WEBVTT 00:00.000 --> 00:13.920 testing one two I said 10 22 10 29 I don't think that worked out quite how I 00:13.920 --> 00:21.980 wanted to but we're gonna manage to make it work you can find me at giggle 00:21.980 --> 00:28.900 and biological.com you can also go to the soapbox link that you can find there 00:28.900 --> 00:34.180 which is also at giggle ohm.bio happy thanks giving everybody happy thanks 00:34.180 --> 00:40.920 giving Pamela Watcherby tense oral strain there's a click there that you can do 00:40.920 --> 00:45.580 to watch the show you can also download the stream deck starting next week we're 00:45.580 --> 00:51.900 gonna have the stream deck up I hope every time I do a stream at least every 00:51.900 --> 00:56.860 week stream and that means that you can start subscribing again if you'd like 00:56.860 --> 01:01.420 to get something like an email and a PDF of the show beforehand it might only 01:01.420 --> 01:06.340 be for Fridays or Wednesdays or whatever day we're gonna make it but 01:06.340 --> 01:10.860 someday at some point we're gonna start that PDF stuff again because these 01:10.860 --> 01:15.260 slides have started to become good enough that I think they're shareable 01:15.260 --> 01:21.060 and useful for people to start to really push these ideas out they haven't been 01:21.060 --> 01:26.140 there in a long time and so I think it's really nice for this to happen 01:26.140 --> 01:31.220 it is a little lonely in this crazy brainwashed world I'll tell you what it's 01:31.220 --> 01:35.860 very lonely my wife feels very lonely 01:38.020 --> 01:47.140 it's yeah it's tough thanks for coming tonight guys I didn't tweet it I didn't 01:47.140 --> 01:51.260 share it I didn't do anything I didn't have time it's Thanksgiving I just wanted 01:51.260 --> 01:57.780 to get a stream out there and keep the streak alive and keep the keep the 01:57.780 --> 02:03.220 sea warm keep the rig warm 02:08.300 --> 02:15.220 I yelled at the basketball team the other night my throat is still a little sore 02:21.260 --> 02:43.980 you schedule for 60 minutes next is going on French British Italian Japanese 02:43.980 --> 02:51.060 television people everywhere are starting to listen to him it's embarrassing yeah 02:51.060 --> 02:55.060 I'm gonna have to start with some more I'm gonna have to start with some more 02:55.060 --> 03:01.820 inspirational some more inspirational stuff in the beginning again I used to 03:01.820 --> 03:06.740 put a lot of work a lot more work into that beginning section of video so I 03:06.740 --> 03:10.260 just didn't think anybody was watching them but that one was a really good one 03:10.260 --> 03:14.140 keep in your arms straight 03:40.260 --> 04:02.620 so you know the drill you can look me up on PubMed and the name of the channel 04:02.620 --> 04:06.980 is derived from the resistance of the recordings that I used to make giga home 04:06.980 --> 04:13.420 resistance high-resistance low-noise information brief let's get this thing 04:13.420 --> 04:15.860 started 04:36.980 --> 04:46.500 I feel like my computer slowed down there for a minute hopefully you guys 04:46.500 --> 04:56.620 didn't lose anything yes happy Thanksgiving everybody good to see 04:56.620 --> 05:02.380 everyone here it's like we lost a few viewers see I told you I told you something 05:02.380 --> 05:08.100 poopy's going on they don't like it our information and our message is 05:08.100 --> 05:13.060 getting too sharp they don't like it at all 05:32.420 --> 05:38.220 yes yes truth bombs at dinner that's an understatement I bet at some of these 05:38.220 --> 05:42.200 places that we've been today thank you very much for joining me this is 05:42.200 --> 05:45.900 giga home biological a high-resistance low-noise information brief brought to 05:45.900 --> 05:50.260 you by a biologist if there's anything wrong with the timing or anything like 05:50.260 --> 05:56.060 that give me a shout out we are still here fighting against the same sort of 05:56.060 --> 06:02.860 nonsense you know the illusion of consensus the Scooby-Doo created by these 06:02.860 --> 06:08.940 people on social media I'm starting to really see what happened I know that 06:08.940 --> 06:13.220 there was this TV stuff and this stuff in Congress and whatever but I'm starting 06:13.220 --> 06:19.300 to understand how these people are all linked and it has a lot to do with these 06:19.300 --> 06:24.780 new social media and new communication platforms that came online during the 06:24.820 --> 06:31.100 pandemic and there's more than one there's actually about five or six and we can 06:31.100 --> 06:35.380 start to look back at when that was all happening when we were all scrambling 06:35.380 --> 06:41.740 during this during this censorship time in 2020 oh my goodness I can see it 06:41.740 --> 06:47.380 also clearly now I'm really excited as we break through some of this stuff on the 06:47.380 --> 06:55.820 18th and 19th of November in Bucharest Romania there was a COVID conference and 06:55.820 --> 07:00.380 some of our friends were at this COVID conference I'm gonna go over here 07:00.380 --> 07:08.660 pull myself down here and see if we can use my mouse to he has so just the 07:08.660 --> 07:14.060 prominent ones for now I'll pop my head down for a second Robert Malone was 07:14.100 --> 07:19.000 there Nick Hudson was there Brett Weinstein was there you can see this 07:19.000 --> 07:24.740 arrow right yeah and Denny Rancor was there among others which is kind of cool 07:24.740 --> 07:31.620 because as you know there is this there is this you know consensus that we're 07:31.620 --> 07:34.460 trying to battle against something and all of us are trying to work together 07:34.460 --> 07:41.700 towards some amorphous goal like health freedom or something like that and there 07:41.740 --> 07:45.840 is this faith in a novel virus this faith that millions of people were 07:45.840 --> 07:50.500 killed by it this faith that millions more were saved from it this faith that 07:50.500 --> 07:55.280 gain a function is real and possibly the source of it and therefore a virus will 07:55.280 --> 08:00.420 come again and this story is basically not been questioned by most of the 08:00.420 --> 08:07.300 people that we have been trying to look to for leadership in this in this fight 08:07.300 --> 08:15.700 to return to you know pre-2019 Western civilization which clearly we're never 08:15.700 --> 08:19.780 going to do the build back better the new normal this was all for real to make 08:19.780 --> 08:27.180 sure that we never thought we could have that back again and so in some ways it 08:27.180 --> 08:35.980 would be it should be somewhat curious if we can't find any leader on this 08:36.020 --> 08:40.900 playing board that doesn't want to say just simply well why don't we just go 08:40.900 --> 08:44.500 back to the way we were doing it about four years ago and start there and then 08:44.500 --> 08:49.540 we can repeat we can we can fix that but first of all we got to go back to that 08:49.540 --> 08:57.340 and everybody that through that out needs to be held to account 08:57.340 --> 09:02.260 everybody that just went against all those rules whence against all those 09:02.300 --> 09:07.460 norms went against all those expectations those people need to be held to 09:07.460 --> 09:11.260 account but that doesn't seem there doesn't seem to be anyone who's 09:11.260 --> 09:17.220 interested in that particular path and in fact yesterday I wanted to point that 09:17.220 --> 09:21.500 out and so that was one of the reasons why we went through that that video from 09:21.500 --> 09:26.700 Stockholm earlier this year so some more people were there that that definitely 09:26.700 --> 09:31.180 as far as I know aren't questioning the narrative that I really like Byron 09:31.180 --> 09:36.660 Bridal a lot and he's a great guy but he is not questioning there being a 09:36.660 --> 09:41.860 novel virus that spread around that's spreading still Jessica Rose hasn't 09:41.860 --> 09:48.220 questioned that even though she has done a selfie with Denny Rancor she has an 09:48.220 --> 09:53.180 acknowledge that that one of the main findings of Denny's data is that there's 09:53.180 --> 09:59.900 no evidence of spread of a novel deadly pathogen Harvey Reich is also another 09:59.980 --> 10:08.260 guy who I've talked to before and not on my show live but on a stream with with 10:08.460 --> 10:12.780 oh I'm not gonna remember her name and shoot that's really annoying Kathy 10:12.780 --> 10:17.500 something I believe I apologize for that if you're watching but it's a one of 10:17.500 --> 10:21.740 these private zoom calls so Harvey Reich is another guy who's not questioning 10:21.740 --> 10:26.100 the narrative there's Ryan Cole I'm not sure how much he's questioning the 10:26.100 --> 10:30.300 spread of a novel virus but I don't think that he's really there yet he's 10:30.300 --> 10:35.300 certainly not there yet with regard to promoting Denny Rancor's data and what 10:35.300 --> 10:41.100 Denny Rancor's data tends to you know it seems to show here's Jill Glasspool 10:41.100 --> 10:46.540 Malone and Meryl Nast you know that those two are also probably not pushing 10:46.540 --> 10:53.380 that narrative and then again what Brett Weinstein Nick and Robert and Denny 10:53.380 --> 11:00.260 Rancor so even if Denny Rancor is there and he presents to all these people and 11:00.260 --> 11:06.580 Robert Malone tweets it out he tweets out that 17 million people were killed 11:06.580 --> 11:13.540 by the vaccine and that he's never seen such shocking accounting of the 17 11:13.540 --> 11:17.700 million people around the world estimated to be killed by the vaccine but he's 11:17.700 --> 11:22.180 not at all shocked by the conclusion that there's no evidence of spread that 11:22.180 --> 11:26.900 the spread respected even borders of counties in the United States it has 11:26.900 --> 11:35.420 correlated much much better to to poverty and mental illness and income than it 11:35.420 --> 11:46.460 is to to like proximity of the previous cases or proximity of of of large 11:46.540 --> 11:51.620 incidences or events of all cause mortality I'm getting really good at 11:51.620 --> 11:55.340 not saying um when I'm doing these things but it's sometimes I have to pause 11:55.340 --> 12:03.700 for a second there um anyway and then I said okay this is all because these 12:03.700 --> 12:08.540 people have participated in this in this illusion of consensus and I don't know 12:08.540 --> 12:12.700 to what extent they were aware of this illusion before the start of the 12:12.700 --> 12:16.140 pandemic but most of them have to be aware of the illusion now because most of 12:16.140 --> 12:21.180 them have heard my my discussion of it our discussion of it over these 12:21.180 --> 12:26.220 this past year or so and most of them know what our contention is and they 12:26.220 --> 12:29.340 don't have a real meaningful argument because the only people that have 12:29.340 --> 12:32.460 tried to meaningfully argue with me are now blocking me on Twitter and no 12:32.460 --> 12:37.340 longer discussing it all and that's because again it really feels very very 12:37.340 --> 12:43.980 positive that that these these people are all involved in 12:44.060 --> 12:48.300 making sure that the worst case scenario of a pandemic caused by a gain of 12:48.300 --> 12:52.940 function virus is never out of your possibility space even if you don't 12:52.940 --> 12:57.020 believe that's what happened this time they need you to believe that that's 12:57.020 --> 13:01.580 what gonna happen next time or could happen next time or or eventually will 13:01.580 --> 13:07.740 happen they need you to believe that maybe in the 13:07.740 --> 13:11.980 same way that they needed us when we were children to really believe that 13:11.980 --> 13:17.100 the Russians had nine thousand warheads and we had six thousand warheads 13:17.100 --> 13:22.300 and they were on a on a hair trigger well two hair keys 13:22.300 --> 13:27.900 so i i don't you know i i have a hard time understanding 13:27.900 --> 13:32.140 anymore what history really is given the fact of all of things that i've 13:32.140 --> 13:36.300 learned over the last three years about how willing and able 13:36.300 --> 13:42.380 this apparatus is to govern us with lies in mythology 13:42.380 --> 13:46.700 and so i think that this illusion of consensus really needs to be inspected 13:46.700 --> 13:51.020 for this mythology and i suspect that we're going to find that it mostly is 13:51.020 --> 13:54.060 and that's why i think you need to think of this as a card game 13:54.060 --> 13:59.500 a card game with a rig deck a card game with a rig deck that a dealer is trying 13:59.500 --> 14:03.260 to steal your money so when you get the cards in order for you to have any 14:03.260 --> 14:07.420 meaningful chance of gambling with success 14:07.420 --> 14:12.300 you have to play knowing that the dealer is trying to get you 14:12.300 --> 14:17.580 to make a mistake so what would that mistake be 14:17.580 --> 14:20.940 how would they how would they communicate to those 14:20.940 --> 14:25.260 inside the game versus those outside the game 14:25.260 --> 14:29.660 what kinds of clues would there be that there's a game being played 14:29.660 --> 14:34.300 i