MicroRNAs and oncolytic viruses.

Available online at www.sciencedirect.com

ScienceDirect MicroRNAs and oncolytic viruses Autumn J Ruiz and Stephen J Russell MicroRNAs regulate gene expression in mammalian cells and often exhibit tissue-specific expression patterns. Incorporation of microRNA target sequences can be used to control exogenous gene expression and viral tropism in specific tissues to enhance the therapeutic indices of oncolytic viruses expressing therapeutic transgenes. Continued development of this targeting strategy has resulted in the generation of unattenuated oncolytic viruses with enhanced potency, broad species-tropisms and reduced off-target toxicities in multipletissues simultaneously. Furthermore, oncolytic viruses have been used to enhance the delivery, duration and therapeutic efficacy of microRNA-based therapeutics designed to either restore or inhibit the function of dysregulated microRNAs in cancer cells. Recent efforts focused on combining oncolytic virotherapy and microRNA regulation have generated increasingly potent and safe cancer therapeutics. Address Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, United States Corresponding author: Russell, Stephen J ([email protected])

Current Opinion in Virology 2015, 13:40–48

roles in regulating cellular processes such as cell cycle, differentiation, metabolism, apoptosis, autophagy, signaling, inflammation, and migration [13–17]. Therefore, miRNA deregulation is commonly associated with disease, including many types of cancer [18–20]. miRNA signatures can distinguish between normal and cancerous cells, as well as different cancer subtypes, specific oncogenic abnormalities, and cancer stem cells [21]. An overview of cancers with miRNA signatures is given in Table 2. Major efforts have been focused on exploiting miRNA regulation for developing new and enhanced therapeutic interventions; as such the fields of miRNAbased therapeutics and oncolytic viruses (OVs) have converged. Although well tolerated, the clinical activity of both of these therapies as stand-alone treatments remains limited [22,23,24]. Increasing evidence suggests that enhanced delivery, increased potency, and combinatorial therapies are necessary for these therapeutics to reach their full potential. Here we review how OVs have been coupled with miRNA regulation to enhance the efficacy and translatability of miRNA-based therapeutics and oncolytic virotherapy and highlight major advances in the field over the past several years.

This review comes from a themed issue on Oncolytic viruses Edited by Grant McFadden and John Bell

http://dx.doi.org/10.1016/j.coviro.2015.03.007 1879-6257/# 2015 Published by Elsevier B.V.

Introduction MicroRNAs (miRNAs) are small, 19–25 nt, non-coding RNAs expressed by all multicellular eukaryotes that mediate post-transcriptional regulation of gene expression [1–5]. miRNAs silence gene expression by binding sequence-complementary target elements in messenger RNAs (mRNAs), commonly found in the 30 untranslated region (UTR), preventing translation or accelerating transcript degradation. Canonical and non-canonical miRNA biogenesis pathways are described in Figure 1, and the reader is referred to Refs. [4,6,7] for more detail. More than 2500 mature miRNAs have been identified in human cells [8–12] and while some miRNAs are ubiquitously expressed, many are enriched within certain tissues (Table 1) and their expression can be lineage-specific, activation, or differentiation stage-specific. They are known to regulate 60% of human genes and play major Current Opinion in Virology 2015, 13:40–48

