Introduction

Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of
diseases.
It is a technique for correcting defective genes responsible for disease development.
The first approved gene therapy experiment occurred on September 14, 1990 in US, when
Ashanti DeSilva was treated for ADA-SCID.
It is essentially an intervention that alters the instruction set of a cell.
There are four approaches: -
1. A normal gene is inserted to compensate for a non-functional/mutated gene.
2. An abnormal gene expression is suppressed.
3. An abnormal gene is repaired through selective reverse mutation.
4. Change the regulation of gene pairs.
Materials DNA, RNA; Cells, Tissues or organs
Delivery Usually required to be delivered into cells (Antisense Oligonucleotides) or
Nucleus (genes)
Mechanisms Usually cure the cause of the disease
Duration of Effect Can be permanent and also can be passed down to next generation in
germline gene therapy.
Ethics Major Issues
principle
putting corrective genetic material into cells alleviates the symptoms of disease. In
practice, considerable obstacles have emerged. But, thanks to better delivery systems, there is
hope that the technique will succeed.
HOW IT WORKS?
A vector delivers the therapeutic gene into a patient's target cell
The target cell become infected with the viral vector
The vector's genetic material is inserted into the target cell
Functional proteins are produced from the therapeutic gene causing the cell to return to a
normal state
PURPOSE & APPROCH OF GENE THERAPY
Monogenic gene therapy

✓ Provides genes for the production of a specific protein

✓Cystic fibrosis, Muscular dystrophy, Sickle cell disease, hemophilia, SCID

Suicide Gene Therapy
Provide 'suicide' genes to target cancer cells for destruction

✓ Cancer
Antisense Gene

✓ Provides a single stranded oligonucleotides in an 'antisense' (backward) orientation to

