggest that oFV is a promising gene delivery platform and that transgenes smaller than 1 kb might be most suitable for oFV arming.
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Terapia Viral Oncolítica , Spumavirus , Animais , Linhagem Celular Tumoral , DNA Complementar , Vetores Genéticos/genética , Humanos , Camundongos , Terapia Viral Oncolítica/métodos , Spumavirus/genética , Timidina Quinase/genética , Transgenes , Replicação ViralRESUMO
A dual microRNA-detargeted oncolytic Mengovirus, vMC24NC, proved highly effective against a murine plasmacytoma in an immunocompetent syngeneic mouse model; however, there remains the concern of escape mutant development and the potential for toxicity in severely immunocompromised cancer patients when it is used as an oncolytic virus. Therefore, we sought to compare the safety and efficacy profiles of an attenuated Mengovirus containing a virulence gene deletion versus vMC24NC in an immunodeficient xenograft mouse model of human glioblastoma. A Mengovirus construct, vMC24ΔL, wherein the gene coding for the leader protein, a virulence factor, was deleted, was used for comparison. The vMC24ΔL induced significant levels of toxicity following treatment of subcutaneous human glioblastoma (U87-MG) xenografts as well as when injected intracranially in athymic nude mice, reducing the overall survival. The in vivo toxicity of vMC24ΔL was associated with viral replication in nervous and cardiac tissue. In contrast, microRNA-detargeted vMC24NC demonstrated excellent efficacy against U87-MG subcutaneous xenografts and improved overall survival significantly compared to that of control mice without toxicity. These results reinforce microRNA-detargeting as an effective strategy for ameliorating unwanted toxicities of oncolytic picornaviruses and substantiate vMC24NC as an ideal candidate for clinical development against certain cancers in both immunocompetent and immunodeficient hosts.
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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.
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Edição de Genes , Integrases/genética , Animais , Sistemas CRISPR-Cas , Proteínas de Fluorescência Verde/genética , Luciferases/genética , CamundongosRESUMO
After centuries of pestilence and decades of global vaccination, measles virus (MeV) genotypes capable of evading vaccine-induced immunity have not emerged. Here, by systematically building mutations into the hemagglutinin (H) glycoprotein of an attenuated measles virus strain and assaying for serum neutralization, we show that virus evolution is severely constrained by the existence of numerous co-dominant H glycoprotein antigenic sites, some critical for binding to the pathogenicity receptors SLAMF1 and nectin-4. We further demonstrate the existence in serum of protective neutralizing antibodies targeting co-dominant fusion (F) glycoprotein epitopes. Lack of a substantial reduction in serum neutralization of mutant measles viruses that retain even one of the co-dominant antigenic sites makes evolution of pathogenic measles viruses capable of escaping serum neutralization in vaccinated individuals extremely unlikely.
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Epitopos de Linfócito B/imunologia , Vírus do Sarampo/patogenicidade , Glicoproteínas de Membrana/metabolismo , Sorogrupo , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Hemaglutininas/genética , Humanos , Vacina contra Sarampo/imunologia , Vírus do Sarampo/genética , Glicoproteínas de Membrana/genética , Testes de Neutralização/métodos , Vacinação/métodosRESUMO
Nonpathogenic retroviruses of the Spumaretrovirinae subfamily can persist long-term in the cytoplasm of infected cells, completing their lifecycle only after the nuclear membrane dissolves at the time of cell division. Since the targeting of slowly dividing cancer cells remains an unmet need in oncolytic virotherapy we constructed a replication competent Foamy Virus vector (oFV) from the genomes of two chimpanzee Simian Foamy Viruses (PAN1 and PAN2) and inserted a GFP transgene in place of the bel-2 open reading frame. oFV-GFP infected and propagated with slow kinetics in multiple human tumor cell lines, inducing a syncytial cytopathic effect. Infection of growth arrested MRC5 cells was not productive, but oFV genomes persisted in the cytoplasm and the productive viral lifecycle resumed when cell division was later restored. In vivo, the virus propagated extensively in intraperitoneal ovarian cancer xenografts, slowing tumor growth, significantly prolonging survival of the treated mice and sustaining GFP transgene expression for at least 45 days. Our data indicate that oFV is a promising new replication-competent viral and gene delivery platform for efficient targeting of the most fundamental trait of cancer cells, their ability to sustain chronic proliferation.Significance: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. In contrast to rapidly proliferating transplantable mouse tumors, slow proliferation is a hallmark of human cancers and may have contributed to the clinical failure of the preclinically promising Murine Leukemia Virus vector Toca511 which failed to show efficacy in a phase 3 clinical trial in patients with glioblastoma. The studies presented in our manuscript show that oncolytic Foamy Virus (oFV) vectors are capable of persisting unintegrated in quiescent cells and resuming their life cycle once the cells start dividing again. This property of oFVs, together with their lack of pathogenicity and their ability to catalyze the fusion of infected cancer cells, makes them an attractive platform for further investigation.
