Rapid generation of long, chemically modified pegRNAs for prime editing.

The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA.

siRNA nanoparticle targeting Usp20 lowers lipid levels and ameliorates metabolic syndrome in mice.

Atherosclerotic cardiovascular disease is closely correlated with elevated low density lipoprotein-cholesterol. In feeding state, glucose and insulin activate mammalian target of rapamycin 1 that phosphorylates the deubiquitylase ubiquitin-specific peptidase 20 (USP20). USP20 then stabilizes HMG-CoA reductase, thereby increasing lipid biosynthesis.

Reducing Oxidative Stress-Mediated Alcoholic Liver Injury by Multiplexed RNAi of , and .

The prevalence of excessive drinking-related alcoholic liver disease (ALD) is rising, yet therapeutic options remain limited. High alcohol consumption and consequent oxidative metabolism by cytochrome P450 (CYP) can lead to extremely high levels of reactive oxygen species, which overwhelm cellular defenses and harm hepatocytes. Our previous investigations showed that inhibiting using RNA interference reduced the incidence of ALD.

Minimizing the ratio of ionizable lipid in lipid nanoparticles for base editing.

Lipid nanoparticles (LNPs) have gained clinical approval as carriers for both siRNA and mRNA. Among the crucial components of LNPs, ionizable lipids play a pivotal role in determining the efficiency of RNA delivery. In this study, we synthesized a series of ionizable lipids, denoted as HTO, with a higher count of hydroxyl groups compared to SM-102.

Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway.

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells.

Lipid nanoparticle delivery of siRNA targeting gene attenuates subacute alcoholic liver injury in mice.

Objectives: To investigate the effect and mechanism of lipid nanoparticle (LNP) delivery of small interfering RNA (siRNA) targeting gene on subacute alcoholic liver injury in mice.

Methods: siRNA targeting gene was encapsulated in LNP (si-Cyp2e1 LNP) by microfluidic technique and the resulting LNPs were characterized. The optimal dose of si-Cyp2e1 LNP administration was screened.

mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation.

preparation of mRNA is a key step for mRNA therapeutics. The widely used T7 RNA polymerase (RNAP) was shown to have many by-products during transcription (IVT) process, among which double-stranded RNA (dsRNA) is the major by-product to activate the intracellular immune response. Here, we describe the use of a new VSW-3 RNAP that reduced dsRNA production during IVT and the resulting mRNA exhibited low inflammatory stimulation in cells.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models.

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic.

Tea leaf-derived exosome-like nanotherapeutics retard breast tumor growth by pro-apoptosis and microbiota modulation.

While several artificial nanodrugs have been approved for clinical treatment of breast tumor, their long-term applications are restricted by unsatisfactory therapeutic outcomes, side reactions and high costs. Conversely, edible plant-derived natural nanotherapeutics (NTs) are source-widespread and cost-effective, which have been shown remarkably effective in disease treatment. Herein, we extracted and purified exosome-like NTs from tea leaves (TLNTs), which had an average diameter of 166.

Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification.

Natural exosome-like nanovesicles from edible tea flowers suppress metastatic breast cancer ROS generation and microbiota modulation.

Although several artificial nanotherapeutics have been approved for practical treatment of metastatic breast cancer, their inefficient therapeutic outcomes, serious adverse effects, and high cost of mass production remain crucial challenges. Herein, we developed an alternative strategy to specifically trigger apoptosis of breast tumors and inhibit their lung metastasis by using natural nanovehicles from tea flowers (TFENs). These nanovehicles had desirable particle sizes (131 nm), exosome-like morphology, and negative zeta potentials.

Cocktail strategy based on a dual function nanoparticle and immune activator for effective tumor suppressive.

Background: Immune checkpoint inhibitor-mediated immunotherapy cannot be carried out on a large scale clinically due to its low universality. In recent years, cyclic guanosine monophosphate synthase/interferon gene stimulating factor (cGAS/STING)-mediated innate immune signaling pathway-mediated immunotherapy has attracted more and more attention. In addition, metabolic inhibitors also show good effects on tumor treatment, but their application is often limited because of their large first pass effect or difficult administration.

Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations.

The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations.

Tumor Microenvironment-Responsive Nanococktails for Synergistic Enhancement of Cancer Treatment via Cascade Reactions.

A combination treatment strategy that relies on the synergetic effects of different therapeutic approaches has been considered to be an effective method for cancer therapy. Herein, a chemotherapeutic drug (doxorubicin, Dox) and a manganese ion (Mn) were co-loaded into regenerated silk fibroin-based nanoparticles (NPs), followed by the surface conjugation of phycocyanin (PC) to construct tumor microenvironment-activated nanococktails. The resultant PC-Mn@Dox-NPs showed increased drug release rates by responding to various stimulating factors (acidic pH, hydrogen peroxide (HO), and glutathione), revealing that they could efficiently release the payloads (Dox and Mn) in tumor cells.

Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing.

The discovery and applications of clustered regularly interspaced short palindromic repeat (CRISPR) systems have revolutionized our ability to track and manipulate specific nucleic acid sequences in many cell types of various organisms. The robustness and simplicity of these platforms have rapidly extended their applications from basic research to the development of therapeutics. However, many hurdles remain on the path to translation of the CRISPR systems to therapeutic applications: efficient delivery, detectable off-target effects, potential immunogenicity, and others.

A HMCuS@MnO nanocomplex responsive to multiple tumor environmental clues for photoacoustic/fluorescence/magnetic resonance trimodal imaging-guided and enhanced photothermal/photodynamic therapy.

Hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) are advantageous for loading small-molecule therapeutic drugs coupled with photothermal ablation for synergistic tumor therapy. However, treatment efficacy mediated by HMCuS NPs is not always satisfactory owing to their insensitivity toward the tumor microenvironment (TME), and unpredictable drug leakage may also result in deleterious systemic toxicity. Here, a novel HMCuS@MnO-based core-shell nanoplatform was developed as a highly efficient TME modulator, which could alleviate tumor hypoxia, deplete the level of intracellular glutathione (GSH) and trigger the dissolution of Mn.

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics.

African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control.

Tuning C-C sp2/sp3 ratio of DLC films in FCVA system for biomedical application.

Diamond like carbon (DLC) films with different C-C sp2/sp3 ratios were prepared by tuning the N2 flow rate in a filtered cathodic vacuum arc (FCVA) system. The increase of N2 flow rate facilitated the increase of C-C sp2/sp3 ratio (1.09-2.

Camellia sinensis and Litsea coreana Ameliorate Intestinal Inflammation and Modulate Gut Microbiota in Dextran Sulfate Sodium-Induced Colitis Mice.

Scope: Polyphenol-enriched herbal extracts have been proved as alternative therapeutic strategies for experimentally induced colitis. The in vivo and in vitro anti-inflammatory effects of Camellia sinensis (green, white, yellow, oolong, black, and dark tea) and Litsea coreana (hawk tea) are comparatively explored.

Methods And Results: HPLC analysis confirms dissimilarities among phytochemical compositions of these teas.

Lycium barbarum lipid-based edible nanoparticles protect against experimental colitis.

Edible plant-derived nanoparticles (NPs) have attracted increasing attention in the treatment of ulcerative colitis (UC). Lycium barbarum (LB), a popular functional fruit, possesses various biological functions. Here, fat-soluble contents were extracted from LB and further processed into LB lipid-derived NPs (LBLNs).

Modulation of drug release by decoration with Pluronic F127 to improve anti-colon cancer activity of electrospun fibrous meshes.

Applications of fibrous meshes for localized chemotherapy have been limited by the lack of optimal carriers that are capable of releasing sufficient drug locally. To overcome this obstacle, we introduced Pluronic F127 (PF127) to the camptothecin (CPT)-loaded poly(lactic acid/glycolic acid) (PLGA) electrospun fibrous meshes, abbreviated as PPC, for modulation of drug release. These PPC meshes had smooth surface and high drug loading (5.

Porous Polymeric Microparticles as an Oral Drug Platform for Effective Ulcerative Colitis Treatment.

Porous microparticles (MPs) have been regarded as a promising vehicle for drug delivery. Herein, porous MPs and their counterparts (nonporous MPs) were produced by a conventional emulsion-solvent evaporation method in the presence and absence of ammonium bicarbonate, and curcumin was encapsulated into these MPs during the preparation process. The obtained MPs possessed desirable diameters of around 1.

Oral administration of colitis tissue-accumulating porous nanoparticles for ulcerative colitis therapy.

To improve the penetration and accumulation of anti-inflammatory drugs in colitis tissue, we functionalized the surface of porous poly(lactic-co-glycolic acid) nanoparticles (NPs) using pluronic F127 (PF127) and loaded curcumin (CUR) into the resulting NPs to obtain porous PF127-functionalized CUR-loaded NPs (porous PF127-NPs). These NPs had an average hydrodynamic diameter of about 270 nm with a highly monodisperse size distribution, slightly negative surface charge and controllable CUR release profile. It was found that they had good biocompatibility and yielded a much higher cellular uptake rate of CUR than porous CUR-loaded NPs without PF127 modification (porous NPs).