MicroRNA regulated transgene expression Introduction of exogenous therapeutic genes often requires cell-specific expression in order to avoid offtarget effects. The idea of exploiting miRNA regulation to restrict gene expression in specific tissues was initially demonstrated using hematopoietic-specific miR-142-3p targets to inhibit lentiviral delivered transgene expression in antigen presenting cells [25,26]. By incorporating miRNA target sequences into the 30 UTR of the transgene, expression was significantly reduced in tissues expressing the specific miRNA, but maintained in those that did not. This concept was quickly adopted for preventing off-target toxicities and increasing tissue specificity of virally encoded genetic cassettes [27,28]. Exogenous gene expression from viral vectors including adenovirus (Ad), adeno-associated virus (AAV), and baculovirus can be controlled and the therapeutic indices of these treatments enhanced by including miRNA response elements (MREs) [29–31]. The utility of this targeting strategy has been widely demonstrated and tissue-specificity has been enhanced for a large number of virally encoded therapeutic genes. Most recently, MREs have been used to control the expression of the TNF-related apoptosis-inducing ligand (TRAIL), a cytokine that selectively triggers apoptosis and that demonstrated robust anticancer activity in preclinical studies. Clinical analysis of TRAIL-receptor agonists revealed that although well tolerated these agents did not have www.sciencedirect.com

MicroRNAs and oncolytic viruses Ruiz and Russell 41

Figure 1

Drosha-independent “Mirtron”

Canonical RNA Pol II

AAAA m7G

m7G

AAAA

exon

m7G

exon

Dicer-independent

AAAA

m7G

AAAA

Spliceosome Drosha

Drosha

DGCR8

DGCR8 RAN-GTP Exportin 5

Ran-GDP

Dicer

Ran-GDP

P Body

TRBP m7G

Deadenylation mRNA degradation Ago 2

PARN

mRNA Storage

Ago 1-4

Passenger Strand Degraded

Ago 2

m7G

AAAA RISC Current Opinion in Virology

Schematic representation of miRNA biogenesis and function. Canonical. miRNAs are transcribed as capped, polyadenylated primary miRNA (primiRNA) hairpins, typically by RNA polymerase II, which are then recognized by the nuclear processor complex (Drosha-DGCR8) and cleaved 22 nt down the miRNA stem. The resulting pre-miRNA hairpins bearing 2 nt 30 overhangs are translocated to the cytoplasm by exportin 5 and are further processed by Dicer-TRBP into double-stranded miRNA duplexes. miRNA duplexes are loaded into Argonaute (AGO)-containing RNAinduced silencing complexes (RISCs), followed by duplex unwinding, mature miRNA retention and passenger strand degradation. Some passengers are functional miRNAs and do not get degraded. The miRNA directs the RISC to partially complementary sites in target mRNAs resulting in translational repression or accelerated transcript degradation by retargeting mRNAs to P bodies. If the miRNA recognizes a sequence of perfect complementarity, the transcript can undergo endonucleolytic cleavage. Drosha-independent. Short intronic hairpins termed mirtons represent the most common alternative miRNA biogenesis pathway. These hairpins are spliced and debranched to form pre-miRNAs, which are subsequently processed through the canonical pathway. Dicer-independent. Pre-miRNAs are produced by Drosha and exported to the cytoplasm, presumably by exportin 5. These pre-miRNAs are loaded on AGO2, which cleaves the pre-miRNA stem, and are then further trimmed by the 30 –50 exonuclease poly(A)-specific ribonuclease (PARN). The mature miRNA-loaded RISC then mediates gene silencing.

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42 Oncolytic viruses

Table 1 MicroRNAs enriched in specific tissues Tissue/cell type Bone Cardiac Endocrine Glands Hematopoietic Kidney Liver Lung Muscle Nervous System

Ovary Pancreas Spleen Testis

Enriched miRNAs

References

miR-377; miR-92a; miR-483 miR-1; miR-126; miR-133a/b; miR-208a; miR-302; miR-367 miR-375 miR-16; miR-142; miR-150; miR-223 miR-10a/b; miR-30c; miR-146a; miR-192; miR-196a/b; miR-200a; miR-204; miR-215; miR-216 miR-92a; miR-122; miR-192; miR-483 miR-126 miR-1; miR-95; miR-128a; miR-133a/b; miR-134; miR-193a; miR-206 miR-7; miR-9; miR-31; miR-33a; miR-93; miR-95; miR-99b; miR-124a; miR-125a/b; miR-128a/b; miR-129; miR-138; miR-149; miR-186; miR-199a/b, miR-212; miR-214; miR-137; miR-143; miR-153; miR-218; miR-323; miR-346; miR-330; miR-708 miR-542-5p miR-216a/b; miR-217; miR-375 miR-146a; miR-223 miR-10b; miR-23a; miR-34b/c; miR-134; miR-187; miR-202; miR-204; miR-449a; miR-506; miR-507; miR-508; miR-509; miR-510; miR-513; miR-514; miR-892b

