OK. So last time I reviewed for you some of the basic cloning techniques.
I failed to show you this slide, which I intended to do, which is a
commercially available plasmid. I told you about plasmids last time,
small circular DNA molecules that are used in the purpose of cloning.
They're derived from nature, but then they've been heavily
manipulated by scientists to be useful for the purposes of cloning.
Recall that they have two, three critical elements, an origin
of replication to allow them to be replicated inside of bacteria,
a selectable marker, a drug-resistance gene,
ampicillin resistance gene for example, and finally,
can you now show the middle slide? He's working on it. And finally
what we call a multiple cloning site, a set of restriction sites,
restriction enzyme recognition sites that allow us to pop
in pieces of DNA. I have dual paper readers.
Dual paper readers? That doesn't happen very often.
You usually have one but not often two in a row. Good.
So these are useful practical tools. Maybe before I forget,
I'll mention, remind you of the quiz on Monday, where you go based on
your last name. There was a discussion last time
about the review session, which is actually not on here but we
didn't change the review session. Oh, because it was last night.
We weren't able to change the review session, which was last night,
but there is a tutoring session which will cover the same material
from today between 4:00 and 6:00. And then there are additional
sessions with the TAs, including up till Monday morning
before the exam. So there should be ample
opportunity to get your questions answered. So plasmids are now sort
of a useful and are a very available tool.
This figure comes from your book. It's a version of what we covered
in our example last time, the cloning of the T gene.
Here in red is the starting material, the DNA from the source.
In our case it was S. pyogenes, that flesh eating bacterium,
but it could be anything. It could be genomic DNA from you.
You take that genomic DNA, whatever the source material is,
you digest it, cut it with a restriction enzyme,
with a particular restriction enzyme. You then mix that DNA with plasmid
DNA which has been cut with the same restriction enzyme,
the sticky ends of the linear molecule anneal with the sticky ends
of the plasmid. You then add DNA ligase to seal
those nicks to produce full covalently closed circular molecules
that are composed of both the plasmid and now an insert
from the DNA sample. You then take those recombinant
plasmids, mix them with bacteria under conditions which allow the DNA
to get into those bacterial cells. This is a process called
transformation. You then plate those bacteria onto
auger plates that contain the antibiotic, in this case ampicillin
in our example. The bacteria that didn't get a
plasmid die. The bacteria that did get a plasmid survive and form
colonies on the dish. And then the question I left you
with was how are we going to find the colony or colonies that carry
the T gene? How are we going to find the gene,
the recombinant plasmid, the colony carrying the recombinant
plasmid with the T gene? I also want to mention a bit of
terminology, which we will come back to later. We often refer to the
collection of colonies, the collection of recombinant
plasmids contained within those bacteria as a library.
Like a library containing books, this is a collection of different
things. In this case, different recombinant plasmids which
you can go back to repeatedly taking isolates from the colony.
In our case we would then grow up the individual bacteria to get ample
amounts of the plasmid of interest. And this term library is used in
molecular biology a lot, a diverse collection of clone
fragments to which you can return depending on your specific needs.
So how are we going to isolate our plasmid of interest?
Well, if you recall, we knew the sequence of the genome
of S. pyogenes. And, therefore,
we knew the sequence of the T gene. So imagine that the T gene had a
particular sequence starting from the 5 prime end of
A-G-G-C-T-G-G-T-G-G-G-A to the 3 prime end.
So this is imbedded within the T gene. The reverse complement on the
other strand reading in this direction from the 5 prime end would
be T, did I say that, what's this? C-C-C-A-C-C-A-G-C-C-T
3 prime. OK? So we know a bit about the thing we're
interested in. We know its sequence.
And we can use this information to our advantage to isolate the plasmid
that carries this fragment. So the bacteria, as shown on this
slide, are present in colonies. Each colony contains a recombinant
plasmid. The first step in isolating the plasmid of interest is
to make a copy of the plate that carries these bacteria to replicate
what's on this plate onto a piece of filter paper.
We call this replica plating.
And in this case we're going to do
it onto a filter, a piece of paper or nylon to make a
copy of the colonies and the pattern of the colonies onto something else
that we can manipulate. So we make an exact copy.
