Dr. Vlad Kalinichenko considered the use of novel pharmacologic treatments that may prevent BPD and/or more effectively treat the manifestations of BPD.
So with that, we are, we're gonna move from clinical to mode of translational. And I would welcome Doctor Beard Kinchen Coo that he is um our director of Phoenix Children's Health Research Institute. He has recently joined us from Cincinnati. He has multiple grants and um we are really excited to um hear next talk about emerging therapies for prevention and treatment of BPD. So please welcome Doctor LA. Um It uh thank you very much for your introduction. Uh So I'll um here, I would like to say that uh my uh finding is uh uh comes from heart lung and blood Institute. So I don't have to disclose it and uh I would uh like to thank Ken who nicely introduced um BPD with Pulmono Vascular disease. And uh I'm kind of bringing back pretty much similar slide from uh Steve Omans to view uh and uh um the just to make sure that you guys, ok. So it's supposed to be around decreased um growth. But uh I guess we would have some shifts in the way. Uh that's why it will go along. But, ok, so about 8 to 10 cases of bronchial pulmonary dysplasia are associated with this, uh, decreased dose of pulmon recla, which is called neonatal lung computer Genesis. And that the one, um, uh, that the cases which with so-called, uh, vascular component, um, most severe cases of PB D and what I'm gonna say later is, uh, related only to that 8 to 10%. And I'm not going to talk about, uh, clinical trials. I'm not, uh, going to talk about off label use of forge proof medications, but I'm gonna kind of uh jump a little bit in the future and I'll tell you what is the vision, uh how um the treatment of um neonatal pulmonary vascular disease in particular DPD uh would be progressed in the uh next few years. So, um the pulmonary vascular disease, a newborn, of course, uh BPT or already been uh extensive in a talk about here. And uh of course, there is a uh C DH, which we didn't talk about, but that also subset of patients which are associated with this decrease of your genesis A CDT VA V diss with an assignment of preliminary veins uh with the um uh associated with Foxon mutations. Uh I was fortunate to uh work with uh Doctor Ro Costa in Chicago who discovered Foxon genes. So, my lab been uh doing a lot of research in Foxon and of course, congenital acino dysplasia was to be exer mutations and uh congenital suffocant abnormalities uh with mutations in Suha associated genes all these diseases are um pretty much have uh potential um for um the use of bronchiogenic therapies. And that's where I am just gonna uh talk about most. Um in my uh it's also shifted but it doesn't matter. Uh So the um it cellular pathway which uh stimulates your genesis is uh vegf signaling pathway. Begf is secreted by multiple cells including the gel stromal cells in the lung and it act on thyra in kinase receptor fok one or KDR on humans, fok one and mice and uh to stimulate uh signaling cascade. Ultimately going through uh the this uh um the cascade of transcription factors start three F one and FF one which regulate each other and form amplifying lobe. So this transcription factors up within the cell, they go to the nucleus and regulate the expression of multiple genes shown here. Uh Gene is important for um the um apoptosis for um proliferation and ultimately, it's increased inio genesis. So, not surprisingly, foxy, one gene is here in this uh amplifying loop is the cause of a capillary dysplasia and that is develop mental deficiency in the uh hormone recap. So, my lab been very active in finding uh Foxon mutation in uh A CD and TV patients. And here is our paper in blue journal which we published a few years ago when we describe uh 29 cases of A CD and TV with 24 new mutations. And altogether we now have about 200 mutations um which are all all Deasy. Uh and uh the this mutation basically cause MA CD MD V. And if you talk about leading experts in the uh in the field, like Pao ST Kviz from Way College of Medicine, uh who discovered the majority of that mutation. So, uh there is more than 90% all, all A CD and DD cases are linked to fox mutations. So, uh some time ago in my lab, we took one of the A CD mutation and knock it and to focus upon gene locus. And here I will show you the newborn mouse lung with red color here, perfused lung capillaries. You can see here that the lung capillaries go all the way to uh uh pleural surface and uh uh that is uh um necessary for efficient gas exchange. And here is the OK. OK. So, uh and here is the mutant of the mouse which favor Foxon uh S 52 F Foxon mutation uh no to mouse gene locus. So, the mutation is uh this region is evolutionary consort. And you see here, the red capillaries only in the proximal region around Fairway. So not even going to uh the uh the capilla um to, to IO region, not surprisingly, there is no religious exchange there. So, um this is to kind of explain why um um mice with a CD and DV. And of course, patients