testing one two I said 10 22 10 29 I don't think that worked out quite how I
wanted to but we're gonna manage to make it work you can find me at giggle
and biological.com you can also go to the soapbox link that you can find there
which is also at giggle ohm.bio happy thanks giving everybody happy thanks
giving Pamela Watcherby tense oral strain there's a click there that you can do
to watch the show you can also download the stream deck starting next week we're
gonna have the stream deck up I hope every time I do a stream at least every
week stream and that means that you can start subscribing again if you'd like
to get something like an email and a PDF of the show beforehand it might only
be for Fridays or Wednesdays or whatever day we're gonna make it but
someday at some point we're gonna start that PDF stuff again because these
slides have started to become good enough that I think they're shareable
and useful for people to start to really push these ideas out they haven't been
there in a long time and so I think it's really nice for this to happen
it is a little lonely in this crazy brainwashed world I'll tell you what it's
very lonely my wife feels very lonely
it's yeah it's tough thanks for coming tonight guys I didn't tweet it I didn't
share it I didn't do anything I didn't have time it's Thanksgiving I just wanted
to get a stream out there and keep the streak alive and keep the keep the
sea warm keep the rig warm
I yelled at the basketball team the other night my throat is still a little sore
you schedule for 60 minutes next is going on French British Italian Japanese
television people everywhere are starting to listen to him it's embarrassing yeah
I'm gonna have to start with some more I'm gonna have to start with some more
inspirational some more inspirational stuff in the beginning again I used to
put a lot of work a lot more work into that beginning section of video so I
just didn't think anybody was watching them but that one was a really good one
keep in your arms straight
so you know the drill you can look me up on PubMed and the name of the channel
is derived from the resistance of the recordings that I used to make giga home
resistance high-resistance low-noise information brief let's get this thing
started
I feel like my computer slowed down there for a minute hopefully you guys
didn't lose anything yes happy Thanksgiving everybody good to see
everyone here it's like we lost a few viewers see I told you I told you something
poopy's going on they don't like it our information and our message is
getting too sharp they don't like it at all
yes yes truth bombs at dinner that's an understatement I bet at some of these
places that we've been today thank you very much for joining me this is
giga home biological a high-resistance low-noise information brief brought to
you by a biologist if there's anything wrong with the timing or anything like
that give me a shout out we are still here fighting against the same sort of
nonsense you know the illusion of consensus the Scooby-Doo created by these
people on social media I'm starting to really see what happened I know that
there was this TV stuff and this stuff in Congress and whatever but I'm starting
to understand how these people are all linked and it has a lot to do with these
new social media and new communication platforms that came online during the
pandemic and there's more than one there's actually about five or six and we can
start to look back at when that was all happening when we were all scrambling
during this during this censorship time in 2020 oh my goodness I can see it
also clearly now I'm really excited as we break through some of this stuff on the
18th and 19th of November in Bucharest Romania there was a COVID conference and
some of our friends were at this COVID conference I'm gonna go over here
pull myself down here and see if we can use my mouse to he has so just the
prominent ones for now I'll pop my head down for a second Robert Malone was
there Nick Hudson was there Brett Weinstein was there you can see this
arrow right yeah and Denny Rancor was there among others which is kind of cool
because as you know there is this there is this you know consensus that we're
trying to battle against something and all of us are trying to work together
towards some amorphous goal like health freedom or something like that and there
is this faith in a novel virus this faith that millions of people were
killed by it this faith that millions more were saved from it this faith that
gain a function is real and possibly the source of it and therefore a virus will
come again and this story is basically not been questioned by most of the
people that we have been trying to look to for leadership in this in this fight
to return to you know pre-2019 Western civilization which clearly we're never
going to do the build back better the new normal this was all for real to make
sure that we never thought we could have that back again and so in some ways it
would be it should be somewhat curious if we can't find any leader on this
playing board that doesn't want to say just simply well why don't we just go
back to the way we were doing it about four years ago and start there and then
we can repeat we can we can fix that but first of all we got to go back to that
and everybody that through that out needs to be held to account
everybody that just went against all those rules whence against all those
norms went against all those expectations those people need to be held to
account but that doesn't seem there doesn't seem to be anyone who's
interested in that particular path and in fact yesterday I wanted to point that
out and so that was one of the reasons why we went through that that video from
Stockholm earlier this year so some more people were there that that definitely
as far as I know aren't questioning the narrative that I really like Byron
Bridal a lot and he's a great guy but he is not questioning there being a
novel virus that spread around that's spreading still Jessica Rose hasn't
questioned that even though she has done a selfie with Denny Rancor she has an
acknowledge that that one of the main findings of Denny's data is that there's
no evidence of spread of a novel deadly pathogen Harvey Reich is also another
guy who I've talked to before and not on my show live but on a stream with with
oh I'm not gonna remember her name and shoot that's really annoying Kathy
something I believe I apologize for that if you're watching but it's a one of
these private zoom calls so Harvey Reich is another guy who's not questioning
the narrative there's Ryan Cole I'm not sure how much he's questioning the
spread of a novel virus but I don't think that he's really there yet he's
certainly not there yet with regard to promoting Denny Rancor's data and what
Denny Rancor's data tends to you know it seems to show here's Jill Glasspool
Malone and Meryl Nast you know that those two are also probably not pushing
that narrative and then again what Brett Weinstein Nick and Robert and Denny
Rancor so even if Denny Rancor is there and he presents to all these people and
Robert Malone tweets it out he tweets out that 17 million people were killed
by the vaccine and that he's never seen such shocking accounting of the 17
million people around the world estimated to be killed by the vaccine but he's
not at all shocked by the conclusion that there's no evidence of spread that
the spread respected even borders of counties in the United States it has
correlated much much better to to poverty and mental illness and income than it
is to to like proximity of the previous cases or proximity of of of large
incidences or events of all cause mortality I'm getting really good at
not saying um when I'm doing these things but it's sometimes I have to pause
for a second there um anyway and then I said okay this is all because these
people have participated in this in this illusion of consensus and I don't know
to what extent they were aware of this illusion before the start of the
pandemic but most of them have to be aware of the illusion now because most of
them have heard my my discussion of it our discussion of it over these
this past year or so and most of them know what our contention is and they
don't have a real meaningful argument because the only people that have
tried to meaningfully argue with me are now blocking me on Twitter and no
longer discussing it all and that's because again it really feels very very
positive that that these these people are all involved in
making sure that the worst case scenario of a pandemic caused by a gain of
function virus is never out of your possibility space even if you don't
believe that's what happened this time they need you to believe that that's
what gonna happen next time or could happen next time or or eventually will
happen they need you to believe that maybe in the
same way that they needed us when we were children to really believe that
the Russians had nine thousand warheads and we had six thousand warheads
and they were on a on a hair trigger well two hair keys
so i i don't you know i i have a hard time understanding
anymore what history really is given the fact of all of things that i've
learned over the last three years about how willing and able
this apparatus is to govern us with lies in mythology
and so i think that this illusion of consensus really needs to be inspected
for this mythology and i suspect that we're going to find that it mostly is
and that's why i think you need to think of this as a card game
a card game with a rig deck a card game with a rig deck that a dealer is trying
to steal your money so when you get the cards in order for you to have any
meaningful chance of gambling with success
you have to play knowing that the dealer is trying to get you
to make a mistake so what would that mistake be
how would they how would they communicate to those
inside the game versus those outside the game
what kinds of clues would there be that there's a game being played
i think one of the clues that that Matt Crawford
of rounding the earth pointed out about two years ago was this
possibility that this covid is actually ovid
ovid being this famous greek set of stories
about various metamorphosis a lot of them are kind of like punishments or
or or gods being vindictive but the point
only being uh in this thing that this is a time of
transformation and some of the the themes
of ovid's metamorphosis in relation to the the fall of rome
and the theme of let's say order being imposed on a world
founded on chaos or based from or or growing from chaos
an order is imposed on that and so in this story
in this idea of covid it could be that in order to transform us into a new
global government in order to transform us into a new global
governance structure based on on digital currencies and digital
IDs and no more national borders that mean anything
they need to have a chaos they need to start from a chaos they can't
