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OCAT
La conferencia de ayer. Varios datos y la noticia cojonuda que llevábamos tiempo esperando: 6 perros con entre 1 y 6 fístulas que acabaron sin ninguna.
Second Cell and Gene Therapy Conference, Philadelphia, PA, September 9, 2015
Erin Kimbrel, Ph.D., Ocata Therapeutics
I'm very excited about being able to share some of our work at Ocata Therapeutics with you today.
Unlike adult stem cells, pluripotent stem cells have the ability to self-renew indefinitely, but also to differentiate into virtually any cell type of the body, many of which can be harnessed for therapeutic purposes. What you may or may not know is that the first wave of pluripotent stem cell-based therapies are actually already in clinical trials, the majority of which are ES cell-derived retinal pigment epithelium, and these are being used for ocular indications such as age-related macular degeneration and Stargardt's disease; however, other cell types are also being used in clinical trial for cardiac progenitors, pancreatic nuclear and also oligodendrocyte progenitors.
Now one of the biggest hurdles in developing pluripotent stem cell-based therapies is the risk of a pluripotent cell contaminant in your differentiated cell product so the risk of tumorigenicity is a great fear. However, the first report suggests that the clinical use of ES-derived RPE is safe, so you can mitigate the risks of tumorigenicity. So a couple of these publications show that among the first three trials, there are a total of 22 patients, they found no tumorous ectopic tissue formation or major adverse effects within one to two years after transplantation. So this is at least encouraging news that pluripotent stem cell-based therapies can potentially be safe for usage.
Now at Ocata Therapeutics, we not only have our ongoing RPE clinical trials, but we also have other ocular products in the pipeline such as photoreceptor progenitors for diseases such as retinitis pigmentosa and ganglion nerve cells. But what I'm going to talk to you about today is another cell type called HMCs, which we are developing for use in autoimmune diseases. So I will share with you a little bit of the characterization of what HMCs are, some in vitro data and then I'll go into some of our preclinical models.
So HMCs are hemangio-derived mesenchymal cells or mesenchymal stem cell-like population derived from ES cells or iPS cells. Starting with your self-renewing pluripotent stem cell population, you can undergo a brief MRA body differentiation followed by this intermediate cell type called the hemangioblast. We then use this intermediate cell type to go on and produce the HMCs, or ES-derived MSCs.
The therapeutic potential of this cell population is tied to their secretion of various bioactive factors, so in response to environmental stimuli, these cells will secrete a variety of different cytokines and growth factors. Ones that are involved in immunomodulation can affect both cells of the innate and adaptive immune system, and we really feel as though it's this property of the HMCs that give rise to their therapeutic potential. HMCs are very similar to tissue-derived MSCs and some of the properties they have. Among the factors that they secrete, they produce indolamine 2, 3-dioxygenase and prostaglandin-2 which are involved in the inhibition of T-cell proliferation and antiinflammatory properties. They also produce exosomes which are filled with a variety of different micro RNAs, RNAs and proteins that can affect cells with the immune system. Looking directly at their inhibition of different cells in the immune system in a dose-dependent manor, we see that HMCs can inhibit T-cell proliferation. They also can inhibit the maturation of dendritic cells, so in response to maturation stimuli, dendritic cells will up regulate expression to CD83. Co-culture with HMCs can reduce them. Likewise dendritic cells will secrete IL-12 p70 in response to maturation stimuli, but co-culture with the MSCs or HMCs can reduce this. These properties that I have just described are very similar to adult MSCs, as well as the expression of various cell surface markers. They are very similar to bone marrow and umbilical cord MSCs.
The MSC populations that are being used in clinical trials are all derived from adult tissue sources, the vast majority of which are bone marrow MSCs. However, as many of you may know, some of the MSC trials have lead to rather disappointing clinical trial results, and that is probably attributed to the fact that there are donor-dependent variability in the cells and also that the cells tend to lose their therapeutic potential if they have been expanded in culture for a very long time. So HMCs may be able to circumvent some of these issues that the adult MSCs have. We have some data that suggests that they may be more potent than the MSCs currently used in clinical trials and I will share some of that with you. The HMC manufacturing process is also readily scalable. By virtue of the fact that you are starting with a replenishable starting material such as embryonic stem cells, you can derive an unlimited number of early passage MSCs thereby avoiding the loss of function that you would normally see with extended in vitro culture.
So some of the molecular differences that we are finding for our HMCs is that they have faster migration than bone marrow or umbilical cord MSCs. This is an in vitro scratch assay showing that after a 6-hour time window that the HMCs will migrate into the red zone, or the empty space, faster than the umbilical cord or bone marrow MSCs. In relation, they also have higher expression of certain migration-associated proteins such as CD24 and MMP9 and this is an ongoing area of investigation. They also have a lower expression of the pro-inflammatory cytokine IL-6 as shown here by an antibody array. They have the lowest scores in comparison to umbilical cord or bone marrow. Not only do they express a lower amount of IL-6, but we also have in vivo data showing that the HMCs can actually reduce the level of disease-associated IL-6 and that's contributing to their therapeutic effects.
