Drug Delivery Principles and Engineering
Prof. Rachit Agarwal
Department of BioSystems Science and Engineering
Indian Institute of Science, Bengaluru
Lecture – 53
Gene Delivery Vectors
Hello everyone, welcome to another lecture
of Drug Delivery Engineering and Principles.
We are at Gene Delivery module and we are
discussing about various aspects of gene therapy
either by cells or without.
So, let us quickly do a recap of what we learned
so far.
So, as I said we are talking about gene therapy.
So, the first thing we talked about is for
gene therapy is let us deliver a cell that
carries the gene of interest.
So, this could be a native cell itself that
is expressing that gene and contains that
gene or this could be a cell that you have
engineered first in laboratory to express
this gene of interest and then deliver it.
So, that is one option and that is very similar
to what we have discussed earlier in the previous
classes where you can encapsulate this cell
in a semi permeable membrane that will prevent
any kind of immune response from acting in,
but the cells can continue to express and
deliver the protein of interest.
The problem with that is it is not fairly
successful because there are several small
components also present in the immune system
that will still be able to go through this
membrane such as cytokines and complement
and they will not let these cells survive.
So, that is one challenge and so what is seen
and we discussed an example in a mouse setting
that if you have an immune deficient mouse
these things survive at least for a few months
and but if you have a healthy functioning
immune system carrying mouse then that those
things do not really survive they within a
month or so we saw that that mouse rejected
the implant.
So, that is a problem.
So, that is where the second strategy would
come in is let us not talk about non-autologous
cell and so any cell that is not from your
own body use the cells that are in your own
body.
So, why cannot we just deliver the gene hopefully
the cell will go the gene will go to the cell
that you wanted to express and that way the
immune system will not really attack the cell
as much.
So, in that regards we discussed several barriers
that are present of course, it is all well
and good to say that you can deliver at DNA,
but then the delivery of the DNA has several
challenges because first of all when you deliver
this there are several accessible barriers.
So, if serum contains lots of enzymes that
can degrade it.
It is hard for this to target the organ that
you want.
So, let us say if you want the expression
to be in lungs it is difficult that all the
DNA will actually go to the lung and then
the immune system again we will clear out
quite a bit of it immune system or you can
also call it the reticular and the reticular
system.
So, all of this we will clear out quite a
bit of it and then there are some of intercellular
barriers again.
So, even if it reaches your target cell how
does it go inside the cell.
So, let us see if this is a cell.
So, first of all you have to figure out how
does your DNA pass the cell membrane because
DNA is a fairly charged in large molecule.
So, it cannot go through.
So, it has to go through endocytosis.
So, if it goes to endocytosis one of the problem
is that lot of it will go to lysosomes and
get degraded.
So, that is one barrier.
Another barrier is how do you if you want
to prevent that how do you burst throughout
this particular endosome to come out in the
cytoplasm and once it is come is out in the
cytoplasm how does it able to move from the
cytoplasm from it’s location all the way
to the nucleus because again it is a large
molecule since the diffusion is fairly limited.
And then once it reaches the nucleus membrane
how does it go beyond that how does it go
inside that because again the same barrier
is present on the nuclear membrane also.
So, several barriers are present.
Nonetheless it is not as bleak as it comes
seems to be people use it all the time and
at least from the literature reports and from
the experiment it does seem that you can get
gene delivery into a cell despite all these
barriers and that is what we are going to
discuss today.
So, let us understand some of the barriers.
So, let us talk about intracellular barrier
the movement it itself and so again you can
characterize the root mean square speed their
velocity here where you are trying to figure
out how quickly the DNA will move in there.
So, it will depend on the diffusion co-efficient
of the DNA in that environment and again you
see that there is quite a lot of variations
you can see the MSDs can vary quite a lot
depending on the different condition used.
So, if it is slow it is like sub diffusive
whereas, if the slope is higher then you are
talking about quite a bit indicates an active
transport.
So, if you have somewhat an active transport
you see that the MSD Mean Square Displacement
is almost.
So, let us let us say let us take at the time
1 second, if somehow we can get an active
transport.
So, we are looking at 10 to the power minus
1 micron square here and 10 to the power minus
4 micron square here.
So, the diffusion has actually increased the
movement is actually increased by 3 orders
of magnitude which is a huge improvement in
translocation.
So, some how you can get the active transport
to work you have much better chance of getting
your cargo into the nucleus.
So, that is what these authors have tried.
What they have done is they have designed
a plasmid that has a binding site as a nuclear
localization signal.
As what this is, this NLS binds to a protein
that can signal to the cell.
So, let us say if a protein gets bound to
this when bound to this DNA will signal the
cell to move it to the nucleus.
