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MODULE 8- LECTURE 1

GENE THERAPY: INTRODUCTION AND METHODS

8-1.1 Introduction

Gene therapy is a novel treatment method which utilizes genes or short oligonucleotide
sequences as therapeutic molecules, instead of conventional drug compounds. This
technique is widely used to treat those defective genes which contribute to disease
development. Gene therapy involves the introduction of one or more foreign genes into
an organism to treat hereditary or acquired genetic defects. In gene therapy, DNA
encoding a therapeutic protein is packaged within a "vector", which transports the DNA
inside cells within the body. The disease is treated with minimal toxicity, by the
expression of the inserted DNA by the cell machinery. In 1990 FDA for the first time
approved a gene therapy experiment on ADA-SCID in the United States after the
treatment of Ashanti DeSilva. After that, approximately 1700 clinical trials on patients
have been performed with various techniques and genes for numerous diseases. Many
diseases such as ADA-SCID, X-linked SCID, Leber's congenital amaurosis (a retinal
disease), Parkinson's disease, multiple myeloma, chronic and acute lymphocytic
leukemia, adrenoleukodystrophy have reported of successful clinical trials. But these are
still not approved by FDA. Some other diseases on which gene therapy based research is
going on are Haemophilia, Tyrosinemia, Hyperbilirubinemia (Criglar-Nijjar Syndrom),
Cystic Fibrosis and many other cancers. After 30 years of research and clinical trials,
only one product called Glybera got approval in November 2012 which may be available
in market in late 2013. It has the ability to cure lipoprotein lipase deficiency (LPLD) a
very rare disease.

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8-1.2 Types of gene therapy

There are several approaches for correcting faulty genes; the most common being the
insertion of a normal gene into a specific location within the genome to replace a non
functional gene. Gene therapy is classified into the following two types:

1. Somatic gene therapy

2. Germ line gene therapy

8-1.2 .1 Somatic Gene Therapy

In somatic gene therapy, the somatic cells of a patient are targeted for foreign gene
transfer. In this case the effects caused by the foreign gene is restricted to the individual
patient only, and not inherited by the patient's offspring or later generations.

8-1.2 .1 Germ Line Gene Therapy

Here, the functional genes, which are to be integrated into the genomes, are inserted in
the germ cells, i.e., sperm or eggs. Targeting of germ cells makes the therapy heritable.

8-1.3 Gene Therapy Strategies

8-1.3.1 Gene Augmentation Therapy (GAT)

In GAT, simple addition of functional alleles is used to treat inherited disorders caused
by genetic deficiency of a gene product, e.g. GAT has been applied to autosomal
recessive disorders. Dominantly inherited disorders are much less amenable to GAT.
Figure 8-1.3.1 shows the GAT strategy

Figure 8-1.3.1: A gene therapy vector has been designed to treat the diseased cells with a gene X. This vector was introduced
inside the diseased cells by various gene transfer methods. After a successful homologous recombination the treated cells will
show the presence of gene X product as well as normal phenotype.

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8-1.3.2 Targeted Killing of Specific Cells

It involves utilizing genes encoding toxic compounds (suicide genes), or prodrugs
(reagents which confer sensitivity to subsequent treatment with a drug) to kill the
transfected/ transformed cells. This general approach is popular in cancer gene therapies.
This is shown in figure 8-1.3.2a & 8-1.3.2b

Figure 8-1.3.2: a) Direct killing of diseased cells by two methods. The first method is the introduction of toxin gene into the
diseased cell which when expresses toxin protein the cells die. The second method involves incorporation of a certain gene (e.g.
TK) in the gene therapy vector which shows a suicidal property on introducing certain drug (e.g. ganciclovir).

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Thymidine kinase (TK) phosphorylates the introduced prodrug ganciclovir which is

further phosphorylated by endokinases to form ganciclovir triphosphate, an competitive
inhibitor of deoxyguanosine triphosphate. Ganciclovir triphosphate causes chain
termination when incorporated into DNA.

