Hitting the Target in Oligonucleotide Delivery

1:01 
Today's session, we'll be focusing on the topic of overcoming the latest, overcoming the latest trends and challenges in targeted oligonucleotide delivery. 

 
1:11 
Let me now introduce you to our session group leaders. 

 
1:14 
Today we have David Evans, who is Head of Drug Discovery and Collaboration at Sirnaomics and his panellist collaborator today will be Soren Otterson, who is Vice President of Research at Sixfold Bio. 

 
1:33 
The session will begin with a short presentation from David followed by a panel discussion. 

 
1:37 
So if you are welcome to use the chat function to post any questions or comments that you have throughout the session. 

 
1:45 
But without any further ado, I'll hand over to David who has a short presentation for us. 

 
1:51 
Thank you very much, Tom. 

 
2:08 
Yep, this slide deck. 

 
2:14 
Thanks for the introductions. 

 
2:18 
This is an interesting topic for everybody involved in oligo nucleotide discovery and their therapeutic use because targeted delivery is showing great promise at the moment to try and reach different tissues and cells in the body. 

 
2:35 
I'm going to start by introducing the success of GalNAc delivery. 

 
2:41 
So we had chemically modified siRNA, which really allowed the development of this concept. 

 
2:53 
So chemically modified siRNA has the use and functionality of siRNA with improved PK and PD, but Triantennary GalNAc attached to the modified siRNA allows binding to the ASGPR that's present on hepatocytes exclusively with very high affinity. 

 
3:13 
The ASGPR is cycled rapidly and enters the cell and GalNAc conjugated siRNA enters the cell by the endosome and is released inside the cell to the cytoplasm where it can silence its target for protracted times, up to several months. 

 
3:29 
It was initially developed by Alnylam and subsequently mimicked by others by attaching GalNAc to novel constructs against additional targets including Arrowheads, Dicerna, Silence and Sirnaomics. 

 
3:38 

Alnylam products are Leqvio, OXLUMO, AMVUTTRA, and Givalaari which are all approved for clinical use. 

 
3:50 
But one of the problems is that GalNAc can induce futile cycling, returning to the cell surface without delivering its payload. 

 
3:57 
So efficiency of GalNAc isn't great and there have been estimates as well as 1% of the siRNA being delivered at any point in time. 

 
4:07 
However, the affinity is so strong that it stays bound to the receptor and may enter when taken up next time. 

 
4:18 
So additional modes of discovery and delivery have been developed and this is a brief introduction to the rest of my talk. 

 
4:25 
So the evidence of success provided by GalNAc provides a search for other tissue specific receptors, to obtain specificity, delivery to specific organs, EG the brain using C-16 modified siRNA delivered intrathecally or IntraCerebroVentricularly. Silences amyloid precursor protein. 

 
4:51 
Also C-16 delivers to the lungs by intranasal delivery and they produce those dose-dependent silencing of SOD1 for approximately 2 months. 

 
5:00 
I'll discuss these later. 

 
5:02 
Delivery to tumours with distinct sites in the body is targeted using up regulated receptors that are specifically on tumour cells, and we can deliver siRNA, PMO and ASO to tissues using antibodies. 

 
5:19 
So delivery to the brain has been the subject of much study. 

 
5:22 
In 2007, Kumar et al published a paper showing the ability of the rabies virus glycoprotein to obtain CNS delivery of unmodified siRNA. 

 
5:31 
In it 29 amino acid peptide nACHR on neuronal cells and IV administration demonstrated transvacuolar delivery and silencing in the brain of Superoxide Dismutase. 

 
5:45 
Silencing was demonstrated with a maximum of two days but again this was using non modified siRNA. 

 
5:53 
This was in 2007. 

 
5:55 
In 2022 Kurt Brown and others from Alnylam demonstrated delivery of siRNA to the brain via intrathecal or IntraCerebroVentricularl injection. 

 
6:08 
The use of C-16 to primal hexadecyl coupled to a modified siRNA and showed a much longer duration of effect, up to three months. 

 
6:18 
In the CNS. 

 
6:19 
They showed silencing the siRNA target amyloid precursor protein and they also showed delivery of C-16 siRNA to the lungs in the eye by a local delivery. 