So, now, you are actually relying on active
transport once you do that.
So, you put this nuclear localization signal
and then the active transport will help it
at least overcome one of the barriers that
is movement first of all into the site through
the cytoplasm.
So, let us say this is a big cell, here is
a nucleus, here is your DNA.
So, all the way from here to here this analyst
first of all will take care of this movement
as well as movement across the nucleus membrane.
So, that is what it is being shown here.
So, if you have different conditions if you
only have the plasmid, you do not really see
quite a lot of production of the proteins.
In this case the protein that is being used
is in this case the protein that is being
used is something that is a luciferase gene.
So, it can be easily measured using light
production and this is on log scale.
So, you see you can change the type of plasmid
here you using the conformation on the plasmid
you do not really get quite a lot of enhancement,
but the moment you put an NLS you actually
see quite a big jump in the localization or
actually the production as this is more a
function assay in the production of the protein
through this.
So, that is one ok.
So, that is good we can overcome one of the
barrier what about the rest of the barrier
what about going into the cell what about
coming out from the endosome and what about
the extracellular barriers the proteases the
nucleases present in the environment how do
you target a particular organ how you target
a particular cell.
So, for that we need a carrier.
So, it is fairly clear that because of so
many barriers if you just inject the DNA by
itself it is probably not going to be able
to reach in sufficient amount to your target
site.
Now that we establish that yes we need a carrier
we have 2 choices; one as we go to the nature
itself and use a virus based carrier and this
is nothing, but a virus that is already adapted
it itself to infect human beings.
So, it has overcome all those challenges if
we talked about.
So, it has a shell of protein shell which
protects it from any extra cellular barrier
it is a different size now.
So, it can translocate it has receptors to
target a particular type of cell in organ
and that can be then used to deliver your
DNA.
So, you can take this virus remove the viral
genome put your gene of interest into the
virus and then let the virus go and deliver
your gene of interest to your target cell
and this is actually very efficient 
in the whole reason is that they are well
adapted to do this.
So, this technology that is being made by
evolution is very efficient, but the problem
is of course, that and we will discuss this
in more detail, but obviously, let us say
if you are not able to remove all the viral
genome you are talking about causing now a
new disease, the other option is to use polymers
and lipids.
So, what we are discussing throughout the
course are non-viral vectors.
So, some kind of material 
and mimic some of these viral strategies either
their size put some targeting ligands on to
this change their charge on the basis of that
get different size particles and then package
these particles with the DNA and then deliver
it.
So, these are two major sort of carriers that
we can use for this application.
So, let us look at the state of the current
science with both the viral and non-viral
vectors.
So obviously, viral vectors as I said has
lots of pros it is a very high transfection
efficiency.
So, you are talking about pretty much more
than 50 percent efficiencies almost more than
half of your cargo will get transfected and
result in functional protein being produced.
So, it has natural tropism which means that
there are viruses existing that infect only
a particular type of cells and then there
are viruses existing that infects many cells
and then also being selective in the type
of cells that they are infecting.
So, that way you can choose what virus infects
your target cell and use that.
So, you do not really have to do much work
you know the nature has already produced all
this to help you do it then these viruses
have actually evolved mechanisms for endosomal
escape.
So, they are very well versed in how to rush
out from the endosome and go back into the
cytoplasm because eventually these viruses
also want to inject their DNA and want the
DNA or the genetic material to go in the nucleus,
so, they can do that.
In fact, one of the strategies that we talked
about the proton sponge effect was something
that we learnt from these viruses.
So, in this case they are coat protein was
such that was acting as a proton sponge and
then they also have mechanisms for transportation
of the DNA into the nucleus.
So, again they are well adapted in overcoming
all these challenges and that is why there
is one of the most ideal systems when you
consider their efficiency in gene transfection;
however, there are some cons and one con again
being that now that these viruses are not
something that is a part of your own body.
So, now, you have introduced a foreign material.
So, your immune system will start acting against
it.
So, you get a very strong immune response
against these viral proteins and that means,
that let us say if you have done it once and
you wanted to do it again and by the time
you are ready to do it again the body is already
aware of how to handle that particular virus
because you essentially vaccinated the body
by giving the first dose and the second time
we do it the body’s going to clear it even
before it can do any of it is function.
So, prohibits multiple administration.
Now the problem is that these viruses even
though they.
So, one of the thing that these virus will
do is remember this chromosome that we have
discussed and let us say this is the gene
that we want to put, but these viruses again
are not very specific in where this gene is
going to go ahead and insert itself.
So, it could happen that this gene will insert
on this chromosome it could happen that this
gene may go and insert in a neighboring chromosome
let us say here.
