mRNA Technology How To Achieve Targeted Delivery to Organs, Cells, Tumors

1. Huateng Pharma https://us.huatengsci.com mRNA Technology: How To Achieve Targeted Delivery to Organs, Cells, Tumors? As a new pharmaceutical technology, mRNA has made breakthroughs in the treatment of infectious diseases and tumors in a short time. At present, one of the major obstacles in clinical mRNA technology is to determine how to deliver mRNA to a specific target cell and protect it from degradation, so the ideal delivery vector must be safe, stable, and organ specific. 1. Nucleic Acid Delivery Technology Since RNA was first discovered, researchers have used a number of methods to deliver it into cells. The initial technique used is naked RNA, which is easily degraded by RNase and causes a strong pro-inflammatory response. Later formulations developed for RNA delivery included carbohydrate polymers, polyethylenimine (PEI), etc. Due to its positive charge and abundance of amines, PEI has a good affinity for nucleic acids, resulting in the formation of positively charged complexes on the surface. In vivo, PEI was successfully used for aerosol gene delivery to the lungs. Although PEI preparations have high transfection efficiency in vitro and in vivo, they are also significantly cytotoxic, partly due to their poor degradation, preventing the wider application of PEI-based vectors in preclinical and clinical settings. Polyesters are another group of materials used for RNA delivery, and the addition of pluripotent F127 reduced the total charge of the nanoparticles and improved their stability. Manipulating F127 levels can lead to lung-specific mRNA delivery, which can be used to treat lung diseases. Natural chitosan is a carbohydrate polymer with biodegradability, biocompatibility, and cationic charge that allows nucleic acid binding. However, it also has limitations such as poor water solubility and limited targeting ability. To date, lipid nanoparticles (LNPs) have been mainly used as RNA therapeutic carriers in clinical. Lipids are organic, water-insoluble lipid compounds with hydrophilic heads and hydrophobic tails that enable LNPs to self-assemble into well-defined structures, such as cell membranes. LNP-RNA systems are formed through hydrophobic interactions in the aqueous environment and electrostatic interactions between negatively charged RNAs and cationic or ionizable lipids. Ionizable lipids are positively charged at low pH (which allows RNA binding) and become neutral at physiological pH, a change that helps reduce the toxicity of LNP-RNA complexes in vivo.

2. Huateng Pharma https://us.huatengsci.com Carriers for Nucleic Acid Delivery: Cargo and Formulation (Adv Drug Deliv Rev. 2020;156:119-132.) 2. Organ-targeted Delivery Using intramuscular injection of LNP-RNA for systemic immunity, how to precisely guide LNP-RNA to target organs? Apolipoprotein E (ApoE) in serum can bind to intravenously injected LNPs, and the liver is the main organ for clearing ApoE-bound lipoproteins. Therefore, systemically delivered LNPs-RNA will bind ApoE and preferentially enter the liver. Excessive liver homing is a significant disadvantage of intravenously administered ionizable LNPs, which can be overcome by selective organ targeting (SORT) strategies. The key to organ-specific delivery is to manipulate the internal and/or external charges of the formulated LNPs. LNPs charge and organ targeting (Pharmaceutics 2021, 13, 1675)

3. Huateng Pharma https://us.huatengsci.com In addition to standard LNP components including ionizable cationic lipids, phospholipids, cholesterol, and PEG, the addition of SORT molecules resulted in lung, spleen, and liver-specific gene delivery. Increasing the percentage of permanently positively charged DDAB and EPC shifted tissue tropism from the liver to the lung. Increasing the negatively charged 1,2-dienoyl-sn-glycero-3-phosphate (14PA) SORT molecule, at 10-40%, resulted in spleen-specific delivery. The appropriate ratio of DODAP and C12-200 as shown in the figure below can also increase liver targeting. Nat Nanotechnol . 2020 Apr;15(4):313-320 3. Cell-targeted Delivery In addition to organ-specific delivery, the researchers also sought to target specific cell subpopulations in the liver by selectively delivering engineered

