Engineered lipid nanoparticles enable therapeutic gene silencing of GTSE1 for the treatment of liver fibrosis.

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix proteins, disrupting normal liver function. Despite its significant health impact, effective treatments remain limited. Here, we present the development of engineered lipid nanoparticles (LNPs) for targeted RNA therapeutic delivery in the liver. We investigated the therapeutic potential of modulating the G2 and S-phase expressed 1 (GTSE1) protein for treating liver fibrosis. Through screening, we identified P138Y LNP as a potent candidate with superior delivery efficiency and lower toxicity. Using these engineered LNPs, we successfully delivered siGTSE1 to hepatocytes, significantly reducing collagen accumulation and restoring liver function in a fibrosis animal model. Additionally, GTSE1 downregulation altered miRNA expression and upregulated hepatocyte nuclear factor 4 alpha (HNF4α). These findings suggest that therapeutic gene silencing of GTSE1 is a promising strategy for treating liver fibrosis by regenerating liver phenotypes and functions.

Development of Lipid Nanoparticle Formulation for the Repeated Administration of mRNA Therapeutics.

During the COVID-19 pandemic, mRNA vaccines emerged as a rapid and effective solution for global immunization. The success of COVID-19 mRNA vaccines has increased interest in the use of lipid nanoparticles (LNPs) for the in vivo delivery of mRNA therapeutics. Although mRNA exhibits robust expression profiles, transient protein expression is often observed, raising uncertainty regarding the frequency of its administration. Additionally, various RNA therapeutics may necessitate repeated dosing to achieve optimal therapeutic outcomes. Nevertheless, the impact of repeated administrations of mRNA/LNP on immune responses and protein expression efficacy remains unclear. In this study, we investigated the influence of the formulation parameters, specifically ionizable lipids and polyethylene glycol (PEG) lipids, on the repeat administration of mRNA/LNP. Our findings revealed that ionizable lipids had no discernible impact on the dose-responsive efficacy of repeat administrations, whereas the lipid structure and molar ratio of PEG lipids were primary factors that affected mRNA/LNP performance. The optimization of the LNP formulation with PEG lipid confirmed the sustained dose-responsive efficacy of mRNA after repeated administrations. This study highlights the critical importance of optimizing LNP formulations for mRNA therapeutics requiring repeated administrations.

Novel piperazine-based ionizable lipid nanoparticles allow the repeated dose of mRNA to fibrotic lungs with improved potency and safety.

mRNA-based protein replacement therapy has received much attention as a novel intervention in clinical disease treatment. Lipid nanoparticles (LNPs) are widely used for their therapeutic potential to efficiently deliver mRNA. However, clinical translation has been hampered by the immunogenicity of LNPs that may aggravate underlying disease states. Here, we report a novel ionizable LNP with enhanced potency and safety. The piperazine-based biodegradable ionizable lipid (244cis) was developed for LNP formulation and its level of protein expression and immunogenicity in the target tissue was evaluated. It was found that 244cis LNP enabled substantial expression of the target protein (human erythropoietin), while it minimally induced the secretion of monocyte chemoattractant protein 1 (MCP-1) as compared to other conventional LNPs. Selective lung targeting of 244cis LNP was further investigated in tdTomato transgenic mice with bleomycin-induced pulmonary fibrosis (PF). The repeated administration of 244cis LNP with Cre recombinase mRNA achieved complete transfection of lung endothelial cells (~80%) and over 40% transfection of Sca-1-positive fibroblasts. It was shown that 244cis LNP allows the repeated dose of mRNA without the loss of activity due to its low immunogenicity. Our results demonstrate that 244cis LNP has great potential for the treatment of chronic diseases in the lungs with improved potency and safety.

Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics.

Several studies have utilized a lipid nanoparticle delivery system to enhance the effectiveness of mRNA therapeutics and vaccines. However, these nanoparticles are recognized as foreign materials by the body and stimulate innate immunity, which in turn impacts adaptive immunity. Therefore, it is crucial to understand the specific type of innate immune response triggered by lipid nanoparticles. This article provides an overview of the immunological response in the body, explores how lipid nanoparticles activate the innate immune system, and examines the adverse effects and immunogenicity-related development pathways associated with these nanoparticles. Finally, we highlight and explore strategies for regulating the immunogenicity of lipid nanoparticles.

Lipid nanoparticles (LNPs) for in vivo RNA delivery and their breakthrough technology for future applications.

RNA therapeutics show a significant breakthrough for the treatment of otherwise incurable diseases and genetic disorders by regulating disease-related gene expression. The successful development of COVID-19 mRNA vaccines further emphasizes the potential of RNA therapeutics in the prevention of infectious diseases as well as in the treatment of chronic diseases. However, the efficient delivery of RNA into cells remains a challenge, and nanoparticle delivery systems such as lipid nanoparticles (LNPs) are necessary to fully realize the potential of RNA therapeutics. While LNPs provide a highly efficient platform for the in vivo delivery of RNA by overcoming various biological barriers, several challenges remain to be resolved for further development and regulatory approval. These include a lack of targeted delivery to extrahepatic organs and a gradual loss of therapeutic potency with repeated doses. In this review, we highlight the fundamental aspects of LNPs and their uses in the development of novel RNA therapeutics. Recent advances in LNP-based therapeutics and preclinical/clinical studies are overviewed. Lastly, we discuss the current limitations of LNPs and introduce breakthrough technologies that might overcome these challenges in future applications.

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

Protein-RNA interaction guided chemical modification of Dicer substrate RNA nanostructures for superior in vivo gene silencing.

Dicer substrate RNA is an alternative gene silencing agent to canonical siRNA. Enhanced in vitro gene silencing can be achieved with RNA substrates by facilitating Ago2 loading of dsRNA after Dicer processing. However, the in vivo use of Dicer substrate RNA has been hindered by its instability and immunogenicity in the body due to the lack of proper chemical modification in the structure. Here, we report a universal chemical modification approach for Dicer substrate RNA nanostructures by optimizing protein-RNA interactions in the RNAi pathway. Proteins involved in the RNAi pathway were utilized for evaluating their recognition and binding of substrate RNA. It was found that conventional chemical modifications could severely affect the binding and processing of substrate RNA, consequently reducing RNAi activity. Protein-RNA interaction guided chemical modification was introduced to RNA nanostructures, and their gene silencing activity was assessed. The optimized RNA nanostructures showed excellent binding and processability with RNA binding proteins and offered the enhancement of in vivo EC50 up to 1/8 of its native form.