have all of this order and then rearrange it
or hope to usefully disassemble it at least that's what they think
that's what they they think that's how how maybe that is how people have been
governed through chaos and then imposed order
and if that's the case then it does seem as though the people that are
governing the globe that want to shift us to this new paradigm
understand it as a necessity for us to go through some kind of chaos
in order for this societal metamorphosis to take place
and so i'm starting to make this argument that there's a fine line between an
announcement and a warning and i want you to start to
think about what people say on our side about where we're going and how we
avoid going there and whether or not it's an
announcement or a warning whether it's an announcement or a
suggestion whether it's an alternative or
an inevitability because i think it's very important it doesn't only have to be
a guy like carol adebach the health guy from germany
or jorston talking about these kinds of things and then for us to know that
oh this is not really a warning or a complaint this is an announcement
when when jorston says that we really need to crack down on misinformation
and the press needs to do their job we understand that fully
that they mean to crack down on misinformation they want us to beg for
that but then when we hear robert melone say that he's been
harassed on on the internet and it's not fun and you wouldn't like it and we've
got to stop using violent speech or they're going to use it against us
is that an warning or an announcement if somebody says we need to build new
underground systems but doesn't ever advocate for us to take back the system
that we have and and remodel it retake it back and use it
the way it's supposed to be restore our republic no he says we need to build new
underground systems it's not very different than what
happened when we got censored back in 2020 and everybody ran to gab
and to telegram
and everybody went on signal and had all these secret groups where they chatted
about i don't know
well everything and everybody went to discord
and how hard was it for these non-encrypted apps to
actually be like honey traps for us in a way all the people that
wanted to march around and be efficient you know or not efficient but uh
evasive of the of the system they went away
they went on gab they went on telegram i'm on signal now
curiously who led us to gab who led us to telegram who led us to signal
who led us to discord who led us to sub-stack
if you hear somebody up on stage say we have a lousy society our children have
been taught all the wrong values it's going to be okay that it all comes
apart is that really a announcement
a warning
is that inevitability
is that somebody trying to help us or is that somebody just trying to keep us
walking down or riding numbly down this neon hallway
covered in lies is that somebody who's pretending to be a part of the
group of mimes stopping this drain it's a really important analogy
try to imagine fooling a young group a group of young kids
and try to see that this guy all by himself with his little chalk dish
he can't fool a group of kids but if you got a bunch of crossfit athletes
and then you fitted them with a bunch of uh
machines that made a lot of noise with wheels and sparks
i guarantee you you could fool a bunch of kids you might even be able to fool
a bunch of teenagers and the more people that you lined up on the train with more
elaborate props and a more elaborate coordinated effort
the more people you could fool the more inspection
they would pass the more the more scrutiny they could withstand
in their act
if you made it really complicated
if you made it with smoke and heat so that people couldn't get very close to the
train as these people stopped it
if you made it really loud so that people were afraid to get near it
the point that i'm trying to make is is that during the pandemic that's what
all of these stories did to us
they made it seem like there was nothing to guess or or wonder about
these people were doing the best that they could to stop a national security
priority a national security threat of never-before-seen proportions
and potential the potential to kill a billion people and we don't have any
treatment we don't know what will work
the best we can do is hope that these people can put out enough pcr's and
lateral flow tests so that we can at least keep track of when we're sick asymptomatically
and so many people on social media seem to agree about this
so many people on social media are arguing whether or not hydroxychloroquine or
ivermectin are crazy or not
and ivermectin and hydroxychloroquine did not save anyone from a ventilator did
not save anyone from rendezivir did not save
anyone from an opioid death
financial incentives to declare covid didn't save anyone from the covid
protocols it actually put more people on them
sending people back with to elderly care homes with pneumonia
and viral pneumonia without antibiotics didn't save anyone
the dismissal of immunology 101 in october of 2020
by a whole list of leaders including wollinski
as in we don't know if our immunity will be good enough it could be like aids
where we build antibodies and that's actually a sign of disease not a sign of
immunity did you know that do you remember that
giant contradiction thanks for reminding me of that pamela or
pointing it out even the value of nutrition in
america was dismissed a place where almost every grown
adult has been malnourished for at least a decade
and many children grow up malnourished over
carbon carbohydrate over sugared over medicated over immunized
they have been doing this train pulling and pushing act for three years
and public health the public health apparatus would like to govern the
world with this act
public health apparatus needed to convert this pattern
into something that they could declare was a pandemic of a novel virus
and in order to convert this very distinct
and reliable pattern this very distinct reliable pattern
in pneumonia deaths into this very indistinct
and novel pattern of pneumonia deaths it required protocols not a novel
virus
it required novel protocols like do not resuscitate people if they're having a
heart attack at home or a stroke let them die
don't bring them back to the hospital pronounce them dead
if you do bring them into the hospital make sure you ventilate in my extra
high pressure to make sure that the virus doesn't come out
antibiotics don't work on viral pneumonia dumbass
and steroids don't either
remdesivir works though it's pretty good we should try it on everybody
and opioid deaths well those people are just covid deaths
we don't even need to talk about them i mean who who even cares about those
people isn't it a waste to even use Narcan on them like
they're just i wonder what people in san francisco would say
death certificate fraud how far should this list go how much longer how much
many more things can we think about they need to go on there
the reason why i want this to happen is because i need you to see
because i need you to see what because i need you to see
what that was an echo there because i had that open
yikes that was scary i need you to see that these people
have really fooled us now this video is a video from one month ago
by a professor
of carny g melon and today i'll be talking mostly
let me get my head out of the way for a second so i can rewind this
and make sure it's all set up correctly
1.5 so this is a video of
a professor from carny g melon university so that's that's in pittsburgh
and she gave this talk one month ago as part of a
honoring peter colas series of lectures so it's about our ln
lnp for r and a delivery which i thought already got the nobel prize
which is usually when something has had you know like a decade of impact
and so she's going to give a lecture about this but i want you to listen
very carefully to the assumptions that she makes
and the assumptions are are monumental
she's going to say what we said at giga ohm biological they were going to do
we said that in 2021 when everybody was all hot and bothered about the
spiky spike being toxic
we said it that they were going to blame the vaccine
and it not working right because they chose the wrong protein or they're
going to make some other excuse and otherwise
otherwise we just proved that r and a and
transfection in general works great in humans
now this is a woman who just gave a talk a month ago and basically
she's going to lead off with that in an extraordinarily aggressive way
r and a's here to stay
so today i wanted to speak with you about some of the work that we've been
doing developing lipid nanoparticles for targets outside of the liver
and i specifically wanted to focus on some of our data regarding delivery to
the pancreas i'm not really sure i just want to
talk to Moderna for a second modified r and a is not biological messenger r and a
i'm not really sure why you're making that point because
we understand that that coat we we recognize that codon optimization
has certain implications and effects we've recognized that the chemical
alterations have certain effects and we're calling it r and a
are calling it transfections so you keep saying that but
i've we've all acknowledged that this is modified r and a
if we call it m r and a or r and a it doesn't really matter it's synthetic
r and a so i mean you're you're making a semantic argument that doesn't
it doesn't really matter if you listen to her talk and you assume that this is
all synthetics then then it's really about what they
can and can't do with them that is the point
they can't control where they go and when they say that they inject you in the
muscle to augment your immune system it's just
absolutely absurd and that's that's what she's going to talk about here with
cartoons so just a little bit about my lab in
general and dr khalis had mentioned this in the introduction
we work in a couple of different areas of drug delivery
so we focus on oral delivery of protein medication so drugs like insulin that
currently have to be injected and trying to find ways of delivering them
in more patient-friendly forms so that might involve oral delivery for
proteins the problem with proteins is that they're
digested by our gastrointestinal tracts and so we're very interested in
studying the transport of these large molecules across the
gastrointestinal tract to facilitate these more patient-friendly means of
delivering proteins. More recently my lab has taken interest in
women's and infant health. I had my first pregnancy at this point about seven
years ago and then another one shortly before the
pandemic happened and there's nothing like firsthand
experience to realize that the state of maternal medicine is rather poor
and there are a lot of things that we can do to try to improve our
understanding of transport processes during pregnancy
to try to create medications that are safe that do not cross into the infant.
