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Gene Therapy and Viral Vector
Lecture 1
"We used to think that our fate was in our
stars, but now we know that, in large
measure, our fate is in our genes, "quotes
James Watson.
Genes, DNA, Alleles
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Our genetic program is made up of thousand of genes,
stretches of DNA, that generally code for different
proteins that do particular jobs in the cells in our body.
The process of decoding a gene into a protein occurs in
two basic stages:
‘Transcription', where specific mRNA molecules are
generated from the DNA stretch concerned; and
‘Translation', where the specific mRNA molecules are
used as a template to make the specific protein.
For any particular gene there are usually two copies
(sometimes the term 'allele' is used instead of copy), one
from the mother and a second passed on from the
father. For most genes, only one normal copy is required
for normal function.
Genetic Disorders
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When genes
are altered so
that encoded
proteins are
unable to
carry out
their normal
functions,
genetic
disorders can
result.
Types of Mutations
Substitution: is a mutation that exchanges one
base for another (i.e., a change in a single
"chemical letter" such as switching an A to a G).
 Insertions: are mutations in which extra base
pairs are inserted into a new place in the DNA.
 Deletions: are mutations in which a section of
DNA is lost, or deleted.
 Frameshift: Since protein-coding DNA is divided
into codons three bases long, insertions and
deletions can alter a gene so that its message is
no longer correctly parsed. These changes are
called frameshifts.
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Classification of Genetic Disorders
Level 1: Single Gene Disorders
 Disorders which result when a mutation causes
the protein product of a single gene to be altered,
differentiated, or missing.
Level 2: Chromosome Abnormalities
 Entire or whole chromosomes, or large segments
of the chromosomes, are missing, duplicated, or
altered.
Level 3: Multifactorial Disorders
 Multifactorial disorders are those which result
from mutations in multiple genes. They’re
complex, often coupled with environmental
causes.
Gene Therapy
Definition:
1.‘The use of genes as medicines’
2. The introduction of normal genes into
cells in place of missing or defective ones in
order to correct genetic disorders
GT ‘will be’ used to treat genetic disorders,
viral infections and mutations in cancer.
How it is done?
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.
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Objectives of a successful GT
Successfull gene therapy should be:
 Efficient (determines the efficacy of gene
delivery)
 Cell Specific
 Safe
“One of the challenges of gene therapy is
the efficient delivery of genes to target cells.
Although the nucleic acids containing the
genes can be generated in the laboratory
with relative ease, the delivery of these
materials into a specific set of cells in the
body is far from simple”.
Basic concept
What are the ways to deliver the
genes?
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Ex-vivo Gene Therapy - In ex-vivo gene therapy, cells are first
cultured or synthesized outside of an organism (for gene therapy),
and then inserted into the organism to provide the treatment. Exvivo gene therapy is more common than in-vivo therapy. Ex-vivo
therapy significantly reduces many risks involved with gene therapy.
At the same time, however, ex-vivo gene therapy holds some
limitations - indirectly introducing the desired-containing cells into
an organism may trigger immune responses. The cells also may not
function as desired, malfunction, or not entirely work at all.
In-vivo Gene Therapy - In in-vivo gene therapy, the gene is
directly delivered to the organism, such as through a vector or
other means. In-vivo gene therapy is less commonly used than exvivo gene therapy. In-vivo gene therapy holds more risks than its
ex-vivo counterpart, such as a possible immune reaction from the
organism.Vectors used in in-vivo gene therapy include viruses,
bacterial plasmids, nanoparticles, and more.
Types of gene therapy
There are two different types of gene therapy
depending on which types of cells are treated:
 Somatic gene therapy: transfer of a section of
DNA to any cell of the body that doesn’t
produce sperm or eggs. Effects of gene therapy
will not be passed onto the patient’s children.
 Germline gene therapy: transfer of a section
of DNA to cells that produce eggs or sperm.
Effects of gene therapy will be passed onto the
patient’s children and subsequent generations.
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Somatic Gene Therapy
Advantages - Somatic gene therapy is relatively
effective as a procedure. Many developments, such
as possible treatments and cures, have been
developed using gene therapy. Also, somatic gene
therapy is significantly less controversial than
germ line gene therapies.
 Disadvantages - If a treatment or cure is
successful (or any other trait modification using
somatic gene therapy, for that matter), it will not
be passed on to the patient/organism's offspring.
