Use of Tissue-Specific MicroRNA to Control Pathology of Wild-Type Adenovirus without Attenuation of Its Ability to Kill Cancer Cells

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 39 UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5610 10 viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral disease

MicroRNA Controlled Adenovirus Mediates Anti-Cancer Efficacy without Affecting Endogenous MicroRNA Activity

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation and stability by binding to complementary sequences usually within the 3′ un-translated region (UTR). We have previously shown that the hepatic toxicity caused by wild-type Adenovirus 5 (Ad5WT) in mice can be prevented by incorporating 4 binding sites for the liver-specific microRNA, mir122, into the 3′ UTR of E1A mRNA. This virus, termed Ad5mir122, is a promising virotherapy candidate and causes no obvious liver pathology. Herein we show that Ad5mir122 maintains wild-type lytic activity in cancer cells not expressing mir122 and assess any effects of possible mir122 depletion in host cells. Repeat administration of 2×1010 viral particles of Admir122 to HepG2 tumour bearing mice showed significant anti-cancer efficacy. RT-QPCR showed that E1A mRNA was down-regulated 29-fold in liver when compared to Ad5WT. Western blot for E1A confirmed that all protein variants were knocked down. RT-QPCR for mature mir122 in infected livers showed that quantity of mir122 remained unaffected. Genome wide mRNA microarray profiling of infected livers showed that although the transcript level of >3900 different mRNAs changed more than 2-fold following Ad5WT infection, less than 600 were changed by Ad5mir122. These were then filtered to select mRNAs that were only altered by Ad5mir122 and the remaining 21 mRNAs were compared to predicted mir122 targets. No mir122 target mRNAs were affected by Ad5 mir122. These results demonstrate that the exploitation of microRNA regulation to control virus replication does not necessarily affect the level of the microRNA or the endogenous mRNA targets

Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins

Design and application of oncolytic viruses for cancer immunotherapy

The approval of the first oncolytic virus (OV) for the treatment of metastatic melanoma and the recent discovery that the use of oncolytic viruses may enhance cancer immunotherapies targeted against various immune checkpoint proteins have attracted great interest in the field of cancer virotherapy. OVs are designed to target and kill cancer cells leaving normal cell unharmed. OV infection and concomitant cancer cell killing stimulate anti-tumour immunity and modulates tumour microenvironment towards less immunosuppressive phenotype. The intrinsic capacity of OVs to turn immunologically cold tumours into immunologically hot tumours, and to increase immune cell and cytokine infiltration, can be further enhanced by arming OVs with transgenes that increase their immunostimulatory activities and direct immune responses specifically towards cancer cells. These OVs, specifically engineered to be used as cancer immunotherapeutics, can be synergized with other immune modulators or cytotoxic agents to achieve the most potent immunotherapy for cancer

MIR-193A MIMICS AS NEW POTENTIAL THERAPEUTIC AGENTS IN CUTANEOUS MELANOMA

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that nega- tively regulate gene and other non-coding transcripts expression. MiR- NAs play relevant functions in cancer biology and “miRNA mimics” may represent new potential innovative therapeutic agents restoring nor- mal function of endogenous tumor suppressive miRNAs1 MiR-193a acts as tumour suppressor in different types of cancer.2–4 In melanoma, it is down-expressed in tissues and in plasma of patients,5,6 but its role as oncosuppressor has not been yet clearly established. Aim of this study was to analyse the effects of miR-193a mimics transfection in melanoma cells to evaluate their potential role as therapeutic agents against cuta- neous melanoma. miR-193a-3p and miR-193a-5p mimics were trans- fected with lipofectamine in three different melanoma cell lines with different B-RAF mutation status. Their effects on cell viability and migration, p-Akt, p-Erk, B-Raf protein levels and epithelial-mesenchy- mal transition (EMT) were evaluated. A significant decrease of cell via- bility and migration ability was induced by both miR-193a mimics in transfected cells. In addition, they decreased B-Raf protein levels and phosphorylation of Akt and Erk proteins. Finally, also Vimentin and E- Cadherin protein levels were significantly changed in transfected-cells: Vimentin expression was significantly reduced, while E-cadherin expres- sion was significantly increased. Overall our results indicate the potential of both miRNA mimics to interfere with melanoma cell proliferation, survival and metastatization, independently from B-RAF mutation status of melanoma cells. So, our data suggest that miR193a mimics may repre- sent potential therapeutic agents reducing melanoma progression. Future experiments will be aimed at investigating this therapeutic strategy in an in vivo cutaneous melanoma model. References: [1] Maitri et al., (2016) EBioMedicine; 12: 34–42. [2] Williams et al., (2015) Oncotarget; 6(27):23480–95. [3] Jian et al., (2016) Tumour Biol.; Jul;37(7):8941–9. [4] Yu et al., (2015) Oncogene; 34(4):413–23. [5] Caramuta et al. (2010) J Invest Dermatol.;130(8):2062–70. [6] Fogli et al., (2017) Tumour Biol.;39(5):1010428317701646