 
6:31 
Antibodies have been used for delivering to other tissues and this example is from Malecova. 

 
6:41 
They described using transferring antibody delivery of siRNA, ASO and PMOs to skeletal and cardiac muscle. 

 
6:50 
The siRNA against the SSB gene, small RNA binding exonuclease protection factor LA showed greater than 75% reduction in mice and monkeys. 

 
7:02 
Transferrin antibodies showed tissue specific delivery and skeletal muscle delivery with an EC50 of 0.3 nanomolar for skeletal muscle and four nanomolar to the heart and 24 nanomolar to the liver. 

 
7:20 
siRNAs have also been delivered to the tumours and various methods have been used. 

 
7:26 
Most like most have focused on tumour specific receptors that are up regulated and one of those is an integrin receptor. 

 
7:34 
Those have long been targeted using RGD peptides, EG solid lipid nanoparticles, polypeptide nanoparticles or even mRNA delivery. 

 
7:44 
Targeting ligands can be coupled to the siRNA directly or to nanoparticles containing siRNA. 

 
7:52 
One of the advantages nanoparticles is they can deliver multiple oligos at a time. 

 
7:57 
These can be the same oligo. 

 
7:58 
Or can be against different targets, often synergistic in the disease model. 

 
8:03 
One example of this is polypeptide nanoparticles HKP plus H combined to receptors including NRP1 and deliver the siRNAs against TGF beta and Cox2 to activated endothelial cells and tumour microenvironment where they show activity against syngeneic orthotopic HCC models blocking the ability for the tumour to grow and augment immune checkpoint inhibitors. 

 
8:34 
Additionally, check one srna was designed with gemcitabine attached to the oligo or incorporated in place for site beans and showed potent activity against pancreatic cancer. 

 
8:45 
CHK1 siRNA was designed with gemcitabine mechanism of action so chemically modified as check one siRNA subsequently made was containing gemcitabine at the same locations as the first paper. 

 
9:00 
So potent activity against pancreatic cancer, non-small lung cancer and triple negative breast cancer and reusing antibodies to deliver this construct to tumours in vivo. 

 
9:12 
Other delivery ligands include aptamers, peptides, centyrins, antibodies, alpha-tocopherol, cholesterol. 

 
9:20 
These can be used to deliver oligos, but it's also important to include endosomal release moieties to get release from the endosome because when you get uptake into the cell, the endosome will entrap the product and as it migrates through the cell it becomes a lysosome and that will degrade the siRNA. 

 
9:46 
So in summary, the ligand-mediated delivery has several challenges; specificity and cell activity. 

 
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We need precise targeting to the disease cells while avoiding healthy cells. 

 
9:59 
Ligands must bind specifically to the receptors overexpressed on the target cells, and they need to show stability. 

 
10:06 
Ligand siRNA should remain stable in circulation and avoid rapid aggravation. 

 
10:11 
Balancing stability with efficient cellular uptake is essential. 

 
10:15 
As I mentioned, endosomal escape is a critical aspect of the delivery vehicle and immune response. 

 
10:23 
You must not generate immunogenicity so that the product can progress through CMC and regulatory approval. Off target effects must be minimised and delivery to non-liver tissues. 

 
10:40 
The most successful ligand-mediated siRNA therapies still currently target the liver. 

 
10:45 
So we've developed therapeutics that go to other tissues but its somewhat of an uphill challenge. 

 
10:54 
Thank you. 

 
10:54 
I will now open it up to questions the audience might have had a chance to think of whilst that presentation was going on. 

 
11:08 
Thanks, David. 

 
11:09 
It's interesting that you managed to basically sum up everything the field has been doing in 10 minutes. 

 
11:15 
And obviously the, the amount of work that's gone into everything here is…I mean, there's a whole industry that does this. 

 
11:24 
The one comment I would add to this is obviously what you presented is a lot of work where researchers have had a concept of a target and a receptor and a mechanism of uptake that they sort of understood. 

 
11:41 
And therefore they could, they could build a mechanism to doing, to getting the uptake. 

 
11:47 
Another, of course the GalNAc approach is a textbook example. 

