A comprehensive analysis of the Bencao (herbal) small RNA Atlas reveals novel RNA therapeutics for treating human diseases.

Cross-kingdom herbal miRNA was first reported in 2012. Using a modified herbal extraction protocol, we obtained 73,677,287 sequences by RNA-seq from 245 traditional Chinese Medicine (TCM), of which 20,758,257 were unique sequences. We constructed a Bencao (herbal) small RNA (sRNA) Atlas ( http://bencao.bmicc.cn ), annotated the sequences by sequence-based clustering, and created a nomenclature system for Bencao sRNAs. The profiles of 21,757 miRNAs in the Atlas were highly consistent with those of plant miRNAs in miRBase. Using software tools, our results demonstrated that all human genes might be regulated by sRNAs from the Bencao sRNA Atlas, part of the predicted human target genes were experimentally validated, suggesting that Bencao sRNAs might be one of the main bioactive components of herbal medicines. We established roadmaps for oligonucleotide drugs development and optimization of TCM prescriptions. Moreover, the decoctosome, a lipo-nano particle consisting of 0.5%-2.5% of the decoction, demonstrated potent medical effects. We propose a Bencao (herbal) Index, including small-molecule compounds (SM), protein peptides (P), nucleic acid (N), non-nucleic and non-proteinogenic large-molecule compounds (LM) and elements from Mendeleev's periodic table (E), to quantitatively measure the medical effects of botanic medicine. The Bencao sRNA Atlas is a resource for developing gene-targeting oligonucleotide drugs and optimizing botanical medicine, and may provide potential remedies for the theory and practice of one medicine.

Evaluation of the potential of ultrasound-mediated drug delivery for the treatment of ovarian cancer through preclinical studies.

Ovarian cancer (OC) has the greatest mortality rate among gynecological cancers, with a five-year survival rate of <50%. Contemporary adjuvant chemotherapy mostly fails in the case of OCs that are refractory, metastatic, recurrent, and drug-resistant. Emerging ultrasound (US)-mediated technologies show remarkable promise in overcoming these challenges. Absorption of US waves by the tissue results in the generation of heat due to its thermal effect causing increased diffusion of drugs from the carriers and triggering sonoporation by increasing the permeability of the cancer cells. Certain frequencies of US waves could also produce a cavitation effect on drug-filled microbubbles (MBs, phospholipid bilayers) thereby generating shear force and acoustic streaming that could assist drug release from the MBs, and promote the permeability of the cell membrane. A new class of nanoparticles that carry therapeutic agents and are guided by US contrast agents for precision delivery to the site of the ovarian tumor has been developed. Phase-shifting of nanoparticles by US sonication has also been engineered to enhance the drug delivery to the ovarian tumor site. These technologies have been used for targeting the ovarian cancer stem cells and protein moieties that are particularly elevated in OCs including luteinizing hormone-releasing hormone, folic acid receptor, and vascular endothelial growth factor. When compared to healthy ovarian tissue, the homeostatic parameters at the tissue microenvironment including pH, oxygen levels, and glucose metabolism differ significantly in ovarian tumors. US-based technologies have been developed to take advantage of these tumor-specific alterations for precision drug delivery. Preclinical efficacy of US-based targeting of currently used clinical chemotherapies presented in this review has the potential for rapid human translation, especially for formulations that use all substances that are deemed to be generally safe by the U.S. Food and Drug Administration.

Impacts and mechanisms of alternative mRNA splicing in cancer metabolism, immune response, and therapeutics.

Alternative pre-mRNA splicing (AS) provides the potential to produce diversity at RNA and protein levels. Disruptions in the regulation of pre-mRNA splicing can lead to diseases. With the development of transcriptome and genome sequencing technology, increasing diseases have been identified to be associated with abnormal splicing of mRNAs. In tumors, abnormal alternative splicing frequently plays critical roles in cancer pathogenesis and may be considered as new biomarkers and therapeutic targets for cancer intervention. Metabolic abnormalities and immune disorders are important hallmarks of cancer. AS produces multiple different isoforms and diversifies protein expression, which is utilized by the immune and metabolic reprogramming systems to expand gene functions. The abnormal splicing events contributed to tumor progression, partially due to effects on immune response and metabolic reprogramming. Herein, we reviewed the vital role of alternative splicing in regulating cancer metabolism and immune response. We discussed how alternative splicing regulates metabolic reprogramming of cancer cells and antitumor immune response, and the possible strategies to targeting alternative splicing pathways or splicing-regulated metabolic pathway in the context of anticancer immunotherapy. Further, we highlighted the challenges and discuss the perspectives for RNA-based strategies for the treatment of cancer with abnormally alternative splicing isoforms.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA.

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Polymeric Nanocarriers with Controlled Chain Flexibility Boost mRNA Delivery In Vivo through Enhanced Structural Fastening.

Messenger RNA (mRNA) shows high therapeutic potential, though effective delivery systems are still needed for boosting its application. Nanocarriers loading mRNA via polyion complexation with block catiomers into core-shell micellar structures are promising systems for enhancing mRNA delivery. Engineering the interaction between mRNA and catiomers through polymer design can promote the development of mRNA-loaded micelles (mRNA/m) with increased delivery efficiency. Particularly, the polycation chain rigidity may critically affect the mRNA-catiomer interplay to yield potent nanocarriers, yet its effect remains unknown. Herein, the influence of polycation stiffness on the performance of mRNA/m by developing block complementary catiomers having polycation segments with different flexibility, that is, poly(ethylene glycol)-poly(glycidylbutylamine) (PEG-PGBA) and PEG-poly(L-lysine) (PEG-PLL) is studied. PEG-PGBA allows more than 50-fold stronger binding to mRNA than the relatively more rigid PEG-PLL, resulting in mRNA/m with enhanced protection against enzymatic attack and polyanions. mRNA/m from PEG-PGBA significantly enhances mRNA in vivo bioavailability and increased protein translation, indicating the importance of controlling polycation flexibility for forming stable polyion complexes with mRNA toward improved delivery.

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA.

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²âº. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.