Gene therapy

 Gene therapy is a technique that uses genes to treat or prevent a disease.

 It involves adding a copy of normal ( or wild type ) gene to genome of

individual carrying defective copies of that gene. The gene that is inserted is
called transgene and organism carrying it is said to be transgenic .

 If gene therapy is successful, the transgene will synthesize the missing gene
product and restore the normal phenotype.
There are two types of gene therapy:

1. Somatic gene therapy-In this the somatic cells receive the

corrected/therapeutic gene. This therapy does not affect the next generation,
however the effect of the therapy is often short lived and therefore requires
repeated administration. This is because the cells of most tissues ultimately die
and are replaced by new cells. All gene therapy to date on humand is only
somatic , such as treating SCID ( Severe Combined Immune Deficiency),
DMD, Cystic Fibrosis etc.

2. Germinal/germ-line gene therapy-In this the germ cells receive the corrected
gene/ therapeutic gene. This therapy would affect the future generation and hence
it is controversial.
Mechanism of gene therapy:
A normal or functional gene can be inserted into the genome by means of
vector or carrier.
A vector is the vehicle that transports or transfuses the transgene into the
host cell.
The most common vectors used in gene therapy are viruses
The viral DNA is manipulated to remove its disease causing genes and insert
the human therapeutic genes.
Target cells such the patients liver, lungs or bone marrow cells are infected
with the viral vector.
The vector then injects transfected DNA ( Viral DNA +therapeutic gene) into
the target cell where the therapeutic gene expresses itself and produces the
functional protein. This restores the target cell to the normal functioning
state.
Types of vectors used in gene therapy
There are basically 2 types of vectors used in gene therapy
1. Viral vectors
2. Non- viral vectors

1. Viral vectors
a) Retroviruses :
 They were the first viruses used as vectors in gene therapy.
 They have as RNA as their genetic material .
 They create copies of ds DNA of the host cell.
 These viruses can invade only actively dividing cells .
 The genes they carry are inserted into the host DNA in a random manner. Retroviral vectors are mostly based on
a mouse viruse (MMLV)

b) Adenoviruses :
 Are a class of viruses with ds DNA they do not usually integrate their DNA into the DNA of the host cells but
leave it free in the nucleus.
 The viral DNA carrying the therauptic gene is transcribed like any other genes.
 The DNA persists in the cell only for few days or weeks. As a result treatment requires readminstration.
 Adenoviruses have been used in treatment of cystic fibrosis, cancer of liver and ovaries.
c) Adeno Associated Viruses (AAV) :
A class of small , non –pathogenic , human ,ssDNA viruses that infects both dividing cells
and non dividing cells .
They can insert its DNA into a specific site on chromosome 19 . It is being used in studies
to treat hemophilia.

d) Herpes Simplex Virus:

These are ds DNA viruses that infect tissues like nerves, muscles, liver and lungs.it is
useful in treating neurodegenerative disorders.
Vector Target cell Cloning capacity Advantage Disadvantage

Adenovirus Lung, respiratory tract 7.5kb Efficient transfection Strong immune

cells response

Adeno associated Fibroblast T-Cells 4.5kb Transfected many cell Small insert size
Virus types

Retrovirus Stem cells and 8kb Prolonged expression Low transfection

proliferating cells efficiency and strong
immune response

Herpes simplex Both nonproliferating and 8kb Prolonged exposure Strong immune
virus proliferating cells response
2. Non-Viral methods:
The therauptic gene can also be delivered into target cell by non-viral
methods.
These methods have certain advantages like low host immunogenicity and
affords large scale production.
1. Naked DNA: This is the simplest method of non-viral transfection.
Naked DNA plasmid is injected intramuscularly.
2. Liposome :these are artificial lipid vesicles that carry the therauptic
gene through the cell membrane of the target cell
3. Direct introduction of the therauptic gene into the target cells like
muscle cells by particle shot gun / gene gun/ biolistic method or
electroporation method / Sonoporation. These microprojectiles literally
fires DNA into the target cells as the DNA is coated on to microscopic
beads of gold or tungsten
Gene therapy can also be classified in two types:
Ex vivo gene therapy: In this method , the cells are removed from the patient, manipulated to
introduce the therauptic gene and then reintroduced into the patient’

It involves the following steps

1. Isolation of the cells with the defective genes from the patient. The use of patents own cells
(autologous cells )ensures that there is no adverse immune responses.

2. Culturing the isolated cells in culture medium to increase the number of these target cells.

3. Transfecting the isolated cells with the therapeutic gene. the therapeutic gene should be stably
maintained and sustainably expressed

4. Selection, growing and testing the transfect cells

5. Transplanting or transfusing the transfected cells back into the patient.

Example for Ex vivo transfer of single gene: ADA -SCID, that is Adenosine deaminase(ADA)
deficiency severe combined immune Deficiency is rare genetic disorder due to a mutant ADA
gene. The defective gene causes the accumulation of adenosine or deoxyadenosine in the blood.

Gene therapy for this disorder involves the use of retroviral vector, namely the Moloney murine
leukaemia virus (MMLV).To create this vector , a cluster of 3 gene namely gal, pol and env are
removed from the viral DNA and replaced by therapeutic human ADA gene.

The recombinant viral DNA has psi(Ψ) sequence required for encapsulation, neomycin resistance
gene (selected marker) and is flanked by long terminal repeat (LTR) sequence that control
transcription and integration into the host genome.

The recombinant viral DNA when packed into the viral protein coats ,they can infest cells but
cannot replicate because of the missing viral genes (gal, pal and env).

