BL5602-L8 mRNA therapeutics 2.pdf

mRNA therapeutics Vaccines save lives and eradicate diseases Smallpox, a highly infectious disease is caused by the variola virus. In 18-century Europe, ~400,000 people died from the disease per year, and 1/3 of all blindness was caused by smallpox. Famous smallpox survivors included Mozart, Bee...

mRNA therapeutics Vaccines save lives and eradicate diseases Smallpox, a highly infectious disease is caused by the variola virus. In 18-century Europe, ~400,000 people died from the disease per year, and 1/3 of all blindness was caused by smallpox. Famous smallpox survivors included Mozart, Beethoven, QE I of England, George Washington and Abraham Lincoln. Smallpox is also thought to be responsible for the demise of the Aztec and Incan empires. https://www.who.int/  Vaccines save lives and eradicate diseases The first successful vaccine in human history. By 1801, through extensive testing, cowpox was shown to effectively protect against smallpox. Social acceptance? Mandatory smallpox vaccination came into effect in Britain and parts of the United States of America in the 1840s and 1850s, as well as in other parts of the world, leading to the establishment of the smallpox vaccination certificates required for travel. By the 1950s, advances in production techniques meant that heat-stable, freeze-dried smallpox vaccines could be stored without refrigeration. In 1958, the World Health Assembly called for the global eradication of smallpox – the permanent reduction to zero cases – without risk of reintroduction. Thanks to the combined efforts of national health agencies, WHO and scientists around the world, smallpox was eliminated from South America in 1971, Asia in 1975 and Africa in 1977. In 1980, WHO declared smallpox officially eradicated. After 3000 years of suffering and death from the disease, there hasn’t been a recorded case of smallpox in almost half a century. A cure was never found for smallpox before eradication. INFECTIOUS DISEASES THAT HAVE BEEN ERADICATED AND COULD BE ERADICATED IN THE FUTURE. https://ourworldindata.org/eradication-of-diseases Vaccine types Live-attenuated vaccines - live vaccines use a weakened (or attenuated) form of the germ that causes a disease Inactivated vaccines - the killed version of the germ that causes a disease. Subunit, recombinant, polysaccharide, and conjugate vaccines - Subunit, recombinant, polysaccharide, and conjugate vaccines use specific pieces of the germ—like its protein, sugar, or capsid (a casing around the germ). Toxoid vaccines - a toxin (harmful product) made by the germ that causes a disease. They create immunity to the parts of the germ that cause a disease instead of the germ itself. That means the immune response is targeted to the toxin instead of the whole germ. Viral vector vaccines – use a modified version of a different virus as a vector to deliver protection. https://www.hhs.gov/immunization/basics/types/index.html  Humans can be infected by adenoviruses. Repeated infection can reduce efficiency of immunization. Viral Vectors for Gene Therapy, Methods and Protocols, Ed. by Otto-Wilhelm Merten 2011 Vaccine types Live-attenuated vaccines - live vaccines use a weakened (or attenuated) form of the germ that causes a disease Inactivated vaccines - the killed version of the germ that causes a disease. Subunit, recombinant, polysaccharide, and conjugate vaccines - Subunit, recombinant, polysaccharide, and conjugate vaccines use specific pieces of the germ—like its protein, sugar, or capsid (a casing around the germ). Toxoid vaccines - a toxin (harmful product) made by the germ that causes a disease. They create immunity to the parts of the germ that cause a disease instead of the germ itself. That means the immune response is targeted to the toxin instead of the whole germ. Viral vector vaccines – use a modified version of a different virus as a vector to deliver protection. Messenger RNA (mRNA) vaccines - mRNA vaccines make proteins in order to trigger an immune response. https://www.hhs.gov/immunization/basics/types/index.html Advantages of mRNA therapeutics mRNA Protein Non-integrating, no potential risk of infection or insertional mutagenesis. mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly. mRNA has the potential for rapid, inexpensive and scalable manufacturing using in vitro transcription reactions. mRNA is easily adapted as personalized and precision drugs. https://www.frontiersin.org/journals/bioengineering-and- biotechnology/articles/10.3389/fbioe.2021.628137/full Therapeutic modalities of mRNA mRNA Protein Replacement therapy, where mRNA is administered to the patient to compensate for a defective gene/protein, or to supply therapeutic proteins; Vaccination, where mRNA encoding specific antigen(s) is administered to elicit protective immunity; Cell therapy, where mRNA is transfected into the cells ex vivo to alter cell phenotype or function, and then these cells are delivered into the patient.  