with a CD and TV, have so severe um uh the uh defect in oxygen action. So, effort to uh increase pulmonary angiogenesis have been weighed uh previously. And uh we's lab with uh also when uh Bernard Tibo was in uh Colorado, uh they tried to develop Bron um um therapies through uh injection of bronchiogenic growth factor like BGF and gela and one stem cell factors. So that um hormones and growth factors are effective in rodent models, but they cannot be translatable to humans because they have a lot of side effects. So the recent advance in bronchiogenic therapies have been done uh with uh looking at meshe stromal cells or M ECs or EXOSOME from M ECs to try to understand their role in um Aron appointment redia with two current ongoing clinical trials, one with uh the DEA in um uh Canada looking at at MS CS therapy in uh uh BPD and another one in Boston with stellar Cambrian looking at the effect of exosome from MS CS uh in the effect of uh uh in the patients with BPD. And the idea is that these cells don't really integrate into the lung tissue, but they secret a lot of uh um factors and uh micro and exosome and lots of them have prone neogene effects. So effects here is basically on degeneris. So in my lab, we've been um trying to develop different avenues how to increase angiograms. And here's the kind of a few directions which we explored in the uh last few years. So, one of them is uh doing um high throughput drug screens or small molecule compound uh to with the idea to identify prone neogen genes. And if you are interested on this site, you can look at uh our recent paper in uh uh blue journal which just published which with identification of some f small molecule compound as a uh prone eogene uh small uh small molecule, which potentially can be used to increase angiogenesis in uh BPD and CDT V patients. We are also interested in the gene therapy here. We've been quite active and I will stop on each therapy a little bit later. And we develop nono particle delivery system which can bring um uh non integrating expression vector to the cells of the lung with very high efficiency. And we published uh multiple manuscripts uh demonstrating that uh delivery of all these three genes which I've been talking about from using the transcription factors have actually um with a nice uh the clinic or benefit on the clinical ones. So um the another idea is to use cell therapy. So you know that there are cells called in theel vital cells. These cells are very interesting because they are the ones with the, the first die after um the and oxygen therapy. So they are the most uh most uh sensitive to hyperoxia. And uh the cells can differentiate in the end the failure in the cells And of course, uh replacement of the cells would be a nice idea for uh severe cases of BPD. And uh of course, uh this uh recent mecca in regenerative medicine, with the question, can we generate the whole lung tissue in the uh somewhere in the um watch animals on the farm. So we can potentially create the lung for transplantation. So, this is something which is I would uh talk about at the end of my dog. But uh this is a future regenerative medicine, not just lung regenerative medicine, but any organ regenerative medicine. All right. So one of the uh kind of brief summary of our paper in blue journal published this year, we identified town fair small molecule compound. And uh what we did was we looked at the um small molecule compounds which can disrupt interaction between v of one transcription factor which is prone biogenic and causative ocular sasia with HT one E three ubiquity and ligase. So, ubiquity and ligase bind to proteins and uh ubiquitin them and basically target them for deprivation and protos soes. Uh a lot of A CD and PV cases uh uh due to loss of one layer of Fox at one. So another layer is still normal. So the idea is if we can prevent uh degradation of existing Fox at one protein produced by um endogen allele, can we have a clinical benefit of that? So, uh this small molecule compound, disruptive interaction and actually results in stabilization of Rongen foxy. It's been done through high throughput, scream of uh 50,000 compounds followed by Mastic screens. So it's ex extensive exercise and the lines and the animals and the main effects are it increase uh angiogenesis and Vitra in increase uh in mouse and human. The dal cells, it also uh results in survival benefit in alveolar display mice mouse models which I showed you previously. It also results in uh increase in vio genesis in vascular organ who is derived from induced prepo and stem cells. So we see D MP D patients which have a loss on Fox 11 fox, one. So um future direction from this studies, we're interested in toxicity studies using the clinical model shows but then go to Montes and we're also interested in the modification of the structure because this chlorine we don't like very much because it can be reactive with oxygen which patients would get through oxygen therapy. And we, we just recently place it