SiRNA-HIF-1α delivered by attenuated Salmonella enhances the efficacy of Lenvatinib against hepatocellular carcinoma.

The treatment of hepatocellular carcinoma (HCC) remains a major challenge in the medical field. Lenvatinib, a multi-target tyrosine kinase inhibitor, has demonstrated anti-HCC effects by targeting and inhibiting pathways such as vascular endothelial growth factor receptor 1-3 (VEGFR1-3). However, the therapeutic efficacy of Lenvatinib is subject to various influences, with the hypoxic microenvironment of the tumor being a pivotal factor. Consequently, altering the hypoxic milieu of the tumor emerges as a viable strategy to augment the efficacy of Lenvatinib. Hypoxia-inducible factor-1α (HIF-1α), synthesized by tumor cells in response to oxygen-deprived conditions, regulates the expression of resistance genes, promotes tumor angiogenesis and cell proliferation, enhances tumor cell invasion, and confers resistance to radiotherapy and chemotherapy. Thus, we constructed a self-designed siRNA targeting HIF-1α to suppress its expression and improve the efficacy of Lenvatinib in treating HCC. The therapeutic efficacy of siRNA-HIF-1α in combination with Lenvatinib on HCC were evaluated through in vivo and in vitro experiments. The results showed that the recombinant Salmonella delivering siRNA-HIF-1α in combination with Lenvatinib effectively inhibited tumor growth and prolonged the survival of tumor-bearing mice. This treatment approach reduced cell proliferation and angiogenesis in HCC tissues while promoting tumor cell apoptosis. Additionally, this combined therapy significantly increased the infiltration of T lymphocytes and M1 macrophages within the tumor microenvironment, as well as elevated the proportion of immune cells in the spleen, thereby potentiating the host's immune response against the tumor.

Recent updates on therapeutic targeting of lipoprotein(a) with RNA interference.

PURPOSE OF REVIEW: RNA interference (RNAi)-based therapies that target specific gene products have impacted clinical medicine with 16 FDA approved drugs. RNAi therapy focused on reducing plasma lipoprotein(a) [Lp(a)] levels are under evaluation. RECENT FINDINGS: RNAi-based therapies have made significant progress over the past 2 decades and currently consist of antisense oligonucleotides (ASO) and small interfering RNA (siRNA). Chemical modification of the RNA backbone and conjugation of siRNA enables efficient gene silencing in hepatocytes allowing development of effective cholesterol lowering therapies. Multiple lines of evidence suggest a causative role for Lp(a) in atherosclerotic cardiovascular disease, and recent analyses indicate that Lp(a) is more atherogenic than low density lipoprotein- cholesterol (LDL-C). These findings have led to the 'Lp(a) hypothesis' that lowering Lp(a) may significantly improve cardiovascular outcomes. Four RNAi-based drugs have completed early phase clinical trials demonstrating >80% reduction in plasma Lp(a) levels. Phase 3 clinical trials examining clinical outcomes with these agents are currently underway. SUMMARY: Currently, four RNAi-based drugs have been shown to be effective in significantly lowering plasma Lp(a) levels. Clinical outcome data from phase 3 trials will evaluate the Lp(a) hypothesis.

Nephrocalcinosis can disappear in infants receiving early lumasiran therapy.

BACKGROUND: Lumasiran is the first RNA interference (RNAi) therapy of primary hyperoxaluria type 1 (PH1). Here, we report on the rapid improvement and even disappearance of nephrocalcinosis after early lumasiran therapy. CASE-DIAGNOSIS/TREATMENT: In patient 1, PH1 was suspected due to incidental discovery of nephrocalcinosis stage 3 in a 4-month-old boy. Bilateral nephrocalcinosis stage 3 was diagnosed in patient 2 at 22 months concomitantly to acute pyelonephritis. Urinary oxalate (UOx) and glycolate (UGly) were increased in both patients allowing to start lumasiran therapy before genetic confirmation. Nephrocalcinosis started to improve and disappeared after 27 months and 1 year of treatment in patients 1 and 2, respectively. CONCLUSION: These cases illustrate the efficacy of early lumasiran therapy in infants to improve and even normalize nephrocalcinosis. As proposed in the 2023 European guidelines, the interest of starting treatment quickly without waiting for genetic confirmation may have an impact on long-term outcomes.

Advances in RNAi therapies for gastric cancer: Targeting drug resistance and nanoscale delivery.

