Good morning, and welcome to us at Circio. My name is Erik Wiklund. I am the CEO at Circio. I have with me today Chief Technology Officer Thomas Hansen, and we will provide you with an R&D update, as well as a summary of our recently completed financing transaction. With that, we move into our presentation for today. We released a press release this morning with some updates. As a brief introduction, we have now validated our results that we have shown previously in heart, that our circVec AAV technology also performs exceptionally well in the eye following local delivery. We have also done extensive additional work in our cardiology program, both repeating as well as expanding on our heart data set.
These data points strongly suggest that our circVec AAV gene therapy technology has a role to play, at least in cardiology and eye genetic disease. We also consider this important validation that we can replicate these findings in more tissues than one, which suggests that the data are reproducible across tissues. It applies more broadly than just in one instance. With that, I move into the presentation. The reason why we are working on this, and particularly excited about it, is that it was, in fact, Thomas and myself, we were colleagues in the lab, 15 years ago, and together we did the early work on circular RNA and published the first papers in the space.
In particular, Thomas' article here in Nature in 2013 is highly cited and probably considered the founding paper of the field that has led to the substantial interest we're now seeing in circular RNA. It's not just Circio working on this. There is other very well-funded companies in the U.S. that are deploying circular RNA technology, and two of these have recently been acquired. In November, BMS bought the company Orbital Therapeutics for $1.5 billion, and Eli Lilly, just a couple of weeks ago, acquired Orna Therapeutics for $2.4 billion. These companies are doing in vitro transcribed or synthetic circular RNA, so it's different from us, but we're both deploying the advantages of circular RNA, but we're not in direct competition.
They were a little bit ahead of Circio in terms of development. None of these companies had initiated clinical trials. Looking at the M&A sums here, it's quite impressive what these companies were able to achieve in terms of valuation for preclinical stage program. It serves to illustrate the potential the industry is seeing in circular RNA. Shortly on circular RNA, this is a naturally occurring class of RNA. Because they are circular, they are resistant to normal degradation pathways inside of the cell. This makes them much more durable. That is why people are excited about circRNA, this extended half-life that is a result of the resistance to degradation. In addition, by being more stable, they're also easier to work with from a molecular biology perspective.
You can engineer them and design various functionalities. I can add that the first circular RNA, so synthetic circular RNA, similar to Orna and Orbital, are in the clinic already. Two Chinese companies started clinical trials in 2025, this is an emerging field, and really only very few patients have been treated to date. We expect this to now quickly expand in the coming year or 2. What makes Circio different? Well, we have a platform technology that we call circVec. The other circRNA companies, they start at the circular RNA. They make the circular RNA and deliver that. What we do is different. We deliver the genetic instructions to the patient's cells to make the circular RNA directly in the patient, and therefore, our products are actually not circular RNA.
Our therapeutics are DNA or viral vectors that carry the instructions for the body to make the circular RNA itself. Here we are really uniquely positioned and differentiated and don't have any direct competition circVec is a platform technology. It enables both enhanced and prolonged gene expression compared to conventional mRNA-based vector systems. We have unique expertise in Thomas and his team, as well as IP and know-how that covers how this is done, for which there really are no competitors around at the moment. We are deploying the circVec technology mainly in two areas, and we'll shortly describe both of these. The first one is to enhance gene therapy, notably the AAV gene therapy format, which is the most popular way to deliver genes into patients.
The other project is in in vivo CAR. This is cell therapy done directly in the patient, which is an emerging and very active area at the moment, with some massive deals having occurred recently. We'll come back to that at the end of the presentation to explain how circVec can fit into the context of in vivo CAR. Starting with AAV gene therapy. We have eight approved gene therapies today. These are therapies that treat genetic diseases. The patient has a mutation in the gene that makes that gene dysfunctional, and basically what you're doing is replacing the mutant gene. That sounds easy, but it's actually very difficult to get those genes delivered effectively into the patient's cells because the body has effectively evolved over millennia, millions of years, to prevent this from happening.
You don't naturally want genetic material from the outside to come in and disturb your cells. The way the industry has been most successful to date in terms of delivering genes is using the AAV vector. You can see here that seven out of the eight approved AAVs on the market are in fact utilizing the AAV vector. It's the vector that works the best, you can manufacture it. It is possible to utilize it to treat patients effectively. However, it has significant caveats, and the big caveat is that it's really hard to get the AAV to express enough of the gene. You can get enough copies in and express enough of it's not trivial. Because the weak gene expression, you need to give extremely high doses.
Those high doses make the products toxic, and they drive up costs. What we're doing with circVec is by switching to circular RNA based expression from the AAV, we can make the AAV more potent, i.e., you can drop down the dose that will make them more safe for patients and cheaper to manufacture. This is the main value proposition for circVec in AAV gene therapy. We believe this can be a disruptive technology for AAV gene therapy. I'll hand you over to Thomas now to explain exactly how we're doing this and show you some of the recent in vivo data.
