Scientist Stories: Annabelle Biscans, Designing siRNAs for Improving their Therapeutic Applications

Welcome everyone to the today's OTS webinar with Dr. Annabel Biskantz, who is working at AstraZeneca at the moment, has recently moved from the core overlap, and she'll be telling us about designing siRNAs. And it should be very interesting because I've seen her talk before. Over to you.

Hopefully. Thank you very much, Petra, for your kind introduction. So I'm really happy to be here online with you to discuss how we can design siRNA to improve siRNA drugs in clinics. So over the past few years, the design of the siRNA have moved from minimally modified and formulated siRNA to formulated-based approaches and to conjugated buzz approach, which are actually the best approach to deliver siRNA to specific tissue. So why the first, why the first siRNA drugs get approved in 2018? This drug consists in a minimally modified siRNA encapsulated in LNP nanoparticles. But as I mentioned, about today The great interest have been to conjugate siRNA to really deliver the siRNA to a specific tissue. And over the last 2 years, already 3 drugs have been approved to treat liver diseases. So I will talk about more how we can design this conjugated base siRNA and how we can achieve robust silencing to develop siRNA in clinic. So, if we take, for example, givosiran, so the second approved ASIN drug to treat acute hepatic porphyria, this drug shows incredible silencing in vivo. And you can see here that after a single injection, the silencing can achieve— can be maintained up to 1 month, and over multiple injections can be maintained even after 6 months. So, this demonstrate that zivosirants, this kind of drug, so this conjugated siRNA drugs, can really achieve an important duration of effect of the compound. So the question here is how we can design the siRNA to achieve this robust duration of effect. And if you look at the, of the design of this Givosirand, we can see, is mainly composed in different parts. So the first one, the first part consists of a fully chemically modified siRNA conjugated to a specific conjugate, here a multivalent GalNAc attached to a pyrrolidine base linker. So we try to demonstrate how we can design the different part of the siRNA to really improve the siRNA efficacy and to develop robust siRNA in clinic. So first of all, when we look at the, uh, sRNA chemical configuration, so as I mentioned, you have really an evolution of the chemical scaffold evolution here. So we, at the beginning, we were working with unmodified sRNA, and after we moved to partially modified sRNA, and now we use, we are using fully chemical sRNAs, stabilized sRNA. So why, why that? So why chemical modification matters? So to answer to that question, as an example here, different siRNA having different chemical modification content have been evaluated. So on the left, you can see a partially modified siRNA having 60% modification, chemical modification, and on the right side a fully chemically stabilized siRNA, meaning that this siRNA is totally chemically modified. Both siRNAs were conjugated to a cholesterol ligand. So after injection of this siRNA and evaluation of this siRNA, while the chemical modification does not prevent least loading or risk activity, it has been observed that having a fully chemically stabilized siRNA is really important to enhanced tissue accumulation compared to a partially modified siRNA. You can see here in red, in the— when you use a fully stabilized siRNA, the tissue after a systemic injection, so liver, kidney, and spleen, we can really see that a large amount of compound accumulates on those tissues compared to the partially modified siRNA. So in order to deliver siRNA to tissues, a fully chemically stabilized siRNA is really essential for system— after systemic delivery. So this is one of the reasons why we need a fully stabilized siRNA, and so why we need to modify siRNA. Another point that I want to make here is that this enhancement of tissue accumulation of the siRNA is not dependent on the conjugate attached to the siRNA, either of the route of administration. For example, in the context of cholesterol conjugates, DHA conjugate, or gamma conjugates, either in IV or subcutaneous injection. This enhancement of accumulation was observed in all of the cases using the fully stabilized siRNA compared to when you use a partially modified siRNA. You, you can see almost no tissue distribution, no tissue exposure at all. So this It's to make the point that we need to stabilize the siRNA to use these conjugated base approaches to deliver efficiently siRNA to tissue. So to stabilize this siRNA, so to do that, we can use chemical modification. And so, and so A lot of chemical modifications have been explored over the past decades, especially explored in the context of antisense oligonucleotides. But, um, so here's a few examples. So you can see you have modification, you can modify the backbone of the siRNA by having a phosphothiuate, for example. You can also modify the 5'-phosphate of the oligonucleotide, or uribose. And you can, for example, substitute your 2' hydroxyl by chemical modification. So in theory, all these modifications can be introduced in siRNA. However, you have to keep in mind that these chemical modifications have been developed and explored in the context of antisense oligonucleotides that do not require risk loading. So some of these modifications may interfere with the risk loading of your siRNA. So some of these modification cannot be used in the context of siRNA. So at the moment in the clinic, uh, 4 modifications have been commonly used. So the phosphotyrate modification for the backbone chemistry, the vinylphosphonate to stabilize the 5' phosphate, and the 2' methyl and 2' fluoro modification to to modify the ribose. So these modifications have been introduced in siRNA in clinic and show that that cannot interfere with the risk loading of the siRNA. However, the points I want to make, uh, take me slow, further, is like the, uh, even if this modification can be used to stabilize siRNA, you have really to be careful when you design your siRNA where where you place this modification within the duplex, and how much of this modification you introduce within the duplex, because it can really impact your activity of your siRNA. As an example, here is a really nice paper from Einay-Lam, which has been published in 2018, and they demonstrate that when you change only one chemical modification in one specific position, you can inhibit your siRNA activity. So here they compare different chemical patterns where they replace a 2' fluoro modification by a 2' O-methyl modification in position 2, 6, or 14 of the antisense, or 11 of the sense strand. And by doing that, they observe that the siRNA is not efficacious anymore. So this demonstrates that having a more sterically demanding modification of specific position is really detrimental for efficacy. So you can use chemical modification, but you are really careful when you design your siRNA to place this modification in a right position. So they also demonstrated in the same paper that having more 2' methyl modification, when you carefully position this modification within the sRNA, you can increase the potency of the sRNA. And here, for example, they, so they evaluate the potency of two different sRNAs, one sRNA having 50% 2' fluoro and one sRNA having only 20% of 2' fluoro. So they increase the O-methyl content of the sRNA, and what they can see by doing that, by increasing this 2' methyl content, they were able to induce