A brief introduction to gene therapy.

1. PRESENTED BY:
SMRITI BHARDWAJ
BIOTECHNOLOGY
HINDUSTAN COLLEGE OF SCIENCE AND TECHNOLOGY

2.  Introduction
 Techniques of Gene therapy
 Applications of Gene therapy
 Immunomodulating Gene therapy
 Cancer Immunotherapy
 Delivery Methods in Gene Therapy
 Conclusion

3.  Gene therapy is a technique in which DNA is introduced into
a patient to treat a genetic disease. The new DNA usually
contains a functioning gene to correct the effects of a
disease-causing mutation.
 Gene therapy may be a promising treatment option for
some genetic diseases, including muscular
dystrophy and cystic fibrosis.
 Gene therapy involves the insertion of a copy of a new
gene, modifying or inactivating a gene, or correcting a gene
mutation. This is done with the help of a vector derived
from a genetically modified virus. Several different viral
vectors are now used for this purpose.

4.  There are several techniques for carrying out gene therapy.
These include:
1.Gene augmentation therapy
 This is used to treat diseases caused by a mutation that stops
a gene from producing a functioning product, such as
a 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.

6.  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 over-
activation 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.

8.  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.

10. 1.METABOLIC DISORDERS
 Gene therapy for most metabolic disorders, unlike therapies
for diseases such as the hemoglobinopathies, will probably
not require strict gene regulation.
 Gene therapy has recently shown great promise as an effective
treatment for a number of metabolic diseases caused by genetic
defects in both animal models and human clinical trials.
 The first federally sanctioned clinical gene therapy trial was
performed at the National Institutes of Health in 1990 to treat
severe combined immunodeficiency caused by adenosine
deaminase deficiency (ADA-SCID).

11. Type of disorder
Glycogen storage
Gluconeogenesis
Galactose metabolism
Aminoacidopathies
Organic acidemias
Fatty acid metabolism
Urea cycle
Example
Glycogen storage
deficiency type 1A
Pepck deficiency
Galactosemia
Phenylketonuria
Maple syrup disease
Tyrosinemia type 1
Methylmalonic acidemia
MCAD
OTC
Citrullinemia
Underlying deficiency
Glucose-6-phosphatase
Phosphoenolpyruvate-
carboxykinase
Galactose-1-phosphate
uridyl transferase
Phenylalanine
hydroxylase
Methyl malonyl-CoA
mutase
Medium chain acyl CoA
dehyodrogenase
Ornithine
transcarbamylase
deficiency

12.  Endocrine disorders, primarily diseases of
the thyroid, parathyroids, pituitary, gonad,
and adrenal glands, constitute a major
health problem in all societies.
 As a result of improved insight into the
diagnosis, pathophysiology, and molecular
bases, it is now possible to diagnose the
disorders earlier in their development, and
the treatment of the deficiency states for
most hormones is successful.

13.  Type 1 diabetes mellitus is an autoimmune disease
resulting from the destruction of pancreatic β cells.
 Current treatments for patients with type 1 diabetes
mellitus include daily insulin injections or whole pancreas
transplant, each of which are associated with profound
drawbacks. Insulin gene therapy, which has shown great
efficacy in correcting hyperglycemia in animal models,
holds great promise as an alternative strategy to treat type
1 diabetes mellitus in humans.
 Insulin gene therapy refers to the targeted expression of
insulin in non-β cells, with hepatocytes emerging as the
primary therapeutic target.

15.  Isolated growth hormone deficiency type-2
(IGHD-2), the autosomal-dominant form of
GH deficiency, is mainly caused by specific
splicing mutations in the human growth
hormone (hGH) gene (GH-1).
 The current treatment of choice for GH-
deficient patients is injection with
recombinant human GH (rhGH). GH is
usually provided as bolus injections, as
frequently as once per day.

16.  Immunomodulating gene therapy has been
developed in an attempt to stimulate cellular
immune responses against tumour specific
antigens.
 One method is to genetically modify the tumour cell
to have it express cytokines ( that attract antigen-
presenting cells such as dendritic cells and
macrophages, and to activate a systemic T-cell
immune response.
 Another method is to genetically modify the tumour
cell itself to become a professional antigen-
presenting cell through the expression of MHC
molecules, co-stimulatory molecules such as B-7,
and cytokines genes necessary for T-cell activation
and proliferation.

17.  Immunotherapy is treatment that uses
certain parts of a person’s immune system
to fight diseases such as cancer. This can be
done in a couple of ways:
 Stimulating your own immune system to
work harder or smarter to attack cancer
cells.
 Giving you immune system components,
such as man-made immune system
proteins.

19.  The main types of immunotherapy now being used to treat
cancer include:
 Monoclonal antibodies: These are man-made versions of
immune system proteins. Antibodies can be very useful in
treating cancer because they can be designed to attack a very
specific part of a cancer cell.
 Immune checkpoint inhibitors: These drugs basically take
the ‘brakes’ off the immune system, which helps it recognize
and attack cancer cells.
 Cancer vaccines: Vaccines are substances put into the body
to start an immune response against certain diseases. We
usually think of them as being given to healthy people to help
prevent infections. But some vaccines can help prevent or
treat cancer.
 Other, non-specific immunotherapies: These treatments
boost the immune system in a general way, but this can still
help the immune system attack cancer cells.

20.  The transfer of genetic material can be
accomplished in vivo through local or systemic
inoculation or ex vivo where the target of
interest is collected and modified outside of the
organism before return to the host.
 Gene transfer via the viral vectors is called
transduction while transfer via the non-viral
vectors is called transfection.
 Retroviruses are RNA viruses that carry a gene
for a reverse transcriptase that transcribes the
viral genetic material into a double stranded
DNA intermediate.

21.  The plasmid DNA of interest is susceptible to rapid
degradation by biological enzymes necessitating
that it be packaged for protection
 Synthetic delivery systems accomplish this using
polycationic polymers and/or cationic lipids which
may be complexed with nucleic acid (polyplex,
liploplex or lipopolyplex systems).
 The use of cationic packaging takes advantage of
the anionic properties of DNA to form an
electrostatic bond between the plasmid of interest
and the protective coating.
 The final DNApackaging construct usually measures
between 100 nanometers to 1 micrometer.

22.  An important considering in gene therapy is
ensuring that the pharmacophore is delivered
to an area that maximises its therapeutic
benefit.
 This can be especially complex in the living
organism due to shared receptors between
tissues, circulatory anomalies and ability of
serum proteins to destabilise synthetic vector
complexes.
 Other targeting techniques include altering the
charge of the synthetic vector-DNA plasmid
particle: cationic liposomes have been shown to
preferentially distribute to the lung after
systemic administration.