RNA Interference Therapeutics Research Articles

The Growing Class of Novel RNAi Therapeutics.

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza{trade mark, serif}), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro{trade mark, serif}), lumasiran (Oxlumo{trade mark, serif}), inclisiran (Leqvio{trade mark, serif}), vutrisiran (Amvuttra{trade mark, serif}), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari{trade mark, serif}), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. Significance Statement The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Model-based meta-analysis to quantify the effects of short interfering RNA therapeutics on hepatitis B surface antigen turnover in hepatitis B-infected mice.

The objective of this study was to compare the efficacy of short interfering RNA therapeutics (siRNAs) inreducing hepatitis B surface antigen (HBsAg) levels in hepatitis B-infected (HBV) mice across multiple siRNA therapeutic classesusing model-based meta-analysis (MBMA) techniques. Literature data from 10 studies in HBV-infected mice were pooled, including 13 siRNAs, formulated as liposomal nanoparticles (LNPs) or conjugated to either cholesterol (chol) or N-acetylgalactosamine (GalNAc). Time course of the baseline- and placebo-corrected mean HBsAg profiles were modeled using kinetics of drug effect (KPD) model coupled to an indirect response model (IRM) within a longitudinal non-linear mixed-effects MBMA framework. Single and multiple dose simulations were performed exploring the role of dosing regimens across evaluated siRNA classes. The HBsAg degradation rate (0.72 day-1) was consistent across siRNAs but exhibited a large between-study variability of 31.4% (CV%). The siRNA biophase half-life was dependent on the siRNA class and was highest for GalNAc-siRNAs (21.06 days) and lowest for chol-siRNAs (2.89 days). ID50 estimates were compound-specific and were lowest for chol-siRNAs and highest for GalNAc-siRNAs. Multiple dose simulations suggest GalNAc-siRNAs may require between 4 and 7 times less frequent dosing at higher absolute dose levels compared to LNP-siRNAs and chol-siRNAs, respectively, to reach equipotent HBsAg-lowering effects in HBV mice. In conclusion, non-clinical HBsAg concentration-time data after siRNA administration can be described using the presented KPD-IRM MBMA framework. This framework allows to quantitatively compare the effects of siRNAs on the HBsAg time course and inform dose and regimen selection across siRNA classes. These results may support siRNA development, optimize preclinical study designs, and inform data analysis methodology of future anti-HBV siRNAs; and ultimately, support siRNA model-informed drug development (MIDD) strategies.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc

BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50–95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

RNA Interference Therapeutics for Chronic Hepatitis B: Progress, Challenges, and Future Prospects.

Chronic hepatitis B (CHB) is a global health challenge that can result in significant liver-related morbidity and mortality. Despite a prophylactic vaccine being available, patients already living with CHB often must engage in lifelong therapy with nucleoside analogues. However, the potential of RNA interference (RNAi) therapeutics as a promising avenue for CHB treatment is being explored. RNAi, particularly using small interfering RNA (siRNA), targets viral RNA that can be used to inhibit hepatitis B virus (HBV) replication. Several candidates are currently being studied and have exhibited varying success in reducing hepatitis B surface antigen (HBsAg) levels, with some showing sustained HBsAg loss after cessation of therapy. The dynamic evolution of RNAi therapy presents a promising trajectory for the development of effective and sustained treatments for CHB. This review highlights recent findings on RNAi therapeutics, including modifications for stability, various delivery vectors, and specific candidates currently in development.

Targeting and monitoring ovarian cancer invasion with an RNAi and peptide delivery system

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.

RNA interference in the era of nucleic acid therapeutics.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.

Identification of Potent siRNA Delivery Peptides Using Computer Modeling.

Peptides with suitable aggregation behavior and electrical properties are potential siRNA delivery vectors. However, identifying suitable peptides with ideal delivery and safety features is difficult owing to the variations in amino acid sequences. Here, a holistic program based on computer modeling and single-cell RNA sequencing (scRNA-seq)is used to identify ideal siRNA delivery peptides. Stage one of this program consists of a sequential screening process for candidates with ideal assembly and delivery ability; stage two is a cell subtype-level analysis program that screens for high in vivo tissue safety. The leading candidate peptide selected from a library containing 12 amino acids showed strong lung-targeted siRNA delivery capacity after hydrophobic modification. Systemic administration of these compounds caused the least damage to liver and lung tissues and has little impact on macrophage and neutrophil numbers. By loading STAT3 siRNA, strong anticancer effects are achieved in multiple models, including patient-derived xenografts (PDX). This screening procedure may facilitate the development of peptide-based RNA interference (RNAi) therapeutics.