 
11:52 
You have a highly expressed, highly specific high turnover receptor with a clearly defined ligand and we all wish there were more of those and there aren't. 

 
12:03 
And so the other part of it is that you go with everything we're going through here is that we're defining a frustratingly small set of cells that we're actually able to address. 

 
12:16 
And we're still left with lots of tissues and diseases that we can't address either because we can't get into the cell we're interested in, or we can get into it, but it's not specific enough. 

 
12:29 
So we're also getting exposures in cells that we're not interested in targeting. 

 
12:34 
And we're getting stuck a little bit with our own understanding of the cells. 

 
12:42 
And maybe the next step is to stop assuming that we know anything about this and try to find methods where we can, where we can search out a bigger space, an infinite space almost where we're looking at ways of analysing and looking at a novel conjugates. 

 
13:02 
Even if we don't know what is the mechanism, even if we don't even know really what these molecules are, but just testing them out in a system where we can get the information. 

 
13:13 
And you’re working with Sirnaomics, you're working with ligands that you've worked with for years. 

 
13:21 
At Sixfold we're trying to get a get beyond that. 

 
13:23 
We're trying to work with an AI mediate method where we can identify the ligands and moieties in a way that isn't dependent on our understanding of the biology. 

 
13:36 
And hoping that that, and I'm hoping we're, we're demonstrating that that can be done in a way that opens up cell types with specificity that we haven't seen with some of these other methods. 

 
13:54 
And that was just my comment to this is that this is very good. 

 
13:57 
And, and all of this we where we're still working with known moieties, known mechanism of uptake and it still has years and decades of additional work to do to get that to work really well. 

 
14:07 
But beyond that, there are still tissues and cells that we still cannot address simply because we don't have the tools to do so. 

 
14:16 
Oh, I agree. 

 
14:17 
I think we've got to take a smaller bite of the apple each time we try and develop a new therapeutic because not only do you have to generate siRNAs against the targets and proof of targets are valuable in that disease model, but then you have to couple those to something to get them delivered specifically to that tissue or cell so that you don't get specificity. 

 
14:45 
We don't require large concentrations of material to be injected possibly causing toxicity etcetera. 

 
14:53 
And so those are very critical questions and obviously in any disease area and we just touched on several right now, but any disease area you've got the wealth of siRNAs that can be designed, whether any of them are synergistic, you know would be very interesting to know. 

 
15:17 
And then you've got also a plan of targets that you could go after to try and deliver those siRNAs. 

 
15:26 
So it becomes somewhat of a combinatorial problem very quickly and not just the little one, an enormous combinatorial problem. 

 
15:36 
I see there's a question in the chat and this is about delivering ASOs to skeletal muscle. 

 
15:51 
And David, I don't know if you have any comments specifically about skeletal muscle. 

 
16:00 
Well, I think the transferrin antibody demonstrated delivery to the skeletal muscle, this would be a very useful approach to an ASO based therapy for adult-onset skeletal muscle wasting disorder is realistic. 

 
16:20 
I'm not sure, I think probably would be because, but again, I'm not sure of what the morphology of that disease would look like. 

 
16:37 
So if the muscle is wasting, maybe it can't take up the same targeted ligands as normal muscle. 

 
16:51 
So there are two things in this. 

 
16:52 
One thing is that you, have to understand your desired endpoint and one endpoint, the absolute gold standard, of course, is a reversal of disease. 

 
17:01 
But sometimes what you're really just looking for is halting the disease. 

 
17:05 
And the hurdle for getting an ASO into a muscle to halt the disease I would think much lower than trying to reverse the disease. 

 
17:19 
I had another comment and now I forgot it I so I'm going to hand that back. 

 
17:32 
Any other comments Tom? 

 
17:41 
We don't have any comments in the in the chat currently. 

 
17:45 
So if anyone does have a comment, oh, we do have one from Sebastian who says what conjugates can be used for improved endosome escape. 

 
17:58 
Well, there, have been a number of studies looking at different entities for endosomal escape. 

 
18:05 
There's obviously chemical means, which is chloroquine, but as regards conjugates which can be attached to peptide ligands or antibodies or other materials when they get inside the cell can release from the endosome. 

 
18:25 
There's cell penetrating peptides. 

