(upbeat music) (soft music) - We have a fantastic list of speakers in this session coming from all parts of California, including Tennessee. I think maybe some of you didn't realize that St. Jude's was part of the California yet, but proud to have Dr. Gottschalk with us. So our three speakers, our first speaker will be Dr. Tippi MacKenzie from UCSF, who'll be talking about her innovative work in terms of developing in utero cellular-based therapies for genetic diseases, in particular alpha thalassemia. And this is very exciting work because many genetic diseases and other diseases begin before the person is born, and the opportunity to intervene before the patient is born, is something that we all look forward to. The second speaker is Dr. Matt Spear, who's a chief medical officer at Poseida Therapeutics I think I got, therapeutics. And he'll be talking about their work in developing CAR-T therapy, so Chimeric Antigen Receptor T cell therapy, to fight various types of cancer and using an approach in which it can, this exciting new therapy might be given to a broad numbers of patients rather than the small number of patients that it still is available to. And then finally we have Dr. Steve Gottschalk from St. Jude's Children's Hospital, where he's the director of the hematopoietic stem cell transplant program. And he'll be talking about a very exciting gene therapy program for a disease called SCID-X1, otherwise known as bubble boy disease. This again is a therapy in which the patient's own cells are modified to give back a healthy gene and then returned to the patient, and the clinical results from this stem cell and gene therapy are simply fantastic. And I look forward to hearing an update from Dr. Gottschalk on that. So, without further ado, let's start with Dr. MacKenzie's talk. So again, thank you very much and thank you for joining us. - Hi everyone, my name is Tippi MacKenzie, and I'm a professor of surgery at UCSF. Today I will speak to you about our clinical trial of fetal stem cell transplantation in fetuses with alpha thalassemia major. So imagine a world where we could treat or even cure a deadly genetic disease before birth. You know, we don't think too much about going to the doctor for various problems we have after birth, but the concept of the fetus as a patient is only several decades old and was actually invented right here at UCSF. So did you know that doctors can actually operate on a fetus in the womb and this ability, which started in the 1980s with fetuses with various anatomic conditions helps us treat fetuses with a number of birth defects. And what we're trying to do is to move this field to now treat genetic conditions. Here's kind of a repertoire of what we're able to do in fetal surgery that involves open fetal surgery, laparoscopic or fetoscopic surgery or a catheter-based interventions. But as I said, what we're focused on is putting in stem cells or other therapeutic agents, just using a needle into the umbilical vein to treat various genetic diseases. And these possibilities exist in this decade, because of the convergence of a number of technologies. So right now we can do rapid genetic diagnosis using whole exome or whole genome sequencing. We have a much better understanding of maternal and fetal biology and our repertoire of being able to treat diseases using the cutting edge methods of gene therapy or gene editing are really exploding. So this gives us a number of techniques to be able to treat fetuses with genetic diseases. So today I'll talk about our work on stem cell transplantation, but there's ongoing work both in our lab and in the labs of our collaborators for additional techniques, such as enzyme replacement or gene therapy, there are a number of single gene disorders that we could potentially treat with these techniques. And today I'll talk about the Fallacemia is, but of course, other single gene disorders, such as lysosomal storage diseases or spinal muscular atrophy to name a few could also be treated. And the main reason to treat the fetus before birth, as opposed to waiting until after the child is born. There are a couple of reasons actually, the number one reason is that the fetus has a very unique immune system, which I'll talk about in a little bit where they can be tolerized to new proteins that they see before birth. You can also of course protect the brain before the blood brain barrier forms. And you can access themselves during their migration. For example, for blood stem cells migration from the fetal liver to the bone marrow. So here's kind of a proposed pipeline of fetal molecular therapies that we're working on at UCSF. Right now, we have a phase one clinical trial of stem cell transplantation for alpha thalassemia major. And we're hoping to expand that to other disease indications once we have more safety and efficacy data. We're working on in utero enzyme replacement therapy for lysosomes storage disorders, and we're speaking to ethics experts and the FDA about in utero gene therapy, of course only in somatic cells, not in the germline. So a lot of this is made possible by this beautiful concept in biology where cells, traffic back and forth between the mother and the fetus, and the fact that you can find fetal cells or DNA in the mother, forms the basis of the whole