Thank you, Erik. Good morning, everyone. Thanks for tuning in to our webcast. My name is Thomas Hansen, I'm the CTO at Circio. I will try to see if I can explain our technology in sort of layman terms, so everybody's on board. Basically, for today's purpose, you can depict here, or you see here the AAV virus. This is what we are working on in our gene therapy program. What you typically do, what all our competitors do, is that they have the genetic material inside that AAV that would encode an mRNA once it reaches the patient cell. That mRNA is linear, as you can tell, as you can see. It's highly unstable, typically degraded within hours by these exonucleolytic enzymes.
You have a very short-lived life for these mRNAs, but still, while they are in the cell, they will encode a protein. You get a fair amount of protein expressed, but not that much due to that instability. It will not accumulate to very high levels, typically. That's, of course, one shortcoming of conventional gene therapy. With our circVec technology, the AAV is the same, but we instruct the AAV then, by changing a little bit the genetic material inside the virus to encode a circular RNA instead. As you can see, the circular structure makes that RNA resistant to the RNA turnover. As a consequence, the RNA is very stable. It will exist in cells for days, weeks, maybe even months, depending a little bit on what tissue you're looking at.
Not only you have a very long-lived RNA, but as a consequence, it also accumulates to very high levels. As you can imagine, you get RNA production from the AAV. It's not being decayed, so it will slowly accumulate over time. You get a very high, steady state level, as we call it, a very high base expression level over time due to that very low decay rate. Consequently, you get very much higher protein yield from that same number of AAV initially. It's a more potent gene expression system by utilizing these stable circular RNA intermediates. We've been working on that for a few years now, and we took advantage initially, on the fact that Erik also said in the beginning, that circular RNAs are natural molecules.
You'll find these circular RNA being produced all the time inside our bodies, most predominantly inside the brain. Our first generation of the circVec vector was basically based on a natural design. We took the genetic sequences, the genetic motifs that were driving circular RNA most effectively inside our cells. We teased out what is the most potent locus naturally occurring. We took those elements, put them into the genetic cassette here that we call circVec. Replaced a little bit the actual sequence that goes into the circular RNA so that it would express the protein of our interest. Then we tested, you know, the performance of that vector. That had a certain, you know, here normalized to the relative level of 1.
That had a certain, a certain effectiveness. Over the course of the past years, we've been able to optimize that, what I would refer to as the best natural design for circular RNA biogenesis. We optimized this IRES elements that is critical to turn the circular RNA into a protein coding RNA. Normally, they will not encode a protein, but if you put in this IRES element, which is short for internal ribosomal entry site, you get protein produced from the circular RNA template. You can find a bunch of different IRES elements with different efficiencies, but I think we've been able to tease out over the course of screening a bunch of these, one IRES elements that's particularly effective across a multitude of different cell lines and tissues.
That was the defining feature of what we call circVec 2.0. We worked on some of these flanking elements. The original flanking elements, as I mentioned, were actually copied from nature, but we were able to shorten them substantially, but also make them more effective in terms of driving the circRNA biogenesis, the production of circRNA from the vector. You can see that also further improve the biogenesis or improve the protein yield from the circVec vector. What was probably the most effective sort of addition to the vector system was actually working on some downstream motifs element that may be in part, a little bit surprisingly, I think we covered that in a, in a, in a webcast last year, that actually drove up expression 4X compared to the generation 2.
This was the generation three defining vectors, these auxiliary elements that we positioned downstream after the cassette. There's a bunch of different elements you can put in here, actually. Lately, we've been working a little bit on what we refer to as UTR elements. Additional elements that you add to the circRNA that also seem to further boost the protein yield. Overall, we've been able to enhance expression almost 40x compared to where we were with the natural design. Even though it's only 1.5x here in the last step, we already had a pretty high protein yield, so you can see it still gives you a substantial boost in expression.
Now, putting these 3.X or generation 3 and generation 4 circVects into an AAV vector, injecting those into mice, we have recently obtained quite a substantial data package as to how this performs. First, I'll show you some recent data on where we inject these AAVs into eyes of mice. And here we've had our vectors inserted into what's referred to as an AAV2. That's a specific AAV serotype that's specifically effective by infecting eye cells or retinal cells. On the top picture here, you see over time, 4 different mice that have been subjected to what's called a intravitreal injection.
You basically inject the AAV into the eye, and then we can monitor expression from our vectors. The vectors, in this case, express a protein called firefly luciferase that lights up, so you can follow expression quite neatly in real time without disturbing the mice much. You can basically see how expression develops and the kinetics of expression over time. This is an experiment that was running for two months, and you can see on the top here, if you express a normal linear mRNA, in this setting, you can detect expression, but it's barely detectable in these eyes using this dose that we have been using, which is quite low, I must also say.
