gene therapy

1. Topic: Gene therapy
Definition: The introduction of normal genes into cells in place of missing or defective ones in
order to correct genetic disorders or it is the therapeutic delivery of nucleic acid polymers into a
patient's cells as a drug to treat disease.
History: In the 1980s, Scientists began to look into gene therapy. The first gene therapywasperformed
on September 14th, 1990. Ashanti DeSilva was treated for SCID (Sever combined immunodeficiency).
Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them back
into her blood stream.This strengthened her immune system but only worked for a few months
Types of gene therapy:There are 2 types of gene therapy.
1. Germ line gene therapy: where germ cells (sperm or egg) are modified by the
introduction of functional genes, which are integrated into their genome. Therefore changes
due to therapy would be heritable and would be passed on to later generation. Theoretically,
this approach should be highly effective in counteracting genetic disease and hereditary
disorders. but at present many jurisdictions, a variety of technical difficulties and ethical
reasons make it unlikely that germ line therapy would be tried in human beings in near
future.
2. Somatic gene therapy: where therapeutic genes are transferred into the somatic cells
of a patient. Any modifications and effects will be restricted to the individual patient only
and will not be inherited by the patients offspring or any later generation.
Gene delivery: In most gene therapy studies, a normal gene is inserted into the genome to
replace an abnormal, disease causing gene. Of all challenges, the one that is most difficult is the
problem of gene delivery i.e. how to get the new or replacement gene into the patient’s target cells.
So a carrier molecule called vector must be used for the above purpose.
Ideal gene delivery vector :
They should be very specific, capable of efficiently delivering one or more genes of the size needed
for clinical application, unrecognized by the immune system and be purified in large quantities at
high concentration. Once the vector is inserted into the patient, it should not induce an allergic

2. reaction or inflammation. It should be safe not only for the patient but also for the environment.
Finally a vector should be able to express the gene for as long as is required, generally the life of
the patient .
Techniques forthe delivery ofvectors : Two techniques have been used to deliver vectors
i.e. ex-vivo and in-vivo.
 ex-vivo method of delivery: It is the commonest method, which uses extracted cells
from the patient. first, the normal genes are cloned into the vector. Next, the cells with
defective genes are removed from the patient and are mixed with genetically engineered
vector. finally the transfected cells are reinfused in the patient to produce protein needed
to fight the disease.
 In-vivo method of delivery : This technique does not use cells from the patient’s
body. Vectors with the normal gene are injected into patient’s blood stream to seek out and
bind with target cell.
Vectors used in gene therapy: Some of the vectors used in gene therapy include:
1. Viral Vector: In order to replicate, viruses introduce their genetic material into the host cell,
tricking the host's cellular machinery into using it as blueprints for viral proteins. Scientists exploit
this by substituting a virus's genetic material with therapeutic DNA. (The term 'DNA' may be an
oversimplification, as some viruses contain RNA, and gene therapy could take this form as well.)
A number of viruses have been used for human gene therapy, including retrovirus, adenovirus,
lentivirus, herpes simplex, pseudotyped viruses, vaccinia and adeno-associated virus.
2. Non-Viral Methods:
Injection of NakedDNA:Simplest method of non-viral transfection is direct DNA injection.
Clinical trials to inject naked DNA plasmids have been performed successfully. There have been
trials with naked PCr products, which have had greater success.
Physical Methods to Enhance Delivery: Research efforts have yielded several non-viral
methods gene transfer such as electroporation (creation of electric field induced pores in plasma
membrane), sonoporation (ultrasonic frequencies to disrupt cell membrane), magnetofection (use

3. of magnetic particle complexed with DNA), gene guns (shoots DNA coated gold particles into
cells by using high pressure), Hydrodynamic deliverymethods are being explored.
Chemical Methods to enhance Delivery: It includes the use of oligonucleotides,
polymersomes, polyplexes, dendrimers, inorganic nanoparticles and cell penetrating peptides.
3 .Hybrid methods: Due to every method of gene transfer having shortcomings, there have
been some hybrid methods developed that combine two or more techniques. Virosomes are one
example; they combine liposomes with an inactivated HIV or influenza virus. This has been shown
to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal
methods alone. Other methods involve mixing other viral vectors with cationic lipids or
hybridising viruses.
Hurdles in gene therapy: Some of the unsolved problems include:
 Short-lived nature : Before gene therapy can become a permanent cure for a condition,
the therapeutic DNA introduced into target cells must remain functional and the cells
containing the therapeutic DNA must be stable. Problems with integrating therapeutic
DNA into the genome and the rapidly dividing nature of many cells prevent it from
achieving long-term benefits. Patients require multiple treatments.
 Immune response: Any time a foreign object is introduced into human tissues, the
immune system is stimulated to attack the invader. Stimulating the immune system in a
way that reduces gene therapy effectiveness is possible. The immune system's enhanced
response to viruses that it has seen before reduces the effectiveness to repeated treatments.
 Problems with viral vectors: Viral vectors carry the risks of toxicity, inflammatory
responses, and gene control and targeting issues.
 Multigene disorders:Some commonly occurring disorders, such as heart disease, high
blood pressure, Alzheimer's disease, arthritis, and diabetes, are affected by variations in
multiple genes, which complicate gene therapy.Some therapies may breach the Weismann
barrier (between soma and germ-line) protecting the testes, potentially modifying the
germline, falling afoul of regulations in countries that prohibit the latter practice.

4.  Insertional mutagenesis: If the DNA is integrated in a sensitive spot in the genome,
for example in a tumor suppressor gene, the therapy could induce a tumor. This has
occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID)
patients, in which hematopoietic stem cells were transduced with a corrective transgene
using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.
One possible solution is to add a functional tumor suppressor gene to the DNA to be
integrated. This may be problematic since the longer the DNA is, the harder it is to integrate
into cell genomes.
 Cost: lipogene tiparvovec or Glybera, for example, at a cost of $1.6 million per patient,
was reported in 2013 to be the world's most expensive drug.
Deaths: Three patients' deaths have been reported in gene therapy trials, putting the field
under close scrutiny. The first was that of Jesse Gelsinger in 1999. One X-SCID patient died
of leukemia in 2003. In 2007, a rheumatoid arthritis patient died from an infection; the
subsequent investigation concluded that the death was not related to gene therapy.
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