High-Precision Synthesis of RNA-Loaded Lipid Nanoparticles for Biomedical Applications.

The recent development of RNA-based therapeutics in delivering nucleic acids for gene editing and regulating protein translation has led to the effective treatment of various diseases including cancer, inflammatory and genetic disorder, as well as infectious diseases. Among these, lipid nanoparticles (LNP) have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA and thereby enhancing the therapeutic potency. These LNP consisting of ionizable lipid, helper lipid, cholesterol, and poly(ethylene glycol)-anchored lipid can stably enclose RNA and help them release into the cells' cytosol. Herein, the significant progress made in LNP research starting from the LNP constituents, formulation, and their diverse applications is summarized first. Moreover, the microfluidic methodologies which allow precise assembly of these newly developed constituents to achieve LNP with controllable composition and size, high encapsulation efficiency as well as scalable production are highlighted. Furthermore, a short discussion on current challenges as well as an outlook will be given on emerging approaches to resolving these issues.

Biochemistry-informed design selects potent siRNAs against SARS-CoV-2.

RNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel COVID-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, and ex vivo assessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September2022). Our approach is easily generalizable to other viruses as long as avariant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.

An Efficacy and Mechanism Driven Study on the Impact of Hypoxia on Lipid Nanoparticle Mediated mRNA Delivery.

Hypoxia is a common hallmark of human disease that is characterized by abnormally low oxygen levels in the body. While the effects of hypoxia on many small molecule-based drugs are known, its effects on several classes of next-generation medications including messenger RNA therapies warrant further study. Here, we provide an efficacy- and mechanism-driven study that details how hypoxia impacts the cellular response to mRNA therapies delivered using 4 different chemistries of lipid nanoparticles (LNPs, the frontrunner class of drug delivery vehicles for translational mRNA therapy utilized in the Moderna and Pfizer/BioNTech COVID-19 vaccines). Specifically, our work provides a comparative analysis as to how various states of oxygenation impact LNP-delivered mRNA expression, cellular association, endosomal escape, and intracellular ATP concentrations following treatment with 4 different LNPs across 3 different cell lines. In brief, we first identify that hypoxic cells express less LNP-delivered mRNA into protein than normoxic cells. Next, we identify generalizable cellular reoxygenation protocols that can reverse the negative effects that hypoxia imparts on LNP-delivered mRNA expression. Finally, mechanistic studies that utilize fluorescence-activated cell sorting, confocal microscopy, and enzyme inhibition reveal that decreases in mRNA expression correlate with decreases in intracellular ATP (rather than with differences in mRNA LNP uptake pathways). In presenting this data, we hope that our work provides a comprehensive efficacy and mechanism-driven study that explores the impact of differential oxygenation on LNP-delivered mRNA expression while simultaneously establishing fundamental criteria that may one day be useful for the development of mRNA drugs to treat hypoxia-associated disease.

RNA Interference Approach Is a Good Strategy against SARS-CoV-2.

COVID-19, caused by SARS-CoV-2, created a devastating outbreak worldwide and consequently became a global health concern. However, no verifiable, specifically targeted treatment has been devised for COVID-19. Several emerging vaccines have been used, but protection has not been satisfactory. The complex genetic composition and high mutation frequency of SARS-CoV-2 have caused an uncertain vaccine response. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control various infectious diseases employing post-transcriptional gene silencing through the silencing of target complementary mRNA. Here, we designed two highly effective shRNAs targeting the conserved region of RNA-dependent RNA polymerase (RdRP) and spike proteins capable of significant SARS-CoV-2 replication suppression. The efficacy of this approach suggested that the rapid development of an shRNA-based therapeutic strategy might prove to be highly effective in treating COVID-19. However, it needs further clinical trials.

Lipid nanoparticle-mediated drug delivery to the brain.