Maybe not that surprising that signal is very dim, but if you then swap to a circVec 4.0 design, it's the same dose, it's the same AAV serotype, but look at the difference here. Now we see a very potent signal. I think almost the signal is saturated in this particular case here. We've quantified the expression over time from mVec and 4.0 circVecs at the 2 different dose level that we tested here. You can see this is how it performs. Depending a little bit on what dose you're looking at, we see almost around a 50x in enhanced expression in the eye if you compare circVec to mVec, but also keep in mind that mVec is maybe very close to background.
A lot of this may actually be background signal that we are in part also quantifying. What is probably even more striking with this experiment is actually, if you look at the solid lines and the dashed lines, which refer to the high dose and low dose respectively, you can actually see that the dashed circVec line here in light blue is 12x higher than the solid mVec line. You have the high dose mVec performing less effectively than the low-dose circVec. In this case, it seems evident that we can reduce dose 10x and still get a more effective protein yield from our circVec vectors compared to conventional gene therapy.
I think this could be quite transformative in ophthalmology gene therapy, potentially even lowering doses more than 10x while still maintaining a good clinical benefit. This is something we're quite excited about, exploring in more detail, you know, what are the single cell granularity of this expression, and we are setting up more therapeutic proteins as we speak, to see if we can reproduce this significant enhancement with a therapeutic protein in a mouse model, specifically within the wet AMD, but I'll maybe come back to that later. We've been working more extensively, it's been going on for a while, with our AAV gene therapy program in heart. This is a little bit of a recap.
This is data we've shown in the past. In this experiment, we use systemic injection of an AAV9, in this case, injecting the same, expressing the same payload. We have mVec expressing firefly luciferase, and then you have two different generations of circVec here. It's an AAV9 that has a pretty broad tropism. It goes effectively to the heart, but in addition to that, we put a heart-specific promoter, so we specifically drive expression selectively in the heart or more selectively in the heart, and that's why we see a very potent heart-specific expression. I guess you can appreciate that we see much higher heart expression when we utilize our circVec cassette compared to the conventional benchmark AAVs. Again, this is something we can quantify, and it's nice to see that we have mVec down here.
We see a substantial improvement from the 2.1, and then an additional improvement from the 3.2, as we also saw in vitro. At least all the exercises that the hardworking R&D team is working on in Stockholm to optimize the cassette in vitro, those endeavors seem to translate well into the mouse model. We see a huge improvement, 40x higher gene expression in the heart when you use circVec 3.2 compared to mVec. We can look at this in a little more detail. We can try to profile what is the expression across the body.
You can see it here in the dark blue, you have circVec 3.2, a very heart-specific signal, as you can see, with very little promiscuous signal in the remainder of the mouse body, and hardly any noticeable signal for mVec. We also tested circVec 4.0, as you can see here in the light blue. Similar to what we see in vitro, you saw the 1.5x improvement in vitro, roughly also what we see in vivo here, a 50% increase in expression. It's not much, but it's still quite noticeable when we these very high levels of luminescence.
Again, very nice to see that all the work we've done in vitro translates to the in vivo work, and we have at least two different vector designs here that greatly outperforms conventional AAV gene therapy in the heart. Moreover, we've characterized, in this case, the 3.2, the performance across a multitude of dose levels. We tried four different dose levels, ranging from 5E12 vg/kg . It may sound like a lot, but this is actually quite a low dose. I think this is one order of magnitude, roughly below what is typically used in the clinic. 5E13 vg/kg , even in some case, 1E14 vg/kg is typically seen in the clinic as being injected.
We were also interested in seeing how does circVec and mVec perform across these different dose levels. What we actually see, we see a consistent and constant fold change when we compare circVec to mVec. This is a little bit of a linear regression across the different time points that we measured, but we see no interaction between the vectors and the dose, meaning that basically, we have the same dose-response relationship for circVec and mVec. As a consequence, this means that, I mean, there's a clear dose-bearing potential here with circVec . The high dose clinical, you know, used dose levels that is currently being used, translates roughly to the protein yield that we see with our low-dose circVec , the 5E12 here.
That was great to see that the circVec performs well across the different dose levels. When a study completes, typically, we will run a study for 2-3 months. In this case, this is 10 weeks, 2.5 months, roughly. We subject the tissue from these mice to more granular, more in-depth characterization. In this first instance, you take out the heart, you will monitor the luminescence, specifically in the heart, just to ensure that the signal you observed across the body of the mice, they were indeed the heart that were emanating that luminescence. We can confirm that we see also roughly the 40X improved here.
