PHRM 413: Pharmacology III 1

Gene Therapy

GENE THERAPY
WHAT IS GENE THERAPY?

James Watson was quoted as saying “we used to think that our fate was in our stars, but now
we know, in large measures, our fate is in our genes”. Genes, the functional unit of heredity are
specific sequences bases that encode instructions to make proteins. Although genes get a lot of
attentions, it is the proteins that perform most life functions. When genes are altered, encoded
proteins are unable to carry out their normal functions, resulting in genetic disorders. Gene
therapy (use of genes as medicines) is basically to correct defective genes responsible for
genetic disorder by one of the following approaches:-

 A normal gene could be inserted into a nonspecific location within the genome to replace
the nonfunctional gene (most common)
 An abnormal gene could be swapped for a normal gene homologous recombination
 An abnormal gene could be repaired through selective reverse mutation
 Regulation (degree to which a gene is turned on or off) of a particular gene could be
altered (enhance the effect of a normally functioning gene, activate the gene that was
shut down during fetal life, deactivates a gene that isn’t functioning properly)

For instance, cancer cells may receive gene therapy to deliver genes that speed the destruction
of cancer cells, supply genes that cause cancer cells to revert back to normal cells, provide
genes that promote or impede the growth of new tissue etc.

Scope of gene therapy is broad with potential in treatment of diseases caused by single gene
recessive disorders (like cystic fibrosis, hemophilia, muscular dystrophy, sickle cell anemia etc),
acquired genetic diseases such as cancer and certain viral infections like AIDS.

Approach

The process of gene therapy remains complex and many techniques need further
developments. The challenge of developing successful gene therapy for any specific condition
is considerable. The challenges are:

 condition in question must be well understood, i.e. diseases and their strict genetic link
need to be understood thoroughly,
 the undying faulty gene must be identified and a working copy of the gene involved must
be available,
 specific cells in the body requiring treatment must be identified and are accessible,
 a means of efficiently delivering working copies of the gene to the cells must be
available.

STEPS IN GENE THERAPY:

1. Identification of the defective gene that is responsible for the disease

2. Cloning of normal healthy gene by recombinant DNA technology
3. Identification of target cell / tissue / organ for gene to be transferred.
4. Insertion of the normal functional gene into the host DNA.

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 2
Gene Therapy

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:

Somatic cell gene therapy Germ line gene therapy

Therapeutic genes transferred into
the somatic cells.
Eg. Introduction of genes into bone marrow
cells, blood cells, skin cells etc.
Will not be inherited later generations.
Affects only the targeted cells in the patient,
and is not passed to future generations.
Short-lived because the cells of most
tissues ultimately die and are replaced by
new cells.
At present all researches directed to correct
genetic defects in somatic cells.
Therapeutic genes transferred into the germ
cells.
Eg. Genes introduced into eggs and sperms.
It is heritable and passed on to later
generations. Possibility of eliminating some
diseases from a particular family.
Result in permanent changes.
For safety, ethical and technical reasons, it is
not being attempted at present.

Germ line gene therapy

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 3
Gene Therapy

Somatic cell gene therapy

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 4
Gene Therapy

Example of different TYPES OF SOMATIC GENE THERAPY

EXAMPLE OF EX VIVO GENE THERAPY

 1st gene therapy – to
correct deficiency of
enzyme, Adenosine
deaminase (ADA).

 Performed on a 4yr old

girl Ashanthi DeSilva, who
was suffering from SCID-
Severe Combined
Immunodeficiency
caused due to defect in
gene coding for ADA.
Deoxy adenosine
accumulates and destroys
T lymphocytes disrupting
immunity, suffer from
infectious diseases and
die at young age.

EXAMPLE OF IN VIVO GENE THERAPY

 Therapy for cystic fibrosis:
In patients with cystic
fibrosis, a protein called
cystic fibrosis
transmembrane regulator
(CFTR) is absent due to a
gene defect.

In the absence of CFTR

chloride ions concentrate
within the cells and it draws
water from surrounding.

This leads to the

accumulation of sticky
mucous in respiratory tract
and lungs.

 Treated by in vivo
replacement of defective
gene using adenovirus
vector.

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 5
Gene Therapy

GENE DELIVERY

Vectors are needed since the genetic material has to be transferred across the cell membrane
and preferably in to the cell nucleus.

