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She's now with Synthgene, but is has moved there from Arranta Bio and she's now the Chief Commercial Officer of Synthgene.
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The title of her talk is novel Cap analogues and modified NTPs to enable therapeutic mRNA development.
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Thank you.
0:18
Thank you, Paloma.
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I'll get people situated moving in and out.
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Well, so you guys can hear me all right.
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I guess the microphone is right there.
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So first of all, I want to thank you to come to my talk and give me opportunity to discuss our novel Cap analogues and modified NTPs to enable therapeutic mRNA development.
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So we switch gears from small solid-state synthesised RNA to mRNA.
0:57
Now mRNA actually have 5 parts start from 5’ end is the cap.
1:04
The primary function of cap is to bind eIF4E to initiate protein translation.
1:11
The Cap is also involved in self versus non-self recognition and also maintain mRNA stability.
1:22
From that the 5’ UTR, 3’ UTR basically regulate expression and your payload is CDS.
1:31
The Poly A is also responsible to maintain mRNA stability.
1:37
So there are two process to add the cap to mRNA one-pot co-capping or enzymatic capping.
1:46
If you do one capping if you use a good cap analogue you can get very high capping efficiency.
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The process is simple.
1:54
They're less impurities because they're less steps and the production cost is relatively low compared to enzymatic capping.
2:04
So if you look at your for example COVID vaccine, the highest cost components is cap analogue followed by modified NTP and then T7 polymerase.
2:18
So this is an enzymatic tapping step.
2:21
There are actually 4 steps.
2:24
First step is to remove that 1P from 5’ end by the vaccinia capping enzyme.
2:33
The second step to add this inverted G, and 3rd step is to add this methyl group here and this is cap 0.
2:41
So if you want to get cap 1, you need to have another enzyme to transfer the methyl group to the 2’ position.
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This is cap 1.
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You also need a methyl donor called SAM, and SAM is on the molecule that is easy to handle.
2:58
By contrast, if you do co-capping all you need is a cap analogue.
3:04
You add it to IDT the this is cap 0.
3:09
So basically see the green dots, there's a methyl group.
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There's OH, here there's no methyl group, right?
3:17
So cap 0 is not found in the higher eukaryotic cells.
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And cap one, here's the methyl group is found in all higher eukaryotes.
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It's basically marked mRNA itself versus your viral RNA.
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And if you have methyl group on the second nucleotide, that's cap 2.
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So cap 2 is also found in 50% of mRNAs.
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The function of cap 2 is still not very clear.
3:47
In mRNA manufacturing, people often use cap 0, that's kind of older generation.
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And now cap one. Cap 2 is not used because the incorporation efficiency is still quite low.
4:02
So cap analogue is not really new.
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It first appeared in literature in 1975 and in 1984.
4:14
In this paper, you actually see this GpppAG cap analogue.
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There's one big paper in 2009 by Ishigawa.
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In that paper, he discussed not only the synthesis of this molecule and also using those trinucleotide Cap analogue to make a mRNA for basic Firefly luciferase.
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It's because of this paper and some other evidence the TriLink pattern was re-examined the last year.
4:53
It's our belief that this molecule, the Cap GpppAG is not covered by the pattern anymore.
5:04
But we didn't know that when we set out to discover novel cap analogues, right?
5:09
We spent lots of time effort to discover some novel cap molecules.
5:16
One reason is to get around the third party IP.
5:21
The other reason is to have a better efficiency.
5:25
So if you look at your cap, this is your first generation cap analogue, it's called ARCA, it's anti reverse capping analogue.
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So basically you have a vessel group here to prevent the, you know, the chain extension from the wrong side.
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So those basically 2 Gs hooked by head to head GPPG.
5:47
And then this is the clean cap training screen cap.
5:55
This version has this sort of ARCA similar structure over here to prevent chain extension on the wrong direction.
6:04
So this cap analogue is actually in the Pfizer COVID vaccine.
6:11
And there's another popular cap.
6:14
It's the methyl group on the M6 position of the A.
6:18
So from now on, all the caps are basically discovered by us.
6:24
So this one, there's no bond over here.
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So it's like an open ring.
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We call it UNA.
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And this one has a double bond over here.
6:32
That's ENE.
6:34
And this one is, you're all familiar with that.
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It's your old friend LNA.
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And then this is like a hexagon instead of Pentagon.
6:43
And here's that.
6:43
You're Morpholino.
6:48
So how did we come up with all those cap analogues?
