I Genetically Engineered *MYSELF* to Fix Lactose Intolerance
Summary
TLDRThis video documents a personal journey to combat lactose intolerance through DIY gene therapy. The creator, once severely affected by lactose, explains the science behind lactose digestion and the enzyme lactase. They detail the process of using an adeno-associated virus (AAV) to deliver a lactase gene to the intestinal tract, successfully restoring their lactose tolerance. The video provides a transparent look into the lab work, the challenges faced, and the successful consumption of dairy products post-treatment, promising a future of enjoying pizza without fear.
Takeaways
- đ· The speaker became lactose intolerant at 15, leading to severe illness from consuming lactose.
- đ Up to 65% of the world's population may lose the ability to digest lactose, causing various symptoms.
- đœïž Lactose is prevalent in many foods, often as a filler or sweetener, due to the dairy industry's by-products.
- đŹ The speaker's personal struggle with lactose intolerance inspired a career in biology, aiming to restore their own lactose tolerance.
- 𧏠Lactose intolerance occurs when the body stops producing lactase, the enzyme that breaks down lactose into glucose and galactose.
- đ A study mentioned used a virus to deliver a working copy of the lactase gene to lactose-intolerant rats, successfully restoring their lactose digestion.
- đ Adeno-associated viruses (AAVs) are preferred for gene therapy due to their safety and reliability.
- đ§Ș The process of creating a virus for gene therapy involves using plasmids and cells in a lab, with careful handling to ensure sterility.
- đ The speaker's DIY gene therapy project aimed to create a treatment in pill form to restore lactose tolerance.
- đ After testing the treatment, the speaker was able to consume dairy products without symptoms, marking a successful initial result.
Q & A
What is the main issue the speaker discusses in the script?
-The speaker discusses their personal struggle with lactose intolerance and their journey to develop a DIY gene therapy to restore their lactose tolerance.
What is the percentage of the human population that may become lactose intolerant at some point?
-Up to 65% of the human population may become lactose intolerant at some point.
What is lactose and why is it problematic for lactose intolerant individuals?
-Lactose is a disaccharide composed of glucose and galactose subunits. It is problematic for lactose intolerant individuals because they lack the lactase enzyme to break it down, leading to gastrointestinal issues when consuming lactose-containing foods.
What is the role of the lactase enzyme in digestion?
-The lactase enzyme, also known as beta-galactosidase, breaks down lactose into its subunits, glucose and galactose, which can then be absorbed by the body for energy.
Why is the adeno-associated virus (AAV) considered the gold standard for gene therapy?
-The AAV is considered the gold standard for gene therapy due to its reliability, long history of safety, and its ability to merge with human DNA in very safe and well-known locations.
What is the purpose of the ITRs in the context of the virus production?
-ITRs, or inverted terminal repeats, are DNA sequences that signal the virus to package the DNA sequence between them into its shell during virus particle formation.
What are plasmids and how are they used in this project?
-Plasmids are small circles of DNA engineered by biologists to contain useful features. In this project, they are used to hold the DNA sequences for the virus shell proteins and the Lac Z gene, which is the target gene for restoring lactose tolerance.
How does the speaker plan to test the effectiveness of the gene therapy?
-The speaker plans to test the effectiveness of the gene therapy by using a color-changing dye called X-Gal, which turns blue in the presence of the lactase enzyme, indicating that the cells are producing the enzyme.
What is the significance of the speaker's successful consumption of pizza after the treatment?
-The successful consumption of pizza, which is high in lactose, without experiencing symptoms of lactose intolerance, signifies that the DIY gene therapy may have successfully restored the speaker's lactose tolerance.
What is the next step for the speaker in terms of this project?
-The next step for the speaker is to continue testing and refining the treatment, monitoring its long-term effects, and potentially sharing updates on the project's progress.