And then finally the subject of today's lecture is in this area of RNA delivery
where I've been working now on lipid nanoparticles for over 15 years
and it's just been wonderful to see how far this field has come since I first
started. My lab is especially interested in how manipulating the chemistry
of these particles which are quite complex chemically.
How those manipulations can affect their functionality in vivo
both in terms of bio distribution where they're working well immunogenicity
and toxicity. So I'll speak a bit more about that today.
So as pretty much all of you probably know at this point these RNA therapies
have gone from niche to mainstream so the first FDA approval of a lipid
nanoparticle formulation and the first FDA approval of an RNA drug
was back in 2018 by Al nylon and they developed this medication for a rare
type of liver disease and this was wonderful and it brought to
fruition decades of research on liposomal and lipid nanoparticle type
materials but even then we still had a very small
population of people who were benefiting from this type of technology
and people were quite cautious about you know using these types of
medications for larger patient populations. So then we had this awful
occasion to need to put these materials to the test
and I think all of us in the delivery field were absolutely delighted to see
how well these materials and how well RNA therapies can work
in you know huge. Was there ever a
humanity or is this a stupid picture like what did they ever have this
actually available or it was always the it was always Pfizer right
it never came out with that I think this is BS right here
anyway this is the statement that I'm trying to make
this is you guys can all see it for what it is this is absolutely absurd
the idea is very clear this was always the plan
there was always going to be the plan it was always going to be
throw the adenavirus under the bus so that we can point to the adenavirus
as well obviously we took it off the market because
it was causing clots and the mRNA doesn't cause clots.
I mean duh the mRNA causes myocarditis but that goes away
myocarditis is fine it only kills 50% of the people that
they get it. It's it's really sad it's really sad
because we said this we said it was going to happen two years ago we said
they were going to do this and not one single person
in that narrative control panel ever even bothered to think out loud that
thought never mind actually speak the words or write a
sub stack about it nobody and for two or three years
almost three years really now that where we are
it's almost three years oh my gosh it's going to be 2024
we have been saying that they are going to blame
some mistake in the methodology some mistake in the choice of protein some
epitope they should have taken out
and then they moved on to impurities then they moved on to
process one versus process two and double stranded DNA and this sv40
enhancer and promoter it's always been the same story
these things work great but there were some problems with the rollout yeah sure
but it was a rush job when we make these things like
fine brewed beer this stuff is amaze balls
that's the story that's the sales pitch that's the tv narrative that's what
skilled tv watchers believe right now that's where we are at
Thanksgiving 2023 just where i said we would be
i'm not thankful for that
which numbers of people and so now with this evidence and hundreds of millions of
people at this technology works well this is really going to open the floodgates
to all sorts of new therapies not just in vaccines but in protein replacement
gene editing and otherwise so think about how naive that is think about
how naive that is that she thinks that those two are equivalent because we
used it for vaccination successfully even if even if
even if
replacing proteins hasn't worked
curing cancer rarely works with mRNA
putting these things in certain places doesn't work
when you change their chemistry and they go places it's a fraction of it right
these people would be very excited if they changed the lipid
the lipid nanoparticle chemical composition and you went from 50 percent of it in the
liver and 50 percent of it in the ovaries to 80 percent of it in the liver and 30 percent
in the ovaries that would be a phd project that would be a very significant result
that would allow them to stand in front of years of lecture halls
saying that these results suggest that in a few years we can have really precisely targeting
lipid nanoparticles based on the chemical alterations of and the properties that
that emerge from those alterations blah blah blah blah blah blah
and so let's listen to this lady explain what she plans to do and explain the
sort of hand waving that she does look at this terrible cartoon already
a protein is just a ball that comes from a comb
I mean so for any trainees in the audience who are a little bit less familiar with this
I wanted to just go through some of the basic molecular biology that
underpins all this RNA work that we're doing so this is the central dogma of molecular biology
that tells us that our permanent blueprint the DNA inside of ourselves this is transcribed
into a temporary copy called messenger RNA which is then translated in the cell cytoplasm
into proteins and so those arrows don't mean anything understanding the DNA polymerase
doesn't mean anything like how it's copied understanding the RNA polymerase
don't worry about that that's just an arrow understanding ribosomes
mRNA protein that's just an arrow who cares about those things
and now she's going to say that proteins are everything proteins are the motor as well
ribosomes are RNA ribosomes are made of RNA and proteins in combination so it's a little
hand wavy to say that proteins do everything when the most important thing the most important
nanomachine that we almost know nothing about is the ribosome
but that's okay you know just just keep us asking the wrong questions keep us focused
on the wrong things instead of the arrows we'll focus on those funny shapes in blue
genes are the doers in our body and because they're the molecules responsible for all function
many diseases are caused by misregulated proteins so either the protein is non-functional
either we have too much of a particular protein or too little and so to treat so a protein is
non-functional we have too much we have too little it has nothing to do with the tertiary
structure of the protein or the tertiary chemical alterations of it that's extraordinary and again
you could say that this is being imprecise but she's talking to a really educated audience you
would think that you'd want to hit this pretty pretty on the money you think you'd want to be
with be pretty sharp with your biology but we're not we're not sharp right now this is one month
ago ladies and gentlemen please we really need to manipulate protein expression such that it goes
back to what it's supposed to be so that first FDA approved drug it contained short interfering
RNA and it's short because it's about 21 base pairs in length as opposed to RNA mRNA which can
be thousands long and it interferes with protein production so you can design this so it would
be specific for a target gene of interest and then you can have reductions in that protein expression
now i'm a little gonna object to the way that this is drawn because it looks like to me
her si RNA is drawn as double-stranded but actually i think correct me if i'm wrong smart people in
the audience but i think RNA is single-stranded and when you put small interfering RNAs in there
they bind to the endogenous RNA and make a double-stranded RNA and that's why they get degraded
because double-stranded RNA ain't allowed in our cells
you see again it's just kind of imprecision you know like
it's just kind of imprecise and it's annoying because these people get paid a crap ton of tax payer
money to teach children to apprentice young biologists into this
into this endeavor
if we want to upregulate protein expression instead of downregulate that's where messenger RNA comes
in and so we can design mRNA's that can for example give us more of a particular gene of or protein
of interest and in the case of the vaccines it's just super cool we can ask our bodies to
generate proteins that our bodies would never normally make so in this case it's generating
the spike protein which is not native to mammalian cells so very powerful technology
i wanted to speak very briefly on our news this week that the noble prize was given for the mRNA
biology that underpinned the covid-19 vaccines so RNA therapeutics really require two parts
so one is functional mRNA that once it goes inside the cell it can produce the protein of interest
effectively and then the other is the delivery system which i think hasn't gotten quite as
much press as the RNA itself but is equally important so today i'll be talking about the delivery
system and the noble prize had to do with this mRNA biology so here are the two people who have
just been awarded so koriko and weissmann who originally did the work at the university of
pennsylvania in philadelphia so they um i like to just tell people they're both really terrific
people i think they could be on a sitcom together or something the two videos that we watched of them
i mean they were lovable people i mean definitely but in a you're not a nobel prize-winning kind
of lovable way i think i think we don't always have um you know sometimes famous scientists aren't
the best models of of collaboration it's been my privilege to collaborate with drew over the years
when i was getting started in the mRNA field it was back in 2017 and RNA is quite expensive