think one of the clues that that Matt Crawford 14:34.300 --> 14:38.540 of rounding the earth pointed out about two years ago was this 14:38.540 --> 14:43.580 possibility that this covid is actually ovid 14:43.580 --> 14:50.140 ovid being this famous greek set of stories 14:50.140 --> 14:55.820 about various metamorphosis a lot of them are kind of like punishments or 14:55.820 --> 15:00.140 or or gods being vindictive but the point 15:00.140 --> 15:04.860 only being uh in this thing that this is a time of 15:04.860 --> 15:08.700 transformation and some of the the themes 15:08.700 --> 15:14.220 of ovid's metamorphosis in relation to the the fall of rome 15:14.220 --> 15:21.100 and the theme of let's say order being imposed on a world 15:21.100 --> 15:25.660 founded on chaos or based from or or growing from chaos 15:25.660 --> 15:33.180 an order is imposed on that and so in this story 15:33.180 --> 15:39.580 in this idea of covid it could be that in order to transform us into a new 15:39.580 --> 15:43.260 global government in order to transform us into a new global 15:43.260 --> 15:47.740 governance structure based on on digital currencies and digital 15:47.740 --> 15:51.980 IDs and no more national borders that mean anything 15:51.980 --> 15:57.180 they need to have a chaos they need to start from a chaos they can't 15:57.180 --> 16:01.740 have all of this order and then rearrange it 16:01.740 --> 16:06.460 or hope to usefully disassemble it at least that's what they think 16:06.460 --> 16:10.460 that's what they they think that's how how maybe that is how people have been 16:10.460 --> 16:15.820 governed through chaos and then imposed order 16:15.820 --> 16:19.340 and if that's the case then it does seem as though the people that are 16:19.340 --> 16:25.340 governing the globe that want to shift us to this new paradigm 16:25.340 --> 16:30.300 understand it as a necessity for us to go through some kind of chaos 16:30.300 --> 16:34.540 in order for this societal metamorphosis to take place 16:34.540 --> 16:38.060 and so i'm starting to make this argument that there's a fine line between an 16:38.060 --> 16:41.980 announcement and a warning and i want you to start to 16:41.980 --> 16:46.860 think about what people say on our side about where we're going and how we 16:46.860 --> 16:51.260 avoid going there and whether or not it's an 16:51.260 --> 16:54.300 announcement or a warning whether it's an announcement or a 16:54.300 --> 16:58.060 suggestion whether it's an alternative or 16:58.060 --> 17:03.500 an inevitability because i think it's very important it doesn't only have to be 17:03.500 --> 17:07.500 a guy like carol adebach the health guy from germany 17:07.500 --> 17:12.140 or jorston talking about these kinds of things and then for us to know that 17:12.140 --> 17:17.020 oh this is not really a warning or a complaint this is an announcement 17:17.020 --> 17:23.340 when when jorston says that we really need to crack down on misinformation 17:23.340 --> 17:27.980 and the press needs to do their job we understand that fully 17:27.980 --> 17:31.820 that they mean to crack down on misinformation they want us to beg for 17:31.820 --> 17:36.460 that but then when we hear robert melone say that he's been 17:36.460 --> 17:41.100 harassed on on the internet and it's not fun and you wouldn't like it and we've 17:41.100 --> 17:47.020 got to stop using violent speech or they're going to use it against us 17:47.020 --> 17:52.060 is that an warning or an announcement if somebody says we need to build new 17:52.060 --> 17:56.300 underground systems but doesn't ever advocate for us to take back the system 17:56.300 --> 18:01.340 that we have and and remodel it retake it back and use it 18:01.340 --> 18:05.100 the way it's supposed to be restore our republic no he says we need to build new 18:05.100 --> 18:09.900 underground systems it's not very different than what 18:09.900 --> 18:15.740 happened when we got censored back in 2020 and everybody ran to gab 18:15.740 --> 18:18.700 and to telegram 18:21.180 --> 18:25.340 and everybody went on signal and had all these secret groups where they chatted 18:25.340 --> 18:29.500 about i don't know 18:29.980 --> 18:35.820 well everything and everybody went to discord 18:35.820 --> 18:39.580 and how hard was it for these non-encrypted apps to 18:39.580 --> 18:45.660 actually be like honey traps for us in a way all the people that 18:45.660 --> 18:51.020 wanted to march around and be efficient you know or not efficient but uh 18:51.020 --> 18:55.180 evasive of the of the system they went away 18:55.180 --> 19:02.460 they went on gab they went on telegram i'm on signal now 19:03.020 --> 19:09.100 curiously who led us to gab who led us to telegram who led us to signal 19:09.100 --> 19:13.820 who led us to discord who led us to sub-stack 19:18.220 --> 19:22.140 if you hear somebody up on stage say we have a lousy society our children have 19:22.140 --> 19:25.420 been taught all the wrong values it's going to be okay that it all comes 19:25.420 --> 19:30.380 apart is that really a announcement 19:30.540 --> 19:33.180 a warning 19:33.660 --> 19:37.020 is that inevitability 19:42.140 --> 19:47.340 is that somebody trying to help us or is that somebody just trying to keep us 19:47.340 --> 19:54.220 walking down or riding numbly down this neon hallway 19:54.220 --> 20:00.620 covered in lies is that somebody who's pretending to be a part of the 20:00.620 --> 20:05.740 group of mimes stopping this drain it's a really important analogy 20:05.740 --> 20:11.580 try to imagine fooling a young group a group of young kids 20:11.580 --> 20:16.620 and try to see that this guy all by himself with his little chalk dish 20:16.620 --> 20:25.340 he can't fool a group of kids but if you got a bunch of crossfit athletes 20:25.420 --> 20:28.700 and then you fitted them with a bunch of uh 20:28.700 --> 20:33.500 machines that made a lot of noise with wheels and sparks 20:33.500 --> 20:36.700 i guarantee you you could fool a bunch of kids you might even be able to fool 20:36.700 --> 20:43.260 a bunch of teenagers and the more people that you lined up on the train with more 20:43.260 --> 20:49.260 elaborate props and a more elaborate coordinated effort 20:49.260 --> 20:54.780 the more people you could fool the more inspection 20:54.780 --> 21:00.300 they would pass the more the more scrutiny they could withstand 21:00.300 --> 21:03.020 in their act 21:03.340 --> 21:07.180 if you made it really complicated 21:07.180 --> 21:11.660 if you made it with smoke and heat so that people couldn't get very close to the 21:11.660 --> 21:14.780 train as these people stopped it 21:14.780 --> 21:21.580 if you made it really loud so that people were afraid to get near it 21:21.580 --> 21:25.100 the point that i'm trying to make is is that during the pandemic that's what 21:25.100 --> 21:28.860 all of these stories did to us 21:28.860 --> 21:34.460 they made it seem like there was nothing to guess or or wonder about 21:34.460 --> 21:38.620 these people were doing the best that they could to stop a national security 21:38.620 --> 21:44.220 priority a national security threat of never-before-seen proportions 21:44.220 --> 21:48.940 and potential the potential to kill a billion people and we don't have any 21:48.940 --> 21:52.540 treatment we don't know what will work 21:52.540 --> 21:56.140 the best we can do is hope that these people can put out enough pcr's and 21:56.140 --> 22:00.540 lateral flow tests so that we can at least keep track of when we're sick asymptomatically 22:00.540 --> 22:09.260 and so many people on social media seem to agree about this 22:09.260 --> 22:13.420 so many people on social media are arguing whether or not hydroxychloroquine or 22:13.420 --> 22:17.580 ivermectin are crazy or not 22:17.580 --> 22:22.460 and ivermectin and hydroxychloroquine did not save anyone from a ventilator did 22:22.460 --> 22:26.380 not save anyone from rendezivir did not save 22:26.380 --> 22:29.900 anyone from an opioid death 22:32.220 --> 22:36.540 financial incentives to declare covid didn't save anyone from the covid 22:36.540 --> 22:39.100 protocols it actually put more people on them 22:43.100 --> 22:47.660 sending people back with to elderly care homes with pneumonia 22:47.660 --> 22:53.500 and viral pneumonia without antibiotics didn't save anyone 22:53.500 --> 22:59.340 the dismissal of immunology 101 in october of 2020 22:59.340 --> 23:03.820 by a whole list of leaders including wollinski 23:03.820 --> 23:09.420 as in we don't know if our immunity will be good enough it could be like aids 23:09.420 --> 23:14.780 where we build antibodies and that's actually a sign of disease not a sign of 23:14.780 --> 23:18.620 immunity did you know that do you remember that 23:18.620 --> 23:23.260 giant contradiction thanks for reminding me of that pamela or 23:23.260 --> 23:27.820 pointing it out even the value of nutrition in 23:27.820 --> 23:33.660 america was dismissed a place where almost every grown 23:33.660 --> 23:37.900 adult has been malnourished for at least a decade 23:37.900 --> 23:42.940 and many children grow up malnourished over 23:42.940 --> 23:50.620 carbon carbohydrate over sugared over medicated over immunized 23:56.060 --> 24:01.260 they have been doing this train pulling and pushing act for three years 24:01.260 --> 24:06.780 and public health the public health apparatus would like to govern the 24:06.780 --> 24:09.900 world with this act 24:10.540 --> 24:17.180 public health apparatus needed to convert this pattern 24:17.180 --> 24:25.020 into something that they could declare was a pandemic of a novel virus 24:27.100 --> 24:30.460 and in order to convert this very distinct 24:30.460 --> 24:35.740 and reliable pattern this very distinct reliable pattern 24:35.740 --> 24:40.300 in pneumonia deaths into this very indistinct 24:40.300 --> 24:45.340 and novel pattern of pneumonia deaths it required protocols not a novel 24:45.340 --> 24:47.740 virus 24:48.540 --> 24:52.780 it required novel protocols like do not resuscitate people if they're having a 24:52.780 --> 24:56.140 heart attack at home or a stroke let them die 24:56.140 --> 25:00.620 don't bring them back to the hospital pronounce them dead 25:00.620 --> 25:03.740 if you do bring them into the hospital make sure you ventilate in my extra 25:03.740 --> 25:08.780 high pressure to make sure that the virus doesn't come out 25:08.780 --> 25:12.780 antibiotics don't work on viral pneumonia dumbass 25:12.780 --> 25:16.700 and steroids don't either 25:17.900 --> 25:23.180 remdesivir works though it's pretty good we should try it on everybody 25:23.740 --> 25:28.700 and opioid deaths well those people are just covid deaths 25:28.700 --> 25:33.340 we don't even need to talk about them i mean who who even cares about those 25:33.340 --> 25:37.420 people isn't it a waste to even use Narcan on them like 25:37.420 --> 25:43.180 they're just i wonder what people in san francisco would say 25:43.180 --> 25:48.380 death certificate fraud how far should this list go how much longer how much 25:48.380 --> 25:51.980 many more things can we think about they need to go on there 25:51.980 --> 25:55.820 the reason why i want this to happen is because i need you to see 25:55.820 --> 25:59.340 because i need you to see what because i need you to see 25:59.340 --> 26:01.980 what that was an echo there because i had that open 26:01.980 --> 26:05.740 yikes that was scary i need you to see that these people 26:05.740 --> 26:15.740 have really fooled us now this video is a video from one month ago 26:15.740 --> 26:19.340 by a professor 26:19.340 --> 26:23.020 of carny g melon and today i'll be talking mostly 26:23.020 --> 26:26.460 let me get my head out of the way for a second so i can rewind this 26:26.460 --> 26:29.660 and make sure it's all set up correctly 26:29.660 --> 26:35.420 1.5 so this is a video of 26:36.140 --> 26:40.620 a professor from carny g melon university so that's that's in pittsburgh 26:40.620 --> 26:47.020 and she gave this talk one month ago as part of a 26:47.020 --> 26:52.300 honoring peter colas series of lectures so it's about our ln 26:52.300 --> 26:56.140 lnp for r and a delivery which i thought already got the nobel prize 26:56.140 --> 27:03.980 which is usually when something has had you know like a decade of impact 27:03.980 --> 27:07.900 and so she's going to give a lecture about this but i want you to listen 27:07.900 --> 27:10.940 very carefully to the assumptions that she makes 27:10.940 --> 27:17.340 and the assumptions are are monumental 27:17.340 --> 27:22.620 she's going to say what we said at giga ohm biological they were going to do 27:22.620 --> 27:29.020 we said that in 2021 when everybody was all hot and bothered about the 27:29.020 --> 27:31.260 spiky spike being toxic 27:35.820 --> 27:41.260 we said it that they were going to blame the vaccine 27:41.260 --> 27:44.620 and it not working right because they chose the wrong protein or they're 27:44.620 --> 27:47.340 going to make some other excuse and otherwise 27:47.340 --> 27:52.300 otherwise we just proved that r and a and 27:52.300 --> 27:56.140 transfection in general works great in humans 27:56.140 --> 28:00.460 now this is a woman who just gave a talk a month ago and basically 28:00.460 --> 28:07.100 she's