Periodic Reporting for period 1 - Yscript (Yeast cell factory for mRNA bioproduction)
Reporting period: 2022-04-01 to 2023-03-31
Messenger RNA (mRNA) is a promising biopharmaceutical for variousmedical applications. Currently, mRNAs are produced by in vitro transcription (IVT). Despite the advantage of IVT being cell-free, reliable large-scale IVT production of long size mRNA, is challenging, and costly.
Yscript radically vision lies in the generation of mRNA bioproduction process in yeast, integrating innovative extraction and purification processes, a complete shift of paradigm compared to IVT production and a revolutionary new use of yeast.
Yscript will provide ground-breaking technological improvements by decreasing the supply chain, increasing yield purity of long mRNA and guaranteeing mRNA stability.
Our consortium brings together unique skill sets and expertise of eight partners. CNRS combines expertise in mRNA therapeutics and yeast mRNA processes. TRON exploits its experience in engineering RNA vectors, in vitro cell reprogramming and immunological in vivo models. IBCH uses its solid experience in optimizing mRNA/protein interaction and accumulation. INRAE leverages its experience as a pre-industrial demonstrator to upscale the process. Specific purification approaches is developed by combining the state-of-the-art expertise of UAVR, UBI and BIASep. UAVR brings its knowledge on the development of integrated extraction-purification ionic liquid (ILs)-based platforms. UBI oversees chromatographic conditions building on their experience in nucleic acids purification and stabilization. Furthermore, BIASep, a leading developer of chromatographic columns, develops ligand-coupling modified mRNA purification columns.
Yscript radically vision lies in the generation of mRNA bioproduction process in yeast, integrating innovative extraction and purification processes, a complete shift of paradigm compared to IVT production and a revolutionary new use of yeast.
Yscript will provide ground-breaking technological improvements by decreasing the supply chain, increasing yield purity of long mRNA and guaranteeing mRNA stability.
Our consortium brings together unique skill sets and expertise of eight partners. CNRS combines expertise in mRNA therapeutics and yeast mRNA processes. TRON exploits its experience in engineering RNA vectors, in vitro cell reprogramming and immunological in vivo models. IBCH uses its solid experience in optimizing mRNA/protein interaction and accumulation. INRAE leverages its experience as a pre-industrial demonstrator to upscale the process. Specific purification approaches is developed by combining the state-of-the-art expertise of UAVR, UBI and BIASep. UAVR brings its knowledge on the development of integrated extraction-purification ionic liquid (ILs)-based platforms. UBI oversees chromatographic conditions building on their experience in nucleic acids purification and stabilization. Furthermore, BIASep, a leading developer of chromatographic columns, develops ligand-coupling modified mRNA purification columns.
Yscript is based on CNRS’s proof of concept (POC) using a yeast strain indicating that a specific chaperone protein is able to recognize and accumulate any mRNAs of a gene of interest (GOI) by forming condensates that can be accumulated and extracted from lysates. Yscript main goal is to implement this bioprocess to reach industrialization.
During this first period, work packages (WPs) 1, 2 and 3 started in agreement with the DoA. The main results obtained so far are detailed below.
WP1: First experiments were dedicated to POC validation and to the identification of parameters allowing to increase the production. INRAE identified strains more suitable for industrial processes with better growth parameters.
During this period, IBCH/PAN studied the interactions between the Chaperone protein and mRNA. IBCH/PAN and CNRS selected two addressing sequences (AS), and experiments are ongoing to validate this. To increase the specificity of Chaperone and mRNA interactions, IBCH is working on the SELEX consensus sequence for the Chaperone that could be added to the designed AS or might act alone. Collected data from this task will allow us to find out the optimal addressing sequence for the mRNA of interest.
CNRS started to produce mRNA –GOI bearing cap1 at their 5’ end. The first approach gave interesting data, produced mRNAs have an improved efficiency both in vitro and in cellulo. Our data are in favor of an improvement of either stability and/or translation efficiency of produced mRNA.
WP2: INRAE constructed strains transiently expressing a plasmid controlling the production of Chaperone and a plasmid allowing mRNA GOI-production. INRAE compared several yeast factories to CNRS strain and found other cell factories to show better production in terms of biomass and/or mRNA. INRAE identified several parameters to modulate mRNA production: culture medium, pH regulation, fed-batch culture and production induction phase after growth phase.
WP3: A set of ILs, namely ammonium-based cations combined with Good’s buffers, amino acids or halides as anions, was synthesized and characterized by UAVR. UAVR and CNRS investigated operating conditions affecting the IL-mediated lysis process.
IL-based supports, synthesized and characterized by UAVR and UBI, are being evaluated for IVT-produced RNA separation. UBI and CNRS demonstrated that supports modified with specific ILs present the ability to eliminate major contaminants. These ILs will be immobilized onto monoliths for mRNA purification. BIASEP evaluated multimodal monolith chromatographic columns (CIM PrimaS and CIM Swiper) for mRNA purification as an alternative. They validated the POC of oligonucleotide-based affinity capture for mRNA of different sizes by the development of Oligo-deoxythymidilic acid (OdT). Affinity-based columns targeting specific sequences in bioproduced mRNA are under development.
BIASEP has successfully implemented the capillary gel electrophoresis method for mRNA qualitative and quantitative analyses.
Conclusions
During the first period, Yscript partners started all tasks as described in DoA. Data gathered from different tasks are promising but still require either validation or improvement. They constitute solid groundwork for the second period in which the performance of the technology to produce intact and efficient mRNAs must be demonstrated.
During this first period, work packages (WPs) 1, 2 and 3 started in agreement with the DoA. The main results obtained so far are detailed below.