Figure 8-1.3.2: b) Assisted killing is another strategy of killing diseased cells. Here one method is to insert a well known foreign
antigen coding gene which induces immune cells for the killing of the diseased cells. Few more methods are based on immune
cells activation in which a certain cytokine encoding gene incorporated into gene therapy vector and inserted into either
diseased cells or non-diseased cells. This will lead to enhanced immune response followed by killing of diseased cells.

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8-1.3.3 Targeted Inhibition of Gene Expression

This is to block the expression of any diseased gene or a new gene expressing a protein
which is harmful for a cell. This is particularly suitable for treating infectious diseases
and some cancers.

Figure 8-1.3.3 To inhibit the target gene expression in diseased cell the antisense mRNA coding gene inserted vector or
triplex-forming oligonucleotides (TFO) or antisense oligonucleotide (ODN) can be introduced which will inhibit the gene
expression either by forming DNA:RNA triplex inside the nucleus or forming RNA:RNA duplex by forming complementary
mRNA strand of disease protein coding mRNA. This may lead to blocking of disease causing protein expression.

8-1.3.4 Targeted Gene Mutation Correction

It is used to correct a defective gene to restore its function which can be done at genetic
level by homologous recombination or at mRNA level by using therapeutic ribozymes or
therapeutic RNA editing.

Figure 8-1.3.5 This is used for disease caused by mutation. The corrected gene will be swapped by the mutant gene X (m).
Then diseased cells will become normal after the correction of mutation by gene therapy.

(Source:
http://www.ncbi.nlm.nih.gov/books/NBK7569/figure/A2871/?report=objectonly)

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8-1.4 Gene Therapy Approaches

8-1.4 .1 Classical Gene Therapy

It involves therapeutic gene delivery and their optimum expression once inside the target
cell. The foreign genes carry out following functions.

Produce a product (protein) that the patient lacks;

Produces toxin so that diseased cell is killed.

Activate cells of the immune system so as to help in killing of diseased cells.

8-1.4 .2 Non-classical gene therapy

It involves the inhibition of expression of genes associated with the pathogenesis, or to
correct a genetic defect and restore the normal gene expression.

8-1.5 Methods of gene therapy

There are mainly two approaches for the transfer of genes in gene therapy:

1. Transfer of genes into patient cells outside the body (ex vivo gene therapy)

2. Transfer of genes directly to cells inside the body (in vivo).

Figure 8-1.5 Gene Therapy using autologous cells: Cells are used, i.e. cells are removed from the patient, cultured in vitro, before
being returned to the patient’s body. In this figure in vivo and ex vivo gene therapy is diagrammatically explained.

(Source:
http://www.ncbi.nlm.nih.gov/books/NBK7569/figure/A2897/?report=objectonly)

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8-1.5.1 Ex vivo gene therapy

In this mode of gene therapy genes are transferred to the cells grown in culture,
transformed cells are selected, multiplied and then introduced into the patient.
The use of autologous cells avoids immune system rejection of the introduced cells.
The cells are sourced initially from the patient to be treated and grown in culture before
being reintroduced into the same individual.
This approach can be applied to the tissues like hematopoietic cells and skin cells which
can be removed from the body, genetically corrected outside the body and reintroduced
into the patient body where they become engrafted and survive for a long period of time.
Figure 8-1.5.1 shows a self explanatory schematic diagram for ex vivo gene transfer.

Figure 8-1.5.1 Ex vivo therapy involves tightly regulated cellular manipulation in harvested cells

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8-1.5.2 In Vivo Gene Therapy

In vivo method of gene transfer involves the transfer of cloned genes directly into the
tissues of the patient.

This is done in case of tissues whose individual cells cannot be cultured in vitro in
sufficient numbers (like brain cells) and/or where re-implantation of the cultured cells in
the patient is not efficient.

Liposomes and certain viral vectors are employed for this purpose because of lack of any
other mode of selection.

