Biosafety assessment of delivery systems for clinical nucleic acid therapeutics

Nucleic acid therapeutics,which involve transferring exogenous genes inside target cells,are a promising clinical treatment option that can regulate gene expression at the transcriptional or post-transcriptional level.Ideally,this kind of treatment modality will not lead to an unwanted immune response.Compared with traditional treatment methods,nucleic acid therapeutics can achieve prolonged and stable curative effects.As an emerging treatment method,nucleic acid therapeutics have played an increasingly important role in clinical settings for the treatment of various conditions,including infectious diseases,cancer,immune-related diseases,and monogenetic diseases.To date,a large number of clinical trials have been conducted,and more than 30 nucleic acid drugs have been approved,highlighting the strong potential of this approach in clinical practice.Diverse carriers are used to protect nucleic acids from being degraded and to help them reach their targets accurately.However,some carriers are known to cause negative effects on the release and expression of nucleic acid drugs as well as adverse effects such as allergic reactions and accumulation in the liver.Therefore,biosafety assessment of delivery systems before their application in clinical settings is critical.In this review,we describe different delivery systems for nucleic acid drugs and discuss their biosafety in both preclinical and clinical studies,with particular focus on the carriers themselves,drug administration method,and overall treatment of the disease.

Application of cationic liposomes for delivery of nucleic acids

Nucleic acid-based bioactive substances have recently emerged as a new class of nextgeneration therapeutics, but their development has been limited by their relatively weakdelivery into target cells. Cationic liposomes have been studied as a means to enhance thestability of nucleic acid therapeutics in the bloodstream and improve their cellular delivery.As nucleic acid therapeutics, siRNA and plasmid DNA have been extensively tested fordelivery using cationic liposomes. This review discusses recent progress in the applicationof cationic liposomes for the delivery of nucleic acid therapeutics.

Analysis of the Effects of Different Synthesis and Processing Methods on Circular RNA Integrity,Protein Expression,and Removal of Immunogenic Impurities

Circular RNAs(circRNAs)are emerging as a promising alternative to messenger RNAs(mRNAs)in gene delivery applications due to their enhanced stability and translation.Developing circRNA-based therapeutic platforms requires efficient manufacturing of circRNA with broad scalability.However,the permuted intron-exon(PIE)-based circRNA production commonly used to date involves complex RNA synthesis,circularization,precursor RNA digestion,and impurity removal steps that have limited practical applications.While co-transcriptional circularization could effectively streamline circRNA production,and both cellulose/phosphatase treatment and high-performance liquid chromatography(HPLC)have demonstrated their reliability in mRNA manufacturing,their potential effects on the quality,translation,and reactogenicity of circRNA remained to be fully investigated.Here,using circRNAs systematically manufactured through three independent workflows,we comprehensively examined the utilities of these RNA synthesis and processing methods in circRNA production by comparing the integrity,translation,and immunogenicity of their circRNA products.We began by manufacturing a mNeonGreen(mNG)-encoding circRNA through these workflows and subsequently assessed circRNA integrity via E-gel EX electrophoresis.Protein expression was then monitored in HEK 293T,A549,and DC2.4 cells at 72 hours post-transfection.Finally,we evaluated the immunogenicity of these circRNAs by measuring their interferon beta(IFN-β)induction in A549 cells at 4 hours post-transfection.Using HPLC purification over cellulose and phosphatase treatment resulted in 10-14%higher circRNA enrichment by reducing nicking associated with processing conditions.Protein expression remained consistent across circRNAs from different workflows(P>0.05),demonstrating that co-transcriptional circularization produces circRNA with translation levels comparable to those obtained from the conventional PIE method.Moreover,both cellulose/phosphatase treatment and HPLC purification effectively minimized IFN-βinduction of the purified circRNAs,confirming their reliability in removing immunogenic impurities introduced during in vitro transcription and their compatibility with the co-transcriptional circularization strategy.Collectively,our results provide valuable insights for improving the production efficiency and scalability of circRNA manufacturing that are crucial for addressing key bottlenecks in the development of circRNA-based therapeutic applications.

Design of synthetic materials for intracellular delivery of RNAs： From siRNA-mediated gene silencing to CRISPR/Cas gene editing

Ribonucleic acids （RNAs） possess great therapeutic potential and can be used to treat a variety of diseases. The unique biophysical properties of RNAs, such as high molecular weight, negative charge, hydrophilicity, low stability, and potential immunogenicity, require chemical modification and development of carriers to enable intracellular delivery of RNAs for clinical use. A variety of nanornaterials have been developed for the effective in vivo delivery of short/ small RNAs, messenger RNAs, and RNAs required for gene editing technologies including clustered regularly interspaced palindromic repeat （CRISPR）/Cas. This review outlines the challenges of delivering RNA therapeutics, explores the chemical synthesis of RNA modifications and carriers, and describes the efforts to design nanomaterials that can be used for a variety of clinical indications.