Gene Therapy

GENE THERAPY
A Molecular Medicine
Presented By
HUIDROM PRADIPKUMAR SINGH
MSc III Sem (BOTANY)
MANIPUR UNIVERSITY
Gene therapy is a novel treatment method which utilizes genes or short oligonucleotide sequences as therapeutic molecules.
This technique is widely used to treat those defective genes which contribute to disease development.
Many diseases such as ADA-SCID, X-linked SCID, Leber's congenital amaurosis (a retinal disease), Parkinson's disease, multiple
myeloma, chronic and acute lymphocytic leukemia, adrenoleukodystrophy have reported of successful clinical trials.
TYPES OF GENE THERAPY
1. Somatic gene therapy 2. Germ line gene therapy
Somatic gene therapy Germline gene therapy

Therapeutic genes transferred into the Therapeutic genes transferred into the
somatic cells (bone marrow cells, blood germ cells. (eggs and sperms).
cells, skin cells etc.)
Will not be inherited later generations. It is heritable and passed on to later

generations.
all researches directed to correct genetic For safety, ethical and technical reasons,
defects in somatic cells. it is not being attempted at present.
METHODS OF GENE THERAPY
1.Transfer of genes into patient cells outside the body (ex vivo gene therapy)
2.Transfer of genes directly to cells inside the body (in vivo gene therapy).
IN VIVO GENE THERAPY
❑ Direct delivery of therapeutic gene into

target cell into patients body.
❑ Requires viral or non viral vector

systems.
❑ It can be the only possible option in

patients where individual cells
cannot be cultured in vitro in
sufficient numbers (e.g. brain cells).
Targeted Delivery
VECTORS : Tools for Genetic Delivery
❑ Facilitate the transfer of genetic information into a cell.
❑ Vectors can be divided into Viral and Nonviral delivery systems.
1. Viral vectors
❑ Viral vectors are built using a blueprint of a virus—not the actual virus itself.
❑ Currently, viral vectors are the most common vehicle used in FDA-approved gene
therapies.(retrovirus, adenovirus and adeno associated virus (AAV).
Can viral vectors cause a viral infection?

Scientists use only certain parts or components of the virus, and they do not use
parts of the gene that cause an infection.
RETROVIRAL VECTORS
❑ Retroviruses are comprised of two copies of a positive single-stranded
RNA(genome of 7 to 10kb).
❑ Their RNA genome is copied into double-stranded DNA,

which integrates into the host cell chromosome and is stably maintained.
❑ Can carry a DNA of size – less than 3.4kb.The recombinant retroviruses have the
ability to integrate into the host genome in a stable fashion.
Adenoviral vectors
❑ The adenovirus is a 36-kb double-stranded linear DNA virus that replicates extrachromosomally
in the nucleus.
❑ Used to transfer genes in vivo into the lung, liver, muscle, blood vessel, synovium, eye, peritoneum, brain, and
tumors in animals.
Major limitation :
❑ the transgene expression is less than one month primarily due to an
immune response to the remaining viral proteins. This targeted specific immune
response rapidly eliminates the transduced cells.
❑ This immune response can also result in severe inflammation at the site of delivery and organ dysfunction.
❑ Furthermore, the vigorous host immune response to the surface proteins of the adenovirus
diminishes the efficacy of repeat administration.
Potential risks :
❑ Development of organ inflammation and dysfunction due to the immune response to adenoviral vector
transduced cells,
❑ Development of tolerance to an adenoviral vector that could result in fulminant disease upon infection
with wild-type virus, and
❑ Development of wild-type virus.
ADENOVIRUS-ASSOCIATED VECTOR
❑ Adenovirus-associated virus (AAV) is a 4.7-kb single stranded DNA virus that
replicates in the nucleus in the presence of helper virus(adenovirus) and integrates
into the chromosome to establish a latent state.
❑ AAV vectors has the ability to transduce nondividing cells.
❑ They are use to transfer genes into a variety of cell types including
hematopoietic stem cells in vitro and hepatocytes, brain, retina, lung, skeletal,
and cardiac muscle in vivo.
❑ Stable expression has been observed for up to one year in several organs.
Potential risks
(1) insertional mutagenesis, (2)generation of wild-type AAV, and (3) administration of
contaminated adenovirus.
Non-viral vectors
Physical methods
It allow genetic materials directly delivered to target cells (Electroporation, Gene Gun, Microinjection, Sonoporation, Photoporation,
etc. ).
Microinjection
❑ Used to introduce DNA or other genetic materials directly into
a cell by using a fine glass micropipette.
❑ This method is widely used for gene transfection in

mammals and it involves delivery of foreign DNA into a
living cell (egg, oocyte, embryos of animals)
❑ No requirement of a marker gene.

❑ It can be used for creating transgenic organisms,
particularly mammals.
Gene gun/Particle Bombardment Method:
a high velocity metal particles (gold or tungsten) that are coated with DNA is use to deliver biologically active DNA into
target cells.
Advantages
Advantages of Gene Gun:
•This method is easy to use, fast and versatile;
•Effective transformation requires only a small amount of nucleic acid and few cells;
•High levels of co-transformation and codelivery of multiple plasmids are possible;
•Can transfer large DNA fragments as well as small interfering RNA (siRNA) for gene
silencing;
•Can transfect many cell types, including non-dividing cells and plant cells;
•Avoid potentially toxic treatments (such as from viruses or chemical and lipid-based
systems).
Chemical means:
Certain charged chemical compounds like Calcium phosphates are mixed with functional cDNA
of desired function.The mixture is introduced near the vicinity of recipient cells.The chemicals
disturbs the cell membrane, widens the pore size and allows cDNA to pass through the cell.
LIPOFECTION :It is a technique used to inject genetic materials into a cell by means of liposomes
(artificial phospholipid vesicles).
ETHICAL ISSUES:
1.Safety: There's a risk of unintended consequences, such as triggering cancer or causing immune responses
against the therapy.
2.Equitable Access: Gene therapy can be expensive and inaccessible to many people, leading to concerns about
disparities between the wealthy and the less privileged.
3.Germline Editing: Editing genes in germline cells (sperm, eggs, embryos) raises ethical questions. Altering the
germline could have permanent effects on future generations, potentially introducing unintended genetic
changes into the human gene pool.
4.Dignity and Identity: Manipulating genes raises philosophical questions about human dignity and identity.
5.Unintended Consequences: Introducing genetically modified organisms into the environment, even for
therapeutic purposes, can have unpredictable ecological consequences.
REFERENCES:
PRINCIPLES OF MOLECULAR MEDICINE J. LARRY JAMESON
An introduction to MOLECULAR MEDICINE AND GENE THERAPY Thomas

F. Kresina, 2001 by Wiley-Liss, Inc.
Online Resources
GENEHOME: https://www.thegenehome.com/how-does-gene-therapy-work/vectors
SCIENCEDIRECT: https://www.sciencedirect.com/topics/biochemistry-genetics-and-
molecular-biology/retrovirus-vector
THANK YOU

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GENE THERAPY

Somatic gene therapy Germline gene therapy

Will not be inherited later generations. It is heritable and passed on to later

❑ Direct delivery of therapeutic gene into

❑ Requires viral or non viral vector

❑ It can be the only possible option in

Can viral vectors cause a viral infection?

❑ Their RNA genome is copied into double-stranded DNA,

❑ AAV vectors has the ability to transduce nondividing cells.

❑ This method is widely used for gene transfection in

❑ No requirement of a marker gene.

An introduction to MOLECULAR MEDICINE AND GENE THERAPY Thomas

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