field of the whole field prenatal genetic diagnosis. And this is what enables us to even diagnose a lot of these genetic diseases before birth. But the trafficking also happens the other way in that there are mother cells in the fetuses. And the very interesting thing is that the fetus actually learns to tolerate these foreign cells and proteins it sees during development. So because it sees maternal cells during development, it just so happens that it learns to tolerate the mother's antigens or proteins on those cells before birth. And a lot of this biology was worked out by Jeff Mold and Mike McCune at UCSF about a decade ago. And what they showed is that the fetuses T cells, which are the main transplant rejecting fight or fighting cells, when they see a new protein, instead of becoming these effector T cells, the red ones, which they would in you or me, they actually are more predisposed to become these green regulatory T cells that confirm tolerance. And that the fact that the fetus is seeing and learning to tolerate these maternal cells forms one of the backbones of kind of the miracle of pregnancy, you know, how the mother and the fetus, what their different genetic backgrounds can tolerate each other. And then if you think about applying this to a situation where the fetus has a birth defect, that requires a bone marrow transplant before birth, you get the logical conclusion that we should be transplanting maternal cells. So these are fetuses with thalassemias or sickle cell disease or Fanconi anemia that could be cured with a bone marrow transplant before birth. And the concept that you can do a bone marrow transplant before birth had been tried some decades before. And it really only works for a small subset of disorders, but it hadn't worked for thalassemias before, but this work shows that we can potentially transplant maternal cells and that it should work if we transplant a very high dose of cells using an intervascular method and using maternal cells in particular. So after a number of years of preclinical experiments, our collaborators in Philadelphia, in Dr. Flakes lab did a seminal study in dogs where they showed that if you transplant maternal cells into fetal dogs, you get good levels of engraftment of the maternal cells, you know, between five to almost 40%. And that these dogs become tolerant now permanently to their mothers. So usually that tolerance to the mother wanes immediately after birth, but in these dogs, they show that they can actually do a kidney transplant from the mother and there's no rejection. So this is a great proof of concept to show that transplantation of the mother cells can resolve in tolerance. And in terms of thinking about these blood disorders, the goal is not necessarily a single shot cure because you're not giving any conditioning into the fetus, which is usually what creates space in the bone marrow to have a cured of a level of cells after a bone marrow transplant. So the idea is that it's a two step process. So you have a fetal transplant that induces tolerance, and then you do a postnatal booster transplant to really increase the levels of engraftment of those maternal cells to definitively cure the disease. And this concept has been shown in mice by Bill Peranteau's lab, again in Philadelphia, where they took mice with sickle cell disease or thalassemia. And you can see here after the initial in utero transplants, you could see the cells, but they weren't really cured of levels of engraftment. But then after birth, they were able to boost the levels to curative levels. So in thinking about applying this to human fetuses, we thought very long and hard about what might be the perfect first disease. And in discussions with international colleagues, we came up with alpha thalassemia major and alpha thalassemia is a very interesting disease. And I'll tell a little bit about fetal hemoglobin biology. So hemoglobin is the molecule in red blood cells. That of course carries oxygen. And in adults, there are two chains, the alpha and beta chains, but in a fetus there's just alpha and gamma chains. And a fetus that has mutations in all four of the alpha globin genes doesn't have any alpha chains. And so it makes these abnormal gamma tetramers. And these are also called hemoglobin Barts. And those fetuses get very sick because those red blood cells, they can't release oxygen to the tissues. So there's low oxygen in the tissues. And we can see this in ultrasounds where the fetus develops fluid around the liver and around the lungs. This is a condition called hydrops, which is fatal, unless you give a blood transfusion to the fetus, it's actually a common disease, multiple populations around the world, including in China and Southeast Asia have a very high carrier rate, but because the fetuses can be treated with blood transfusions before birth, we thought it was an ideal disease to do an in utero stem cell transplant using maternal cells, because you already have to do an intervention on the fetus. So you get rid of any additional technical issues. So families, interestingly, with a pregnancy with alpha thalassemia major are often counseled to end the pregnancy because of several concerns. You know, if the fetus has hydropic and they don't get any therapy at all, that's a very dire situation. The