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The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients.
Assuntos
Anticorpos Neutralizantes/imunologia , Soros Imunes/imunologia , Vírus do Sarampo/genética , Proteína Cofatora de Membrana/metabolismo , Mieloma Múltiplo/terapia , Terapia Viral Oncolítica/métodos , Neoplasias Ovarianas/terapia , Animais , Vírus da Cinomose Canina/genética , Feminino , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Humanos , Proteína Cofatora de Membrana/imunologia , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Ligação Proteica , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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Vírus da Cinomose Canina/imunologia , Vírus do Sarampo/imunologia , Terapia Viral Oncolítica/métodos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Camundongos , Camundongos Nus , Células VeroRESUMO
Infectious nucleic acid has been proposed as a superior formulation for oncolytic virus therapy. Oncolytic picornaviruses can be formulated as infectious RNA (iRNA), and their unwanted tropisms eliminated by microRNA (miRNA) detargeting. However, genomic insertion of miRNA target sequences into coxsackievirus A21 (CVA21) iRNA compromised its specific infectivity, negating further development as a novel oncolytic virus formulation. To address this limitation, we substituted a muscle-specific miRNA response element for the spacer region downstream of the internal ribosomal entry site in the 5' non-coding region of CVA21 iRNA, thereby preserving genome length while avoiding the disruption of known surrounding RNA structural elements. This new iRNA (R-CVA21) retained high specific infectivity, rapidly generating replicating miRNA-detargeted viruses following transfection in H1-HeLa cells. Further, in contrast with alternatively configured iRNAs that were tested in parallel, intratumoral administration of R-CVA21 generated a spreading oncolytic infection that was curative in treated animals without associated myotoxicity. Moreover, R-CVA21 also exhibited superior miRNA response element stability in vivo. This novel formulation is a promising agent for clinical translation.
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Genetically modified vesicular stomatitis virus (VSV) is an attractive agent for cancer treatment due to rapid intratumoral replication and observed clinical responses. Although VSV selectively kills malignant cells and can boost antitumor immunity, limited induction of intratumoral immune infiltration remains a barrier to efficacy in some cancer models. Here we engineered the oncolytic VSV platform to encode the T cell chemokine CXCL9, which is known to mediate the recruitment of activated CD8+ cytotoxic T cells and CD4+ T helper cells, and demonstrates conserved protein function between mice and humans. Chemotactic activity of the virally encoded chemokine was confirmed in vitro. Intratumoral concentration of CXCL9 was shown to increase after VSV therapy in three different cancer models, but to a much greater degree after VSV-CXCL9 therapy as compared with VSV control viruses. Despite a steep chemokine gradient from the tumor to the bloodstream, tumor trafficking of adoptively transferred and endogenous T cells was not measurably increased following VSV-CXCL9 therapy. Our results indicate that oncolytic VSV infection promotes release of CXCL9 in the tumor microenvironment, but further boosting of the functional chemokine gradient through virus engineering has little incremental impact on intratumoral immune cell infiltration in mouse and human tumor models.