WP1: First experiments were dedicated to POC validation and to the identification of parameters allowing to increase the production. INRAE identified strains more suitable for industrial processes with better growth parameters.
During this period, IBCH/PAN studied the interactions between the Chaperone protein and mRNA. IBCH/PAN and CNRS selected two addressing sequences (AS), and experiments are ongoing to validate this. To increase the specificity of Chaperone and mRNA interactions, IBCH is working on the SELEX consensus sequence for the Chaperone that could be added to the designed AS or might act alone. Collected data from this task will allow us to find out the optimal addressing sequence for the mRNA of interest.
CNRS started to produce mRNA –GOI bearing cap1 at their 5’ end. The first approach gave interesting data, produced mRNAs have an improved efficiency both in vitro and in cellulo. Our data are in favor of an improvement of either stability and/or translation efficiency of produced mRNA.
WP2: INRAE constructed strains transiently expressing a plasmid controlling the production of Chaperone and a plasmid allowing mRNA GOI-production. INRAE compared several yeast factories to CNRS strain and found other cell factories to show better production in terms of biomass and/or mRNA. INRAE identified several parameters to modulate mRNA production: culture medium, pH regulation, fed-batch culture and production induction phase after growth phase.
WP3: A set of ILs, namely ammonium-based cations combined with Good’s buffers, amino acids or halides as anions, was synthesized and characterized by UAVR. UAVR and CNRS investigated operating conditions affecting the IL-mediated lysis process.
IL-based supports, synthesized and characterized by UAVR and UBI, are being evaluated for IVT-produced RNA separation. UBI and CNRS demonstrated that supports modified with specific ILs present the ability to eliminate major contaminants. These ILs will be immobilized onto monoliths for mRNA purification. BIASEP evaluated multimodal monolith chromatographic columns (CIM PrimaS and CIM Swiper) for mRNA purification as an alternative. They validated the POC of oligonucleotide-based affinity capture for mRNA of different sizes by the development of Oligo-deoxythymidilic acid (OdT). Affinity-based columns targeting specific sequences in bioproduced mRNA are under development.
BIASEP has successfully implemented the capillary gel electrophoresis method for mRNA qualitative and quantitative analyses.
Conclusions
During the first period, Yscript partners started all tasks as described in DoA. Data gathered from different tasks are promising but still require either validation or improvement. They constitute solid groundwork for the second period in which the performance of the technology to produce intact and efficient mRNAs must be demonstrated.
Yscript has many potential impacts: i) decreasing the supply chain for mRNA production and purification processes; ii) increasing the purity yield of long mRNA; iii) guaranteeing mRNA stability; iv) reducing production cost; and v) ensuring easy scale-up.
Yscript uniquely takes into account industrialisation considerations in the methodology. This is a key asset to ensure results exploitability and accelerate translation from research to innovation. Yscript relies on yeast cultures, which are a simple, widely used model organisms. Necessary infrastructures are already in place, fostering the diffusion and large adoption of the technology. The following challenges will be addressed:
Large and agile production of RNA vaccines during pandemics. While mRNA-based pharmaceuticals have demonstrated great potential to quickly combat global threats, the complexity and cost-effectiveness of large-scale production are currently major obstacles to mass vaccination. Yscript methodology could tackle both challenges. Yscript process will ease local production of RNA vaccines: the developed yeast clone will be easily distributed among existing yeast production infrastructures worldwide, thereby limiting cold-chain related issues.
Reduce inequities: affording therapies for low-/mid-income populations. Yscript will allow cost- reduction of mRNA by avoiding expensive reagents; integrating extraction and purification steps; enabling simultaneous several mRNAs production and using yeast cost-effective cell-factory. Yscript will thus make mRNA therapies widely accessible, a major target of the UN Sustainable Developmental Goal 3.
Pave the way for new therapies. Yscript’s technology will facilitate open access to affordable, high yield and pure mRNA production, enable simple and rapid production of any types of mRNAs and allow the use of long size mRNAs, which is currently difficult to produce. Thus, it will open mRNA approaches for therapies requiring multiple protein production or long-lasting protein expression.
Greener processes: The processes will be developed under sustainability concepts, reducing energy consumption, amounts of solvents used and wastes generated.
Yscript uniquely takes into account industrialisation considerations in the methodology. This is a key asset to ensure results exploitability and accelerate translation from research to innovation. Yscript relies on yeast cultures, which are a simple, widely used model organisms. Necessary infrastructures are already in place, fostering the diffusion and large adoption of the technology. The following challenges will be addressed:
Large and agile production of RNA vaccines during pandemics. While mRNA-based pharmaceuticals have demonstrated great potential to quickly combat global threats, the complexity and cost-effectiveness of large-scale production are currently major obstacles to mass vaccination. Yscript methodology could tackle both challenges. Yscript process will ease local production of RNA vaccines: the developed yeast clone will be easily distributed among existing yeast production infrastructures worldwide, thereby limiting cold-chain related issues.
Reduce inequities: affording therapies for low-/mid-income populations. Yscript will allow cost- reduction of mRNA by avoiding expensive reagents; integrating extraction and purification steps; enabling simultaneous several mRNAs production and using yeast cost-effective cell-factory. Yscript will thus make mRNA therapies widely accessible, a major target of the UN Sustainable Developmental Goal 3.
Pave the way for new therapies. Yscript’s technology will facilitate open access to affordable, high yield and pure mRNA production, enable simple and rapid production of any types of mRNAs and allow the use of long size mRNAs, which is currently difficult to produce. Thus, it will open mRNA approaches for therapies requiring multiple protein production or long-lasting protein expression.
Greener processes: The processes will be developed under sustainability concepts, reducing energy consumption, amounts of solvents used and wastes generated.