Gastric cancer poses a significant health challenge, and exploring innovative therapeutic strategies is imperative. RNA interference (RNAi) has employed as an important therapeutic strategy for diseases by selectively targeting key pathways involved in diseases pathogenesis. Small interfering RNA (siRNA), a potent RNAi tool, possesses the capability to silence genes and downregulate their expression. This review provides a comprehensive examination of the potential applications of small interfering RNA (siRNA) and short hairpin RNA (shRNA), supplemented by an in-depth analysis of nanoscale delivery systems, in the context of gastric cancer treatment. The potential of siRNA to markedly diminish the proliferation and invasion of gastric cancer cells through the modulation of critical molecular pathways, including PI3K, Akt, and EMT, is highlighted. Besides, siRNA demonstrates its efficacy in inducing chemosensitivity in gastric tumor cells, thus impeding tumor progression. However, the translational potential of unmodified siRNA faces challenges, particularly in vivo and during clinical trials. To address this, we underscore the pivotal role of nanostructures in facilitating the delivery of siRNA to gastric cancer cells, effectively suppressing their progression and enhancing gene silencing efficiency. These siRNA-loaded nanoparticles exhibit robust internalization into gastric cancer cells, showcasing their potential to significantly reduce tumor progression. The translation of these findings into clinical trials holds promise for advancing the treatment of gastric cancer patients.

RNAi-Based Therapy: Combating Shrimp Viral Diseases.

Shrimp aquaculture has become a vital industry, meeting the growing global demand for seafood. Shrimp viral diseases have posed significant challenges to the aquaculture industry, causing major economic losses worldwide. Conventional treatment methods have proven to be ineffective in controlling these diseases. However, recent advances in RNA interference (RNAi) technology have opened new possibilities for combating shrimp viral diseases. This cutting-edge technology uses cellular machinery to silence specific viral genes, preventing viral replication and spread. Numerous studies have shown the effectiveness of RNAi-based therapies in various model organisms, paving the way for their use in shrimp health. By precisely targeting viral pathogens, RNAi has the potential to provide a sustainable and environmentally friendly solution to combat viral diseases in shrimp aquaculture. This review paper provides an overview of RNAi-based therapy and its potential as a game-changer for shrimp viral diseases. We discuss the principles of RNAi, its application in combating viral infections, and the current progress made in RNAi-based therapy for shrimp viral diseases. We also address the challenges and prospects of this innovative approach.

RNAi therapies: Expanding applications for extrahepatic diseases and overcoming delivery challenges.

The era of RNA medicine has become a reality with the success of messenger RNA (mRNA) vaccines against COVID-19 and the approval of several RNA interference (RNAi) agents in recent years. Particularly, therapeutics based on RNAi offer the promise of targeting intractable and previously undruggable disease genes. Recent advances have focused in developing delivery systems to enhance the poor cellular uptake and insufficient pharmacokinetic properties of RNAi therapeutics and thereby improve its efficacy and safety. However, such approach has been mainly achieved via lipid nanoparticles (LNPs) or chemical conjugation with N-Acetylgalactosamine (GalNAc), thus current RNAi therapy has been limited to liver diseases, most likely to encounter liver-targeting limitations. Hence, there is a huge unmet medical need for intense evolution of RNAi therapeutics delivery systems to target extrahepatic tissues and ultimately extend their indications for treating various intractable diseases. In this review, challenges of delivering RNAi therapeutics to tumors and major organs are discussed, as well as their transition to clinical trials. This review also highlights innovative and promising preclinical RNAi-based delivery platforms for the treatment of extrahepatic diseases.

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 SIGNIFICANCE: RNA 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.

RNAi therapeutics for diseases involving protein aggregation: fazirsiran for alpha-1 antitrypsin deficiency-associated liver disease.

INTRODUCTION: Therapeutic agents that prevent protein misfolding or promote protein clearance are being studied to treat proteotoxic diseases. Among them, alpha-1 antitrypsin deficiency (AATD) is caused by mutations in the alpha-1 antitrypsin (SERPINA1) gene. Fazirsiran is a small interfering RNA (siRNA) that is intended to address the underlying cause of liver disease associated with AATD through the RNA interference (RNAi) mechanism. AREAS COVERED: This article describes the role of misfolded proteins and protein aggregates in disease and options for therapeutic approaches. The RNAi mechanism is discussed, along with how the siRNA therapeutic fazirsiran for the treatment of AATD was developed. We also describe the implications of siRNA therapeutics in extrahepatic diseases. EXPERT OPINION: Using RNAi as a therapeutic approach is well suited to treat disease in conditions where an excess of a protein or the effect of an abnormal mutated protein causes disease. The results observed for the first few siRNA therapeutics that were approved or are in development provide an important promise for the development of future drugs that can address such conditions in a specific and targeted way. Current developments should enable the use of RNAi therapeutics outside the liver, where there are many more possible diseases to address.