In case of viral vectors such type of cultured cells were often used which have been
infected with the recombinant retrovirus in vitro to produce modified viral vectors
regularly. These cultured cells will be called as vector-producing cells (VPCs)). The
VPCs transfer the gene to surrounding disease cells.

The efficiency of gene transfer and expression determines the success of this approach,
because of the lack of any way for selection and amplification of cells which take up and
express the foreign gene.

Figure 8-1.5.2 shows various steps of in vivo gene transfer.

Figure 8-1.5.2 various steps of in vivo gene transfer

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Table 8-1.5.1: Differences between In Vivo and Ex Vivo gene therapy

Difference Between in vivo and ex vivo Gene Delivery Systems

In vivo Ex vivo

Less invasive More invasive

Technically simple Technically complex

Vectors introduced directly No vectors introduced directly

Safety check not possible Safety check possible

Decreased control over target cells Close control possible

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8-1.6 Target sites for Gene Therapy

Therapeutic genes have to be delivered to specific target sites for a specific type of
disease. This table describes the list of such disease and their target sites for gene therapy.

Table 8-1.6.1: Target cells for gene transfer

Target cells for gene transfer

Disease Target Cells

Cancer Tumor cells, antigen presenting

cells (APCs), blood progenitor
cells, T cells, fibroblasts, muscle
cells

Inherited monogenic disease Lung epithelial cells, macrophages,

T cells, blood progenitor cells,
hepatocytes, muscle cells

Infectious disease T cells, blood progenitor cells,

antigen presenting cells (APCs),
muscle cells

Cardiovascular disease Endothelial cells, muscle cells

Rheumatoid arthiritis Sinovial lining cells

Cubital tunnel Syndrome Nerve cells

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8-1.7 Vectors for gene therapy

Vectors for gene therapy can be classified into two types:

1. Viral vectors

2. Non-viral

Note: Table 2 shows vectors used in gene therapy. It is adapted From AR Prabhakar in
Gene Therapy and its Implications in Dentistry. International Journal of Clinical Pediatric
Dentistry, 2011; 4(2):85-92

Table 8-1.7: Vectors used in gene therapy

Vectors used in gene therapy

Viral Vector Non-viral Vectors

Adenovirus Lipid complex

Retrovirus Liposomes

Adeno- Associated Peptide/protien

Virus

Lentivirus Polymers

Vaccinia virus

Herpes simplex virus

Direct gene transfer methods like mechanical, electroporation, gene

gun are also ued to transfer genes into target cells.

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8-1.7.1 Viral vectors

Retroviruses, adenoviruses and adeno-associated viruses (AAV) some commonly used

viral vectors whereas some less commonly used viral vectors are derived from the Herpes
simplex virus (HSV-1), the baculovirus etc.

Adenoviral vectors

Adenoviruses are large linear double-stranded DNA viruses that are commonly used for
preparing gene transfer vectors. Adenovirus vectors are known to be the second most
popular gene delivery vector for gene therapy of various diseases like cystic fibrosis and
certain types of cancer. Figure 8-1.7.1.1 shows how the adenoviruses enter cells by
receptor-mediated endocytosis. A primary cellular receptor binds to viral fiber then the
virus interacts with secondary receptors which are responsible for its internalization.
Coxsackie and Adenovirus Receptor (CAR), Heparan sulphate glycosaminoglycans,
sialic acid, CD46, CD80, CD86, alpha domain of MHC I are the primary receptors which
are specific for specific strains of adenovirus. Integrins are the secondary receptors which
helps in the internalization of viral particles. Some adenovirus directly interacts with
integrins like in the case of fiber deficient Ad2 virions.