going to lead off with that in an extraordinarily aggressive way 28:07.100 --> 28:10.380 r and a's here to stay 28:13.420 --> 28:16.700 so today i wanted to speak with you about some of the work that we've been 28:16.700 --> 28:20.540 doing developing lipid nanoparticles for targets outside of the liver 28:20.540 --> 28:23.740 and i specifically wanted to focus on some of our data regarding delivery to 28:23.740 --> 28:26.380 the pancreas i'm not really sure i just want to 28:26.380 --> 28:31.180 talk to Moderna for a second modified r and a is not biological messenger r and a 28:31.180 --> 28:34.380 i'm not really sure why you're making that point because 28:34.380 --> 28:40.460 we understand that that coat we we recognize that codon optimization 28:40.460 --> 28:45.580 has certain implications and effects we've recognized that the chemical 28:45.580 --> 28:50.860 alterations have certain effects and we're calling it r and a 28:50.860 --> 28:55.260 are calling it transfections so you keep saying that but 28:55.260 --> 29:00.220 i've we've all acknowledged that this is modified r and a 29:00.220 --> 29:05.820 if we call it m r and a or r and a it doesn't really matter it's synthetic 29:05.820 --> 29:11.260 r and a so i mean you're you're making a semantic argument that doesn't 29:11.260 --> 29:14.540 it doesn't really matter if you listen to her talk and you assume that this is 29:14.540 --> 29:18.300 all synthetics then then it's really about what they 29:18.300 --> 29:21.500 can and can't do with them that is the point 29:21.500 --> 29:25.740 they can't control where they go and when they say that they inject you in the 29:25.740 --> 29:29.180 muscle to augment your immune system it's just 29:29.180 --> 29:33.500 absolutely absurd and that's that's what she's going to talk about here with 29:33.500 --> 29:36.940 cartoons so just a little bit about my lab in 29:36.940 --> 29:39.180 general and dr khalis had mentioned this in the introduction 29:39.180 --> 29:41.900 we work in a couple of different areas of drug delivery 29:41.900 --> 29:46.380 so we focus on oral delivery of protein medication so drugs like insulin that 29:46.380 --> 29:49.740 currently have to be injected and trying to find ways of delivering them 29:49.740 --> 29:53.580 in more patient-friendly forms so that might involve oral delivery for 29:53.580 --> 29:55.660 proteins the problem with proteins is that they're 29:55.660 --> 29:58.700 digested by our gastrointestinal tracts and so we're very interested in 29:58.700 --> 30:01.260 studying the transport of these large molecules across the 30:01.260 --> 30:05.420 gastrointestinal tract to facilitate these more patient-friendly means of 30:05.420 --> 30:09.740 delivering proteins. More recently my lab has taken interest in 30:09.820 --> 30:12.780 women's and infant health. I had my first pregnancy at this point about seven 30:12.780 --> 30:15.100 years ago and then another one shortly before the 30:15.100 --> 30:17.100 pandemic happened and there's nothing like firsthand 30:17.100 --> 30:21.100 experience to realize that the state of maternal medicine is rather poor 30:21.100 --> 30:23.660 and there are a lot of things that we can do to try to improve our 30:23.660 --> 30:26.700 understanding of transport processes during pregnancy 30:26.700 --> 30:31.660 to try to create medications that are safe that do not cross into the infant. 30:31.660 --> 30:35.580 And then finally the subject of today's lecture is in this area of RNA delivery 30:35.580 --> 30:38.780 where I've been working now on lipid nanoparticles for over 15 years 30:38.860 --> 30:41.820 and it's just been wonderful to see how far this field has come since I first 30:41.820 --> 30:45.420 started. My lab is especially interested in how manipulating the chemistry 30:45.420 --> 30:47.820 of these particles which are quite complex chemically. 30:47.820 --> 30:50.700 How those manipulations can affect their functionality in vivo 30:50.700 --> 30:54.380 both in terms of bio distribution where they're working well immunogenicity 30:54.380 --> 30:57.580 and toxicity. So I'll speak a bit more about that today. 30:57.580 --> 31:01.100 So as pretty much all of you probably know at this point these RNA therapies 31:01.100 --> 31:05.420 have gone from niche to mainstream so the first FDA approval of a lipid 31:05.420 --> 31:09.180 nanoparticle formulation and the first FDA approval of an RNA drug 31:09.180 --> 31:14.140 was back in 2018 by Al nylon and they developed this medication for a rare 31:14.140 --> 31:17.260 type of liver disease and this was wonderful and it brought to 31:17.260 --> 31:21.260 fruition decades of research on liposomal and lipid nanoparticle type 31:21.260 --> 31:24.700 materials but even then we still had a very small 31:24.700 --> 31:27.820 population of people who were benefiting from this type of technology 31:27.820 --> 31:30.940 and people were quite cautious about you know using these types of 31:30.940 --> 31:34.620 medications for larger patient populations. So then we had this awful 31:34.620 --> 31:37.740 occasion to need to put these materials to the test 31:37.740 --> 31:41.500 and I think all of us in the delivery field were absolutely delighted to see 31:41.500 --> 31:44.780 how well these materials and how well RNA therapies can work 31:44.780 --> 31:46.940 in you know huge. Was there ever a 31:46.940 --> 31:51.500 humanity or is this a stupid picture like what did they ever have this 31:51.500 --> 31:55.980 actually available or it was always the it was always Pfizer right 31:55.980 --> 32:01.340 it never came out with that I think this is BS right here 32:01.820 --> 32:05.740 anyway this is the statement that I'm trying to make 32:05.740 --> 32:11.260 this is you guys can all see it for what it is this is absolutely absurd 32:11.260 --> 32:16.780 the idea is very clear this was always the plan 32:16.780 --> 32:21.100 there was always going to be the plan it was always going to be 32:21.100 --> 32:25.100 throw the adenavirus under the bus so that we can point to the adenavirus 32:25.100 --> 32:28.380 as well obviously we took it off the market because 32:28.380 --> 32:35.500 it was causing clots and the mRNA doesn't cause clots. 32:35.500 --> 32:40.140 I mean duh the mRNA causes myocarditis but that goes away 32:40.140 --> 32:44.940 myocarditis is fine it only kills 50% of the people that 32:44.940 --> 32:50.540 they get it. It's it's really sad it's really sad 32:50.540 --> 32:54.220 because we said this we said it was going to happen two years ago we said 32:54.220 --> 33:00.060 they were going to do this and not one single person 33:00.060 --> 33:05.420 in that narrative control panel ever even bothered to think out loud that 33:05.420 --> 33:09.100 thought never mind actually speak the words or write a 33:09.100 --> 33:16.620 sub stack about it nobody and for two or three years 33:16.620 --> 33:19.420 almost three years really now that where we are 33:19.420 --> 33:23.900 it's almost three years oh my gosh it's going to be 2024 33:23.900 --> 33:27.980 we have been saying that they are going to blame 33:27.980 --> 33:33.740 some mistake in the methodology some mistake in the choice of protein some 33:33.740 --> 33:37.580 epitope they should have taken out 33:38.860 --> 33:42.860 and then they moved on to impurities then they moved on to 33:42.860 --> 33:48.700 process one versus process two and double stranded DNA and this sv40 33:48.700 --> 33:55.500 enhancer and promoter it's always been the same story 33:55.500 --> 34:00.620 these things work great but there were some problems with the rollout yeah sure 34:00.620 --> 34:04.620 but it was a rush job when we make these things like 34:04.620 --> 34:09.900 fine brewed beer this stuff is amaze balls 34:12.620 --> 34:16.860 that's the story that's the sales pitch that's the tv narrative that's what 34:16.860 --> 34:21.020 skilled tv watchers believe right now that's where we are at 34:21.020 --> 34:26.780 Thanksgiving 2023 just where i said we would be 34:30.940 --> 34:32.220 i'm not thankful for that 34:36.540 --> 34:39.980 which numbers of people and so now with this evidence and hundreds of millions of 34:39.980 --> 34:43.180 people at this technology works well this is really going to open the floodgates 34:43.180 --> 34:47.340 to all sorts of new therapies not just in vaccines but in protein replacement 34:47.340 --> 34:52.060 gene editing and otherwise so think about how naive that is think about 34:52.060 --> 34:56.380 how naive that is that she thinks that those two are equivalent because we 34:56.380 --> 35:02.700 used it for vaccination successfully even if even if 35:02.700 --> 35:05.260 even if 35:06.620 --> 35:11.980 replacing proteins hasn't worked 35:13.260 --> 35:18.060 curing cancer rarely works with mRNA 35:22.540 --> 35:26.300 putting these things in certain places doesn't work 35:28.380 --> 35:33.340 when you change their chemistry and they go places it's a fraction of it right 35:35.180 --> 35:38.220 these people would be very excited if they changed the lipid 35:39.100 --> 35:45.180 the lipid nanoparticle chemical composition and you went from 50 percent of it in the 35:45.180 --> 35:51.180 liver and 50 percent of it in the ovaries to 80 percent of it in the liver and 30 percent 35:51.180 --> 35:56.380 in the ovaries that would be a phd project that would be a very significant result 35:57.100 --> 36:00.460 that would allow them to stand in front of years of lecture halls 36:01.100 --> 36:06.860 saying that these results suggest that in a few years we can have really precisely targeting 36:07.020 --> 36:11.980 lipid nanoparticles based on the chemical alterations of and the properties that 36:11.980 --> 36:16.060 that emerge from those alterations blah blah blah blah blah blah 36:18.460 --> 36:22.940 and so let's listen to this lady explain what she plans to do and explain the 36:23.500 --> 36:26.780 sort of hand waving that she does look at this terrible cartoon already 36:28.700 --> 36:31.340 a protein is just a ball that comes from a comb 36:32.300 --> 36:36.940 I mean so for any trainees in the audience who are a little bit less familiar with this 36:36.940 --> 36:40.060 I wanted to just go through some of the basic molecular biology that 36:40.060 --> 36:44.380 underpins all this RNA work that we're doing so this is the central dogma of molecular biology 36:44.380 --> 36:48.700 that tells us that our permanent blueprint the DNA inside of ourselves this is transcribed 36:48.700 --> 36:53.500 into a temporary copy called messenger RNA which is then translated in the cell cytoplasm 36:53.500 --> 36:59.500 into proteins and so those arrows don't mean anything understanding the DNA polymerase 36:59.500 --> 37:03.420 doesn't mean anything like how it's copied understanding the RNA polymerase 37:04.380 --> 37:08.060 don't worry about that that's just an arrow understanding ribosomes 37:08.940 --> 37:12.860 mRNA protein that's just an arrow who cares about those things 37:15.340 --> 37:19.500 and now she's going to say that proteins are everything proteins are the motor as well 37:20.620 --> 37:28.220 ribosomes are RNA ribosomes are made of RNA and proteins in combination so it's a little 37:29.500 --> 37:35.580 hand wavy to say that proteins do everything when the most important thing the most important 37:35.580 --> 37:39.100 nanomachine that we almost know nothing about is the ribosome 37:42.140 --> 37:47.260 but that's okay you know just just keep us asking the wrong questions keep us focused 37:47.260 --> 37:53.340 on the wrong things instead of the arrows we'll focus on those funny shapes in blue 37:54.300 --> 37:59.100 genes are the doers in our body and because they're the molecules responsible for all function 37:59.100 --> 38:04.860 many diseases are caused by misregulated proteins so either the protein is non-functional 38:04.860 --> 38:09.660 either we have too much of a particular protein or too little and so to treat so a protein is 38:09.660 --> 38:13.820 non-functional we have too much we have too little it has nothing to do with the tertiary 38:13.820 --> 38:19.740 structure of the protein or the tertiary chemical alterations of it that's extraordinary and again 38:20.620 --> 38:26.860 you could say that this is being imprecise but she's talking to a really educated audience you 38:26.860 --> 38:33.740 would think that you'd want to hit this pretty pretty on the money you think you'd want to be 38:33.740 --> 38:39.020 with be pretty sharp with your biology but we're not we're not sharp right now this is one month 38:39.020 --> 38:44.620 ago ladies and gentlemen please we really need to manipulate protein expression such that it goes 38:44.700 --> 38:49.820 back to what it's supposed to be so that first FDA approved drug it contained short interfering 38:49.820 --> 38:55.340 RNA and it's short because it's about 21 base pairs in length as opposed to RNA mRNA which can 38:55.340 --> 38:59.900 be thousands long and it interferes with protein production so you can design this so it would 38:59.900 --> 39:05.020 be specific for a target gene of interest and then you can have reductions in that protein expression 39:05.660 --> 39:12.140 now i'm a little gonna object to the way that this is drawn because it looks like to me 39:12.700 --> 39:20.620 