No abstract

mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.

Efficient endosomal escape is the most essential but challenging issue for siRNA drug development. Herein, a series of quaternary ammonium-based amphiphilic triblock polymers harnessing an elaborately tailored pH-sensitive hydrophobic core were synthesized and screened. Upon incubating in an endosomal pH environment (pH 6.5–6.8), mPEG45-P­(DPA50-co-DMAEMA56)-PT53 (PDDT, the optimized polymer) nanomicelles (PDDT-Ms) and PDDT-Ms/siRNA polyplexes rapidly disassembled, leading to promoted cytosolic release of internalized siRNA and enhanced silencing activity evident from comprehensive analysis of the colocalization and gene silencing using a lysosomotropic agent (chloroquine) and an endosomal trafficking inhibitor (bafilomycin A1). In addition, PDDT-Ms/siPLK1 dramatically repressed tumor growth in both HepG2-xenograft and highly malignant patient-derived xenograft models. PDDT-Ms-armed siPD-L1 efficiently blocked the interaction of PD-L1 and PD-1 and restored immunological surveillance in CT-26-xenograft murine model. PDDT-Ms/siRNA exhibited ideal safety profiles in these assays. This study provides guidelines for rational design and optimization of block polymers for efficient endosomal escape of internalized siRNA and cancer therapy.

Extracellular vesicles (EVs) have shown great potential in drug delivery, disease diagnosis, and treatment owing to their versatile native features and functions. RNA interference (RNAi) therapeutics that block the programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) pathway have attracted increasing interest for the treatment of various cancers. Here, immunoregulatory EVs are developed by decorating M1-macrophagederived EVs (M1 EV) with vesicular stomatitis virus glycoprotein (VSV-G), a pH-responsive viral fusion protein, and electroporating anti-PD-L1 siRNA (siPD-L1) into the EVs. After administration to CT26 tumor-bearing mice, this virus-mimic nucleic acid engineered EVs (siRNA@V-M1 EV) can target tumor tissues, which is attributed to the natural tumor-homing property of M1 EV. Then, the fusion of VSV-G with cells facilitates the direct release of siPD-L1 into the cytoplasm and triggers robust gene silencing, leading to the efficient block of PD-L1/PD-1 interaction and then the elevation of CD8 + T cell population. Meanwhile, the M1 EVs and IFN-γ secreted by the CD8 + T cells promote the repolarization of M2 tumor-associated macrophages to M1 macrophages. The combination of PD-L1/PD-1 pathway blocking, T cell recognition reconstructing, and M1 macrophage repolarization via multifunctional EVs can achieve satisfactory antitumor efficacy in this tumor model, showing potential as a new modality to fight cancers.

Pancreatic cancer is currently one of the deadliest of the solid malignancies, whose incidence and death rates are increasing consistently during the past 30 years. Ribonucleotide reductase (RR) is a rate-limiting enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides, which are essential for DNA synthesis and replication. In this study, 23 small interfering RNAs (siRNAs) against RRM2, the second subunit of RR, were designed and screened, and one of them (termed siRRM2), with high potency and good RNase-resistant capability, was selected. Transfection of siRRM2 into PANC-1, a pancreatic cell line, dramatically repressed the formation of cell colonies by inducing remarkable cell-cycle arrest at S-phase. When combining with doxorubicin (DOX), siRRM2 improved the efficacy 4 times more than applying DOX alone, suggesting a synergistic effect of siRRM2 and DOX. Moreover, the combined application of siRRM2-loaded lipid nanoparticle and DOX significantly suppressed the tumor growth on the PANC-1 xenografted murine model. The inhibition efficiency revealed by tumor weight at the endpoint of the treatment reached more than 40%. Hence, siRRM2 effectively suppressed pancreatic tumor growth alone or synergistically with DOX. This study provides a feasible target gene, a drug-viable siRNA, and a promising therapeutic potential for the treatment of pancreatic cancer.