And this done by literally placing the piece of filter paper onto the
Petri dish, as shown here. Here is the Petri dish with the
colonies. You place a filter on top of those
colonies. They'll stick to it. Some of the cells in those colonies
will stick to the filter. You then pull the filter off and
you get some of the colonies, some of the cells sticking in
exactly the place where they were on the original plate.
You can actually place this on another plate and allow those cells
to grow directly on the piece of filter paper. So you make an exact
copy of what was here onto a piece of paper. Now,
what are you going to do with that? Well, we're going to use this
information that we have about the sequence of the gene to isolate the
colony of interest. First thing we do is to lyse the
bacteria that are now present on this filter and denature the DNA.
What I mean by denature the DNA is
to pull it apart. It's double-stranded when it's
present in the bacteria. To denature DNA is to make it
single-stranded. And typically the way we do that in
these applications is to place a piece of filter paper under high pH
conditions, and it causes the DNA strands to melt apart.
So now the plasmids, which were double-stranded in these bacterial
cells, were double-stranded here have now been pulled apart into
single-stranded molecules. So, on this piece of filter paper
there are cells with unwound DNA, single-stranded DNA in them in this
same pattern, which I won't try to reproduce here.
And the question is how are we going to identify which colony
carries a piece of the T gene? How are we going to do that? Does
anybody have a clue? All you need to know is present up
here. Yeah?
So you're thinking of the example from the book in which the,
in the example in the book they used a plasmid that had two selectable
markers. And you were interested in those that grew under one antibiotic
selection and didn't grow under a different antibiotic selection.
In our case, in our plasmid, we actually didn't have two selectable
markers. We only had one. So we're not going to use that
strategy. We have to rely on some of the information I've
given you already. Well, we know the sequence of the T
gene. We know, for example, this little bit of the
T gene, so we can make some that we call a probe. A probe,
for this application, is an oligonucleotide,
a small segment of nucleotides that we can synthesize.
You can actually make any DNA sequence you want using
chemical synthesis. This has now been fully automated.
So you can just punch the sequence into a computer,
attach it to one of these machines, the machine will go off and make a
linear piece of DNA of any sequence you want. It's like a typewriter
almost. You type it in, and what you get back out is a piece
of DNA that is the sequence that you typed in. So we can make any DNA we
want such that we could make a piece of DNA that had the sequence
A-G-G-C-T-G-G-T-G-G-G-A.
OK? We could make that piece of DNA. Moreover, we could modify that
piece of DNA with enzymes to add a radioactive phosphate at this 5
prime end. So we could make a radio labeled probe.
So now that we have that, what are we going to do? Anybody?
Yes. Yes.
Right.
Exactly. So this is now complementary to the other strand.
The DNA within these cells that have been lysed and now stuck onto
this filter are also single-stranded. So this complementary strand is
going to be sitting in a single stranded conformation ready to
anneal, hybridize with this probe. So you apply this radioactive probe
to this filter. It will bind somewhat
indiscriminately at first, but it will bind very tightly to
that complementaryary strand from the T gene. And remember there are
probably a million bacterial cells within this colony,
so there will be a million copies of the probe anneal to this colony.
You do have to do a little washing to get rid of the nonspecific
binding which will take place. So you wash the cells, or wash the
filter, get rid of the nonspecific binding.
The specific binding will withstand the wash. And then you apply a
piece of x-ray film. And what you'll end up with is a
faint image of the filter, some very faint signal from the
colonies that don't hybridize, and a very strong signal from the
colonies that did hybridize properly to the probe. OK?
So now you know, based on the position on that filter,
which colony it is that carries the T gene. And in this case it's that
one right there. So we can go back to that master
plate that we've kept, use a toothpick, pluck off that
colony and grow it up into high concentrations successfully having
completed our cloning project of the ever toxic T gene.
And that's gone through here in this figure, if you want to look at
it again. You take this filter that has the colonies.
It's actually the step here where you grow the cells up on
the filter itself. You then lyse the cells,
denature the DNA, add the radioactive probe.
