Oisin Biotechnologies is one of many companies that have appeared in our community of supporters and researchers related to the Methuselah Foundation and SENS Research Foundation .
Oisin representatives are working on a platform that can selectively destroy cells based on the internal expression of specific proteins by these cells. Their initial goals are senescent cells and cancer cells. First, they are going to bring anticancer therapy to clinical trials, since successful cancer treatments are easier to regulate than many other therapies.This will allow them to reuse the technology, preparing for subsequent tests of senolytic therapy , capable of cleansing many senescent cells from different tissues. At the Undoing Aging conference organized by the SENS Research Foundation and Forever Healthy Foundation in early 2018, Oisin Biotechnologies CSO John Lewis talks about their technology and new results.
Performance
Good evening everyone! It is very nice to be at this meeting. I sincerely thank Aubrey de Gray and Michael Greve for the invitation. I couldn’t even ask for a better sequence, because the previous speaker did a fantastic job of highlighting the reasons why senescent cells are important, and why they are such a problem. I am an academic scientist working in oncology and a beginner in the field of aging, although I have a lot of experience in killing cells, and I would like to talk about this now: how Oisin Biotechnologies develop a very selective therapy to destroy senescent cells and cancer.
I won’t talk much about what senescent cells are, or what they do. In a few words, these are cells that arise in the body, as a reaction to external stresses - oxidative stress , genotoxic stress , and they mainly impede the development of cancer. It is also important to note that as the cells become senescent, they also send signals that propagate in the body . I repeat that we do not have universal markers of senescent cells. There are common features that we can use in their recognition.
For example, the accumulation of active enzymes in lysosomes . We can stain active β-galactosidase . But actually it is a very heterogeneous population, and they arise in different ways. I do not want to dwell on the connections, I just emphasize that the initiation of completion of the cell cycle is regulated by several factors: p16 , p21 , p53 . We thought about it and asked: what are the common ways that we could aim to create a targeted therapy that eliminates these cells? I do not need to repeat that while senescent cells are involved in the aging process, there are also very specific diseases that are symptoms of aging and which can be treated clinically with the same techniques.
It was an important point in the last conversation that p16 may not be the whole story. But you need to look at the data that has been studied over the past few years - and it really convinced me - that, of course, all senescent cells may not express p16, but in the mouse model it is very clear that if you design it in such a way that you could selectively destroy all cells expressing p16, you will get phenomenal changes in the phenotype. For me it was very impressive.
For example, the work of John van Deyrsen , in which there were genetically modified mice expressing a suicide gene driven by the p16 promoter , using the so-called INK-ATTAC caspase-9 system , which allows them to use a dimerizer that activates apoptosis in cells that express p16. And these mice showed phenomenal changes in their phenotype. Significant improvements in health, a 25% increase in average life expectancy, 50% less cancer, as well as functional changes: a decrease in cataract formation, a decrease in weakness , and a decrease in hair loss.I will show, as an introduction to my lecture, some data that has recently been released. They made me think that it would be worthwhile as a therapy - this is the work of Peter de Keyser , published last year, using p53 and FOXO4 mechanisms. He was able to use an accelerated model of aging, mice that lose hair become weak, and showed that removing senescent cells after these changes have already occurred can change them. This for me really confirmed the fact that the destruction of senescent cells is the right path of development.
Therefore, this is the foundation of Oisin technology. As an engineering group, we thought, even during the development of the therapy that we can use in the treatment of a specific disease, we thought about aging in general, and how this can be used in the future after we test the effectiveness clinically. Thus, we wanted to use a similar strategy, using modern animal models developed in our time. We wanted to develop something that has a low toxicity profile that is well tolerated, and something that can be repeated cyclically. Something that is non-immunogenic and has no side effects. Obviously, many aging phenotypes are tissue-specific, and the ability to target therapy to different tissues would be an advantage.
