Discovery and development of single-subunit RNA polymerases for efficient RNA manufacturing

Project Summary AbstractRNA plays key roles in cells and organisms including as a carrier of protein coding information and as a regulator of gene expressionRNA therapeutics and RNA based vaccineswhich exploit the natural functions of RNA in creating physiological responses designed to prevent or treat diseasehave received increasing attention in the past few yearsparticularly for developing novel vaccines and for curing rare diseases caused by heritable genetic defectsThe expanded effort in RNA therapeutics and RNA vaccines has created a new demand for RNA molecules manufactured in large quantities to precise specificationsIn particularthe need to create RNA molecules andgtkb in length and to incorporate modified nucleotides for more efficient deliveryhigher stability and better clinical efficacyhas compounded this manufacturing problemAlthough RNAs have been produced enzymatically in vitro for several decades with the use of bacteriophage RNA polymerasesthe enzymes traditionally used to produce RNA for Randamp D purposes are not suited for the demanding specifications that apply to RNA molecules intended for RNA therapeuticsA new class of enzymeshighly optimized for synthesis of long RNAs with specific sequences and structuresneed to be created to meet this new demandIn this projectPrimordial Genetics aims to expresspurify and characterize known but so far untested single subunit RNA polymerases that can be used as starting reagents and genetic building blocks in the development of specialized RNA manufacturing enzymesWe will testdifferent enzymesrepresenting the natural diversity of bacteriophage RNA polymerasesfor their ability to meet the critical requirements for in vitro RNA synthesisincluding efficienthighyield RNA synthesisincorporation of non natural nucleotides and high RNA qualityThe two best enzymes will be improved by mutagenesis based on structural modelingusing the structural and functional information available for this class of enzymesThe proposed work is a feasibility study for isolating and developing novel enzymes suitable for RNA manufacturingand also for creating an enzyme development pipeline that can meet the varied needs for manufacturing a diversity of RNA sequencessizes and chemical structures represented in RNA vaccines and RNA therapeutic products under developmentThe enzymes discovered and improved in this work will be directly useful for RNA manufacturing applicationsand can be licensed or sold to companies developing RNA vaccines and therapeutics as well as companies building RNA manufacturing capabilities Project Narrative The principal aim of this project is to develop novel and improved RNA polymerasesenzymes used for manufacturing RNARNA vaccines and RNA medicines have steadily gained attention and investment as potentially revolutionary ways of protecting against disease and treating rare diseases caused by genetic defectsOne of the challenges with RNA based medicines is the production of clinical quantities of intacthigh quality RNA that is modified by incorporation of non natural building blocks that serve to stabilize the RNA and increase its efficacy in the human bodyDevelopment of novel RNA polymerases will help accelerate the development and production of this novel and highly promising class of therapeutics and vaccinesThis project can impact the prevention and treatment of viral diseases such as HIV and Hepatitis Band genetic diseases such as cystic fibrosis