Figure 8-1.7.1.1a
Adapted and modified from: http://www.ncbi.nlm.nih.gov/books/NBK7569/figure/A2918/?report=objectonly

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The adenoviral DNA has inverted terminal repeats (ITRs) and a terminal protein
(TP) is attached covalently to 5’ termini. The adenoviral genome is classified as early and
late regions based on the proteins they express. Proteins encoded by early region (E1, E2,
E3, E4) genes are involved in viral DNA replication, cell cycle modulation and defense
system. The late region genes (L1, L2, L3, L4, L5) encodes the viral structural proteins.
Three classes of adenoviral vectors namely first, second and third generation viral vectors
are developed for gene therapy purpose.

Figure 8-1.7.1.1b Map of Adenoviral genome and construction of different types of adenoviral vectors

Adapted and Modified from: R Alba, A Bosch and M Chillon (2005). Gutless adenovirus: last-generation adenovirus for gene
therapy. Gene Therapy, 12, S18-S27

First generation adenoviral Vectors

These vectors are constructed by replacing the E1/E3 expression cassette and
inserting our candidate gene of 3-4kb size. E1 encodes proteins responsible for
expressions of other viral genes required for viral growth. So cell lines that can
provide E1 proteins in trans are required for the replication of the E1 deleted viral
vectors.

Advantages:

• They are human viruses produced at very high titers in culture.

• They can infect a wide range of human cell types including non- dividing cells.

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• They enter into cells by receptor mediated endocytosis with a very high
transduction efficiency reaching upto 100% in vitro

• Their large size enables them to accept large inserts.

Disadvantages:

• Expression of foreign gene is for short period of time as they do not integrate into
the chromosome.

• These vectors may generate immune response causing chronic inflammation.

Second generation adenoviral Vectors

These vectors have been developed to overcome these difficulties. Here of E1/E2
or E3/E4 expression cassettes are called deleted and replaced. The E1/E2 or
E3/E4 proteins are required for viral DNA replication. Similar to first generation
viral vector, cell lines which can complement both E1and E2 or E3 and E4 are
needed. It can carry DNA insert upto 10.5kb

Advantages:

It has improved safety and increased transgene expression.

Disadvantages:

These viral vectors are associated with immunological problems.

Construction of these vectors is difficult.

Third generation adenoviral Vectors

These vectors are otherwise called as gutless adenovirus. These are also known
as helper dependent adenovirus as they lack all the coding sequences and require
helper virus which carries all the coding sequences. Helper virus for example
AAV, or artificially disabled viruses provide the viral functions needed for
successful infection like viral DNA replication, viral assembly and infection of
new cells etc. The size of insert DNA can be 36kb and hence called as high

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capacity adenoviruses. They carry only 5’ inverted terminal repeats (ITR) and 3’
packaging signal

Advantages:
These are non-integrative and high-capacity vectors.
It can be produced in high titer and the construction of these vector is easy.
It shows longer stability and reduced immune response.
Disadvantages:
Helper virus contamination contamination can cause diseases like conjunctivitis,
pharyngitis, cold and respiratory disease.
Adeno- Associated Virus (AAV)

Adeno-associated viruses (AAVs) are a group of small, single-stranded DNA viruses

which cannot usually undergo productive infection without co-infection by a helper virus,
such as an adenovirus.

• The insert size for AAV is 4.5 kb, with the advantage of long-term gene
expression as they integrate into chromosomal DNA.
• AAVs are highly safe as the recombinant adeno associated vectors contains only
gene of interest and 96% viral genes are deleted.
Adeno-associated viruses are explained in detail in Module 5-Lecture 1.
Retroviral Vectors

Retroviruses are RNA viruses which possess a reverse transcriptase activity, enabling
them to synthesize a complementary DNA. Following infection (transduction),
retroviruses deliver a nucleoprotein complex (pre-integration complex) into the
cytoplasm of infected cells. The viral RNA genome is reverse transcribed first and then
integrates into a single site of the chromosome.

