As Industry And Academic Researchers Move Forward In Their Quest To Enhance Their Understanding Of Human Development And Diseases, Discover Novel Therapeutic Compounds And Develop Improved Tests For Toxicity, So Does The Demand To Move Toward A More Relevant Source Of Research Materials. This Panel Will Discuss New Developments In Gene Editing And That Anticipates Gene Editing In Combination With Human Cells That Will Have Significant Positive Impact On Advancements In Relevant Models And Stem Cell Based Assays.
► STEVEN L. STICE, PHD, REGENERATIVE BIOSCIENCE CENTER
► COLIN EDWARD BISHOP, PHD, WAKE FOREST INSTITUTE FOR REGENERATIVE MEDICINE
► JAN A. NOLTA, PHD, UC DAVIS
► THOMAS P. ZWAKA, MD, PHD, ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
Welcome today to this session our session on the power of gene editing the implications for future of medicines we've got very short presentation that. I'm gonna go through some of my thoughts on you know just sort of overview of where things are normal move them into work done by Thomas Watkins lab. They wear and then we'll talk with general terms will talk from UC Davis called the ship from Wake Forest. So we've got a good breadth of knowledge in this area that we're going covered today it is gonna be interactive so we really want your questions and comments and so that we can all really learn from each other so we'll we'll get started he re think the real key question in front of many of us today in regenerative medicine area drug development side the whole area of stem cell therapy. Is show do we get to do clinical trials most quickly through compound development nonclinical testing to the India and so forth so. I really foresee that the future of gene editing is one really trying to be getting us there more quickly so can we develop better models better representation of disease models with Jean editing and the other side of things. I think is obviously the hope that in the future that we can correct certain diseases with gene editing practices along with that comes a whole set of ethical questions about gene editing and the ability to should we be doing this somatic cells are in the germ line as well so. I just throw up a set of examples here couple of things different areas there's been some work lot of work and Jean editing overcast couple of years and it's just exploded so example of correcting gene mutation is a number of those are out there today and a lot of work is going on in the area and in addition there's lots of work developing better models they can you develop a cardiac ship where are those that was using induced plenipotentiary stem cells with say a mitochondrial defect or so for us so a lot of published work in the last couple of year slot of good work that's going on out there this session these sessions are meant to be really general we're not supposed to get into depth in any great detail with their research but in order just to get people up to speed on taking this slide from our collaborators at transpose agenda they have a whole suite of different ways of gene editing by far they're not the only people out there's a lot of different groups offering different gene editing technologies from talents at crisper to Nicks next generation systems they're going on out there so one of the questions that are opposed to the speakers today is how can we most effectively get this technology moving forward what's the what's the cheapest what's the fastest what's the most reliable systems out there going forward and then beyond that to whether the same of the easiest gene modifications whether. It's in this case Mail calendar urging addition type of technology and we'll get into some of that hopefully in the discussion part as well certainly there's a lot of interest in different gene editing has been around for some time some people have called a gene targeting in the past so there's a mall against recombination decree locks recombination type of technology so there's there's advantage sand disadvantages to a number of different technologies out there and hopefully we can discuss some of those as well for instance the Cree lock system as it is thought to have less flexibility than some of the crisper and talons technology and less mutagen them some of these new generation gene editing technologies so something further to discuss during the presentations as well. I think it's also alluded to this previously is the choice of cells are we gonna be doing this type of gene editing in stem cells nonproliferation stem cells and differentiated cells are restarting to work with more differentiated cells or progenitor cells for a better maybe tissue-specific and I'm and eventually came we'd be doing this officially in tissues and possibly a whole organism as well so those awesome of the areas that we need to think about as well or are you gonna Angie Medina's is how to use some of our sales going forward and how will be able touche gene editing and stem cells traditionally most people think about Gina modifications in the very potent stem cell state them selecting expansiveness and then for differentiation and 3d culture systems or adhering culture system and then for our interest in the neural cells and full disclosure here as we're interested in this company side as well or in the bio medical is a group that I work with as well so one of the things that we're interested in is again to use it in play potent stem cells so that we could possibly save time and using them in progenitor cell lines we avoid some of the complications. If you're looking in particularly neural imaging applications therefore eliminating the non-neuronal contamination side of