Project
Gene Therapy
Certificate
Year: 2013-14
This is to certify that OV Shashank a student of
RN Podar School, of class XII- D, Roll
No: , has completed his full semester
project in the fulfilment of curriculum All India
Senior Secondary Examination
The project work entitled Gene Therapy, is the
original work done by him during his above
full semester project.
Date:
_____________________ ___________________
Internal Examiner Principal
______________________
External Examiner School Stamp
Acknowledgement
1
I take this opportunity to express my sincere gratitude
to the honourable Principal Mrs Avnita Bir of RN
Podar School for her deep interest and guidance
provided to me during the course.
I am most grateful to our Biology teacher Mrs
Padmavathi for her great help in the completion of
this project.
Students Signature
__________________________
Table of Contents
2
Introduction
Gene Therapy
Targets
Types of gene therapy
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
3
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.
Infections
These are diseases caused due to invasion of a
foreign parasitic organism. They are temporary
because the immune system of organisms can fight
such pathogens (disease causing organisms) to a
certain extent hence helping in prevention of the
disease. The immune system can also be aided
with the use of several drugs. Apart from easy treatment they can also
be easily prevented.
Lifestyle Diseases
Lifestyle diseases (also sometimes called diseases of
longevity or diseases of civilization interchangeably) are diseases that
4
appear to increase in frequency as countries become more industrialized
and people live longer. Diet and lifestyle are major factors thought to
influence susceptibility to many diseases. Drug abuse, tobacco smoking,
and alcohol drinking, as well as a lack of exercise may also increase the
risk of developing certain diseases, especially later in life. These
diseases can be prevented completely by living a healthy lifestyle.
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.
These diseases are totally random and difficult to prevent as they are not
caused by external agents. Also as their root cause lies in the genome of
the organism their cure was thought to be impossible until the
breakthrough research unlocking the secrets of DNA leading to the
development of biotechnology and hence gene therapy.
5
Gene Therapy
We can think of a medical
condition or illness as a
"broken window." Many
medical conditions result
from flaws, or mutations, in
one or more of a person's
genes. Mutations cause
the protein encoded by
that gene to malfunction.
When a protein
malfunctions, cells that
rely on that protein's
function can't behave
normally, causing problems for whole tissues or organs.
Medical conditions related to gene mutations are called
genetic disorders.
6
approaches, which may treat the problem, but which do
not repair the underlying genetic flaw.
7
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.
8
Types of Gene
Cell types
Gene therapy may be classified into two types:
Somatic
In somatic cell gene therapy (SCGT), the therapeutic
genes are transferred into any cell other than
a gamete, germ cell, gametocyte or undifferentiated stem
cell. Any such modifications affect the individual patient
only, and are not inherited by offspring. Somatic gene
therapy represents mainstream basic and clinical
research, in which therapeutic DNA (either integrated in
the genome or as an external episome or plasmid) is used
to treat disease.
Germline
In germline gene therapy (GGT), germ
cells (sperm or eggs) are modified by the introduction of
functional genes into their genomes. Modifying a germ cell
causes all the organism's cells to contain the modified
gene. The change is therefore heritable and passed on to
later generations. Australia, Canada, Germany, Israel,
9
Switzerland and the Netherlands prohibit GGT for
application in human beings, for technical and ethical
reasons, including insufficient knowledge about possible
risks to future generations and higher risks versus
SCGT. The US has no federal controls specifically
addressing human genetic modification (beyond FDA
regulations for therapies in general).
The first step is to find and isolate the gene that will be
inserted into the genetically modified organism. Finding
the right gene to insert usually draws on years of
scientific research into the identity and function of useful
genes. Once that is known the DNA needs to be cut at
specific locations to isolate the gene of interest. This can
be done by using restriction enzymes also known as
molecular scissors which cut DNA at specific sites
containing palindromic DNA sequences. But in order to cut
the DNA with restriction enzymes, it needs to be in pure
form, free from other macro-molecules.
Until the early 1970s DNA was the most difficult
cellular molecule for the biochemist to analyse.
Enormously long and chemically monotonous, the string of
nucleotides that forms the genetic material of an
organism could be examined only indirectly,
by protein or RNA sequencing or by genetic analysis.
Today the situation has changed entirely. From being the
most difficult macromolecule of the cell to analyse, DNA
has become the easiest.