block the production of harmful proteins

✓ AIDS/HIV
TYPES OF GENE THERAPY
Somatic cell gene therapy
Germ line gene therapy
Current gene therapy is exclusively somatic cell gene therapy which involves the
introduction of genes into somatic cells of an affected individual.
The prospect of human germline gene therapy is currently not sanctioned.
Germline gene therapy targets the reproductive cells, meaning any changes made to the DNA
will be passed on to the next generation. The effects of gene therapy are too unpredictable.
Any additional mutations that are introduced as a result will be passed on to the next
generation.
Germ line gene therapy
In germline gene therapy, DNA is inserted into the reproductive cells (eggs or sperm) in the
human body. Germline gene therapy will correct the genetic variants of the reproductive cells
of an individual, and this would be passed down to future generations. This therapy removes
a hereditary disorder from a family line forever. Hereditary disorders occur at human’s are
possibly inherited from the germline cells. However, curing these diseases is possible only
through modifying their nuclear and mitochondrial DNA mutations in preimplantation
embryos, which is commonly known as germline gene therapy
New genes have been successfully introduced into the germ lines of other mammals, but with
very low efficiency. At the same time, pre-implantation genetic diagnosis allows parents to
choose embryos based on their genetic variations, as long as the parents themselves produced
the desired variations. If not, donated eggs or sperm would be a much safer and easier way to
introduce the desired genes than somatic cell gene therapy. Germ line gene therapy may turn
out to be most important as a barrier to somatic cell gene therapy. If germ line gene therapy
were banned, researchers using somatic gene therapy might need to make the difficult
showing that the transplanted genes could not ‘infect’ the patient's germ cells and thus
constitute inadvertent germ line gene therapy.
Or
In germline gene therapy, germ cells (sperm or eggs) are modified by the introduction of
functional genes, which are integrated into their genomes.
Germ cells will combine to form a zygote which will divide to produce all the other cells in
an organism and therefore if a germ cell is genetically modified then all the cells in the
organism will contain the modified gene. This would allow the therapy to be heritable and
passed on to later generations
Somatic cell gene therapy
Somatic cell gene therapy involves the placement of a human gene into a living person's
somatic cells—cells that do not produce the eggs and sperm that in turn produce the next
generation. Somatic cell gene therapy would aim to cure a disease only in the patient, not in
the patient's descendants. It was initially conceived as introducing a properly functioning
copy of a gene into a person who had a genetic disease as a result of inheriting only
improperly functioning copies. Different types of somatic cell gene therapy have since been
investigated for the treatment of diseases that are not primarily caused by inherited genes,
such as AIDS and cancer. Over 100 clinical trials of somatic cell gene therapy have taken
place; very few have, thus far, shown any success.
The genetic aspects of somatic cell gene therapy have been largely uncontroversial. In
essence, the gene therapy is merely another drug delivery system, a different way to get a
normal human protein to the right place in the body. Somatic cell gene therapy therefore
stands in the same position as most experimental therapies. Like such therapies, it has
prompted concerns that desperate patients are not truly giving informed consent and that the
possible benefits of the treatment are exaggerated. Gene therapy may face the latter problem
to a greater extent than most experimental treatments because of the exaggerated public view
of the power of anything genetic.
or
In somatic gene therapy, the therapeutic genes are transferred into the somatic
cells (non sex- cells), or body, of a patient. Any modifications and effects will be restricted to
the individual patient only, and will not be inherited by the patient's offspring or later
generations. Somatic gene therapy represents the mainstream line of current basic and clinical
research, where the therapeutic DNA transgene (either integrated in the genome or as an
external episome or plasmid) is used to treat a disease in an individual
Most of these trials focus on treating severe genetic disorders, including immunodeficiency,
haemophilia, thalassemia, and cystic fibrosis. These disorders are good candidates for
somatic cell therapy because they are caused by single gene defects. The replacement of
multiple genes in somatic cells is not yet possible.
This is off 2 types
1.EX VIVI GENE TRANSFER
2.IN VIVO GENE TRANSFER
1. EX VIVO GENE TRANSFER
Transfer of cloned genes into cells grown in culture.
Those cells which have been transformed successfully are selected, expanded by cell culture
in vitro, then introduced into the patient.
To avoid immune system rejection of the introduced cells, autologous cells are normally
used: the cells are collected initially from the patient to be treated and grown in culture before
being reintroduced into the same individual.
Clearly, this approach is only applicable to tissues that can be removed from the body, altered
genetically and returned to the patient where they will engraft and survive for a long period of
time (e.g. cells of the hematopoietic system and skin cells).
This type of gene therapy involves transplantation of autologous genetically modified cells
and so can be considered a modified form of cell therapy.
Isolate cells with genetic defect from a patient

Grow the cells in culture

Introduce the therapeutic genes.

Select genetically corrected cells and grow.

Transplant the modified cells to the patient.