things and tenon-uniform differentiation process and potential that being able to do it. Ina stem cell-based system is that we have availability and consistencies and all the advantages of doing that in the stem cells side of things are in the plural progenitor state so we'll we've been doing some work as. I said with transpose agenda and trying to genetically engineered metal progenitor cells here and we've added piggyback system that is amenable to G mail engineering and we'Veblen able to closely isolate these cells and select for particular genes of interest and then differentiate those into differentiated neurons so that is just to give you a flavor of some of the things that are going on interests in this area are widespread and look forward to the rest of the panel giving some thoughts and then we'll move right into discussion and questions so far this skill come on up and get started with your so. I'm Thomas Bach and I am at the black family Stem Cell Institute in at Mount Sinai in New York a movie. I moved three years ago and I wanted to give you an idea we have appeared current years we are not many developing gene editing techniques activity we have more time to use that site by and telling you one particular study and hopefully be able to illustrate that but how much has changeover the last perhaps ten years so. I started my post don't come in 2001 and Jamie Thompson snap and the major projects actually to the gene targeting. In human embryonic stem cells and we basically moderate apart from car that was used for a number of years and the mouse which is a classical gene targeting replacement affect us all year long commodity arms and both sides and you have the peace in the Middle Ages counterexample selection cassette put in and so this project and. I specific regulatory time that doctor for GFP to create both a report I said. I guess where this headline but can be selected going besides express talked floor and just in bed time that he particularly actually about the Select UK said because he part maybe in the future one day we will be able to take transcription factors and open expression and when you should like talk to a connection with program into embryonic stem cell-like says and. I think he speaks ISO 22000 vision Jamie had already in the nineties not only to develop stem cells as a technology but also thinking the real in my opinion years and years ahead of Liverpool and then ended up in 2006 so they could do targeting it took me over a year to create a target impact 02 cyclopedias get him back to and wrapping and now they year to do the gene targeting in human embryonic stem Sousa two years could put a single gene and no prospect of 22 2010. I was at that time and day 9 we had a project to knock out a particular gene you interested in going in and we want to do is a human embryonic stem cells and learning from the experience. I had but also what led to the effect that 90 proposed tax when you wanted to do this with classic indeed time to time we turn to add technology bed with their bedtime that missus and from the mediated gene targeting the idea of any attention to do the same thing that you helped yes airspace introducing a double-stranded break in the DNA in this site specific fashion so they said this had a number of advantages but. I get him back to West much simpler so let me get taken on vellum oMG. I'm so much on the downside was the same thing that's needed to be developed a company in this case in with Cigna and will publish his service he took that problem. I'm still crude but unless and thinking that $25,000 and so what's more convenient and fast and sexy also pretty expensive. I think in order to make it took us about six months to the knockout Betty was successful and so when. I was asking how soon can you help us with this session here. I walked into been mad and just randomly picked to post EPS and told them to Budapest about the Kremlin Christmas and this just tells you. I think that now regular number of them stayed here he says not everyone is going Chris by at least be screwed. I am end-to-end and it just in my opinion changed every thing so well let me tell you a lot about how it works but that one could argue to you in New York on pic of the day period a couple of weeks ago that s heavy things and not only of a science which we all know about that but also background about the personal of the two people involved in this it's a fascinating story in case you're interested so it's all about big time between projects moving doing the first one was a mutation in a unified receptor gene could we wanted to create a gain-of-function mutation 378 addition it has been found in humans could cause a particular disease.. I thought I'd rather get to damage displays had so we wanted to do it to interviews with mutation into yes says and some beer a healthy do but you have to design the sky down they say these are very standardized and extremely straight forward and takes essentially a day they knew what went to happen you seriously and not only wanted to because it tells her fishnet only make the mutation but also had to stop colon to create 10 9 mutation so we immediately created a number of guide on Facebook provides gene targeting experiment and he's been targeting scheme against the same thing as a replacement back to the chart horology arms who died on a and the crisp and destroying the cut and this is how it looked be experimenting it was incredibly efficient you see ya baby they targeting frequency address. I guess what about 50% and homologous GMT targeting effects with 25 percent roughly 5 percent so far this experiment took us a few him to do and we both can