The ACTC-Team has a plate full of benefits. The more i read this the more it sinks in. "Let this sink in"... Lanza, ASM 10/22/13
RPE
Of course we have the pigment of eye disease that is our primary project. That includes retinal pigment epithelium for the clinical trials for macular degeneration, but we are also studying a range of other cell types for a number of other diseases such as glaucoma, advanced blindness, uveitis and corneal repair. We also have our single blastomere program. We have now created an entire Master Cell Bank that hopefully can be used for future clinical trials, that would be ethically compliant and xeno free, as well as our induced pluripotent stem cell project. We are in the process right now of creating a Master Cell Bank for our Phase I clinical trial using those.
We also are continuing with our blood program and in particular the megakaryocytes and platelets, and we also have our mesenchymal stem cell program and we are currently testing those right now in both rodents and large animals. So for the ocular programs, of course you are all aware of our RPE for macular degeneration. We also have FDA approval to begin trials now for myopia. We are also working on the retinal ganglia progenitors for glaucoma. There is some interesting preliminary data on that as well, corneal epithelium for corneal disease, mesenchymal stem cells for a range of diseases, including uveitis and glaucoma, as well as the retinal neural progenitors, photoreceptor replacement and neural protection.
So just briefly, just to update you on the RPE clinical trials, of course we have clinical trials going here in the US at Jules Stein Eye Institute, Wills Eye Institute, Bascom Palmer Eye Institute and Mass Eye and Ear. We have treated now 12 patients in dry AMD; 3 have received 50,000 cells, 6 have received 100,000 cells, 3 of those includes the better vision patients, and three patients with 150,000 cells. We have also treated 9 patients so far with Stargardt's disease, 3 with 50,000 cells, 4 with 100,000 cells; one of those was a better vision patient, and two patients have received 150,000 cells. We are of course also continuing our European clinical trials at Moorsfield Eye Hospital where they have treated 9 patients including 3 at 50,000, 3 at 100,000 and another 3 at 150,000.
Early last year we published data in the Lancet on our first two patients. We have now of course treated another 28 patients and as Gary mentioned, we now are summarizing this data. To date we have not seen any major safety concerns related to stem cell treatment. There are clear signs of long-term engraftment and survival. You can see here in the left panel. That is the injection site on Day 1; you don’t see any pigmented patches. By two months you can see the appearance of these pigmented sections and that continues to increase even at half a year.
So what you are seeing is very robust engraftment in many if not most of these patients. During the one-year follow-up period, patients in both the Stargardt's and the dry AMD trials have shown significant improvement in visual acuity in the RPE treated eye. Vision in one of the patients for example improved from 20/400 to 20/40 in the first month. By contrast the fellow, the untreated eye, has remained unchanged or continued to show decline in visual acuity during that same time period.
So the next step, as Gary had mentioned briefly, we are planning to complete and publish the results of the Phase I clinical trial. We’ve already had some advanced preparation for design of the Phase II clinical trials. We have created the NED-7 Master Cell Bank. That's the line that was created from our single blastomere technology, so no embryos were destroyed. There were no mouse feeders so it is no longer a xeno product and just to give you an idea of numbers here, our Master Cell Bank is 500 vials. We took just a few of those to create a working cell bank that has 260 vials and from that we took just 2 vials to create 800 bottles of RPE and that is enough for 800 patients and we could have easily made more from just those 2 vials. And so we are currently now waiting for manufacture and regulatory sign off on that.
MMD
And we are also hoping before the end of the year, to begin the Phase I clinical trial at UCLA to treat myopia.
BLOOD/PLATELET
We are continuing, of course, with our megakaryocyte and platelet program. Megakaryocytes of course are the multi-nucleated giant cells that create platelets and pro-platelets, so we have been able to now create very large numbers of those, not only from embryonic stem cells but also from iPS cells, and we can now, as you can see in the lower left panel, get very high purity levels of those platelets.
So we have been able to show that not only the ES but also the iPS cells participate in clot formation. They also incorporated into the mouse thrombus after laser-induced arterial injury and we have been able to show that both the iPS and the ES platelets are delayed in mice that have been macrophage depleted. We have examined these platelets using electron microscopy for both the morphology and ultra-structures, and in the upper panel here you are seeing the cross section of a normal platelet. Remember they are biconcave, so there are actually two planes here. And this is a cross section of the platelets from iPS cells and as you can see they are virtually identical. You can see the alpha granules, the dense tubular system, the glycogen granules and of course all of the appropriate cellular organelles, including mitochondria.