Lipid nanoparticles (LNPs) have revolutionized the field of drug delivery through their applications in siRNA delivery to the liver (Onpattro) and their use in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While LNPs have been extensively studied for the delivery of RNA drugs to muscle and liver targets, their potential to deliver drugs to challenging tissue targets such as the brain remains underexplored. Multiple brain disorders currently lack safe and effective therapies and therefore repurposing LNPs could potentially be a game changer for improving drug delivery to cellular targets both at and across the blood-brain barrier (BBB). In this review, we will discuss (1) the rationale and factors involved in optimizing LNPs for brain delivery, (2) ionic liquid-coated LNPs as a potential approach for increasing LNP accumulation in the brain tissue and (3) considerations, open questions and potential opportunities in the development of LNPs for delivery to the brain.

Low-density lipoprotein receptor-related protein 1 (LRP1) as an auxiliary host factor for RNA viruses.

Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that are resistant to Rift Valley fever virus (RVFV). This screen returned the low-density lipoprotein receptor-related protein 1 (LRP1) as a top hit, a plasma membrane protein involved in a wide variety of cell activities. Inactivation of LRP1 in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the human cell line HuH-7, LRP1 also promoted early infection stages of sandfly fever Sicilian virus and La Crosse virus, but had a minor effect on late infection by vesicular stomatitis virus, whereas encephalomyocarditis virus was entirely LRP1-independent. Moreover, siRNA experiments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

From a genome-wide screen of RNAi molecules against SARS-CoV-2 to a validated broad-spectrum and potent prophylaxis.

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen of over 16,000 RNAi triggers against the SARS-CoV-2 genome, using a massively parallel assay to identify hyper-potent siRNAs. We selected Ten candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity (IC50 < 20 pM) and strong blockade of infectivity in live-virus experiments. We further enhanced this activity by combinatorial pairing of the siRNA candidates and identified cocktails that were active against multiple types of variants of concern (VOC). We then examined over 2,000 possible mutations in the siRNA target sites by using saturation mutagenesis and confirmed broad protection of the leading cocktail against future variants. Finally, we demonstrated that intranasal administration of this siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the gold-standard Syrian hamster model. Our results pave the way for the development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Research Status and Prospect of Non-Viral Vectors Based on siRNA: A Review.

Gene therapy has attracted much attention because of its unique mechanism of action, non-toxicity, and good tolerance, which can kill cancer cells without damaging healthy tissues. siRNA-based gene therapy can downregulate, enhance, or correct gene expression by introducing some nucleic acid into patient tissues. Routine treatment of hemophilia requires frequent intravenous injections of missing clotting protein. The high cost of combined therapy causes most patients to lack the best treatment resources. siRNA therapy has the potential of lasting treatment and even curing diseases. Compared with traditional surgery and chemotherapy, siRNA has fewer side effects and less damage to normal cells. The available therapies for degenerative diseases can only alleviate the symptoms of patients, while siRNA therapy drugs can upregulate gene expression, modify epigenetic changes, and stop the disease. In addition, siRNA also plays an important role in cardiovascular diseases, gastrointestinal diseases, and hepatitis B. However, free siRNA is easily degraded by nuclease and has a short half-life in the blood. Research has found that siRNA can be delivered to specific cells through appropriate vector selection and design to improve the therapeutic effect. The application of viral vectors is limited because of their high immunogenicity and low capacity, while non-viral vectors are widely used because of their low immunogenicity, low production cost, and high safety. This paper reviews the common non-viral vectors in recent years and introduces their advantages and disadvantages, as well as the latest application examples.

Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection.

RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) lung infection in vivo. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The study presented here demonstrates an attractive alternate delivery strategy for the prophylactic treatment of both future and emerging respiratory viral diseases.

Progress in Respiratory Gene Therapy.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

Effect of Cholesterol Content of Lipid Composition in mRNA-LNPs on the Protein Expression in the Injected Site and Liver After Local Administration in Mice.

Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure-function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure-activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pKa between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure-activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression.