knock out to space and mutation ABC specific mutation came back in the second example systems already in bed by then but the mutation was introduced after it's something we're doing right now and it is needed here in BC scrutiny a bunch of mutations in and people deeply and disease-specific you're saying and again this is Stacy Nibbler 26 meet project and then the class essentially no cost once you have it back to us and the guide on ABC's now no additional thing you need to do so that's where we'recurrently things stand. I think and then that. I hope this will trigger some discussion of a trained in this technology so. I don't know of a sudden he came into the law during this and. I think that's awesome beauty of it is so simple that already chopped liver shuts that organized everywhere with different companies involved and so on but we've been going to talk about this it was said he come to move with you know me wanting to do these knockouts and then been coming and saying Christmastime and it's became john Dunn really Chris bring things you can you let me see if we create a mic questions overseas pro-football Apr on what can she do we do then and other things very relevant of course this is purely experimental and say so the question is of course about off-target effects do we introduce a mutation because we essentially to inspect and replace into says right and then from my perspective it just works and if you have the right controls but from a basic research perspective it does n't matter so much when we have to dig a bit deeper into visit when once to find out if Albert off-target effects of those Justin from designing just last week of this week as science la detained version of the enzyme that but seems to be more specific but then again it's not my research area and it's just a basic science perspective where you can say having the right controls you don't have to worry so much about things so you just say go ahead and Chris Barrett but there's a big comeback and say well it'd be easier to do it with talents these days for obviously not sing fingers but they say. I can't do this is to go against the hostile to do i don't know. I would n't have that's probably possible but this is what they come back with right and they talked to a job and they every one president in papers and. I think this way to speed everything in terms of efficiency and simplicity my day. I think it's pretty clear or any other specific questions for Thomas for off-target effects is very new please has been used for a while that's still like in terms of actually analyzing right there are some you see things a newly designed so that predicts off-target effects babe big and that's not my area of expertise. I can tell you okay as themselves gene therapy and we are using stem cells to deliver the gym editing platform so. I think that's why I'm here to get a different take on things so we have several therapeutic stem cell gene therapy options the most well-known is adding abnormal gene copy to restore function and buttocks themselves are currently best without long-term therapy we can do so replacement strategies now so transplantation in healthy progenitors or stem cells and here we most often think of the June modified and patient induced plenipotentiary stem cell-derived and sells decides neurons and this is where their Christmas geoengineering will be used a lot in this health care platform so used to hearing committee in genesis in neurosis hymeneal stem cells and. I worked with for thirty years are very good at that just on their own they secrete factors that promote healing over inflammation and scarring and easily be modified and then using those cells used a MSG is to carry the cargo criticizing and Jean editing friend dominate mutant allele and that's what. I'm going to talk about today is our work nonworking toward a therapy for Huntington's disease is specifically juvenile Huntington's disease by using the embassies or other platforms to change in editing cargo into the brain ambitious so we have an amazing team working on different treatment moralities for Huntington's disease and. I just wanted to point out to him and this is our fearless leader Dr. Vicki you like who is the AMD who treat these patients she follows three hundred and fifty families with this rare disease they moved to the Sacramento area to be followed by her and her partner in the clinic very tempting and we have seventeen children with juvenile Huntington's disease in the area sow are very focused on that this is a forest they have a low hanging fruit type therapy that we could pose a 404 adult Huntington's disease the team had team is directed by my doctor Dr. Kyle thinking. I'm really presenting his work today because I was asked to be on the panel and then it was OK he shows his legs like OK. I circles where he's really do you still really great stuff with his team and again these young people they know those backward sand forwards in we get there early it has a different funding for this team and we thank all of those donors it's mostly through donors and fellowships right now sincere is attractive target there are CAG repeat the normal member under 39 CAG repeats a gray area 3238 Huntington's disease occurs if they're over 38 CAG repeats in the in Huntington's gene and so it makes a horrible mutant protein is necessary makes a formal mutant protein that really clubs the cells causes these inclusions that killed in neurons and causes and I'm straddle Los primroses in late eighty years a healthy stratum and there is the Huntington'stratum so we're trying to do something about this triple treat aids causing Juvenile Haiti needs to be cut out