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Tumor-selective oncolytic vesicular stomatitis viruses (VSVs) are being evaluated in clinical trials. Here, we report that the MPC-11 murine plasmacytoma model is so extraordinarily susceptible to oncolytic VSVs that a low dose of virus leads to extensive intratumoral viral replication, sustained viremia, intravascular coagulation, and a rapidly fatal tumor lysis syndrome (TLS). Rapid softening, shrinkage and hemorrhagic necrosis of flank tumors was noted within 1-2 days after virus administration, leading to hyperkalemia, hyperphosphatemia, hypocalcemia, hyperuricemia, increase in plasma cell free DNA, lymphopenia, consumptive coagulopathy, increase in fibrinogen degradation products, decreased liver function tests, dehydration, weight loss, and euthanasia or death after 5-8 days. Secondary viremia was observed but viral replication in normal host tissues was not detected. Toxicity could be mitigated by using VSVs with slowed replication kinetics, and was less marked in animals with smaller flank tumors. The MPC-11 tumor represents an interesting model to further study the complex interplay of robust intratumoral viral replication, tumor lysis, and associated toxicities in cases where tumors are highly responsive to oncolytic virotherapy.
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Terapia Viral Oncolítica/efeitos adversos , Plasmocitoma/terapia , Síndrome de Lise Tumoral/etiologia , Vírus da Estomatite Vesicular Indiana/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Transplante de Neoplasias , Vírus Oncolíticos/genética , Resultado do TratamentoRESUMO
Tyrosinemia type I (TYRSN1, TYR I) is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects approximately one in 100,000 individuals worldwide. Pathogenic variants in FAH cause TYRSN1, which induces cirrhosis and can progress to hepatocellular carcinoma (HCC). TYRSN1 is characterized by the production of a pathognomonic metabolite, succinylacetone (SUAC) and is included in the Recommended Uniform Screening Panel for newborns. Treatment intervention is effective if initiated within the first month of life. Here, we describe a family with three affected children who developed HCC secondary to idiopathic hepatosplenomegaly and cirrhosis during infancy. Whole exome sequencing revealed a novel homozygous missense variant in FAH (Chr15(GRCh38):g.80162305A>G; NM_000137.2:c.424A > G; NP_000128.1:p.R142G). This novel variant involves the catalytic pocket of the enzyme, but does not result in increased SUAC or tyrosine, making the diagnosis of TYRSN1 problematic. Testing this novel variant using a rapid, in vivo somatic mouse model showed that this variant could not rescue FAH deficiency. In this case of atypical TYRSN1, we show how reliance on SUAC as a primary diagnostic test can be misleading in some patients with this disease. Augmentation of current screening for TYRSN1 with targeted sequencing of FAH is warranted in cases suggestive of the disorder.
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Carcinoma Hepatocelular/genética , Hidrolases/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Mutação de Sentido Incorreto , Tirosinemias/diagnóstico , Adolescente , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Domínio Catalítico , Linhagem Celular Tumoral , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Heptanoatos/metabolismo , Humanos , Hidrolases/química , Lactente , Cirrose Hepática/complicações , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Linhagem , Análise de Sequência de DNA , Tirosina/metabolismo , Tirosinemias/complicações , Tirosinemias/genéticaRESUMO
UNLABELLED: Mengovirus, a member of thePicornaviridaefamily, 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, vMC24, 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 vMC24NCRs. 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 fitnessin vivo. In vivotoxicity 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 vMC24-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 [TCID50]) or intravenous (10(7)to 10(8)TCID50) injection into BALB/c mice bearing MPC-11 plasmacytomas. Overall survival of vMC24-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.
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Mengovirus , MicroRNAs/genética , Mieloma Múltiplo/terapia , Terapia Viral Oncolítica , Animais , Infecções por Cardiovirus/etiologia , Infecções por Cardiovirus/prevenção & controle , Linhagem Celular , Efeito Citopatogênico Viral , Feminino , Marcação de Genes , Instabilidade Genômica , Humanos , Hospedeiro Imunocomprometido , Mengovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Mieloma Múltiplo/imunologia , Síndromes Neurotóxicas/prevenção & controle , Síndromes Neurotóxicas/virologia , Terapia Viral Oncolítica/efeitos adversos , RNA não Traduzido/genética , Replicação ViralRESUMO
Antitumor efficacy of oncolytic virotherapy is determined by the density and distribution of infectious centers within the tumor, which may be heavily influenced by the permeability and blood flow in tumor microvessels. Here, we investigated whether systemic perfusion pressure, a key driver of tumor blood flow, could influence the intratumoral extravasation of systemically administered oncolytic vesicular stomatitis virus (VSV) in myeloma tumor-bearing mice. Exercise was used to increase mean arterial pressure, and general anesthesia to decrease it. A recombinant VSV expressing the sodium iodide symporter (NIS), which concentrates radiotracers at sites of infection, was administered intravenously to exercising or anesthetized mice, and nuclear NIS reporter gene imaging was used to noninvasively track the density and spatial distribution of intratumoral infectious centers. Anesthesia resulted in decreased intratumoral infection density, while exercise increased the density and uniformity of infectious centers. Perfusion state also had a significant impact on the antitumor efficacy of the VSV therapy. In conclusion, quantitative dynamic radiohistologic imaging was used to noninvasively interrogate delivery of oncolytic virotherapy, highlighting the critical importance of perfusion pressure as a driver of intratumoral delivery and efficacy of oncolytic viruses.