with uh uh fluoride and uh hope and we have a similar effect and that's what we're gonna be moving. And that also uh there is a four inks, maybe this compounds were hydrophobic. So what we are doing here is we are developing nanoparticle carriers to deliver this small molecule compounds into the uh lung and the paal cells in the Eva. Ok. So moving to gene therapy and uh this is very interesting. And uh so nanoparticles are um delivery vehicles which uh can be used potentially for multiple things. So for gene therapy to deliver non integrated expression vectors, for drug delivery, to deliver specific drugs into specific cell time. And using this, you can decrease off targets effect. And uh compared to uh the um for example, um to um systemic administration, that's very, very um uh good for drugs which have a lot of side effects. And of course recent uh uh success story with development of vaccines. So, you know that Pfizer and Moderna COVID-19 vaccine, this is nanoparticle which deliver RN A. So tells you two things. Number one nanoparticles are very effectively useful in the medical field. And number two, they are quite uh he good uh safety profile, at least many of them, but a lot of studies are needed for that. So we d uh we developed uh about three years ago, we uh we developed a nanoparticle formulation called uh uh Pay ma P A pay space for um P in the mean and P for P in me the case to a case uh um three aces. And uh so um the what is we found here that this particular nanoparticles can deliver um the expression reactors into the cells. And that is the flowy dory from the mouse lungs and which we take different cell types and see in which cell types. Uh um no, our nanoparticle end up and you can clearly that very nice target of cells here. And uh uh we demonstrated these nanoparticles, nontoxic and highly effective mouse models of bed and A CDT V. We are now moving uh this particular status to monkeys. Uh hopefully, we can get good uh toxicity profile uh on our road, try to get the things. So the, so um the nanoparticles dock to the A L cells and uh because it has a negative charge, nanoparticles are aic and that gets internalized uh inside of the cells. So they fuse with endos, then they fuse with woes and basically, they uh pump proton in. And what happens is the RSO breaks down and uh the um expression vector comes out and then depending what you're delivering, you can deliver uh RNAGM ASE or AC ASE. So you can even either increase or decrease gene expression. So um the uh we published manuscript and blue journal three years ago in which we used um the uh mouse uh DPD model which is uh um in mice, it's a treatment with uh a neonatal hyperoxia which would give seven days after in between uh postnatal day one and I seven nanoparticles are given A P two through a facial vein of neonatal mice. This is pretty unique job and they have the microscope because as a small babies, we have a mice, the one we by the small for, especially for intravenous injections. So here is uh uh what we found is that not surprisingly hyperoxia poison oversimplification and delivery of uh A FM one or F one expression vectors are pretty improved. Uh the um size of alveoli and improve lung function, uh which we um measured by uh flex. And uh as a result here is the PV loop area. And if you're interested in other functional biochemical androgen expression analysis, you can look at our manuscripts, it's all there. Uh And uh so it basically tells you that delivery of prone Gegen transcription factors can uh prevent development of BRCA preliminary dysplasia in the mouse model. What we are now, U I used this particular paper as a um the preliminary data for Ro one grant. And now we have found it next uh for years to look at whether uh this um uh treatment can be, can improve, uh can actually treat BPD in which we are extending our hyperoxia period to two weeks. And these nanoparticles would be given after um the b pulmonary dys dysplasia over deform. And we are looking at variety of factors including pulmonary hypertension weight and life. So which is important uh for this particular one. So this is pretty mild exposure and preventive strategy, not clinical relevant. But uh we, we, we're gonna go with more severe uh uh effects. So here is uh looking on that manuscript, you can see here, this magenta dots which are in the cells labeled with PM one or CD 31. Uh And uh that is green comes from GFPGFP is green fluorescent protein which is uh uh encoded by a plasmid delivered by nanoparticles. So, what does it tell you if we have expression of GFP that the plasmid was delivered specifically to the A L cells? And uh the um uh and a plasma not degraded and uh GFP was produced, transcribed, translated and correctly folded to produce functional fluorescents. What we also do, we can do flow cytometry and look at how many details also targeted by nanoparticles. And this experiment is 80% and that is uh our control uh with no injections. So this is a very, very nice number considering