if you purchase it and i was giving a seminar at pen and drew was a stranger to me and we had a
meeting and he offered to make me whatever mRNA i wanted and send it to me and people didn't believe
too much in RNA therapies at that time there was still a lot of hesitation so i was taken aback
and confused why a stranger would offer to help me in this way given the expense so it took me a
couple of years to actually approach him and then when i did he was true to his work and he's provided
me with with RNA since that time and has otherwise been a great collaborator i've also met
katie koriko briefly and she's given me some nice advice so just such a pleasure to see good
people um reward it for for their work so um in terms of what they did they discovered that
base modifications of RNA are needed for protein translation and mammals so here is the molecule
uridine which is one of those canonical nucleic acids that are present in RNA and the problem
when we make RNA typically with these molecules such as uridine is that our cells recognize them
as foreign and that's because our cells make RNA to incorporate some base modifications so if it
sees RNA without base modifications it mounts a bit of an immune response that shuts down protein
production so that's part of why nobody thought RNA therapies would become a reality because
when we made RNA in lab and introduced it it just would not make protein so what they found was that
they could make modifications to these different nucleobases and so here's um one version of this
this is um n1 methyl pseudo uridine the ones that are used in the vaccines are just called
pseudo uridine so i don't think this is i'm sorry i had to i had to put something in my ear and
clean it from my i'm wearing my headphones again tonight i don't think this is right and i hope i
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
nonsense i don't think that base modifications are necessary for protein translation in mammals i
think they're necessary for sustained protein translation in mammals for them to get a signal
that they can point to and say look it works but if you put non uh methyl pseudo uridine
RNA in your lipid nanoparticle you'll get very transient expression
very transient expression absolutely but that that's not quite right what they what this is is
what this is is somebody who is i think over their skis saying what they've been told is right
they probably practiced this talk and somebody said it's fine but essentially she's not saying
this correctly i i really don't think so i know that we have base alterations in our RNA
and they are used presumably for RNA regulation but for purposes of a
transfection tool that's true for purposes of how RNA functions endogenously in your cells
that's true to an extent because actually again the transient nature of RNA being degraded quickly
in your cells is how protein expression is regulated at least as far as we guess
and that's why small interfering RNAs that are complementary to your own
mRNAs and circulation are an interesting way to interfere with protein expression
somebody put in the chat that they're double stranded RNAs i'm going to look it up i'm not sure
i think the mechanism is how i've described it though um
um and so i think this is a bit of a hand-waving exercise to say that this is how it works but
she's specifically talking about transfection not about about translation of proteins and
endogenous proteins so we have these modifications and the cells are more willing to recognize these
externally introduced RNAs as self and then they are willing to produce that protein so all the
work that i'm showing you today is made with RNA that's been base modified so that it works well
so that it works well see so so that it works well in its transfection excuse me so to go back to
the delivery vehicle part of the equation RNA is large it's on the order of a hundred thousand
grams per mole it's negatively charged and so it doesn't readily cross negatively charged cell
membranes and if you were to inject it into the bloodstream it would be both degraded and
negatively charged on the order of a hundred thousand delivery vehicle part of the equation
RNA is large it's on the order of a hundred thousand grams per mole it's negatively charged
and so it doesn't readily cross negatively i get to go back a little bit further and
previous of delivery barrier it's been base modified so that it works well
so in terms of delivery barriers excuse me so to go back to the delivery vehicle part of the
equation RNA is large it's on the order of a hundred thousand grams per mole it's negatively
charged and so it doesn't readily cross negatively charged cell membranes and if you were to
inject it into the bloodstream it would be both degraded and quickly cleared from our system so
we need a vehicle that's going to package it up and protect it and take it to the cells of interest
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
what she says because that assumption is wrong listen carefully you inject it into the bloodstream
it would be both degraded and quickly cleared from our system so if you injected it into the
bloodstream it would be degraded and very quickly cleared did she mean that without lip
and nanoparticle i think that must be what she means right because
wow that that must be just naked RNA but we need a vehicle that's going to package it
yeah okay and take it to the cells of interest so this diagram depicts a couple of barriers that
it needs to overcome if we were to do IV injection so once it's injected into the bloodstream it
needs to avoid clearance by macrophages once it exits the bloodstream it needs to
it needs to avoid clearance by macrophages it also needs to avoid
transfecting endothelial cells and avoid transfecting blood cells and as it goes through
capillaries that will be more and more difficult because the then that's where this is all going
to occur right that's where this size scale comes into play
it's not going to happen in the big vessels it's going to happen in the capillaries and capillaries
that this this size scale is wrong this cartoon is wrong and avoiding the transfection of endothelial
cells is much harder than one might think diffuse through the tissue of the organ that you want to
target it then needs to be up taken into the cell of interest and this happens through a
process called endocytosis where the cell membrane reaches up and around that particle
and it brings it inside and so at this point the nanoparticle is in this Waldorf compartment
and the cell keeps it there because it's not sure what it is and it looks and says this is
something I want to see that the the whole anytime a biologist anthropomorphizes the
operations of a cell and I don't even know I don't think you can ever do that and if you do
it you need to be very very careful in this case it's ridiculous it's held in an endosome until
it does the cell decides what to do with it because it's not sure what it is
are you kidding this is a chemical covered in other chemicals
the hell is she talking about
and typically a lipid nanoparticle is not on its wish list so the endosome begins to acidify
and would otherwise degrade these lipid nanoparticles so part of the design of the chemistry behind
these particles is that they need to mediate this process of endosomal escape and that's one of
the most difficult parts of this process so once the RNA is into the cell cytoplasm that's when
it can interface with the protein translational machinery and make our protein of interest
so years ago we asked if we could develop a potent and degradable RNA delivery system
potent for obvious reasons and degradable because many of these therapeutics will need to be
repeat dose I mean seriously jittery and maker jason all of these arrows are pretty magical
all of these arrows are pretty magical this arrows very magical this arrow what is this
like is this an an electron going down from two different energy levels what the hell is this
I mean did they take and are they trying to convert a photosynthesis diagram into a
I mean even this is silly this part you know like okay now we have RNA I guess but it's
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
I don't know how to overcome this I mean could I get her to come to
giga ohm biological and let me give her an hour lecture like this but then say completely
the opposite what would she do it's shocking isn't it it's just shocking this B arrow this
C arrow what is that the part of the design of the chemistry behind these particles is that they
need to mediate this process of endosomal escape and that's one of the most difficult
parts of this process so once the RNA is into the cell cytoplasm that's when it can interface
with the protein translational machinery and make our protein of interest see if you can say all
those words together that's when it can interface with the protein the cells protein machinery or
whatever I mean that that doesn't make you a commander of this knowledge it doesn't make you
understanding the way that this complex system manifests it's just a joke
she was anthropomorphizing the cell as holding the liposome in an endosome because it didn't
know what it was and it wasn't sure what to do with it that was her explanation for these other PhDs
so years ago we asked if we could develop a potent and degradable RNA delivery system
potent for obvious reasons and degradable because many of these therapeutics will need to be
repeat dosed we will not necessarily create a cure but a treatment and we don't want this material
building up over time so the materials that we work with in my lab they're lipid-like materials