[74] [74,75] [76] [75,76] [74,75] [74,75] [74] [74–76] [74–76]

[75] [74,75] [74] [74,75]

*Given the volume of manuscripts involved in obtaining the above data, reviews were cited instead of original reports to limit the number of references.

robust antitumor activity. One possible explanation for this was that relatively weak agonists were chosen because of initial concerns over potential hepatotoxicity. Therefore, stronger agonists may provide a better clinical outcome as long as the off-target toxicities are prevented. Several groups showed that MREs could confer tumorspecificity and reduce toxicity of TRAIL therapy against

glioma, uveal melanoma, prostate, and osteosarcoma models [32,33]. MREs can also be used to facilitate the manufacture of viruses expressing transgenes. Savdaminova et al. [34] used MREs to generate modified Ads expressing nucleases for cell type specific genome editing. Nuclease

Table 2 Cancers with miRNA signatures Cancer Breast Bladder CLL Colon Colorectal Cancer Esophagus Glioblastoma

Down-regulated miRNA Let-7; miR-31; miR-34; miR-200; miR-126; miR-29 miR-34; miR-143; miR-133a/b miR-15/16; miR-29 miR-Let-7a-2; miR-34, miR-107 miR-143, miR-145

Kidney Leukemia Liver

miR-221/222; miR-34; miR-7; miR-124; miR-128; miR-218 miR-34 miR-29 # miR-34; miR-122a; miR-26a;

Lung

miR-Let-7; miR-34; miR-29

Lymphoma Myeloma Ovarian

miR-155; miR-29

Pancreas Prostate Stomach Thyroid

miR-Let-7a-2; miR-31; miR-34; miR-143; miR-199a; miR-200 miR-107; miR-96; miR-196 miR-Let-7c; miR-125b; miR-145; miR-15/16 miR-Let-7a-2; miR-31; miR-126;

Up-regulated miRNA miR-21; miR-221/222; miR-10b; miR-17-92; miR-155 miR-21; miR-155 miR-21; miR-196; miR-17-92; miR-155 miR-31 miR-21; miR-10b; miR-196 miR-21; #miR-10b; miR-196

miR-21; miR-155 # miR-21; #miR-221/222; miR-181b miR-21; miR-221/222; miR-17-92; miR-155 miR-155 miR-21, miR-181b miR-21, miR-200a/b/c

References [20,77] [75,77] [75,77,78] [20,77] [75,78] [20] [20,77,78,79] [77] [77,78] [20,75,77,78] [20,75,77,78]

[75,77,78] [20,77] [20,75,77]

miR-21 miR-21

[20,77] [75,77]

miR-21 miR-221/222; miR-146

[20,77] [77]

*Given the volume of manuscripts involved in obtaining the above data, reviews were cited instead of original reports to limit the number of references. miRNA-based therapeutics currently undergoing preclinical and clinical analysis.

#




expression was inhibited in the producer cells allowing virus rescue without restricted expression in target cells.