It won't hybridize strongly to those colonies that don't carry the
clone of interest. It will hybridize strongly to the
colonies that do have the clone of interest. And then through this
x-ray film method you can visualize where those colonies are and then go
back and pick the colonies from the master plate. OK?
So that's how it works. It's fairly straightforward and
works extremely well to give you the cells and clones of
interest. Woops.
So that's what we call clone identification by hybridization
using a radioactive probe. And I should mention, because it
will come up again later, that we use these radioactive probes
extensively for molecular biology. And we use non-radioactive probes,
oligonucleotide synthesized in the same way that I mentioned for all
sorts of molecular biology applications in this era.
And you'll see examples later in the lecture. I want to briefly
mention another technique known as cloning by complementation.
In this instance, we're going to clone by the function of the plasmid
that we've introduced into the cells.
Not its sequence. By its function. And the example
I'm going to give you is to clone a gene that you might know about based
on the biochemistry of the organism, but you haven't identified the gene
that encodes that enzyme. The example I'm going to give you
is to clone an enzyme which is responsible for catalyzing the
breakdown of lactose, a disaccharide. And this is
accomplished by an enzyme called beta-galactosidase.
And it converts glucose. It converts lactose to glucose and
galactose. And this is an important enzyme in bacteria and also in you.
The enzyme is encoded by the lacZ gene of bacteria.
OK? And this exercise that we're going to go through is to try to
isolate the lacZ gene through this method called cloning by
complementation. We need to use a trick,
which I'll show you on this slide. And that is that there's an
artificial substrate for this enzyme which is called in the field X-gal.
It's related to lactose. The important thing is that when X-gal
gets cleaved by beta-galactosidase, it releases galactose and this
molecule which precipitates and turns blue. So we have a visual
indicator of beta-galactosidase activity by placing in the medium,
either in liquid medium or on a Petri dish, this substance X-gal.
We know whether or not the cells have beta-galactosidase activity by
virtue of whether the cells or the colonies turn blue.
OK. So the first thing that we're going to do in this technique is to
take some wild type E. coli.
If we take a liquid culture of wild type E. coli and we plate them onto
a tissue culture, a Petri dish that contains X-gal,
this substrate that in the presence of beta-galactosidase will turn blue,
will get colonies. And what color will those colonies
be? Blue. These are normal E.
coli. So they're able to metabolize the X-gal and produce this blue
pigment. OK? Now, I'm interested in the enzyme,
the gene that encodes the enzyme that carries out this activity.
And the way that I'm going to get to it is first to isolate mutant E.
coli. It cannot do this reaction. I'm going to try to isolate a mutant
strain that's deficient in beta-galactosidase activity.
And the way that I do that is to mutagenize the wild type E.
coli. I can do this by physical methods or more commonly by just
adding a chemical mutagen to the broth that the E.
coli is growing in. I mutagenize the cells and then I
plate them onto a plate that has X-gal.
Now, most of the cells that got mutagenized will have mutated some
other gene, not the beta-galactosidase gene,
some other gene, or maybe no gene, such that what color will those
colonies be? They'll be blue because they still have a functional
beta-galactosidase enzyme. They still have a wild type lacZ
gene. So most of the colonies will be blue. But what if the cell
incurred a mutation in the lacZ gene that knocked out lacZ function?
What color will the colony be? Not blue. It will be white. OK?
And we're going to assume, for the sake of argument,
that if you get a white colony it means that the cells have a mutation
in this gene lacZ. That's actually not a safe
assumption. It could have a mutation in some other gene,
but we're not going to worry about that today. Just say that if we get
a white colony we're going to assume that the cells have a mutation
in the lacZ gene. So now we want to clone the lacZ
gene. Our goal is to clone the lacZ gene. What are we going to do?
What are we going to do? Well, I'll give you the first clue. We're
going to make a library. We're going to make a library of
fragments of DNA some of which will carry the lacZ gene.
From what are we going to make that library? What will be our source
DNA to make that library? Will it be these cells isolated
from this colony or will it be some other cell? Do these cells have a
functional lacZ gene? No. Do these cells?