we call them lipidoids and they're a variation of the cationic lipids that have been used for
decades at this point for different types of nucleic acid delivery first a lot of the work
was done in the DNA delivery field so here's one of these cationic lipids if you were to put it
into aqueous medium you would find that they would assemble into these liposomal structures
where we have the hydrophilic head group on the outside of the aqueous media and then we would
have these hydrophobic tails on the inside and you can load then hydrophilic drugs into the interior
so we wanted to make something like this to hold on to our RNA and we also wanted to
generate materials where we could look at a lot of different chemistries and try to understand
I want you to see how how extraordinary it is that she talks as though we wanted to do we wanted to
do we wanted to do like it's something you know it's almost like saying you know I wanted to put
together a bunch of pages and I'm gonna call it a book and I really wanted to make a book so it was
my idea to bunch a bunch of pages together and then bind them at the back
she's talking about a a technology that she started her talk by saying that it had gone from
niche to mainstream so what does she need to explain it for what's so special about hers
and why does she need to explain it on this level she's giving a talk with Peter Coles in the
background it's extraordinary I find this extraordinary it is the lack of
appropriate audience I mean it's it's just oh it drives me crazy this is what used to drive me
crazy about about neuroscience you could have a room full of people talking about supposedly
about the highest level of discussion about our understanding of how micro circuits in the brain
work and you could have somebody in the back room raise their hand and say I don't I don't
could you briefly explain what a G protein coupled receptor is
and you might have been talking about a type of receptor that is a G protein coupled receptor
and you may have even given an introduction that was reasonable about G protein coupled
receptors and this person would still have the audacity to raise their hand and ask that question
only in biology
you
couldn't go into a physics lecture and say hey can you briefly explain to me what you mean by that
I'm not sure I get it you couldn't go into an organic chemistry lab or lecture and say you know
can you briefly explain to me what that category of molecule is I'm not really familiar with that
but for some reason in biology everybody any any poser that wants to can go and join a lab
and do a PhD project and there you go
you did some programming as a PhD student and now you're up now you're going to do a postdoc in biology
and since you did some T cell stuff I guess you're an immunologist now
oh you put some silt bubbles and some RNA together and you followed the recipe
and then you followed another recipe successfully and after following six recipes successfully that
you basically adapted from other people's recipes you're a biologist now
and the depth of your understanding of the concepts that you purport to wield is terrifying
the chemistry of these lipidoids would affect their functionality and try to derive structure
functional relationships from that so we use a high throughput type of chemistry called Michael
addition in which we take an alkalamine so we need some combination and say primary means a
secondary means and we would react them with alkal acrylate tails here it's an easy reaction
you put the two together in a scintillation vial there's no solvent you can mix them for a
couple of days at high temperature and out the other side comes something that's not too dissimilar
from this cationic lipid up here so we can vary the chemistry of both this amine and the tail
to generate large numbers of materials the degradability comes in from the use of these
acrylate materials with which result in an ester in the final form of these lipids and those esters
degrade under physiological conditions so these lipids are ionizable meaning that under reduced
pH conditions as we form these particles they are going to have this nice electrostatic interaction
with the negatively charged RNA which will help to catalyze the formation of these particles
so the particles are made through a nanoprecipitation process in which we need rapid mixing
so we have a rapid mixing of two streams to form our nanoparticles one of which is going to be
RNA that's in a saline solution and then i do have to say that microfluidics is really cool
they can do a lot of really cool things with microfluidic devices we have a variety of lipids
that are dissolved in ethanol and they are rapidly mixed you can use microfluidic devices
t-mixers and you can also use pipettes which are relatively straightforward
pipettes are not appropriate for scale up or anything like that but they work well in the
lab when you don't have a lot of money for microfluidic systems so you mix these two streams together
and you get materials that you know you have you have lipids both on the exterior of the
particle like we had with liposomes but you also have lipids throughout the material
so in terms of the ingredients in these particles they include the ionizable lipid or lipidoid that
i just told you about this tends to be where a lot of the IP is in the lipid nanoparticle space
many labs or companies attempt to create their own chemistry and intellectual property with
these synthetic materials they also contain helper lipids so these are often phospholipids that
naturally occur in our cell membrane they can help with stability of the particles and also
their ability to help with endosomal escape we have a lot of cholesterol in these particles
which adds stability and prevents the particles from falling apart very similar to the way we
have a lot of cholesterol in our cell membranes we would all be piles of boo if it were not for
our cholesterol in our particles would be too then we have our polyethylene glycol lipid so the lipid
anchor here will insert on the inside of the particle and then we have polyethylene glycol
which is a hydrophilic polymer which decorates the outside of the particle it can quench that
nanoparticle formation process so help us obtain particles of a nice size it can also improve
stability and decrease the amount of unwanted immune cell uptake that we get
unwanted immune cell uptake i thought you wanted immune cell uptake
or maybe not for their maybe they're maybe they're not trying to do vaccines maybe they're trying
to do something else so over the years we've made and screened thousands of these ionizable lipidoids
for RNA delivery and we found a number that we really like and we've used them for numerous
applications so here's one of our favorite lipidoids that we use in my lab
it's a four-tailed lipidoid it has a couple of these nitrogen groups which help it
protonate in the endosome and endosome doing endosomal escape we have our esters here for
degradability and then these materials are interesting because they have these branch
tails and the branch tails seem to change the structure of these lipids so that they can
interface with endosomal membranes differently and aid that fusion and escape process so this
is a great lipid and so the work i'm going to show you today all of it involves this ionizable lipid
so standard nanoparticles where the state of the field is at this point is that we can achieve
delivery to a couple of locations so the liver has been the easiest location i don't want to say
it's easy but it's been the easiest because when you inject particles the liver is the organ that
will naturally clear them from the bloodstream and so if it doesn't clear them from the bloodstream
then those particles go elsewhere they go to any place they want to go they go to endothelial
cells they go to the ovary they go to the testes they go to your brain they can go anywhere they
want to if if you have perforated endothelial barriers so this concept that it can be
it can be remain in the muscle and look how they label it immune cells and muscle now i'm sorry i'm
not trying to be a creep here but the immune system isn't really organized to protect the
interior of your deltoid there may be some resident immune cells there there may be a lymph
vessel that runs through there the immune system is present but the immune system is
oriented around barriers there is no question about it and barriers with the outside world
there is a barrier with the outside world that runs through your body there's a barrier with
the outside world that is in your lungs and there's a barrier in the outside world on the
outside of your body and all three of these barriers are manned are our outward facing
epithelial barriers epithelial cells are necessarily dividing barriers
and the immune system is organized around them and so this assumption that she makes that we
can inject it in in a muscle and it stays there and it's expresses protein and the immune system
comes and it's not sure what to do but eventually it decides that what what the intention of this
protein is is to make me make immune to a memory to it or something like this whenever they would
talk through it and that first FDA approved product was to treat a liver condition and it's a to treat
a liver condition because that's where the lipid nanoparticles go necessarily because the liver
cleans your blood not because they're chemically altered to go to the liver but because that's
where the majority of them go all of them don't go there
the ones that don't transfect something else first get pulled out in the liver