 
18:28 
There are various sequences our sequences histidine, lysine, polypeptide that the nanoparticles formed from and the histidine has a PK about 6 so as the endosome acidifies goes down to PH 5, the histidine protonates and that helps the siRNA escape from the endosome. 

 
18:51 
There are also ligands. 

 
18:56 
Polyarginine has been shown to have effects, but that suffers from possible toxicity and other reagents have been studied as well. 

 
19:11 
So there are a number of different ways that you can improve endosomal escape, and it depends somewhat on the ligands that you're looking at targeting. 

 
19:22 
So for example peptide like RGD delivering to tumour cells, you would probably want to include a short cell penetrating peptide there or something else. 

 
19:39 
Cholesterol has been used to get uptake into the cells and get release. 

 
19:44 
And for lipid nanoparticles, it's the ionic lipids that allow the endosomal escape of oligos. 

 
19:54 
Antisense and even mRNA. 

 
20:00 
Anything to add to that? 

 
20:05 
This is a conversation that's been in the field forever about what happens when you the perturb the endosomal escape and whether there's a benefit of that gives you this nice long duration of action that you see in oligonucleotides whether its ASOs or siRNAs. 

 
20:27 
But I think the desire to be able to modify the rate of endosomal escape is such a value that I think we still need to keep developing that. 

 
20:46 
There's another question these if chloroquine depends on spread on sponge effects, the suspect they will still work as conjugates. 

 
20:55 
An siRNA, I think we've used chloroquine as an adjunct. 

 
21:04 
So when we've used delivery agents to see if they do produce endosomal escape and improve delivery we use chloroquine just as a control to compare against the delivery agent on its own to see how well it behaves. 

 
21:27 
And we can show effects better than chloroquine in our models. 

 
21:39 
There's another question. 

 
21:40 
Do companies work on specific delivery to skin targeting to skin cells? 

 
21:46 
Well, we have an example. 

 
21:49 
So we have STP 705 which is a therapeutic product delivering TGF beta and Cox2 to the skin for treatment of non-Melanoma skin cancer cell carcinoma and basal cell carcinoma during phase one and phase 2-3 studies. 

 
22:10 
Right now, as I mentioned earlier, HKP has a mechanism to activate endothelial cells around such tumours and we delivered TGF beta and Cox2, which then reduce the those means in the vicinity of the tumour. 

 
22:33 
And we believe by reducing TGF beta, we see uptake of T cells into the tumour environment, and we also see silencing our potentiation of antitumor efficacy. 

 
22:56 
Is that a local administration or systemic administration to these tumours? 

 
23:00 
That's a local administration. 

 
23:02 
So that's injected intratumorally or around the vicinity of the tumour. 

 
23:11 
For ASO's angle because that's what I what my background is in. 

 
23:19 
I know that there is a natural distribution to some cells in the skin, but the question also you have to ask the next question, which is what are the cells you're interested in? 

 
23:29 
Because skin is a complex tissue. 

 
23:32 
So you need to know which cells you're looking for. 

 
23:34 
And also by systemic administration, as soon as you're looking at tissues where you get lower exposure, you have to take into account the systemic exposure to the major tissues which is where you're going to see the toxicity in the kidney and the liver. 

 
23:49 
So can you get all, can you get an oligonucleotides to go to the skin? 

 
23:54 
Probably. 

 
23:55 
But you're still going to have to find a way to enrich it either by local administration as David's talking about. 

 
24:02 
But more likely you're going to have to find another way of either avoiding the liver and kidney entirely or find a general uptake mechanism that is appropriate for the skin. 

 
24:15 
And I don't think we have that just on the on the table right now, but it’s definitely something that's, worthwhile because it's just as we were talking about earlier, these are tissues that are not otherwise addressable by this mechanism of action. 

 
24:30 
And maybe the focus ought to be on trying to enable these tissues. 

 
24:38 
I think I think you're right. 

 
24:40 
If you're delivering the product locally, it's much easier to target something like a tumour where you know where it is and you can biopsy it and define the tumour type, other skin diseases which are general psoriatic disease or something along those lines. 

 
25:00 
I can't imagine there would be anything better than a small molecule to approach that. 

