0:33 Thank you for the introduction and the opportunity to present this.

0:40 From the first presentation, I will zoom into the mRNA platform by presenting the streamlined IVT workflow to maximize RNA yield and decrease process development time from template design to synthesis.

1:02 A bit about us: Takara Bio is based around the world, originally from Japan, but also in the US and Europe, including Paris. Historically, we are enzyme developers, working on enzymes for PCR, cloning, and more. Over time, we have evolved to propose more enzymatic systems, including those required to synthesize mRNA. We are also a CDMO facility, involved in the synthesis of a self-amplifying vaccine for COVID in Asia. This dual aspect helps us come up with better ways of making mRNA.

2:08 The success of the mRNA platform during COVID shows it could be a new platform of choice for emerging diseases. However, there are inherent issues in vaccine development, such as long process development times and high immunogenicity of mRNA. During COVID, we learned to replace uridine to reduce immunogenicity and produce large-scale mRNA quickly. Double-strained RNA was made during the IVT process, but this was not an issue for mRNA vaccines because it functions as an adjuvant. We could reproduce billions of doses. This experience suggests that the mRNA platform can be a choice for upcoming diseases like Disease X. When we look at CEPI’s ambitious target to produce a vaccine in 100 days from the emergence of disease, RNA seems to be a platform worth considering.

4:22 Although making RNA is not easy, previous knowledge from outbreaks and the presence of a spike protein helped us. For unknown diseases, more research is needed to identify targets and sequences. Lead times can vary, especially for personalized medicine like cancer vaccines, where development must be quick due to patient survival. Other applications like cell and gene therapy also present challenges.

5:42 To summarize, we need to reduce the time to achieve good RNA sequences and reduce immunogenicity. Today, I will focus on shortening the IVT process from template preparation to mRNA synthesis.

6:19 The goal is to fast-track template design, cloning, amplification, and in vitro transcription. To

make this more efficient we start with template preparation. We need to achieve the coding sequence of the gene of interest, add 5 prime and 3 prime UTRs, transcription start sites, and T7 promoters into a plasmid. This requires a lot of work. With the CDS we have to do codon optimisation which requires large synthetic fragments over 1.5kb, multiple fragments, and mutagenesis.

7:49 Selecting and cloning UTRs involves inserting the, immediately before or after CDS. We propose a solution with an already linearized plasmid containing T7 promoter with AGG start sequence, 5' and 3' UTRs, and a poly-A tail. Researchers only need to add their coding sequence. This simplifies the process, reducing cloning time from months to days.

10:14 The digestion site for Hind III ensures mostly adenosines in the poly-A tail, sometimes there are a few additional nucleotides, but this does not affect RNA expression. The process involves PCR preparation, insertion with infusion, transformation, amplification, and plasmid purification, ready for IVT in two days. Sometimes cloning can take 3 months. We conduct PCR using one of our flagship products called In-Fusion this allows you to insert a gene of interest (GOI) in any vector anywhere. The researcher needs to design primers that are complementary to both ends of the plasmid, then you amplify by PCR. The enzyme then ‘chews back’ some nucleotides at the three prime end of it, leaving space for recombination by homologous recombination.

11:24

The cells of interest are then transfected. This is compatible with multiple fragments and mutagenesis. It is super-fast; it only requires 15 minutes incubation.

12:04 The system is flexible, compatible with CleanCap analogues and vaccine capping enzymes but the CleanCap is the gold standard. For those using ARCA or other analogues, modifications to the plasmid are needed. The infusion technology allows easy switching and modification of sequences. The idea is to give flexibility after you have the plasmid because if you need to modify nucleotides in the gene of interest, it can be done, giving you flexibility to play around with your sequence.

13:47 Once the plasmid is ready, proceed to in vitro transcription. The system is compatible with various protocols, including post-transcriptional capping. It also has a poly-(A)-tail included. One thing to note from this synthesis is that the process is pretty much the same from the 20 micro-litre reaction to the reaction in the 30 litre bioreactor. The yield remains consistent across different construct sizes, from small to large sequences.

16:19 Our focus is on improving yield to reduce costs. So we did a benchmarking exercise with our IVT pro mRNA synthesis kit. The kit includes optimised enzyme mixes for high yields, achieving 200 micrograms per 20-microlitre reaction, scalable up to 200 microlitres. It does not require additional enzymes. However we do not include capping analogs but the enzymes we use are compatible with CleanCap. This yield is double what was achieved during COVID vaccine synthesis. Th aim of this is to produce a solution that ‘fits all’.

19:12 The system is compatible with clean cap and vaccine capping enzymes. ARCA and other cap analogs require modifications and using ARCA can decrease yield because it competes with the GTP in the reaction, so we recommend clean cap for large-scale synthesis. CleanCap incorporation does not affect the yield. For cost reduction, vaccinia capping enzymes and 2'-O-methyltransferase can be used. We compared the expression profile with CleanCap which remains the gold standard, the vaccinia capping and 2’-O-methyltransferase do not perform as well but they are useful for reducing the cost of production.

21:17 The goal is to make RNA synthesis easier and faster, maintaining high yields and flexibility. The system supports various capping systems and modified nucleotides, ensuring high mRNA yield per reaction.

22:32 In summary, the synthesis system includes a cloning kit and IVT synthesis kit, compatible with existing plasmids and scalable for large-scale manufacturing. Enzymes are available in research, high-quality, and GMP grades for clinical manufacturing.