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.
 Was suffering from SCID- Severe Combined Immunodeficiency.
 Caused due to defect in gene coding for ADA.
 Deoxy adenosine accumulate and destroys T lymphocytes.
 Disrupts immunity, suffer from infectious diseases and die at young age.
IN VIVO GENE TRANSFER
The cloned genes are transferred directly into the tissues of the patient.
This may be the only possible option in tissues where individual cells cannot be cultured in
vitro in sufficient numbers (e.g. brain cells) and/or where cultured cells cannot be re-
implanted efficiently in patients.
Liposomes and certain viral vectors are increasingly being employed for this purpose.
It is often convenient to implant vector-producing cells (VPCs), cultured cells which have
been infected by the recombinant retrovirus in vitro: in this case the VPCS transfer the gene
to surrounding disease cells.
As there is no way of selecting and amplifying cells that have taken up and expressed the
foreign gene, the success of this approach is crucially dependent on the general efficiency of
gene transfer and expression.
Or
 Direct delivery of therapeutic gene into target cell into patients body.
 Carried out by viral or non viral vector systems.
 It can be the only possible option in patients where individual cells cannot be cultured
in vitro in sufficient numbers (e.g. brain cells).
 In vivo gene transfer is necessary when cultured cells cannot be re-implanted in
patients effectively.
Example of in vivo gene therapy
 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 by adenovirus vector.
TARGETS OF GENE THERAPY
 Central Nervous System (Huntington's Disease)
 Respiratory Tract
 Other solid organs (Liver)
 T cells and Hematopoeitic cells
 Muscles
 Vascular system (Lymphocytes)
Vectors in gene therapy
To transfer the desired gene into a target cell, a carrier is required. Such vehicles of gene
delivery are known as vectors.
2 main classes
 Viral vectors
 Non viral vectors
Viral vectors
1) RETROVIRUS VECTOR SYSTEM
 The recombinant retroviruses have the ability to integrate into the host genome in a
stable fashion.
 Can carry a DNA of size - less than 3.4kb
 Replication defective virus particles
 Target cell dividing
ADENO VIRUS VECTOR SYSTEM
Adeno virus with a DNA genome - good vectors.
Target- non dividing human cell.
Eg. Common cold adenovirus.
3) ADENO ASSOCIATED VIRUS VECTOR
It is a human virus that can integrate into chromosome 19.
It is a single stranded, non pathogenic small DNA virus.
AAV enters host cell, becomes double stranded and gets integrated into chromosome.
4) HERPEX SIMPLEX VIRUS VECTOR
Viruses which have natural tendency to infect a particular type of cell.
They infect and persist in nervous cells.
NON VIRAL VECTOR SYSTEM
1. PURE DNA CONSTRUCT
Direct introduction of pure DNA construct into target tissue.
Efficiency of DNA uptake by cells and expression rather low.
Consequently, large quantities of DNA have to be injected periodically.
2. LIPOPLEXES
Lipid DNA complexes; DNA construct surrounded by artificial lipid layer.
Most of it gets degraded by lysosomes.
3) DNA MOLECULAR CONJUGATES
Commonly used synthetic conjugate is poly-L-lysine bound to specific target cell receptor.
Therapeutic DNA is then made to combine with the conjugate to form a complex.
It avoids lysosomal breakdown of DNA.
4) HUMAN ARTIFICIAL CHROMOSOME
Can carry a large DNA ie, with one or more therapeutic genes with regulatory elements.
METHODS OF GENE DELIVERY PHYSICAL METHODS
Gene Gun
Employs a high-pressure delivery system to shoot tissue with gold or tungsten particles that
are coated with DNA
Microinjection
Process of using a glass micropipette to insert microscopic substances into a single living
cell.
Normally performed under a specialized optical microscope setup called a micromanipulator.
CHEMICAL METHODS
 USING DETERGENT MIXTURES
 Certain charged chemical compounds like Calcium phosphates are mixed with
functional CDNA of desired function.
 The mixture is introduced near the vicinity of recipient cells.
 The chemicals disturbs the cell membrane, widens the pore size and allows cDNA to
pass through the cell.
LIPOFECTION
 It is a technique used to inject genetic materials into a cell by means of liposomes.
 Liposomes are artificial phospholipid vesicles used to deliver a variety of molecules
including DNA into the cells.
ADVANTAGES
 Gene therapy has the potential to eliminate and prevent hereditary diseases such as
cystic fibrosis, ADA-SCID etc.
 It is a possible cure for heart disease, AIDS and cancer.
 It gives someone born with a genetic disease a chance to life.
 It can be used to eradicate diseases from the future generations.
DISADVATAGES
 Long lasting therapy is not achieved by gene therapy; Due to rapid dividing of cells
benefits of gene therapy is short lived.
 Immune response to the transferred gene stimulates a potential risk to gene therapy.
 Viruses used as vectors for gene transfer may cause toxicity, immune responses, and
inflammatory reactions in the host.
 Disorders caused by defects in multiple genes cannot be treated effectively using gene
therapy.