Distinguishing High from Low Concentrations of RNA Cancer Marker: Five‐Input DNAzyme Thresholding Gate

AbstractRNA‐cleaving DNAzymes are promising agents for gene therapy that can overcome the low selectivity of other therapeutic nucleic acid agents, including CRISPR/Cas9, antisense oligonucleotides, and RNA interference agents, in protein‐independent RNA cleavage. To achieve this, DNAzyme gene therapy agents should accurately distinguish cancer from healthy cells. Often, cancer RNA markers are expressed in healthy tissue at lower concentrations. Previously, we developed a DNAzyme thresholding gate (DTh), which must bind to three cancer marker molecules prior to cleaving the RNA target. This article presents the next step in the development of DTh by converting a signal from five microRNA inputs into an output cleavage of the RNA model using an advanced system of molecular logic. We demonstrate that it is possible to broaden the available threshold range by increasing the number of marker recognition modules. The threshold gates can be easily adjusted to cleave a broad range of RNA targets. This approach can be used in cancer diagnostics as well as the computational component of DNA nanorobots, autonomous DNA devices for anti‐cancer gene therapy.

Ionizable Lipid Nanoparticles with Integrated Immune Checkpoint Inhibition for mRNA CAR T Cell Engineering.

The programmed cell death protein 1 (PD-1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment. While clinical approaches to inhibiting the PD-1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD-1 suppression, increasing the risk of autoimmune reactions. This study reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic gene expression and RNA interference (RNAi)-mediated transient gene knockdown in T cells. In developing this platform, interesting interactions are observed between the two RNA cargoes when co-encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co-delivery platform is adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD-1 to primary human T cells ex vivo and strong CAR expression and PD-1 knockdown are observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including the development of improved cancerimmunotherapies.

Direct cytoplasmic delivery of RNAi therapeutics through a non-lysosomal pathway for enhanced gene therapy

The first approved RNAi therapeutics, ONPATTRO, in 2017 moves the concept of RNA interference (RNAi) therapy from research to clinical reality, raising the hopes for the treatment of currently incurable diseases. However, RNAi therapeutics are still facing two main challenges—susceptibility to enzymatic degradation and low ability to escape from endo/lysosome into the cytoplasm. Therefore, we developed disulfide-based nanospheres (DBNPs) as universal vehicles to achieve efficient RNA delivery to address these problems. Notably, the DBNPs possess unique and desirable features, including improved resistance to nuclease degradation, direct cytoplasmic delivery through thiol-mediated cellular uptake, and cytosolic environment-responsive release, greatly enhancing the bioavailability of RNA therapeutics. Additionally, DBNPs are superior in terms of overcoming formidable physiological barriers, including vascular barriers and impermeable tumor tissues. Owning to these advantages, the DBNPs exhibit efficient gene silencing effect when delivering either small interfering RNA (siRNA) or microRNA in various cell lines and generate remarkable growth inhibition in the zebrafish and mouse model of pancreatic tumors as compared to traditional delivery vectors, such as PEI. Therefore, DBNPs have potential application prospect in RNAi therapy both in vitro and in vivo. Statement of significanceRNA interference (RNAi) therapeutics could target and alter any disease-related mRNA translation, thus have great potential in clinical application. Delivery efficiency of RNA modalities into cell cytoplasm is the main problem that currently limit RNAi therapeutics to release their full potential. Most of the known delivery materials suffer from the endo/lysosomal entrapment and enzymatic degradation during endocytosis-dependent uptake, resulting unsatisfied efficiency of the cytoplasmic release. Here, we developed disulfide-based nanospheres could directly transfer RNA modalities into the cytoplasm and significantly enhance the delivery efficiency, thus holding great potential in RNAi therapy.