a better silencing and a more prolonged effect of the siRNA. So what is important here when you design? So you have to stabilize your siRNA by using chemical modification, but you have to be really careful about the position and the content of the chemical modification. And it seems like having a rich siRNA, having 2'-methyl modification, is more beneficial to develop robust siRNA in clinic. So in addition to the chemical modification content, also a stabilization of your siRNA is really required to achieve this robust silencing in tissue. And to demonstrate that, here is two examples of two different compounds developed by Anylam, so Revuzirant and Rivuzirant. So Rivuzirant have been developed a few years ago, but it has been discontinued because this compound showed an imbalance in mortality between the drug-treated patient and the placebo group. At contrary, Givosiran is an approved drug, like I mentioned previously, to treat acute hepatic porphyria. So both of these two drugs have two prime, or methyl two prime fluoromodifications. So why one was discontinued, and why one was really efficacious and is no use, is not approved? So if we look more closely, if you look at this pattern, we can see like in the ribosirants, this compound doesn't have 5' stabilisation of the strand. In contrary, in givosiran, you can see here in red, they have this phosphothiowate modification, so meaning you stabilise more your duplex. So in this context of no 5' stabilisation and 50% 2' o-methyl modification, rivuzirant was injected at a dose of 25 grams per year per patient. Which is, with seduce, is a really large dose. So this can explain the toxicity observed with this compound. So at contrary, in, in gerbosirans, more so, you have this 5' stabilization of the strand as well as more 2' methyl content, so 55% of 2' methyl. So as I mentioned previously, having a more methyl content in your siRNA allows a better silencing, better directional effect. So this is an improvement of the design. And you can see as well that at the 5' end of the antisense strand, you have a vinylphosphonate to stabilize this 5' phosphate. So why we need a 5' phosphate in the first place? So this 5' phosphate is really necessary on the 5' of the antisense strand to be able to load the sRNA into the RISC. So normally when you use and modify sRNA, the sRNA go inside the cells and the 5' position, so of the antisense strand, get phosphorylated by a kinase inside the cells, and so the sRNA get loading into RISC. However, when you modify, totally modify, your sRNA your strands become a poor substrate to this kinase, and so the phosphorylation of this 5' end is limited. So you limit your RISC loading, you limit the, the mRNA enter to the RISC. So to avoid that, you can introduce chemically a 5' phosphate, but however, when you do that, this 5' phosphate is not stable enough in tissue to be, to be maintained. And so you will lose this 5' phosphate, and so you will decrease your risk, your siRNA loading into the RISC. So a lot of groups, labs, and companies have been working to stabilize this 5' phosphate using analogs. So these analogs is just a few examples here. So you can use, for example, an analog that instead of having bridging oxygen here, you can replace by carbon. You substitute as well in the 5' position with methyl. You can use a phosphothiowate, or you can use this ibonylphosphonate here. And what is, is really interesting with this kind of modification is that they stabilize this 5' end of the antisense strand, but they also enhance the interaction with the ago protein to the RISC. So they enhance the the capability of the siRNA to be loading into RISC. And what has been demonstrated using this, this 5' stabilization is like, for example, here, if you use a vinylphosphonate instead of 5' phosphates, or instead of 5' OH, no phosphate at all, you can increase your activity of your siRNA in vivo. So this shows that having this kind of stabilization is really important to maximize mRNA performance. So having all these kind of stabilizations, so the 5' stabilization in the rosarant at the, of the strand, 55% of 2' methyl, and this is vinylphosphonate, allows to decrease the dose less than 1 gram per year per patient. So only by designing very well the siRNA, so by stabilizing the siRNA, we went from 20 grams per year per patient to 1 gram per year per patient. So it's— the difference is huge. So this shows how much is important to really well design and modify the right— to put the right modification into your siRNA. So the next question here is, okay, so it's really important to stabilize your siRNA, but if you stabilize even further the siRNA, can we improve even further the activity of the siRNA? So what have been done here is, is like, uh, the content of phosphotyrate chemical modification have been, have been increased. So moving from 8 phosphotyrate modifications in total to 13 phosphotyrate modifications. And so the goal is, is to understand the impact of the content of phosphorus-thiocyte modification on siRNA distribution and efficacy. And the goal is to see if we can increase the stability, we can increase more the activity a bit further. So after injection into mice of these two compounds systematically, what you have been observed, that's the phosphothioryl content does not impact much siRNA accumulation in tissue, but it does impact silencing. And it has been demonstrated that having a high phosphothioryl content is detrimental for efficacy. So meaning that it's pretty nice, really good to have chemical modification to stabilize your siRNA to be able to achieve robust silencing in tissue. But if you induce too, too much of one kind of modification, for example, phosphorothioid, you really impact sRNA activity and you will lose some activity. So these results can be due to the fact that phosphorothioid content, we know that it can interact with different proteins, and so having a lot of phosphorothioid in your design can, and can change your interaction with protein inside the cells and can just change your activity of your siRNA. So this is another example that why you have to really be careful when you design your siRNA. When you add your chemical modification, you need to choose which modification, which position, and how much you want to include in your siRNA. Another point I would like also to mention is that, okay, chemical modifications are crucial to be designed in a careful way, but also the structure of the siRNA is really important. So here you have an example of different structures that have been explored with different labs and companies, and you can see that, for example, Annalum used this structure where they have the two nucleotide overhang at the three prime end of antisense strand, but some companies use also overhangs of both ends of the sRNA. Some use blunt compounds, whether some use dicer sRNA having this, this tetraloop into that structure. So I will not go into detail of all the structure, but I will just give one example to show you how the structure is really important when you design your sRNA. So you have to choose the right structure according your conjugate you have and your tissue you target. So here, as an example, 3 different siRNA having 3 different structures have been evaluated. So 3 of them are fully stabilized siRNA having lipids as a conjugate. And the difference between these 2 structures is like having either an overhang, so 5 nucleotide overhang, or 2 nucleotide overhang, or not overhang at all. And so the goal of this study was to see if the structure impacts siRNA tissue