When certain proteins in the body are not produced correctly, it can result in disease. Examples of such diseases include alpha-1 antitrypsin deficiency (AATD), hereditary transthyretin amyloidosis (ATTR), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease, where misfolded proteins can form harmful aggregates. Research is underway to develop new therapies such as small interfering RNA (siRNA), antisense oligonucleotides, monoclonal antibodies, clustered regularly interspaced short palindromic repeats (CRISPR), and small molecule protein modulators to prevent protein aggregates. An example of an experimental siRNA therapy for patients with AATD is fazirsiran, designed to prevent the production of mutated alpha-1 antitrypsin (Z-AAT) and allows the liver to clear previously accumulated Z-AAT protein, which is believed to be responsible for causing AATD liver disease. This review summarizes the development of fazirsiran and describes how it helps our understanding and treatment of diseases associated with protein aggregation.

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.

BACKGROUND & AIMS: 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 target all major HBV transcripts. METHODS: 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, 200 mg of VIR-2218 (total n = 24) vs. placebo (n = 8), given 4 weeks apart, in participants with cHBV infection. RESULTS: 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 200 mg 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. CONCLUSIONS: 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. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. IMPACT AND IMPLICATIONS: 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.

Bioengineered Bacteriophage-Like Nanoparticles as RNAi Therapeutics to Enhance Radiotherapy against Glioblastomas.

Since glioblastomas (GBMs) are radioresistant malignancies and most GBM recurrences occur in radiotherapy, increasing the effectiveness of radiotherapy by gene-silencing has recently attracted attention. However, the difficulty in precisely tuning the composition and RNA loading in nanoparticles leads to batch-to-batch variations of the RNA therapeutics, thus significantly restricting their clinical translation. Here, we bioengineer bacteriophage Qß particles with a designed broccoli light-up three-way junction (b-3WJ) RNA scaffold (contains two siRNA/miRNA sequences and one light-up aptamer) packaging for the silencing of genes in radioresistant GBM cells. The in vitro results demonstrate that the cleavage of de novo designed b-3WJ RNA by Dicer enzyme can be easily monitored in real-time using fluorescence microscopy, and the TrQß@b-3WJLet-7gsiEGFR successfully knocks down EGFR and IKKα simultaneously and thereby inactivates NF-κB signaling to inhibit DNA repair. Delivery of TrQß@b-3WJLet-7gsiEGFR through convection-enhanced delivery (CED) infusion followed by 2Gy X-ray irradiation demonstrated that the median survival was prolonged to over 60 days compared with the 2Gy X-ray irradiated group (median survival: 31 days). Altogether, the results of this study could be critical for the design of RNAi-based genetic therapeutics, and CED infusion serves as a powerful delivery system for promoting radiotherapy against GBMs without evidence of systemic toxicity.

Cation-free siRNA-cored nanocapsules for tumor-targeted RNAi therapy.

Cation-associated cytotoxicity limits the systemic administration of RNA delivery in vivo, demanding the development of non-cationic nanosystems. In this study, cation-free polymer-siRNA nanocapsules with disulfide-crosslinked interlayer, namely T-SS(-), were prepared via the following steps: 1) complexation of siRNA with a cationic block polymer cRGD-poly(ethylene glycol)-b-poly[(2-aminoethanethiol)aspartamide]-b-poly{N'-[N-(2-aminoethyl)-2-ethylimino-1-aminomethyl]aspartamide}, abbreviated as cRGD-PEG-PAsp(MEA)-PAsp(C=N-DETA), 2) interlayer crosslinking via disulfide bond in pH 7.4 solution, and 3) removal of cationic DETA pendant at pH 5.0 via breakage of imide bond. The cationic-free nanocapsules with siRNA cores not only showed great performance (such as efficient siRNA encapsulation, high stability in serum, cancer cell targeting via cRGD modification, and GSH-triggered siRNA release), but also achieved tumor-targeted gene silencing in vivo. Moreover, the nanocapsules loaded with siRNA against polo-like kinase 1 (siRNA-PLK1) significantly inhibited tumor growth without showing cation-associated toxicity side effects and remarkably improved the survival rate of PC-3 tumor-bearing mice. The cation-free nanocapsules could potentially serve as a safe and effective platform for siRNA delivery. STATEMENT OF SIGNIFICANCE: Cation-associated toxicity limits the clinical translation of cationic carriers for siRNA delivery. Recently, several non-cationic carriers, such as siRNA micelles, DNA-based nanogels, and bottlebrush-architectured poly(ethylene glycol), have been developed to deliver siRNA. However, in these designs, siRNA as a hydrophilic macromolecule was attached to the nanoparticle surface instead of being encapsulated. Thus, it was easily degraded by serum nuclease and often induced immunogenicity. Herein, we demonstrate a new type of cation-free siRNA-cored polymeric nanocapsules. The developed nanocapsules not only showed capacities including efficient siRNA encapsulation, high stability in serum, and cancer cell targeting via cRGD modification, but also achieved an efficient tumor-targeted gene silencing in vivo. Importantly, unlike cationic carriers, the nanocapsules exhibited no cation-associated side effects.