Modifying virus tropism MicroRNA targeting can also be used to regulate oncolytic viral tropism to enhance tumor specificity and eliminate undesirable toxicities [35]. This was originally demonstrated by incorporating muscle-specific MREs into the 30 UTR of Coxsackievirus A21 (CVA21), which normally causes fatal myositis [36]. Inclusion of MREs eliminated muscle toxicity but did not compromise oncolytic activity. This strategy has since been applied to many viruses and may facilitate clinical translation of OVs with enhanced potency. The safety of therapeutic viruses can be improved by deleting essential viral genes, however this is often associated with viral attenuation and decreased potency [37–40]. By contrast, MREs can control gene expression without resulting in attenuation. Deletions within the E1A and E1B genes are often used to generate tumor-selective conditionally replicating Ads, but result in viral attenuation. Yao et al. [41] constructed an oncolytic Ad with a MRE controlled E1A gene. This virus, OA-4MREs, resulted in increased viral output and cytotoxicity in cultured and primary glioma cells compared to ONYX-015, a clinically tested Ad with an E1B region deletion. By contrast to ONYX-015, OA-4MREs inhibited neuro-toxicity and hepatotoxicity, displayed enhanced antitumor activity and prolonged survival of tumor-bearing mice. Callegari et al. [42] targeted miR-199 to regulate oncolytic Ad replication for improved antitumor activity and reduced hepatotoxicity in both tumorbearing, newborn mice treated intratumorally and in immune-competent miR-221 transgenic mice with diethylnitrosamine induced liver cancer treated intravenously (IV). The importance here is that few preclinical studies are conducted in IV-treated, tumor-bearing, immune competent mice, a model that most closely mimics a clinical setting. While preclinical data suggest miRNA targeting may be sufficient to eliminate toxicities in animal models, combining targeting strategies, especially for unattenuated viruses may prove more prudent in humans. Placing an E1A gene under liver-specific miR-122 control in Ad6, which has reduced liver toxicity, significantly improved safety versus unmodified Ad5 or Ad6 and allowed administration of elevated doses [38]. The tropism of unattenuated oncolytic herpes simplex viruses (oHSVs) can also be modified by MREs [43]. Most recently, Mazzacurati et al. [44] developed a wild-type oHSV modified to bind epidermal growth factor receptor and epidermal growth factor receptor variant III and encode a miR-124 regulated ICP4 gene. Replication in normal brain tissue and toxicity were reduced in nude mice and antitumor activity against primary human glioblastoma multiforme tumors was maintained. www.sciencedirect.com

Multi-tissue detargeting Multiple tissue tropisms can be regulated simultaneously by targeting a ubiquitous miRNA or multiple different miRNAs. The Let-7 family of miRNAs function as tumor suppressors and are down-regulated in a large number of cancers. miR-Let-7 response elements have been used for broad control of live attenuated poliovirus vaccines, vesicular stomatitis virus (VSV), vaccinia virus, Ad, and oHSV [41,44,45–47]. However, Let-7 levels in different tissues are variable and target recognition is redundant among family members. Thus, even though one family member is down-regulated, other members may have ample expression levels to reduce therapeutic efficacy in the tumor and increase the possibility of miRNA saturation in normal cells. Several studies have used multiple miRNA targets simultaneously to redirect viral tropism and this has proven just as efficacious as using ubiquitous targets [26,41,48,49,50]. This may prove the preferred method as incorporation of different miRNA targets decreases the potential for saturating a single miRNA. Furthermore, the ability to simultaneously eliminate various off-target toxicities increases the potential for clinical translation of OVs with increased potency. Fu et al. [51] combined miR-Let-7, miR-122, and miR-124 targets to control the glycoprotein H gene of oHSV, with a liver-specific promoter to generate a highly targeted, potent oHSV.