mother can get sick as the fetus gets sick, that's called mirror syndrome because the brain hasn't had oxygen during development, you can have poor neurologic outcomes and the child after birth and the alpha thalassemia, it was felt previously to be a very severe chronic disease with an untenable medical burden. So we first set out to understand, you know, are these valid concerns? Is this a disease where it's ethical to think about fetal therapy? Can there be meaningful survival and how can we improve outcomes for these families? Well, it turns out that there are multiple reports of good outcomes in fetuses with alpha thalassemia major. If you do these blood transfusions before birth, and if you do them very rigorously. So this is our series that we published in 2014 of around 20 patients. There was an international registry that was published around the same time, and then one from Hong Kong. And the upshot of all these series is that if you do fetal blood transfusions, the hydrops resolves, the mom does not get sick. The babies can be born near term or term, and they have in many cases, excellent neurological outcomes. So even if these fetuses were initially sick in utero, some of them do very, very well. And some of them can even be cured after getting a bone marrow transplant after birth. So to really understand at a larger level how these patients are doing, we've started a UCSF international registry to understand both the neurological outcomes of fetuses who had adequate transfusions and really understanding patient and provider attitudes regarding fetal therapy versus ending the pregnancy. So what happens to patients with alpha thalassemia major afterbirth? Well, it turns out it's a very similar disease to beta thalassemia major, where there are basically two kinds of therapies. You can either have chronic transfusions once a month or every three to four weeks. And the complications there can be iron overload, or now there are medicines for culation, but there can be toxicity from that. Or you can have a bone marrow transplant. And as I mentioned, this can be a definitive cure. However, there is about a 10% mortality because of all the medicines that we need to give, to prepare the bone marrow, to accept the transplants, it can be difficult to find a suitable donor. The mom has only the perfect donor before birth and patients can have graft versus host disease, et cetera. So we think that in utero stem cell transplantation may decrease the burden of disease. And that's the idea behind our fetal bone marrow transplantation clinical trial. So in this phase one clinical trial, we're giving a stem cell transplant from the mother before birth. And the way that it works is we do an initial video consultation. And if the patient meets the inclusion criteria, we do the transplant and then evaluate the chimerism after birth. So I'll tell you a little bit about our first patients. They were written up in the New York times after birth. They did very well, although they presented with hydrops at 18 weeks, they improved with in utero transfusions and transplanted at 23 weeks. And we did five total in utero transfusions, and the hydrops resolved with careful transfusions. We saw that the clinical protocol is feasible and appeared safe, and the fetal transplant can establish long lasting tolerance. So these are some pictures of how we do the bone marrow transplants. And here you see the bone marrow harvest. We select the cells and then infuse them back into the fetus using ultrasound guidance. And as I mentioned, you know, we have to do further work to expand the indications and also to optimize the efficacy. There are a number of ethical considerations, of course, for non-directive counseling and patient representation is really critical for planning the therapies. So the main lessons we're learning is that the moms can tolerate the bone marrow harvesting well. The fetuses are doing well so far, and really that patients with alpha thalassemia can be given multiple pregnancy options, including fetal therapy. And as I mentioned, we are currently recruiting for enrollment. All of this is made possible at the Center for Maternal-Fetal Precision Medicine which I co-direct with Mary Norton and our clinical arm, which is the Fetal Treatment Center. And I'd like to acknowledge both funding from CIRM which is funding this phase on clinical trial, as well as my multiple colleagues at the Precision Medicine Center, as well as our thalassemia team, particularly Dr. Vicinsky, who's a thalassemia expert. And thank you for your participation for our patients, thank you. - Hi, I'm Matt Spear, the chief medical officer at Poseida Therapeutics. We're a cell and gene therapy company located in San Diego California. Poseida has developed a broad portfolio of cell and gene therapy technologies, particularly transposon-based gene transfer such as the super piggyBac system and Cas-CLOVER, a novel gene editing system. The piggyBac system for gene transfer is unique in that instead of using virus to put new genes into cells it uses a transposon on which is a plasmid that carries a cargo, i.e. the gene to be into cells that is cut out by an enzyme called transposase and paste it into the genome to express the new gene in the target cells. This has particularly