So we are continuing to optimize our platelet production and the functionality in vitro. We are working right now on using microfluidics and bioreactive systems to scale that process up. We are continuing to test those in animals, not only for efficacy and biodistribution, but also safety and toxicology and tumorgenicity, and we are in the process of completing the transfer to our GMP manufacturing facility. So we will obviously hopefully be filing the appropriate regulatory applications including an IND.
CORNEAL REPAIR
We also have our program on corneal repair, so we have been able to make corneal endothelium to rise from our embryonic stem cells. There are over 10 million people who have corneal blindness. The cornea is the most transplanted organ and a third of all of those are as a result of corneal endothelial failure. The solutions, of course, involve the transportation of the entire cornea, but more popular and recently just the transplantation of the corneal endothelium on Descemet's membrane. We have been able to characterize these now quite extensively. We have shown that there is 01 positive for tight junctions. They have the ATP, the sodium FNNP pump staining. We have been able to optimize cryopreservation so we can now ship the corneal epithelium worldwide, and we have carried out global gene analysis showing that the corneal endothelium we created from our pluripotent stem cells are nearly identical to adults, so you can see the gene profiles very closely cluster. So the next steps here are to continue testing these corneal endothelial cells in our in-vivo model; that's a rabbit model. We are hoping to increase the purity up to over 99%. We are currently also testing and designing a hydrogel sheet that we will be not only flexible and transparent but also biodegradable. In fact, we will then test those scaffolds with the corneal endothelium in a rabbit model or cul-de-sac.
We have, as I mentioned earlier, a number of other cell types that we are studying in the eye field, including our retinal neural progenitors, photoreceptor progenitors, and also ganglion progenitors. As regard to the retinal neural progenitors, we have looked at these cells in animals that have retinal degeneration and we can look at the ERG, the electrical activity for instance of the "A" wave that represents the cones and the rods and we can see very significant rescue of their activity. Similarly, when you look at the cytology of these animals and the untreated animals here on the left, you can actually see that the outer nucleated layer is pretty much almost gone – just a very narrow layer. By contrast, these animals that have been treated with these progenitor cells, you can see the outer nucleated layer, again those are the photoreceptors, very robust rescue. And when we look in the eyes of these animals with PCR analysis, we were able to show that there was no human, the probe was negative, and it was only mouse, there were no human cells present in the eye so it was very clear that these cells were having an endocrine, a paracrine effect from a distance. And so we have now engaged in looking at the differences between those cells that are active and nonactive and we have now identified different molecules using these antibody arrays that differ, so that we hopefully can then proceed with a cocktail that will have no cell component.
This is another study that we carried out in the RCS rat, again one of the gold standards for macular degeneration and we used our retinal neural progenitors in those animals and what you are seeing here on the left is an untreated animal PDS treatment and if you stain for rhodopsin, which is for the rod, you can see that it is completely missing in those animals; however a single injection of our progenitors, whether it was just systemic tail vein injection or intravitreally, you can see here the green, very robust rescue of those rods.
We also have a program that we are pursuing using these photoreceptor progenitors. When you inject these into animals subretinally, what we were able to actually see here in the first week, in red, that is anti-human staining, those are the cells incorporating into the retina and within only three weeks you can see them moving into the outer nucleated layer and integrating.
So the next steps there will be to test these cells both in vitro and in animal models. We have collaborations currently underway with various leading groups worldwide. We are continuing to identify the endocrine and paracrine factors using different methodologies, including antibody arrays, gel analysis, mass effect, and we are also testing, again, the cell free lysates, or the specifically identified factors that we have gotten from these studies.
We are also studying the photoreceptor progenitors. We are also looking to see if we can recover visual function and retinal structure using those. We also want to test those both in vitro and in vivo in terms of using conditioned media in the secreted factors. We are also studying our ganglial progenitors and we are continuing to look at those in animals for prolonged term survival of the transplanted cells, as well as for the protection or replacement of the host ganglial cells. We are also looking at using these cells in the optic nerve regeneration model.
MSC
And excitingly, our MSC program, as you know MSCs are normally found in bone marrow, adipose tissue, umbilical cord, tooth buds and they can differentiate into fat, bone and cartilage. They are, in particular for clinical trials, immune privileged, so you don't need to worry about matching and they migrate to the site of injury. They can exert immunosuppressant effects and facilitate tissue repair. There are over 200 clinical trials that are listed on clinicaltrials.gov for a range of different indications and virtually all of those applications involve the use of bone marrow or other primary sources of MSCs.
So why use ES or iPS-derived MSC versus these adult sources. The reason is pretty obvious. We can have off-the-shelf therapy available for immediate use. We can create a virtually unlimited supply of these cells. They are very easy to derive. They can be expanded to very large numbers in vitro. In fact we can get 30,000 times more units of the ES-derived MSCs than you can get from bone marrow MSCs. They are more youthful. They live longer and we can… these hemangioblast-derived MSCs yield exponentially greater yields than other methods.