Outlines
đ„¶ Lactose Intolerance Struggles and DIY Gene Therapy
The speaker shares their personal journey with lactose intolerance, which began at the age of 15, causing frequent illness. They explain how lactose, a sugar found in milk products, affects those who cannot produce lactase, the enzyme required to break it down. The lack of lactase results in symptoms like bloating and diarrhea when consuming dairy products. The speaker discusses the prevalence of lactose in various food items due to its use as a filler and sweetener. They also delve into their motivation to study biology to find a solution to their lactose intolerance, aiming to restore their ability to consume dairy without repercussions. The narrative culminates in their pursuit of DIY gene therapy as a means to treat their condition, with a focus on transparency and education about the process.
𧏠Understanding Lactose and Gene Therapy
This section delves into the science behind lactose intolerance and the proposed gene therapy treatment. Lactose is described as a disaccharide composed of glucose and galactose, which is broken down by the lactase enzyme. When lactase is absent, lactose reaches the colon, causing gastrointestinal issues. The speaker outlines a method using a virus to deliver a functional lactase gene to the intestinal tract, as demonstrated in a study on rats. The video aims to demystify DIY gene therapy, emphasizing safety and clarity in the process. It also introduces adeno-associated virus (AAV) as a preferred vector for gene therapy due to its safety and efficiency. The process of creating the virus involves using specific DNA sequences and cellular machinery to package the therapeutic gene into the viral shell.
đŹ Lab Work: Cultivating Cells and Preparing for Gene Therapy
The speaker describes the laboratory procedures involved in preparing for gene therapy. This includes the cultivation of human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, which are used for producing viral particles. The cells are grown in a sterile environment using specialized media and equipment. The process involves the use of lip effect amine to facilitate the transfer of DNA into the cells, which is a critical step in the production of the viral vector. The speaker also details the steps for transducing the cells with the viral particles carrying the lactase gene, including the use of a color-changing dye (X-gal) to confirm successful gene expression. The video provides a behind-the-scenes look at the lab work and the challenges faced during the experiment.
đ Testing the DIY Gene Therapy: A Personal Trial
In the final paragraph, the speaker recounts their personal experience testing the DIY gene therapy. After a period of allowing the virus to deliver the lactase gene, the speaker consumes dairy products to test the effectiveness of the treatment. They report a successful outcome, with no adverse reactions to lactose after taking the gene therapy. The speaker expresses their excitement and relief at being able to enjoy dairy products without the previous symptoms of intolerance. They acknowledge that the project is ongoing and that further testing and refinement are necessary. The video concludes with a call to action for viewers to support future videos and a note of gratitude to patrons who have contributed to the project.
Mindmap
Keywords
đĄLactose intolerance
đĄLactase
đĄAdenoviral-associated virus (AAV)
đĄGene therapy
đĄLactose
đĄLac Z gene
đĄX-Gal
đĄLipid-mediated transfection
đĄPlasmids
đĄInverted terminal repeats (ITRs)
đĄBiosafety
Highlights
Individual developed lactose intolerance at 15, experiencing severe symptoms from consuming lactose.
Up to 65% of the human population may lose the ability to digest lactose, leading to various symptoms.
Lactose is a disaccharide found in many food products, including those using lactose as a filler or sweetener.
Lactase enzyme is crucial for breaking down lactose; without it, lactose intolerance symptoms occur.
Individual pursued biology to find a solution to restore lactose tolerance, aiming for personal dietary freedom.
DIY gene therapy is used to develop a treatment to restore lactose tolerance, using adeno-associated virus (AAV).
AAV is a safe and reliable vector for gene therapy, commonly found in humans without causing illness.
Plasmids are used to carry the DNA sequences for virus shell proteins and the Lac Z gene for lactase production.
Lipid-based transfection agents, like Lipofectamine, are used to deliver DNA into cells for virus production.
Two types of cells, HEK and CHO, are used for virus production, with different growth characteristics.
X-Gal assay is used to test for the presence of the Lac Z gene, indicating successful gene delivery and expression.
Virus harvesting involves pelleting cellular debris and precipitating the virus with ammonium sulfate.