Barely any detectable signal in mVec, as you can see, a lot of light emanating from circVec hearts. On top of that, you can take all the tissues that you collected. You can see how much signal do we actually get from the heart compared to other tissues. Here we just see 2 out of a whole panel of different tissues, but actually across that panel, 80% of the signal that we observed was seen in the heart. It was only 40% for mVec. In contrast, there's a lot of mVec expression in the liver, where we see very little expression from our circVec cassette in the liver. There seems to be not necessarily detargeting of the liver, but it seems our cassette has a deexpression profile in the liver.
Much more on tissue expression is seen, much less off-target expression in the liver, and this is definitely what people are looking for. Liver expression is not wanted if it's not a liver-related indication that you're trying to treat. Moreover, we can quantify these samples. How much RNA do we see in this sample? How much circular RNA, and how much mRNA do we actually see? On this level, the fold change is even more impressive, almost two orders of magnitude difference between the RNA that's being seen in the cell from circVec compared to mVec. This underscores and emphasizes that it is at the RNA level that the difference is happening, moreover, it is changing to a circular RNA instead of mRNA.
That basically explains the difference between the low-yield expression in mVec and the high-yield expression in circVec. You could claim that, "Maybe it's just some weird difference in the AAV, so you have more effective transduction of heart cells with circVec than mVec." I would just emphasize that it is the same AAV, it is the same serotype. On the surface, these two AAVs that we are comparing should be completely identical. It's just the cargo inside that differs, and this is also being confirmed here, where we actually quantify how many viral particles is then actually being delivered to the heart cells, and you do that by quantifying the viral DNA, and there we see no difference.
We get the same level of transduction, but then expressing the circular RNA instead of the mRNA drives up this huge difference in expression. Also, when you then look at how many cells are we then actually targeting, how many cells show expression, that's another critical feature. If you wanna treat a heart, rare indication, you need to reach most of the cardiomyopathy. The heart cells, sorry. And as you can see here, this is using a technology that's called RNAscope. This is basically a technology that will detect individual RNA transcripts. It's a single molecule detection technique that we use to detect the gene that is being expressed from our AAV. We've done that for, again, the circVec 3.2 and the mVec.
Again, the difference is quite remarkable. We see a broad distribution of RNA across the whole heart. We actually been able to quantify how many cells have a positive signal for RNA, and it's 80%, which is quite remarkable for the low dose that we are using. I mean, we also see a quite good coverage here, it's just difficult to see the single dots here because there are so few with mVec. There's also quite a nice coverage here. It's just remarkable to see that broad distribution with a low dose. We believe that with the low dose we're using, we get high protein yield, full coverage within the heart.
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We see more specific expression, we see more expression in the heart compared to sort of other tissues, such as the liver, which is a great point in terms of the safety and then toxicity. Moreover, and I didn't go into the details here, not only do we have the dose sparing, that we're able to reduce dose at least tenfold while maintaining the at least similar, if not higher expression. We actually have also been able to profile the stress activity inside heart cells. This is using a high-throughput technique called RNA sequencing, where we basically profile all genes expressed. We can actually see there that when you use conventional gene therapy, the mVec, in this case, you see activation of different stress pathways, and you see that more so than you do with circVec.
On top of the dose sparing, we also believe that using a circRNA-based gene expression technology is less stressful for the cell. Maybe because we are now basing the accumulation on stability and not on production. We don't need necessarily high production levels because we rely on the fact that they are simply staying in the cells for a very long time, and they accumulate. You can have a very gentle, slow production that is less stressful for the cells, and then you still accumulate very high levels to achieve these impressive fold changes in protein yields. Hopefully that was informative and positive. I think we are very excited about the data ourselves, and I think with that, I'll pass the word back to Erik. That will finalize the webcast.
Thank you for your attention.
Thank you, Thomas. Why is this important? I'll give you an example here in a heart clinical trial that is ongoing. There is a company, Rocket Pharmaceuticals, they're developing a series of AAV-based gene therapies for genetic heart diseases. Very unfortunately, earlier this year, they had a patient die in one of their trials, directly linked to the high dose of AAV that is required. Now, interestingly, it's very clear that their gene therapy has a clinical benefit. It works, the patient gets better. These are patients that typically die or require a heart transplant in their 20s. You have this treatment that is effective, however, it's so toxic that you risk dying if you take it.
If you can bring down the dose for these patients, for this gene therapy, that would be a massive advantage. Our data suggests that our AAVs are up to 40-fold better or more potent in heart. The dose response curve that Thomas showed you shows that at least 10-20-fold, we expect the dose can be reduced. If you can get the dose down by 10x, that, or even more, this should substantially reduce the tox. It should bring down the cost and make these gene therapies better, safer, and cheaper, more accessible for patients. We are not the only ones working on enhanced AAV gene therapies. This has been a field where the classic approach with conventional AAVs has struggled.