So one of the models, I just want to give you a sampling of some of the in vitro efficacy. One of the studies involved multiple sclerosis and this is in the EAE model. In the animals that are untreated, normally they have clinical score of 2-4. That means that they are partially or complete leg paralysis. By contrast, in the animals that got an injection of our ES MSCs, we saw that the clinical score was less than one and there was no paralysis.
MSC AND PAPER
Also, interestingly, when we did, in the same model, bone marrow derived MSCs, we saw that the impact was variable or minimal and those animals still showed paralysis. So just in summary here, I can't go into too many details because we’re preparing this for publication, but the ES-derived MSCs dramatically reduced the clinical score in the EAE model and this is showing both prophylactic and therapeutic inhibition. We also see inhibition of T-cell function, in vitro or in the laboratory. We also see a differential cytokine expression between our ES-derived MSCs and the bone marrow MSCs. We also see differential ability of both cells to migrate into the damaged tissue close to the CNS. So as I mentioned, we are currently preparing a publication and the review had suggested some follow-up studies which we are now completing. And so based on these data we think that MS is a potentially important candidate for clinical translation.
MSCs AND UVEITIS
Another example in the eye for instance is the use of these MSCs to treat uveitis. Uveitis is an inflammatory autoimmune condition of the uvea. It accounts for 10% of blindness in the United States, mostly in patients between the ages of 20-40 years old. In the mouse model, there is something known as the experimental autoimmune uveitis of the UAE model that very closely resembles human uveitis. And this disease state can be assessed using fundoscope imaging that is also used on other methods, and we have now completed the first two studies. Using these cells, the preliminary studies show that they are effective indeed in treating uveitis.
MSCs AND LUPUS
We also have a program using the ES-derived MSCs in lupus and also lupus nephritis. Systemic lupus erythematosus, as you may know, is an autoimmune disease that can affect virtually every organ or system in the body. There is no cure for lupus. It most often affects, for anyone who is aware of a loved one that may have it, it affects their heart, their joints, their skin, lungs, blood vessels and kidneys, it is a pretty devastating disease. Fifty percent of those patients develop lupus nephritis. This is an immune complex deposition in the kidney glomerulates that leads to kidney function failure. If you look at this picture here you see on the left, that is a normal glomerulus; here on the right what you are seeing is that in a lupus patient who has had these immune complexes. There is an animal model, the NZB mouse, that spontaneously develops lupus nephritis, which is the gold standard and it is very similar to human lupus. And we have now studied some of our cells, the ES MSCs, in these animals and we have seen that they have a dramatic effect on morbidity including proteinuria, kidney function. It protects from death and showed a very marked improvement in survival. So based on this data we also consider this probably one of our lead clinical indications in this program.
MSCs AND PAIN
So we have another program that is sort of exciting and that is using the MSCs to treat pain. So pain can actually be measured in rodents using equipment using this reward/conflict paradigm. In using our ES MSCs, we have been able to significantly reduce the chemotherapy-induced neuropathy in response to Taxol. So again, this is another potential indication -- essential candidate for clinical translation.
So I just mentioned a few of the potential applications of MSCs, but again, there are literally hundreds of potential applications. There are over a hundred autoimmune diseases including MS, osteoarthritis, lupus, aplastic anemia and of course a whole range of other systems including the liver, kidney and pulmonary diseases. But importantly, these cells are ideal for clinical translation because there is no need for immunosuppression. You can have off-the-shelf allogeneic-supplied cells. They persist transiently so no issue in terms of long-term tumorigenicity for instance, and they also can be irradiated and still have functionality.
MSCs IN CANINE INDICATIONS
And finally I just want to mention that we are also studying these MSCs in the canine indications. We have a program for treating orthopedic disease including osteoarthritis and disc disease. We are also studying several inflammatory diseases including sepsis and acute pancreatitis and we are also looking at a number of immune-mediated diseases including irritable bowel syndrome disease, chronic hepatitis, glomerulonephritis and Crohn's disease. So we have filed, as Gary had mentioned, an INAD with the FDA to treat 10 of these indications listed here and again, naturally induced diseases in large animals such as dogs provide an excellent model of human conditions and they may provide a more robust assessment of safety and therapeutic end points than we would obtain from inbred rodent models. We have initiated a collaboration with Tufts University School of Veterinary Medicine. They have a very large population of suitable animals and they are world experts at regenerative medicine. We have already received IACUC approval to begin the first of these studies and in addition to the obvious veterinary applications, these studies will help inform and optimize our human medical trials.
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