Novel siRNA therapeutics demonstrate multi-variant efficacy against SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that causes COVID-19 disease, with an estimated global mortality of approximately 2%. While global response strategies, which are predominantly reliant on regular vaccinations, have shifted from zero COVID to living with COVID, there is a distinct lack of broad-spectrum direct acting antiviral therapies that maintain efficacy across evolving SARS-CoV-2 variants of concern. This is of most concern for immunocompromised and immunosuppressed individuals who lack robust immune responses following vaccination, and others at risk for severe COVID and long-COVID. RNA interference (RNAi) therapeutics induced by short interfering RNAs (siRNAs) offer a promising antiviral treatment option, with broad-spectrum antiviral capabilities unparalleled by current antiviral therapeutics and a high genetic barrier to antiviral escape. Here we describe novel siRNAs, targeting highly conserved regions of the SARS-CoV-1 and 2 genome of both human and animal species, with multi-variant antiviral potency against eight SARS-CoV-2 lineages - Ancestral VIC01, Alpha, Beta, Gamma, Delta, Zeta, Kappa and Omicron. Treatment with our siRNA resulted in significant protection against virus-mediated cell death in vitro, with >97% cell survival (P < 0.0001), and corresponding reductions of viral nucleocapsid RNA of up to 99.9% (P < 0.0001). When compared to antivirals; Sotrovimab and Remdesivir, the siRNAs demonstrated a more potent antiviral effect and similarly, when multiplexing siRNAs to target different viral regions simultaneously, an increased antiviral effect was observed compared to individual siRNA treatments (P < 0.0001). These results demonstrate the potential for a highly effective broad-spectrum direct acting antiviral against multiple SARS-CoV-2 variants, including variants resistant to antivirals and vaccine generated neutralizing antibodies.

Autophagy-Interfering Nanoboat Drifting along CD44-Golgi-ER Flow as RNAi Therapeutics for Hepatic Fibrosis.

The upregulated autophagy fuels the activation of hepatic stellate cells (HSCs) to promote hepatic fibrosis. However, the lack of specific inhibitors targeting autophagy and high requirements for cell targeting impede the application of antifibrotic therapy that targets autophagy. RNA interference (RNAi)-based short interfering RNA (siRNA) provides an approach to specifically inhibit autophagy. The therapeutic potential of siRNA, however, is far from being exploited due to the lack of safe and effective delivery vehicles. The cytoplasmic delivery of siRNA is essential for RNAi, and the intracellular trafficking pathway of vehicles determines the fate of siRNA. Unfortunately, the lysosomal degradation pathway, the intracellular fate of most gene vehicles, impedes RNAi efficiency. Inspired by the trafficking pathway of some viruses infecting cells, KDEL-grafted chondroitin sulfate (CK) was designed to alter the intracellular delivery fate of siRNA. The well-designed CD44-Golgi-ER trafficking pathway of CK was realized by triple cascade targeting including (1) CD44 targeting mediated by chondroitin sulfate, (2) Golgi apparatus targeting mediated by the caveolin-mediated endocytic pathway, and (3) endoplasmic reticulum (ER) targeting mediated by coat protein I (COP I) vesicles. CK was adsorbed on the complex of cationic liposomes (Lip) encapsulating siRNA targeting autophagy-related gene 7 (siATG7) to afford Lip/siATG7/CK. Lip/siATG7/CK functions as a drifting boat that follows the CD44-Golgi-ER flow and travels downstream to its destination (ER), bypassing the lysosomal degradation pathway and endowing HSCs with excellent RNAi efficiency. The efficient downregulation of ATG7 leads to an excellent antifibrotic effect both in vitro and in vivo.

