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By - Jayasmita Mahapatra (IGE/IGMGS)

Genetic Modification



Direct manipulation of an organism's genome
One of the most promising uses for genetic
modification being eyed in the future is on the field
of medicine
...

The process involves a group of
technologies that enable the intentional
transfer of specific exogenous genetic
information into cells and the
application of these technologies for
pharmaceutical development
...

The idea behind this technology is to treat disease by the
administration of DNA (rather than a drug), which will
produce an appropriate amount of gene product (usually a
protein) to correct the condition
...


In a broad sense therefore, gene therapy can be viewed as a
natural progression in the application of biomedical science to
medicine
...


Gene therapy



The term “gene delivery” or “polynucleotide
delivery” has been the preference of many, rather
than “gene therapy,” which might tend to indicate a
proven therapeutic benefit and be reserved for
clinical medicine
...


In the tissue, the genetic material should be efficiently endocytosed
across the lipid bilayer and directed to the host cell nucleus
...


Gene Therapy
It derives its name from the idea that DNA can be
used to supplement or alter genes within an
individual's cells as a therapy to treat disease
1
...


3
...


It is the most common approach
The abnormal gene would be swapped
by homologous recombination
Would cause a return to normal function
Control expression of genes, similar to
epistasis, when one gene affects the
expression of another gene

Approaches For Gene Therapy


Germ-line Gene Therapy
It produces a permanent transmissible
modification
 Might be achieved by gamete modification




Somatic Gene Therapy
It aims to modify specific cells or tissue in
patient
 All current Gene Therapy trails and protocols
are Somatic cell Therapy


Somatic gene therapy

Germ-line Gene Therapy






In the case of germ
line gene therapy, germ cells,
i
...
, sperm or eggs, are
modified by the introduction
of functional genes, which are
ordinarily integrated into their
genomes
the change due to therapy
would be heritable and would
be passed on to later
generations
Banned in many countries for
ethical reasons







In the case of somatic gene
therapy, the 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
patient's offspring or later
generations
It represents the mainstream
line of current basic and
clinical research to treat a
disease





Somatic gene therapy is a logical and natural progression in the
application of fundamental medical science to medicine and
offers great potential in the long term for the management and
correction of inherited/acquired human disorders, cancers, and
acquired immunodeficiency syndrome (AIDS)
...


Steps involved in the Development of GT
in Humans
1
...


3
...


In vitro experiments and
research
on
laboratory
animals (Pre-clinical trials)
Phase I trials with a small
number (5-10) of human
subjects to test safety of the
product
Phase II trials with more
human subjects to assess
whether the product is helpful
Phase III trials in large human
samples for a final and
comprehensive analysis of the
safety and efficacy of the
product

Phases of Gene Therapy Clinical Trials

Recombinant DNA Advisory Committee (RAC) is the
supervisory body of the National Institute of Health, USA, that
clears proposals on experiments involving Gene Therapy
A list of some important diseases, which are currently under various
stages of gene therapy trials

Disease
-Severe combined immunodeficiency (SCID)
-Cystic Fibrosis
-Hemophilia B
-Thalassemia
-AIDS
-Head and Neck Cancer
-Breast Cancer

Gene Therapy
Adenosine Deaminase
CFTR
Factor IX
alpha & beta Globin
rev and env
p53
Multidrug resistance I

Gene Augmentation Therapy (GAT)

1
...
Targeted killing of specific cells


Genes are directed to the target cells and then expressed so as to cause
cell killing (Popular in cancer gene therapies)



inserted genes are expressed to produce a lethal toxin (suicide genes)



Indirect cell killing uses immunostimulatory genes to provoke or
enhance an immune response against the target cell

3
...
Targeted mutation correction


Done at different levels: at the gene level (e
...
by gene targeting
methods based on homologous recombination); or at the RNA
transcript level (e
...
by using particular types of therapeutic
ribozymes or therapeutic RNA Editing)

Targeted Inhibition


Ribozymes




can cleave (or repair) mRNA

Triple helix

Oligonucleotides




Antisense oligos




block gene transcription
block mRNA translation

SiRNA (Knockdown)

Techniques to prevent the production
of a mutated protein

Ribozymes




RNA molecules that act as enzymes
...
E
...
, spliceosomes are believed to be a type
of ribozyme
Ribozyme gene therapy involves the following steps:
 Delivery of RNA strands engineered to function as ribozymes
...


2
...


The genetic material is transferred directly into the
body of the patient
More or less random process; small ability to
control; less manipulations
Only available option for tissues that can not be
grown in vitro; or if grown cells can not be
transferred back

IN VIVO GENE THERAPY
•

•

•

Liver, muscle, skin, spleen, lung, brain and blood cells
Gene delivery can be carried out by viral or non
viral vector systems
The success depends on:
–

–

–

The efficiency of uptake of the therapeutic gene by
target cells
Intracellular degradation of gene and its uptake by
nucleus
The expression capability of the gene

IN VIVO GENE THERAPY


In vivo gene therapy research focuses on perfecting
specific vectors for targeted cells and tissues,
increasing the efficiency of gene transfer, increasing
the consistency of gene delivery to an exact
location in the patient’s DNA sequence and
researching new disease-fighting applications for
this technique
...
g
...
Disorders inherited on
the X chromosome appear only in males
...
This means she
may pass the mutated gene on to her children
...
(In other words, the child has two copies of the same
gene mutation
...

In most cases, no one knows what causes the mutation to appear the first
time
...


Types of SCID


There are several types of SCID
...
It is inherited on the X chromosome and
appears only in boys

ADA deficiency SCID

— About 15% of patients with
SCID have adenosine deaminase (ADA) deficiency
...
Lack of ADA leads to low
numbers of T cells and B cells
...
These cells were then injected
back into her body, and began to express a normal enzyme
...

 Transgene can result into continuous production of a
therapeutic protein that normally has a short half life
...

 Gene therapy can improve patient's compliance and
decrease cost of therapy on long term bases












Understanding of the disease process
Structure/function of gene to be introduced
Efficient delivery of gene
Control of gene expression
Prevention/control of immune responses
Animal model & assessment of function
Clinical trial



To be continued

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