Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk.

Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing.

Previous work has demonstrated that the epitranscriptomic addition of m6A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m6A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m6A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.

Influenza A virus-derived siRNAs increase in the absence of NS1 yet fail to inhibit virus replication.

While the issue of whether RNA interference (RNAi) ever forms part of the antiviral innate immune response in mammalian somatic cells remains controversial, there is considerable evidence demonstrating that few, if any, viral small interfering RNAs (siRNAs) are produced in infected cells. Moreover, inhibition of RNAi by mutational inactivation of key RNAi factors, such as Dicer or Argonaute 2, fails to enhance virus replication. One potential explanation for this lack of inhibitory effect is that mammalian viruses encode viral suppressors of RNAi (VSRs) that are so effective that viral siRNAs are not produced in infected cells. Indeed, a number of mammalian VSRs have been described, of which the most prominent is the influenza A virus (IAV) NS1 protein, which has not only been reported to inhibit RNAi in plants and insects but also to prevent the production of viral siRNAs in IAV-infected human cells. Here, we confirm that an IAV mutant lacking NS1 indeed differs from wild-type IAV in that it induces the production of readily detectable levels of Dicer-dependent viral siRNAs in infected human cells. However, we also demonstrate that these siRNAs have little if any inhibitory effect on IAV gene expression. This is likely due, at least in part, to their inefficient loading into RNA-induced silencing complexes.

Gene Editing: A New Tool for Viral Disease.

The emergence of the CRISPR/Cas system of antiviral adaptive immunity in bacteria as a facile system for gene editing in mammalian cells may well lead to gene editing becoming a novel treatment for a range of human diseases, especially those caused by deleterious germline mutations. Another potential target for gene editing are DNA viruses that cause chronic pathogenic diseases that cannot be cured by using currently available drugs. We review the current state of this field and discuss the potential advantages and problems with using a gene editing approach as a treatment for diseases caused by DNA viruses.

Partial reconstitution of the RNAi response in human cells using Drosophila gene products.

While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of ∼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.

Viral Epitranscriptomics.

Although it has been known for over 40 years that eukaryotic mRNAs bear internal base modifications, it is only in the last 5 years that the importance of these modifications has begun to come into focus. The most common mRNA modification, the addition of a methyl group to the N6 position of adenosine (m6A), has been shown to affect splicing, translation, and stability, and m6A is also essential for embryonic development in organisms ranging from plants to mice. While all viral transcripts examined so far have been found to be extensively m6A modified, the role, if any, of m6A in regulating viral gene expression and replication was previously unknown. However, recent data generated using HIV-1 as a model system strongly suggest that sites of m6A addition not only are evolutionarily conserved but also enhance virus replication. It is therefore likely that the field of viral epitranscriptomics, which can be defined as the study of functionally relevant posttranscriptional modifications of viral RNA transcripts that do not change the nucleotide sequence of that RNA, is poised for a major expansion in scientific interest and may well fundamentally change our understanding of how viral replication is regulated.

Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer.

Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus-but not poliovirus-replication in human cells.

Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators.

Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such "synergistic activation mediators" can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.

The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.

A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs.

While lentiviral expression vectors are widely used in many facets of molecular biology, due to their ability to stably express heterologous genes in both dividing and non-dividing cells, they suffer from the disadvantage that introns inserted into the vector genome are generally rapidly lost by splicing in packaging cell lines. The presence of an intron, if achievable, has the potential to facilitate the expression of transgene cDNAs, as splicing has been extensively shown to facilitate mRNA biogenesis and function. Moreover, if a stable intron could be introduced into a lentiviral vector, this could greatly facilitate the expression of microRNAs (miRNAs), and especially miRNA clusters, as the introduction of pri-miRNA stems into the exonic region of a lentiviral vector can strongly reduce both vector titer and the expression of any miRNA-linked indicator gene due to cleavage of the vector RNA genome by cellular Drosha. Here, we describe a novel lentiviral vector design in which transgenes and/or miRNAs are expressed using an antisense-orientated, inducible promoter driving an expression cassette bearing a functional intron. We demonstrate that this lentiviral vector, called pTREX, is able to express higher levels of both transgenes and pri-miRNA clusters when compared with a closely similar conventional lentiviral vector.

Derivation and characterization of Dicer- and microRNA-deficient human cells.

We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr cofactor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its cofactors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs vs. passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells.

Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.

RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease.

High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. Importance: Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.

Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs.

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.

Effect of ribonucleotides embedded in a DNA template on HIV-1 reverse transcription kinetics and fidelity.

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.

Frequent incorporation of ribonucleotides during HIV-1 reverse transcription and their attenuated repair in macrophages.

Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.

Seneca Valley virus replicons are packaged in trans and have the capacity to overcome the limitations of viral transgene expression.

Oncolytic viruses (OVs) promote the anti-tumor immune response as their replication, and the subsequent lysis of tumor cells, triggers the activation of immune-sensing pathways. Arming OVs by expressing transgenes with the potential to promote immune cell recruitment and activation is an attractive strategy to enhance OVs' therapeutic benefit. For picornaviruses, a family of OVs with clinical experience, the expression of a transgene is limited by multiple factors: genome physical packaging limits, high rates of recombination, and viral-mediated inhibition of transgene secretion. Here, we evaluated strategies for arming Seneca Valley virus (SVV) with relevant immunomodulatory transgenes. Specificially in the contex of arming SVV, we evaluated transgene maximum size and stabiltity, transgene secretion, and the impact of transgene inclusion on viral fitness. We find that SVV is not capable of expressing secreted payloads and has a transgene packaging capacity of ∼10% of viral genome size. To enable transgene expression, we developed SVV replicons with greater transgene size capacity and secretion capabilities. SVV replicons can be packaged in trans by virus in co-infected cells to express immunomodulatory transgenes in surrounding cells, thus providing a means to enhance the potential of this therapeutic to augment the anti-tumor immune response.

Abundant non-canonical dUTP found in primary human macrophages drives its frequent incorporation by HIV-1 reverse transcriptase.

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nm), compared with activated CD4(+) T cells (2-5 µm). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 µm) is ∼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2',3'-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.

Development of intravenously administered synthetic RNA virus immunotherapy for the treatment of cancer.

The therapeutic effectiveness of oncolytic viruses (OVs) delivered intravenously is limited by the development of neutralizing antibody responses against the virus. To circumvent this limitation and to enable repeated systemic administration of OVs, here we develop Synthetic RNA viruses consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. For two Synthetic RNA virus drug candidates, Seneca Valley virus (SVV) and Coxsackievirus A21, we demonstrate vRNA delivery and replication, virus assembly, spread and lysis of tumor cells leading to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. Synthetic-SVV replication in tumors promotes immune cell infiltration, remodeling of the tumor microenvironment, and enhances the activity of anti-PD-1 checkpoint inhibitor. In mouse and non-human primates, Synthetic-SVV is well tolerated reaching exposure well above the requirement for anti-tumor activity. Altogether, the Synthetic RNA virus platform provides an approach that enables repeat intravenous administration of viral immunotherapy.