Evaluation of RNAi therapeutics VIR-2218 and ALN-HBV for chronic hepatitis B: Results from randomized clinical trials

Current therapy for chronic hepatitis B virus (cHBV) infection involves lifelong treatment. New treatments that enable HBV functional cure would represent a clinically meaningful advance. ALN-HBV and VIR-2218 are investigational RNA interference therapeutics that targetall major HBV transcripts. We report on: i) the safety of single doses of VIR-2218 (modified from ALN-HBV by enhanced stabilization chemistry plus technology to reduce off-target, seed-mediated binding while maintaining on-target antiviral activity) and ALN-HBV in humanized mice; ii) a cross-study comparison of the safety of single doses of VIR-2218 and ALN-HBV in healthy human volunteers (n= 24 and n= 49, respectively); and iii) the antiviral activity of two doses of 20, 50, 100, 200mg of VIR-2218 (total n= 24) vs. placebo (n= 8), given 4 weeks apart, in participants with cHBV infection. In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration of VIR-2218 compared with ALN-HBV. In healthy volunteers, post-treatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in participants receiving 200mg was 1.65 log IU/ml. The HBsAg reduction was maintained at 0.87 log IU/ml at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose-dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. A significant unmet need exists for therapies for chronic HBV (cHBV) infection that achieve functional cure. We report clinical and non-clinical data on two investigational small-interfering RNAs that target HBx, ALN-HBV and VIR-2218, demonstrating that incorporation of enhanced stabilization chemistry plus technology in VIR-2218 reduces its propensity to cause ALT elevations relative to its parent compound, ALN-HBV. We also show that VIR-2218 reduces hepatitis B surface antigen levels in a dose-dependent manner in participants with cHBV infection. These studies support the continued development of VIR-2218 as part of therapeutic regimens for cHBV infection, with the goal of a functional cure, and are important for HBV researchers and physicians.

RNA interference (RNAi)-based therapeutics for treatment of rare neurologic diseases.

Advances in genome sequencing have greatly facilitated the identification of genomic variants underlying rare neurodevelopmental and neurodegenerative disorders. Understanding the fundamental causes of rare monogenic disorders has made gene therapy a possible treatment approach for these conditions. RNA interference (RNAi) technologies such as small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA), and other oligonucleotide-based modalities such as antisense oligonucleotides (ASOs) are being developed as potential therapeutic approaches for manipulating expression of the genes that cause a variety of neurological diseases. Here, we offer a brief review of the mechanism of action of these RNAi approaches; provide deeper discussion of the advantages, challenges, and specific considerations related to the development of RNAi therapeutics for neurological disease; and highlight examples of rare neurological diseases for which RNAi therapeutics hold great promise.

Chitosan and chito-oligosaccharide: a versatile biopolymer with endless grafting possibilities for multifarious applications.

Chito-oligosaccharides (COS), derived from chitosan (CH), are attracting increasing attention as drug delivery carriers due to their biocompatibility, biodegradability, and mucoadhesive properties. Grafting, the process of chemically modifying CH/COS by adding side chains, has been used to improve their drug delivery performance by enhancing their stability, targeted delivery, and controlled release. In this review, we aim to provide an in-depth study on the recent advances in the grafting of CH/COS for multifarious applications. Moreover, the various strategies and techniques used for grafting, including chemical modification, enzymatic modification, and physical modification, are elaborated. The properties of grafted CH/COS, such as stability, solubility, and biocompatibility, were reported. Additionally, the review detailed the various applications of grafted CH/COS in drug delivery, including the delivery of small drug molecule, proteins, and RNA interference therapeutics. Furthermore, the effectiveness of grafted CH/COS in improving the pharmacokinetics and pharmacodynamics of drugs was included. Finally, the challenges and limitations associated with the use of grafted CH/COS for drug delivery and outline directions for future research are addressed. The insights provided in this review will be valuable for researchers and drug development professionals interested in the application of grafted CH/COS for multifarious applications.

The Role of RNA Interference Therapeutics in Hypercholesterolemia and Implications for Practice

An estimated 80% of patients with dyslipidemia do not reach their targeted low-density lipoprotein-cholesterol goals. Despite the use of traditional lipid-lowering therapies, challenges exist in getting patients to goal. Long-acting RNA interference (RNAi) therapeutics represent the next generation of medication that can provide up to 50% reduction of low-density lipoprotein-cholesterol, with few adverse effects for patients who need additional therapy or are intolerant or nonadherent to statin therapy. Its twice-yearly dosing profile offers a treatment option that may help in overcoming treatment barriers. Nurse practitioners need to be knowledgeable about novel pharmacologic agents to treat high-risk populations not meeting treatment goals.