Radiation-Triggered Selenium-Engineered Mesoporous Silica Nanocapsules for RNAi Therapy in Radiotherapy-Resistant Glioblastoma.

Radiotherapy-resistant glioblastoma (rrGBM) remains a significant clinical challenge because of high infiltrative growth characterized by activation of antiapoptotic signal transduction. Herein, we describe an efficiently biodegradable selenium-engineered mesoporous silica nanocapsule, initiated by high-energy X-ray irradiation and employed for at-site RNA interference (RNAi) to inhibit rrGBM invasion and achieve maximum therapeutic benefit. Our radiation-triggered RNAi nanocapsule showed high physiological stability, good blood-brain barrier transcytosis, and potent rrGBM accumulation. An intratumoral RNAi nanocapsule permitted low-dose X-ray radiation-triggered dissociation for cofilin-1 knockdown, inhibiting rrGBM infiltration. More importantly, tumor suppression was further amplified by electron-affinity aminoimidazole products converted from metronidazole polymers under X-ray radiation-exacerbated hypoxia, which sensitized cell apoptosis to ionizing radiation by fixing reactive oxygen species-induced DNA lesions. In vivo experiments confirmed that our RNAi nanocapsule reduced tumor growth and invasion, prolonging survival in an orthotopic rrGBM model. Generally, we present a promising radiosensitizer that would effectively improve rrGBM-patient outcomes with low-dose X-ray irradiation.

Acyclic (S)-glycol nucleic acid (S-GNA) modification of siRNAs improves the safety of RNAi therapeutics while maintaining potency.

Glycol nucleic acid (GNA) is an acyclic nucleic acid analog connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base-pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

Population Pharmacokinetic Analysis of the RNAi Therapeutic Givosiran in Patients with Acute Hepatic Porphyria.

BACKGROUND AND OBJECTIVE: Givosiran, approved for the treatment of acute hepatic porphyria (AHP), is the first subcutaneously administered RNAi therapeutic. This analysis was undertaken to describe the plasma pharmacokinetics (PK) of givosiran and its active metabolite, AS(N-1)3' givosiran, and to identify factors that contribute to intersubject PK variability. METHODS: A population PK model was developed using data from givosiran clinical trials that enrolled patients with AHP or who were asymptomatic chronic high excreters (CHEs) of toxic heme intermediates. Givosiran and AS(N-1)3' givosiran PK were modeled simultaneously using non-linear mixed-effects modeling. RESULTS: Plasma PK of givosiran was best described by a two-compartment model. Givosiran absorption after subcutaneous administration and conversion of givosiran to AS(N-1)3' givosiran were incorporated as first-order processes. Hepatic clearance was the major route of elimination from the central compartment, with renal clearance accounting for < 20% of the total clearance. Body weight, East Asian ethnicity, and renal impairment were significant covariates in the model; however, none of the covariates evaluated resulted in clinically meaningful differences in plasma exposures of givosiran and AS(N-1)3' givosiran. The model adequately described observed concentrations and variability across a wide range of dose levels. Model-derived simulations showed similar exposures for givosiran and its active metabolite in adults and adolescents. CONCLUSIONS: The PK of givosiran and its active metabolite were not significantly affected by demographic or clinical parameters that would require adjustment from the approved body weight-based dose of givosiran 2.5 mg/kg once monthly.

Liver cancer cells as the model for developing liver-targeted RNAi therapeutics.