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Figure 8-1.6.1.1c
Adaptedand modified from: http://www.ncbi.nlm.nih.gov/books/NBK7570/figure/A2357/?report=objectonly

Tumor retroviruses, example Moloney's murine leukemia virus (MoMuLV), is

widely used for the generation of recombinant vectors. these are produced at low
titers as all the viral genes are deleted.
Oncoretroviruses: The cells that divide shortly after infection can only be
transduced by oncoretrovirus. The preinitiation complex is excluded and their
entry is restricted in to the nucleus as they can only enter when nuclear
membrane dissolves during cell division the target cells for this viral vector is
limited

Recombinant lentiviruses are being developed that are non- pathogenic to

humans and have the ability to transduce stationary cells.

Other Viral Vectors:

These include herpes simplex virus vectors and baculovirus.

Herpes simplex virus vectors: Herpes simplex virus-1 (HSV-1) is a 150 kb double-
stranded DNA virus with a broad host range that can infect both dividing and non-
dividing cells. the insert size is comparatively larger (>20kb) but have a disadvantage of
short-term expression due to its inability to integrate into the host chromosome

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Baculovirus: They can take up very large genes and express them highly efficiently.
They help in recombinant protein expression in insect cell. They can infect hepatocytes as
an only mammalian cell type and the gene expresses under the control of either
mammalian or viral promoter.

Simian Virus 40 Vectors (SV40): SV40 are icosahedral papovavirus with a circular
double stranded DNA of 5.2kb size as genetic material. The genome encodes for early
proteins viz; large T antigen (Tag) and small t antigen (tag) and late protein viz; a
regulatory protein agnoprotein and three structural proteins (VP1, VP2, VP3). The Tag
gene is removed as it is responsible for inducing immunogenicity in the recombinant
SV40 vector. All the structural proteins except the major capsid protein VP1 is removed
resulting in a genome of 0.5kb size which includes origin of replication (ori) and
encapsidation sequence. Recombinant SV40 vectors allows expression of transduced
gene

8-1.7.2 Non- viral vectors

It involves chemical and physical methods such as direct injection of naked plasmid
DNA (particle bombardment), receptor-mediated endocytosis and gene transfer through
liposomes, polymers, nano particles etc.

Some non viral methods

Direct injection/particle bombardment:

DNA can be injected parenterally which can be considered for Duchenne muscular
dystrophy (DMD). An alternative approach uses particle bombardment (‘gene gun’)
technique, in which DNA is coated on to metal micro particles and fired from a ballistic
gun into cells/tissues. This technique is used to transfer the foreign DNA and its transient
expression in mammalian cells in vitro and in vivo as well. It can cross the physical
barriers like skin, muscle layer for which it is used for vaccination. Particle bombardment
is used to deliver drugs, fluorescent dyes, antigenic proteins etc.

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Advantage: Simple and comparatively safe.

Disadvantage:

• Poor efficiency of gene transfer.

• A low level of stable integration of the injected DNA. Repeated injection may
cause damage in the proliferating cells.

Microinjection needle
Host cell containing foreign
DNA

cytoplasm

Holding pipette nucleus

MICROINJECTION

Figure 8-1.7.2.1 Microinjection process

Microinjection involves the delivery of foreign DNA, by the help of glass micropipette
into a living cell. The cell is held against a solid support or holding pipette and micro
neeedle containing the desired DNA is inserted into the cell. The tip of the pipette used is
about 0.5 to 5 micro meter diameter which resembles an injection needle. For this, glass
micropipette is heated until the glass becomes somewhat liquefied and is quickly
stretched to ressemble a injection needle. The delivery of foreign DNA is done under a
powerful microscope (micromanupulator).

