Gene Therapy
Gene Therapy
Gene Therapy
Introduction
Gene Therapy
Targets
Isolation of gene
Gene Targeting
Gene Delivery
Case Study Cystic Fibrosis
The Disease
Is it a good Target
Choosing Vectors
History
Challenges
Ethical Issues
Recent Upcoming
CRISPR
Conclusion
Bibliography
Websites
Books
Introduction
Diseases
The term disease broadly refers to any condition that impairs normal function, and is
therefore associated with dysfunction of normal homeostasis. When the functioning
of one or more organs or systems of the body is adversely affected, characterised by
various signs and symptoms, we say that we are not healthy, i.e., we have a
disease.
Health can be defined as a state of complete physical, mental and social well-being.
When people are healthy, they are more efficient at work. This increases productivity
and brings economic prosperity. Health also increases longevity of people and
reduces infant and maternal mortality.
Genetic Disorders
A genetic disorder is an illness caused by one or more abnormalities in the
genome, especially a condition that is present from birth (congenital). They are
medical disorders related to gene mutation.
Genetic disorders are heritable, and are passed down from the parents' genes. Other
defects may be caused by new mutations or changes to the DNA. In such cases, the
defect will only be heritable if it occurs in the germ line.
The same disease, such as some forms of cancer,
may be caused by an inherited genetic condition in
some people, by new mutations in other people, and
by non-genetic causes in still other people.
So, if a flawed gene caused our "broken window," can we "fix" it? What are our
options?
1. Stay silent: ignore the genetic disorder and nothing gets fixed.
2. Try to treat the disorder with drugs or other approaches: depending on the
disorder, treatment may or may not be a good long-term solution.
3. Put in a normal, functioning copy of the gene: if you can do this, it may solve
the problem!
If it is successful, gene therapy provides a way to fix a problem at its source. Adding
a corrected copy of the gene may help the affected cells, tissues and organs work
properly. Gene therapy differs from traditional drug-based approaches, which may
treat the problem, but which do not repair the underlying genetic flaw.
What role does the protein encoded by the gene play within the cells of that
tissue?
4. Adding a normal copy of the gene should fix the problem in the affected
tissue. This may seem like obvious, but it's not. What if the mutated gene
encodes a protein that prevents the normal protein from doing its job? Mutated
genes that function this way are called dominant negative and adding back the
normal protein won't fix the problem.
How accessible is the tissue? Is it fairly easy (skin, blood or lungs), or more
difficult to reach (internal organs)?
The techniques of biotechnology have made it possible to isolate the required gene
in the laboratory and also deliver the gene.
Isolation of DNA
Since the DNA is enclosed within the membranes, we have to break the cell open to
release DNA along with other macromolecules such as
RNA, proteins, polysaccharides and also lipids. This can
be achieved by treating the bacterial cells/plant or animal
tissue with enzymes such as lysozyme (bacteria),
cellulase (plant cells), chitinase (fungus). Genes are
located on long molecules of DNA intertwined with
proteins such as histones. The RNA can be removed by treatment with ribonuclease
whereas proteins can be removed by treatment with protease. Other molecules can
be removed by appropriate treatments and purified DNA ultimately precipitates out
after the addition of chilled ethanol. This can be seen as collection of fine threads in
the suspension.
Cutting of DNA
PCR or polymerase chain reaction is then used to create multiple copies of the gene
of interest. In this reaction, multiple copies of the gene (or DNA) of interest is
synthesised in vitro using two sets of primers (small chemically synthesised
oligonucleotides that are complementary to the regions of DNA) and the enzyme
DNA polymerase. The enzyme extends the primers using the nucleotides provided in
the reaction and the genomic DNA as template. If the process of replication of DNA
is repeated many times, the segment of DNA can be amplified to approximately
billion times, i.e., 1 billion copies are made.
Gene Targeting
Gene delivery is one of the biggest challenges in the field of gene therapy.
3. INTEGRATING the gene in the cells. The gene must stay put and continue
working in the target cells. If so, it must be ensured that the gene integrates into, or
becomes part of the host cell's genetic material, or that the gene finds another way to
survive in the nucleus without being rejected.
There is no "perfect vector" that can treat every disorder. Like any type of medical
treatment, a gene therapy vector must be customized to address the unique features
of the disorder. We have learnt the lesson, of transferring genes into plants and
animals from bacteria and viruses, which have known this for ages how to deliver
genes to transform eukaryotic cells and force them to do what the bacteria or viruses
want.
Part of the challenge in gene therapy is choosing the most suitable vector for treating
the disorder. Some vectors commonly used are:
Viruses
Usually when we think of viruses, we think of them causing diseases such as the
common cold, the flu, and HIV/AIDS. When faced with the problem of gene delivery,
scientists looked to viruses. Why reinvent the wheel if there's a perfectly good one
out there? If we can modify viruses to deliver genes without making people sick, we
may have a good set of gene therapy tools.
Non-Viral Vectors
Although viruses can effectively deliver genetic material into a patient's cells, they do
have some limitations. It is sometimes more efficient to deliver a gene using a non-
viral vector, which has fewer size constraints and which won't generate an immune
response.
Non-viral vectors are typically circular DNA molecules, also known as plasmids. In
nature, bacteria use plasmids to transfer genes from cell to cell.
Scientists use bacteria and plasmids to easily and efficiently store and replicate
genes of interest from any organism.
Vectors used at present, are engineered in such a way that they help easy linking of
foreign DNA and selection of recombinants from non-recombinants.
These are not the only way to introduce alien DNA into host cells.
In a method known as micro-injection, recombinant DNA is directly injected into the
nucleus of an animal cell. In another method, suitable for plants, cells are
bombarded with high velocity micro-particles of gold or tungsten coated with DNA in
a method known as biolistics or gene gun.
Case Study
Cystic Fibrosis
Challenges
Some the factors that have kept gene therapy from becoming an effective treatment
for genetic diseases are:
Problems with viral vectors - Viruses, while the carrier of choice in most
gene therapy studies, present a variety of potential problems to the patient --
toxicity, immune and inflammatory responses, and gene control and targeting
issues. In addition, there is always the fear that the viral vector, once inside
the patient, may recover its ability to cause disease.
Websites
http://en.wikipedia.org/wiki/Gene_therapy
http://www.trip2medi.com/treatmentCGeneTherapy.php
http://learn.genetics.utah.edu/content/tech/genetherapy/
http://ghr.nlm.nih.gov/handbook/therapy/
http://cystic-fibrosis.emedtv.com/cystic-fibrosis/cystic-fibrosis-gene-therapy.html
http://en.wikipedia.org
Books
12th NCERT Biology
Stryer Biochemistry