Versatility of miRNA targeting DNA and RNA viruses are both amenable to miRNA targeting. To date, the majority of miRNA-targeted RNA viruses are positive-sense, single-stranded viruses. These viruses have proven highly responsive to this targeting strategy, likely because both viral genomes and mRNAs are targeted. By contrast, only a few negative-sense RNA viruses including influenza A virus (IAV), VSV and measles virus (MV) have been targeted using MREs. miRNAtargeting of negative-strand RNA viruses is hypothesized to be more difficult as the accessibility of MREs will likely be hindered because encapsidation of the viral genome occurs concomitantly with transcription and replication. MREs oriented to target the viral genome of VSV or IAV did not result in miRNA-mediated silencing [52,53]. Therefore miRNA responses in negative sense RNA viruses are likely limited to mRNAs, increasing the burden for miRNA targeting by allowing continual production of mRNAs and increasing the potential for saturation of miRNAs. MicroRNA-targeting has been applied to IAV to generate a safe and effective live-attenuated vaccine as well as a virus that is transmissible among ferrets, but not among humans [54,55]. Additionally, Chua et al. [56] used miRNA-targeting technology to facilitate mechanistic studies on IAV replication. Interestingly, miRNA-targeting of the non-structural protein 1 resulted in significantly down-regulated expression both on an mRNA and proCurrent Opinion in Virology 2015, 13:40–48


tein level, but did not affect virus replication in vitro or in vivo. This demonstrates the importance of selecting genes whose expression level dramatically affects viral replication for optimal targeting. MV has also proven amenable to miRNA targeting [57]. Most recently a multi-tissue detargeted MV regulated by miR-7, miR-122 and miR-148a was generated [49]. Viral spread in the presence of these miRNAs was restricted, but antitumor activity against pancreatic carcinoma xenografts was unaltered. Unfortunately, the utility of this targeting strategy is difficult to assess because MV cannot replicate in murine cells. However, this study may have clinical relevance as MV has shown clinical efficacy at extremely high doses [58]. As the dose and potency of this virus are elevated to maximize therapeutic efficacy, toxicities in off-target tissues may be an issue. Studies focused on redirecting the tropism of VSV demonstrated that miRNA targeting of the polymerase gene (L) was more efficacious than targeting the gene encoding the matrix protein (M). This could be due to several different factors including, firstly, lower levels of L protein due to the transcriptional gradient of VSV; secondly, larger impact on viral replication that may mimic targeting of viral genomes; or thirdly, higher accessibility of the miRNA target. VSV tropism was redirected and neurotoxicity reduced when miR-125b response elements were used to control L protein expression. 90% of mice injected with this virus were protected against neuropathogenesis following an intracerebral injection of a lethal dose for unmodified VSV. However, the one mouse that developed encephalitis did not have mutations within the target sequences indicating that this MRE configuration was insufficient for eliminating neurotoxicity. The in vitro and in vivo results of this targeted virus were inferior to that observed with other targeted viruses and this may be attributed to various factors. miRNA silencing is more efficient when viral genomes are targeted compared to viral mRNAs, however this has not been achieved in negative-sense RNA viruses. VSV is a rapidly replicating virus and mRNAs will continually be synthesized increasing the potential for miRNA recognition escape and or saturation. Targeting of a single gene may not be sufficient and control of various genes simultaneously may be warranted. Location within the 30 UTR may not be optimal for target insertion and enhanced control may be achieved by incorporating the miRNA target within the open reading frame of the gene as was demonstrated for Sindbis virus [59]. Finally, the miRNAs chosen may not be optimal. It was recently demonstrated that inhibition of several miRNAs including miR-125b increased susceptibility of human bronchial epithelial cells to VSV infection [60]. The affects of viruses and targeted miRNAs on the cellular transcriptome, antiviral state and inflammatory response need to be determined in order to engineer safe and effective miRNA-targeting strategies. Current Opinion in Virology 2015, 13:40–48