her si RNA is drawn as double-stranded but actually i think correct me if i'm wrong smart people in 39:20.620 --> 39:28.140 the audience but i think RNA is single-stranded and when you put small interfering RNAs in there 39:28.140 --> 39:36.140 they bind to the endogenous RNA and make a double-stranded RNA and that's why they get degraded 39:36.460 --> 39:41.740 because double-stranded RNA ain't allowed in our cells 39:45.020 --> 39:47.900 you see again it's just kind of imprecision you know like 39:50.300 --> 39:55.900 it's just kind of imprecise and it's annoying because these people get paid a crap ton of tax payer 39:55.980 --> 40:05.260 money to teach children to apprentice young biologists into this 40:06.940 --> 40:07.980 into this endeavor 40:11.580 --> 40:16.300 if we want to upregulate protein expression instead of downregulate that's where messenger RNA comes 40:16.300 --> 40:22.380 in and so we can design mRNA's that can for example give us more of a particular gene of or protein 40:22.380 --> 40:27.020 of interest and in the case of the vaccines it's just super cool we can ask our bodies to 40:27.020 --> 40:31.180 generate proteins that our bodies would never normally make so in this case it's generating 40:31.180 --> 40:36.780 the spike protein which is not native to mammalian cells so very powerful technology 40:36.780 --> 40:42.300 i wanted to speak very briefly on our news this week that the noble prize was given for the mRNA 40:42.300 --> 40:48.380 biology that underpinned the covid-19 vaccines so RNA therapeutics really require two parts 40:48.460 --> 40:53.340 so one is functional mRNA that once it goes inside the cell it can produce the protein of interest 40:53.340 --> 40:57.260 effectively and then the other is the delivery system which i think hasn't gotten quite as 40:57.260 --> 41:01.900 much press as the RNA itself but is equally important so today i'll be talking about the delivery 41:01.900 --> 41:07.500 system and the noble prize had to do with this mRNA biology so here are the two people who have 41:07.500 --> 41:12.060 just been awarded so koriko and weissmann who originally did the work at the university of 41:12.060 --> 41:16.940 pennsylvania in philadelphia so they um i like to just tell people they're both really terrific 41:16.940 --> 41:22.620 people i think they could be on a sitcom together or something the two videos that we watched of them 41:22.620 --> 41:32.700 i mean they were lovable people i mean definitely but in a you're not a nobel prize-winning kind 41:32.700 --> 41:37.980 of lovable way i think i think we don't always have um you know sometimes famous scientists aren't 41:37.980 --> 41:43.420 the best models of of collaboration it's been my privilege to collaborate with drew over the years 41:43.500 --> 41:48.700 when i was getting started in the mRNA field it was back in 2017 and RNA is quite expensive 41:48.700 --> 41:53.500 if you purchase it and i was giving a seminar at pen and drew was a stranger to me and we had a 41:53.500 --> 41:59.500 meeting and he offered to make me whatever mRNA i wanted and send it to me and people didn't believe 41:59.500 --> 42:03.100 too much in RNA therapies at that time there was still a lot of hesitation so i was taken aback 42:03.100 --> 42:07.020 and confused why a stranger would offer to help me in this way given the expense so it took me a 42:07.020 --> 42:10.940 couple of years to actually approach him and then when i did he was true to his work and he's provided 42:10.940 --> 42:15.980 me with with RNA since that time and has otherwise been a great collaborator i've also met 42:15.980 --> 42:20.060 katie koriko briefly and she's given me some nice advice so just such a pleasure to see good 42:20.060 --> 42:26.460 people um reward it for for their work so um in terms of what they did they discovered that 42:26.460 --> 42:31.980 base modifications of RNA are needed for protein translation and mammals so here is the molecule 42:31.980 --> 42:38.140 uridine which is one of those canonical nucleic acids that are present in RNA and the problem 42:38.140 --> 42:43.180 when we make RNA typically with these molecules such as uridine is that our cells recognize them 42:43.180 --> 42:49.500 as foreign and that's because our cells make RNA to incorporate some base modifications so if it 42:49.500 --> 42:54.220 sees RNA without base modifications it mounts a bit of an immune response that shuts down protein 42:54.220 --> 42:58.860 production so that's part of why nobody thought RNA therapies would become a reality because 42:58.860 --> 43:03.580 when we made RNA in lab and introduced it it just would not make protein so what they found was that 43:03.580 --> 43:09.740 they could make modifications to these different nucleobases and so here's um one version of this 43:09.740 --> 43:14.140 this is um n1 methyl pseudo uridine the ones that are used in the vaccines are just called 43:14.140 --> 43:19.420 pseudo uridine so i don't think this is i'm sorry i had to i had to put something in my ear and 43:19.420 --> 43:24.460 clean it from my i'm wearing my headphones again tonight i don't think this is right and i hope i 43:24.460 --> 43:32.060 don't so i don't care if i do it's it's thanksgiving and i'm streaming i i think that this is absolute 43:32.140 --> 43:38.380 nonsense i don't think that base modifications are necessary for protein translation in mammals i 43:38.380 --> 43:43.820 think they're necessary for sustained protein translation in mammals for them to get a signal 43:43.820 --> 43:53.020 that they can point to and say look it works but if you put non uh methyl pseudo uridine 43:53.820 --> 43:57.500 RNA in your lipid nanoparticle you'll get very transient expression 43:58.460 --> 44:08.860 very transient expression absolutely but that that's not quite right what they what this is is 44:10.460 --> 44:18.620 what this is is somebody who is i think over their skis saying what they've been told is right 44:19.740 --> 44:25.420 they probably practiced this talk and somebody said it's fine but essentially she's not saying 44:25.500 --> 44:33.580 this correctly i i really don't think so i know that we have base alterations in our RNA 44:34.860 --> 44:43.340 and they are used presumably for RNA regulation but for purposes of a 44:43.340 --> 44:53.900 transfection tool that's true for purposes of how RNA functions endogenously in your cells 44:54.620 --> 45:02.460 that's true to an extent because actually again the transient nature of RNA being degraded quickly 45:02.460 --> 45:08.060 in your cells is how protein expression is regulated at least as far as we guess 45:08.780 --> 45:15.260 and that's why small interfering RNAs that are complementary to your own 45:15.980 --> 45:21.500 mRNAs and circulation are an interesting way to interfere with protein expression 45:23.020 --> 45:27.020 somebody put in the chat that they're double stranded RNAs i'm going to look it up i'm not sure 45:27.660 --> 45:30.940 i think the mechanism is how i've described it though um 45:31.500 --> 45:37.980 um and so i think this is a bit of a hand-waving exercise to say that this is how it works but 45:37.980 --> 45:45.100 she's specifically talking about transfection not about about translation of proteins and 45:45.100 --> 45:49.820 endogenous proteins so we have these modifications and the cells are more willing to recognize these 45:49.820 --> 45:54.620 externally introduced RNAs as self and then they are willing to produce that protein so all the 45:54.620 --> 45:59.420 work that i'm showing you today is made with RNA that's been base modified so that it works well 45:59.580 --> 46:07.580 so that it works well see so so that it works well in its transfection excuse me so to go back to 46:07.580 --> 46:12.940 the delivery vehicle part of the equation RNA is large it's on the order of a hundred thousand 46:12.940 --> 46:17.180 grams per mole it's negatively charged and so it doesn't readily cross negatively charged cell 46:17.180 --> 46:21.340 membranes and if you were to inject it into the bloodstream it would be both degraded and 46:21.340 --> 46:25.260 negatively charged on the order of a hundred thousand delivery vehicle part of the equation 46:25.260 --> 46:30.460 RNA is large it's on the order of a hundred thousand grams per mole it's negatively charged 46:30.460 --> 46:34.060 and so it doesn't readily cross negatively i get to go back a little bit further and 46:34.060 --> 46:37.900 previous of delivery barrier it's been base modified so that it works well 46:39.100 --> 46:45.100 so in terms of delivery barriers excuse me so to go back to the delivery vehicle part of the 46:45.100 --> 46:50.780 equation RNA is large it's on the order of a hundred thousand grams per mole it's negatively 46:50.780 --> 46:54.940 charged and so it doesn't readily cross negatively charged cell membranes and if you were to 46:55.020 --> 46:59.500 inject it into the bloodstream it would be both degraded and quickly cleared from our system so 46:59.500 --> 47:04.220 we need a vehicle that's going to package it up and protect it and take it to the cells of interest 47:04.220 --> 47:11.020 wow so that's to me we've got to hear that for what she says it is right you got to hear it for 47:11.020 --> 47:16.620 what she says because that assumption is wrong listen carefully you inject it into the bloodstream 47:16.620 --> 47:21.740 it would be both degraded and quickly cleared from our system so if you injected it into the 47:21.740 --> 47:27.180 bloodstream it would be degraded and very quickly cleared did she mean that without lip 47:27.180 --> 47:32.700 and nanoparticle i think that must be what she means right because 47:34.060 --> 47:38.780 wow that that must be just naked RNA but we need a vehicle that's going to package it 47:38.780 --> 47:44.860 yeah okay and take it to the cells of interest so this diagram depicts a couple of barriers that 47:44.860 --> 47:49.100 it needs to overcome if we were to do IV injection so once it's injected into the bloodstream it 47:49.100 --> 47:53.580 needs to avoid clearance by macrophages once it exits the bloodstream it needs to 47:53.580 --> 47:57.980 it needs to avoid clearance by macrophages it also needs to avoid 47:58.940 --> 48:06.940 transfecting endothelial cells and avoid transfecting blood cells and as it goes through 48:06.940 --> 48:13.260 capillaries that will be more and more difficult because the then that's where this is all going 48:13.260 --> 48:16.140 to occur right that's where this size scale comes into play 48:19.180 --> 48:23.740 it's not going to happen in the big vessels it's going to happen in the capillaries and capillaries 48:23.740 --> 48:34.460 that this this size scale is wrong this cartoon is wrong and avoiding the transfection of endothelial 48:34.460 --> 48:42.460 cells is much harder than one might think diffuse through the tissue of the organ that you want to 48:42.460 --> 48:47.020 target it then needs to be up taken into the cell of interest and this happens through a 48:47.020 --> 48:50.940 process called endocytosis where the cell membrane reaches up and around that particle 48:50.940 --> 48:55.100 and it brings it inside and so at this point the nanoparticle is in this Waldorf compartment 48:55.740 --> 48:58.780 and the cell keeps it there because it's not sure what it is and it looks and says this is 48:58.780 --> 49:05.340 something I want to see that the the whole anytime a biologist anthropomorphizes the 49:05.340 --> 49:14.060 operations of a cell and I don't even know I don't think you can ever do that and if you do 49:14.060 --> 49:21.260 it you need to be very very careful in this case it's ridiculous it's held in an endosome until 49:21.260 --> 49:25.660 it does the cell decides what to do with it because it's not sure what it is 49:26.940 --> 49:32.700 are you kidding this is a chemical covered in other chemicals 49:33.260 --> 49:36.220 the hell is she talking about 49:39.180 --> 49:43.900 and typically a lipid nanoparticle is not on its wish list so the endosome begins to acidify 49:43.900 --> 49:47.820 and would otherwise degrade these lipid nanoparticles so part of the design of the chemistry behind 49:47.820 --> 49:51.980 these particles is that they need to mediate this process of endosomal escape and that's one of 49:51.980 --> 49:56.380 the most difficult parts of this process so once the RNA is into the cell cytoplasm that's when 49:56.380 --> 49:59.980 it can interface with the protein translational machinery and make our protein of interest 50:00.460 --> 50:06.300 so years ago we asked if we could develop a potent and degradable RNA delivery system 50:06.300 --> 50:10.700 potent for obvious reasons and degradable because many of these therapeutics will need to be 50:10.700 --> 50:18.220 repeat dose I mean seriously jittery and maker jason all of these arrows are pretty magical 50:18.220 --> 50:22.860 all of these arrows are pretty magical this arrows very magical this arrow what is this 50:22.860 --> 50:28.860 like is this an an electron going down from two different energy levels what the hell is this 50:30.540 --> 50:36.540 I mean did they take and are they trying to convert a photosynthesis diagram into a 50:41.900 --> 50:48.860 I mean even this is silly this part you know like okay