NIR Light‐Mediated Mitochondrial RNA Modification for Cancer RNA Interference Therapeutics

AbstractMitochondrial RNA (mtRNA) plays a critical role in synthesis of mitochondrial proteins. Interfering mtRNA is a highly effective way to induce cell apoptosis. Herein, we report a near‐infrared (NIR) light‐mediated mitochondrial RNA modification approach for long‐term imaging and effective suppression of tumors. A tumor‐targetable NIR fluorescent probe f‐CRI consisting of a cyclic RGD peptide, a NIR fluorophore IR780, and a singlet oxygen (1O2)‐labile furan group for RNA modification was rationally designed and synthesized. This probe was demonstrated to dominantly accumulate in cellular mitochondria and could be covalently conjugated onto mtRNA upon 808 nm irradiation resulting in prolonged retention in tumors. More notably, this covalent modification of mtRNA by f‐CRI could perturb the function of mitochondria leading to remarkable tumor suppression. We thus envision that our current approach would offer a potential approach for cancer RNA interference therapeutics.

NIR Light-Mediated Mitochondrial RNA Modification for Cancer RNA Interference Therapeutics.

Mitochondrial RNA (mtRNA) plays a critical role in synthesis of mitochondrial proteins. Interfering mtRNA is a highly effective way to induce cell apoptosis. Herein, we report a near-infrared (NIR) light-mediated mitochondrial RNA modification approach for long-term imaging and effective suppression of tumors. A tumor-targetable NIR fluorescent probe f-CRI consisting of a cyclic RGD peptide, a NIR fluorophore IR780, and a singlet oxygen (1O2)-labile furan group for RNA modification was rationally designed and synthesized. This probe was demonstrated to dominantly accumulate in cellular mitochondria and could be covalently conjugated onto mtRNA upon 808 nm irradiation resulting in prolonged retention in tumors. More notably, thie covalent modification of mtRNA by f-CRI could perturb the function of mitochondria leading to remarkable tumor suppression. We thus envision that our current approach would offer a potential approach for cancer RNA interference therapeutics.

Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the β-alanine (βA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-βA-βA-W4-βA-βA-R4-CO-NH2 and Ac-K-βA-βA-W4-βA-βA-R4-CO-NH2, with only a difference in N-terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component's self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.

Cholesterol-conjugated siRNAs silence gene expression in mucosal dendritic cells in cervicovaginal tissue in mice.

RNA interference (RNAi) provides a powerful way to investigate the role of genes in disease pathogenesis and modulate gene expression to treat disease. In 2018, the FDA approved patisiran, the first RNAi-based drug, hence paving the way for a novel class of RNAi therapeutics. Harnessing RNAi to inhibit vaginal HIV transmission requires effective gene silencing in immune cells, which remains difficult. Knockdown in accessible mucosal tissues may be easier than systemic gene silencing. Vaginally applied cholesterol-conjugated small interfering RNAs (chol-siRNAs) blocked herpes simplex virus transmission in mice without tissue damage or immunostimulation. To investigate using flow cytometry, confocal microscopy, and quantitative imaging if chol-siRNAs silence gene expression in vaginal immune cells in mice. Although chol-siRNAs and lipoplexed-siRNAs silence gene expression in dendritic cells (DCs) in vitro, most internalized siRNAs concentrate within multivesicular bodies, where they are inaccessible to the cellular RNAi machinery. When applied intravaginally in vivo, chol-siRNAs penetrate the vaginal mucosa, including the lamina propria, and are efficiently internalized by intraepithelial (IE) and lamina propria (LP) DCs, and CD11b+ CD45+ cells, but not by T cells. Chol-siRNAs induce partial gene silencing in IE and LP DCs throughout the genital mucosa in vivo but are inactive in F4/80+ CD11b+ macrophages and T cells. As mucosal DCs play an essential role for mucosal viral entry and dissemination, chol-siRNAs could be harnessed to target various host factors that are critical for viral uptake, DC migration and trans-infection of virions to T cells, hence allowing the development of a preventive vaginal HIV microbicide. Furthermore, chol-siRNAs could help elucidate the pathways of HIV transmission and understand the immunologic function of DCs in the genital tract.