Guidelines for optimal targeting Many factors contribute to targeting efficacy [61]. Elevated miRNA expression levels correlate with increased regulatory activity and certain miRNAs are more efficient at gene silencing than others [52,61]. Completely complementary target sequences also promote miRNA-gene regulation and reduce the potential for miRNA saturation because perfectly complementary sequences can be endonucleolytically cleaved allowing rapid recycling of the miRNA. This does require that the miRNA associate with an Argonaute protein with endonucleolytic activity. Increasing the copy number of target sequences generally will enhance targeting efficiency, however this is not always the case and defining an optimal target number for each miRNA is recommended. The level of targeted gene expression, attributed to the replication kinetics and transcription gradient of the virus, can affect the regulatory capacity of the MREs as will the level of competing mRNAs targeted by the miRNA being utilized. Finally, the insertion site is a major factor in dictating suppressive activity. While most MRE elements are found within the 30 UTR of mRNAs, relocation to the 50 UTR or the open reading frame of a gene or genome can be more efficacious [59]. Insertion sites need to allow high accessibility of the miRNA target, which can be hindered by secondary structures normally formed by the virus or additional structures introduced by the incorporated target sequences. Studies in our laboratory revealed that while various MREs inserted into the 30 UTR of Mengovirus regulate viral replication, recognition of Let-7a inserted at the same site is prohibited (Ruiz et al. unpublished data). In general, selecting a miRNA with high expression levels, and incorporating 4–6 copies of MREs with complete complementarity, in a site with low secondary structure and high conservation is an optimal starting place when ablation of gene expression is desired [61]. However, trial and error, both at the level of viral genome engineering to make stable recombinant viruses, and at the whole organism level in a relevant model is absolutely required for evaluating and optimizing miRNA regulation.

Potential caveats While miRNA targeting offers many advantages to traditional strategies, it is not without potential caveats. Given the amplification potential and high error rates of replication-competent viruses, there are two common concerns when using this technology. The first is the potential evolution of escape mutants, which has been demonstrated with miRNA-targeted CVA21, poliovirus, and Dengue virus [36,45,62]. Using perfectly complementary target sequences in tandem and at various stable locations throughout the genome decreases the potential for the evolution of escape mutants [59,61]. Additionally, MREs are differentially effective and susceptible to different selective pressures to generate escape mutations, which may be target and/or tissue-specific [63]. www.sciencedirect.com


Figure 2

Normal Cell TS-miRNA

Viral protein expression Transgene expression Viral genome transcription

No targeted miRNA

TS promoter

Host miRNA mimic No targeted genes

Viral protein expression Transgene expression Viral replication

Viral gene Transgene MREs

Malignant Cell

miRNA-RISC

OncomiR

Viral replication Transgene expression Tumor cell lysis Antitumor immune response Viral genome transcription

miRNA decoy/sponge

miRNA restoration

Viral replication Transgene expression Tumor cell lysis Antitumor immune response

Current Opinion in Virology

Overview of miRNA regulated oncolytic viruses and viral delivery of miRNA therapeutics. Tissue-specific (TS) MREs can be built into nuclear or cytoplasmic replicating viruses to control expression of viral genes/genomes or therapeutic transgenes in normal cells expressing the respective TS-miRNAs, but allowing replication, lysis, and immune response induction in malignant cells. DNA and RNA viruses can be engineered to express artificial miRNA mimics to restore cellular homeostasis, target oncogenes, enhance OV destruction, or facilitate the antitumor immune response. OVs can also be engineered to express miRNA decoys or sponges that are recognized by specific miRNAs. Sequestration of miRNAs can allow expression of genes to enhance OVs or facilitate the immune response. miRNA sponges can also function to sequester oncomiRs. Encoding miRNA-based therapeutics in miRNA-targeted OVs can further prevent off-target toxicities.