advantages over viruses and the ability to curate very large numbers of genes, i.e. large cargo capacity. And preferential favoritism for a number of cell types, such as T stem cell memory cells or Tscm The Cas-CLOVER gene editing system is a novel gene editing system, which is similar to CRISPR and zinc fingers combined. It uses to guide RNs to bring together the dCas9 protein along with a Clo51 endonuclease to execute the cleavage site that is targeted to be edited in the gene. This has the advantage of having a very low to no off target editing because of the utilization of to guide RNA's. Also it has the ability to edit resting T cells, a particularly utility in creating CAR T cells. We have used this technology to create a broad portfolio of potential CAR T cells and gene therapy products, particularly focusing on CAR T cells at the beginning, as a reminder of CAR T cells are T cells that have been modified with the insertion of a gene for a CAR or chimeric antigen receptor that targets the T cells towards cancer. It started out with autologous CAR T cells. These are T cells that are taken from an individual patient and then modified with the piggyBac transposon system to carry a CAR, to target these T cells to cancer, such as multiple myeloma and prostate cancer. Subsequently using gene editing system, we have been able to create allogeneic CAR T cells where one donor can provide T cells to multiple patients. We're falling this on with a number of gene therapy products to treat inborn errors of metabolism, such as OTC or ornithine transcarbamylase deficient. To go into a bit more detail as to what Tscm or T stem cell memory T cells are T cells are infection or cancer fighting cells in the body. They originate from memory cells, which may stay in the body for a long period of time, and then differentiate into effector cells, which actually do the work of killing infections such as viruses or tumor cells. So you can see why it would be useful to have a CAR T cells that originates from a TSCM and stays in the body for a long period of time. You could also imagine were utilizing TSCM T cells to create CAR T cells could be particularly useful in the treatment of solid tumors. In that as a T cell differentiates into multiple waves of effector T cells, it could continually eat into a solid tumor. As I mentioned earlier, Poseida got started creating autologous CAR T cells targeting multiple myeloma. In particular, P-BCMA-101, which targets the myeloma tumor associated antigen BCMA. Myeloma, as you know, is a common blood cancer that affects many patients in the United States every year. BCMA is an antigen expressed on almost all multiple myeloma cells Poseida developed the CAR T cells targeting BCMA for the treatment of multiple myeloma several years ago, initiated a clinical trial in 2017 and has escalated through multiple cohorts, multiple dose levels, and was awarded an RMAT status by the FDA based off of the results of this study. This is a description of the study, a very typical first in man cancer study, utilizing CAR T cells. The study has been conducted at a large number of academic institutions, particularly a significant number of institutions that are CIRM Alpha Stem Cell Center institutions in California. The protocol was supported in part by CIRM. And this allowed us to begin this protocol several years ago, to start treating patients with this new CAR T cells cell therapy. The results of the clinical trial have been quite good to date. Cytokine release syndrome, which is a common toxicity associated with CAR T cells has been relatively low and response rates have been quite good. Poseida the next moved on to targeting PSMA for the treatment of prostate cancer with autologous CAR T cells called P-PSMA 101. Like BCMA, PSMA is expressed on prostate cancer cells broadly. Prostate cancer, as you know, affects many men in the United States. And many of those patients will go on to develop metastatic castrate-resistant, prostate cancer, failing all prior therapies. And thus there's a high unmet medical need for new therapies such as this. In this case, the CAR I mentioned earlier was created to target PSMA instead of BCMA that's prostate cancer cells. Thus far, this has been tested in mice having a model of prostate cancer called LNCap. Quite successfully, you can see that the P-PSMA-101 CAR T cells, eliminated tumors in these mice on the right hand side of this graph recently we initiated a clinical trial with P-PSMA-101 and patients with metastatic castrate resistant, prostate cancer. Again, the studies leading up to this trial we're funded in part by CIRM, and there are a number of CIRM sites, alpha stem cell center sites involved in the conduct of clinical trial. The next frontier in the CAR T cells cell space is allergenic cartese cells. These are CAR T cells that have had their MHC class one antigen and TCR removed so that one donor can create CAR T cells to treat many patients as opposed to each donor by each patient having to have their own T cells manufactured to treat themselves. And you could imagine where this could bring the therapy to patients much quicker with much more reproducible and consistent quality of CAR T cells. So Poseida is using its transposon-based technology to again place the CARs targeting various