Messenger RNA in lipid nanoparticles rescues HEK 293 cells from lipid-induced mitochondrial dysfunction as studied by real time pulse chase NMR, RTPC-NMR, spectroscopy.

Analytical tools to study cell physiology are critical for optimizing drug-host interactions. Real time pulse chase NMR spectroscopy, RTPC-NMR, was introduced to monitor the kinetics of metabolite production in HEK 293T cells treated with COVID-19 vaccine-like lipid nanoparticles, LNPs, with and without mRNA. Kinetic flux parameters were resolved for the incorporation of isotopic label into metabolites and clearance of labeled metabolites from the cells. Changes in the characteristic times for alanine production implicated mitochondrial dysfunction as a consequence of treating the cells with lipid nanoparticles, LNPs. Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. The methodology is applicable to all cultured cells.

Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery.

Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.

Anticipating the Next Chess Move: Blocking SARS-CoV-2 Replication and Simultaneously Disarming Viral Escape Mechanisms.

The COVID-19 pandemic initiated a race to determine the best measures to control the disease and to save as many people as possible. Efforts to implement social distancing, the use of masks, and massive vaccination programs turned out to be essential in reducing the devastating effects of the pandemic. Nevertheless, the high mutation rates of SARS-CoV-2 challenge the vaccination strategy and maintain the threat of new outbreaks due to the risk of infection surges and even lethal variations able to resist the effects of vaccines and upset the balance. Most of the new therapies tested against SARS-CoV-2 came from already available formulations developed to treat other diseases, so they were not specifically developed for SARS-CoV-2. In parallel, the knowledge produced regarding the molecular mechanisms involved in this disease was vast due to massive efforts worldwide. Taking advantage of such a vast molecular understanding of virus genomes and disease mechanisms, a targeted molecular therapy based on siRNA specifically developed to reach exclusive SARS-CoV-2 genomic sequences was tested in a non-transformed human cell model. Since coronavirus can escape from siRNA by producing siRNA inhibitors, a complex strategy to simultaneously strike both the viral infectious mechanism and the capability of evading siRNA therapy was developed. The combined administration of the chosen produced siRNA proved to be highly effective in successfully reducing viral load and keeping virus replication under control, even after many days of treatment, unlike the combinations of siRNAs lacking this anti-anti-siRNA capability. Additionally, the developed therapy did not harm the normal cells, which was demonstrated because, instead of testing the siRNA in nonhuman cells or in transformed human cells, a non-transformed human thyroid cell was specifically chosen for the experiment. The proposed siRNA combination could reduce the viral load and allow the cellular recovery, presenting a potential innovation for consideration as an additional strategy to counter or cope COVID-19.

Development of lipid nanoparticles and liposomes reference materials (II): cytotoxic profiles.

Lipid based nanocarriers are one of the most effective drug delivery systems that is evident from the recent COVID-19 mRNA vaccines. The main objective of this study was to evaluate toxicity of six lipid based formulations with three surface charges-anionic, neutral or cationic, to establish certified reference materials (CRMs) for liposomes and siRNA loaded lipid nanoparticles (LNP-siRNA). Cytotoxicity was assessed by a proliferation assay in adherent and non-adherent cell lines. High concentration of three LNP-siRNAs did not affect viability of suspension cells and LNP-siRNAs were non-toxic to adherent cells at conventionally used concentration. Systematic evaluation using multiple vials and repeated test runs of three liposomes and three LNP-siRNA formulations showed no toxicity in HL60 and A549 cells up to 128 and 16 µg/mL, respectively. Extended treatment and low concentration of LNPs did not affect the viability of suspension cells and adherent cells at 96 h. Interestingly, 80% of A549 and HL60 cells in 3D conditions were viable when treated with cationic LNP-siRNA for 48 h. Taken together, anionic, cationic and neutral lipid formulations were non-toxic to cells and may be explored further in order to develop them as drug carriers.