So, on the basis of where it is in where it
is getting integrated this may lead to several
consequences first of all the expression may
change, the second problem is what if now
gene you have a promoter in a gene as well.
So, that it can express what it is carrying.
What if downstream you have some oncogene.
So, now, along with your own protein you are
also making the cell cancerous.
So, that is a big problem actually there was
a lot of enthusiasm for these viral vectors
and they went even in human clinical trials,
but in one of those trials what they found
out is more than 50 percent of the patient
actually developing cancers and the patient
actually died because of that.
So, that decreased the enthusiasm when using
this evaluator of course, this was quite a
long time ago and technology has since improved
quite a lot, but there is still a concern
as to something like this could happen and
lead to more serious disease then what are
you trying to cure.
It is a fairly complicated synthesis process
it is not trivial to take a virus and then
load it with your own DNA and let it go and
do whatever you want it to do.
It also is a limitation on the gene size that
you can put.
So, this virus coat protein that you may have
it may only be of a certain size and there
is a limit.
So, let us say the virus genome is about 10
kilo base pair then you can’t really load
more than 20 or so I mean it may have some
stretch ability.
So, you can add maybe few kilo base pairs,
but eventually you cannot tell you put 100
kilo base pair in there.
It is just too big for this to be loaded into
that space that is given in the viral shell.
So, if you if your gene is big you have to
look for some other way.
And again there is toxicity associated with
this because the immune system there is also
chance that you may contaminate your thing
with the live virus and if you do that you
again cause a different disease and then what
you are trying to cure and that is never good.
So, there are few cons that exist with these
viral based vectors and so that is why the
field has now also tried to look into other
alternatives to what these viral vectors offer.
And so, once again as we discussed are the
non-viral vectors.
These are these polymers and lipid-based vectors.
And one of the major advantages is unlike
viruses they have fairly low immunogenicity
we have seen this throughout this course that
these polymers and lipids, there are so large
libraries of it that you can choose fairly
low immunogenic polymers and lipid.
They would not typically develop an immune
response against these proteins or these polymers
and lipids, there is a big library.
So, you can use non-toxic biomaterial, they
are fairly easy to synthesize.
So, we know how to synthesize this very well
and there is all kind of chemistry and you
can do a very easy mass production.
So, and that is a big plus because if you
want to let us say use it in clinics and you
expect to get 1,000 patients walking in every
day in a clinical setting, you want quite
a lot of this polymer and you can get that
you can potentially target that.
So, you can once you form the particle you
can conjugate it with targeting ligands 
and that would be enough to help with some
targeting.
There is no limit to the plasmid size, you
can get as much plasmid size as you want.
So, if you want 10 kilo base pair that is
feasible you want 100 kilo base pair that
is feasible.
So, large genes are easy to take it through
unlike the viral vectors that you are saying
there is a limitation to the gene size and
they do not really integrate
So, in this case if let us say this is the
nucleus and these are your chromosome.
So, the viral vectors would not integrate
they will just reside separately and will
produce gene from the separate plasmid.
So, you do not have to worry about onco genes
being activated and other things like that.
So, they can be administered fairly without
concern of causing cancer and other diseases.
However, they like the viral vector there
are some cons associated with these systems
as well and let us look at what some of these
cons are.
So, the major the biggest con is it is a fairly
low transfection efficacy.
So, now, here you are talking about more than
50 percent of the cargo transfecting here
the efficiency is actually very low.
We have been trying to improve that, but we
have picked as good as the nature is in increasing
this efficiency.
So, this could be as low as 1 percent actually
it just depends on what you are using and
that means, that almost 99 percent or more
of your drug is getting inactivated or is
wasted.
Most of the vehicles that we use are fairly
toxic at high doses and the reason we need
high doses is goes back to the transfection
efficiencies now that because we have such
a low transmission efficiency we want to deliver
more of it.
So, that at least even if 1 person goes we
have enough dose, but then at those high doses
you can have toxicity associated with these
vesicles and these non-viral vectors and then
there is no natural tropism.
So, you have to worry about how do we target
a particular cell, how do you target different
cells endosomal escapes become a challenge.
So, we said we adopted some of the proton
sponge effect, but obviously, we have not
come to the technology which is as good as
the viruses in proton sponge and then also
the nuclear transport mechanism became an
issue because earlier we were talking about
DNA molecule having problems diffusing in
the cytoplasm.
Now, we are saying that this DNA molecule
is in a large particle.
So, it is even larger now how this is going
to diffuse in the cytoplasm is another bigger
challenge.