But there's still a lot of activity happening in the area, both in academia and in industry, and we see a number of deals happening. All the deals that are happening here are for second-generation type AAV technology. Technologies to make AAV safer, more specific, more potent. Here is just some examples of recent transactions, financing, or BD deals that have happened in this area of AAV. All these use different tweaks to try and make their AAVs better, and in most cases, these would be complementary with circVec. Maybe in the future, you can merge what you call engineered capsid with better targeting, enhance other expression enhancement systems, safety systems, plus circVec, and you can get a completely different order of magnitude of potency for AAVs....
You may also note here that 3 of these transactions are in ophthalmology. Ophthalmology is an area of particular interest for AAV gene therapy. That's why we're also moving into that direction, are particularly excited about our eye data. What we have shown you today is our new data in eye. This is a earlier readout. We haven't got as far as in the heart, but we show the best expression advantage we have seen to date, 50 times improved expression level for circVec 4.0 when you deliver locally to the eye. In heart, we see around a similar advantage, 40X, and this is with systemic delivery. With the heart, we deliver into the bloodstream of the mouse systemically, get this very high specificity in heart. With the eye, we can deliver it locally.
With two different delivery routes, we see roughly the same type of advantage, and we see it in two different tissues. Very important for us to get confidence that our findings are broadly applicable biologically. What we're doing now is trying to identify what are suitable disease areas, or specific diseases to move forward in these two, tissue areas, heart and eye. We talked about Danon disease, just now. That's a very attractive prospect, scientifically, but it's also a very small disease. It's only about 1,500 patients worldwide, so it may be too small to be commercially viable. We're exploring, now, systematically what are relevant, diseases to move into, where there is a good scientific fit, industry interest, and also, it makes sense commercially and competition-wise.
Here is some examples of what we're looking at. We are also working on CNS, so Central Nervous System. This is a tissue with normally very high levels of naturally occurring circular RNAs, and we've done some early work here. It's a bit trickier to work with CNS. Early indications have shown about four times improved activity, but this is for circVec 2.1. We have ongoing work with 3.2 and 4.0. The 2.1 for X is roughly similar to what we saw in heart and eye at this stage. We're encouraged about this, and here we also have an ongoing collaboration with a big pharma company, where we're testing specific designs for the partners.
We look forward to updating you in the future here, but these are crystallizing as the three areas that we will be prioritizing for our gene therapy program. I'll briefly now touch on our in vivo CAR approach. This is an area of massive investments at the moment. You almost every week see a big pharma moving into the space. You can see we summarize from the middle of last year until now, transactions by the big players to acquire in vivo CAR approaches. Conventional cell therapy requires the use of cells that are taken out of the patients or from a donor, and then get manipulated or genetically engineered outside of the patient, propagated up, and then given back to the patient.
This is a relatively cumbersome and expensive way to do it, but it's what's available now, and it works really well. What everyone's trying to do now is make this happen in the patient. Can you do the cell engineering directly inside of the patients? The data we see in monkey studies have been highly encouraging. That's why you get these big investments into the space, and several clinical trials are now ongoing, and we will expect data later this year from the first in vivo CAR approaches. I think the one here by AbbVie and Capstan is the first in line, and this can completely revolutionize how cell therapy happens. Now, circVec can be deployed here in a unique way, and I'll explain to you why.
In this case here, we move away from the AAV, this is no longer a viral vector. Here we use a synthetic DNA construct, and these synthetic DNAs then carry our circVec insert to express genetic payload using circRNA, and we deliver it by LNP. This is the same way that, for example, the COVID vaccines with mRNA are also delivered using LNP. When you give a DNA vector that express use mVec, so express the payload, the gene using mRNA, we always see the same pattern, where we get high expression in the liver. It all goes to the liver, and lots of expression there, and then it quickly disappears. You can see from here, day 14, day 23, so week 2 or 3, not much expression left. The mice sort of stay with limited expression for the remainder of the experiment.
However, you switch to circVec using the circRNA expression, as Thomas showed before, circular RNA is not stable in liver. It's not good for liver expression. Actually avoids the liver expression. We completely eliminate this expression in liver. What happens from week three onwards is that we start seeing signal in spleen. Spleen is the organ where the immune cells reside. B cells and T cells that you may have heard of, are important immune cells in the case of in vivo CAR, immune-enriched T cells. These are all present in the spleen, and what we see is from week three here, we start getting expression in these cell types. We confirm that this occurs both in B cells and T cells in this case.
More importantly, the expression actually lasts for up to six months. We can deliver one dose, we get expression for six months, in specifically the immune cells, whereas with mVec, only expression in liver, and it only lasts for two weeks. A completely different biology happens when you switch to the circRNA expression. Why is this important? All of these deals I showed you in the, in the space are occurring for RNA-based approaches to in vivo CAR, either mRNA or using circular RNA. This gives you expression for few days. mRNA will express for one to two days, circular RNA for a week, then it's gone. The only other alternative is to use something that is called a lentivirus, which permanently integrates, or makes a permanent change to the T cells.