Initial testing of the DIY gene therapy involved consuming lactose-rich food to test for restored tolerance.
Individual successfully consumed pizza and milk without symptoms, indicating potential success of the treatment.
The project is ongoing, with plans for further testing and refinement to ensure long-term effectiveness.
Individual encourages viewers to subscribe for updates on the project's progress and success.
Transcripts
[Music]
and in media and all these my stomach
release the virus into the virus and go
into my intestinal lining
when I was 15 I started getting sick all
the time at first it was pretty
infrequent but I remember the day that
it started I didn't know at the time
that I'd suddenly become
lactose-intolerant so all the food I was
eating was hurting me
laugh it off but in my case it wasn't
just a bit of gas I was getting
violently ill if I ate even a minut
amount of lactose and symptoms would
start in less than an hour and I'm not
alone this happens to up to 65% of the
human population at some point your
ability to eat milk just vanishes and
you start getting sick symptoms severity
varies but the extreme cases like mine
can really get in your way
most people don't actually think about
how much lactose is in the food we eat
as a society we actually produce so much
milk for you some cheese making that
it's literally a waste product so all
the components are separated out and
used and resold for a variety of
purposes the sugars specifically lactose
are refined and used as filler in flavor
in an enormous amount of products most
pharmaceutical manufacturers uses to
bulk up drugs used to make pills so the
tiny amount of drug that they need to
carry can be pressed into a
comparatively large pill whey is used as
a sweetener butter is in everything as
his cheese and cream I basically had to
build a whole skill set around making
sure food was safe to eat Irie taught
myself to cook and learn how to bake so
that I didn't have to miss out on too
much good food but all of this is also
the reason I went into biology from the
day I decided on the university program
I was going to apply for I've said the
same thing
one day I'll fix this when I was asked
if I was going to biology to end world
hunger or some other grand gesture my
response has always been now dude I just
want to eat pizza again and not need to
check every label on everything and I
can happily say that that day is finally
here after six years I finally have
access to the tools and know-how to
build a permanent treatment that should
restore my lactose tolerance and fix
this once and for all now I know the
idea of the DIY gene therapy can seem
intimidating or scary so let me set the
record straight
while some users make the application of
DIY gene therapy looks sloppy and
sketchy I'm hoping to change that with
this video I'm going to be as
transparent and make sure everything is
as clear as possible so you know exactly
what I'm doing how the thing works and
why it's not scary also this project is
still in the process of being developed
this video is just the first
demonstration of the process I'll be
continuing to refine it over the next
little while also
is going to be a long ish video so if
you'd like to skip to the testing and
results go to the time you see on screen
before we get into the lab and get
working let's go over how this is going
to work first we need to understand both
lactose and the enzyme that breaks it
down lactose is a disaccharide or simple
sugar that has two subunits one glucose
and one galactose subunit lactase also
known as beta galactosidase the enzyme
that breaks it down splits the two
subunits apart and then the individual
pieces can actually be absorbed by your
body and used for energy if you don't
produce the lactase enzyme the sugar
will make it all the way through your
small intestine and into your colon your
colon is full of a lot more bacteria
especially gas producing bacteria and so
they feed on the influx of extra
available food because the lactose
wasn't broken down and absorbed also
lactose attracts and holds on to water
so a lot less water is absorbed in your
colon that is supposed to be a DIN
irritation you've got all the classic
symptoms of lactose intolerance so what
do we do about it
well we need to make my small intestine
produce the lactase enzyme again
specifically in what's called the brush
border the brush border is a layer of
cells covered in tiny hairlike
protrusions these increase the surface
area of the small intestine
significantly these protrusions are also
packed full of enzymes that interact
with the material passing through and
helps break them down we could use the
human gene for lactase but it's actually
standard practice to use lactase enzyme
from E coli called Lac z lac c is used
all throughout biology and in
combination with a color changing dye
can be used as a marker to indicate if
cells are doing what they're supposed to