10
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
Restriction enzyme digestions are
performed by incubating purified
DNA molecules with the
restriction enzyme, at the optimal
conditions for that specific
enzyme. The cutting of DNA by
restriction endonucleases results
in the fragments of DNA. These fragments can be
separated by a technique known as gel electrophoresis.
Since DNA fragments are negatively charged molecules
they can be separated by forcing them to move towards
the anode under an electric field through a
medium/matrix. The separated bands of DNA are analysed
11
for the required gene and then it is cut out from the
agarose gel and extracted from the gel piece. This step is
known as elution.
12
Gene Targeting
Gene targeting (also, replacement strategy based on
homologous recombination) is a genetic technique that
uses homologous recombination to change
an endogenous gene. The method can be used to delete a
gene, remove exons, add a gene, and introduce point
mutations. Gene targeting can be permanent or
conditional. Conditions can be a specific time
during development / life of the organism or limitation to a
specific tissue.
13
immune response against it. If the body develops immunity
against a specific gene delivery vehicle, future rounds of
the therapy will be ineffective. For gene delivery to be
successful, foreign DNA must survive long enough in the
host cell to integrate into its genome. This requires foreign
DNA to be synthesized as part of a vector, which is
designed to enter the desired host cell and deliver
the transgene to that cell's genome .Vectors utilized as
the method for gene delivery can be divided into two
categories, non-viral and viral.
Viruses
14
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.
The host cell will carry out these instructions and produce
additional copies of the virus, leading to more and more
cells becoming infected. Some types of viruses insert
their genome into the host's cytoplasm, but do not
actually enter the cell. Others penetrate the cell
membrane disguised as protein molecules and enter the
cell.
15
Viruses can cause immune responses in patients,
resulting in two potential outcomes:
Non-Viral Vectors
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
16
coated with DNA in a method known as biolistics or gene
gun.
The first way is to inject the vector into the body and
specifically target affected cells. This is called an in vivo
approach. The second way, called ex vivo, is to deliver the
gene to cells while they're outside the body by:
Case Study
Cystic Fibrosis
17
The Disease A Genetic Disorder
18
To check this some questions must be answered:
Will adding a normal copy of the gene fix the problem in the affected tissue? Yes.
While the mutated CFTR gene encodes a non-functional ion channel
protein, it will not prevent a normal CFTR channel protein from
working properly. Therefore, adding a normal copy of the CFTR gene
should fix the problem
Is it feasible to deliver the gene to the cells of the affected tissue? Yes, in part.
Treating the lungs of patients with CF might be feasible, since the
lung surfaces are exposed to the air and somewhat easy to reach.
Because the digestive system is less accessible, however, it might
be a more difficult region to treat.
Choosing vectors
The vectors that are most suitable for gene therapy are:
Retrovirus
Retroviruses are enveloped viruses that replicate in a host cell
through the process of reverse transcription. It is a single-stranded
RNA virus that stores its nucleic acid in the form of an mRNA
genome targets. Once inside the host cell cytoplasm the virus uses
its own reverse transcriptase enzyme to produce DNA from its RNA
genome, the reverse of the usual pattern, thus retro (backwards).
This new DNA is then incorporated into the host cell genome by an
integrase enzyme, at which point the retroviral DNA is referred to as
a provirus. The host cell then treats the viral DNA as part of its own
genome, translating and transcribing the viral genes along with the
cell's own genes, producing the proteins required to assemble new
copies of the virus.
19
One drawback of retroviruses, such as the Moloney retrovirus,
involves the requirement for cells to be actively dividing for
transduction. As a result, cells such as neurons are very resistant to
infection and transduction by retroviruses.
But the airway cells which are affected by the disease cystic
fibrosis and must be targeted divide infrequently. Hence Retrovirus
is not a suitable vector for this disease.
Adenovirus
Adenoviruses (members of the family
Adenoviridae) are medium-sized (90
100 nm), nonenveloped (without an
outer lipid bilayer) viruses with
anicosahedral nucleocapsid
containing a double stranded DNA
genome.
20
virus. Herpes Simplex can be spread through contact with saliva,
such as sharing drinks.
But these viruses only affect the cells of the nervous system and
cannot infect the airway cells and hence not suitable.
Adeno-Associated Viruses
Hence it is the best choice for gene delivery in the case of Cystic Fibrosis.
