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Module 1: Gene Delivery e Altre Varianti

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Video:

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.
(Refer Slide Time: 00:46)

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.
(Refer Slide Time: 02:37)

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.
(Refer Slide Time: 04:53)

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.
(Refer Slide Time: 06:46)

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.
(Refer Slide Time: 08:49)

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.

(Refer Slide Time: 11:26)

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.
(Refer Slide Time: 17:08)

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.
(Refer Slide Time: 19:34)

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.

(Refer Slide Time: 22:01)

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.
(Refer Slide Time: 24:22)

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.
(Refer Slide Time: 27:43)

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.