distribution and efficacy in tissue after systemic administration. So when we look at the data here, we saw that having an overhang or not an overhang does not impact siRNA accumulation in extrahepatic tissue in the context of lipid-conjugated siRNA. However, it does impact activity, and we have observed that, uh, sRNA having an overhang induce better silencing than blunt sRNA, again in this context. So this shows that, uh, other than the chemical pattern of the sRNAs, the structure of the sRNAs is really important when you design sRNA. So outstanding chemical scaffolds show robust, efficient, and prolonged efficacy due to the right optimization of the chemical composition, the structure of the siRNA, and the content of the, of the chemical composition of the siRNA. However, we can wonder if this chemical stabilized siRNA will be toxic because it's they're not, they're heavy chemically modified. So we can ask, oh, maybe this, this modification can impact the toxicity, and we can, so we can increase toxicity in tissue. So I will talk about 3 points here. So we can worry like the 2'-fluoromodification induce toxicity. Indeed, like few years ago, Ionis published some papers with a demonstration demonstrated that 2' furrow modification can be toxic in the context of the antisense oligonucleotide. Whether this kind of toxicity induced for— induced due to the 2' furrow has never been demonstrated in the context of double-stranded siRNA. So the reason that is that because having a chemically stabilized, uh, siRNA using this kind of, uh, this kind of compound, the dose you use, uh, to treat patients is really low. So the risk to see toxicity due to chemical modification is really limited. But with that said, uh, we have to be careful to, to see if this kind of modification impacts, I mean, induce some toxicity at long term. So we don't know that yet, so it's maybe something to look, to watch for in the future. Another effect that can induce toxicity is off-target effects. So when you use the siRNA compounds, you can target your gene of interest, but also it's possible that you target another gene, so you reduce the expression of the protein, so you induce toxicity. So to avoid these off-target effects, we have tools, we have bioinformatics tools. So when you design your siRNA, you sequence, you make sure that your sequence that you selected is not, it will not interact with another genome in your, in your cell of interest. We have also, uh, we can also use chemical modification to reduce this off-target effect. And Ann Alam published a beautiful paper demonstrating that if you introduce a specific chemical modification at position 7 of your antisense strand, such as a GNA modification, you can destabilize the pairing in the seed region. And so you, you decrease your off-target effect. So this is really something, of course, always when you design an siRNA, you have to be careful of this off-target effect because it's the most common sign, the most common, yeah, sign of toxicity. So you have to watch for that, but we have tools to decrease this. Another point I want to make here is we have seen that using this chemically stabilized siRNA, we can induce a directional FFA up to 6 months, even 1 year after injection. So this durability of your, of the compound have been investigated by Annayalam, and they published a beautiful paper in 2020 that showed that in the context of GalNAc-sRNA conjugate, this sRNA can accumulate into an acidic intracellular compartments and act like a depot. To, to really be able to load the siRNA to RISC and to help and to have this long duration of effect. So we can have a concern here, maybe because we know if the RISC is content— if you continuously load your RISC, you can interfere with mRNA homeostasis. However, it's never been shown in the context of siRNA, and the risk of that is really low because when you load your RISC using this GalNAc siRNA, you load less than 5% of your RISC complex available in the cells. So it's unlikely that the toxicity due to this depot in the cells will be observed. So, uh, so for now I would say the chemical stabilized siRNA developed in clinic looks pretty safe. Okay, so, uh, we have, I'm seeing that, uh, fully chemically modified siRNA is really important to design, uh, to increase, uh, efficacy of your siRNA tissue. But what about also the conjugates? It's another part of the siRNA and is as important of the siRNA chemical configuration. So can we design this conjugate to increase the siRNA activity? So we have seen like in gerosiran and in the other drugs approved so far, the GalNAc, the multivalent GalNAc conjugate have been used. And this GalNAc chemistry has really revolutionized the field for liver disease, to treat liver diseases. It's incredible how it's really efficient to deliver an RNA to the liver. However, it's not one way to conjugate, to conjugate, to put the GalNAc attached to the siRNA. It's a multiple way to attach GalNAc, and you can have a multivalent GalNAc, cluster GalNAc. You can have also monomeric GalNAc in series, or you can have different number of GalNAc at different position, 5' end of the sense strand, 3' end of the sense strand, etc. So a lot of labs and companies are exploring this design of the GalNAc to see if we can really improve even better the, the siRNA activity in liver. So here is What is some few examples? I will not go through all of them because it's a lot in the literature, but again, I will show an example. So this data is from Silence Therapeutics. They published a nice paper last year in 2020, and they— what they do in the paper, they screen different GalNAc-conjugated siRNA where they change the position of the GalNAc and the number of the GalNAc moieties attached to the siRNA, and they demonstrate that it does matter. Like, you can change the position, you can affect the efficacy as well as the numbers. So this is an example to show that even if GalNAc is really, is really nice to deliver siRNA to liver, we can still optimize this to even achieve a better silencing in liver. So now what about the other ligands? Because GalNAc can be just used to deliver siRNA to liver only. So you have been a lot of efforts from academics, lab, company to try to discover different other ligands than GalNAc that targets different tissue. So here's some example. You can use peptide conjugate siRNA. So AstraZeneca in collaboration with Ionis show beautiful data when they the GLP-1 peptide to deliver specific catecholamine nucleotide into pancreatic cells. Santorin conjugate have been also developed by Arrow Biotherapeutics, and they show that the Santorin conjugate can really bind tightly with a specific protein in different cell types. Antibody conjugated also have been developed to deliver siRNA to muscle. Avidi Biosciences GCT Biosciences and Dyne Therapeutics show really nice data about that. So I really recommend to watch their presentation on that. And a lot of papers also have been published using ATAMER conjugates, where they show really promising data to use this strategy to deliver oligonucleotides to a specific cell type and tissue. So other than this targeting motif, you have also lipophilic conjugates to be able to deliver functional siRNA into extrahepatic tissue. So I will talk a little bit more in detail of this lipophilic conjugate because it was my main postdoctoral project in Anastasia Vorova's lab. So what we have done to understand if we can design in a particular way the lipids to really achieve functional delivery in astrohepatic tissue here. So we have designed different lipid