through June editing technology down to a normal size or the resulting mutant RNA and protein need to be silenced soon of those two strategies to make an impact upon this deadly neurological disease is always fatal and we have no other treatments so this working on thesis beautiful in addition we can add a day gene editing let everyone every library working the talons right now because the smaller Christmas when available we started this but whatever we had in the days it's really a great as. I'm going to so you at the techniques in a moment but how do how to get this into the brain and how is it going to work in the brain challenges for treating these single gene disorders in the CNS 3G meditating really comes down to deliberate at this point amazing tools amazing accuracy target is thanks but how are we going to get it into the site where it's needed in then in the patient or in the animal model and so how do we get these DNA molecules into it and nothing else opportunities to have an impact and we both will be selective survival of correcting neural progenitors the methods that we're considering and comparing it and no associated vector 89 in particular can really carry things but it's not gonna be able to make these guys are in there so it's not going to be perfect for this and other particles could encapsulate them take them in for a one-time administration into the neurons and then whatever we get into the neurons which would be an air together as they call it the cargo because it's the protein and RNA and the guide RNA we can just correct idea see sex vivo and then transplant though this and those might have an effect in Huntington other CNS diseases and then what we're hoping to do is to use these MSG media and exercise of micro particles encapsulating the gene editing cargo and so we achieving MSG's because they have several just in aid effect if they do. I'm around just innate talents I will stay and I can do appreciate these tell us more and more every day is over to these cells for almost 30 years so they do secrete contraband fact is they reduce inflammation need is programmed cell death in his connections between neurons in they reduced cell toxicity so they're already a good candidate for treating Huntington's disease they can be readily engineered using viral vectors to robustly deliver cargo so for the gene silencing for instance remain in the bay for about one to two month sand they migrate as they do this so they can contact a lot of. Iran's they do not require images depression between humans they are very strong safety profound human clinical trials and there's demonstrated deliveryman interest-only contents to neurons in other cells so they can be very easily genetically engineered or loaded with this cargo delivery can be through secretion and he's micro particles or direct transfer via telephone interviews they do secret twosomes or micro particles and factors been normally confined to the cytoplasm can be transferred to the target cells and so we do have a patent on this for RNA inhibition MSC is moving around in the dish communicating with one another you can see they form an energy bill here and you can see the dots transferring between those are the mitochondria going from one cell to the other so there is done to sell transfer or organelles record best area to sell a release a whole bunch of micro particles that then get snapped up by the other cell so you can see you right here on this is Micah particles are coming along and the NHL comes in ingest them gets extra factors ATP in my country and then divides and then goes on about its business so this is a very good at talking to other cells transferring things into other cells and if we let them up and send them into a tissue they can do this in vivo and gather their content through the trail that they've done through or into the other damaged tissue and we have we are doing extensive studies in the brain with other Huntington's work you human amnesties labeled in red as a tomato red and then my bus in green and sharing their communication with each other this video was taken on the bus station so frame every 30 seconds and you can see that they really do live around quite a bit they leave a lot of particles become care packages for the other cells to take and get a lot of cells the cell transfer going on this particular video is in its not showing up very well that this particular video is online now in a human gene therapy methods just accept it silences de gene knockdown we can use cell and gene therapy to deliver genes that would shut down the production of or destroyed immune attar name is jointly owned and that could but then the devastating in protein-protein produce din the neurons in this is particularly important in rapidly progressing at juvenile HDD and this one is just showing us the more. I saw this telecommunication and I don't think it's loaded game play so this one shows the green legitimacy is chasing this damage neuron and starting to stand be particles down into the neuron and so he noted we've engineered the MSC is to make our nation inhibition short strands of RNA and then send them into these Huntington's disease affecting neuronal cells' reduction of hunting tin gene and protein we could get a significant reduction in the news story in the ATT protein this is through direct co-culture in the dishwasher now looking at this in animal models of Japan LHD but heading is what we really are here to talk about so moving forward for gene silencing which is easy to engineering MSC to do that through antiviral vector which stably integrated in their dunno and they did are making tons of this island in m RNA that