Yes. So you want to make a library from wild type E.
coli. So that you have a bunch of plasmids that carry fragments of the
E. coli genome some of which carry the lacZ gene.
What am I going to do with that library? What am I going to do with
that collection of clones?
I'm going to transform it en masse all the different clones to a
population of cells. What cells will I transform it into?
Which bacteria? Would I put those ones into the
lacZ mutants?
And then I plate that transformation onto plates that contain amp.
I need the drug to identify those cells that have picked up any
plasmid because I don't care about cells that haven't picked up any
plasmid so I use amp plates. And what else do the plates have in
them? The indicator of lacZ activity,
X-gal. So I'm going to take these transformants,
which were derived from these cells, and I'm going to plate them out onto
a plate. What color will most of the colonies be?
Will most of them, raise your hand. Will most of them
be white? Will most of them be blue? In fact, most of them will be white
because most of the cells did not pick up a plasmid that carries the
functional lacZ gene. Most of the cells picked up a
plasmid that carried some other piece of the E.
coli genome. OK? So most of the cells will not be,
will not have functional lacZ activity, they'll be white.
However, at some frequency a clone, a cell will pick up a recombinant
plasmid that does carry the lacZ gene and that colony
will turn blue. And that's the colony that we're now
interested in because it has reconstituted lacZ activity.
The mutant, the recombinant DNA has complemented the mutation.
So I could isolate this plasmid and
sequence it. And based on the sequence I might have confidence
that indeed it is the lacZ gene. Remember I told you that there was
an assumption built in here that this mutation really did affect the
lacZ gene? And that's why the cells were white.
That was an assumption. And to confirm that assumption we
could look at the sequence of the clone that we isolated and figure it
out, figure out whether it's the right enzyme. OK?
So I find that teaching this is always a little confusing.
I tried to simplify it today and go through it slowly.
Hopefully you took notes and you can think about it on your own.
It is relatively straightforward and it's a useful concept to
understand that A) you could isolate mutations, and B) you can find the
genes that were mutated by complementing those mutations
through cloning. OK?
OK. So now that I've spent a lecture and a half talking about
cloning, I'm now going to tell you that cloning, while it was
incredibly important and still is, has almost been superceded by
another technique. It's not that we don't use cloning.
We actually use it all the time. But it's been joined by another
technique known as PCR which stands for the polymerase chain reaction.
And any of you who have worked in
molecular biology labs have been exposed to this because it is a
totally pervasive technology. Everybody uses it. And it's not
just in molecular biology labs. Forensic labs. Archeology labs.
Anybody who's interested in generating large amounts of DNA from
a small amount of DNA uses this technique called PCR.
It was invented only a few years ago, but it's incredibly important.
In fact, the guy who invented it, who was not a very well known
scientist, won the Nobel Prize just a couple of years after he invented
it because it became so powerful so quickly. It's a very,
very simple technique. It's one of these ah-ha techniques that after
it's been invented and described everybody says,
oh, I could have thought of that. But, actually, nobody did until
this guy, Kary Mullis. So I'm going to show it to you
briefly on the board. And then we're going to go through
a movie that shows it again. So hopefully you'll get how it
works. Importantly, it relies on having some information
about the DNA that you want to amplify up in large quantities.
Again, the goal here is to amplify up a piece of DNA of interest to
large quantities. This technique is so powerful that
you can use a single DNA molecule to start. You can use the amount of
DNA that you get from hair from a crime scene, for example.
And that's mostly the way, you know.
What is it? CSI Miami and stuff. They use this technique all the
time. So you can use minuscule amounts of DNA.
Even a single molecule is enough. But you do need to know a little
bit of known sequence.
So if you know a little bit of known sequence you can use PCR.
So the first thing you do is to denature the DNA.
And in this case we do that by heat.
If you heat up a DNA molecule it will also separate from itself.
And we typically heat it up to about 94 degrees.
And the consequence of this is this double-stranded piece of DNA
molecule will separate into two single-stranded pieces of DNA.
And at that point we anneal, hybridize onto the DNA --
I'm not about to do another demonstration here.
We anneal onto the DNA oligonucleotides,
synthetic little fragments of DNA that are complementary to this known
sequence at this end and at this end. So I synthesize a short
oligonucleotide which will anneal to this strand at this position. OK?