conjugates with distinct physicochemical properties. So conjugation of different classes of conjugates: fatty acids, sterols, vitamins, incorporation of polar groups such as phosphocholine group, or modulation of the valency of the conjugates. So have been explored to understand the relationship between the structure of the conjugate and the PK/PD property of the So, uh, so these different lipid conjugates have been attached to the fully stabilized, uh, siRNA, and these have been injected into mice via a single subcutaneous injection. And after 2 days post-injection, so spatial distribution and antisense strand quantification in 15 tissues have been performed, and later on some silent significancy have been done in order to see if we can deliver efficiently these conjugates in several tissues. So, first of all, when we look at the distribution of the different lipid conjugated siRNA in liver and kidney, what we observe is that by only changing the chemical structure of the conjugates, we can alter the liver kidney distribution. And we observe a correlation between the hydrophobicity of the conjugate and the liver kidney distribution. Less hydrophobic conjugate, as hydrophobic compound accumulates more in kidney than liver, and hydrophobic compound accumulates more in liver than kidney. Though this difference in liver-kinney distribution can be explained in part by the capability of the siRNA to bind spontaneously with different lipoprotein in serum, In general, uh, hydrophobic compounds bind with LDL and less hydrophobic compounds bind with HDL. So explaining why tissue, for example, rich in LDL receptors such as liver tend to accumulate more hydrophobic, um, siRNA such as cholesterol siRNA, for example. So this was really interesting to see that by only changing the chemical structure of the conjugates, the conjugate can be designed and engineered to achieve predictable partitioning into lipid transport pathway, and liver kidney distribution and clearance kinetics can be predicted. So while the, the nature of the conjugate, the hydrophobicity of the conjugate, drives the overall distribution in kidney and liver, we observe a really interesting thing is like the structure of the conjugate impacts the cell distribution within the tissue. Here, for example, in kidney, if we look at EPA conjugated sRNA and PCAPA conjugated sRNA, we can see that these two conjugated sRNAs accumulate in all— to the stem extend to the kidney, but Using ePSRNA, we can deliver siRNA to glomerulus cells, where when we use PCAPA, we don't. So by only having this phosphocholine polar group in the conjugates, we inhibit this distribution in this specific, specific cell type. So this is really to show you that you can really design your conjugate to really tune your distribution within the tissue. The same was observed in the liver. So cholesterol PCTS and DC conjugated siRNA has an overall tissue distribution in liver, but they have different cell distribution profile. Cholesterol accumulates everywhere, mostly in hepatocyte cells, but DC sRNA accumulate only in Kupffer cells and endothelial cells. So we look also at other tissues than liver and kidney. We look at extrahepatic, extrarenal tissue, and we have demonstrated that also we can deliver efficiently conjugated siRNA in different tissue, heart, muscle, lung, and fat, for example, we can reach significant silencing on those tissues. So again, it's used to make the ponsites by engineering and designing your conjugates. So having saturated or unsaturated fatty acids, having a phosphocholine group, so a poly group, or changing the balancing of the conjugate, you can modulate your distribution of your siRNA tissue. To be able to achieve functional silencing in this, in those tissues. So we have seen that both the, the, the siRNA can be engineered, can be designed to maximize siRNA efficacy, as well as the conjugates. But what about the linker between the conjugate and the siRNA? Can be also designed in a certain way to improve siRNA activity? So in the context of GalNAc, in the Gevozerant here, a specific linker has been used, the pyrrolidine-pyrazolide linker. And so we can ask if it's the best linker to improve siRNA, I mean, to achieve potent silencing. So what has been published 2 years ago, 3 years ago now, is like they evaluated different linker chemistry using this GalNAc conjugate, and they use two different approaches. The cluster-based approach of the GalNAc, that means the GalNAc, it just have this multivalency. So these three GalNAc attached to some linker, and this attached to the siRNA. Or they also use a monomer approach where they— the GalNAc have been attached in series along the RNA. And they studied different linkers using these two different approaches. So the anilam linker, the pyrrolidine-based linker, and a linker having a TAG spacer. And for the monomer approach, they use either a phosphate, phosphodiester linker, or a TAG spacer as well. When these different compounds have been injected into mice via systemic administration, what they observed first is that by changing the chemical structure of the linker, you using this cluster GalNAc, it doesn't impact much the activity of the siRNA. At high dose, you can see between the red and the light blue, a slightly, a slightly improvement of your efficacy using the annihilator, let's call it like that. But because the GalNAc is so potent, you don't see a huge gap of improvements of efficacy when you change your linker. They also observe like having this monomer approach, you can decrease the silencing, the activity of the siRNA compared to the cluster approach, especially at high dose if you compare the red and the dark blue here. So this shows that In this example, of course, you can change your linker chemistry and you will impact slightly your, uh, your siRNA activity. But because the GalNAc is so potent, you will not see maybe too much change when you change the linker, the chemistry of your linker in the GalNAc context. But what about in lipid-conjugated siRNA? Because the silencing is not as good, uh, as the GalNAc. So maybe here we will see a more like impact of the chemistry of the linker of siRNA activity. And yes, it is the case. So when two different siRNA lipid siRNA have been evaluated into mice via sub-q injection, they compare here a stable linker and a cleavable linker between so the conjugates, the lipids and the siRNA, and they have been demonstrated that having a cleavable linker is really beneficial for siRNA activity. So this means that the linker can really be optimized, designed in a way to improve siRNA activity, and it seems like in this context of lipid-conjugated siRNA, a cleavable linker is preferred. So this can be mainly due like to the fact like if you cleave your ligand to your siRNA inside the cells, you can maybe improve your endosomal escape, and so you can improve your siRNA activity. Okay, so to conclude, so you have, like, in the past decades, a lot of effort have been done to really design and optimize each part of the siRNA. So the chemical composition, the chemical content, the linker chemistry, and the conjugate to really achieve robust silencing in tissue. There is a lot of room of improvement here, of course, it's not perfect. We can still optimize even more the siRNA, and the first thing I think we need to understand is how this siRNA interact intracellularly with different proteins to really understand which part of the assay we have to really optimize further. So I will stop there. So thank you very much, and I will be happy to take any questions. Okay.