Assuntos
Pressão Arterial , Mieloma Múltiplo/terapia , Vírus Oncolíticos/fisiologia , Simportadores/genética , Vesiculovirus/fisiologia , Administração Intravenosa , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Camundongos , Mieloma Múltiplo/fisiopatologia , Terapia Viral Oncolítica , Perfusão , Simportadores/metabolismo , Células VeroRESUMO
Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte (HC) transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted HCs in a rodent model of inherited liver disease: hereditary tyrosinemia type 1. Wild-type C57Bl/6J mouse HCs were transduced ex vivo with a lentiviral vector containing the mouse Slc5a5 (NIS) gene controlled by the thyroxine-binding globulin promoter. NIS-transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS-transduced HCs were transplanted into congenic fumarylacetoacetate hydrolase knockout mice, and this resulted in the prevention of liver failure. NIS-transduced HCs were readily imaged in vivo by single-photon emission computed tomography, and this demonstrated for the first time noninvasive 3-dimensional imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary HC spheroids engrafted in the liver. With the NIS reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, and this thereby demonstrated a novel strategy for HC transplantation. This work is the first to demonstrate the efficacy of NIS imaging in the field of HC transplantation. We anticipate that NIS labeling will allow noninvasive and longitudinal identification of HCs and stem cells in future studies related to liver regeneration in small and large preclinical animal models.
Assuntos
Hepatócitos/transplante , Imageamento Tridimensional/métodos , Falência Hepática/prevenção & controle , Regeneração Hepática , Simportadores/metabolismo , Tomografia Computadorizada de Emissão de Fóton Único , Tirosinemias/cirurgia , Microtomografia por Raio-X , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Sobrevivência de Enxerto , Hepatócitos/metabolismo , Hidrolases/deficiência , Hidrolases/genética , Falência Hepática/diagnóstico , Falência Hepática/genética , Falência Hepática/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imagem Multimodal , Valor Preditivo dos Testes , Simportadores/genética , Fatores de Tempo , Transdução Genética , Transfecção , Tirosinemias/diagnóstico , Tirosinemias/genética , Tirosinemias/metabolismoRESUMO
Systemically administered oncolytic viruses have the ability to cause tumor destruction through the expansion and coalescence of intratumoral infectious centers. Efficacy is therefore dependent upon both the density and intratumoral distribution of virus-infected cells achieved after initial virus infusion, and delivery methods are being developed to enhance these critical parameters. However, the three-dimensional (3D) mapping of intratumoral infectious centers is difficult using conventional immunohistochemical methodology, requiring painstaking 3D reconstruction of numerous sequential stained tumor sections, with no ability to study the temporal evolution of spreading infection in a single animal. We therefore developed a system using very high-resolution noninvasive in vivo micro single-photon emitted computed tomography/computed tomography (microSPECT/CT) imaging to determine the intratumoral distribution of thyroid radiotracers in tumors infected with an oncolytic virus encoding the thyroidal sodium-iodide symporter (NIS). This imaging system was used for longitudinal analysis of the density, distribution, and evolution of intratumoral infectious centers after systemic administration of oncolytic vesicular stomatitis virus in tumor-bearing mice and revealed heterogeneous delivery of virus particles both within and between tumors in animals receiving identical therapy. This study provides compelling validation of high resolution in vivo reporter gene mapping as a convenient method for serial monitoring of intratumoral virus spread that will be necessary to address critical barriers to systemic oncolytic virus efficacy such as intratumoral delivery.