RNAi is a sequence-specific gene regulation mechanism that involves small interfering RNAs (siRNAs). RNAi therapeutic has become a new class of precision medicine and has shown great potential in treating liver-associated diseases, especially metabolic diseases. To facilitate the development of liver-targeted RNAi therapeutics in cell model, we surveyed a panel of liver cancer cell lines for the expression of genes implicated in RNAi therapeutics including the asialoglycoprotein receptor (ASGR) and metabolic disease associated genes PCSK9, ANGPTL3, CIDEB, and LDLR. A high-content screen assay based on lipid droplet staining confirmed the involvement of PCSK9, ANGPTL3, and CIDEB in lipid metabolism in selected liver cancer cell lines. Several liver cancer cell lines have high levels of ASGR1 expression, which is required for liver-specific uptake of GalNAc-conjugated siRNA, a clinically approved siRNA delivery platform. Using an EGFP reporter system, we demonstrated Hep G2 can be used to evaluate gene knockdown efficiency of GalNAc-siRNA. Our findings pave the way for using liver cancer cells as a convenient model system for the identification and testing of siRNA drug candidate genes and for studying ASGR-mediated GalNAc-siRNA delivery in liver.

RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies.

RNA interference (RNAi) provides researchers with a versatile means to modulate target gene expression. The major forms of RNAi molecules, genome-derived microRNAs (miRNAs) and exogenous small interfering RNAs (siRNAs), converge into RNA-induced silencing complexes to achieve posttranscriptional gene regulation. RNAi has proven to be an adaptable and powerful therapeutic strategy where advancements in chemistry and pharmaceutics continue to bring RNAi-based drugs into the clinic. With four siRNA medications already approved by the US Food and Drug Administration (FDA), several RNAi-based therapeutics continue to advance to clinical trials with functions that closely resemble their endogenous counterparts. Although intended to enhance stability and improve efficacy, chemical modifications may increase risk of off-target effects by altering RNA structure, folding, and biologic activity away from their natural equivalents. Novel technologies in development today seek to use intact cells to yield true biologic RNAi agents that better represent the structures, stabilities, activities, and safety profiles of natural RNA molecules. In this review, we provide an examination of the mechanisms of action of endogenous miRNAs and exogenous siRNAs, the physiologic and pharmacokinetic barriers to therapeutic RNA delivery, and a summary of the chemical modifications and delivery platforms in use. We overview the pharmacology of the four FDA-approved siRNA medications (patisiran, givosiran, lumasiran, and inclisiran) as well as five siRNAs and several miRNA-based therapeutics currently in clinical trials. Furthermore, we discuss the direct expression and stable carrier-based, in vivo production of novel biologic RNAi agents for research and development. SIGNIFICANCE STATEMENT: In our review, we summarize the major concepts of RNA interference (RNAi), molecular mechanisms, and current state and challenges of RNAi drug development. We focus our discussion on the pharmacology of US Food and Drug Administration-approved RNAi medications and those siRNAs and miRNA-based therapeutics that entered the clinical investigations. Novel approaches to producing new true biological RNAi molecules for research and development are highlighted.

Molecularly engineered siRNA conjugates for tumor-targeted RNAi therapy.

RNA interference (RNAi) is a major cellular mechanism regulating gene expression in which short double-stranded RNA molecules called small interfering RNA (siRNA) mediate sequence-specific mRNA degradation. RNAi technology has recently emerged as a promising therapeutic platform for the effective treatment of various diseases caused by inappropriate gene activity, such as cancer. However, the clinical translation of siRNA therapeutics has been hampered by the major hurdles associated with biological instability and limited delivery efficiency. Based on the various efforts, recent siRNA delivery strategies using cationic lipids and polymers allowed to enhance pharmacokinetics and delivery efficiency, resulting in potent and liver-targeted RNAi therapy. However, non-specific protein adsorption, high liver accumulation, and severe toxicity of cationic nanocarriers still limit the possibility of transfer of siRNA therapeutics from the laboratory to the clinic. One of the promising delivery strategies to overcome the limitations of siRNA therapeutics is carrier-free bioconjugation which is chemically modified and connected with biocompatible molecules such as lipids, peptides, antibodies, aptamers, and polymers. These molecularly engineered siRNA conjugates can be utilized for RNAi delivery to tissues beyond the liver, providing opportunities for clinical translation. This review focused on introducing the recent progress in molecularly engineered siRNA conjugates and their applications toward overcoming the limitations of siRNA for tumor-targeted delivery and therapy.