In addition, the methodology of somatic gene
therapy, such as the use of viral vectors, is difficult.
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Germline gene therapy
Advantages - Germ line gene therapy is done
before the organism has grown or developed;
therefore, the cure is inherited by future generations
of that organism. Also, germ line gene therapy allows
for a desired gene to become fully incorporated into
the organism before activation; therefore, one of the
factors influencing unwanted immune responses (to
the gene) is removed.
 Disadvantages - Germ line gene therapy is very
controversial. In addition, due to this controversial
nature and other limiting factors, germ line gene
therapy is not fully pursued for development. Germ
line gene therapy also holds numerous risks, such as a
margin for possible error during the gene 'transplant‘.
Banned in countries throughout the world
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Types of Gene Therapy
Gene Therapy Techniques
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1. Gene augmentation therapy
This is used to treat diseases caused by a mutation that stops
a gene from producing a functioning protein.
This therapy adds DNA containing a functional version of the
lost gene back into the cell.
The new gene produces a functioning product at sufficient
levels to replace the protein that was originally missing.
This is only successful if the effects of the disease are
reversible or have not resulted in lasting damage to the body.
For example, this can be used to treat loss of function
disorders such as cystic fibrosis by introducing a functional
copy of the gene to correct the disease.
Cell with non-functioning gene
Cell with functioning gene
2. Gene inhibition therapy
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Suitable for the treatment of infectious diseases, cancer and
inherited disease caused by inappropriate gene activity.
The aim is to introduce a gene whose product either:
inhibits the expression of another gene
interferes with the activity of the product of another gene.
The basis of this therapy is to eliminate the activity of a gene
that encourages the growth of disease-related cells.
For example, cancer is sometimes the result of the overactivation of an oncogene (gene which stimulates cell
growth). So, by eliminating the activity of that oncogene
through gene inhibition therapy, it is possible to prevent
further cell growth and stop the cancer in its tracks.
3. Killing of specific cells
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Suitable for diseases such as cancer that can be
treated by destroying certain groups of cells.
The aim is to insert DNA into a diseased cell that
causes that cell to die.
This can be achieved in one of two ways:
the inserted DNA contains a “suicide” gene that
produces a highly toxic product which kills the
diseased cell
the inserted DNA causes expression of a protein that
marks the cells so that the diseased cells are attacked
by the body’s natural immune system.
It is essential with this method that the inserted
DNA is targeted appropriately to avoid the death of
cells that are functioning normally.
How is DNA transfer done?
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A section of DNA/gene containing
instructions for making a useful protein is
packaged within a vector, usually a virus?,
bacterium? or plasmid? Or nanoparticles.
The vector acts as a vehicle to carry the
new DNA into the cells of a patient with a
genetic disease.
Once inside the cells of the patient, the
DNA/gene is expressed by the cell’s normal
machinery leading to production of the
therapeutic protein and treatment of the
patient’s disease.
Challenges of Gene Therapy
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Delivering the gene to the right place and
switching it on:it is crucial that the new gene
reaches the right cell
delivering a gene into the wrong cell would
be inefficient and could also cause health
problems for the patient
even once the right cell has been targeted
the gene has to be turned on
cells sometimes obstruct this process by
shutting down genes that are showing
unusual activity.
Avoiding the immune response:
The role of the immune system is to fight off
intruders.
 Sometimes new genes introduced by gene
therapy are considered potentially-harmful
intruders.
 This can spark an immune response in the
patient, that could be harmful to them.
 Scientists therefore have the challenge of
finding a way to deliver genes without the
immune system ‘noticing’.
 This is usually by using vectors that are less
likely to trigger an immune response.
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Making sure the new gene doesn’t
disrupt the function of other genes:
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Ideally, a new gene introduced by gene
therapy will integrate itself into the genome
of the patient and continue working for the
rest of their lives.
There is a risk that the new gene will insert
itself into the path of another gene,
disrupting its activity.
This could have damaging effects, for
example, if it interferes with an important
gene involved in regulating cell division, it
could result in cancer.
The cost of gene therapy:
Many genetic disorders that can be
targeted with gene therapy are extremely
rare.
 Gene therapy therefore often requires an
individual, case-by-case approach. This may
be effective, but may also be very
expensive.
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