antigens, but starting with PCMA again into donor's T cells, but also using the Cas-CLOVER gene editing system to remove the TCR and MHC class I antigens to prevent the CAR T cells from being rejected by the patient. They are transfused into as well as eliminating the TCR to avoid a reaction against that patient's normal cells by the CAR T cells. There's another novel technology Poseida has developed to help create these allogeneic CAR T cells called a Booster Technology, which allows far more doses of CAR T cells to be produced with a single donor's T cells. These CAR T cells, allogeneic CAR T cells now are targeting BCMA have been tested in a multiple myeloma preclinical model, and likewise demonstrated elimination of tumors in that multi-myeloma mouse model. And we hope to bring this into a clinical trial in the near future. There are a number of other CAR T cells programs Poseida is currently working on targeting other tumor associated antigens to treat other types of cancer. Such as MUC1C, which is a unique antigen, which is preferentially expressed in cancer cells of epithelial origin. So this has many common types of cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, and others. MUC1 is normally expressed in many cells in the body, but MUC1C which these new CAR T cells target is selectively expressed in tumors. And you can see below where there is significant specific antitumor activity in vitro for a number of these tumor types. And as you've seen in prior slides, these CAR T cells also eliminate those tumor types in mouse models in particular here a triple negative breast cancer and ovarian cancer. The last thing I want to highlight is the ability of the transposon technology to create multi-CARs because of the large cargo capacity or the large number of genes, a transposon can carry, we can put many different CARs into a single CAR T cells cell. This is an example of four different CARs or four different targets being put into a single cell, a single CAR T cells cell, and those CAR T cells being able to subsequently target multiple different tumor types, expressing those different antigens. At Poseida we hope to develop these into multi targeted or by targeted CAR T cells, dual CAR T cells in the next several years. So overall we've been very excited with what we have accomplished thus far with CIRM's funding and CIRM's assistance and partnership with CIRM and look forward to upcoming potential advantages and advances in the field using this novel technology platform, thank you. - Thank you so much for giving me this opportunity to update you on our gene therapy study for X-linked SCID. Before I start, I want to highlight that it's a effort which involves multiple groups, including at UCSF, which is spearheaded by Morton Cowen and Jennifer Puck and then at our institution in particular Ewelina Mamcarz X-linked SCID is a profound immunodeficiency, also known as bubble boy disease and infants normally succumb to severe infections within the first year of life. The only curative option currently is a bone marrow transplant. However, over the last 20 years, gene therapy approaches also have been explored in particular retroviral vectors have been used and while they were very successful to correct part of the immunodeficiency. Also side effects were noted in the form of leukemias in subset of treated patients. The late Brian Sorrentino at St. Jude developed a new lentiviral vector with enhance safety features having insulators at both ends of the vector. And initially had conducted a study in older patients together with Harry Malech at the NIH and had shown some initial safety as well as efficacy This then led to the current study in which newly diagnosed patient with X SCID are treated. We take marrow from these patients in the laboratory. The stem cells are then genetically modified with a lentiviral vector and then patients receive low dose chemotherapy before the genetically modified stem cells are infused into the patient. Up to now, we have infused 16 patients on this protocol, their age vary between two months to 14 months. And some majority of patients had medical problems, which were in line with the underlying diagnosis of X-linked SCID. We were successful in generating solid products for all of these patients and mean vector copy number of the infused product range between .16 to 1.74 copies per cell. As mentioned the patient received low dose busulfan, which was overall very well tolerated. And we only observed very mild side effects effect in three of the 16 patients. Importantly, none of the patients required any blood product transfusion, although we observed the transient decline of neutrophils as well as their platelet count. So then we wanted to ask three main questions. Do patients develop functional T cells and B cells? Is our gene therapy approach durable and safe? And do patients benefit from it? First, we looked at the development of normal T cells and as you can see here, all patients developed high levels of T cells except patients number one, and say, not only develop normal T cells, but if you look closely, they also developed so-called naive T cells, which is very important for a normal function immune system patient also developed T cells hat had left the thymus again, that is very important to make