4. Huateng Pharma https://us.huatengsci.com ionizable lipid nanoparticles into liver cells and hepatic sinusoidal endothelial cells (LSEC). Increased PEG content enhanced hepatocyte targeted delivery, while addition of mannose increased hepatic sinusoidal endothelial cell (LSEC) delivery. Cell-targeted delivery (Sci. Adv. 2021, 7, eabf4398.) 4. Tumor targeted Delivery Heterogeneity is one of the important markers of high malignancy of cancer, so efforts to eradicate cancer by targeting a single receptor are likely to fail, and the loss of this specific targeted receptor will lead to the growth of drug-resistant cancer cells. Although monoclonal antibodies that bind specifically to cell surface receptors are widely used, chemical coupling of multiple different monoclonal antibodies to LNPs is inefficient and requires careful optimization of each monoclonal antibody. A recent new LNPs engineering strategy, ASSET, is a membrane-anchored lipoprotein that is integrated into RNA-loaded lipid nanoparticles and interacts with the antibody Fc domain. It consists of two functional domains: an N-terminal signal sequence followed by a short CDQSSS peptide NlpA motif that undergoes lipidylation in bacteria, (clone RG7/1.30)9 which binds to the Fc constant region of an IgG2a antibody. This lipification strategy, originally used to demonstrate proteins anchored to the inner lining of E. coli, allows the purified recombinant lipified ASSET to be inserted into any lipid vesicle. An mCherry domain and His tag were fused to the C-terminal of ASSET for tracking LNPs absorption (for experimental use) and ASSET purification. and an scFv of a monoclonal antibody

5. Huateng Pharma https://us.huatengsci.com Nat Nanotechnol . 2018 Mar;13(3):214-219. After the cells to be targeted are selected, only specific target antibodies or antibody combinations of corresponding cells need to be injected, and LNPs can self-assemble into targeted LNPs in vivo by binding with antibody Fc. The targeting specificity and effectiveness were confirmed by mouse model experiments (Reference [4]). 5. Commercialization Progress ReCode Therapeutics in the United States is promoting the commercialization of SORT LNPs (currently in preclinical stages), and completed its Series B financing of $80 million on October 21, 2021, led by Pfizer Ventures and EcoR1 Capital, with participation from Sanofi Ventures, and the funds will be used for SORT LNPs technology for primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) clinical research. 6. Conclusion How to accurately deliver mRNA to the target area is the main strategy to maximize the effect of mRNA and minimize toxicity. According to the physical and chemical characteristics of the delivery vector itself, targeted delivery of lung, liver and spleen has been realized, and even specific cells can be accurately targeted. The recently developed self-assembled LNPs self-assemble into targeted LNPs in vivo with specific targeted antibodies or groups of targeted antibodies to achieve accurate targeting of specific cancer tissues or inflammatory tissues. Organ targeted delivery of mRNA the focus of the next generation of mRNA technology development, and ReCode Therapeutics is ready to enter clinical trials. can will be mRNA has emerged as a new class of therapeutic agent for the prevention and treatment of various diseases, such as mRNA vaccines. As a professional PEG derivatives and supplier, Huateng Pharma is able to provide some PEG products which used pharmaceutical intermediates

6. Huateng Pharma https://us.huatengsci.com as ingredients in mRNA vaccines for your mRNA delivery from mg to kg. For more information, please read the page PEGs for mRNA Delivery. References: [1]. Alexandra S Piotrowski-Daspit et al，Polymeric vehicles for nucleic acid delivery，Adv Drug Deliv Rev . 2020; 156:119-132. [2]. Qiang Cheng et al，Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene Apr;15(4):313-320. [3[. Kim, M.; Jeong, M.; Hur, S.; Cho, Y.; Park, J.; Jung, H.; Seo, Y.; Woo, H.A.; Nam, K.T.; Lee, K.; et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Sci. Adv. 2021, 7, eabf4398. [4]. Ranit Kedmi et al ， A modular platform for targeted RNAi therapeutics ， Nat Nanotechnol . 2018 Mar;13(3):214-219. [5]. Zak, M.M.; Zangi, L. Lipid Nanoparticles for Organ-Specific mRNA Therapeutic Delivery. Pharmaceutics 2021, 13, 1675 editing ， Nat Nanotechnol . 2020