This emphasizes the importance of trial and error when engineering miRNA-regulated OVs. This issue may be less of a concern in an immune-competent host when the virus can be cleared before evolution. The second concern is the potential for off-target effects on the host miRNA machinery. Saturation of the miRNA machinery could allow toxicity to reoccur or alter the cellular transcriptome and result in disease. Again, for redirecting viral tropism, using completely complementarity targets allowing rapid recycling of the miRNA, using different MREs, and determining the minimal number of miRNA targets needed will reduce the potential for miRNA saturation [61,64]. Introducing miRNA-based therapeutics into a host cell can similarly saturate the proteins involved in miRNA biogenesis (Figure 1) and create bottlenecks in the pathway if their expression levels are not calibrated. Overexpression of pre-miRNAs can result in accumulation of small RNAs within the nucleus by overloading the exportin proteins involved in translocation from the nucleus to the cytoplasm. Additionally, overexpression of miRNAs can cause a buildup of RNAinduced silencing complex (RISC) substrates by overloading the available RISC complexes and result in cellular toxicities. Therefore, miRNA biogenesis and pro-inflammatory responses in normal tissues need to be evaluated in clinically relevant animal models when analyzing the safety of OVs encoding miRNA-based therapeutics. Viruses have the ability to regulate miRNA biogenesis in infected cells and this may inhibit their ability to deliver miRNA-based therapeutics. Adenoviruses express a noncoding RNA, VA1, which saturates the function of exportin 5 preventing miRNA biogenesis. Poxviruses induce the degradation of all host miRNAs, but 20 O-methylated siRNAs are protected. These factors need to be taken into consideration when designing OV delivery vehicles and the reader is referred to [65] for an in-depth discussion on the potential constraints associated with virally delivered sRNAs.

Oncolytic virus delivery of miRNAtherapeutics Major efforts are focused on developing miRNA-based therapeutics to either restore or inhibit function of deregulated miRNAs and several have recently advanced into clinical testing [24,66]. These agents include synthetic miRNAs that can be processed by host cellular machinery and mimic normal miRNA function (miRNA mimics), or overexpressed RNAs encoding miRNA complementary target sites that compete for miRNA recognition (miRNA decoy/sponge). Studies suggest OVs can enhance delivery, duration, and efficacy of some miRNA-based therapeutics [67–69]. Lou et al. [70] developed an oncolytic Ad coexpressing miR-34a, a miRNA known to silence the antiapoptotic Bcl-2 gene, and the tumor-suppressor protein, IL24. This virus had significantly improved antitumor activity Current Opinion in Virology 2015, 13:40–48

against hepatocellular carcinoma xenografts compared to viruses expressing miR-34a or IL-24 alone. Moshiri et al. [71] modified the Ad-199T virus [42] and a recombinant AAV vector to express a decoy cassette that bound miR-221. This virus resulted in a reduction of miR-221 and an increase in miR-221 target protein, however, neither vector was analyzed in vivo. While a lot of focus has been on engineering DNA viruses for miRNA therapeutic delivery, it was recently discovered that RNA viruses, including IAV, Sindbis virus, and VSV, could also deliver miRNA mimics and decoys [53,72,73]. This is especially interesting as oncolytic RNA viruses are yielding promising results in clinical trials and by incorporating miRNAs that target proinflammatory cytokines or facilitate the antitumor immune response the therapeutic efficacy may be enhanced. While intriguing, caution must be taken when combining therapies as viral expression of miRNAs can cause toxicity in normal tissues if not properly controlled [65].

Conclusions miRNA-targeting can increase the therapeutic index of OVs and gene therapy by precisely detargeting expression in a species and cell-specific manner. As such an arsenal of OVs and therapeutic genes showing enhanced potency and reduced toxicity in preclinical models have been generated. As more tissue and cancer-specific miRNA signatures are identified, defining optimal configurations that can be tailored to individual patients becomes possible. It will also facilitate combination therapies that currently would prove far too toxic for patients. For example, it is now theoretically possible to generate an unattenuated OV encoding an immune stimulatory transgene, and an miRNA targeting an immune inhibitory protein with no off-target toxicities. If such viruses prove efficacious and safe in clinically relevant animal models, the future of cancer therapeutics will then include combinatorial therapy consisting of single-shot formulations (Figure 2).

Acknowledgments Al and Mary Agnes McQuinn, the Richard M. Schulze Family Foundation, and Mayo Clinic support studies in the Russell lab. We apologize to colleagues whose work was not cited due to space limitations.

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