Is this going to be available online later? Yes, it will be. Yes, Annabelle has agreed to make the PDF available. How do you deal with the stereoisomers RPS or SPS? Do you select, purify the stereoisomers with high activity?

So in the context, it's true in the context of antisense oligonucleotides, to have this stereoisomer SPS dramatically change your activity and the stability of your antisense. So in the context of siRNA, it has been tested where the S isomer has been proved to have better stability, but the advantage in the context of siRNA to have this this stereoisomer compound compared to this, the diosomer, the normal PS, does not improve much activity. So in the context of siRNA, I will— I don't think it's necessary to use this stereoisomer. So to do select— to to increase, I mean, you have to be more worked on that, of course, because it's always a balance between stability and activity, right? So if you use one stereoisomer that stabilizes your siRNA but is detrimental for activity, and you use the other isomer it's more active, but it changes stability. It's a little bit tricky, but in the context of siRNA, it's not that important. With iso-oligonucleotide, I would say, I would say yes.

I don't know if I answered. We have another question from Fabrice Leclercq. How many phosphosiloate modifications can be incorporated without any significant toxicity in terms of numbers or ratio modified and unmodified?

So in terms of toxicity, I don't know. In terms of efficacy, as I mentioned, the optimal scaffold is to have like these two phosphotyrate at each end, at the 5' end of the antisense strand. So to achieve the efficacy, we need to really understand which protein binding interactions, the PS content in the context of SRI RNA we have, because we know in the context of antisense oligonucleotide but not in context of siRNA.

Um, Michaela Denti would like to know the data on toxicity of 2'-fluorometabolites. I think you already referred to the Janus paper from Al-Nalam on that, so I think that contains all of their data. Any known off-target effect associated with the 5' phosphate modifications used for stabilization?

So, uh, yes, of course, when you stabilize your 5' phosphates, so the goal is to load into RISC-U sRNA, but you want to load your antisense strand. So, uh, you have been, uh, some work, uh, when you stabilize this 5' phosphate, you have, you have to introduce a chemical modification. That do not interfere with AGO binding, AGO2 binding. If you do that, if you, for example, instead of even if phosphonate, you introduce a Z-phosphonate, so other configuration, you will not, you will not block your antisense strand. So you can have off-target effect but only your sense strand.

Okay, where is GalNAc located, at 3' terminus of sense strand? Instead of the 5' terminus?