much of the transfection occurs at the liver
and in fact it's very likely that when you get injected with an mRNA in your muscle and it leaks
out the transfection occurs in the liver the transfection occurs in the capillaries of the heart
the capillaries of the lungs maybe the capillaries of your skin
I think we've seen it many many times where it's the capillaries of the skin where we get a massive
transfection and therefore a massive inflammation a massive neutrophil attack on the endothelial cells
we've seen this all around the world we've seen what happens we know this is what's going on
there's no other biological alternative and these people know even as she disingenuously describes
the nature of targeting the liver they called them liver targeting lipid nanoparticles because
the liver pulls them out and the majority of them showed up there
I mean I just hope you can see how disingenuous that is
I just hope you can see it
oops
we also are capable of vaccination so delivery to the immune cells and the muscle cells within
the muscle tissue to mount the immune response that we need but the question really is is we're
thinking about the future of RNA therapies is how we will deliver these materials beyond the liver
so as you can imagine most of the diseases in the body are outside of the liver and there are
many different places we'd like to go so my lab works on delivering these materials to a couple
of these particular organs and today I'll be talking mostly about our ability to deliver to
the pancreas so for this work we're going to be focusing on swapping out the helper lipids
in these particles and looking at how the influence of helper lipid charge in particular
can direct these particles to different cell types so there have been a couple of people who
have reported the influence of charge on the delivery process so Dan Seagard's lab was one
of the first to show that changing the charge either cationic or negative compared to some of
the neutral helper lipids can shift delivery from the liver to either the spleen or the lungs
and we found something similar in our lab so in this case I'm showing you so DOPE is the standard
phospholipid that we use in my lab it carries a net neutral charge and you can see if we look at
organ distribution of protein expression so that's the blue and purple signal here you can see that
we have expression in the liver and a bit in the spleen it's more it's more in the liver than the
spleen just because the liver is larger so here's the quantified signal where we're looking at
luminescence expression which is a model protein that we were trying to create so here we have
mostly liver and a little bit in the spleen and then if we change our helper lipid to DOPS
which is a negatively charged lipid you can see now that we have improved delivery in the spleen
some shifts there whereas if we include a positively charged helper lipid now we have a
significant shift shifts to the lung tissue so this is just evidence that the charge of these
materials can be quite influential and this is just one of the the molecules out of four types of
lipids that are present in these particles so we wanted to know if we could use a similar approach
in our delivery to the pancreas so we're going to manipulate two parameters to try to get good
delivery to the pancreas one is that chemistry so specifically the helper lipid and its charge
and the other is the route of administration so you can imagine that depending on where you are
injecting materials into the body you're going to go different places so we are going to swap out
some of the typical IV injections for interperitoneal injections which is into the cavity that holds
the liver and the pancreas and some other organs so for all of our experiments here that i'm going
to show you today we're looking at the expression so interperitoneal would be like to lift the skin
of your of your gut area and then poke through the skin but not poke into an organ and then squirt
it in there it's like the space if you were to cut open the skin of your belly and open it up to
see your intestines and then squirt it under there that that's the interperitoneal if you do that on
a mouse or or a dog he would just pick up the skin of the belly and then put it into the triangle of
skin that you have there and then you'd be in the space and you'd inject it in of a model protein
so firefly vociferase and we're able to visualize protein expression if we have good delivery so
our mRNA encodes firefly vociferase we'll put that into our particles we will deliver them into
mice and then a few hours later we'll be able to take out their organs and to image them for
luminescence so they take them out a few hours later you don't know where it goes after that you
don't know if where it was is where it's going to stay you don't know anything that's the whole
point of all of these experiments with mice is that there's necessarily an end point you're
not going to cover all time lengths you're not going to cover all and then you're going to draw big
conclusions and so imagine the extraordinary hubris that's going on here okay we already know
they're safe because we tested them on three billion people so if they don't go everywhere we want
them to all the time it doesn't matter because we know they're safe
expressing proteins in the wrong place and the body doesn't cause any harm at all because we
know they're safe from the vaccines don't you see otherwise we would know from the five billion
shots that we rolled out don't you see we can target the liver because that's where they all go when
you put them in as Mark said it's like a smart ball that when no matter which way you throw it
it goes right back to the ground no matter what you inject in your body and into your blood it
goes to your liver wow that's pretty brilliant you are so smart expression so here what i'm showing
you first is if we take some of our favorite particles and we deliver them IV and this is
with a neutral helper lipid and you can see as i showed you before that most of our expression
is in the liver what my postdoc found when she quantified this was a very small amount of
expression in the pancreas so note this is a exponential scale here so 10 to the 7th versus
you know a couple of orders of magnitude more in the liver but she noticed there was a little
something and she wondered if she could turn that little something into something more substantial
so the first thought was okay let's change our route of administration and let's deliver
these same particles intreparitinial injection and you can see here that we do have some shifts
away from the liver and into the pancreas and if we quantify this here you can see the pancreas
expression has jumped up in order of magnitude and we do see some reductions in the amount of liver
i say give this woman a NIH grant i say payer for five more years right i mean that's
hugely significant holy p-values Batman expression that we have and the overall efficacy is a bit
lower this total bar is a bit lower but if our goal is to deliver to the pancreas that's not a
problem so then she wondered about the charge of that helper lipid so here again are her data with
the overall net neutral helper lipid which when she delivers it IP we have quite a bit in the
pancreas then if she swaps this out for a negatively charged helper lipid we don't really see significant
changes there there's certainly not more in the pancreas if anything there's a little less and
then if she uses a positive helper lipid instead you can see something interesting happens so we
we have the same amount of expression in the pancreas but what we've done is is to almost
you know not completely but we've significantly reduced the amount of expression in the liver
so where the hell does it go does she think it disappears it's degraded
or is it in the endothelial cells or is it in the ovaries or is it in the brain
there's only four organs listed here there's only four places they're looking
and so this is really nice because ultimately if we want to do things in the pancreas you usually
don't also want to manipulate protein expression in the liver so it's nice if we can do something
more targeted so just to summarize what we've been able to do with these two different manipulations
if we have IV injection of our neutral helper lipid we have less than 1% of signal in the pancreas
if we change that to IP administration now we have 15% in the pancreas and if we then swap that
neutral helper lipid out for something that's positively charged now we flip the ratio of
pancreas to liver and we have about 60% and the rest we don't know what happens to who cares
because you know we gave five billion shots to people and they're all fine so obviously it can't
hurt holy balls i can't believe it so our next question was where in the pancreas was this
expression occurring and what my postdoc found was that interestingly it's occurring in the
eyelet cells so the eyelet cells are not a whole lot of the total tissue i forgot what it is but
it's less than 10% of total pancreas tissue are these eyelets which contain both alpha and beta
cells and so what she's trying to transfect the pancreas and it has two basic cell types and
she's not even sure what the ratio is like i'm not an expert in the pancreas i'm not trying to
transfect i'm not i'm not asking for grant money for the pancreas but if you don't know that ratio
off the top of your head and you are supervising a postdoc doing a
showing you here is some staining of luciferase expression so these are our control mice
which have not received lipid nanoparticles and you can see that they don't have any brown