1993
In 1993, the first experimental CF gene therapy treatment was given
to a patient with cystic fibrosis. Researchers modified a common
cold Adenovirus to act as a delivery vehicle by carrying normal
genes to the CFTR cells in the nasal passages. Researchers chose
nasal passages as the site of delivery because they are easier to
access and measure gene activity than the lung airway. Later trials
delivered the vector to patients lung airways.
21
In the earlier trials, it had looked like the virus had entered cells and
that the CTFR gene was working. But later trials with different
patients showed levels of VFTR gene activity that were too low to
make any difference. Researchers came to think that the adenovirus
cant easily enter airway cells, especially in the low doses that were
being given. In the earlier trials, they speculated, gene activity
resulted from the damage to the cells during delivery allowing the
virus to enter easily.
Hence when higher doses of the virus were tried, the immune
system of the patients started mounting immune responses and
fighting off the virus. This caused a blockage in the trials until 1998.
1998
Trials using Adeno-associated virus to deliver the CTFR gene began in
1998. Unlike the adenovirus, the Adeno-associated virus caused no
immune response or adverse side effects in patients.
But unlike the researchers predictions, the adeno-associated virus did not
enter cells efficiently and integrate into calls genomic DNA. They
produced only low and fleeting amounts of CFTR gene activity.
Researchers are still working to figure out what caused the viruses to fail.
Challenges
Some the factors that have kept gene therapy from becoming an
effective treatment for genetic diseases are:
22
and stable. Problems with integrating therapeutic DNA into the
genome and the rapidly dividing nature of many cells prevent
gene therapy from achieving any long-term benefits. Patients
will have to undergo multiple rounds of gene therapy.
Issues regarding
Gene Therapy
What are the possible implications of gene therapy research to
society? All of us - researchers, policymakers and the public - have a
23
responsibility to explore the potential effects of gene therapy
research on our lives so that we can make informed decisions.
What if we could alter human traits not associated with disease? Would it be
okay to use gene therapy to improve or enhance a person's genetic
profile?
24
Who will have access to gene therapy, treatments and long-term follow-
ups? Will gene therapy and genetic enhancements create an
advantage for those who can afford it?
Recent Upcoming
CRISPR
CRISPR stands for clustered
regularly interspaced short
palindromic repeats. These RNA
sequences serve an immune
function in archaea and bacteria,
but in the last year or so,
scientists have seized upon them
to rewrite genes. The RNA
sequence serves as a guide to
target a DNA sequence in, say, a
zygote or a stem cell. The guide
sequence leads an enzyme,
Cas9, to the DNA of interest. Cas9 can cut the double strand, nick it,
or even knock down gene expression. After Cas9 injures the DNA,
repair systems fix the sequence - or new sequences can be
inserted.
It isn't the first or only method of gene repair therapy thats been
developed, but the CRISPR technology, says Ramesar, is so special
because, unlike previous methods which were more laborious and
could only target one kind of cell in the body, it appears to be a "one
size fits all delivery", adaptable for different tissues. The procedure
also seems relatively simple to perform.
Exciting as the development may be, CRISPR wont be delivering
instant cures just yet.
Ramesar says, from his initial impressions of the literature, that it
would seem that localised, accessible abnormal tissue (as in the
retina or skin) could be targeted more easily.
25
Conditions affecting the body more systemically, however, such as
certain developmental syndromes, or central nervous system
disorders, might be problematic in terms of getting the repair
technology into enough of the target cells in that tissue to make an
effective difference.
"It may also depend on the stage one attempts to carry out the
therapy, in terms of the patients age and level of advancement of
the disease," says Ramesar.
Conclusion
Although early clinical failures led many to dismiss gene therapy as
over-hyped, clinical successes since 2006 have bolstered new
optimism in the promise of gene therapy. These include successful
treatment of patients with the retinal disease Leber's congenital
amaurosis, X-linked SCID, ADA-SCID, adrenoleukodystrophy, chronic
lymphocytic leukaemia (CLL),acute lymphocytic
leukaemia (ALL),multiple myeloma, haemophilia and Parkinson's
disease. These recent clinical successes have led to a renewed
interest in gene therapy, with several articles in scientific and
popular publications calling for continued investment in the field.
Bibliography
Websites
http://en.wikipedia.org/wiki/Gene_therapy
http://www.trip2medi.com/treatmentCGeneTherapy.php
26
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
27