functional T cells and reassuring these T cells now responded to PHA as showing unequivocally that we've generated functional T cells within these patients. If you look at a B cells, we use the production of antibodies, which B cells normally do as a surrogate marker. And as you can see, The patient's B cells started to produce IGA or IGM within a year after gene therapy. And more importantly they also responded to vaccines like normal children. If you now look at the vector copy number in the detected T cells, NK cells and B cells, you will notice that we can readily detect genetically modified cells longterm in these patient. And while there is a little bit fluctuation over time. The level is actually very constant for each individual patient. In addition, we could also detect genetically modified myeloid cells, and we also were able to detect genetically modified cells longterm in bone marrow samples. If you look a little bit more closer at gene marking, you realize that there is actually a higher group and lower group for T cells and K cells and B cells. And this really correlates with the number off VCN in the infused graft. Children who got graph with low VCN tend to have lower VCN in their T cells and K cells and B cells, whereas children's who have high VCN have higher levels. The question of course, is does it have any functional consequence? And fortunately it does not. If you look here, again, shows the VCN per T cell number. But if you now look at the T cell count of this patient, regardless if say I have gotten a low or a high VCN graft, they still develop normal T cells. In addition to normal T cells, if you now zoom in, in the first year, it might be that higher VCN graphs results in the foster T cell reconstitution. But that is really not the case, because if you look here regardless of getting low or high VCN graphs, these patients develop or had the same kinetics off a T cell recovery, if you now look a little bit closer within the genome where our lentivirus integrated, we found a very consistent pattern. And just to orient, you normally the genome is presented in a circle where you have here's the X chromosomes, the Y chromosome and then it goes from one all the way around to 22. And you observed these peaks, which are very consistent across NK cells, myeloid B and T cells. And if you do a correlative analysis of all the integration sides, you will notice that the integration sides overlap between all these lineages indicating that our gene therapy approach, we in the end genetically modified very, very early hematopoietic progenitor, or hematopoietic stem cells. In regards to safety, I just want to highlight one feature again, showing here a chromosome wheel and highlighting consistent integration sides across patients. So we use this data now, and to looked in the literature at other studies and reassuringly, we find the same integration pattern as reported by others. Here is chromosomes three years, chromosome three, here's chromosome 11, here is chromosome 11, highlighting that in these very young patient who receive genetically modified product. The integration site pattern is exactly the same, like in older patients who had no side effects with these type of therapies. In regards to clinical outcome, all our patients are doing very well. And I sorted some again for you into the low and high VCN group. Again, there is no difference in clinical outcome. All of these patients did very well except for patient number one who needed a gene therapy boost one year after his original infusion, but since then has done very well. So in conclusion, our lentiviral vector in combination with low dose exposure busulfan is well tolerated and results in the development of a functional normal immune system. So far, we have not observed evidence of malignant transformation. And the belief is that our approach presents a promising alternative to current therapies. And of course our study is ongoing to determine longterm safety, as well as sustained efficacy. With this I want to close. And I would like to thank again, all our collaborators and also in particular CIRM for supporting this clinical study. Thank you so much for your attention. - Dr. McKenzie in your patient, obviously very good safety profile. I'm curious about whether there's any update on any evidence of chimerism now in the peripheral blood or bone marrow of the trace patients who are a patient who is treated prenatally. - Yeah, thanks for the question, Matt. So we've treated two patients so far and they both did well in terms of tolerating the transplantation on the multiple in utero transfusions that they needed to have. And we've seen low levels of engraftment in both patients. So micro chimerism probably related to the fact that we're not giving any bone marrow conditioning in utero, but the very encouraging thing is that we have seen tolerance to the maternal donor for the entire year of the study in one of those patients, meaning that we should be able to do a booster transplant using some conditioning, but not necessarily needing to do any immunosuppression because the recipient would already be tolerant to the donor. So we've offered that to the family who had that outcome, but they're actually very happy getting their monthly transfusions. And so they haven't