 
25:10 
And then you accept the complete distribution throughout the body of this molecule and it has to be tolerable beyond the local tissues. 

 
25:23 
And then of course, you often you deal with skin disease with a topical administration. 

 
25:28 
And I'm not sure that that's amenable for an organ nucleotide. 

 
25:32 
I don't think I've ever seen anyone propose a topical administration of an oligonucleotide. 

 
25:39 
We have topical administration for wound healing. 

 
25:42 
But when you have a wound, you've got easy administration direct to the underneath. 

 
25:48 
So I agree with you. 

 
25:49 
I think it would be very challenging. 

 
25:52 
That actually reminds me of the thing I forgot just before about the muscle disease, which is that when you're going to have muscle wasting or even lung fibrosis or whatever the tissue is, you're going to be looking at a tissue that is altered. 

 
26:06 
So whatever you try to develop for natural healthy tissue may not be amenable for these diseases. 

 
26:14 
Or if you're looking at a muscle wasting disease, you're also stuck in the situation where you can, where you have to take your disease model into account, which is a complexity for our understanding of normal human biology and disease biology. 

 
26:38 
Absolutely. 

 
26:42 
We’ve seen differences even in liver fibrosis uptake of HKP at a normal level where you get distribution everywhere else but hepatocytes. 

 
27:01 
But the fibrotic liver takes up the HKPs probably slightly more so that that's a promising outcome for fibrosis treatments. 

 
27:15 
And so that argument goes where everything, including the skin diseases, if you're looking at any kind of abnormal skin manifestation, even your screening protocol, your method by that of identifying your preferred delivery ligand is going to depend on whether you have a truly faithful disease model that accurately replicates that uptake. 

 
27:41 
And sometimes that's a difficult thing to find. 

 
27:47 
Even just finding receptors that are differentially expressed between disease tissue and normal tissue that will deliver the siRNA or the oligo into the cell is somewhat of a challenge. 

 
28:05 
Then one of the other problems is when you've got a receptor and you start putting peptides on your siRNA to deliver them, degradation of those peptides becomes an issue as well and you get a corona of other proteins built up around a lot of these constructs. 

 
28:27 
But then that doesn't behave the same way in vivo as it did in vitro. 

 
28:32 
So by migrating from in vitro to in vivo, you have to have, you obviously will get some attrition and that can be a big problem. 

 
28:43 
But you address enough of those problems at once and try and drive a product forward based on attrition. 

 
28:55 
Hopefully you can do it. 

 
29:02 
And so we're getting to the end of the talk, but we've talked very little about tolerability. 

 
29:09 
But tolerability becomes even more of an issue when you're going into a local tissue. 

 
29:14 
And you can certainly in the local disease tissue. 

 
29:16 
If you're going into say an asthmatic lung or something where there is a higher state of inflammation. 

 
29:24 
And you're bringing in some of these molecules that are based on oligonucleotides that for better or worse have a propensity for immune activation. 

 
29:34 
You're also in this situation that while you may be able to replicate the uptake, you may be able to replicate the mechanism of action, you may be able to get into the right cells. 

 
29:43 
But certainly once you're in the diseased state, you get an altered tolerability profile. 

 
29:49 
And that's also something that you really that you need to think into your process. 

 
29:54 
Way ahead. 

 
29:58 
Yeah, there's been a lot of studies performed in academia and in industry over the last 20 years, even since siRNA and ASOs were discovered and used first of all. 

 
30:13 
But as we've seen only recently there have been papers showing these targeted delivery molecules are now starting to work. 

 
30:22 
So I hope we've got a handle on it, and we can take some comfort from the things that have been shown to work by Alnylam and other companies, because one thing we've noticed is obviously the GalNAc story was an interesting one because Alnylam was the first to develop that. 

 
30:44 
But now everybody has a GalNAc conjugate to the siRNA 

 
30:49 
It might not look the same, but there's a lot of copycats out there that are developing therapeutics that have great promise using that GalNAc approach. 

 
31:05 
So I hope others can copy from people and the others have novel delivery vehicles and will try and validate their siRNAs in disease models using antibodies or C-16 approaches to deliver the siRNAs. 

 
31:36 
I think with that, we can wrap it up there.