now we have RNA I guess but it's 50:48.860 --> 50:55.180 double stranded or something what is this you know I don't I don't I don't I don't know what to say 50:56.140 --> 51:01.100 I don't know how to overcome this I mean could I get her to come to 51:01.820 --> 51:06.620 giga ohm biological and let me give her an hour lecture like this but then say completely 51:06.620 --> 51:17.820 the opposite what would she do it's shocking isn't it it's just shocking this B arrow this 51:17.820 --> 51:23.580 C arrow what is that the part of the design of the chemistry behind these particles is that they 51:23.660 --> 51:27.100 need to mediate this process of endosomal escape and that's one of the most difficult 51:27.100 --> 51:31.500 parts of this process so once the RNA is into the cell cytoplasm that's when it can interface 51:31.500 --> 51:36.140 with the protein translational machinery and make our protein of interest see if you can say all 51:36.140 --> 51:41.100 those words together that's when it can interface with the protein the cells protein machinery or 51:41.100 --> 51:48.140 whatever I mean that that doesn't make you a commander of this knowledge it doesn't make you 51:48.140 --> 51:53.260 understanding the way that this complex system manifests it's just a joke 51:55.900 --> 52:02.060 she was anthropomorphizing the cell as holding the liposome in an endosome because it didn't 52:02.060 --> 52:08.940 know what it was and it wasn't sure what to do with it that was her explanation for these other PhDs 52:09.260 --> 52:17.500 so years ago we asked if we could develop a potent and degradable RNA delivery system 52:17.500 --> 52:21.980 potent for obvious reasons and degradable because many of these therapeutics will need to be 52:21.980 --> 52:26.860 repeat dosed we will not necessarily create a cure but a treatment and we don't want this material 52:26.860 --> 52:32.620 building up over time so the materials that we work with in my lab they're lipid-like materials 52:32.620 --> 52:37.180 we call them lipidoids and they're a variation of the cationic lipids that have been used for 52:37.260 --> 52:41.340 decades at this point for different types of nucleic acid delivery first a lot of the work 52:41.340 --> 52:45.820 was done in the DNA delivery field so here's one of these cationic lipids if you were to put it 52:45.820 --> 52:50.940 into aqueous medium you would find that they would assemble into these liposomal structures 52:50.940 --> 52:57.660 where we have the hydrophilic head group on the outside of the aqueous media and then we would 52:57.660 --> 53:02.460 have these hydrophobic tails on the inside and you can load then hydrophilic drugs into the interior 53:03.020 --> 53:07.420 so we wanted to make something like this to hold on to our RNA and we also wanted to 53:07.420 --> 53:11.260 generate materials where we could look at a lot of different chemistries and try to understand 53:11.260 --> 53:17.420 I want you to see how how extraordinary it is that she talks as though we wanted to do we wanted to 53:17.420 --> 53:24.700 do we wanted to do like it's something you know it's almost like saying you know I wanted to put 53:24.700 --> 53:31.180 together a bunch of pages and I'm gonna call it a book and I really wanted to make a book so it was 53:31.180 --> 53:34.860 my idea to bunch a bunch of pages together and then bind them at the back 53:36.780 --> 53:45.660 she's talking about a a technology that she started her talk by saying that it had gone from 53:45.660 --> 53:54.060 niche to mainstream so what does she need to explain it for what's so special about hers 53:55.020 --> 54:01.580 and why does she need to explain it on this level she's giving a talk with Peter Coles in the 54:01.580 --> 54:09.260 background it's extraordinary I find this extraordinary it is the lack of 54:13.180 --> 54:19.820 appropriate audience I mean it's it's just oh it drives me crazy this is what used to drive me 54:20.220 --> 54:29.660 crazy about about neuroscience you could have a room full of people talking about supposedly 54:29.660 --> 54:34.940 about the highest level of discussion about our understanding of how micro circuits in the brain 54:34.940 --> 54:39.500 work and you could have somebody in the back room raise their hand and say I don't I don't 54:40.140 --> 54:43.420 could you briefly explain what a G protein coupled receptor is 54:43.980 --> 54:51.340 and you might have been talking about a type of receptor that is a G protein coupled receptor 54:54.780 --> 54:59.980 and you may have even given an introduction that was reasonable about G protein coupled 54:59.980 --> 55:04.380 receptors and this person would still have the audacity to raise their hand and ask that question 55:07.180 --> 55:08.380 only in biology 55:09.100 --> 55:10.620 you 55:10.620 --> 55:15.580 couldn't go into a physics lecture and say hey can you briefly explain to me what you mean by that 55:17.900 --> 55:25.100 I'm not sure I get it you couldn't go into an organic chemistry lab or lecture and say you know 55:25.100 --> 55:30.460 can you briefly explain to me what that category of molecule is I'm not really familiar with that 55:31.180 --> 55:38.860 but for some reason in biology everybody any any poser that wants to can go and join a lab 55:38.860 --> 55:41.340 and do a PhD project and there you go 55:45.580 --> 55:51.340 you did some programming as a PhD student and now you're up now you're going to do a postdoc in biology 55:53.180 --> 55:56.940 and since you did some T cell stuff I guess you're an immunologist now 56:00.540 --> 56:05.020 oh you put some silt bubbles and some RNA together and you followed the recipe 56:05.740 --> 56:10.780 and then you followed another recipe successfully and after following six recipes successfully that 56:10.780 --> 56:15.340 you basically adapted from other people's recipes you're a biologist now 56:18.860 --> 56:24.540 and the depth of your understanding of the concepts that you purport to wield is terrifying 56:25.420 --> 56:29.980 the chemistry of these lipidoids would affect their functionality and try to derive structure 56:29.980 --> 56:33.900 functional relationships from that so we use a high throughput type of chemistry called Michael 56:33.900 --> 56:38.940 addition in which we take an alkalamine so we need some combination and say primary means a 56:38.940 --> 56:43.580 secondary means and we would react them with alkal acrylate tails here it's an easy reaction 56:43.580 --> 56:46.940 you put the two together in a scintillation vial there's no solvent you can mix them for a 56:46.940 --> 56:50.780 couple of days at high temperature and out the other side comes something that's not too dissimilar 56:50.860 --> 56:55.900 from this cationic lipid up here so we can vary the chemistry of both this amine and the tail 56:55.900 --> 57:00.620 to generate large numbers of materials the degradability comes in from the use of these 57:00.620 --> 57:06.060 acrylate materials with which result in an ester in the final form of these lipids and those esters 57:06.060 --> 57:12.540 degrade under physiological conditions so these lipids are ionizable meaning that under reduced 57:12.540 --> 57:17.660 pH conditions as we form these particles they are going to have this nice electrostatic interaction 57:17.660 --> 57:21.980 with the negatively charged RNA which will help to catalyze the formation of these particles 57:22.700 --> 57:27.580 so the particles are made through a nanoprecipitation process in which we need rapid mixing 57:28.540 --> 57:33.820 so we have a rapid mixing of two streams to form our nanoparticles one of which is going to be 57:33.820 --> 57:39.020 RNA that's in a saline solution and then i do have to say that microfluidics is really cool 57:39.020 --> 57:44.380 they can do a lot of really cool things with microfluidic devices we have a variety of lipids 57:44.460 --> 57:48.700 that are dissolved in ethanol and they are rapidly mixed you can use microfluidic devices 57:48.700 --> 57:52.620 t-mixers and you can also use pipettes which are relatively straightforward 57:52.620 --> 57:55.820 pipettes are not appropriate for scale up or anything like that but they work well in the 57:55.820 --> 58:00.140 lab when you don't have a lot of money for microfluidic systems so you mix these two streams together 58:00.620 --> 58:05.580 and you get materials that you know you have you have lipids both on the exterior of the 58:05.580 --> 58:09.100 particle like we had with liposomes but you also have lipids throughout the material 58:09.100 --> 58:13.740 so in terms of the ingredients in these particles they include the ionizable lipid or lipidoid that 58:13.740 --> 58:18.300 i just told you about this tends to be where a lot of the IP is in the lipid nanoparticle space 58:18.300 --> 58:22.860 many labs or companies attempt to create their own chemistry and intellectual property with 58:22.860 --> 58:27.340 these synthetic materials they also contain helper lipids so these are often phospholipids that 58:27.340 --> 58:31.180 naturally occur in our cell membrane they can help with stability of the particles and also 58:31.180 --> 58:35.660 their ability to help with endosomal escape we have a lot of cholesterol in these particles 58:35.660 --> 58:40.220 which adds stability and prevents the particles from falling apart very similar to the way we 58:40.300 --> 58:43.900 have a lot of cholesterol in our cell membranes we would all be piles of boo if it were not for 58:43.900 --> 58:49.580 our cholesterol in our particles would be too then we have our polyethylene glycol lipid so the lipid 58:49.580 --> 58:53.420 anchor here will insert on the inside of the particle and then we have polyethylene glycol 58:53.420 --> 58:57.740 which is a hydrophilic polymer which decorates the outside of the particle it can quench that 58:57.740 --> 59:02.700 nanoparticle formation process so help us obtain particles of a nice size it can also improve 59:02.700 --> 59:06.460 stability and decrease the amount of unwanted immune cell uptake that we get 59:07.900 --> 59:12.700 unwanted immune cell uptake i thought you wanted immune cell uptake 59:14.700 --> 59:18.940 or maybe not for their maybe they're maybe they're not trying to do vaccines maybe they're trying 59:18.940 --> 59:23.340 to do something else so over the years we've made and screened thousands of these ionizable lipidoids 59:23.340 --> 59:27.900 for RNA delivery and we found a number that we really like and we've used them for numerous 59:27.900 --> 59:31.980 applications so here's one of our favorite lipidoids that we use in my lab 59:31.980 --> 59:35.500 it's a four-tailed lipidoid it has a couple of these nitrogen groups which help it 59:35.500 --> 59:40.620 protonate in the endosome and endosome doing endosomal escape we have our esters here for 59:40.620 --> 59:44.220 degradability and then these materials are interesting because they have these branch 59:44.220 --> 59:49.260 tails and the branch tails seem to change the structure of these lipids so that they can 59:49.260 --> 59:54.540 interface with endosomal membranes differently and aid that fusion and escape process so this 59:54.540 --> 59:58.940 is a great lipid and so the work i'm going to show you today all of it involves this ionizable lipid 59:59.820 --> 01:00:04.140 so standard nanoparticles where the state of the field is at this point is that we can achieve 01:00:04.140 --> 01:00:08.780 delivery to a couple of locations so the liver has been the easiest location i don't want to say 01:00:08.780 --> 01:00:13.420 it's easy but it's been the easiest because when you inject particles the liver is the organ that 01:00:13.420 --> 01:00:18.380 will naturally clear them from the bloodstream and so if it doesn't clear them from the bloodstream 01:00:18.380 --> 01:00:23.580 then those particles go elsewhere they go to any place they want to go they go to endothelial 01:00:23.580 --> 01:00:29.260 cells they go to the ovary they go to the testes they go to your brain they can go anywhere they 01:00:29.260 --> 01:00:37.740 want to if if you have perforated endothelial barriers so this concept that it can be 01:00:37.740 --> 01:00:45.180 it can be remain in the muscle and look how they label it immune cells and muscle now i'm sorry i'm 01:00:45.180 --> 01:00:51.660 not trying to be a creep here but the immune system isn't really organized to protect the 01:00:51.660 --> 01:00:58.540 interior of your deltoid there may be some resident immune cells there there may be a lymph 01:00:58.540 --> 01:01:07.340 vessel that runs through there the immune system is present but the immune system is 01:01:07.340 --> 01:01:12.620 oriented around barriers there is no question about it and barriers with the outside world 01:01:12.620 --> 01:01:17.340 there is a barrier with the outside world that runs through your body there's a barrier with 01:01:17.340 --> 01:01:22.860 the outside world that is in your lungs and there's a barrier in the outside world on the 01:01:22.860 --> 01:01:31.820 outside of your body and all three of these barriers are manned are our outward facing 01:01:31.820 --> 01:01:38.140 epithelial barriers epithelial cells are necessarily dividing barriers 01:01:38.300 --> 01:01:48.860 and the immune system is organized around