I should have written up the polarities because people often get
confused. Here's the 5 prime end of this DNA molecule,
the 3 prime end of that DNA molecule, the 3 prime end of that one,
5 prime end of that one. So if I'm thinking about the top strand here,
the 5 prime end here, the 3 prime end here, and that little
oligonucleotide, which in this context we call a
primer, would have its 5 prime end here and its 3 prime end here.
Thus, the arrow because this is the direction of DNA synthesis.
If we're going to synthesize DNA off of this piece of DNA it's going to
go in that direction. And then we likewise order up and
anneal onto an oligonucleotide that binds to this strand of this
sequence. And, again, to show the polarities,
here's the 5 prime end. Ah, thank you very much.
Here's the 5 prime end, here's the 3 prime end, so that the
oligonucleotide primer has its 5 prime end here, its 3
prime end here. OK.
And now these primers are sitting in such a way, annealed to a template
piece of DNA with a 3 prime end facing in this direction,
that are ready to be extended by DNA polymerase.
Sorry. I'm getting confused about
my colors here. You can't see them on the board?
You can't see the blue. OK. All right. So the blue,
sorry about this. The blue will now be orange. OK?
And this one will be pink. OK? So I'm going to extend this
pink oligonucleotide with a polymerization reaction to
fill in this strand. And I'm going to extend the orange
guy likewise to fill in this strand. OK? So this is accomplished by
addition of DNA polymerase and the nucleotide precursors that are
needed for DNA synthesis, which we abbreviate dNTPs.
And if I incubate that in that way I'll get extension from this primer
in this direction, extension from this primer in this
direction. And what I've done here is to go from one DNA molecule to
two DNA molecules. I've duplicated this piece of DNA
in a test-tube. The beauty of PCR is that you then
go through this process again and again and again and again.
And each time you get an exponential increase in the amount of DNA.
You go from one to two to four to eight and so on and so on and so on
and so on. And in a relatively short time you can now have millions,
billions or more copies of your DNA. OK? So that's the basic principle.
And, again, we'll go through it in a little more detail in the movie.
As I said, the technique was invented by Kary Mullis,
an investigator at a company called Cetus.
Kary Mullis is an interesting character. I don't have time to
tell you the full stories of Kary Mullis. As I told you,
he won the Nobel Prize. He's a real California kind of a surfer dude.
Very laid back.
So, anyway, you can use this technique for lots of applications,
as shown here. It's been commercialized in many ways.
We now have machines that will go through these various stages of
denaturation, annealing and synthesis using a temperature
regulated block that will go from 94 degrees to 68 degrees to 72 degrees
to allow denaturation, annealing and polymerization around
this circle many, many, many times. And if you do this,
for example, 30 times, if you go through this cycle 30
times you make two to the thirtieth copies of your DNA,
which is ten to the ninth DNA molecules, which is a ton.
And that can be done in just a couple of hours using a machine such
as this. OK. So let me take you through this movie.
I'm going to cut it short because the process is gone through
in great detail. The narrator is actually Paul
Matsudaira who is a professor here at MIT. And Paul really sort of
drags it on, but I'm going to --
Basically all he says is what I said, which is you take an individual DNA
molecule for which you know sequences, and you run through this
reaction multiple times. And each time you do you get a
duplication of the DNA sequence. And your book goes through it, too.
So I will get this thing posted on the Web. It's not on Monday's quiz
so you don't have to worry about that.
We can learn about it in the future. Maybe I'll fix it so that on
Monday's lecture or Wednesday's lecture, Professor Sive who is
giving that lecture can go through it with you. So sorry about this.
I also wanted to mention briefly that the enzyme that we use to carry
out the polymerization, the particular DNA polymerase that
we use is isolated from what we call a thermophilic bacterium isolated
from the hot springs. And that's important because we
carry out this reaction at a very high temperature.
You'll notice that the polymerization was done at 72
degrees. Now, your DNA polymerases won't work at
72 degrees but this organism grows at very, very high temperatures so
its DNA polymerase can function there. And this,
again, has turned into a cottage industry. There are hundreds of
millions of dollars worth of this particular polymerase called Taq
polymerase sold for this application. OK.