they're sending out in these packages care packages into the neurons that we really want to use this platform pre-jean editing so this is Kyle is a leader in the gene editing team this is his work along with Peter day his the grad student that works for both their lives works in both of us and a DNA students on his team really brilliant team assisted by Dave Sega who works on molecular therapy for Angel man syndrome and other money Janis neurological disorders at our mind. Institute at UC Davis and so using the town's with nucleate for a double strand break and deletion to turn on enhanced gene expression and then finally to blocked you expression in and the first thing that they're trying to do is to get a CD collapse so to try to get fewer CAG is until there's finally a strict hindrance between these so they did the normal size it would be theoretically as 16 CH en no more cuts could happen there is all silencing did they are working out with the Arab demands to stop the expression and so the MSC is a good dad be expressing he's admitted in protein and tagged with Imperial able and we can recover them identify them in the build and moving forward with that its files work in hopefully will be published soon weakened and several abstracts on this presented neurological society meetings and we also have our exercise team led by Jonathan Anderson and his wonderful team in this is a collaboration with the Carolinian. Institute and we have a paper clearly embargo but coming out in stem cells very soon discussing the content of these twosomes that the kind of stem cells make in their what they can do so just back down to the question and delivery how do you get it into the tissues that we want and I think that's really the overriding question what do you think of this work would apply for nucleotide repeat diseases and there are quite a few of this triplet repeat disorders in so we are forging ahead it's really interesting and we have a big Huntington's disease team at UC Davis in international collaborators and just really wanted to a specially the Huntington's disease patient and the kids patients and families that work with us moving toward our first planned clinical trials for embassy making brain-derived neurotic factor this really takes a village a lot of teamwork we have a lot of course in Ind enabling studies for a lot of different academic and industry partners just. I love going into work every day and staying with these bright young students have come up with in June editing is one of them so that's it thanks you for inviting me that's a good question in Vivo as I understand it is about 20% but we're always trying to improve that and this is what the loading and to answer you come up against the ethical issues of Jeanette team and that's saying juvenile. I need to say is that question coming up these days your research and wants to do to you we talked about it a lot that would mean I think the question is for when the gene editing molecules are being delivered. I'd be there to be together in editing or transaction of this rumor eggs and that is what they always worried about is this is being modified if we put any better into any patient and so that's the main concern and again with this and that's the question that we get delivering them directly into the central nervous systems we're doing that reduces although does n't completely eliminate the worry about that but should he care of kids with juvenile Huntington's disease. I think everybody's pretty much on the same day defense is that with that it's a terrible disease and going to the level of modernity well. I'modifying the German level now we are not considering that just delivering into the brain trying to introduce some controversy here yeah maybe someday in the future because there's a lot of prenatal diagnosis Judy that's done for Huntington's disease thank you so. I'm calling the ship on professor at Wake Forest Institute of regenerative medicine. I'd like to apologize I don't have any science to show you I didn't quite realize it was before my both but I'd like to talk to a little bit about the way we intend to LA and continuing to do gene editing livestock large animals and they doing this in collaboration with will i Still need to Virginia Tech in fact there before Institute for Regenerative Medicine have recently formed a joint Institute of regenerative medicine and as Thomas was saying some time ago it was extremely difficult and time-consuming to make modifications in mice and rats it was doable and many people did it but it was even worse in large animals like pigs and cows and things like that why would you want to do it in Beijing says it's because they actually represented quite a good model for many human diseases especially cardiac disease which all terribly well reflected in Mex models or road removals in general but in fact jean editing and it in large animals was restricted because even to this day in pigs and cattle yes lines of not being developed for some reason they have not been able to generate good your lines from these large agricultural animals so will the genetic changes that were made will buy some Coltrane's Genesis which you don't have a lot of control over you put a gene into a pig embryo or OKs whatever you wanna do and it lands somewhere and you get good expression you get bad expression it's it is a little bit hit and miss and then of course there they came the ability to actually clone animals which was a great surprise that showed much roman Reloaded the text books he liked and showed that in fact that excels terminally differentiated somatic cells if they will put into developing and nucleated a