What I'm going to tell you today is the Oisin technology that we have developed. It is called the SENSOlytic platform. This is a lipid nanoparticle platform (LNP) that contains a non-integrable DNA plasmid . It is designed to be activated by a chemical dimeriser that initiates a very fast and irreversible apoptotic response. Perhaps the most important part of this is the drug delivery system — the ability to deliver plasmid DNA systemically to different tissues without significant toxicity. Oisin has developed a multipurpose plasmid-based technology, and, unlike interference RNA or messenger RNA , plasmids can be elegantly designed to only activate when a specific pathway, such as p16, p21 or p53, is activated. But they can also be designed with enhancers or repressors and target specific tissues or diseases. We have created a system and library of designs that are active in various conditions. Two of them, which I will show you today, are versions of the p16 and p53 promoter, including suicidal genes.We have created a library of plasmids, which are basically a specific selective promoter associated with the iCas9-induced suicidal gene, which is then incorporated or dimerized using a chemical dimerizer. Many of you may have seen this before, but iCas9 is a modified caspase, so it has been truncated, the inclusion domain has been removed and replaced with the FKBP dimerization domain . These areas interact very strongly with the chemical dimerizer AP20187 or its clinical analogue AP1903, which is safe, as was shown during phase II clinical trials. What is really good in this system is that you only temporarily express the genes of your plasmid in cells with active expression of p16 or p53 in our case. And nothing happens until you add a dimerizer. The low molecular weight dimerizer is very well tolerated, saturates all tissues within a few minutes and causes an irreversible apoptotic response. iCas9 dimerizes under these conditions, self-cleaves, activates the formation of apoptosomes and causes very rapid cell death within 2 to 3 hours. Cells cannot resist this. They cannot evolve or otherwise resist it.
Some of our in vitro studies used the placental myofibroblast cell line IMR-90. In this case, we induced senescence using 10 degrees of radiation and transfected cells using iCas9. iCas9 is slightly less than caspase-9 and can be detected with antibodies to caspase-9. In cells that were not irradiated, there was no expression of iCas9. In those cases where the cells become senescent by expressing p16, iCas9 induction begins, and when we add a little dimerizer to these cells, it disappears. Culture is very quickly cleared of these cells. Then, when we look at the ability to kill these cells, we see in viability tests that every cell that is successfully transfected with a plasmid dies. We conducted other experiments. I am just showing an example in which we use flow cytometry and we confirm that we are causing apoptosis in these cells.
Thus, we have a plasmid that is very selective for cells expressing p16. We can kill them very quickly by adding a dimeriser. The question is how do we turn this into a therapy that works in humans. Need a delivery mechanism, very efficient and safe. We decided to use lipid nanoparticles. Lipid nanoparticles have been used for many years, and I would say that there have been many promises and investments, and very few successes. Alnylam Pharmaceuticals in Boston recently conducted a successful Phase III trial with an mRNA preparation, and the problem is that lipid nanoparticles usually accumulate in the liver and their nucleic acid delivery mechanism to cells uses a positive charge. This is a very simple technology. They created lipids with a positive charge. If you use a positive charge, they very easily pierce holes in the membranes , and you can effectively deposit material into cells, except that they are very toxic. Thus, they have a very low safe dose.
In response to this, several companies have developed the so-called conditionally cationic lipid . This lipid , usually neutral in the bloodstream, enters the endosomes and becomes cationic in an acidic environment. They are the subject of ongoing programs that go through clinical trials of lipid nanoparticles. They work, but they are still very toxic. An ideal delivery system is one that can use neutral lipids with an alternative mechanism for cell delivery of nucleic acids. I am going to tell you a little about how we came to him. If you have a lipid nanoparticle, and it needs to get inside the cell, it must pass through the plasma membrane with all its protection. Viruses have evolved over millions of years to solve this problem and have developed many fusogenic proteins . These proteins are beautiful, gigantic and elegant, and the way they connect the membranes together, create pores and mix lipids is really fantastic, but you cannot attach them to a lipid nanoparticle, because they are large proteins with giant active centers and high immunogenicity.