This is an approach which is then last forever, but it carries a lot of safety issues, and it can't be reversed. This is really not favored. Our expression that I just showed you of 6 months, puts circVec in the middle. We offer something completely different. It will be non-integrating, non-genome integrating, so much safer than the lentiviral, but it will last much longer than the mRNA and circRNA approach. 6-month duration gives you a completely different type of therapeutic dynamic. In addition, it could be re-dosable and avoids liver expression. Where we believe this fits is in vivo CAR for cancer applications. Most of these RNA approaches to in vivo CAR, they're used in autoimmune disease, where short expression probably is sufficient.
The short expression of these RNA approaches is less likely to be impactful in cancer because you're not going to be able to eliminate all the cancer cells in that short expression window. Here we see a unique opportunity to move into this space. We have active research programs in this area, and we will expect to update the market on our progress here during the second quarter. Earlier stage than our gene therapy approach, but very high potential, given the importance of this space currently in the biopharma industry. I'll wrap up with a short summary of our financing and plans going forward. As most of you, I'm sure are aware, we just raised the capital.
We announced the transaction back in December, a rights issue, where we had strong support from our existing shareholders. We had roughly half of the transaction secured by pre-subscriptions, NOK 24.2 million. Then we targeted a raise of NOK 50 million, and we had a guarantee on top that brought the secured amount to 90% or 88% of the NOK 50 million. The total subscriptions were more than 50% over the NOK 50 million. Unfortunately, we were not able to meet all the subscriptions. There were more than 1,000 subscribers in total, and several of these were larger orders, which we see as good because that brings in more larger and investors with capacity to support the company in the future.
We had a structure where we could use a directed issue to place some of these amounts. In total, following all these transactions, we raised close to NOK 70 million or $7 million in this fundraising, which is substantially over what we were aiming for. The deal came with warrants. Everyone who subscribed in the transaction got one warrant for each shares allocated, and these warrants gives the right to buy a new share in Circio from 26th of May to 9th of June. That's the exercise period of the warrants, and that enables the holder to buy a share at a 20% discount to market price at that time. This provides an upside for the investors who participated, and also it provides us an opportunity to raise more capital.
The current funds will give the company roughly a 12-month runway from today, and we expect to extend that runway by 6-12 months from the exercise of warrants. This, of course, depends on the situation at the time and the investor demand, but this is where we expect to end up following the transaction, or the exercise of warrants in June. We're guaranteed funding for one year. The funding may last as long as into 2028, based on this transaction structure. On business development, we've made important progress. We did initiate a feasibility study with a big pharma, one of the top 5 in the world.
This was signed and presented, announced to the market in November of last year, was, of course, an important component of a successful financing. This project is ongoing. We can't say too much about it, but we're testing circVec AAV in a specific disease area. If this is successful, it may lead to a subsequent licensing of the technology. We expect second half of the year, we should be able to provide some updates from this project. We do have several other ongoing collaborations. Many of these are 50/50 collaborations, where we try to combine technologies that can be complementary. Specifically for our in vivo CAR program, we are exploring delivery systems. We are a vector company, a circRNA company.
We're not a drug delivery system, and not a drug delivery company. Here, we rely on external partners to enable us to get the circVec vectors for in vivo CAR therapy delivered to the right cell types. Here we have several collaborations that we are either already ongoing or starting, and the idea here is that we test several approaches, and we will pick our favorite to move forward with. Again, we expect, hopefully during Q2 or Q3, we will provide an update on this work. Finally, I point out that we are still actively searching for more collaborations. We do not have any formal collaborations yet with our heart and eye gene therapy programs. We will be exploring opportunities for that, establishing similar big pharma collaborations in these areas....
or with companies that have complementary AAV enhancing technologies. That's another interesting aspect. For example, targeted capsids, as we say here, capsids, viruses that are designed to more specifically infect the target cell, like the heart cells. What if we combine that with circVec ? Can we make it even better? That's another approach that we will form collaborations and test in the future. Milestones going forward, we have broad activities. The funding allows us now to accelerate activities and do more studies in parallel. This means we'll have exciting readouts coming up. We showed you already the circVec 3 and 4 data in heart and eye. We will have more of that. The data package in heart is very expansive now.
We're now doing that same work in eye, so we'll understand more precisely how it works there, then we expect to release CNS, Central Nervous System data later in the second quarter. The next step is to move to disease constructs. Now we're doing what we call reporters, these luciferase genes that light up, that are good for experimental purpose. The next step is to do actual disease genes, and that's work that is ongoing. It's being tested in vitro at the moment, then we're gonna go in vivo with that soon. Again, we expect data to be presented during Q2. The T-cell delivery for in vivo CAR is also actively ongoing, and that should deliver data during Q2.