that uh it basically um uh we are targeted specifically in basilio cells. So we can look at PC R looking on exogenous F one RN A and we can see it's been delivered to the lung. Uh We also, most importantly, we can uh look at a capillary network and uh uh visualize that quantitate and to show on the delivery of we focus and one fon um plasmids can improve uh um and uh neonatal angiogenesis here is in 3D staining with diu area which is marker fundal cells or we can inject in the L before which is basically a way of perfuse capillary only because it binds to Luminal shorts of fundo cells. And uh so basically, it tells you that we increase angiogenesis in Eva uh through nanoparticle therapy. And what is interesting if we isolate and the paleo cells containing nanoparticles and compare them with the ones which do not have nanoparticles. And look at how many uh cells in the different phases of um in the cell cycles are present in the um one versus the other population. We can clearly see that only the population containing um the nanoparticles uh with the bronchiogenic genes, we have increased and the proliferation clearly uh saying that uh in at least in the mouse mo in the mouse model of BPD, um the delivery of Fox M one F one expression vector directly into circulation, stimulating el and your gene and mouse model of DPD delivery has to be intravenous if we deliver it in the tr uh this another particles do not go even to epithelial cells not to mention to endothelial cells. So it's very, very specific uh trend. So now we're kind of making the next step and the next step is delivering um getting another nanoparticle formulation which targets only one. So what I told you before, this is the specific nanoparticles but goes to the cells everywhere in the body except brain because they don't across the paper. And every time we've been uh having conversation with the companies and uh of course, we get the reviewer comments from our paper, but they say they go everywhere to the PC. So there would be a big potential for target effects. So now we have another formulation which we published here already one month ago. And this is one of the top nanoparticle journals by a materials. And here is a new, new permeation fluorinated the PB A EPA PG nanoparticles which contain uh three different uh nanoparticle polymers. And uh these nanoparticles go on only in the lung tissue as a a very few nanoparticles end up on different tissue. This is uh orders of magnitude and by on a large scale. And uh this is called by the imaging and the plasma is luciferase. So, this is luciferase uh which are delivered through the um plast. And another method we use the micro CT, this is called by the imaging called iris. And this micro CT also show you signal coming from the lung only but not through other cells. And when you look at this lung and look at the with the fibr meta negative cells through flow cytometer using multiple markers to identify cell types. This is very nice uh signal from endothelial cell. And uh if we are looking which endothelial cell actually get a nanoparticle or nanoparticles, which are here in the, the blue dots in alveolar regions, none of them in the uh vascular of watch arteries or veins. So, we basically all this 90% of cells coming from the um the um capillary in theel cells. And of course, recently, we know that uh the capillary in theel cells come with two flavors, capillary, one or gaps, general capillary cells and capillary two which are um aeroc sites and both of them are targeted here with a very high percentage of. So we are delivering um the uh our plasma specifically into one capillary enden cells without targeting any other cells in the body. And now I'm using this preliminary data to get another big grant from my age, hopefully kind of weight next step for the treatment of PPT. But with a new approach, uh so cell therapy is basically an interesting approach as I told you, which would be based on the transplantation of endothelial progenitor cells into the lung. And uh so what is interesting here is that um in order to identify the endothelial progenitor cells, we need to understand what are endothelial cells in the lung. So we are using method called single cell sequencing in which we are, we can sequence multiple thousands of cells coming from the lung. And that is paper from a Stanford group which they um basically discovered this capillary one and capillary two cells c two or airy. They are present. They are very big in the paleo cells which are spinning several alveolar region. They are involved in just exchange most because they form the uh type uh membrane with the type one epithelial al piel cells. And this is also another uh group of uh uh endothelial cells, general capillary cells, which are far less complex, but these cells are endothelial pregenital cells, which has been demonstrated by multiple groups now including my. So uh the uh OK. So here is supposed to be showing here. You can see here, alveola capilla or gab cells have like