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We started with AI assisted structure design.
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So those are building blocks.
6:56
You can modify that.
6:58
So once we have the idea, we did a molecular docking simulation, basically stick the molecule in eIF4E to see which one give you highest binding.
7:09
From that we then look at the synthesis.
7:12
So if the molecule is very difficult to synthesise, there's no commercial value, right?
7:17
And once we have a candidate, we synthesise those cap analogues and then we did a whole suite of assays and to come up with a candidate and then we repeat the cycle.
7:30
So basically design, test, resolve and learn.
7:36
So first we look at the IVP performance.
7:40
We use clean cap as a benchmark and then we put them the cap analogues.
7:48
This is our version of the GpppAG and those are the three new cap analogues.
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We put them in 20 microliter IVT reaction.
7:57
We look at the yield, the very similar integrity, capping efficiency and double strand RNA.
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We saw, OK, those are good cap analogues and then we look at how resistant are they to decapping enzyme.
8:14
So we use different cap analogue to make mRNA.
8:19
So I think those are GFP.
8:21
And then we incubate the capped mRNA with different analogues with a decapping enzyme at 37° C for 30 minutes.
8:33
And then we look at how much mRNA remains capped.
8:39
So if you look at your benchmark, they're about 20%.
8:43
And we're really thrilled by this.
8:46
If you use UGA, you still have like 80% capping efficiency.
8:51
So if you remember from previous slide, it started like 98%.
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The ENE is marginally better.
8:59
We're really surprised by this because in literature people report LNA can be more resistant to decapping.
9:09
And then we did some study in cells and animals.
9:13
I'm really impressed with this because I work for different like two companies and most company can't even do cell based assays.
9:23
They not only can do cell based assays, they actually can do animal work.
9:27
So in cells, if you look at it GFP expression.
9:32
So this is your benchmark.
9:33
You see this one seem to give you a higher expression, right?
9:37
And then in cells, I mean, animals, we inject the luciferase, right?
9:44
We're really impressed with that.
9:46
So if you look at your benchmark at 48 hours, you don't have much luciferase left, but this one actually give you a longer persistent expression.
9:56
So we figured there got to be opportunity applications like this.
10:01
You don't need very high expression to start with, but you need your protein to last longer time.
10:10
And then we study the biosafety.
10:13
So we use your control that GpppAG cap analogue and also the UNA we make mRNA basically luciferase we inject into mice, we inject 50 microgram, we also inject saline and just cap analogue along not our RNA, right.
10:39
And then we measure the animal weight.
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They track really well same as saline.
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And then we did blood chemistry.
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They all look very similar.
10:48
We did tissue biopsy of liver, kidney, lung, spleen.
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The tissue stain also looks very similar.
10:57
So those are cap analogues.
10:59
We also have like over 150 different modified NTPs including N1-methyl-pseudo-U, pseudo-U and di-label UTPs.
11:11
You want to track where your mRNA goes in cells or in animals.
11:16
And not only that, we also have some new NTPs.
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This is like you hear people talking about like phosphothioate.
11:25
This will give you phosphothioate mRNA.
11:30
So we heard talk about that the phosphothioate is more resistant to nuclease, right?
11:41
There's also a paper, I think two years ago they discovered by incorporating the phosphothioate on the 5’ UTR.
11:54
It also helped to improve the protein synthesis by accelerating translation initiation.
12:01
So basically we made this because some.
12:11
So there's some customers say we want this.
12:14
So we're actually in the middle of optimising conditions because you can't, you know, substitute all your NTPs with this molecule.
12:24
So we're in the process of optimising the IDT condition.
12:31
So this is comparing our UTPs with a benchmark.
12:38
So they're very similar.
12:40
And the main story is basically this right by using MMS or pseudouridine and also pseudouridine you get higher GFP expression than wild type.
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So we come up with novel cap analogues and also nucleotides.
12:59
Our goal is really to support the clinical and commercial manufacturing of mRNA.
13:05
So we build the scale and we also support that with the Type 2 DMF filing.
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We did that last year.
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So if you use our raw material making your mRNA you file IND, you can site our DMF number rather than write the whole section.
13:29
So that's our GMP manufacturing facility.
13:32
We have 20,000 square feet GMP with multiple clean room for different process.
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We have like for chemical synthesis or prep, Chrome prep, large scale prep for purification.
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We used animal free starting material.
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We have strict burden control and we basically follow ICH Q7 guideline guidelines for GMP manufacturing.
14:04
So set that manufacturing.