I think all of these readouts here, we expect to have before the warrant exercise period in June. Looking at the second half of the year, we are aiming to generate disease model data. The first step is to just show we can make constructs with disease-carrying genes, and then you wanna put these in mouse models where you can look at efficacy. Does it actually not just express a lot of the protein, but give you an effect on a parameter that is predictive of efficacy in a patient's actual clinical benefits? These, as I said before, looking at what diseases are most attractive, what disease models are the best, and the second half of the year, we'll start seeing readouts from disease model data.
To summarize, AAV circVec, that's our gene therapy approach. We can get up to 50x improved gene expression, and this technology has, in our view, disruptive potential in AAV gene therapy. You may need to have this in the future in order to have a competitive gene therapy, at least in heart and eye, and we expect CNS. The in vivo cell therapy approach is a new and differentiated way to extend the expression window. We believe we can occupy a niche share that is not yet taken in an area of extremely high industry interest at the moment. Our pipeline is rich, multiple shots on goal. For you are no biotech, I think what differentiates Circio is that we are a platform company.
We generate preclinical data. This is not a company that hinges on one clinical readout. You're not funding an expensive clinical trial that will be either positive or negative two years into the future. Here we have continuous readouts. There is not one single data point that is gonna make or break it for us, at least not in the short to mid-term future. That means we can also do multiple deals on the platform and still retain activity for ourselves. We intend to continue like this, be preclinical, keep the cost lower, and generate value for shareholders through partnerships.
Next step, as I mentioned, heart and eye, disease model data in gene therapy, as well as T-cell delivery for our in vivo CAR program. We already did one partnership with the big pharma. We aim to add more during this year. With that, I wrap up the formal part of the presentation. I have a slide here at the end where you can see we are broadly covered in the media. I recently featured on the podcast here in BioSpace that you can listen to. Otherwise, there are some articles that I think nicely cover circular RNA and also Circio. We are starting to get noticed internationally and in the industry for having a uniquely positioned technology to enhance gene and cell therapy.
With that, I wrap up, and we've received some questions, and maybe we start with those here in the chat, and maybe, Thomas, this one is for you: Do you know if there are ongoing research on mVec tech that is likely to bring the mVec tech closer to the expression of circVec?
I think that's a brilliant question. Obviously, other companies working on mRNA-based technologies are also working on improving that. I would say most of that work is either when you have a DNA vector, would either focus on optimizing the promoter, either for tissue specificity or for production. I would claim, and this is what we have observed so far with the stuff we've done, that that optimization would apply equally well to circVec. If you have a more potent promoter expressing an mRNA, that would also more potently express the circRNA. I think those kind of optimizations would benefit us equally well.
Another approach that's being done quite a lot, and particularly in recent years, at least in the published literature, is what is referred to as codon optimization. If you're familiar with the degeneracy of the genetic code, namely, that you can encode the same proteins, with different RNA sequences, and those different RNA sequences may be translated with different efficiencies. What you refer to as codon optimization is find that RNA sequence that most effectively translates into a protein. This is also stuff we've been working quite a lot on, and we've actually, at least in vitro, compared sort of our preferred codon-optimized approach with actually benchmark sequences that are going into the clinic, and we consistently see better performance for circVec .
Again, I think this is an approach where some of these optimizations may very likely be translatable to circVec as well as mVec. We are following the literature, we are talking to people. Of course, if anything emerges that we believe could be of interest to circVec , then we will test it immediately and while still adhering to our own optimization pipeline. But I think it's a brilliant question, and this is something that's definitely constantly on our radar. Thank you for that.
Another one here for you, Thomas. You say you are working on circVec AAV disease constructs for eye and heart. Could you elaborate on what this actually means?
Yeah. Specifically, we had as Erik mentioned, previously, we had a focus on Danon disease, based on the fact that toxicity issues were observed in the clinic. You know, you had a good clinical benefit. It seemed to be a good fit for our technology scientifically, but there are some financial business aspects that may and some patient population indication sizes that unfortunately may make it less attractive. But in this particular case, what we do is for Danon disease, it's a protein called LAMP2B that you need to express in heart. We are now still as a proof of concept, generating circVec vectors that would express LAMP2B.
We see very good performance of these vectors in vitro, so that protein is being expressed much more effectively from circVec compared to mVec. We're still setting up a small in vivo study, where we then try to see the performance of these LAMP2B expression vectors in the heart, where we compare CircVec to mVec, similar to what we've done with the firefly luciferase, but just with another protein. That would be something that potentially could be where there's a clear path forward if the data readout is as expected, that is almost a clinical candidate right there. That would be if we decide on re-engaging with Danon disease.