wanted to, you know, have any sort of prolonged hospital stay. But in terms of a proof of concept for doing kind of a two step protocol, which is an in utero transplant to establish a long, long lasting tolerance and that a postnatal single-shot booster we're encouraged by the results of these patients so far. - Yeah, thank you. And maybe one more question that goes into the details as you and probably many on listening in are aware there's a transition of hematopoiesis from the fetal liver to the bone marrow. I forget when you gave the cells with respect to this transition, whether that might have any ability to get engraftment in the bone marrow. Yeah, I think you probably understand what I'm trying to get at. - Yeah, I think that's a great question. And so I think we're kind of asking for a lot to take these adult bone marrow cells from the mother and, you know, manipulate them all day and then put them into the fetus' circulation where we expect them to go home to the fetal liver for a couple of weeks and then sort of join the migration over to the bone marrow. So I think what you're probably getting at is how can we manipulate the donor bone marrow so that it's better suited to survive and sort of out-compete the host's hematopoietic stem cells in that environment. And I think we have multiple colleagues that are working on that important question. So I think both some manipulation of the donor bone marrow and some manipulation of the host hematopoietic stem cell niche will be necessary to create a more effective transplantation regimen if we want to have a single shot cure for these patients. But as I said, you know, we're just starting out and even seeing the long lasting tolerance has been a dream. - That's fantastic and of course, I'll give a shout out to CIRM for supporting some of the most innovative work in creating niche space without having to use chemotherapy, which might really dovetail nicely to the strategy- - Absolutely, absolutely. - That you're developing. I have a question for Dr. Spear. You mentioned one of the real advantages of the transposon system is that you could put multiple CARs into one cargo, which I clearly have some technical advantages I'd like you to maybe discuss though the pros and cons of having multiple CARs on one T cell versus multiple T cells with each, with a different CAR and how that might play out in terms of efficacy, safety, and maybe pharmacodynamics. 'Cause I'm sure you've done a lot of thinking about that. - Yeah, it's a very good question. Most people are trying to do all CARs or more that have the single CAR on, or sorry, multiple CARs on one T cells opposed to multiple T cells each with different CAR. There's a couple of reasons for that, it's not an absolute, but one you have economy of scale in terms of production CAR T cells, unfortunately very, very expensive and difficult to produce. So by doing one production run, as opposed to multiple production runs for a patient, you could see some utility there. The second possibility or second issue, is there some interaction or some interaction I should say, has been suggested overall several studies between multiple binding epitopes in that you may actually get a better outcome. If each CAR T cell can be bind to and be activated by two epitopes, as opposed to one, whether it's two binding regions that bind the same antigen or different epitopes and the same antigen or two different CARs that bind to different antigens two different tumor associated antigens. So largely hypothetical at this point, but some good rationale behind it. - There's a question from our audience, which is of course we're using retroviral vectors or lentiviral vectors. The integration profile is semi random, and actually we saw some of that data from Dr. Gottschalk in hematopoietic stem progenitor cells. How would you compare the piggyBac integration profile to these viral based integration profiles and any thoughts on safety or efficacy implications of the different integration profiles? - Yeah, always an issue. One of the things we published over the years is with regards to the integration and profile transpose as in general one what we've studied with regards to those is what, as well as what others have studied with regards to that. So it is also semi random. It has a consensus site, but that consensus site is relatively broad throughout the genome. B one advantage is that it's not completely random. That consensus site does tend to be in genetically less disadvantageous locations. So from the times we've done walks the genome where you find this consensus site unlikely to be upstream from an oncogene, for example, in the middle of a tumor suppressor gene, for example. Again, hypothetical, that's where the preclinical data suggests at this point. I wouldn't spend one of good things overall, is that with regards to CAR T studies at least there hasn't been a oncogenic transformation event yet. So there may be some dependence of the cell type or patient's disease that affects the potential for oncogenic events in gene therapies of which of course our T cells are a type. - Yup, that, that very good point. And of course, a comment that the integration into that too is both a double edged sword that Carl June and his group reported, thank you. I have looking no