Fortunately, there is a Canadian scientist who has studied these fusogenic orthoreviruses all his life, and found that they do not use the fusogenic protein to penetrate the cells, but as soon as they enter the cells, they cause all the cells around them to merge quickly. He spent his career characterizing this class of fusion-related transmembrane proteins , which are two orders of magnitude smaller than the smallest fusogenic protein expressed by other viruses, but are sufficient to initiate the fusion of cells and, most importantly, lipid nanoparticles and cells.
When you include these proteins in a platform based on neutral lipid nanoparticles, you will find that neutral lipids alone are extremely poor in load delivery. In our example, we use the mCherry plasmid against cancer cells, and therefore without fusogenic proteins there is no delivery at all, and with them we get a fantastic delivery. Thus, they increase the delivery of a neutral lipid particle by 80-350 times, and they are well tolerated in vivo . In an experiment with luciferase, we inject mRNA expressing luciferase into the tail vein. We get luciferase expression throughout the body. We notice an accumulation in the lungs and liver, but we get good expression in many tissues, including skin and soft tissues throughout the body.
We use our platform to deliver payloads against senescent cells. The platform is called Fusogenix . It uses a neutral lipid nanoparticle, which is non-toxic and well tolerated. And she uses these fusogenic proteins to enter the cell. I do not want to go into all the little things. It took us three years to create antibodies against these proteins, they are really non-immunogenic. The reason for this is that most of them are transmembrane domains. They are lipophilic , so they pack lipids around themselves, and they are not immunogenic. We spent a lot of time improving these fusogenic proteins to make them better. I’m not going to delve into all the little things, but now we have a platform for their industrial production in large quantities and lyophilization , and we can send them on request for use.
Let me show you the data obtained in the experiment - in which they took activated p16 caspase-9 and introduced it into mice. In this case, we conducted an experiment with 16 mice, an elderly mouse group of 80 weeks. We divided them into three groups, we introduced them the control LNP, without a dimerizer, or two doses, 5 and 10 mg / kg - and 10 mg / kg - this is a large dose. We treated these animals once by injection into the tail vein. We waited for 96 hours, and then introduced a dimerizer, also intravenously. Then we waited two more days, collected tissues, blood, performed sensitive RT-PCR , and controlled it with several genes. We received a convincing dose-dependent decrease in p16 expression in various tissues.
I'm going to show you a couple of images where we spend a lot of time optimizing β-gal staining in mice. These are the best images that we have, but in several tissues we received a dose-dependent decrease in β-galactosidase expression. Very encouraging information. Of course, working in the laboratory is good, but if we are going to translate it into people, there are many things that need to be clarified. Toxicology is extremely important. This is important for a drug that you are about to perform cyclically to ensure that your body does not form neutralizing antibodies. Thus, we conducted many studies on repeated dosing, and we did not record any antibodies, so we can give it in repeated doses without any decrease in effectiveness. CARPA is something that I recently learned about, a complementarily activated pseudo-allergy , an immune response that many patients receiving therapy with nanoparticles such as doxil can have . We ran all the analyzes and they have a lower profile than doxil, so they are very well tolerated.
I am pleased to say that we conducted several pilot studies with primates, we administered them a ten-fold maximum human dose, and it was very well tolerated. These monkeys actually received treatment, both p53 and p16, separately and in combination, and a dimerizer, so we are counting on good data. Now we are working on them.The tolerance of these particles is very important. I am showing this slide because it shows all the efforts made in clinical trials of lipid nanoparticles and why they failed. If you look at the first three programs, they were promising ten years ago using cationic liposomes and lipid nanoparticles. You can see that their maximum tolerated dose is less than 1 mg / kg, and these programs have all failed due to liver toxicity. The second generation, conditionally cationic lipids, they were transferred to a greater or lesser extent, and some of these programs were successful and will lead to the approval of drugs, but all goals are the liver. As lipid nanoparticles accumulate in the liver, you will see dose-limiting toxicity if you are not using a neutral lipid composition. Then you can see the work using a neutral lipid composition like ours, and they could not find the maximum tolerated dose in one study. Based on our studies of non-human primates, we expect our particles to be equally tolerable.