them and so this assumption that she makes that we 01:01:48.860 --> 01:01:56.460 can inject it in in a muscle and it stays there and it's expresses protein and the immune system 01:01:56.460 --> 01:02:03.980 comes and it's not sure what to do but eventually it decides that what what the intention of this 01:02:04.060 --> 01:02:09.420 protein is is to make me make immune to a memory to it or something like this whenever they would 01:02:09.420 --> 01:02:15.580 talk through it and that first FDA approved product was to treat a liver condition and it's a to treat 01:02:15.580 --> 01:02:20.940 a liver condition because that's where the lipid nanoparticles go necessarily because the liver 01:02:20.940 --> 01:02:27.420 cleans your blood not because they're chemically altered to go to the liver but because that's 01:02:27.420 --> 01:02:31.900 where the majority of them go all of them don't go there 01:02:35.020 --> 01:02:40.060 the ones that don't transfect something else first get pulled out in the liver 01:02:42.620 --> 01:02:46.060 much of the transfection occurs at the liver 01:02:49.260 --> 01:02:54.700 and in fact it's very likely that when you get injected with an mRNA in your muscle and it leaks 01:02:54.700 --> 01:03:03.020 out the transfection occurs in the liver the transfection occurs in the capillaries of the heart 01:03:03.020 --> 01:03:06.940 the capillaries of the lungs maybe the capillaries of your skin 01:03:08.380 --> 01:03:13.340 I think we've seen it many many times where it's the capillaries of the skin where we get a massive 01:03:13.340 --> 01:03:21.980 transfection and therefore a massive inflammation a massive neutrophil attack on the endothelial cells 01:03:21.980 --> 01:03:28.700 we've seen this all around the world we've seen what happens we know this is what's going on 01:03:28.700 --> 01:03:38.940 there's no other biological alternative and these people know even as she disingenuously describes 01:03:39.580 --> 01:03:48.540 the nature of targeting the liver they called them liver targeting lipid nanoparticles because 01:03:48.540 --> 01:03:52.060 the liver pulls them out and the majority of them showed up there 01:03:59.180 --> 01:04:02.540 I mean I just hope you can see how disingenuous that is 01:04:07.180 --> 01:04:08.460 I just hope you can see it 01:04:09.340 --> 01:04:11.340 oops 01:04:13.260 --> 01:04:18.300 we also are capable of vaccination so delivery to the immune cells and the muscle cells within 01:04:18.300 --> 01:04:22.780 the muscle tissue to mount the immune response that we need but the question really is is we're 01:04:22.780 --> 01:04:28.220 thinking about the future of RNA therapies is how we will deliver these materials beyond the liver 01:04:28.220 --> 01:04:32.620 so as you can imagine most of the diseases in the body are outside of the liver and there are 01:04:32.620 --> 01:04:36.860 many different places we'd like to go so my lab works on delivering these materials to a couple 01:04:36.940 --> 01:04:41.020 of these particular organs and today I'll be talking mostly about our ability to deliver to 01:04:41.020 --> 01:04:47.580 the pancreas so for this work we're going to be focusing on swapping out the helper lipids 01:04:47.580 --> 01:04:52.540 in these particles and looking at how the influence of helper lipid charge in particular 01:04:52.540 --> 01:04:57.660 can direct these particles to different cell types so there have been a couple of people who 01:04:57.660 --> 01:05:03.100 have reported the influence of charge on the delivery process so Dan Seagard's lab was one 01:05:03.100 --> 01:05:07.660 of the first to show that changing the charge either cationic or negative compared to some of 01:05:07.660 --> 01:05:12.540 the neutral helper lipids can shift delivery from the liver to either the spleen or the lungs 01:05:12.540 --> 01:05:18.860 and we found something similar in our lab so in this case I'm showing you so DOPE is the standard 01:05:18.860 --> 01:05:23.340 phospholipid that we use in my lab it carries a net neutral charge and you can see if we look at 01:05:23.340 --> 01:05:27.900 organ distribution of protein expression so that's the blue and purple signal here you can see that 01:05:27.900 --> 01:05:32.380 we have expression in the liver and a bit in the spleen it's more it's more in the liver than the 01:05:32.380 --> 01:05:36.380 spleen just because the liver is larger so here's the quantified signal where we're looking at 01:05:36.380 --> 01:05:41.580 luminescence expression which is a model protein that we were trying to create so here we have 01:05:41.580 --> 01:05:46.700 mostly liver and a little bit in the spleen and then if we change our helper lipid to DOPS 01:05:46.700 --> 01:05:50.860 which is a negatively charged lipid you can see now that we have improved delivery in the spleen 01:05:50.860 --> 01:05:55.580 some shifts there whereas if we include a positively charged helper lipid now we have a 01:05:55.580 --> 01:06:00.380 significant shift shifts to the lung tissue so this is just evidence that the charge of these 01:06:00.460 --> 01:06:05.420 materials can be quite influential and this is just one of the the molecules out of four types of 01:06:05.420 --> 01:06:10.780 lipids that are present in these particles so we wanted to know if we could use a similar approach 01:06:10.780 --> 01:06:15.740 in our delivery to the pancreas so we're going to manipulate two parameters to try to get good 01:06:15.740 --> 01:06:20.460 delivery to the pancreas one is that chemistry so specifically the helper lipid and its charge 01:06:20.460 --> 01:06:24.620 and the other is the route of administration so you can imagine that depending on where you are 01:06:24.620 --> 01:06:28.860 injecting materials into the body you're going to go different places so we are going to swap out 01:06:28.940 --> 01:06:34.540 some of the typical IV injections for interperitoneal injections which is into the cavity that holds 01:06:34.540 --> 01:06:39.660 the liver and the pancreas and some other organs so for all of our experiments here that i'm going 01:06:39.660 --> 01:06:45.500 to show you today we're looking at the expression so interperitoneal would be like to lift the skin 01:06:45.500 --> 01:06:53.660 of your of your gut area and then poke through the skin but not poke into an organ and then squirt 01:06:53.740 --> 01:06:59.580 it in there it's like the space if you were to cut open the skin of your belly and open it up to 01:06:59.580 --> 01:07:05.420 see your intestines and then squirt it under there that that's the interperitoneal if you do that on 01:07:05.420 --> 01:07:12.620 a mouse or or a dog he would just pick up the skin of the belly and then put it into the triangle of 01:07:12.620 --> 01:07:18.860 skin that you have there and then you'd be in the space and you'd inject it in of a model protein 01:07:18.860 --> 01:07:23.900 so firefly vociferase and we're able to visualize protein expression if we have good delivery so 01:07:23.900 --> 01:07:29.260 our mRNA encodes firefly vociferase we'll put that into our particles we will deliver them into 01:07:29.260 --> 01:07:34.300 mice and then a few hours later we'll be able to take out their organs and to image them for 01:07:34.300 --> 01:07:39.660 luminescence so they take them out a few hours later you don't know where it goes after that you 01:07:39.660 --> 01:07:44.300 don't know if where it was is where it's going to stay you don't know anything that's the whole 01:07:44.380 --> 01:07:51.020 point of all of these experiments with mice is that there's necessarily an end point you're 01:07:51.020 --> 01:07:57.580 not going to cover all time lengths you're not going to cover all and then you're going to draw big 01:07:57.580 --> 01:08:06.540 conclusions and so imagine the extraordinary hubris that's going on here okay we already know 01:08:06.540 --> 01:08:11.180 they're safe because we tested them on three billion people so if they don't go everywhere we want 01:08:11.260 --> 01:08:14.940 them to all the time it doesn't matter because we know they're safe 01:08:18.460 --> 01:08:22.780 expressing proteins in the wrong place and the body doesn't cause any harm at all because we 01:08:22.780 --> 01:08:27.980 know they're safe from the vaccines don't you see otherwise we would know from the five billion 01:08:27.980 --> 01:08:37.180 shots that we rolled out don't you see we can target the liver because that's where they all go when 01:08:37.660 --> 01:08:45.260 you put them in as Mark said it's like a smart ball that when no matter which way you throw it 01:08:45.260 --> 01:08:52.220 it goes right back to the ground no matter what you inject in your body and into your blood it 01:08:52.220 --> 01:09:00.940 goes to your liver wow that's pretty brilliant you are so smart expression so here what i'm showing 01:09:00.940 --> 01:09:05.420 you first is if we take some of our favorite particles and we deliver them IV and this is 01:09:05.420 --> 01:09:09.260 with a neutral helper lipid and you can see as i showed you before that most of our expression 01:09:09.260 --> 01:09:14.860 is in the liver what my postdoc found when she quantified this was a very small amount of 01:09:14.860 --> 01:09:19.900 expression in the pancreas so note this is a exponential scale here so 10 to the 7th versus 01:09:19.900 --> 01:09:24.140 you know a couple of orders of magnitude more in the liver but she noticed there was a little 01:09:24.140 --> 01:09:28.060 something and she wondered if she could turn that little something into something more substantial 01:09:28.060 --> 01:09:31.660 so the first thought was okay let's change our route of administration and let's deliver 01:09:31.660 --> 01:09:36.540 these same particles intreparitinial injection and you can see here that we do have some shifts 01:09:36.540 --> 01:09:40.780 away from the liver and into the pancreas and if we quantify this here you can see the pancreas 01:09:40.780 --> 01:09:45.500 expression has jumped up in order of magnitude and we do see some reductions in the amount of liver 01:09:46.140 --> 01:09:52.060 i say give this woman a NIH grant i say payer for five more years right i mean that's 01:09:52.060 --> 01:09:58.140 hugely significant holy p-values Batman expression that we have and the overall efficacy is a bit 01:09:58.140 --> 01:10:01.900 lower this total bar is a bit lower but if our goal is to deliver to the pancreas that's not a 01:10:01.900 --> 01:10:07.340 problem so then she wondered about the charge of that helper lipid so here again are her data with 01:10:07.340 --> 01:10:12.700 the overall net neutral helper lipid which when she delivers it IP we have quite a bit in the 01:10:12.700 --> 01:10:18.060 pancreas then if she swaps this out for a negatively charged helper lipid we don't really see significant 01:10:18.060 --> 01:10:21.660 changes there there's certainly not more in the pancreas if anything there's a little less and 01:10:21.660 --> 01:10:26.300 then if she uses a positive helper lipid instead you can see something interesting happens so we 01:10:26.300 --> 01:10:29.980 we have the same amount of expression in the pancreas but what we've done is is to almost 01:10:30.700 --> 01:10:34.380 you know not completely but we've significantly reduced the amount of expression in the liver 01:10:34.380 --> 01:10:39.260 so where the hell does it go does she think it disappears it's degraded 01:10:40.300 --> 01:10:45.340 or is it in the endothelial cells or is it in the ovaries or is it in the brain 01:10:46.780 --> 01:10:52.300 there's only four organs listed here there's only four places they're looking 01:10:56.940 --> 01:11:03.180 and so this is really nice because ultimately if we want to do things in the pancreas you usually 01:11:03.180 --> 01:11:06.620 don't also want to manipulate protein expression in the liver so it's nice if we can do something 01:11:06.620 --> 01:11:12.380 more targeted so just to summarize what we've been able to do with these two different manipulations 01:11:12.380 --> 01:11:17.260 if we have IV injection of our neutral helper lipid we have less than 1% of signal in the pancreas 01:11:17.260 --> 01:11:22.300 if we change that to IP administration now we have 15% in the pancreas and if we then swap that 01:11:22.300 --> 01:11:25.900 neutral helper lipid out for something that's positively charged now we flip the ratio of 01:11:25.900 --> 01:11:31.660 pancreas to liver and we have about 60% and the rest we don't know what happens to who cares 01:11:31.660 --> 01:11:37.340 because you know we gave five billion shots to people and they're all fine so obviously it can't 01:11:37.340 --> 01:11:47.260 hurt holy balls i can't believe it so our next question was where in the pancreas was this 01:11:47.260 --> 01:11:52.300 expression occurring and what my postdoc found was that interestingly it's occurring in the 01:11:52.300 --> 01:11:57.020 eyelet cells so the eyelet cells are not a whole lot of the total tissue i forgot what it is but 01:11:57.020 --> 01:12:01.900 it's less than 10% of total pancreas tissue are these eyelets which contain both alpha and beta 01:12:01.900 --> 01:12:08.540 cells and so what she's trying to transfect the pancreas and it has two basic cell types and 01:12:08.540 --> 01:12:19.100 she's not even sure what the ratio