RNA emerges as a promising therapeutic agent and is becoming an increasingly popular tool for delivery ofgenetic information to cultured cells and living organisms. Notably, mRNA is used as a basis for new vaccinedevelopment and personalized gene therapy and is replacing DNA vectors in a variety of applications. The highcost of mRNA production currently limits the widespread use of mRNA-based therapeutics, such as introductionof RNA-based flu vaccine or coronavirus vaccine for general population. For all research and medicalapplications, mRNA is produced by in vitro transcription of linear DNA templates with single-subunit RNApolymerases (RNAPs) from bacteriophages. The requirement for mRNA capping complicates its straightforwardproduction. Co-transcriptional capping, with RNAP incorporating a cap analogue during transcription initiation,compromises efficiency of both transcription and capping, resulting in significantly decreased mRNA yield.Alternatively, mRNA can be purified from transcription reaction and then modified post-transcriptionally withcapping enzymes, which are also expensive to produce and purify. RNA polymerases and mRNA modifyingenzymes are irreversibly denatured and destroyed during mRNA purification. Development of a technology thatallows reusing of the enzymes will significantly decrease the mRNA manufacturing costs, thus supporting morewidespread therapeutic uses of mRNA. We propose to create a sequential pipeline for mRNA production, inwhich the enzymes are immobilized and used in multiple consecutive cycles of in vitro transcription, mRNAcapping, and polyadenylation. First, we will synthesize mRNA encoding influenza virus haemagglutinin (HA) andSARS-CoV-2 spike (S) protein using immobilized T7 RNAP. We will establish the conditions for RNAPimmobilization, regeneration, and repeated transcription cycles which, compared to a batch reaction in solution,will significantly increase the mRNA yield per unit of RNAP. Next, the HA and SARS-CoV-2 S protein mRNAswill be capped using the vaccinia virus capping enzyme immobilized via its catalytic subunit. Repeated cycles ofcapping using the same preparation of the immobilized enzyme will be used to determine its robustness, rigor,stability and the limits of the enzyme recycling. The successful completion of the proposed Phase I research willserve as a foundation for the complete pipeline of functional mRNA production. It will increase the mRNA yieldand promote purification of the final product, eliminating the need for protein destruction after each enzymaticcycle. It is applicable in various fields of biomedical research and medicine relying on the in vitro synthesis ofmRNA and, particularly, will enhance the cost-effectiveness of mRNA-based vaccine manufacturing.
Public Health Relevance Statement: NARRATIVE
mRNA encoding for clinically relevant proteins can serve as a safe and effective therapeutic agent in
personalized medicine, gene therapy, and vaccines. Production of mRNA at current state of the art is prohibitively
expensive when it comes, for example, to production of seasonal flu vaccine or a coronavirus vaccine for millions
of people. We propose to immobilize RNA polymerase and other enzymes needed for mRNA manufacturing and
re-use them in multiple cycles of mRNA synthesis and modification to reduce the cost of mRNA synthesis and
make it available for various public health applications.
Project Terms: absorption ; Affect ; Bacteriophages ; Phages ; bacterial virus ; Biomedical Research ; Cultured Cells ; cost effectiveness ; Deoxyribonuclease I ; DNA Endonuclease ; DNase I ; Pancreatic DNase ; Thymonuclease ; Disease Outbreaks ; Outbreaks ; DNA ; Deoxyribonucleic Acid ; Superhelical DNA ; Supercoiled DNA ; Supertwisted DNA ; Enzymes ; Enzyme Gene ; Immobilized Enzymes ; Escherichia coli ; E coli ; E. coli ; Foundations ; gene therapy ; DNA Therapy ; Gene Transfer Clinical ; Genetic Intervention ; gene-based therapy ; genetic therapy ; genomic therapy ; Immobilization ; orthopedic freezing ; In Vitro ; Influenza ; Grippe ; influenza virus vaccine ; Influenza Vaccines ; flu vaccine ; flu virus vaccine ; vaccine against flu ; vaccine against influenza ; Lysine ; L-Lysine ; Medicine ; Methylation ; Methyltransferase ; EC 2.1.1 ; methylase ; transmethylase ; Mole the mammal ; Moles ; Mutation ; Genetic Alteration ; Genetic Change ; Genetic defect ; genome mutation ; Organism ; living system ; Polynucleotide Adenylyltransferase ; ATP-RNA Adenylyltransferase ; Poly A Polymerase ; Polyadenylate Polymerase ; Polyadenylate Synthetase ; Riboadenylate Transferase ; Production ; Proteins ; Publishing ; Reagent ; Natural regeneration ; Regeneration ; regenerate ; Research ; Plant Resins ; resin ; Risk ; RNA ; Non-Polyadenylated RNA ; RNA Gene Products ; Ribonucleic Acid ; DNA-Directed RNA Polymerase ; DNA-Dependent RNA Polymerases ; RNA Polymerases ; Messenger RNA ; mRNA ; Sepharose ; Agarose ; Technology ; Testing ; Time ; Genetic Transcription ; Gene Transcription ; RNA Expression ; Transcription ; Translations ; Vaccines ; Vaccinia ; Vaccinia virus ; Poxvirus officinale ; recombinant vaccinia virus ; Virus ; polyadenylated messenger RNA ; Poly(A)+ mRNA ; Polyadenylated mRNA ; T7 RNA polymerase ; base ; Solid ; Phase ; Medical ; analog ; Recycling ; Therapeutic ; Therapeutic Agents ; tool ; Protocol ; Protocols documentation ; Reaction ; dimer ; protein purification ; develop a vaccine ; development of a vaccine ; vaccine formulation ; vaccine development ; mRNA capping ; Catalytic Core ; Catalytic Region ; Catalytic Site ; Catalytic Subunit ; Catalytic Domain ; Exclusion ; General Public ; General Population ; Manpower ; personnel ; Human Resources ; Abscission ; Extirpation ; Removal ; Surgical Removal ; resection ; Excision ; Modeling ; RNA purification ; Functional RNA ; Non-Coding ; Non-Coding RNA ; Non-translated RNA ; Noncoding RNA ; Nontranslated RNA ; noncoding ; Untranslated RNA ; RNA Polyadenylation ; Polyadenylation ; Transcription Initiation ; protein expression ; Therapeutic Uses ; Influenza Virus ; influenzavirus ; his6 tag ; Mammalian Cell ; Public Health Applications ; Public Health Applications Research ; Vaccine Production ; produce vaccines ; Enzymatic Biochemistry ; Enzymology ; Preparation ; Process ; Modification ; Development ; developmental ; vector ; cost ; clinically relevant ; clinical relevance ; novel vaccines ; new vaccines ; next generation vaccines ; induced pluripotent stem cell ; iPS ; iPSC ; iPSCs ; large scale production ; seasonal influenza ; seasonal flu ; personalized medicine ; personalization of treatment ; personalized therapy ; personalized treatment ; genetic information ; RNA vaccine ; RNA-based vaccine ; mRNA vaccine ; mRNA-based vaccine ; 2019-nCoV ; 2019 novel corona virus ; 2019 novel coronavirus ; COVID-19 virus ; COVID19 virus ; CoV-2 ; CoV2 ; SARS corona virus 2 ; SARS-CoV-2 ; SARS-CoV2 ; SARS-associated corona virus 2 ; SARS-associated coronavirus 2 ; SARS-coronavirus-2 ; SARS-related corona virus 2 ; SARS-related coronavirus 2 ; SARSCoV2 ; Severe Acute Respiratory Distress Syndrome CoV 2 ; Severe Acute Respiratory Distress Syndrome Corona Virus 2 ; Severe Acute Respiratory Distress Syndrome Coronavirus 2 ; Severe Acute Respiratory Syndrome CoV 2 ; Severe Acute Respiratory Syndrome-associated coronavirus 2 ; Severe Acute Respiratory Syndrome-related coronavirus 2 ; Severe acute respiratory syndrome associated corona virus 2 ; Severe acute respiratory syndrome corona virus 2 ; Severe acute respiratory syndrome coronavirus 2 ; Severe acute respiratory syndrome related corona virus 2 ; Wuhan coronavirus ; coronavirus disease 2019 virus ; hCoV19 ; nCoV2 ; mRNA delivery ; coronavirus vaccine ; vaccine against coronavirus ; SARS-CoV-2 spike protein ; 2019-nCoV S protein ; 2019-nCoV spike glycoprotein ; 2019-nCoV spike protein ; COVID-19 S protein ; COVID-19 spike glycoprotein ; COVID-19 spike protein ; COVID19 S protein ; COVID19 spike glycoprotein ; COVID19 spike protein ; SARS-CoV-2 S protein ; SARS-CoV-2 spike glycoprotein ; SARS-CoV2 S protein ; SARS-CoV2 spike glycoprotein ; SARS-CoV2 spike protein ; Severe acute respiratory syndrome coronavirus 2 S protein ; Severe acute respiratory syndrome coronavirus 2 spike glycoprotein ; Severe acute respiratory syndrome coronavirus 2 spike protein ; coronavirus disease 2019 S protein ; coronavirus disease 2019 spike glycoprotein ; coronavirus disease 2019 spike protein ; therapeutically effective ;