We are currently evaluating various constructs to find out which one is best used on humans, and we obviously talked about this at this conference - the creation of biomarkers that are good indicators of clinical trials, as well as in animal models for evaluating effectiveness. We really want to talk with any scientists who have a good biomarker. We have groups of mice in which we look at the lifespan and health of these mice. We are thinking about moving into the clinical phase when we get the GMP and GLP toxicity analysis .
Therefore, I am going to switch to cancer, because it is our main way to the clinic. My main job is studying prostate cancer . One thing that really intrigued me with the connection between aging and cancer is the activation of the p53 pathway. The p53 gene is the most mutated gene in cancer, and there are many types of cancer that have a high mutational load in p53. I take the prostate because it has low rates, on average they make up 10%, most prostate cancers are low-mobility. As soon as you get metastatic disease , this mutation rate is over 50%. Therefore, it is a good goal in the treatment of cancer. Although p53 protein itself has not yet been targeted in cancer therapies, I believe that they are very promising. In p53, you can get two types of mutations. During cell replication or mutations, they activate p53 to either repair the damage or undergo apoptosis. Therefore, the cells either mutate or get rid of p53 to bypass it. As a result, the actual activation paths are very regulated. So can this activation be used to kill cancer cells?
I skip all the information in vitro , because I have only three slides left, and I want to show you some information in vivo , as it is really important. In this case, we use very similar constructions, as I showed earlier. There is a developed p53 promoter that drives the suicidal iCas9 gene, placed in a neutral nanoparticle. In this example, we grow giant prostate cancer tumors in immunocompromised mice. Mice NOD / SCID line . We grow them up to 500 mm 3 . We make one intratumoral injection of nanoparticles, wait three days, and then do a systemic injection of a dimerizer. We saw that most tumors shrank 90-95% in 48 hours - and this is not surprising for intratumoral injection, but I was very happy because it means that our plasmid successfully worked in most tumor cells, which is very interesting.The real proof is the ability to make systemic injections, and we conducted these studies. This is just an example of four mice in this group. We grew these very large tumors, growing them to a size of 500 mm 3 . In this case, we make four daily injections of LNP into the tail vein, and on the fifth day we give them one dose of the dimerizer systemically. Again, we saw remarkable results: a tumor reduction of 50-98% in just two days. This led to a significant increase in survival after a single injection in these animals. On average, in six mice per group, tumor volumes are reduced by almost 70%.
A thing that is very important: primary tumors do not kill patients in most cases. This is a metastatic disease, so we are very interested to know if we can cure metastatic cancer. Obviously, we will take such cases in the first clinical trials. Thus, we have made a number of models studying the ability of LNP to control metastatic disease, in this case we have a model of systemic metastasis of prostate cancer and a model of immunocompetent melanoma . In both cases, in the reusable mode, we were able to effectively control this disease.
We are on the right track. Clearly, Oisin's promising goal is to develop tools that kill senescent cells. But I am sure that in the short term it is very important, not only for nanoparticle technology, but for the entire platform, to show its safety and effectiveness in the clinic. So, we have already developed clinical trials of phase I / phase IIb for cancer treatment, and now we are preparing to test the toxicology of GLP, which will facilitate these studies. We hope for a CI per person in early 2019. We are pleased to accelerate the use of this technology. I will also mention an important thing: there are many types of cancer in which it can work. In Canada, we can conduct Phase I trials with all types of cancer, mainly colorectal, prostate, lung, and others. We will seek the greatest success in treatment and the most significant cancer in order to be able to expand this group, and then perform phase II.