14:06
The actual manufacturing is only half of the GMP, the other half are documentation and control.
14:14
So we have ISO9001 certificate, we have over 30 people in QC and QA department and our documentation are compliant with GMP requirement.
14:34
We are actually, we have been audited by clients and we also in addition to the control I mentioned before are all our analytical equipment are data integrity compliant.
14:54
I work for two different CDMOs making mRNA.
14:57
So I understand there's some areas that maybe people have gaps.
15:03
1 area is the analytics.
15:06
And so this is from USP.
15:08
So if you want to make mRNA DS to release, you have to run all those assays, right?
15:14
One challenging assay is capping efficiency because you need to design cleavage probe to cut the 5’ end off.
15:24
If you don't do that, if your mRNA is like one KB, you can't differentiate one nucleotide, right?
15:30
So that's why you have to design cleavage probe and then you run LC-MS and those are expensive equipment.
15:39
And so to help customers get around this, we can design Cleavage probe for you.
15:47
So to end up enable you to run your own assay, we can develop the method tech transfer the method to you or we can just run the method for you.
15:58
We also make paired capped and uncapped mRNA standard.
16:04
To my knowledge, we're the only company who does that for PolyA.
16:09
We also do method development transfer for double strand RNA.
16:15
We actually offer ELISA detection kit.
16:19
They're in strips of there.
16:21
It's kind of 96 well plate, but they come in strips of eight, right.
16:26
So you have a capture anybody on the bottom, you added a sample detection and then you measure a double stranded RNA.
16:33
We also have control double stranded RNA standard not only the one with wild type U, but also with N1-methyl-pseudoU and pseudo uridine.
16:47
And we have different kits for detecting residue enzyme from the IVP reaction.
16:55
So if you move forward, so the cap we make analogue, they're pretty pure.
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They're like 90%, 98% pure.
17:03
However, you do have impurities once you move to the later phase in your clinical journey, you will need to validate your impurity assay, right?
17:16
So for that, we actually synthesise the different cap impurity standards we supply to customers.
17:24
So this is actually the capped and uncapped standard I was talking about.
17:29
So this one is basically 22 nucleotide mRNA plus the G, so that's why it's 23 and this one is 22 nucleotides added with 5’ PPP.
17:43
So the people synthesis oligo, they know the one the 5’ and it's not PPP.
17:49
So we have to add that to them.
17:51
So we have the capped versus uncapped standard, we mix them 25%, 50%, 75% and then we check.
18:01
They correlate pretty well.
18:03
So this is a tool for people to qualify, validate their capping assay.
18:09
A few words about the company.
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I like to tell people I've never worked for a Chinese company before.
18:15
This is the first Chinese company I've ever worked for, but I think there is.
18:19
Founded by a group of passionate nucleic acid chemists with submission to democratise mRNA vaccine, right?
18:28
The company is five years old, and they have over 100 patent applications, including the first cap analogue patent in China.
18:40
We have raised 400 plus yuan in funding.
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We're raising more.
18:47
The company has 400 people, 60% in R&D.
18:51
So that's what attract me to this company.
18:54
Our products also used by 200 people in involved with vaccine manufacturing.
19:04
So in addition to mRNA, we also supply phosphoramidite for making guide RNA, or siRNA, or oligonucleotide.
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We also have a small group making IVD.
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So I hope by now you get the impression that we are raw material suppliers.
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We want to help people with that.
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But along the way we recognise that not everybody is all up here, right.
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There's some gaps in the skills in different areas.
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We're here.
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We're here to help the customers to fill those gaps so that you get to the clinical stage faster and use our raw materials.
19:48
So for sorry.
19:49
So for example, we are we even offer sequence design.
19:52
We have our own algorithm.
19:55
You just need to give us amino acid sequence.
19:58
We'll give you a construct, we give you 2 to 3 candidates with UTRs and codon optimised.
20:06
We can also synthesise mRNA.
20:10
For you, if you want to do your own study or do encapsulation study for LNP we do encapsulation service.
20:18
We also have our own ionisable lipid.
20:23
They come in a 96 well plate so you can take it, rehydrate, add your mRNA and screen.
20:31
Which one give you best performance.
20:34
We do PD work for IVP.
20:38
So if you need service there, we can develop the process, take transfer to you and then eventually hopefully you get to the GMP manufacturing, use our material.
20:50
And then if you file IND, we also provide IND filing support.
20:55
So with that, I'm ready for a question.
20:58
Thank you.