signal which would correspond to luciferase expression and now this is the treated sample
and you can see that the eyelets are expressing a lot of this luciferase there's also some
outside here in the asinar tissue as well and if we quantify how important these beta cells are
for protein expression we see that they are predominantly responsible for the protein
expression so here if we look at wild type mice we have a certain amount of protein expression
around 10 to the eighth and then if you treat these mice with stz which is a molecule that
will deplete beta cells in the body you can see that we have about an order of magnitude reduction
in the signal that we have in the animal suggesting that most of the protein expression
is occurring in these beta cells so how on earth is this happening how are we transmitting these
eyelets it's it would be very strange if the particles were penetrating the outer capsule
of the pancreas my collaborator who's a pancreatic surgeon says you know that's quite unlikely
so we wanted to understand more about how this was happening and what my postdoc did first is
she looked at the bio distribution of these particles in the peritoneal cavity so she used
fluorescently labeled mRNA so it was labeled with a red dye and she isolated different cell types
so B cells T cells and macrophages and she looked at where these particles were accumulating and
what she found was that they did not enter T cells but they were entering B cells and macrophages
so this is not complete delivery we're not looking at protein expression here we're looking at
which cells took up these particles so since both B cells and macrophages took up the particles
she then asked if either one of these were involved in the process so here is an efficacy experiment
in which she has wild-type mice and she compared the expression of luciferase here in our wild-type
mice to mice in which those B cells have been depleted and what we see is no difference in
expression so this suggests that the B cells are taking up the particles but they're not producing
protein however we see something do you understand what she's testing here she she's testing whether
the B cells the take-up protein are responsible for the signal that she saw on the spleen the liver
or the lung or the pancreas and so she's depleting lymphocytes
to show that there's no change in fluorescence
to demonstrate that the fluorescence that she sees in her target organs is fluorescence that's
not caused by these immune cells but it's caused by a transfection of those cells in those tissues
now
I just don't know what to say about this
I don't know what it means I don't know what she's showing I don't know why she would call this
an experiment that would answer that question but that's what she said that's what she explained
and she's saying that if you deplete the macrophages that you get decreased
efficacy of transfection decreased
in different with the macrophages in the peritoneal cavity
when we use a molecule that depletes these macrophages we see about an order of magnitude
reduction in the expression that we're getting suggesting that macrophages are very important
for this delivery process and as we dug a little bit deeper we wondered how macrophages were involved
so there is some literature showing the process of or the concept of horizontal gene transfer
by which cells in our body the macrophages in particular
holy shit holy shit do you hear this what she does is she transfects the macrophages
and the macrophages produce exosomes and she's calling them extracellular vesicles
they are exosomes filled with that mRNA that they just got squirted with
and those exosomes are then causing the transfection are you hearing this this is not facilitation
I'm really I'm really I'm wow I don't even know what to say
the type of communication to other cells that are nearby and we wondered if these extracellular
vesicles might have some kind of role in this process so we did a rather complex experiment
I'm going to try to walk you through it so again we have particles that are expressing
our luciferase we're containing our luciferase mRNA we're going to inject them
interparitoneally into mouse number one and then after a little bit of time goes past we're going
to do a peritoneal wash so that's where you introduce some saline into the interparitoneal cavity
and then you pull it out and it's going to contain the immune cells that are in that IP space
so we'll take that wash and we'll put them into a cell culture dish and then we are going to
allow these cells to produce extracellular vesicles which will bud off of them into the media
and then we will collect them okay so now we have these EVs from the immune cells that are in mouse
number one why are they calling them EVs when they're exosomes they're extracellular vesicles
they are exosomes number one that have been treated with lmp's we take these EVs and we inject those
interparitoneally into mouse number two so this mouse has not seen any lipid nanoparticles
it's just seen these extracellular vesicles and then we do imaging to look at this luciferase
expression and the results we found are quite interesting so we see that these EV treated mice
so those are mouse or mice number two they do not have statistically significant differences
in delivery compared to our lipid nanoparticle treated mice although the averages here are a bit
we might have you know the averages about two times that of these EV treated mice so it's really
very interesting that these EVs seem to be responsible for at least part of the delivery
that we're seeing to the pancreas and as next steps we're going to try to figure out how exactly
that's happening and why so finally I'll just share a little bit of safety data I always like to say
when it comes to looking at immune responses and the toxicity there are lots of different ways
that toxicity can happen and we've looked at only a subset of those measures and so more testing
as well in this case we've looked at after our injections of these particles we look at some
innate cytokines that can be expressed and we don't want to see significant differences there
for the most part and certainly not elevation of these inflammatory cytokines then we also
looked at antibody expression for IgG and IgM and we don't see any significance there
after injection either what IgM and IgG antibodies would you be looking for what epitopes did you
look for just serum IgG changes or what do you expect to see a serum IgG change
that's a specific or was this specific for an epitope that's weird what a weird statement
what a weird wow we also did some histology of all of these pertinent organs and compared to
I mean then what does it mean like I don't understand does it activate the immune system then or not
because how I guess it doesn't it only does it when they want it to they're so good at this
wow it's impressive we also did some histology of all of these pertinent organs and compared to
our PBS controls we don't see significant differences in the tissue or infiltration of
immune cells so encouraging for now but as I said there's always more work so they were able to do
histological inspection of the pancreas liver spleen heart kidney and lungs for immune
infiltration and let's just think about this a second let's just think where are we here I'm
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
bit of play where is that diagram where is that diagram well back here farther I guess
where's the diagram there was a diagram where she told us the pro this the the the
where she told us the actual experiment and how long was it going to take right here okay
let's go back
I want to go back because I want to hear how long they left the mice before they went looking for
problems come on you demon don't mess with me demon come on later we'll be able to take out
their organs and to image them for mice codes firefly with sipperase we'll put that into our
particles we will deliver them into mice and then a few hours later we'll be able to take out
their organs and to image them for luminescence expression so here what I'm showing you first a
few hours later a few hours later that can't be when they did it for this one at 33 no way no way
no way that can't be but you know that is it's the same
they waited like an hour
it's EV treated mice so it's really very interesting that these EVs seem to be responsible for at least
part of the delivery that we're seeing to the pancreas and as next steps we're going to try
to figure out how exactly that's happening and why so finally I'll just share a little bit of safety
data I always like to say when it comes to looking at immune responses and and then of course we
reviewed the safety data toxicity can happen and then of course we reviewed the safety data
more testing is always needed in this case we've looked at after our injections of these particles
we look at some innate cytokines that can be expressed and we don't want to see significant
differences there for the most part and certainly not elevation of these inflammatory cytokines
then we also looked at antibody expression for IgG and IgM and we don't see any significance
there after injection either we also did some histology of all of these pertinent organs
boy I'd like to know how long this was before they did it see significant differences in
the tissue or infiltration of immune cells so after how many days there's always more work to be done
so in summary what I've shown you today is that we can take our favorite ionizable lipid
and we can formulate particles in which we tune both the helper lipid charge and the route of
