Gene therapy involves inserting a normal gene into a patient's cells to replace a defective gene that causes disease. There are two main types of gene therapy: somatic cell therapy, which treats the individual patient, and germline therapy, which could affect future generations. The two most common methods of delivery are ex vivo therapy, where cells are removed and modified before being returned, and in vivo therapy through direct injection. Retroviruses and adenoviruses are the most widely used vectors for transporting genes, but each has advantages and disadvantages for successful gene expression and integration. While promising for treating many diseases, gene therapy still faces challenges from issues like gene silencing, immunogenicity, and potential safety risks that require more research.

1. Dr. IFTHAQAR. H. F

2.  Definiton: An experimental technique for
correcting defective genes that are
responsible for diseases by replacing
them.
 Several approaches to gene therapy:
1. Inserting a normal gene to replace an
abnormal gene.
2. Inactivating, or “knocking out,” a mutated
gene that is functioning improperly.
3. Introducing a new gene into the body to help
fight a disease.

3. STEPS IN GENE THERAPY:
 Identification of the defective gene.
 Cloning of normal healthy gene.
 Identification of target cell / tissue / organ.
 Insertion of the normal functional gene into
the host DNA.

4. PRINCIPAL:
Introduction of FUNCTIONAL GENES into
appropriate cells
Transferred gene (TRANSGENE)
encodes & produces proteins
The Proteins encoded by Transgene
corrects the disorder

5. TYPES OF GENE THERAPY:
1. SOMATIC CELL THERAPY
2. GERM LINE THERAPY

6. 1.SOMATIC CELL THERAPY:
 Insertion of therapeutic gene into somatic
cells.
 like fibroblasts, myoblasts, epithelial cells,
nervous cells, glial cells etc.
 This can correct the genetic defect in the
patient.
 Transgene cannot be passed on to the
siblings.

7. 2. GERMLINE THERAPY:
 Introduction of the foreign gene into germ
cells like sperm / ovum / fertilized egg.
 Results in expression of modified features in
both somatic as well as germ cells of the
offspring.
 Considered unethical, and is not advocated
in humans.

8. TECHNIQUE OF GENE
THERAPY
1. Ex vivo
2. In vivo
Two distinct strategies are used to achieve long-term
gene expression:
one is to transduce stem cells with an integrating
vector, so that all progeny cells will carry the
donated gene;
the other is to transduce long-lived cells such as
skeletal muscle or neural cells

9. 1. Ex vivo approach:
-Target cells are removed from the
body and grown in vitro.
-The gene is then introduced into the
cultured cells.
-These cells are then re-introduced
into the same individual.
-Examples: Fibroblast cells,
Hematopoietic cells.

11. 2. In vivo approach: (Direct Gene
Transfer)
-Cloned therapeutic gene is introduced
directly into the affected tissue.
-Specially designed vehicles are
needed.
-Examples are: Lungs, Brain

13. DELIVERY:
1. PHYSICAL METHODS: carrier-free gene delivery
-Parenteral injection
-Microinjection
-Electroporation
-Gene gun
2. CHEMICAL METHODS: synthetic vector-based
gene delivery
-Calcium phosphate
-Liposomes
3. BIOLOGICAL METHODS:
Viral Vectors like
-Retrovirus -Adenovirus
-HSV -AAV
Non
viral
vectors
viral vectors

14. To be successful, a vector
must:
 TARGET the right cells. If you want to deliver a
gene into cells of the liver, it shouldn't wind up in
the big toe.
 ACTIVATE the gene. A gene must go to the cell's
nucleus and be "turned on," & the protein that is
produced must function properly.
 INTEGRATE the gene in the cells. You need to
ensure that the gene integrates into the host cell's
genetic material.
 AVOID harmful side effects.

15. COMMON VECTORS USED FOR
GENE THERAPY:
1.Retro viruses
2. Adeno viruses
3. Liposomes
'gag', 'pol' &
'env' genes
replaced by the
genes to be
transferred.

16. 1. RETRO VIRUSES:
 Retroviruses are used ONLY in EX VIVO
THERAPY.
Advantages:
 Chromosomal integration & stable
modification of target cells.
Disadvantages:
 Uncontrolled integration; May be oncogenic.
 Cannot infect non-dividing cells.

18. 2. ADENO VIRUSES:
 Second most commonly used.
Advantages:
 Can infect non-dividing cells, thus suitable for gene
therapy of Cystic fibrosis, DMD.
 Can be produced at high titres in cultures.
 Non-integration to chromosome. Avoids the risks
of uncontrolled integration.
Disadvantages:
 Transient expression of gene due to episomal
integration.
 Provokes immune response.
Alternative & better – AAV for 3 reasons

21. 3.LIPOSOMES:
 These are lipid bilayers surrounding an
aqueous vesicle.
 Can be used to introduce foreign DNA into a
target cell.
Advantages:
 Safer when compared to Viral vectors.
 Can carry large DNA molecules.
Disadvantages:
 Inefficient transfer.
 Transient expression.

23. Possible applications of Gene
Therapy:
 Cystic fibrosis: The limitation is that airway
epithelial cells are rapidly shed off.
 Severe combined immunodeficiency disease
(SCID):
 Growth hormone deficiency: by implanting
cultured myoblasts transfected with GH gene.
 Familial hypercholesterolemia: by introducing
LDL receptor gene into hepatocytes.

24.  Lesch-Nyhan syndrome: by introducing HPRT
gene.
 Stroke, head injury, multiple sclerosis: by
delivering nerve growth factor gene.
 Duchenne muscular dystrophy: by
administering muscle dystropin gene
 Sickle cell anaemia: by introducing beta/ delta
sickle cell inhibitor hybrid gene.

25.  Haemophilia: by introducing factor VIII gene.
 DM - 1: by introducing insulin-1 gene into
liver.
 HIV infection: by injecting fibroblasts
expressing HIV envelope glycoprotein gene to
augment immunity against HIV.
 Alzheimer's disease, Huntington's chorea,
Gaucher's disease.

26. Cancer:
(i) By genetic introduction of an enzyme (viral
thymidine kinase) into tumour cells followed by a
prodrug that is converted to the toxic metabolite,
tumour cells are selectively killed.
(ii) By inserting TNFa, IL-2 and other cytokine genes
into tumour cells to increase their immune
recognition and destruction by tumour infiltrating
lymphocytes.

27. (iii) By introducing promoter 'antisense' gene or
'suppressor' gene which negatively regulate
tumour growth.
(iv) By introducing multidrug resistance MDR-1
gene into bone marrow cells and render them
less susceptible to destruction by
myelosuppressant drugs, toxicity of many
anticancer drugs can be overcome.

29. Potential Complications of Gene
Therapy:
 Gene silencing
 Genotoxicity: complications arising from
insertional mutagenesis.
 Phenotoxicity: complications arising from
overexpression or ectopic expression of the
transgene.
 Immunotoxicity: harmful immune response to
either the vector or transgene.
 Risks of horizontal transmission: shedding of
infectious vector into environment.
 Risks of vertical transmission: germline
transmission of donated DNA.

30. References
 Essentials of Medical Pharmacology, 6th Edition
by KD TRIPATHI.
 Harrisons Text book of medicine – 18th edition.
 Centre for Genetics Education.
www.genetics.edu.au
 Genetics Home Reference
(http://ghr.nlm.nih.gov/)
 Google images.

31. TAKE HOME MESSAGE:
 Very promising prospect to cure many diseases.
 Two things to remember
2 types
2 vectors
2 methods
 Should be restricted to treat diseases not change
nature.
 Still lot of research needs to be done.