Intra-Tumoral Activation of Endosomal TLR Pathways Reveals a Distinct Role for TLR3 Agonist Dependent Type-1 Interferons in Shaping the Tumor Immune Microenvironment.

Toll-like receptor (TLR) agonists have received considerable attention as therapeutic targets for cancer immunotherapy owing to their ability to convert immunosuppressive tumor microenvironments towards a more T-cell inflamed phenotype. However, TLRs differ in their cell expression profiles and intracellular signaling pathways, raising the possibility that distinct TLRs differentially influence the tumor immune microenvironment. Using single-cell RNA-sequencing, we address this by comparing the tumor immune composition of B16F10 melanoma following treatment with agonists of TLR3, TLR7, and TLR9. Marked differences are observed between treatments, including decreased tumor-associated macrophages upon TLR7 agonist treatment. A biased type-1 interferon signature is elicited upon TLR3 agonist treatment as opposed to a type-2 interferon signature with TLR9 agonists. TLR3 stimulation was associated with increased macrophage antigen presentation gene expression and decreased expression of PD-L1 and the inhibitory receptors Pirb and Pilra on infiltrating monocytes. Furthermore, in contrast to TLR7 and TLR9 agonists, TLR3 stimulation ablated FoxP3 positive CD4 T cells and elicited a distinct CD8 T cell activation phenotype highlighting the potential for distinct synergies between TLR agonists and combination therapy agents.

Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses.

Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving "undruggable" targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205(+) DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR.

Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells.

BACKGROUND: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes. METHODS: High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSß heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSß (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells. RESULTS: This study identified distinct genetic profiles for MutSα and MutSß, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers. CONCLUSION: Our findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors.

Aptamer-targeted antigen delivery.

Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8α(+) dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8(+) T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8(+) T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257-264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-γ and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines.

Optimizing siRNA delivery to the genital mucosa.

RNA interference (RNAi) describes a highly conserved pathway, present in eukaryotic cells, for regulating gene expression. Small stretches of double-stranded RNA, termed small interfering RNAs (siRNAs), utilize this pathway to bind homologous mRNA, resulting in site-specific mRNA cleavage and subsequent protein degradation. The ubiquitous presence of the RNAi machinery, combined with its specificity and efficacy, makes it an attractive mechanism for reducing aberrant gene expression in therapeutic settings. However, a major obstacle to utilizing RNAi in the clinic is siRNA delivery. Administered siRNAs must make contact with the appropriate cell types and, following internalization, gain access to the cytosol where the RNAi machinery resides. This must be achieved so that silencing is maximized, whilst minimizing any undesirable off-target effects. Recently, the utility of siRNAs as a microbicide, usually applied to the genital mucosa for preventing transmission of sexually transmitted diseases including HIV-1 and HSV-2, has been investigated. In this review we will describe these studies and discuss potential strategies for improving gene silencing.

siRNA-based topical microbicides targeting sexually transmitted infections.

Sexually transmitted infections (STIs) are a major cause of morbidity and mortality worldwide. Although a vaccine is available for HPV, no effective vaccines exist for the HIV-1 and HSV-2 viral pathogens, and there are no cures for these infections. Furthermore, recent setbacks in clinical trials, such as the failure of the STEP trial to prevent HIV-1 infection, have emphasized the need to develop alternative approaches to interrupt the transmission of these pathogens. One alternative strategy is represented by the use of topically applied microbicides, and such agents are being developed against various viruses. RNAi-based microbicides have recently been demonstrated to prevent HSV-2 transmission, and may be useful for targeting multiple STIs. In this review, microbicides that are under development for the prevention of STIs are described, with a focus on topically applied microbicidal siRNAs.