other questions. So I'll continue with Dr. Gottschalk. So some things I noticed was that it seemed that the cell numbers and VCN seemed to improve over time. Was that a result of overt changes in the manufacturing and inferences process, or is it simply that this is such a new field that center just get better and better as they do it more and more? - No, as I showed on one of the slides, we changed the vector prep after the first aid patient using what we call the second generation producer cell line and with that, our transduction efficiencies improve. - [Matthew] Gotcha. - It probably has to do with the VSV-G content on the cell surface on the virus not to get too technical, but it also allowed us actually to go from a two hit to one hit transduction, by increasingly VCN. - Yup, fantastic, sorry I missed that, that'll be great. And I guess though, that highlights that even as we're developing these therapies, that there will be continual improvements in vector production, transduction, stem cell harvesting stem cell transplantation. I like to ask a followup question and then go back to a question for Dr. Spear, which is, you know, across different diseases. People have been using different doses of busulfan in terms of conditioning. And you've described in this trial that you're using a low dose of busulfan and yet it appeared that the VCN in the CD34 hematopoietic stem shell progenitor population was relatively well-maintained even compared to some of the trials we're using full dose busulfan. So like you to comment what you think about that really I would call it surprising maintenance, so the VCN and the semi-progenitor cell compartment. - I think it's very hard as you pointed out, it's probably very disease specific- - Yep. - You know, dealing with an immunodeficiency. So we really do not have, you know, like so clearly I, you know, I think we cannot do the experiment now taking busulfan away. - No. - As you know, it was originally introduced because if you look at a lot of other studies, you know, there was never good immune reconstitution, the B cell compartment. - [Matthew] Right. - And I don't think that's a lentiviral vector is magically gives you B cell immune reconstitution which a retroviral vector does not. - [Matthew] Yeah. - So we really believes that the busulfan is important to kind of prepare, you know, as a soil, you know, for minimal- - [Matthew] Yeah. - Engraftment and differentiating the cells. So, but I think it might be, you might be able to replace it, you know, it was, you know, I know you're involved in some secret antibody studies - [Matthew] Yeah. And I think everyone, you know, in the non-malignant gene therapy field would love to get completely way from chemotherapy, but I think we need some sort of conditioning. - I agree, great I see, we have another question for Dr. Spear, but I think he'll be able to clarify. The question is, what other criteria determine the target sites for the transposon. But I think you mentioned in the prior answer that in fact, the transposons do go in and in a random fashion, but perhaps you want to expand that on the idea that it's not targeted integration, but it's a different mechanism of integration in the genome. - Yes the transposons do go in broadly they have TTA consensus sequence, but again, a fairly broadly found sequence in the genome. The Cas-CLOVER, the gene editing on the other hand is very, very specific. Because you used guide RNA's target to a specific sequence and confirm that that sequence and overlap sequences don't exist in the rest of the genome. - And maybe another question for you, Steve, there was a, and I may be getting in the weeds here, but I'm super curious is you mentioned the idea that the integration site suggests that you've modified the hematopoietic stem cell because it's giving rise to multi-lineages. And yet the VCN across NK cells, T cells and B cells were a little bit different. So just get maybe a little bit more discussion on what you think about the, what stem or progenitor cells actually in grafting and how that relates to the clonality of what we're seeing at the T and K and B cell level. - I think you see an enrichment in the committed lineages, which require the common gamma chain. - Yeah. - And I think what these, I think VCN increase, highlights that you probably need at least one, if not two copies of the introduced gene to generate functional immune cells. - Yep. - Whereas in the myeloid lineage, you have absolutely no select pressure- - [Matthew] Yep. - And therefore in the end the VCN or the incoming craft is maintained. - Yep, okay, well, I don't know about the rest of you, but, you know, zoom fatigue is real, so I sure everyone would love to get a few minutes off before we move on to the next session, but I want to, again, thank CIRM for their tremendous support over the last 16 years for a variety of cell and gene therapy programs, three of which we heard about today, like to thank the speakers for as again, they're very clear, concise, and informative presentations on how really selling gene therapy is gonna transform the lives of patients, both with unmet medical needs and rare diseases, as well as patients with much more common diseases. So with that, I will log off and again, thank you very much, everyone. (soft music)