Unit 2 - Molecular and genetic factors in disease
16
Lecture 4 - Gene Therapy
Aim of the lecture
After completion of this lec the student must know
1. The concept of Gene therapy
2. Types of Vector
3. Types of gene therapy
4. Concept of personalized Medicine &Pharmacogenomics
Gene therapy is a form of treatment that involves
introducing genetic material into a person’s cells to fight
or prevent disease A gene can be delivered to a cell using
a carrier known as a “vector.” The most common types of
vectors used in gene therapy are viruses. The viruses used
in gene therapy are altered to make them safe, with
introducing a therapeutic gene in the vector which will be
then transferred to the patient.
Although some risks still exist with gene therapy. The
technology has been used with some success.
Gene therapy for had been tried for a number of diseases,
such as severe combined immunodeficiencies,
hemophilia ,Parkinson's disease, cancer and even HIV.
Several approaches to gene therapy are being tested,
including:
Replacing a mutated gene that causes disease with a
healthy copy of the gene
Inactivating, or “knocking out,” a mutated gene that is
functioning improperly
Introducing a new gene into the body to help fight a
disease
In general, a gene cannot be directly inserted into a
person’s cell. It must be delivered to the cell using a
carrier, or vector.
Vector systems can be divided into:
1) Viral vectors
2) Non-viral vectors
The most common type of vectors is viruses that have
been genetically altered to carry normal human DNA .
Viruses have evolved a way of encapsulating and
delivering their genes to human cells in a pathogenic
manner. It has been tried to use this ability by
manipulating the viral genome to remove disease-causing
genes and insert therapeutic genes.
Target cells such as the patient's liver or lung cells are
infected with the vector. The vector then unloads its
genetic material containing the therapeutic human gene
into the target cell. The generation of a functional protein
product from the therapeutic gene restores the target cell
to a normal state.
Gene therapy can be split into two categories:
1)
, which means exterior (where cells are modified
outside the body and then transplanted back in again). In
some gene therapy clinical trials, cells from the patient’s
blood or bone marrow are removed and grown in the
laboratory. The cells are exposed to the virus that is
carrying the desired gene. The virus enters the cells and
inserts the desired gene into the cells’ DNA. The cells grow
in the laboratory and are then returned to the patient by
injection into a vein. This type of gene therapy is called ex
vivo because the cells are treated outside the body.
2)
, where genes are changed in cells still in the body,
This form of gene therapy is called in vivo, because the
gene is transferred to cells inside the patient’s body.
Types of Gene Therapy
All cells in the human body contain genes, making them
potential targets for gene therapy. However, these cells
can be divided into two major categories: somatic
cells (most cells of the body) or cells of
the germline (eggs or sperm). In theory it is possible to
transform either somatic cells or germ cells.
1)
Gene therapy using germ line cells
results in
permanent changes that are passed down to subsequent
generations. The application of germ line gene therapy is
its potential for offering a permanent therapeutic effect for
all who inherit the target gene. Successful germ line
therapies introduce the possibility of eliminating some
diseases from a particular family, and ultimately from the
population, forever. However, this also raises controversy.
Some view this type of therapy as unnatural, Others have
concerns about the technical aspects. They worry that the
genetic change propagated by germ line gene therapy may
be harmful, with the potential for unforeseen negative
effects on future generations.
2)
Somatic cell therapy
is more conservative and safer
approach because it affects only the targeted cells in the
patient, and is not passed on to future generations.
However, the disadvantage of this type of therapy is that
the effects of somatic cell therapy are short-lived. Because
the cells of most tissues ultimately die and are replaced by
new cells, repeated treatments over the course of the
individual's life span are required to maintain the
therapeutic effect. Transporting the gene to the target cells
or tissue is also problematic. Regardless of these
difficulties, however, somatic cell gene therapy is
appropriate and acceptable for many disorders,
including cystic fibrosis, muscular dystrophy, cancer, and
certain infectious diseases, the results of any somatic gene
Unit 2 - Molecular and genetic factors in disease
17
therapy are restricted to the actual patient and are not
passed on to his or her children. All gene therapy to date
on humans has been directed at somatic cells, whereas
germline engineering in humans remains controversial .
Choices of Vectors
The ideal vector should have the following characteristics:
1) An adequate carrying capacity (some genes are large) .
2) To be undetectable by the immune system.
3) To be non-inflammatory.
4) To be safe to the patients.
5) Efficient
6) To have long duration of expression and or the ability
to be safely readministred.
Viral Vectors
Viruses attack their hosts and introduce their genetic
material into the host cell as part of their replication cycle.