good actually form alive and healthy animal and so that opens the possibility that one could genetically manipulate fibroblasts and then through somatic cell nuclear transfer actually cloned a modified appear in court Koch and several years ago. I decided I had a graduate student very clever girl and together with where we decided that we would try and use the carrot as a bio reactor to makebiopharmaceuticals and one of the primacy of proteins we're interested in is human serum albumin which right now is obtained from donors from human donors and design in short supply in the world actually so we decided that we could not shoot the bovine serum albumin gene replace it with human serum albumin gene and also put a promoter from to see the human serum albumin genes it also expressed in the milk we would in fact a character which would express human serum albumin in the blood and also in milk or meat we could derive a clinical product and at the time tolerance square just becoming the best way to do this and so we use talon sand we were able not only what we wanted of course to do was not only just mutate to June and inactivated little wanted to do was use gene editing all these talents to make a double strand break which vastly increases the efficiency of a homologous recombination so we made the double strand break in the exact place that we wanted to in the bovine genome am very pleased to introduce a 10Kb piece of DNA reaching which we had engineered to contain human serum albumin many genes and some promoters and stuff and we have been that and then we ran out of moneyso we have these things engineered and ready to do some addicts so nuclear transfer then of course Christmas became available and they are so much more efficient so much easier to make sure that. I wouldn't even not use the lines I've got now but make new ones even better one some fun stuff through the use of Christmas so we have been making models or beginning to make models of human diseases of immunodeficiency diseases which is simply a way of all we had to do was not in the first instance Casino immune deficiency gene and instead of going through fibroblast you know the team in the fibroblast going through somatic cell nuclear transfer in fact you can inject the Christmas directly into the one cell e g and we got approximately if. I remember correctly around about 60%mano a man o a lot of kids and about 20 Sep 20 percent by 11 hits which is absolutely phenomenal. I'm sure with more practice session will get even better and I think that we're actually involved in right now is trying to use this perhaps we'll touch on the controller see you like to introduce and I get to regenerative medicine and in particular reproductive biology for many years and particularly in male infertility and so will. I decided that we would see if it was possible tore generate the germ line from an infantile person many people's presented the clinic with male infertility and the sequencing of the genome net which is coming down too much not much more than a thousand dollars and of course exomesequencing and so on more and more patients the actual mutation which is causing infertility is being discovered their patients with mutations in my exchange like SYC P 300patients with me deletions of diesel the Y chromosome in soil and most of these deletions they don't lead to a lack of spermatogeneses but they actually leave the stem cell the spermatogonia stem cell pool intact so we have policies that if we could take stem cells from infertile men then collect that mutation by using up perhaps it includes a simple mutation we could easily correct along only go and then expand to those stem cells including them in this is the important part if you have to use in human you can actually claim they stem cells and you could if you wanted to you could in sequence the entire genome to make sure that you have any off-target hits or you would rearrange some of the chromosomes or there was something going on we're not about to do that in the human know so we decided to make a pig mobile so we know we are in we haven't finished this yet but we are proposing to locate the bombs a gene which leads to infertility in mice and do you also know at least to infertility in several other animals so very highly conserved gene once we have that models we going to take the spermatogonia stem cells which is still left expand them fix them and put them back into the same in fact I'll pick and hopefully hypothesis will be there will be able to regenerate the germ cells stem the germ cell line and of course this is one this is this crash to be done through usingspermatogonia stem cells it couldn't be done any other way you with any sort oft he gene corrections you could think of perhaps injecting Christmas into the guys testes and maybe a small deletion or something but you have absolutely no control over it you would know what you were doing all day many sperms and being prepared correctly it would really know people civil so we're doing it in animal models and. I think I believe that they're doing some other things actually in animal models but one thing that. I would throw out there that discussion is I mean regenerative medicine and many of my colleagues and I'm sure love you here trying to make artificial pancreas sees pancreatic kidneys livers hearts that can be put back into people which suffered damage of those organs but for many years know there's been several companies it's changed hands many times have said we'd like to be able to do with some a transplantation from paying into humans caused the pig organs are pretty much like us and it's been slowly building a snow day one gene