is like i'm not an expert in the pancreas i'm not trying to 01:12:19.100 --> 01:12:25.340 transfect i'm not i'm not asking for grant money for the pancreas but if you don't know that ratio 01:12:25.340 --> 01:12:29.180 off the top of your head and you are supervising a postdoc doing a 01:12:33.660 --> 01:12:38.060 showing you here is some staining of luciferase expression so these are our control mice 01:12:38.700 --> 01:12:42.380 which have not received lipid nanoparticles and you can see that they don't have any brown 01:12:42.380 --> 01:12:46.540 signal which would correspond to luciferase expression and now this is the treated sample 01:12:46.620 --> 01:12:51.420 and you can see that the eyelets are expressing a lot of this luciferase there's also some 01:12:51.420 --> 01:12:57.500 outside here in the asinar tissue as well and if we quantify how important these beta cells are 01:12:57.500 --> 01:13:01.980 for protein expression we see that they are predominantly responsible for the protein 01:13:01.980 --> 01:13:06.460 expression so here if we look at wild type mice we have a certain amount of protein expression 01:13:06.460 --> 01:13:11.020 around 10 to the eighth and then if you treat these mice with stz which is a molecule that 01:13:11.020 --> 01:13:15.820 will deplete beta cells in the body you can see that we have about an order of magnitude reduction 01:13:15.820 --> 01:13:19.740 in the signal that we have in the animal suggesting that most of the protein expression 01:13:19.740 --> 01:13:26.460 is occurring in these beta cells so how on earth is this happening how are we transmitting these 01:13:26.460 --> 01:13:30.540 eyelets it's it would be very strange if the particles were penetrating the outer capsule 01:13:30.540 --> 01:13:34.620 of the pancreas my collaborator who's a pancreatic surgeon says you know that's quite unlikely 01:13:34.620 --> 01:13:40.220 so we wanted to understand more about how this was happening and what my postdoc did first is 01:13:40.220 --> 01:13:45.020 she looked at the bio distribution of these particles in the peritoneal cavity so she used 01:13:45.500 --> 01:13:50.860 fluorescently labeled mRNA so it was labeled with a red dye and she isolated different cell types 01:13:50.860 --> 01:13:55.980 so B cells T cells and macrophages and she looked at where these particles were accumulating and 01:13:55.980 --> 01:14:00.700 what she found was that they did not enter T cells but they were entering B cells and macrophages 01:14:00.700 --> 01:14:03.980 so this is not complete delivery we're not looking at protein expression here we're looking at 01:14:03.980 --> 01:14:09.260 which cells took up these particles so since both B cells and macrophages took up the particles 01:14:09.260 --> 01:14:13.900 she then asked if either one of these were involved in the process so here is an efficacy experiment 01:14:13.900 --> 01:14:18.940 in which she has wild-type mice and she compared the expression of luciferase here in our wild-type 01:14:18.940 --> 01:14:24.220 mice to mice in which those B cells have been depleted and what we see is no difference in 01:14:24.220 --> 01:14:28.460 expression so this suggests that the B cells are taking up the particles but they're not producing 01:14:28.460 --> 01:14:34.940 protein however we see something do you understand what she's testing here she she's testing whether 01:14:34.940 --> 01:14:45.260 the B cells the take-up protein are responsible for the signal that she saw on the spleen the liver 01:14:45.260 --> 01:14:52.300 or the lung or the pancreas and so she's depleting lymphocytes 01:14:54.700 --> 01:14:57.740 to show that there's no change in fluorescence 01:14:57.900 --> 01:15:09.260 to demonstrate that the fluorescence that she sees in her target organs is fluorescence that's 01:15:09.260 --> 01:15:15.580 not caused by these immune cells but it's caused by a transfection of those cells in those tissues 01:15:17.740 --> 01:15:23.740 now 01:15:24.140 --> 01:15:28.940 I just don't know what to say about this 01:15:30.860 --> 01:15:35.420 I don't know what it means I don't know what she's showing I don't know why she would call this 01:15:35.420 --> 01:15:40.860 an experiment that would answer that question but that's what she said that's what she explained 01:15:46.620 --> 01:15:50.780 and she's saying that if you deplete the macrophages that you get decreased 01:15:51.020 --> 01:15:57.260 efficacy of transfection decreased 01:16:01.260 --> 01:16:03.900 in different with the macrophages in the peritoneal cavity 01:16:03.900 --> 01:16:08.780 when we use a molecule that depletes these macrophages we see about an order of magnitude 01:16:08.780 --> 01:16:12.620 reduction in the expression that we're getting suggesting that macrophages are very important 01:16:12.620 --> 01:16:19.580 for this delivery process and as we dug a little bit deeper we wondered how macrophages were involved 01:16:19.660 --> 01:16:24.140 so there is some literature showing the process of or the concept of horizontal gene transfer 01:16:24.140 --> 01:16:27.260 by which cells in our body the macrophages in particular 01:16:27.260 --> 01:16:35.020 holy shit holy shit do you hear this what she does is she transfects the macrophages 01:16:35.020 --> 01:16:39.740 and the macrophages produce exosomes and she's calling them extracellular vesicles 01:16:39.740 --> 01:16:44.140 they are exosomes filled with that mRNA that they just got squirted with 01:16:44.860 --> 01:16:55.580 and those exosomes are then causing the transfection are you hearing this this is not facilitation 01:17:03.580 --> 01:17:07.340 I'm really I'm really I'm wow I don't even know what to say 01:17:07.340 --> 01:17:11.740 the type of communication to other cells that are nearby and we wondered if these extracellular 01:17:11.820 --> 01:17:16.220 vesicles might have some kind of role in this process so we did a rather complex experiment 01:17:16.220 --> 01:17:19.500 I'm going to try to walk you through it so again we have particles that are expressing 01:17:19.500 --> 01:17:24.300 our luciferase we're containing our luciferase mRNA we're going to inject them 01:17:24.300 --> 01:17:29.980 interparitoneally into mouse number one and then after a little bit of time goes past we're going 01:17:29.980 --> 01:17:34.620 to do a peritoneal wash so that's where you introduce some saline into the interparitoneal cavity 01:17:34.620 --> 01:17:38.460 and then you pull it out and it's going to contain the immune cells that are in that IP space 01:17:39.180 --> 01:17:43.980 so we'll take that wash and we'll put them into a cell culture dish and then we are going to 01:17:43.980 --> 01:17:48.620 allow these cells to produce extracellular vesicles which will bud off of them into the media 01:17:48.620 --> 01:17:54.460 and then we will collect them okay so now we have these EVs from the immune cells that are in mouse 01:17:54.460 --> 01:17:59.660 number one why are they calling them EVs when they're exosomes they're extracellular vesicles 01:17:59.660 --> 01:18:06.460 they are exosomes number one that have been treated with lmp's we take these EVs and we inject those 01:18:06.540 --> 01:18:10.780 interparitoneally into mouse number two so this mouse has not seen any lipid nanoparticles 01:18:10.780 --> 01:18:15.340 it's just seen these extracellular vesicles and then we do imaging to look at this luciferase 01:18:15.340 --> 01:18:20.220 expression and the results we found are quite interesting so we see that these EV treated mice 01:18:20.220 --> 01:18:25.020 so those are mouse or mice number two they do not have statistically significant differences 01:18:25.020 --> 01:18:29.660 in delivery compared to our lipid nanoparticle treated mice although the averages here are a bit 01:18:29.660 --> 01:18:35.100 we might have you know the averages about two times that of these EV treated mice so it's really 01:18:35.100 --> 01:18:39.020 very interesting that these EVs seem to be responsible for at least part of the delivery 01:18:39.020 --> 01:18:43.500 that we're seeing to the pancreas and as next steps we're going to try to figure out how exactly 01:18:43.500 --> 01:18:48.700 that's happening and why so finally I'll just share a little bit of safety data I always like to say 01:18:48.700 --> 01:18:53.580 when it comes to looking at immune responses and the toxicity there are lots of different ways 01:18:53.580 --> 01:18:57.980 that toxicity can happen and we've looked at only a subset of those measures and so more testing 01:18:57.980 --> 01:19:03.340 as well in this case we've looked at after our injections of these particles we look at some 01:19:03.340 --> 01:19:07.740 innate cytokines that can be expressed and we don't want to see significant differences there 01:19:07.740 --> 01:19:12.460 for the most part and certainly not elevation of these inflammatory cytokines then we also 01:19:12.460 --> 01:19:17.020 looked at antibody expression for IgG and IgM and we don't see any significance there 01:19:17.020 --> 01:19:24.700 after injection either what IgM and IgG antibodies would you be looking for what epitopes did you 01:19:24.700 --> 01:19:32.940 look for just serum IgG changes or what do you expect to see a serum IgG change 01:19:32.940 --> 01:19:38.620 that's a specific or was this specific for an epitope that's weird what a weird statement 01:19:38.620 --> 01:19:45.260 what a weird wow we also did some histology of all of these pertinent organs and compared to 01:19:45.260 --> 01:19:50.220 I mean then what does it mean like I don't understand does it activate the immune system then or not 01:19:50.220 --> 01:19:57.740 because how I guess it doesn't it only does it when they want it to they're so good at this 01:19:57.740 --> 01:20:03.580 wow it's impressive we also did some histology of all of these pertinent organs and compared to 01:20:03.580 --> 01:20:08.940 our PBS controls we don't see significant differences in the tissue or infiltration of 01:20:08.940 --> 01:20:13.500 immune cells so encouraging for now but as I said there's always more work so they were able to do 01:20:13.500 --> 01:20:21.020 histological inspection of the pancreas liver spleen heart kidney and lungs for immune 01:20:21.660 --> 01:20:28.060 infiltration and let's just think about this a second let's just think where are we here I'm 01:20:28.060 --> 01:20:39.420 going to look at the time the time is 33 19 okay so let's look let's just play shall we play a little 01:20:39.500 --> 01:20:47.340 bit of play where is that diagram where is that diagram well back here farther I guess 01:20:48.540 --> 01:20:55.900 where's the diagram there was a diagram where she told us the pro this the the the 01:20:57.180 --> 01:21:00.940 where she told us the actual experiment and how long was it going to take right here okay 01:21:02.220 --> 01:21:03.020 let's go back 01:21:03.020 --> 01:21:13.660 I want to go back because I want to hear how long they left the mice before they went looking for 01:21:13.660 --> 01:21:25.580 problems come on you demon don't mess with me demon come on later we'll be able to take out 01:21:25.580 --> 01:21:30.700 their organs and to image them for mice codes firefly with sipperase we'll put that into our 01:21:30.700 --> 01:21:35.980 particles we will deliver them into mice and then a few hours later we'll be able to take out 01:21:35.980 --> 01:21:42.860 their organs and to image them for luminescence expression so here what I'm showing you first a 01:21:42.860 --> 01:21:52.140 few hours later a few hours later that can't be when they did it for this one at 33 no way no way 01:21:52.460 --> 01:21:59.100 no way that can't be but you know that is it's the same 01:22:02.140 --> 01:22:03.420 they waited like an hour 01:22:04.860 --> 01:22:10.220 it's EV treated mice so it's really very interesting that these EVs seem to be responsible for at least 01:22:10.220 --> 01:22:14.220 part of the delivery that we're seeing to the pancreas and as next steps we're going to try 01:22:14.220 --> 01:22:19.420 to figure out how exactly that's happening and why so finally I'll just share a little bit of safety 01:22:19.420 --> 01:22:23.980 data I always like to say when it comes to looking at immune responses and and then of course we 01:22:23.980 --> 01:22:29.420 reviewed the safety data toxicity can happen and then of course we reviewed the safety data 01:22:29.420 --> 01:22:34.380 more testing is always needed in this case we've looked at after our injections of these particles 01:22:34.380 --> 01:22:39.100 we look at some innate cytokines that can be expressed and we don't want to see significant 01:22:39.100 --> 01:22:43.900 differences there for the most part and certainly not elevation of these inflammatory cytokines 01:22:43.900 --> 01:22:48.540 then we also looked at antibody expression for IgG and IgM and we don't see any significance 01:22:48.540 --> 01:22:53.660 there after injection either we also did some histology of all of these pertinent organs 01:22:53.660 --> 01:22:58.380 boy I'd like to know how long this was before they did it see significant differences in 01:22:58.380 --> 01:23:04.060 the tissue or infiltration of immune cells so after how many days there's always more work to be done 01:23:05.020 --> 01:23:09.500 so in summary what I've shown you