Evaluation of monocytes as carriers for armed oncolytic adenoviruses in murine and Syrian hamster models of cancer

Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs

Recombinant adenoviral vectors turn on the type I interferon system without inhibition of transgene expression and viral replication

Recombinant adenovirus administration gives rise to transgene-independent effects caused by the ability of the vector to activate innate immunity mechanisms. We show that recombinant adenoviruses encoding reporter genes trigger IFN-alpha and IFN-beta transcription from both plasmacytoid and myeloid mouse dendritic cells. Interestingly, IFN-beta and IFN-alpha5 are the predominant transcribed type I IFN genes both in vitro and in vivo. In human peripheral blood leukocytes type I IFNs are induced by adenoviral vectors, with a preponderance of IFN-beta together with IFN-alpha1 and IFN-alpha5 subtypes. Accordingly, functional type I IFN is readily detected in serum samples from human cancer patients who have been treated intratumorally with a recombinant adenovirus encoding thymidine kinase. Despite inducing functional IFN-alpha release in both mice and humans, gene transfer by recombinant adenoviruses is not interfered with by type I IFNs either in vitro or in vivo. Moreover, IFN-alpha does not impair replication of wild-type adenovirus. As a consequence, cancer gene therapy strategies with defective or replicative-competent adenoviruses are not expected to be hampered by the effect of the type I IFNs induced by the vector itself. However, type I IFN might modulate antitumor and antiadenoviral immune responses and thus influence the outcome of gene immunotherapy

Effect of adenovirus-mediated RNA interference on endogenous microRNAs in a mouse model of multidrug resistance protein 2 gene silencing

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery

An oncolytic adenovirus controlled by a modified telomerase promoter is attenuated in telomerase-negative cells, but shows reduced activity in cancer cells

The promoter for human telomerase reverse transcriptase (hTERTp) is preferentially active in malignant cells. It was recently used to control the expression of the adenoviral E1A gene for the development of oncolytic adenoviruses. To ensure maximal repression in normal cells, the inclusion of additional E-boxes in the proximal region of the core promoter was described. We found that the transcriptional activity of this artificial sequence (T-255-4DEB) is minimal in normal cells, but it is also reduced in all the cancer cell lines tested. The cancer specificity of a new oncolytic adenovirus based in this promoter (AdTE1) was evaluated by direct comparison with wild-type adenovirus type 5 (AdWT) in vitro and in vivo. In all the parameters tested, AdTE1 was attenuated in normal cells, but the efficacy in cancer cells showed a parallel reduction, suggesting a lack of specificity. However, the cytotoxicity of AdTE1 was repressed in senescent cells compared to AdWT. Therefore, we conclude that AdTE1 is preferentially attenuated only in cells that are permanently devoid of telomerase expression such as senescent cells. Further modifications in the telomerase-based promoters should be introduced in order to combine maximal attenuation of oncolytic adenoviruses in normal tissues and enhanced activity in tumors

<em>Rbfox1</em> Downregulation and Altered <em>Calpain 3</em> Splicing by <em>FRG1</em> in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)

<div><p>Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (<em>FRG1</em>) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. <em>FRG1</em> has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in <em>FRG1</em> mice. We find that splicing perturbations parallel the responses of different muscles to <em>FRG1</em> over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of <em>FRG1</em>-affected splicing events. <em>Rbfox1</em> knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the <em>Rbfox1</em> RNA and decreases its stability. Consistent with this, <em>Rbfox1</em> expression is down-regulated in mice and cells over-expressing <em>FRG1</em> as well as in FSHD patients. Among the genes affected is <em>Calpain 3</em>, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In <em>FRG1</em> mice and FSHD patients, the <em>Calpain 3</em> isoform lacking exon 6 (<em>Capn3 E6–</em>) is increased. Finally, <em>Rbfox1</em> knockdown and over-expression of <em>Capn3 E6-</em> inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of <em>FRG1</em>, reducing <em>Rbfox1</em> levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.</p> </div