Another, in the eye, we are very currently working on wet AMD, this is a indication where you have too much vascularization in the eye. The treatment there is typically to express a protein, called anti-VEGF, that restricts this vascularization. There's a few different ways you can do this. What's most popular is to express a protein called aflibercept, this is also a protein that is in Phase III clinical trials. A little bit similar as a proof of concept, we are now setting up circVec expression in vitro, testing that it works, that we get the expression level that we need, and we see that the protein is functional.
Then, there are some very well-established mouse models where you can then inject those, circVec AAV vectors intravitally into the eye, and you see how that impacts the vascularization actually in real time, so you don't have to need to sacrifice the mice to get that readout. I don't know whether that answers the question, but these are some of the therapeutically relevant proteins that we are setting up in vitro, characterizing the expression, putting it into an AAV, and then, we aim to see how it performs in a mouse model. That's our goal in the near-term future to get some of these data points.
Thanks, Thomas. We also have a question around remove and replace. We have previously talked about remove and replace as a technology, and the question centers around where and how this can be applied. Maybe you can comment on that as well, Thomas.
The remove and replace technology is basically where we have a bimodal cassette, so we still express the circRNA as we've shown, but then we can insert an additional element that encodes a knockdown moiety. Basically, in a target-specific manner, could deplete your RNA of interest. It is applicable to a subset of diseases where you may have some toxic accumulation of a mutated gene. We had a focus on AATD in the past where that was namely what was observed, toxic accumulation of a toxic mutated mRNA. There's a few indications in the eye where you also have this autosomal dominant inheritance. You get the accumulation of a toxic transcript that could potentially benefit from a remove and replace.
You remove that toxic RNA, and then you replenish with a circRNA-based expression of your, of a wild type or a functional, non-toxic counterpart. It's still something we are looking into, and we think our technology is particularly well-suited for these scenarios. Not all indication would fit into that remove and replace would be relevant for remove and replace, but definitely something we are looking into. I think there's also a few cases in with heart diseases that could potentially benefit from actually removing that mutated gene to improve the benefit of the treatment.
This is definitely a really cool approach, and we can do both, and this is unique for circRNA and circVec that you have this dual activity. We're also mindful that we want to minimize the technology risk of development. You don't want to do too many things at the same time. We consider it the more prudent and smarter pathway to start by simply express a gene, pick an indication where that really high and durable protein expression is what you need, and then we may, in the future, add on other features once you establish that the first one works to, what we say, reduce the technology risk. Do one thing at a time. This is absolutely a unique feature of the platform that can be an exciting component in the future.
We have another question here in the chat. What about the competitors' technology? By competitors, I'm assuming the question relates to Orna and Orbital and how circVec compares to it. Again, I'd like to stress that these other circular RNA companies, compared to us, we're both utilizing the advantage of circular RNA to make more durable RNA and more durable expression. Using synthetic RNA formulated in an LNP and using what we're doing, DNA or viral vectors, is completely different. They have completely different use areas. There is very limited overlap. You should not consider these competing technologies. These are more complementary. In certain situations, it's better to use the RNA strategy of Orbital and Orna Therapeutics. They can never be relevant in the genetic disease where you need permanent expression.
You have to go with something much more durable. In that case, an AAV with circVec is relevant to use. Of course, it shows these deals that are happening in that space, that this is a format that is considered highly promising, and we're the only ones deploying it in the way we're doing. Instead of competing head-to-head against those guys, we identify a way to do it differently and own a niche in the circRNA space. That's how we like to see it. I also received a question here that we can wrap up with around the cash burn and expected the runway and whether we are scaling up or not after the fundraise.
As many of you know, who have followed the company over time, we have been on quite a tight financing and therefore scaled our operations accordingly, and we've been operating on a burn rate of around NOK 4 million per month, or less, NOK 3.5 million , for the past couple of years. The new funding here allows us to accelerate. We're adding scientists to our team in Stockholm. We're going to do more things in parallel. We're considering to more quickly move into primate studies, utilizing these funds. But we're not going to go overboard, and we're very prudent about how we do our development. I would say expect that we increase our burn rate short term by 20%-30%.
Second half of the year, maybe we'll increase it by about 50%. We're also going to pay close attention to developments in the market, what our data looks like, how the warrant exercise period materializes, and scale up step by step. With that, I think we covered the questions. We came to the top of the hour. I hope this was a helpful run-through of our technology and progress to date. I would like to stress that we are extremely excited about this data in eye. This looks really promising. And like Thomas showed you, we got further in heart. We have a very substantial, I think, convincing data set, mechanistically demonstrating what happens. It's reproducible, and it all sort of stacks up. That heart package is very solid.
Next step now will be to do the same type of groundwork for the eye and understand exactly what's happening at the cellular, at the RNA, and at the vector number. In totality, I think this starts now to become a robust, broad data package that becomes impossible to ignore for prospective partners, and therefore, we expect increasing interest in collaborating and doing deals with Circio in the future. With that, we sign out from Circio, thank you guys for tuning in and listening. Bye-bye.