today is that we can take our favorite ionizable lipid 01:23:09.500 --> 01:23:14.780 and we can formulate particles in which we tune both the helper lipid charge and the route of 01:23:14.780 --> 01:23:21.020 administration to deliver RNA to the pancreas and it seems that the eyelets are where most 01:23:21.020 --> 01:23:26.700 but it was mostly the the adjustment of where you injected it it had very little to do with 01:23:26.700 --> 01:23:31.820 what you did to the lipid did you notice that when you injected into the blood it didn't really 01:23:31.820 --> 01:23:35.580 change very much of where it went it was when you injected it into peritonealia and 01:23:35.900 --> 01:23:44.780 this is they didn't show it they didn't it's like this is so typical for an 01:23:44.780 --> 01:23:51.660 academy vision to say that these results are something because my p-values say it's something 01:23:54.300 --> 01:24:00.060 it's just unbelievable protein expression is happening and then we see that peritoneal 01:24:00.060 --> 01:24:04.300 macrophages are doing something very interesting to help in this process perhaps by engaging in 01:24:04.300 --> 01:24:10.540 this horizontal gene transfer mediated perhaps by engaging in horizontal gene transfer but the 01:24:10.540 --> 01:24:17.420 only thing they have as evidence that macrophages do something are this one figure 01:24:21.980 --> 01:24:29.900 this figure is not proof the difference between these six animals and these six 01:24:29.900 --> 01:24:36.220 animals right here is the proof that macrophages do something like horizontal gene transfer 01:24:38.140 --> 01:24:43.980 you understand that right this whole story with cartoons about macrophages 01:24:45.420 --> 01:24:46.300 at the end here 01:24:46.700 --> 01:24:54.140 the do something this cartoon hello it's me what what a horse no 01:24:58.860 --> 01:25:06.540 it's just unbelievable how how far this has gone how unbelievable it's it's it's totally harmless 01:25:08.700 --> 01:25:10.460 if you're doing experiments about 01:25:10.700 --> 01:25:17.340 soil microbes and just trying to figure out how that whole ecosystem works and you're never going 01:25:17.340 --> 01:25:22.780 to do any you know genetic manipulation and you're just measuring stuff and whatever that's great 01:25:23.980 --> 01:25:29.100 or if you work somewhere on some remote island on some interesting tuber 01:25:31.660 --> 01:25:36.940 but if you're making medical therapeutics if you're making biologics if you're making 01:25:36.940 --> 01:25:41.820 things that you intend to save the world with you better be freaking more complicated and more 01:25:42.460 --> 01:25:44.460 more humble in your biology than this 01:25:47.580 --> 01:25:52.540 you better have more command over the details than this you better get the details better than this 01:25:55.340 --> 01:26:03.180 this is terrible this is somebody with a very long narrow sliver of expertise 01:26:03.180 --> 01:26:11.900 a very long sliver of expertise that extends right to the edge of understanding of how 01:26:11.900 --> 01:26:20.300 lipid nanoparticles can be targeted to specific tissues but not nearly broad enough to understand 01:26:20.300 --> 01:26:29.020 how dumb a question it is if she had this kind of an understanding of biology she might understand 01:26:29.020 --> 01:26:34.060 how kind of ridiculous it is what she's trying to do if she had this broad of an understanding of 01:26:34.060 --> 01:26:43.820 biology she would not even be in this field a guy like me would have never found himself 01:26:43.820 --> 01:26:47.260 in this lab because this lab is even worse than 01:26:49.500 --> 01:26:57.500 wow someday i'm going to do like a a week on patch clamp physiology so that we can talk about 01:26:57.980 --> 01:27:02.140 what we can and can't do with it and more importantly we can talk about what we did with 01:27:02.700 --> 01:27:10.060 the mice before we did patch clamp experiments and how the in vivo manipulations of the behavior 01:27:10.060 --> 01:27:18.780 of the animal were read out in the the micro circuit i think i heard one member of the clown 01:27:18.780 --> 01:27:25.420 show talking the other day about how i might not know anything about anything because i've only 01:27:25.500 --> 01:27:33.660 ever worked in vitro and under a microscope a good majority of my work is under a microscope 01:27:33.660 --> 01:27:38.860 a good majority of my work in pitzburg was in a condor a confocal microscope a good majority of 01:27:38.860 --> 01:27:45.420 my work in trolenheim was and in the Netherlands was with live animals that were manipulated before 01:27:45.420 --> 01:27:52.780 the recordings were made either molecularly or behaviorally or both 01:27:52.940 --> 01:27:59.820 so i've got plenty of in vitro and in vivo experience and more importantly i've got that 01:27:59.820 --> 01:28:09.740 nice combination but you know it's these kind of limp-risted attacks that are happening all the 01:28:09.740 --> 01:28:17.020 time now and we're just not going to look backwards we're not going to look backwards in the cave 01:28:17.100 --> 01:28:20.380 we're not going to listen to the people saying hey wait for us 01:28:23.500 --> 01:28:32.140 we're out our flashlights are out pointed out we are climbing out we are actually probably 01:28:32.140 --> 01:28:39.500 outside of the hole helping other people out we're not climbing back in the cave 01:28:39.580 --> 01:28:48.220 let's see if there's any more for her let's see any more for her perhaps by engaging in this 01:28:48.220 --> 01:28:53.980 horizontal gene transfer mediated by extracellular vesicles so with that i'd like to thank all of 01:28:53.980 --> 01:28:57.740 the members of my group and particularly the people who were the most hands-on with this work 01:28:57.740 --> 01:29:02.380 so sam lakresti was the postdoc who looked at some of our initial charged helper-lifted work 01:29:02.380 --> 01:29:06.060 in the spleen and lungs and then the bulk of the work i showed you today was performed by 01:29:06.060 --> 01:29:11.420 jillian melamed who so this is jillian and this is sam and then our collaborator george 01:29:11.420 --> 01:29:14.940 get us who's at the university of pitzburg he's been a huge help he's a pancreatic surgeon and was 01:29:14.940 --> 01:29:19.820 able to assist us with some of our experiments um so uh then i'll just mention that i had the 01:29:19.820 --> 01:29:24.700 privilege of giving a ted talk a couple of years ago so if any of you want to see a lay version of 01:29:24.700 --> 01:29:29.100 some of what i've described here you can find that they're called fatballs for the purposes of 01:29:29.100 --> 01:29:35.420 uh psychom to lay audiences and then oh my gosh for the purposes of science communication they call 01:29:35.420 --> 01:29:43.020 them fatballs we almost should really look that that thing up right i i almost have to look at that 01:29:43.020 --> 01:29:51.580 that talk up um because you know it's going to be even worse than this um anyway uh who's the funders 01:29:52.300 --> 01:29:58.220 oh DARPA that's nice oh that's sweet isn't that nice then i age in DARPA good good good i mean 01:29:58.300 --> 01:30:06.700 what else you know what else what else could it be what else could it be it could only be that 01:30:09.260 --> 01:30:14.140 ladies and gentlemen sometime in 2019 they started telling us a story about a lab leak 01:30:14.140 --> 01:30:19.500 bat cave zoanosis maybe it came from a bat cave maybe it come from a laboratory maybe it came from 01:30:19.500 --> 01:30:24.140 the stitching done in the laboratory maybe they took it to the market after they sold animals 01:30:24.140 --> 01:30:30.220 there it doesn't matter this multi-colored rainbow of RNA covered the world the last three 01:30:30.220 --> 01:30:35.740 years and there is an illusion of consensus about this that the EUA and lockdowns were a bad idea 01:30:36.460 --> 01:30:41.340 but millions of people were killed by this novel virus and millions more were saved from it 01:30:41.900 --> 01:30:46.220 we should focus on regulating gain of function viruses because that's almost certainly where this 01:30:46.220 --> 01:30:55.020 came from and this faith in the fidelity of PCR and in the the lack of relevance of the 01:30:55.020 --> 01:31:03.260 protocols this is what we need to dismiss because the faith is a lie it was a conflated background 01:31:03.260 --> 01:31:14.460 signal or conflated background signals as my uh my my the clown car behind me said you know it's 01:31:14.460 --> 01:31:21.900 all clones now it wasn't all clones a year ago but it's all clones now the only real debate is 01:31:21.900 --> 01:31:29.420 whether clones spread or not which of course we've known that that's then the debate all along 01:31:29.420 --> 01:31:33.180 but these children are just catching up ladies and gentlemen so we're not going to look back 01:31:33.180 --> 01:31:35.980 we're not going to worry about it we're going to keep our arms straight we're going to keep 01:31:35.980 --> 01:31:41.500 our flashlights pointed forward and we are going to keep helping people find the way out 01:31:44.540 --> 01:31:48.460 because we are at a time point where our kids could end up enslaved 01:31:50.060 --> 01:31:55.820 and the way out is not to bug out the way out is not to get a cabin in the woods and don't look 01:31:55.900 --> 01:32:03.900 back or sign up for for Elon Musk satellite internet why would we do that 01:32:06.940 --> 01:32:11.420 I mean I almost feel like I have to start putting my money where my mouth is why would I do that when 01:32:11.420 --> 01:32:19.500 I could just take over Pittsburgh when everybody in Pittsburgh knows this immunology why would I 01:32:19.580 --> 01:32:26.620 have to leave when everybody at your church knows this immunology why would we need to leave 01:32:28.860 --> 01:32:33.580 when everybody knows this biology why would we need to leave 01:32:38.140 --> 01:32:43.580 making an underground network of bad of good guys is not going to solve the problem 01:32:43.580 --> 01:32:51.340 burning down the whole system is not going to solve the problem it's not fine if this whole 01:32:51.340 --> 01:32:58.780 thing falls apart because our kids were taught bad morals it's time for the adults to put their 01:32:58.780 --> 01:33:05.420 pants on and do the work that they should have been doing for the last 20 years it's time for 01:33:05.420 --> 01:33:12.620 people like you and me in our 50s to start doing the things that we should have been doing that our 01:33:12.620 --> 01:33:16.380 parents should have been doing but didn't do because we left it to Mitch McConnell 01:33:17.660 --> 01:33:21.580 and we left it to Nancy Pelosi and we left it to Joe Biden 01:33:25.820 --> 01:33:32.380 my dad could be president right now if he wanted to for the last 40 years but he certainly could have 01:33:32.380 --> 01:33:38.940 been a senator he certainly could have been a representative and he certainly would have had 01:33:38.940 --> 01:33:41.260 a hell of a lot better than some of the people that we've had 01:33:43.660 --> 01:33:47.900 it's a little weird talking from that way from Wisconsin because we did have Russ Feingold when 01:33:47.900 --> 01:33:52.220 I was a kid and when my dad would have been a senator and so that would have been a ridiculous 01:33:52.220 --> 01:33:56.620 person to run against because Russ Feingold was the last good guy we've had in Wisconsin 01:33:58.540 --> 01:34:03.580 I don't know about the the the young guy though he's pretty good Gallagher seems like a good guy 01:34:03.580 --> 01:34:07.500 stop all transfections and healthy humans please ladies and gentlemen because they are trying to 01:34:07.500 --> 01:34:12.460 eliminate the control group by any means necessary I don't know if this was a very good stream or 01:34:12.460 --> 01:34:19.180 not but I thought it was really important to show you how how far the assumptions have gone this is 01:34:19.180 --> 01:34:27.420 totally gone from niche to mainstream and that niche to mainstream is a lie because intramuscular 01:34:27.420 --> 01:34:31.820 injection of any combination of substances with the intent of augmenting the immune system is dumb 01:34:32.380 --> 01:34:38.940 and specifically transfection in healthy humans is criminally negligent viruses are not pattern 01:34:38.940 --> 01:34:46.220 integrity neither are exosomes neither are extracellular vesicles neither are lipid nanoparticles 01:34:46.220 --> 01:34:52.780 filled with mRNA none of them are patterned integrity and they all basically behave roughly the same 01:34:54.380 --> 01:34:59.260 let's just start to get this once you start to get this we're gonna get them we're gonna get them 01:34:59.260 --> 01:35:03.500 we're gonna win I assure you the book's not even out yet 01:35:06.220 --> 01:35:09.340 I know I sound like a broken record but man oh man 01:35:11.100 --> 01:35:12.620 holy p-values Batman 01:35:13.260 --> 01:35:15.260 hmm 01:35:18.700 --> 01:35:21.020 thanks guys I'll see you again tomorrow 01:35:21.020 --> 01:35:24.460 it's friday gotta keep working 01:35:42.620 --> 01:35:44.620 you 01:36:04.300 --> 01:36:09.820 are you from Wisconsin I'm from cadat up by Chippewa Falls good to see you thanks for joining 01:36:09.820 --> 01:36:12.460 see you tomorrow