The transfer of recombinant nucleic acid into target cells—a special instance of the ‘drug
distribution’ problem—is critical to the success of gene therapy. Nucleic acid must pass from the
extracellular space across the plasma and nuclear membranes and it must then be incorporated
into the chromosomes. Because DNA is negatively charged and single genes have
molecular weights around 104 times greater than conventional drugs, the problem is of a
different order from the equivalent stage of routine drug development.
There are several important considerations in choosing a delivery system; these include:

• the capacity of the system (e.g. how much DNA it can carry)
• the transfection efficiency (its ability to enter and become utilised by cells)
• the lifetime of the transfected material (determined by the lifetime of the targeted cells)
• the safety issue , especially important in the case of viral delivery systems.

Different carrier systems are used for gene delivery-

1) Viral systems (biological methods)

 Retroviruses
 Adeno viruses
 Adeno associated viruses
 Herpes simplex viruses

2) Non viral systems

Methods of non-viral gene delivery have also been explored using physical (carrier-free gene
delivery) and chemical approaches (synthetic vector-based gene delivery).

 Physical approaches, including Needle injection (microinjection), Electroporation, Gene

gun etc. These employ a physical force that permeates the cell membrane and facilitates
intracellular gene transfer.

 Chemical methods: Liposomes (Plasmid liposome complex, Uncovered plasmids,

ligand linked liposomes), microspheres etc.

COMMON VECTORS USED FOR GENE THERAPY:

1.Retro viruses 2. Adeno viruses 3. Liposomes

RETROVIRUS:

 class of viruses that have RNA as genetic material

 can create double stranded DNA copies with the enzyme reverse transcriptase
 These copies of its genome can be integrated into the chromosome of host cell by
another enzyme carried the virus called integrase
Now the host cell has been modified to contain a new gene. If such modified host cells divide
later, their descendants will contain the new genes.

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 6
Gene Therapy

Problem:
 Uncontrolled integration; May be oncogenic: integrase enzyme can insert genetic
material of the virus into any arbitrary position in the genome of the host, which can lead
to insertional mutagenesis (if insertion is in the middle of the gene) or uncontrolled cell
division (if gene happens to be one regulating cell division) leading to cancer
 Cannot infect non-dividing cells:
 Retroviruses are used ONLY in EX VIVO THERAPY: since many retroviruses show little
specificity, they could infect germ or non-target cells and produce undesired effects if
administered in vivo. For this reason, retroviruses have been used mainly for ex vivo
gene therapy.

ADENOVIRUS:

 Class of viruses that have double stranded DNA as genetic material. When these
viruses infect a host cell, they introduce their DNA molecule into the host. The genetic
material of the adenovirus is not incorporated into the host cell’s genetic material. The
DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra
DNA molecule are transcribed just like any other gene
 Can infect non-dividing cells
 genetic material of the adenovirus is not incorporated into the host cell’s genetic
material, thus avoids the risks of uncontrolled integration (DNA molecule is left free in
the nucleus of the host cell, and the instructions in this extra DNA molecule are
transcribed just like any other gene)

Problems:

 More likely to be attacked by the patient’s immune system, and the high levels of virus
required for treatment often provoke an undesirable inflammatory response.
 Transient expression (producing temporary effects)

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 7
Gene Therapy

LIPOSOMES:

 These are lipid bilayers surrounding an aqueous vesicle that can be used to introduce
foreign DNA into a target cell. Liposomes containing DNA have been shown to undergo
cellular uptake through endocytosis, with subsequent transient exogenous gene
expression.

Advantages:
 Safer when compared to viral vectors.
 Can carry large DNA molecules.

Disadvantages:
 Inefficient transfer to cell.
 Transient expression as there is no host genome integration.

ADVANTAGES OF GENE THERAPY OVER CONVENTIONAL THERAPY:

 Can replace dysfunctional or deficient gene that actually causes the disease.
 Can result in continuous production of therapeutic protein, which otherwise by
conventional therapy would require frequent drug dosing.
 Avoids side effects as the genes are targeted to specific site.
 Can avoid therapeutic failures due to non-compliance.

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016

 PHRM 413: Pharmacology III 8
Gene Therapy

PROBLEMS WITH GENE THERAPY

 Short Lived
 Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of
cells prevent gene therapy from long time
 Would have to have multiple rounds of therapy

 Immune Response
 new things introduced leads to immune response
 increased response when a repeat offender enters makes it difficult for gene therapy
to be repeated in patient

 Problems with viral vectors

 patient could have toxic, immune, inflammatory response and gene control and
targeting issues
 may recover its ability to cause disease

 Multigene Disorders
 Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to
treat because you need to introduce more than one gene.

 Insertional mutagenesis :
 Virus may target the wrong cells. If the DNA is integrated in the wrong place in the
genome, for example in a tumor suppressor gene, it could induce a tumor.

Reference: Rang and Dale’s Pharmacology

Farah Shahjin, Sr.Lecturer, DOP, EWU Fall 2016