The T-cell from the patient are isolated, cultured and transfected by the recombinant viral DNA
carrying the hDNA gene integrated into the chromosome of the cell. The transfected T-cell then
isolated, cultured and then transfused or infused back into the body of the patient.
Vector with
hADA gene
2. In vivo gene therapy

 In this method , the gene is directly transferred to cells inside the patient’s
body. This method also uses vectors like Adenovirus, retrovirus as well as
the non-viral methods to transfer the therapeutic gene.

 In vivo gene therapy has been tried for ADA deficiency SCID and muscular
dystrophy.
Problems of gene therapy:

 In many cases of gene therapy the effect is short lived and it has to
be readministered several times for the therapy to be effective .

 Problems with viral vectors –viruses may pose a variety of threats

to the patient: like toxicity, immune responses and the ability to
cause diseases.

 Chance of inducing tumours-if the DNA is integrated in the wrong

place in the genome it could induce a tumour.
 Multiple factor gene therapy for cancer

 Cancer research and genomics have experienced considerable advancements over the last two
decades.

 In 2001 the human genome sequence and the identification of approximately 30000 genes
were published.

 As a expected this was followed by extensive research in molecular genetics with advanced
tools used to dissect gene function and explore the biological processes involved in causing
genetic aberration and malignant transformation.

 The recent proliferation of knowledge of cancer has led to the development of novel
therapeutic approaches in cancer management, particularly gene therapy.

 Concerning cancer, initial efforts to deactivate oncogenes and replace non-functioning tumour
suppressor genes were barely successful.
 Subsequently, new approaches have been developed to transfer genetic
materials (transgenes) directly into the target cells, aiming to change their
phenotype.
 Target cells may be normal cells, cancerous cells, immune mediated cells or
pluripotent stem cells.
 Once the transgene enters the cancer cell, it can then assists in its death or
restore normal cellular functions, whereas for normal cells, the transgene can
protect them from drug-induced toxicities, or activate an immune cell to get rid
of the cancer cell.
 Gene and vector- based molecular therapies for cancer comprise a wide range of
treatment modalities to modify cancer cell, normal cells, and/ or a tumour
microenvironment
Gene faults and cancer
Many genes changes that may make a cell become cancerous are caused by environmental or lifestyle
factors, such as smoking. But some people have inherited damaged genes cause between 2 and 3 out of
every 100 cancers. Getting genes into cancers cells is one of the most difficult aspects of gene therapy.
Types of gene therapy

Researchers are looking at different ways of using gene therapy, including

1. Boosting the immune response
Some type of gene therapy aim to boost the body’s natural ability to attack cancer cells.
Our immune system has cells that recognize and kills harmful things that can cause disease, such
as cancer cells. There are many different types of immune cells to destroy cancer cells, some
types of therapy and genes to patient’s immune cells to make them better at finding or
destroying particular types of cancer. There are a few trials using this type of gene therapy in
UK.

2. Gene therapies to make cancer treatments work better

some gene therapies put genes into cancer cells to make the cells more sensitive to particular
treatments such as chemotherapy or radiotherapy. This type of gene therapy aims to make
the other cancer treatments work better
3. Pro drug gene therapy
Some types of gene therapy deliver genes into the cancer cells that allow the cells to
change drugs from an inactive form to an active form. The inactive form of the drug is
called pro drug. After giving the carrier containing the gene, the doctor gives the patient
the pro drug. The pro drug may be a tablet or capsule that swallow, or you may have it
into the bloodstream. The pro drug circulates in the body and doesn’t harm normal cells.
But when it reaches the cancer cells, the genes activates it and the drug kills the cancer
cells.

4. Blocking process that protect cancerous cells.

Some gene therapies block processes that cancer cells use to survive. For example : most cells in
the body are programmed to die if their DNA is damaged beyond repair this is called
programmed cell death or apoptosis. But cancer block this processes so they don’t die even when
they are suppose to . Some gene therapy strategies aim to reverse this blockage. Doctors hope that
this new types that of treatment will make the cancer cells die.
5. Using altered viruses
Some viruses infect and kill cells. Researchers are working on ways to change these viruses so
that they only target and kill cancer cells living healthy cells alone this sought treatment uses
viruses to kill cancer cells directly rather than to deliver genes. So it is not cancer gene
therapy in true sense of the world but doctors some times refer to it as gene therapy. One
Example : uses the herpes simplex virus. The changed virus is called oncovex.it has been tested
in early clinical trials for advanced melanoma, pancreatic cancer and head and neck cancers
Ethical issues on gene therapy

 The prospects of altering the blue print of human life raised the question about ‘ playing God’.
Some had hopes of eliminating almost all the diseases: however, some saw the spectre of
unintended consequences.
 Somatic gene therapy many feel that the somatic gene therapy was an extension of conventional
therapies poising few ethical issues. However, the early concerns was that the altered genetic
material could find its way into the gametes thus affecting their progeny.
 The efficiency of somatic cell gene therapy has not had the anticipated success; effectiveness of
treatment of most diseases has been disappointing and encouraging for few .
 Germinal or Germ-line gene therapy is more controversial as it involves the use of gametes and
pre-embryos so it is open ended therapy extending indefinitely into the future.

 It creates and destroys embryos at the will of the researcher and this has tremendous ethical and
legal issues as many feel the pre embryo has its own identity and therefore has the right to life
like any other individual so killing this embryos in the name of science to paramount to murder.

 Germinal gene therapy raises issues like tampering with human nature and misused to enhance
human traits like intelligence, athletic body and other traits of interest this gives the opportunity
to manipulate the traits of the children by the parents to create designer children