at least two populations. And we were interested, what other sub class application would we have? And that is very interesting that this second population of cells, this is neonatal lung in the mouse. You can see here that that's where fif one transcription factor is expressed, which is biogenic. That's where another um marker from the th progenitor cells of heat is expressed and that's where proliferation occurs. So that is on the thal progenitors. And we can use this sophisticated computational analysis to predict that this uh folk have one positive CT positive on the AC would differentiate into arterial viol and uh uh mature capillary cells. And uh uh of course, the prediction is nice. A lot of people now predict what can happen from riva. But uh to prove it, it's far more complicated. So which we did in a paper published in a blue journal two years ago, what we did was we knocked out GFP into fox F one endogenous locus fusing this with uh uh so basically GFP would express only in Fox if one producing cells. So we can isolate them and look at the function uh rather than just describe what gene expression do they have. And uh we spend a lot of uh time showing that our GSP comes from the same self as uh um uh fox one antibody identify uh endogenous protein. And that's the early embryos in which Fox one expression is whatever passed from uh different organs. So, of course, by flowy dome, we can isolate this population. This is our double positive cells which we uh can generate. And uh uh we also give colony information as say to demonstrate the fon positive CT positive uh endo cells have a large colonies, suggestion that they have colony forming and they are potentially in the brain of the cells. And so what we did then we crossed this mice into mice which contain g meal. So we have a two red and green label. And then we saw the cells uh based on this labels and inject them with the vascular of the uh the uh ff one deficient mouse, which I introduced previously because they are the most effective way to check for angiogenesis. And then we trace them who they become. And here the result before uh injection, they know on the daily progenitors and now they become arter and the cat cells pretty much demonstrating that these are in the progenitor cells. So what they do is after transplantation, they integrate into the uh perfuse lung was shown here with the blue, which is uh labeled of um comes from isolation before and that the cells proliferate and expand they form a colonies. And uh this is a very nice way to prove that we are getting uh extra progeny of uh the uh injected cells. And most importantly, we have uh by far robust capillary network compared to the uh untreated mice or mice. Three, the salin and increase angiogenic is actually causing increase in oxygenation, which is very nice and throwing function. And it also increased that the increased alveolar size. And that's consistent with the, with the idea that angiogenesis alveolar organization is a process which are very tightly coordinate. And we also look at one function to see uh that and to show that a transplantation of uh this material progenitor cells uh does increase lung function that's demonstrated by B pairing and L compliance as uh determined by um flex small, small animal vent. So we identify this interesting population uh which are pulmonary. So uh which are a result in the pulmonary circulation which will cause now, ct positive, focus upon positive a capillary display or C positive, focus up on positive uh general cap C which can differentiate the multiple cell types. Uh We've proven that and uh that these cells can be affected in the mouse models. And we now interested to look at toxicity p ability of the therapy in preclinical models and to identify tissue sources for potential transplantation of end progenitor cells. And we have pretty good idea and we're working on that, that we can get the placenta from procuring kids to isolate this cell. So, and these cells are functional So uh the idea would be if we can use it to center of procuring heat with a high risk of developing PPP. If that particular they own cells can be used for cell therapy to prevent BPD or to um uh decrease the incidence of PD. So, and finally, uh I spent two minutes here, a generation of embryonic stem cells derived tissues. So this is a far more complicated than I just previously spoken. So uh the idea is you inject embryonic stem cells into Blais of the embryo. And uh what you do is this embryonic stem cells are going through developmental stage because the embryonic, the this plastic would be implanted in the viva into the pregnant female. And uh all these embryonic stem cells give rise to the lung. And that is uh just uh to a demonstration that uh that to create chimeras. So the chimera is the um animal which have uh uh cell types from the um from the one animal and another and they coexist together, right. So uh the this particular idea can be used for organ regeneration as the uh the um paper from naka