So now we'll go into the preclinical animal models using our HMCs, first in comparison to adult-derived MSCs and also proof of principle in other experiments. The first one I would like to share with you is the EAE model for multiple sclerosis. It's an induced model, whereby you can induce paralysis in the animals, and what you're looking at is the clinical severity scores. The higher the number here, the more severe the paralysis is. And what you can see is that in comparison to PBS controls, bone marrow MSCs had very little therapeutic activity and were not able to reduce the disease severity. Umbilical cord MSCs did have a therapeutic effect, but this effect was lost over time. However, our HMCs, either live or irradiated, were able to inhibit the disease process and lower the disease severity for the extent of the time period. And also in this EAE model, we looked at the migration of our HMCs into the inflamed CNS tissue. So you're looking at time focal images, either volume or iso surface rendered showing that GFP labeled HMCs were able to extravasate out of the vasculature and into the inflamed tissues. However, bone marrow MSCs also labeled with GFP, while they homed to the inflamed tissue, they were not able to extravasate out of the vessels and they remained trapped in the vasculature. So we think this migration phenomenon that we are observing could relate to improved therapeutic activity of HMCs.
Jumping to another autoimmune disease model, we have looked at the effects of our HMCs in lupus-prone mice. Here we are using the New Zealand black and New Zealand white F1 generation mice, which develop immune complex mediated glomerulonephropathy and they have a reduced lifespan because of this lupus nephritis effect. So as the control animals will die by around 35 weeks of age, if they are treated with either a single or two doses of HMCs, we can see they have a significant effect on the survival of these animals. They also can maintain kidney function as evidenced by blood urea nitrogen, serum creatinine and proteinuria levels. You can see that as the mice age the kidney function begins to fail; however, if the animals are treated with HMCs, we see a reduction in this disease-associated process.
If you look at the histopathology of these animals, you see that going from a pre-diseased to a diseased state, you undergo a process whereby there is hyperproliferation of mesangial cells and a loss of the Bowman space surrounding the glomeruli. When you treat the animals with the ES MSCs or HMCs, you see preservation of the kidney architecture. In addition, you do not see the protein casts that normally appear as the disease manifests itself if you treat with the HMCs. You also see a reduced number of infiltrating T-cells when you treat with the HMCs. A blinded veterinary histopathologist has scored the animals and what we can see is that in three different categories, the glomeruli, protein cast and interstitial scores, the HMCs had this therapeutic effect.
A third model that we have investigated is the EAU model for uveitis, which is an inflammatory eye disorder. You could see in the untreated animals that undergo the disease process, you see an infiltration of immune cells into the vitreous cavity and also a disruption of the retinal architecture. However, treating the animals with the HMCs you see much fewer infiltrating immune cells and preservation of the retinal architecture. Also, scored by a blinded histopathologist, there is an improvement in the histology and funduscope scores.
The last model I would like to share with you is actually a large animal model using canines. In collaboration with Tufts University Cummings School of Veterinary Medicine, we treated six dogs that had cyclosporin-resistant anal furunculosis with our HMCs, and we found that while the animals have between 1 and 6 fistulas upon presentation, after three months after the HMC injection there was an absence of fistulas in these dogs, which was a remarkable finding considering that they had basically undergone resistant cyclosporin-resistant fistula appearance for quite some time. Six months afterwards, two of the six animals did have a small recurrence of fistulas suggesting that perhaps more than one injection of the HMCs would be needed for a sustained effect, and yet the results were quite promising.
So the last part of the talk, I would like to touch upon the commercial scale of manufacturing that we have for our HMCs. The HMC name refers to this hemangioblast intermediate cell type that we have. This intermediate is highly expandable. It is also cryopreservable, so we can generate a large working cell bank with these hemangioblasts and then use them to generate several manufacturing lines, which provides greater consistency for our final HMC product.
Again, getting back to the issue of tumorigenicity from a pluripotent stem cell-derived product, QC lot release criteria are heavily focused on safety, so not only do we screen the HMCs for a variety of different viruses, mycoplasma, sterility, we also ensure they have a normal karyotype, and then we look at the expression of pluripotency-related genes in the final HMC product, and what you can see is there is a strong down regulation of pluripotency-associated genes as there is an up regulation of the MSC-associated genes. We can also detect the absence of pluripotency-associated cell surface markers trial 160 and trial 181 in the final HMC product.
Just the take-home message from the talk today, the HMCs are ES-derived mesenchymal stem cells that we are deriving through a prior observable hemangioblast intermediate. They are showing unique molecular properties, and they have a greater in vivo potency, at least in the EAE model, than tissue-derived MSCs. I have shared with you some of the evidence in four different preclinical models of autoimmune disease, that they have therapeutic potential. Our platform is scalable for commercial manufacturing in the capacity that we don't have to expand them very much in order to do this, so we can maintain the potency. The QC lot release criteria are heavily focused on safety and ensuring lack of tumorigenicity in the final product, and what I didn't touch upon, but which is important, obviously intellectual property surrounding the composition of matter and method of manufacturing for this product.
Lastly, I would just like to thank members of the Ocata research team, as well as our collaborators at Tufts University and UCLA. Thank you.
Q: What was the source of the human embryonic stem cells?
Erin: We used our FDA-approved MA09 embryonic stem cell line, which is derived from a single blastomere technology. It's a technology that unlike most technologies that derive the cells from the inner cell mass, single blastomere technology isolates cells from an 8-cell stage so you are not essentially destroying the embryo in the process.
Q: There's nothing to do with Geron?
Erin: No.
Q: I wonder if testing does compare it to MSCs, the effectiveness or efficacy or are you now just looking at your own product?
Erin: We made a lot of the comparisons in the EAE model, but currently we're looking at the lupus nephritis models and doing direct head to heads with umbilical cord MSCs.
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