Challenges in mRNA therapeutics mRNA Protein mRNA stability- RNase is ubiquitously found in the environment and tissues. Delivery into target cells- negatively charged RNA across hydrophobic membranes. Cytotoxicity- host cell innate immune responses to exogenous RNA, Expression levels. Instability, inefficient delivery and low expression efficiency à more mRNA introduced à greater cytotoxicity. Key advances in mRNA therapeutics https://www.nature.com/articles/s41392-022-01007-w  Main steps of mRNA therapeutics In vitro transcription/ processing Delivery into cells Cell membrane Protein products  5’ CAP m7G 5’ cap stabilizes mRNA by making the 5’ end resistant to exonucleases. recruits translation initiation factors eg. eIF4E, for protein translation. decapping enzymes (DCPs) targets the triphosphate bridge to remove the 5’ cap.  5’ CAP- biotechnology implications m7G 5’ cap stabilizes mRNA by making the 5’ end resistant to exonucleases. recruits translation initiation factors eg. eIF4E, for protein translation. decapping enzymes (DCPs) targets the triphosphate bridge to remove the 5’ cap. Modification of m7G and the triphosphate bridge to increase binding to eIF4E and resistance to DCPs. Commonly used 5’ CAP modifications (base+ribose) 3’ poly A 3’ poly A added to the 3’ of mRNA without a template (in vivo). stabilizes mRNA. binds to poly A-binding protein (PABP), which then interacts with eIF4G to form a “closed loop” structure for translation initiation. 3’ poly A modifications in biotechnology 3’ poly A Optimization of poly A length to minimize mRNA decay and enhance translation. Usually longer poly A leads to greater stability and higher translation, but not always. poly Ts are incorporated into the plasmid template.  ORF (open reading frame) mRNA sequence in the ORF are decoded by ribosomes/tRNA to form polypeptides. ORF engineering Codon optimization: replace rare codons with common ones without changing amino acids, but increasing protein yield. Modified nucleotides such as 5-methylcytidine (m5C) and pseudouridine (Ψ) are used to minimize immunogenicity. Other modifications The 5’- and 3’- UTRs: optimizing secondary structures to enhance stability and translation. Inclusion of functional peptides into the ORF: signal peptide, membrane penetration peptide etc.  Main steps of mRNA therapeutics In vitro translation/ processing Delivery into cells Cell membrane Protein products mRNA delivery by lipid nanoparticles Structure of membrane lipids Self organization of lipids in aqueous solution micelle liposome mRNA delivery by lipid nanoparticles mRNA delivery by lipid nanoparticles mRNA delivery by lipid nanoparticles The lipid nanoparticles— protect mRNA or other drugs from degradation in tissue. can be engineered for targeted delivery. can be readily uptaken by cells, usually via endocytosis. RNA drug payload must escape from the endosomes For efficient delivery, the nucleic acid payloads must be released into the cytosol before the maturation of late endosomes to lysosomes, where the foreign materials are degraded enzymatically. Most endocytic cargos are degraded or recycled But how does RNA drug payload escape from the endosomes? Schematic representation of the bilayer to hexagonal phase transition of LNPs. Under the acidic pH of endolysosomal compartment, ionizable lipids are positively charged and interact with the anionic lipids present on the inner leaflet of the endosomal membrane. This interaction leads to the transition from bilayer to hexagonal phase transition resulting in endosomal membrane damage and cargo release. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10945858/  Approach to study RNA drug payload escape from the endosomes Negative 6 different LNPs control GFP mRNA https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8666849/  A comparison between Pfizer and Moderna mRNA vaccines Pfizer Moderna Luciferase mRNA https://www.nature.com/articles/s41541-023-00751-6 A comparison between Pfizer and Moderna mRNA vaccines TEV, a control mRNA backbone Fluc, firefly luciferase https://www.nature.com/articles/s41541-023-00751-6  Advantages of mRNA therapeutics mRNA Protein Non-integrating, no potential risk of infection or insertional mutagenesis. mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly. mRNA has the potential for rapid, inexpensive and scalable manufacturing using in vitro transcription reactions. mRNA is easily adapted as personalized and precision drugs. https://www.frontiersin.org/journals/bioengineering-and- biotechnology/articles/10.3389/fbioe.2021.628137/full  mRNA therapy- present and future https://www.grandviewresearch.com/industry-analysis/mrna-therapeutics-market-report