here and they've looked at the hyper acutejeanswearspeed incredibly slow and of course the big problem is all the endogenous retrovirus system runs in the butt in the last yearGeorge church in Harvard has managed with using Christmas to knock a sixty 60jeans so he has a pig running around with sixty gene knockouts net took him one year to do there are no more genes that need to be knocked out but I would it be. I mean I would sort of put on in five or 10 years if you need a heart transplant or kidney it might just come from a genetically engineered or crispy engineered pig and I guess we'll leave it at that it be that big actually human pancreas two times out that's needed on in China but you may have to make scream in Jay Hawkins to correct him good Ok questions for was a show of hands out about I think it is roughly about it at least have a viewer saying that really like to do Jeanette team you know what is one oft he barriers there and they asked in China men as well we're we're redoing mortgages at the cost is that the expertise but that's what are the barriers to gene entity toxicity target effects in a particular application or is it OK for an in vivo delivery type of system jam did you want to address them insanely it's a worry but there are improvements made in these systems on the time and people make it better and better and more specific trends in with any there be used to benefit ratio so. I think it really does depend on what you're trying to treat the disease versus are you just trying to modify something that might make someone's life a little easier so that's just my take on it darkness you were saying is very inexpensive to do these things you've got sets put together and tell us what part figured if you wanted to try to do this particular cells expensive well as PIAA didn't notice his action on the budget so. I don't know it's a plan and the components out really nothing in comparison to those here illegally plans made its inexpensive PCR-based that's what it is remove barriers or for a little modification or really more on the embryo side that the barriers here of course is relatively simple to do anything so blind that you know you want to modify it's a relatively simple even simpler and sent in a sense just to inject into the embryo and their families reborn when you're dealing with pigs ok as you need the whole agricultural set up it's its expensive business yeah and it's not for the faint-heartedgroups out there though it suggests there may be a need you don't even need nuclear transfer clearly you just if you want to put a big piece of DNA and you probably believe. I don't know I don't know how efficient that would be my simple injection into the embryo because you are asking me to make the double strand break but then you're asking for the piece of DNA to go in and probably a piece of a few hundred base pairs is nothing but if you trying to put something on the final six or seven maybe 10 Kb in its gonna be a lot more difficult thing that brings up to the talk but whether you knock out her kid and what's what's easier where the pitfalls and then he just cannot connect to similar question and in this respect today and we were of course alone with the Huntington's patients advocates and there is in the United States we are you dictated by in what we can do by the FDA we have to be compliant with the FDA so. It's important for the patient advocate the biggest scientists to do it with 22 let the FDA know when they accept as. It is a risk versus benefit ratio and then it can be considered you know the science test to be there has to be strong it has to be efficacious in we have to show safety said that that is on you know the patient and the kids it's important for them to get scientists interest did but the FDA is the final has the final say we can't do it no matter how much the patients with it. If it's not FDA approved and I think with the patient and the kids can do most is due it does get funding and do fund raising events because without offending many this gets done and even get started the really important thing to him and treating children with a semi or city correcting it in the end because there in this pre-implantation genetic diagnosis around which you know if the parent shave in over 25 or 50 percent chance depending on the Internet Explorer bit you can choose between 12 in plans going on all the time but of course you know treating children having a discussion about that. It did arrest night specifically against Huntington's there is that but it's not paid for by insurance that's another important discussion with modified the genome mean so that children would also have a 50% chance is turning right Huntington disease is the dominant soon if you choose is you choose the embryo to implant they did not get the expanded an ill right then it would not be passed further. It's very expensive anything and everything you would if you we retreating in the brain you would't be very online at do anything so just help the patients who are suffering from Genesis Huntington's could be the first of all that's going to be cured. I doubt it because again at all times in defending still here less than amazing tormented post appears and helps us get there and he does not be the first time but we are still a proceeding do you have some thoughts are put on the spot here will be the first. I think probably because it may contain them. It's less scary to people in modifying the brain and they tend to be. In a jerk reaction to modifying the brain and spinal cord but that contained have been pioneered denies that we have a great. I agreed to do some pretty cool stuff and yeah you can see it.