Small-molecule GSDMD agonism in tumors stimulates antitumor immunity without toxicity.

Gasdermin-mediated inflammatory cell death (pyroptosis) can activate protective immunity in immunologically cold tumors. Here, we performed a high-throughput screen for compounds that could activate gasdermin D (GSDMD), which is expressed widely in tumors. We identified 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB) as a direct and selective GSDMD agonist that activates GSDMD pore formation and pyroptosis without cleaving GSDMD.

Tumor editing suppresses innate and adaptive antitumor immunity and is reversed by inhibiting DNA methylation.

Cancer cells edit gene expression to evade immunosurveillance. However, genome-wide studies of gene editing during early tumorigenesis are lacking. Here we used single-cell RNA sequencing in a breast cancer genetically engineered mouse model (GEMM) to identify edited genes without bias.

NOP16 is a histone mimetic that regulates Histone H3K27 methylation and gene repression.

Post-translational modifications of histone tails alter chromatin accessibility to regulate gene expression. Some viruses exploit the importance of histone modifications by expressing histone mimetic proteins that contain histone-like sequences to sequester complexes that recognize modified histones. Here we identify an evolutionarily conserved and ubiquitously expressed, endogenous mammalian protein Nucleolar protein 16 (NOP16) that functions as a H3K27 mimic.

The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor. Blockage of NKp46 inhibits NK killing of many cancer targets.

Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2 orthotopic, metastatic, and genetically engineered mouse breast cancer models.

Combinatorial siRNA Polyplexes for Receptor Targeting.

As synthetic small interfering RNA (siRNA) against antitumoral gene targets show promise for cancer treatment, different siRNA delivery systems have sparkled intense investigations. To develop tumor-specific carriers for cytosolic and systemic siRNA delivery, our laboratory has recently generated folate-conjugated targeted combinatorial siRNA polyplexes based on sequence-defined oligomer platform compatible with solid-phase-supported synthesis. These polyplexes presented efficient siRNA-mediated gene silencing in folate receptor-expressing tumors in vitro and in vivo.

Folate receptor-directed orthogonal click-functionalization of siRNA lipopolyplexes for tumor cell killing in vivo.

The delivery of small interfering RNA (siRNA) and its therapeutic usage as an anti-cancer agent requires a carrier system for selective internalization into the cytosol of tumor cells. We prepared folate-bearing formulations by first complexing siRNA with the novel azido-functionalized sequence-defined cationizable lipo-oligomer 1106 (containing two cholanic acids attached to an oligoaminoamide backbone in T-shape configuration) into spherical, ∼100-200 nm sized lipopolyplexes, followed by surface-functionalization with various folate-conjugated DBCO-PEG agents. Both the lipo-oligomer and the different defined shielding and targeting agents with mono- and bis-DBCO and varying PEG length were generated by solid phase supported synthesis.

Systemic Delivery of Folate-PEG siRNA Lipopolyplexes with Enhanced Intracellular Stability for In Vivo Gene Silencing in Leukemia.

Protection of small interfering RNA (siRNA) against degradation and targeted delivery across the plasma and endosomal membranes to the final site of RNA interference (RNAi) are major aims for the development of siRNA therapeutics. Targeting for folate receptor (FR)-expressing tumors, we optimized siRNA polyplexes by coformulating a folate-PEG-oligoaminoamide (for surface shielding and targeting) with one of three lipo-oligoaminoamides (optionally tyrosine-modified, for optimizing stability and size) to generate ∼100 nm targeted lipopolyplexes (TLPs), which self-stabilize by cysteine disulfide cross-links. To better understand parameters for improved tumor-directed gene silencing, we analyzed intracellular distribution and siRNA release kinetics.

Precise redox-sensitive cleavage sites for improved bioactivity of siRNA lipopolyplexes.

Lipo-oligomers have been proven as potent siRNA carriers based on stable electrostatic and hydrophobic complex formation and endosomal membrane destabilization. Although high stability of siRNA polyplexes is desirable in the extracellular space and cellular uptake, intracellular disassembly is important for the cytosolic release of siRNA and RNA-induced silencing complex formation. To improve the release, bioreducible sequence-defined lipo-oligomers were synthesized by solid-phase assisted synthesis using the disulfide building block Fmoc-succinoyl-cystamine for precise positioning of a disulfide unit between a lipophilic diacyl (bis-myristyl, bis-stearyl or bis-cholestanyl) domain and an ionizable oligocationic siRNA binding unit.

Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes.

Small interfering RNA (siRNA) promises high efficacy and excellent specificity to silence the target gene expression, which shows potential for cancer treatment. However, systemic delivery of siRNA with selectivity to the tumor site and into the cytosol of tumor cells remains a major limitation. To achieve this, we generated oligoaminoamide-based sequence-defined polycationic oligomers by solid-phase assisted synthesis, which can form polyplexes with anionic siRNA by electrostatic interaction to serve as siRNA carrier.

Dual antitumoral potency of EG5 siRNA nanoplexes armed with cytotoxic bifunctional glutamyl-methotrexate targeting ligand.

Synthetic small interfering RNA (siRNA) is a class of therapeutic entities that allow for specific silencing of target genes via RNA interference (RNAi) and comprise an enormous clinical potential for a variety of diseases, including cancer. However, efficient tissue-specific delivery of siRNA remains the major limitation in the development of RNAi-based cancer therapeutics. To achieve this, we have synthesized a series of sequence-defined oligomers, which include a cationic (oligoethanamino)amide core (for nanoparticle formation with siRNA), cysteines (as bioreversible disulfide units), and a polyethylene glycol chain (for shielding of surface charges) coupled to a terminal targeting ligand.

Sequence-defined oligoaminoamides for the delivery of siRNAs.

Since it was found that synthetic small interfering RNA (siRNA) can invoke RNA interference (RNAi) responses in mammalian cells, it has gained enormous attention as a tool for gene silencing in basic science and as a novel therapeutic modality. To develop carriers for cytosolic and systemic siRNA delivery, our laboratory has recently developed a sequence-defined polymer platform compatible with solid-phase-supported synthesis. These polymers have displayed efficient siRNA-mediated gene silencing in vitro and in vivo.