administration to deliver RNA to the pancreas and it seems that the eyelets are where most
but it was mostly the the adjustment of where you injected it it had very little to do with
what you did to the lipid did you notice that when you injected into the blood it didn't really
change very much of where it went it was when you injected it into peritonealia and
this is they didn't show it they didn't it's like this is so typical for an
academy vision to say that these results are something because my p-values say it's something
it's just unbelievable protein expression is happening and then we see that peritoneal
macrophages are doing something very interesting to help in this process perhaps by engaging in
this horizontal gene transfer mediated perhaps by engaging in horizontal gene transfer but the
only thing they have as evidence that macrophages do something are this one figure
this figure is not proof the difference between these six animals and these six
animals right here is the proof that macrophages do something like horizontal gene transfer
you understand that right this whole story with cartoons about macrophages
at the end here
the do something this cartoon hello it's me what what a horse no
it's just unbelievable how how far this has gone how unbelievable it's it's it's totally harmless
if you're doing experiments about
soil microbes and just trying to figure out how that whole ecosystem works and you're never going
to do any you know genetic manipulation and you're just measuring stuff and whatever that's great
or if you work somewhere on some remote island on some interesting tuber
but if you're making medical therapeutics if you're making biologics if you're making
things that you intend to save the world with you better be freaking more complicated and more
more humble in your biology than this
you better have more command over the details than this you better get the details better than this
this is terrible this is somebody with a very long narrow sliver of expertise
a very long sliver of expertise that extends right to the edge of understanding of how
lipid nanoparticles can be targeted to specific tissues but not nearly broad enough to understand
how dumb a question it is if she had this kind of an understanding of biology she might understand
how kind of ridiculous it is what she's trying to do if she had this broad of an understanding of
biology she would not even be in this field a guy like me would have never found himself
in this lab because this lab is even worse than
wow someday i'm going to do like a a week on patch clamp physiology so that we can talk about
what we can and can't do with it and more importantly we can talk about what we did with
the mice before we did patch clamp experiments and how the in vivo manipulations of the behavior
of the animal were read out in the the micro circuit i think i heard one member of the clown
show talking the other day about how i might not know anything about anything because i've only
ever worked in vitro and under a microscope a good majority of my work is under a microscope
a good majority of my work in pitzburg was in a condor a confocal microscope a good majority of
my work in trolenheim was and in the Netherlands was with live animals that were manipulated before
the recordings were made either molecularly or behaviorally or both
so i've got plenty of in vitro and in vivo experience and more importantly i've got that
nice combination but you know it's these kind of limp-risted attacks that are happening all the
time now and we're just not going to look backwards we're not going to look backwards in the cave
we're not going to listen to the people saying hey wait for us
we're out our flashlights are out pointed out we are climbing out we are actually probably
outside of the hole helping other people out we're not climbing back in the cave
let's see if there's any more for her let's see any more for her perhaps by engaging in this
horizontal gene transfer mediated by extracellular vesicles so with that i'd like to thank all of
the members of my group and particularly the people who were the most hands-on with this work
so sam lakresti was the postdoc who looked at some of our initial charged helper-lifted work
in the spleen and lungs and then the bulk of the work i showed you today was performed by
jillian melamed who so this is jillian and this is sam and then our collaborator george
get us who's at the university of pitzburg he's been a huge help he's a pancreatic surgeon and was
able to assist us with some of our experiments um so uh then i'll just mention that i had the
privilege of giving a ted talk a couple of years ago so if any of you want to see a lay version of
some of what i've described here you can find that they're called fatballs for the purposes of
uh psychom to lay audiences and then oh my gosh for the purposes of science communication they call
them fatballs we almost should really look that that thing up right i i almost have to look at that
that talk up um because you know it's going to be even worse than this um anyway uh who's the funders
oh DARPA that's nice oh that's sweet isn't that nice then i age in DARPA good good good i mean
what else you know what else what else could it be what else could it be it could only be that
ladies and gentlemen sometime in 2019 they started telling us a story about a lab leak
bat cave zoanosis maybe it came from a bat cave maybe it come from a laboratory maybe it came from
the stitching done in the laboratory maybe they took it to the market after they sold animals
there it doesn't matter this multi-colored rainbow of RNA covered the world the last three
years and there is an illusion of consensus about this that the EUA and lockdowns were a bad idea
but millions of people were killed by this novel virus and millions more were saved from it
we should focus on regulating gain of function viruses because that's almost certainly where this
came from and this faith in the fidelity of PCR and in the the lack of relevance of the
protocols this is what we need to dismiss because the faith is a lie it was a conflated background
signal or conflated background signals as my uh my my the clown car behind me said you know it's
all clones now it wasn't all clones a year ago but it's all clones now the only real debate is
whether clones spread or not which of course we've known that that's then the debate all along
but these children are just catching up ladies and gentlemen so we're not going to look back
we're not going to worry about it we're going to keep our arms straight we're going to keep
our flashlights pointed forward and we are going to keep helping people find the way out
because we are at a time point where our kids could end up enslaved
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
back or sign up for for Elon Musk satellite internet why would we do that
I mean I almost feel like I have to start putting my money where my mouth is why would I do that when
I could just take over Pittsburgh when everybody in Pittsburgh knows this immunology why would I
have to leave when everybody at your church knows this immunology why would we need to leave
when everybody knows this biology why would we need to leave
making an underground network of bad of good guys is not going to solve the problem
burning down the whole system is not going to solve the problem it's not fine if this whole
thing falls apart because our kids were taught bad morals it's time for the adults to put their
pants on and do the work that they should have been doing for the last 20 years it's time for
people like you and me in our 50s to start doing the things that we should have been doing that our
parents should have been doing but didn't do because we left it to Mitch McConnell
and we left it to Nancy Pelosi and we left it to Joe Biden
my dad could be president right now if he wanted to for the last 40 years but he certainly could have
been a senator he certainly could have been a representative and he certainly would have had
a hell of a lot better than some of the people that we've had
it's a little weird talking from that way from Wisconsin because we did have Russ Feingold when
I was a kid and when my dad would have been a senator and so that would have been a ridiculous
person to run against because Russ Feingold was the last good guy we've had in Wisconsin
I don't know about the the the young guy though he's pretty good Gallagher seems like a good guy
stop all transfections and healthy humans please ladies and gentlemen because they are trying to
eliminate the control group by any means necessary I don't know if this was a very good stream or
not but I thought it was really important to show you how how far the assumptions have gone this is
totally gone from niche to mainstream and that niche to mainstream is a lie because intramuscular
injection of any combination of substances with the intent of augmenting the immune system is dumb
and specifically transfection in healthy humans is criminally negligent viruses are not pattern
integrity neither are exosomes neither are extracellular vesicles neither are lipid nanoparticles
filled with mRNA none of them are patterned integrity and they all basically behave roughly the same
let's just start to get this once you start to get this we're gonna get them we're gonna get them
we're gonna win I assure you the book's not even out yet
I know I sound like a broken record but man oh man
holy p-values Batman
hmm
thanks guys I'll see you again tomorrow
it's friday gotta keep working
you
are you from Wisconsin I'm from cadat up by Chippewa Falls good to see you thanks for joining
see you tomorrow