So as I mentioned, it's a different way to conjugate the GalNAc to sRNA. So mostly on the sense strand, so 3'-5', and the 3' end of the antisense strand. So what you want to avoid is the 5' of your antisense strand, because you want this strand phosphorylated to be able to load the strand into the RISC. But, uh, so depends of the depending on lab, so you have different positions where you can conjugate the GalNAc.

And then, I guess, in most of your works from Professor Anastasia Kroer's lab, the length of siRNA is 20-20. Why not 21?

Uh, yes, I mean, at my lab, you 23, we use 20. Uh, you have to be between, so you don't want You don't want something too short because if you're too short, you will have a lot of unspecific effects. So you want to be between 19 and 25. If it's too long, you will have a lot of off-target effect and it will recognize as well as your immune response will be stimulated. So it will, you will have toxicity. So we have to be on this 19-25 length, so 20 is on the range, you can use 23 if you want, it's not like set rule.

Yi Yin would like to know that if you have one structure showed, uh, give us a run with VP modification but the other showed no VP, can you confirm if VP modification has been used in the approved siRNA drug give us Yes, yeah, I go for that. How are siRNAs formulated in general?

Formulated, can the person like can specify, is mean like in nanoparticles or injection in mice? So if you just inject without any nanoparticle, you just dilute your siRNA PBS, for example, at certain concentration, and you inject directly the mice with your solution. So you don't need to formulate it because you have, you have this, the siRNA is chemically modified, so it's stable. It will not be degraded by enzyme, and you have the conjugate that help to improve the half-time of the distribution of the tissue of the conjugate. So you don't need any formulated agent.

What are these new tools for off-targets you mentioned?

So for the off-targets, I mentioned the bioinformatics tools. So this is more BLAST tools. So you can have— so basically you have your sequence and you can identify if your sequence sequence target other gene in your genome, your 5' UTR in your cells. So this can be one tool. The other tool I mentioned here is to introduce chemical modification in specific position, and my lab showed that having this destabilization of this, of the pairing in the seed region of the 7th position of the antisense strand, allowed to decrease this, uh, the off-target effect. So these are the two tools you can use to decrease that.

Yeah, there's also a reason, very recent paper, I'll post a link that is about one of those tools that is free, made freely available.

Thanks.

Is there any software for asymmetric siRNA design?

Available online, not yet. So when we were using this asymmetric siRNA in the Khorova lab, the lab designed the tools, the software based on— so basically we screen a lot of asymmetric siRNA and we set up some rules and the algorithm has been built on that. So at the moment is not available online, if I'm not mistaken.

Uh, Michaela Denti: I assume most of the rules you have talked about apply for sRNAs delivered through intravenous injection. Is there something different in toxicity, efficacy, efficiency, and so on when injecting into the CNS?

Uh, so, uh, Subcutaneous injection versus IV injection is the same rules. So the difference, you will have not the same pharmacokinetics of your compound. I mean, when you do IV injection, your compound will go through the blood, so it will distribute more quickly in your tissue. So, so this rule can apply for both, I would say. For the CNS, so for the CNS, yes, it's mostly you inject directly in the brain. In terms of toxicity, we don't see any difference. I recommend to read the really nice paper in Biotech Technology that Anastasia published on this bivalent siRNA, or they show like the toxicity using this stabilized, fully stabilized siRNA is not an issue at all, and you can have an efficacy up to 6 months in certain regions of the brain. So I don't think the issue in CNS using the stabilized chemical siRNA.

This is a comment, I guess, by Jan Weiler. Inclisiran does not carry a VP and is working great.

Yes, it is, but yes, I mean, it's a different way to design a siRNA, right? You can put other modifications. People are investigated to have different instead of phosphothioids, no, it's maybe the PS2, or it can be something else. Of course, you have different way to stabilize stabilize the siRNA. So I just mentioned the VP because it's something we know and something can be important, but I think if you find a way to stabilize, another way to stabilize the siRNA, it works as well, it's working as well.

David Slack would like to talk about why GalNA targets liver and companies like Avidity and Dyna are targeting neurological and muscle tissues. What approaches show promise for delivery to solid tumor cancers?

Uh, I think they also show in tumor that antibodies approaches can be used, because this company use antibodies for muscle delivery, and they show really nice data. But if you engineer your antibody to target solid tumor cancer, you can target this cancer as well. So I will suggest maybe to look a little bit more on what IDT is doing on that.

Valentina Funk here. How will this modification influence encapsulation efficiency, efficacy? Uh, you don't encapsulate.

Yeah, you don't exactly, you don't encapsulate because you don't need to If you want to, I mean, because the goal of to have all this modification is not, is not to use a particle, nanoparticle, because you want really avoid, you want to deliver your siRNA in different tissues and liver, for example. So when you encapsulate, it's how to really deliver to siRNA to a pretty specific tissue other than liver and kidney, but I don't see why this chemical modification will impact encapsulation, because they're not something, they're not something like will interact with the membrane. And we'll see, like in patisirans, they have some modification. It's not fully chemically modified, but they have some modification. The sRNA have some modification. Using the LNP, so I don't see any influence on that.

I think the list of questions is just getting longer and longer. We've got a question here about a company that can produce docosahexaenoic acid conjugation and the VP modification. I'm not sure that's an appropriate answer here, because we're not endorsing any of of the companies. Yeah, I would just look at some of the top companies that produce oligonucleotides, and if they don't have it on the website, just please contact them and ask. They're usually able to accommodate if you have the money to pay for it.