Harnessing therapeutic viruses as a delivery vehicle for RNA-based therapy

Messenger RNA (mRNA) and microRNA (miRNA)-based therapeutics have become attractive alternatives to DNA-based therapeutics due to recent advances in manufacture, scalability and cost. Also, RNA-based therapeutics are considered safe since there are no risk of inducing genomic changes as well as the potential adverse effects would be only temporary due to the transient nature of RNA-based therapeutics. However, efficient in vivo delivery of RNA-based therapeutics remains a challenge. We have developed a delivery platform for RNA-based therapeutics by exploiting the physicochemical properties of enveloped viruses. By physically attaching cationic liposome/RNA complexes onto the viral envelope of vaccinia virus, we were able to deliver mRNA, self-replicating RNA as well as miRNA inside target cells. Also, we showed that this platform, called viRNA platform, can efficiently deliver functional miRNA mimics into B16.OVA tumour in vivo

Novel personalized cancer vaccine platform based on Bacillus Calmette-Guèrin

Background Intratumoral BCG therapy, one of the earliest immunotherapies, can lead to infiltration of immune cells into a treated tumor. However, an increase in the number of BCG-induced tumor-specific T cells in the tumor microenvironment could lead to enhanced therapeutic effects. Methods Here, we have developed a novel cancer vaccine platform based on BCG that can broaden BCG-induced immune responses to include tumor antigens. By physically attaching tumor-specific peptides onto the mycobacterial outer membrane, we were able to induce strong systemic and intratumoral T cell-specific immune responses toward the attached tumor antigens. These therapeutic peptides can be efficiently attached to the mycobacterial outer membrane using a poly-lysine sequence N-terminally fused to the tumor-specific peptides. Results Using two mouse models of melanoma and a mouse model of colorectal cancer, we observed that the antitumor immune responses of BCG could be improved by coating the BCG with tumor-specific peptides. In addition, by combining this novel cancer vaccine platform with anti-programmed death 1 (anti-PD-1) immune checkpoint inhibitor (ICI) therapy, the number of responders to anti-PD-1 immunotherapy was markedly increased. Conclusions This study shows that intratumoral BCG immunotherapy can be improved by coating the bacteria with modified tumor-specific peptides. In addition, this improved BCG immunotherapy can be combined with ICI therapy to obtain enhanced tumor growth control. These results warrant clinical testing of this novel cancer vaccine platform

Characterization of a novel OX40 ligand and CD40 ligand-expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform

Ylösmäki et al. develop an oncolytic adenovirus expressing immunostimulatory cytokines OX40L and CD40L. This virus was tested in a peptide-based cancer vaccine platform called PeptiCRAd. Intratumoral administration of PeptiCRAd induced systemic and intratumoral tumor-specific T cell responses, reduced tumor growth, and sensitized tumors to immune checkpoint inhibitor therapy

Circulating microRNAs as biomarkers for early diagnosis of cutaneous melanoma

Introduction: Cutaneous melanoma is the deadliest form of skin cancer, with a dramatic increase in the incidence rate worldwide over the past decade. Early detection has been shown to improve the outcome of melanoma patients. The identification of noninvasive biomarkers able to identify melanoma at an early stage remains an unmet clinical need. Circulating miRNAs (c-miRNAs), small non-coding RNAs, appear as potential ideal candidate biomarkers due to their stability in biological fluids and easy detectability. Moreover, c-miRNAs are reported to be heavily deregulated in cancer patients. Areas covered: This review examines evidence of the specific c-miRNAs or panels of c-miRNAs reported to be useful in discriminating melanoma from benign cutaneous lesions. Expert opinion: Although the interesting reported by published studies, the non-homogeneity of detection and normalization methods prevents the individuation of single c-miRNA or panel of c-miRNAs that are specific for early detection of cutaneous melanoma. In the future, prospective wide and well-designed clinical trials will be needed to validate the diagnostic potential of some of the c-miRNA candidates in clinical practice

Exploiting preexisting immunity to enhance oncolytic cancer immunotherapy

Because of the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T-cell repertoire. Although CD8þ antitumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4þ T-cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4þ T-cell memory populations could be reengaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I–restricted tumor-specific peptides and developed it further by introducing pathogen-specific MHC-II–restricted peptides. Mice preimmunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid- and tumor-specific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced antitumor efficacy and induced TT-specific, CD40 ligand-expressing CD4þ T helper cells and maturation of antigen-presenting cells. Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4þ memory T-cell repertoires when designing immunotherapeutic treatment regimens. Finally, the antitumor effect was even more prominent when combined with the immune checkpoint inhibitor anti–PD-1, strengthening the rationale behind combination therapy with oncolytic viruses. Significance: These findings establish a novel technology that enhances oncolytic cancer immunotherapy by capitalizing on pre-acquired immunity to pathogens to convert a weak antitumor immune response into a much stronger one