slab demonstrated. And then what they did was they injected uh rat embryonic stem cells into the mice which lack a PDX one gene. And PGX one gene is the master regulator of the pancreatic develop. And so, without PDX one gene, there is no uh pancreas at all in the uh knuckles mice And uh so in BRYN stem cells which are donor go to this area in which endogenous embryo cannot develop pancreas and then they make pancreas. So they've been able to generate rat pancreas and the PTX knockout mice and mouse pancreas and PTX knockout rats, which is pretty cool if you think about how they can be used for human. They suggested this and improve with that experimental in the nature paper that what they do is they take diabetic mouse, they make induced prepotent stem cells from this mice. They injected them into the rat embryo and actually make rats with the mouse pancreas being created by the patient. I I call it patient mouse specific uh pancreas. They, they isolate pancreat from these rats and then they injected back into the mice and the rest to diabetes. So uh the idea is here is that you can use your own cells. And if you can generate organs somewhere on the farm, that would be really nice to uh have your own organ grown um in the on the farm, let's put it this way. So I'm not saying this experiment can be done now. No, they can. So uh two years ago, my lab together with two other labs, one bill from Caro's lab from Columbia University and uh Kitahara S lab from Japan generated uh lung tissue in the uh in the mouse embryos which lack um with lung A genesis. So in our case, we use the lung. Uh We use the um TT F knockout TT F uh uh or nex 2.1 mice do not develop lung or thyroid. We injected uh donor embryonic stem cell and we generated uh donor lung and donor thyroids. And uh they were uh very, very, nicely formed uh proven by multiple um uh um results. So, uh and what we are doing now, what we are trying to do because it's never been done yet to generate mouse lung in red. So at that, we inject embryonic stem cells into red lo the systems. And we are creating this uh animals which have both mouse and red cells and uh very nice integration of the cells. And what we just to summarize which we've been able to do besides making mouse lung in the mouse, which we published in the um blue journal and generate and the progenitor cells from uh from embryonic stem cells, which are already published from blue journal. And we recently generated whole bone uh mouse bone marrow from embryonic stem cells in the red. Uh You can look our way live paper and now we have a paper in the blue journal in which is uh pretty much um wing mouse lung in the red. And uh what we do is we use genome editing technique to knock out and connect 2.1 gene in the ray at all sites. And then we do a transplantation and show the pregnant rate, then we harvest blastocyst and the blastic stage, we inject now donor cells. So at this stage, we knocked out the gene important for lung development. And then we uh complement this embryos with the donor cells which do have that gene. And then we transplant it back um in utero and we make a rat carrying mouth lump. And you can see here that genome editing of the rat of the gene. This is tha cavity, you have a uh ventricles arterials and no lung, right. So this is a control, it's not a nice lung and uh present if we complement that animals with stem cells here, the lung appears and the lung are pretty good. Have alveola alveolar sacs that have vascular full of eys, it have all necessary cell types. And most importantly, it is green. The green means that comes from the mouse and all epithelial cells which express 2.1 pretty much comes from the mouse cells. And we even do single cell sequencing showing that all cell types can come uh can are present in the um this particular um lung created from the uh re and bruni stem cells in the uh in the uh from miles embryonic stem cells in the red and look at that numbers. So we are getting 98.6% of mao cells in the whole lung and only 1.4% of red sox, which are endogenous. So that would be transplantable lung if we can get it uh applicable to him. So, of course, these experiments cannot be done for a variety of uh ethical, religious, political and about by seeing of other reasons. But we need to pretty much be ready where uh the uh web that can be available for us to move forward. And we need to protect our technology and organs first. And we also need to prevent integration of the uh donor cells into um brain sensory organs, reproductive organs, which we cannot do at this moment. So, and finally, it is just uh road to uh pronic therapy through uh pronic genes through gene therapy with pro uh the biogenic small molecule compounds. So, gene therapy with uh Bango genes, cell therapy was the data brain cells and generation the whole edition. And uh so it's my funding. I have three large ro one grants from NH MP I and lots of collaborators in USA and all over the world. So lots of people have been involved, Benjamin.