Yes, thank you, Petra.

Did you evaluate the administration via inhalation? For example, can you tell about the way RNAs are prepared and administered?

Inhalations, yes, we did some work in inhalation during the COVID time because we were working on COVID's siRNA targeting the mRNA of the, of the virus. So you doesn't need to prepare like another way you do for systemic administration because you just, you just, so depends what you call by inhalation. If it's just like intratracheal administration, you don't need anything. If you want to use like some nebulizer, I'm not an expert at all on this field, so I guess you have to prepare in a way like you have to do some like powder, maybe dry powder of your siRNA, but I'm not sure, I'm not an expert on this field, so I don't know that.

Um, how many siRNAs are in the clinic that do not use a delivery system, and what is the toxicity?

I think that's quite a big question here, a lot A lot in clinic, it's a lot of clinical trials using Calnex RNA. I don't know the number, more than 20, 30, I don't know, I don't know the number. And regarding the toxicity, I mean, for those have been accepted, so have been approved, is all about the FDA or the European Union. So it's 3 of them using the conjugated base strategy, and they show no toxicity at all. So, but the other that are in clinic, maybe it's too soon to, to see if there are some talks associated to that. And yeah, I don't know that.

I would like to say that, well, I, I, Alnylam, um, Seqana, there's quite a few companies who have things in clinic clinical trials, so it's worthwhile to look at their websites and check out the listings that they have of the, what they have in testing. What is the most effective, efficient approach behind conjugation chemistry? Could you please provide examples?

So, so you can conjugate different ways, so you can use solid supports that already have your ligand on it and grow your oligonucleotide on it. So this is one way. You can also use a phosphoramidate strategy or use a phosphoramidate to, to put your ligands on your siRNA. But this has to be— it's a bit tricky because the chemistry of your conjugate have to be really adaptable with the condition when you use, that you use during the synthesis. And the other way is to do post-synthetic reaction. So you can attach your conjugate after you, in solution when you have your siRNA. So you do, you know, you can do click or an HSS-tag strategy to conjugate that. The most efficient approach, I would say I like to use the one they use, uh, function like the conjugate on the CpG, because when you do post-synthesis reaction, it's not total, it's not, it's not perfect. So using this CpG with your ligand on it, you'll be at the end when you purify, you're sure that you have 100% of your siRNA that is conjugated. So, but it's different way. As an example, I mean, all the lipids that I show, like what we did in Anastasia's lab, we use conjugate that we use CpG with the ligand on it. But in the GalNAc, like a lot of companies such as Silence, for example, Therapeutics, they use the monomeric approach where they use the phosphoramidate. So I think it depends on what you like. The most. And yeah, I think it's not like a better or the worst, but you have to make sure— it depends why you want to conjugate, because you have to be sure that your ligand is compatible with the chemistry you want to do. So this is the only thing you have to be careful of.

That, um, I think we need to maybe speed up a little bit because The messages, questions keep piling up. Is it possible that siRNA conjugated with cholesterol may have, may interfere with the LDL system when you systemically administer?

Yes, so using cholesterol conjugated siRNA, this has been the paper 2006 or 2007 in Nature Communications, they show that the cholesterol interferes with LDL lipoprotein. Yes.

Do the lipid siRNA form micelle structures?

Uh, no. So, uh, we check that, and when you have only one lipid, it doesn't. When you change the valency of your lipid, so when you have three, uh, three fatty acids, for example, it do some— you can see some micelle structure. Yes.

Is a cleavable linker benefit observed in siRNA conjugated to anything other than lipids?

So in the literature, so no, because for example for antibodies, avid tissue, they don't have any cleavable linker. In the context of a GalNAc, so depends if it's siRNA or only of antisense, but the siRNA, I mean, analog at this, as this phosphate, so it can, can, it can be cleavable, right? So maybe under gall light is also beneficial. Yeah.

Can you comment a little bit on the administration method for the DIS RNA, the work from the Corral lab?

Uh, yes, so it's administered locally in the, directly in the CNS. Yes, it's locally injection, it's not systemic administration, it doesn't cross the brain barrier, so it's directly injected.

Alexandre Debacca, thank you Annabelle, amazing talk, I fully agree to that. What would you be, what you recommend, what would be your recommended path to develop a fully modified siRNA? Would you start by screening on modified sequences, then screen different fully modified pattern scaffolds on the working sequences, or would you, from the, would you go from the beginning fully modified sRNAs using your preferred fully modified pattern to identify the best?

Yes, so, uh, I will start screening modified sRNA because we have to show like when you use algorithm, sorry, if you use one type that you can use for unmodified sequence and the one you use, we use for modified sequences, they do not correlate at all. So you have to be careful when you design, when you screen, when you use the first screen. I will definitely go with the modification first, including new design.

By siRNA structure, you mean sequence? Do you mean sequence?

No, I mean I mean, the— if we have like, for example, here an asymmetric sRNA, or you have like a blunt, so it's more like the length of the strand is more in that way. The sequence is nothing to do with it.

Yeah, I think we've had that question. Yes, the recording will be available as usual. It is available from the OTS website as usual. Well, in 2-3 days. Okay, what cleavable linker would you recommend for lipid siRNAs?