So let's carry onto the next topic then. So what we've been talking
about now is transferring DNA in the case of cloning or amplifying DNA
for various applications. For some applications that's not
good enough. For certain therapeutic applications,
for example, taking human genes and cloning them to make therapeutic
proteins or to do gene therapy you cannot do standard cloning.
And the reason is that human genes are usually way too big.
They have exons and introns, which you learned about, and they
can be hundreds of thousands and sometimes millions of nucleotides in
length. And that's way too big to fit into one of these plasmid
cloning vectors and to efficiently introduce it to bacteria.
And so for the purposes of generating clones from human genes
we often use a technique called cDNA cloning.
And I'll take you through this
briefly. Recall that human DNA, human genes are broken up into
coding elements that we call exons. And we usually number these exons
from left to right, one, two, three. During the process
of transcription a copy of this gene is made in the form of a precursor
mRNA which has the same sequence. It has both the exons and the
introns present. And then before this is translated
into protein the information in the introns has to be removed in a
process called splicing. And that produces a mRNA which
carries the exons lined up next to each other. So the introns are
removed, the exons are joined, and it's this mRNA that then gets
translated into protein. As I said, this can be very,
very large. But this is rather short. And so if we want to make a
copy of a gene we can also make a copy of its mRNA.
And that might be more efficient and more useful. And this is a
technique called cDNA cloning. It was made possible by an
invention at MIT, a discovery at MIT of an enzyme that
was not known to exist, an enzyme which violated the
so-called central dogma. The central dogma being that DNA is
transcribed into RNA and that's translated into protein.
David Baltimore discovered an enzyme called reverse
transcriptase.
Which can take the information in RNA and convert it to a DNA form.
And he actually won the Nobel Prize for that. So this is the principle.
You can take an mRNA which has polarity 5 prime to 3 prime.
This mRNA that I produced up there, for example.
You can anneal onto it an oligonucleotide primer.
This oligonucleotide primer is made of DNA. It has a 5 prime end and a
3 prime end. And then you add this enzyme reverse transcriptase,
which I'll abbreviate RT. Reverse transcriptase is unique in
its ability to copy from an RNA template a DNA strand.
And so you'll get a duplex which has RNA on the top and
DNA on the bottom.
You then get rid of the RNA.
You hydrolyze the RNA.
So all you're left with is that single strand of DNA.
You now anneal on another primer, another oligonucleotide primer which
now has a polarity 3 prime to 5 prime in this direction because this
one went 5 prime to 3 prime in this direction. And then you
add DNA polymerase.
Which will extend from this primer and make a full duplex of DNA where
both strands are made out of DNA. And this we call a cDNA clone, a
copy of the mRNA. cDNA clone. And you can make
libraries of cDNA clones from all the RNAs in your cells,
for example. And then you can look,
using various techniques, at different clones within that
library for ones that function in different ways.
So I want to take you through a couple of examples of how we use
cDNAs in the context of gene therapy. This one comes out of your book.
This is an application where there's an enzyme called TPA which
will dissolve clots. It's called tissue plasminogen
activator. And it's used now to treat people
who've had heart attacks or strokes. So what was done was to make a cDNA
copy of the TPA gene, introduce that into a plasmid vector
with appropriate sequences in red and yellow to allow the expression
of that gene in bacteria, transfer it into E. coli by
transformation. Now, the E. coli will pump out this
enzyme TPA. You can then purify that enzyme and
inject it into this happy looking stroke patient to help dissolve the
clots that it formed in the formation of the stroke.
OK? So that's an example of genetic engineering one of our genes
into bacteria to turn them into little protein factories from which
you can purify the enzyme and hopefully mitigate the consequences
of the disease.
A second example, as opposed to putting in a
therapeutic protein, you might want to put in a
therapeutic gene. So the example here I'll give you
is cystic fibrosis.