be doing when I was looking into this I
found a paper that uses a special virus
to deliver a working copy of lac z to
the intestinal tract of rats a week
after the researchers applied the virus
they challenged two sets of rats that
had previously been completely lactose
intolerant with a lactose only diet the
rats that have been given the virus
maintained their weight and their blood
glucose levels went up after they ate
the lactose indicating that they had
started to produce the lactase enzyme
and was able to use it for calories the
virus they used is called an AAV or
adeno-associated virus this virus is the
gold standard for gene therapy because
its reliability and long history of
safety the wild virus can be found in
almost everyone and in most tissue
samples that have ever been tested so
it's extremely common but it doesn't
cause any sort of illness it just kind
of hangs out it can't even replicate on
its own for it to spread you need to be
infected with its second actual
adenovirus hence why it's called an
Deno associated virus it just sort of
hitches a ride also it has the useful
property that the DNA that gets packaged
inside of it often merges with your DNA
but it does so only in a couple of very
well-known locations that have been
shown to be very very safe so how do you
make the virus and how do you choose
what DNA ends up on the inside well it
all works on a very clever system to
make a virus particle you need all of
the sequences of DNA that code for all
the proteins that make the shell of the
virus to be transcribed in the same cell
the various pieces will float around
until they bump into the rest of the
pieces and then they sort of snap
together to form the virus particle in
order for the virus to know which DNA is
owned and what to package into the shell
it uses two DNA sequences called ITRs or
inverted terminal repeats if you put
these on either side of some other DNA
sequence everything between the ITRs get
packaged normally this would be in the
virus packages DNA that contains all of
the information to make all of its own
pieces but what biologists did is break
their DNA into three separate pieces the
first to contain all of the sequences to
make all of the proteins but lack the
ITR sequences the last contains the ITRs
and whatever DNA we want a package by
the way these three pieces of DNA are
just called plasmids they're basically
just small circles of DNA that
biologists have engineered to have some
useful features I like to think of them
like CDs for a computer they hold a bit
of code that contain the instructions
for a piece of software when you run it
the program executes and something
happens in this case when the plasmid
makes it to the nucleus of a cell it
starts being translated into RNA and
then that gets translated into protein
let's take a closer look at the plasmids
to see what's actually going on on the
first two plasmids you can see that
proteins that make up the shell of the
virus and the rest of the proteins that
help it move and copy its DNA and
package everything you'll notice that
both have something called an Ori and a
gene that codes for ampicillin
resistance that's how you can put this
DNA into bacteria and use them as tiny
copying machines to make a literal
bucket worth of DNA to be used in future
experiments we'll be exploring that
process in a future video
the last plasmid is where all of the
important parts are here you can see the
ITR as I mentioned before this part is
called a promoter
it's a sequence that encourages certain
proteins to bind to that spot and start
transcribing the DNA
to RNA to get the process started then
right after it we have the Lac Z gene
that we want and finally something
called a poly a signal this is just a
part that's necessary for when the
protein is actually being made for both
the promoter and poly a signal there are
a ton of options to choose from but this
particular plasmid uses the CMV promoter
and an sv40 poly a signal the CMV
promoter is really good for when you
want lots of protein to be produced
wherever the DNA happens to be running
to actually make the virus particles you
take all three pieces of DNA and using a
special agent to help it enter cells you
apply it to a flask of mammalian cells
the cells will start producing the virus
particles and filling them with the DNA
and then they exit the cell all we need
to do is harvest the virus purify it
into a safe to consume form and package
it into a pill okay now that we know how
this works let's get to work
I'm here in my friend's genetics lab and
he's letting me use his equipment and
materials so I can do this project for
this we'll need a few things first is
the three pieces of DNA the two virus
plasmids come as a kit and I bought the
lac C plasmid separately we'll be using
two kinds of cells HEC or human
embryonic kidney and Cho or Chinese
hamster ovary heck are the standard for
making things like viruses and proteins