This genetic material contains basic 'instructions' of how to
produce more copies of these viruses, 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 genes into the
host's genome. This incorporates the genes of that virus
among the genes of the host cell for the life span of that
cell.Viruses like this could be used as vehicles to carry
'good' genes into a human cell. First, the virus genes that
cause disease must be removed then those genes are
replaced with genes encoding the desired effect
This procedure must be done in such a way that the genes
which allow the virus to insert its genome into its host's
genome are left intact.
Non-Viral Vectors
Non-viral methods present certain advantages over viral
methods, with simple production and low host
immunogenicity. Previously, their disadvantage was low
levels of transfection and expression of
the gene however, recent advances in vector technology
have generate molecules and techniques with transfection
efficiencies similar to those of viruses.example: .Naked
DNA &Liposome
1) Naked DNA
This is the simplest method of non-viral transfection,
intramuscular injection of a naked DNA plasmid have
occurred with some success; however, the expression has
been very low in comparison to other methods of
transfection. In addition to trials with plasmids, there have
been trials with naked PCR product, which have had
similar or greater success. This success, however, does
not compare to that of the other methods, leading to
develop more efficient methods for delivery of the naked
DNA such the use of a "gene gun", which shoots DNA
coated gold particles into the cell using high pressure gas.
2) Liposome
Liposome is an artificial lipid sphere with the therapeutic
DNA in the aqueous core it is capable of passing the DNA
through the cell membrane
Advantage of non-viral vector
• DNA/lipid complexes are easy to prepare
• there is no limit to the size of genes that can be delivered
• Carrier systems lack proteins; they may evoke much less
immunogenic responses.
• Much less risk of generating the infectious form or
inducing tumorigenic mutations because genes delivered
have low integration frequency and cannot replicate or
recombine.
Antisense therapy
Oligonucleotides
Is a form of treatment for genetic disorders or infections.
When the genetic sequence of a particular gene is known
to be cause of a particular disease, it is possible to
synthesize a strand of nucleic acid (DNA, RNA or a
chemical analogue) that will bind to the messenger RNA
(mRNA) produced by that gene and inactivate it,
effectively turning that gene "off
The use of synthetic oligonucleotides in gene therapy is to
inactivate the genes involved in the disease process. There
are several methods by which this is achieved. One
strategy uses antisense specific to the target gene to
disrupt the transcription of the defective gene.
Another strategy uses double stranded
oligodeoxynucleotides as a trick for the transcription
factors that are required to activate the transcription of the
target gene. The transcription factors bind to the it instead
Unit 2 - Molecular and genetic factors in disease
18
of the promoter of the defective gene, which reduces the
transcription of the target gene, lowering expression.
Problems with vectors:
1) The new gene might be inserted in the wrong location in
the DNA, possibly causing harmful mutations to the DNA
or even cancer.
2) 2.The possibility that transferred genes could
be overexpressed,producing so much of the
missing protein as to be harmful;
3) The viral vector could cause an immune reaction;
4) The viral vector could be transmitted from the patient to
other individuals or into the environment
• Current uses of gene therapy focus on treating or curing
existing conditions. In the future, the focus could shift to
prevention. As more of the human genome is understood,
medicine will know more about which genes contribute to
or cause disease. With that knowledge in hand, gene
therapy could be used to head off problems before they
occur
Is Gene therapy totally safe ???
Although gene therapy is a promising treatment option for
a number of diseases (including inherited disorders, some
types of cancer, and certain viral infections), the technique
remains risky and is still under study to make sure that it
will be safe and effective. Gene therapy is currently only
being tested for the treatment of diseases that have no
other cures
How can the patients receive: "the right medication, in
the right amount, in the right form, at the right time,
for the right disease
The answer is by the Personalized Molecular Medicine
Pharmacogenomics
Genetic testing for assessment of drug response may
predict the best specific drugs and dosages for individual
patients based on genetic profiling: so-called
‘personalized medicine’
Polymorphic mutations within genes can affect individual
responses to some drugs, such as loss-of-function mutations
Example:CYP2D6 gene is part of a large family of highly
polymorphic genes encoding cytochrome P450 proteins,
mostly expressed in the liver, which determine the
metabolism of a host of specific drugs. Polymorphisms in
the CYP2D6 gene determine codeine activation, while
those in the CYP2C9 gene affect warfarin inactivation
This. Polymorphisms in these and other drug genes
determine the persistence of drugs and, therefore,should
provide information about dosages and toxicity
Pathway medicine
The ability to manipulate pathways that have been altered
in genetic disease has therapeutic potential for Mendelian
disease, but a firm understanding of both disease
pathogenesis and drug action at a biochemical level is
required. An example of this has been the discovery that
the vascular pathology associated with Marfan’s
syndrome is due to the defect in fibrillin molecules
causing up-regulation of transforming growth factor
(TGF)- signalling in the vessel wall. Losartan is an
antihypertensive drug also acts as a partial antagonist of
TGF-signalling and is effective in preventing aortic
dilatation in Marfan’s syndrome