So, all of these combined result in quite
a lot of pros and cons for each of these strategies
and depending on what is it that is your application
and concern you choose one or the other; obviously,
this is a drug (Refer time: 21:40) course
and we have been talking about polymers.
So, we are going to focus on non-viral vectors.
The viral vectors for gene delivery are still
being used in research and they have gone
to clinics in the past as well.
So, this is another field, but we are not
going to talk about the viral vectors much
we will focus on the non-viral vectors.
Okay so, gene delivery where and how?
So, let us look at microscopically what is
happening.
So, you go to a clinic the doctor injects
you something.
So, these could be injections these could
be in nasal spray orally you take something
they put it under the skin muscle whatever
then if you zoom into it as to what is happening
there you have some particles.
You use either some targeting or these particles
inherently go into the cell.
They go into the endosomes and eventually
if you have used proton sponge effect or something
else, they burst this endosome and come out
into the cytoplasm.
Once they come out in the cytoplasm typically
the best strategy is to let your DNA come
out because this particle as I said in the
previous slide is too big.
Now we are talking about 100 nanometer earlier
the DNA was maybe 1 to 10 nanometer, now you
are talking about 100 nanometer big particle
moving in the cytoplasm that is going to be
a big challenge.
So, most vectors you can design in such a
way that once they come out from the endosome
the DNA can release and we will talk about
how this release can happen, but we talk we
expect the carriers to release in cytoplasm.
And then either using NLS or some other strategy
we hope that this DNA will translocate to
the nuclear membrane and go into the nuclear
membrane.
And once it goes there we hope that this is
still functional and start secreting the protein.
So, this is the whole overview of gene delivery
in a nutshell; obviously, it has some advantages
you are not actually now delivering cell.
So, this is your own cell and so immune system
is not going to act on that which is great.
So, this is autologous order it is not really
an implant.
So, you are in this case now your drug is
gene instead of being cell.
So, we want to engineer some novel carriers
that will facilitate this process and make
it better.
So, we then go to polymers as we have been
doing throughout this course.
So, mostly cationic polymers are used and
why cationic is because DNA itself is highly
negatively charged.
So, as I said the DNA is composed of nucleotides
which are composed of phosphate and this is
several negative charge is present on each
of those nucleotides and so you can imagine
100 kilo base pair DNA will have quite a lot
of negative charge on it’s surface or on
in the structure and so you can use cationic
polymers and that way these DNA molecule interact
quite strongly ionically with your cationic
polymer.
So, these end result in formation of complexes.
So, here is one DNA then you let us say you
take up a polymer which is positively charged
and you mix them in the right ratio or you
vary the ratio and at some ratio what will
happen is they will start precipitating out
and form these complexes or create packets
or particles.
And depending on the ratio you can change
the size and all and then further you can
modify these polymers with various ligands
to target a cell receptor or to target some
other function.
Then these particles are big enough that they
can be taken up through endocytosis and once
they are taken up through endocytosis you
rely on their escape from the endosome to
and then eventually release of this DNA.
So, how does the release work?
So, outside the cell, so, let us say this
is the cell you have cytoplasm and you have
extra cellular.
So, the extracellular environment is typically
low ionic concentration is present, but in
the cytoplasm you have high ionic concentration.
So, what that does mean what does that mean?
That means, that since there is high ionic
concentration the dielectric of this particular
cytoplasm is high 
and because of that this ion-ion based interaction.
According to Coulomb’s law, dielectric is
in the denominator since it is high it is
going to decrease and so; that means, that
the bond is not going to be as strong and
this can separate out.
So, that is how when this particles will come
out in the cytoplasm from the after the endosomal
escape they will cause a release of the DNA
and then the DNA can go in to the nucleus
and transfect and start producing what you
wanted.
So, this is the whole concept of using polymers.
There are a few polymers that are particularly
good at this and some of these are poly lysine,
poly ethyleneimine, chitosan some polyamidoamine,
dendrimers.
So, here are some examples that I am giving
you.
You can use other polymers also it does not
have to be cationic polymer, but it is just
seen that cationic polymers are much better
at this than any other type of the polymers.
And so, you can have a big polymer such as
let us say PLGA, put your gene inside 
and when the PLGA degrades this thing can
come out, but you then run into the problem
that they do not really have good encapsulation
efficiency, not a whole lot of DNA is going
inside your particles and then the release
is also just relying on degradation rather
than triggering when it’s out in the cytoplasm.
So, that is one issue but then you will still
use it.
These can slowly release this gene once they
going to the body, you can make various sizes
as we discussed already with the PLGA particle.
You can make matrices, you can make gels and
some of the polymers that are used is PLGA,
PVP etc. for all these applications.
We will stop here and we will continue rest
in the next class.
Thank you.