I've mentioned to you in earlier lectures that cystic fibrosis is a
genetic disease in which the individuals have a defect in a
transporter, an ion transporter, so that in contrast to normal cells,
here is a normal cell which has this CFTR transporter which will allow
chloride ions to move in and out of the cell, as is a wild
type individual. In the case of CF,
that transporter is missing. That leads to an inability of the
cells to properly regulate their water content,
and this leads to disease. So what could you do about this
disease? Well, why don't you just put the
defective gene back in? We could make a cDNA copy of the
cystic fibrosis gene, build it into a vector and introduce
it not into bacteria but back into these cells. So we could clone a
CFTR cDNA back into these cells. They would, in fact, pick up that
DNA and begin to express it. They would make this protein.
And that would allow them to transport chloride properly.
And they might be ìnormalî. This actually works extremely well in the
tissue culture dish. It doesn't work nearly so well in
the context of a human being. Even though we can make viral
versions of this, viruses that carry cDNAs,
recombinant viruses, and I'll draw that up here.
Using the same methods of
recombinant DNA technology, I can make a recombinant virus,
like a cold virus that now carries a piece of this cDNA for CFTR,
and I could use that to infect a person who has CF.
That's a person and they have CF so
they're not happy. I could introduce into their nasal
passage, they could breath in this virus that carries the gene.
And if the gene, if the virus infected the cells of the lung,
remember the problem occurs in the lung, maybe, if this were very
efficient, the person would be happy, we could cure them.
The problem is that this introduction of the virus is very
inefficient in vivo, in the person. It doesn't work very
well. You cannot get enough virus in to infect enough cells to correct
the disease in the person. So although gene therapy is very
attractive for many diseases such as CF, it actually hasn't worked very
well. But there is one example of a cure using gene therapy.
And I want to just end by telling you that story.
To cure the disease you probably heard about, the disease that causes
ìthe boy in the bubbleî syndrome, which is severe combined
immunodeficiency, or at least one form of it,
these individuals have a defect in an enzyme called ADA,
adenosine deaminase. And this results in a very severe
immunodeficiency. They don't have their immune cells.
They cannot fight infection. So the kids have to stay inside
protective chambers to avoid exposure to pathogens,
the bubble. For a long time they really weren't well treated.
They could be treated with having a little bit of the enzyme.
I guess that's not shown here but on the next slide.
If you add the enzyme back, even to their blood, you can get
some protection. But, nevertheless,
the kids did not do terribly well and often died from infections.
Here's the disease mechanism. It's caused by a defective enzyme that
normally breaks down adenosine. This leads to a buildup of a
precursor, or rather a metabolite that kill cells of the immune system,
and the individuals have immunocompromised state.
If you can just hang on for one or two more minutes.
So what's done here, in contrast to the failed example
for CF where I said that it's very difficult to get the virus into the
cells in the person, what's done in the case of this
disease is to take the cells out of the person. And this is called ex
vivo gene therapy where you isolate cells from the bone marrow of one of
these kids, the stem cells that give rise to the cells of the blood
system, and then you infect those cells in vitro with the virus that
carries the cDNA of the ADA gene. You isolate those cells that have
that gene and then you introduce the cells back into the person.
And you can control that process much more carefully,
much more efficiently, and you have a much greater
concentration of cells that are doing the right thing now.
This goes through it in some more detail. It's a version of a slide
from your book so you can look at it.
It's gene therapy. And I want to point out that
although it can work, and it does work, it has one risk,
namely that the virus that carries the therapeutic gene actually
integrates into the DNA of the cells. And if it integrates into a gene
that's important that integration could actually cause a mutation.
So there's a risk associated with gene therapy.
And, in fact, although this was successful, as you can see here,
two kids with SCID, or actually a number of kids with SCID were cured
through this ex vivo gene therapy approach, and now they can run
around like everyday kids, unfortunately, in this first study
of 11 patients, all of whom were ìcuredî,
later two of the kids ended up getting leukemia.
And they did because the virus had inserted itself,
the genome of the virus had inserted itself next to a gene that when it
becomes too active it causes the cells of the immune system to
proliferate abnormally leading to leukemia.
So although there was a benefit there was also a risk.
In this case, families of these kids actually are willing to take
that risk because it's such an awful disease. But you have to be aware
that in all of these kinds of therapies there's a risk-benefit
analysis that has to go on.