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Gene gun

Plastic disc with DNA coated gold

particle

Disc stopped by screen

DNA coated gold
particle

Target plant cell

GENE GUN MEDIATED GENE TRANSFER

Figure 8-1.7.2.2 Gene gun mediated gene transfer

In particle bombardment method, the tungsten or gold particles (micro projectiles) are
coated with the foreign DNA. Micro-projectile bombardment uses high-velocity metal
particles to deliver biologically active DNA into the target cells. The macroprojectile is
coated with the coated particles and is accelerated with air pressure and shot into plant
the target tissue. A perforated plate is used, which allows the micro-projectiles to pass
through to the cells on the other side of the plate and stops the macropojectile. Particle
coated with the foreign gene releases the foreign gene when enters into the target cell and
integrates into the chromosomal DNA. This technique is also used to transfer genes in
mammalian cells. Mammalian cell lines like HEK 293, MCF7 showed gene expression
when transfected with luciferase and green fluorescent genes and their gene expression
was dependent on helium pressure, size and amount of gold particle and DNA load on
each particle. Cell viability depends on helium pressure.
(See lecture 3 of module 5 for advantages and limitations)

Liposomes Mediated

Liposomes are spherical vesicles which are made up of synthetic lipid bilayers which
imitate the structure of biological membranes. DNA to be transferred is packaged into the
liposome in vitro and transferred to to the targeted tissue. The lipid coating helps the
DNA to survives in vivo and enters into the cell by endocytosis. Cationic liposomes,

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where the positive charge on liposomes is stabilized by binding of negatively charged

DNA, are popular vehicles for gene transfer in vivo. For more detail see lecture 2 of
module 5.

Figure 8-1.7.2.4: In vivo liposome mediated gene transfer- (A) formation of lipid bilayer in water (B) Structure of anionic and
cationic liposome (C) Use of liposome to transfer genes into cells.

Adapted and modified from: http://www.ncbi.nlm.nih.gov/books/NBK7569/figure/A2924/?report=objectonly

Advantage:

• The liposomes with the foreign DNA are easy to prepare.

• There is no restriction in the size of DNA that is to be transferred.

Disadvantage:

• Efficiency of gene transfer is low and transient expression of the foreign gene is
obtained as they are not designed to integrate into the chromosomal DNA.

Electroporation

In electroporation, the external electric field is applied to the protoplast, which changes
the electrical conductivity and the permeability of cell membrane; and thus the exogenous
molecules found in the medium are taken up to either the cytoplasm (transient
transfection) or into the nucleus (stable transfection).The efficiency of electroporation
can be increased by giving the cell a heat shock, prior to the application of electric field

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or by using small quantity of PEG while doing electroporation. See lecture 3 of module 5
for detailed explanation.

Advantage:

By electroporation large numbers of cells can be processed at once, and thus the
amount of time spent processing cells can be cut down.

Disadvantages:

If the voltage applied is not calculated properly, the cells may damage.
If electroporation does not occur in controlled environment, the potentially
harmful substances can enter the cell or the impurities from solution may enter.
This is because there is no way to control what enters the cell membrane.

ELECTROPORATION

Before pulse On application After pulse application

of Electric field

Increase in cell membrane

Cell membrane electrical conductivity and Cell healed with
Foreign DNA permeability foreign DNA inside

Introduction of voltage
by electic field

Figure 8-1.7.2.5 Electroporation

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Sleeping Beauty Transposition

This is a non viral method of gene transfer which offer the advantage of stable
DNA integration into the chromosomes of vertebrates. The sleeping beauty
transposition system consists of a sleeping beauty transposon and a sleeping
beauty transposase. The SB transposon consists of two terminal inverted repeats
present each at the ends of the gene of interest and the SB transposase mediates
excision of the SB transposon and its integration into a site of chromosome
having a dinucleotide dimer TA by cut and paste mechanism.

Figure 8-1.7.2.6 Cut and paste mechanism of a DNA transposon by a transposase (txp gene)

Adapted and modified from: http://www.discoverygenomics.net/pdf/SBT_ProlongedExpressions.pdf

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For gene therapy transposase gene is replaced by our gene of interest and transposase is
provided in trans as shown in Figure8-1.7.2.7.