but Cho are equally good
HEC cells or at least the kind that we
use our suspension cells and like to
float around whereas Cho cells are
adherent and actually stick to the
culture flask to feed the cells and keep
them happy we'll need special media I
won't show you how to prepare the media
as well save that for a future video and
finally to get the DNA into the cells
we'll be using a special chemical called
lip effect amine first things first we
need to start some cells all of our work
will be taking place in a biosafety to
laminar flow hood this keeps everything
super sterile and prevents ourselves
from getting contaminated or other
viruses getting in also we use
antibiotics in the media to further
limit contamination specifically we use
a mixture of penicillin and streptomycin
before entering the hood to spray down
the surface with alcohol put on gloves
and then spray your arms up to the elbow
with the alcohol before bringing any
materials into the hood they must be
sterile and the container needs to be
sprayed with alcohol before we can use
the media we need to warm it up to body
temperature to keep the cells happy
we're using a water bath for this and
check the temperature until the media is
at least 32 degrees
but no warmer than 37 degrees Celsius
when it's a temp we can dry it off
sterilize the bottle and bring it into
the hood to move the media around we're
using special serological pipettes that
have the wide opening and come pre
sterilized and individually wrapped
we're also using an auto pipettor that
makes it easy to draw and release
liquids we'll be growing ourselves in a
special tissue culture flask that comes
pre sterilized in this case we'll be
using a small 25 centimeter flask that
holds a maximum of 5 milliliters of
liquid the first step is always to label
your flask with the cell type the
experiment and the date first let's look
at what we do for the cho cells we can
remove the lid of the flask and their
lid of the media placing the lids
facedown on the work surface then set up
the auto pipette or with a fresh pipette
and draw up four mils of media for cho
cells that means F 12 K media
supplemented with 10% fetal bovine serum
pick up the flask tilt it and dispense
the liquid it's good practice to limit
things touching as much as physically
possible then disposes the pipette and
then tilt the dish back and forth until
the media covers the bottom with the
flask prepared we can add ourselves I'm
using a regular P 1000 micropipette and
I'm adding the cells that we grew and
prepared in advance I'll show how we did
that in a future video for the HEC cells
we did basically the same thing but
because their suspension cells we just
grew them to the density we wanted in a
larger flask and then pulled off 5
milliliters worth of suspension and
transfer that to a new flask to be used
immediately the cho cells need more time
to grow though so those get transferred
to a co2 humidified incubator set at 37
degrees Celsius and 5% co2 since the HEC
cells are already ready we can go
through the transduction procedure for
this we'll need the lip effect amine
which comes in two parts the media for
the HEC cells and the three DNA
solutions to set this up we first put
500 microliters of HEC media into two
sterile DNA free EPI tubes we want five
micrograms of DNA total which works out
to 1.6 micrograms of each type of DNA or
6.8 microliters of each DNA solution add
that to one of the tubes and to the same
tube add 5 microliters of lip effect
amine plus reagent to the other tube we
add 5 microliters of regular lip effect
amine give those mix then mix the DNA
solution into the lip effect amine not
the other
way around allow that to incubate for
five minutes lip effect Amin is sort of
an oil that forms a bubble called lipids
ohms around the DNA that allows it to
get across the cell membrane it takes a
few minutes for the lipids ohms to form
hence the incubation time after the
incubation all that we have do is add
the solution to the flask of cells with
that done the flask can be transferred
to the incubator the reason we're using
two cell types is because I actually
tried this experiment twice the first
time was with the HEC cells but the
cells died because the live effect Amin
is kind of aggressive and can damage the
cells the second time the procedure was
the same but i use the cho cells which
because they're adhered to the dish have
the added benefit that 12 hours after
the transfection I can change the media
to fresh lip effect amine free media
this keeps the cells much healthier the
cho cells work so we let them sit and
produce as much virus as possible for
three whole days did you have to see if
this worked we'll be using a
color-changing guy called ex Cal ex
Cal's a dye molecule fused to a
galactose molecule so that if lactase is
present the two gets split the dye
becomes active and turns blue and we
know that the thing is working I made a
