Dr. IFTHAQAR. H. F

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.

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.

PRINCIPAL:

Introduction of FUNCTIONAL GENES into

appropriate cells

Transferred gene (TRANSGENE) encodes & produces proteins

The Proteins encoded by Transgenecorrects the disorder

TYPES OF GENE THERAPY:

1. SOMATIC CELL THERAPY

2. GERM LINE THERAPY

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.

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.

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

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.

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

METHODS OF GENE

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

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.

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.

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.

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 episomalintegration.

Provokes immune response.

Alternative & better – AAV for 3 reasons

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.

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.

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.

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.

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.

(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.

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.

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.

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.