Investigation of the ionic conditions in SiRNA-mediated delivery through its carriers in the cell membrane: a molecular dynamic simulation.

SiRNA is a new generation of drug molecules and a new approach for treating a variety of diseases such as cancer and viral infections. SiRNA delivery to cells and translocation into cytoplasm are the main challenges in the clinical application of siRNA. Lipid carriers are one of the most successful carriers for siRNA delivery. In this study, we investigated the interaction of siRNA with a zwitterionic bilayer and how ion concentration and lipid conjugation can affect it. The divalent cation such as Mg2+ ions could promote the siRNA adsorption on the bilayer surface. The cation ions can bind to the head groups of lipids and the grooves of siRNA molecules and form bridges between the siRNA and bilayer surface. Our findings demonstrated the bridges formed by divalent ions could facilitate the attachment of siRNA to the membrane surface. We showed that the divalent cations can regulate the bridging-driven membrane attachment and it seems the result of this modulation can be used for designing biomimetic devices. In the following, we examined the effect of cations on the interaction between siRNA modified by cholesterol and the membrane surface. Our MD simulations showed that in the presence of Mg2+, the electrostatic and vdW energy between the membrane and siRNA were higher compared to those in the presence of NA+. We showed that the electrostatic interaction between membrane and siRNA cannot be facilitated only by cholesterol conjugated. Indeed, cations are essential to create coulomb repulsion and enable membrane attachment. This study provides important insight into liposome carriers for siRNA delivery and could help us in the development of siRNA-based therapeutics. Due to the coronavirus pandemic outbreak, these results may shed light on the new approach for treating these diseases and their molecular details.

Noncoding RNAs Emerging as Drugs or Drug Targets: Their Chemical Modification, Bio-Conjugation and Intracellular Regulation.

With the increasing understanding of various disease-related noncoding RNAs, ncRNAs are emerging as novel drugs and drug targets. Nucleic acid drugs based on different types of noncoding RNAs have been designed and tested. Chemical modification has been applied to noncoding RNAs such as siRNA or miRNA to increase the resistance to degradation with minimum influence on their biological function. Chemical biological methods have also been developed to regulate relevant noncoding RNAs in the occurrence of various diseases. New strategies such as designing ribonuclease targeting chimeras to degrade endogenous noncoding RNAs are emerging as promising approaches to regulate gene expressions, serving as next-generation drugs. This review summarized the current state of noncoding RNA-based theranostics, major chemical modifications of noncoding RNAs to develop nucleic acid drugs, conjugation of RNA with different functional biomolecules as well as design and screening of potential molecules to regulate the expression or activity of endogenous noncoding RNAs for drug development. Finally, strategies of improving the delivery of noncoding RNAs are discussed.

Cheminformatics Modeling of Gene Silencing for Both Natural and Chemically Modified siRNAs.

In designing effective siRNAs for a specific mRNA target, it is critically important to have predictive models for the potency of siRNAs. None of the published methods characterized the chemical structures of individual nucleotides constituting a siRNA molecule; therefore, they cannot predict the potency of gene silencing by chemically modified siRNAs (cm-siRNA). We propose a new approach that can predict the potency of gene silencing by cm-siRNAs, which characterizes each nucleotide (NT) using 12 BCUT cheminformatics descriptors describing its charge distribution, hydrophobic and polar properties. Thus, a 21-NT siRNA molecule is described by 252 descriptors resulting from concatenating all the BCUT values of its composing nucleotides. Partial Least Square is employed to develop statistical models. The Huesken data (2431 natural siRNA molecules) were used to perform model building and evaluation for natural siRNAs. Our results were comparable with or superior to those from Huesken's algorithm. The Bramsen dataset (48 cm-siRNAs) was used to build and test the models for cm-siRNAs. The predictive r2 of the resulting models reached 0.65 (or Pearson r values of 0.82). Thus, this new method can be used to successfully model gene silencing potency by both natural and chemically modified siRNA molecules.