solution of two milligrams of X Cal in
five milliliters of DMSO this has the
benefit that DMSO will carry the X gal
into cells that we exposed to it also
DMSO is one of the only things that can
actually dissolve ex Cal because the cho
cells were adhered before we do the
tests we can collect all the media which
is where most of the virus is going to
be save this in a separate sterile
Falcon tube then we can test the cells
themselves first we need to make them
let go of the dish so that we can take a
sample and test them to do that we'll be
using two milliliters of pre-made
trypsin solution allow the trypsin to
sit in the dish until the solution
starts to get a bit cloudy this is
evidence that the cells have let go and
are floating around when this happens I
use a pipette to collect all of the
cells and transfer them to a falcon tube
these then get loaded into a centrifuge
and spun down at 5000 rpm for five
minutes two pellet them onto the bottom
so that we can remove the trypsin when
that's done we need to reach sterilize
everything and bring it back into the
hood so that we can take our sample I
removed the trypsin solution and
resuspended the cells in two milliliters
of media and then took a two hundred
microliters sample and transferred it to
a small tube I added an equal volume of
x-gal and then let everything sit for a
while to give it time for the reaction
to occur if any lactase is present after
the incubation I took everything to the
micro
scope so that we can see what actually
happened it was really hard to capture
with my camera but the cells were
definitely blue and with that I knew
that everything had worked and that we
can finally harvest the virus for this
very first round of testing we'll
actually be using a really
straightforward method to harvest our
virus first things first the media full
of virus were that we saved has to be
spun down at 2,000 rpm to pellet any
cellular debris the virus is super light
so it'll just stay floating around
collect the supernatant and leave any
salts behind to get the virus out of the
solution we can add ammonium sulfate
solution this is what's known as salting
out ideally you'd do this in two steps
to first remove the residual protein and
then pellet the virus but I'm just going
to be doing this all in one step I'm
adding an equal volume of 4 molar
ammonium sulfate solution this sudden
influx of salt makes the virus
precipitate out so that we can actually
collect it let that sit for an hour and
then spin it down at 4,000 rpm to
collect the product now I know this will
be contaminated with other proteins but
for this test I judged that to be an
acceptable risk based on all of the
possible protein contamination in the
future this will be refined much further
and more purification steps will be in
place to make sure the virus is truly
the only thing in here remove the
supernatant and discard it then Reda's
all've the viral pellet in 800
microliters of sterile phosphate
buffered saline and that's most of it
done all that's left is to package this
into a pill to do that and mix the
solution with a minimal amount of micro
crystalline cellulose and then put that
into gel caps using this handy gel cap
jig machine thing ideally I should have
freeze-dried the powder before packaging
but again for the initial test I decided
I didn't want to wait after taking the
pills I waited three days to give the
virus time to deliver the DNA and give
my cells time to start producing lactase
and then it was time to test it we
ordered the milk iasts cheesiest pizza
we could
try to be less scary here is the mind
killer is it good can you not tell now
I'll be honest this was maybe one of the
most stressful moments in recent memory
willingly eating something that would
normally make me extremely sick scared
the hell out of me
but I had ate several pieces and then
settled into a movie after an hour I
felt fine after a whole night's sleep I
still felt fine and after 48 hours I was
completely fine I couldn't believe it
but the therapy seemed to have actually
worked in two weeks as you can see got
more pizza I've enjoying just the most
copious amounts of lactose filled foods
for last two weeks it's been wonderful I
feel better than I have in eight years
at first when I was testing this I would
still get a little bit of gas but that's
completely gone away now I think it just
took a while for more protein to be
expressed and for my microbiome to
readjust to the radical change in diet
as I said this project is still in the
development stage and more testing will
need to be done I'll be taking notes to
see if the effect wears off after some
time and I'll be sure to keep you
updated if this project interested you
and you want to see more I'd really
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patrons who helped make these videos
possible that's all for now and I'll see
you next time
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