So what I've been done here is just having like DNA, for example. So it's like what I showed, the result with the cleavable versus non-cleavable lipid, is just a DNA linker. So I will really recommend that. It's cheap, easy to, easy to synthesize. So I will use that.

Any recommendation for macrophage delivery? I think I darkly remember an answer delivery on that, remember the paper.

Yes, I think I'm not lying, I'm not lying, so I have the paper on that. It's just I've been published 2020. I think they use, uh, I think they use as well GalNAc. Uh, no, I'm not sure. I'm not sure about that. I don't want Facebook. Um, sorry, I don't know.

How is the future for oligonucleotides, siRNAs? What's your opinion?

It's, uh, we have seen over the past 2 years, we have 3 drugs, uh, get approved in clinic and way more are in the path, like in the pipeline, is like where it will get approved. So I think the future is, is really promising and it's really great. It will expand and we will use this technology for multiple, uh, these to treat multiple diseases in multiple extrahepatic tissues. So I'm really optimistic about this technology.

Yeah, if you, if you want to find more about oligonucleotides and siRNAs, then please check out the OTS website. I'll post the link in a moment. Is position important for phosphonates, for example the vinylphosphonate, in sense or antisense strand of the sRNA?

Uh, yes, it is important. I think really soon it will be a paper published from Anastasia group, or they place different phosphonates linkage in different, in a different position of the siRNA. So I will invite you to read that paper, it will be out soon.

Do you know if those lipid siRNA conjugates have a mycelium? No, we've had this question. Why do you modify the sense strand? Which strand loads into RISC complex? That's so basic.

So yes, so it's an antisense strand on the RISC, we will load it. If you RISC, I mean, both can be loaded, right, it's two strands. But if you want to target your, like, on-target effects, you have to load the antisense strands that you designed for your targets. Yes.

Toxicity of the phosphonate modification of the sRNA?

So depends again, depends how many phosphotylenes where you put the phosphonates on your siRNA. So depends on your, of your structure. It can be toxic at different position, I mean, or if you put too much. Yes, depends, I guess.

Have you ever observed upregulation then rather than knockdown when using certain modifications?

Uh, no, never. Uh, no, I mean, no, I mean, if you design really well, uh, when you do like the bioinformatics, when you design your sequence, yeah, it should knock down and not upregulate. So I never upset that, but I guess it could be possible.

Yeah, if you, if you look at one, uh, if you look at small activating RNAs, particular Mina Technologies here in London do this. It's slightly different mechanism, so, but it's sort of very similar structure to start off with of the RNA. Yeah, are there any examples of LNA modification siRNAs in clinical trials? Not to my knowledge.

No, not to my knowledge as well.

Yeah, uh, Christopher Brown says that VP is not being used in Give Us a Run.

Oh, okay, sorry about that. Okay, I thought it was. Thanks.

2' modifications are base or position dependent? Um, 2' modification refers to the 2' sugar.

Yes, exactly. So by 2' modification, it's just you replace your 2' hydroxyl with the modification. So Yeah, I'm not sure.

Okay, would you have any bioconjugate recommendations for delivery to skeletal muscle?

Uh, so yes, uh, I mean, uh, in the lab we demonstrate, we publish a paper this year that's using fatty acids, uh, DCA, you can deliver functional siRNA to skeletal muscles, so I invite you to read that.

I would— oh, sorry, someone commented GLUT4 ligands if they exist as a recommendation. What's the percentage of lipid conjugates that will go to other tissues not to liver and kidneys with respect to GalNAc conjugates? Is the dose you need to get an appreciable silencing much higher?

Yes, uh, this is a good point. When you use, uh, lipid conjugates. So depends of your lipid, so depends where you distribute your tissue, as your siRNA, sorry, you can reach like 5% of your injecting dose in, for example, using this TCA conjugate that I mentioned before for muscle, you can reach 5% of the injecting dose on, on this tissue, on muscle tissue. And to do that, the dose is much more higher. You need like 10 mg/kg, 20 mg/kg. So of course, GalNAc is way more efficacious to deliver assignate to liver. I mean, it's really specific because using a lipid is not, is not a targeting moiety you're using.

Okay, I think we've made it to the last question. Between hydrogelates and nanoparticles for oligonucleotides, is there a competition between conjugates and nanoparticles for oligonucleotides delivery? Probably each technology is most suitable for a specific purpose.

Uh, yes, I mean, uh, depends, I guess, your tissue you want to target. If you want to target, uh, because having your nanoparticle, you can engineering, I guess, to deliver different tissue, but you have— you increase the size of your, of your drug. So you cannot treat any, any tissue. So I agree, can be, it can be complementary here depending on the tissue you want to target.

Yeah, but also need to point out, if you use a nanoparticle, then that has to be produced and tested, and the whole formulation has to go through the whole approval process, so it makes it a bit more expensive and more complicated. Okay, I think we've reached the end of the questions, and there's lots of thank-yous from, from all the participants.

Thank you, thank you all.

Yeah, I would like to point out that yes, the recording will be available soon from the website. I've posted the link on the chat, and I would like to point out that we have the next presentation with Professor Stanley Crook, and he'll tell us all about the N. Laurum Foundation, about n=1 ASO trials, and how they're helping patients with that. And that would be on the 9th of February. And again, you can find the details on the OTIS website. Thank you very much, Annabel.

Thank you so much. Yeah, it was a great pleasure.

Thank you very much.

Thank you. Bye-bye.

Bye-bye.