Figure8-1.7.2.7 Mechanism of gene transfer by sleeping beauty transposition

Adapted and modified from: http://www.discoverygenomics.net/pdf/SBT_ProlongedExpressions.pdf

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RNA-DNA Chimera

This RNA- DNA chimera also known as chimeroplast is used to correct point
mutations by mismatch repair. This 68 nucleotides long double stranded nucleic
acid molecule comprises of one strand DNA and another strand consisting of two
10 nucleotides long 2’-O methyl RNA stretch separated by a 5 nucleotide long
DNA stretch as shown in Figure 8-1.7.2.8. The pentameric chimeric DNA carries
the mismatch and the other DNA strand has its complementary bases.
Additionally this RNA-DNA chimera consists of two hairpin loop and a GC
clamp.

Figure 8-1.7.2.8: Structure and mechanism of action of RNA DNA chimera

Source: http://www.sciencedirect.com/science/article/pii/S0925443902000686

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Receptor-Mediated Endocytosis

It can be both viral and non-viral mediated gene transfer. Viral vectors attach to the
surface receptors through viral surface components and internalized. In non viral mode of
receptor mediated endocytosis DNA is first coupled to a ligand that binds specifically to a
cell surface receptor and causes transfer of the DNA into cells by endocytosis. Coupling
is done by linking the receptor molecule with polylysine followed by reversible binding
of the negatively charged DNA to the positively charged polylysine component.
Transferrin receptor which is comparatively abundant in proliferating cells and
hematopoietic cells is utilized as a target and transferin as a ligand in this approach.

Advantage: Gene transfer efficiency may be high.

Disadvantage:

• It does not allow integration of the transferred genes. Also, the protein— DNA
complexes are not stable in serum.

Coupling of inactivated adenovirus to the DNA-transferin complex can increase gene

transfer efficiency which help in receptor mediated endocytosis and lysosomal escape.

Figure 8-1.7.2.3 Receptor-Mediated Endocytosis

Adapted and modified from: http://www.ncbi.nlm.nih.gov/books/NBK7569/figure/A2927/?report=objectonly

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8-1.8 Endosomal/Lysosomal Escape

In gene therapy the gene becomes entrapped in the endocytic pathway and is degraded by
the hydrolytic enzymes which are a major limiting step in gene therapy. There are various
approaches to escape this endocytic pathway for both viral and non-viral gene delivery
systems.

For viral gene delivery system

Enveloped virus can penetrate through endosome membrane and non enveloped
viruses escape by forming pores or lyse the vesicular membrane.

For non-viral gene delivery system

There are various strategies developed non-viral vector system for endosomal escape.

o The ‘proton sponge’ hypothesis: ‘Proton sponge’ effect has been seen in
some cationic polymers which contain protonable amine groups with pKa
close to endosomal/lysosomal pH can be used for gene delivery During
acidification of endosomes the cationic polymers become protonated so
that the H+ influx is increased and thereby Cl- concentration is increased
inside the endosome leading to water influx. As a result the endosome
swells and eventually ruptures.

o Flip-Flop mechanism: Due to electrostatic interaction of cationic

lipoplexes and anionic lipids of endosomes facing towards cytoplasm
there is lateral diffusion of lipids resulting in displacement of nucleic acids
from lipoplexes to cytoplasm.

o Pore formation: In a peptide based gene delivery system a peptide named

GALA which can undergo conformation change at low pH forms a pore in
the vesicular membrane when incorporated.

o Photochemical internalizaton: This technique uses photosensitizers

which can bind to and localize in the plasma membrane. These
photosensitizers become confined to endosomal membrane during

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endocytosis and remain inactive. When irradiated with light of a particular

wavelength they produce reactive oxygen species resulting in lysis of
endosomes.

8-1.9 Advantages of Gene Therapy

Gene therapy can cure genetic diseases by addition of gene or by removal of gene
or by replacing a mutated gene with corrected gene.

Gene therapy can be used for cancer treatment to kill the cancerous cells.

Gene expression can be controlled.

Therapeutic protein is continuously produced inside the body which also reduces
the cost of treatment in long term.

Bibliography

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