GENE THERAPY

Dept of Pediatrics, Dr.SMCSI Medical College, Karakonam, India

DR.PADMESH.V

Dr.Padmesh. V

Definition of gene therapy: “ Novel approach to treat,cure or

ultimately prevent a disease by changing the expression of a person’s genes”

Dr.Padmesh. V

Alright,tell me how you want to change…!

Gene

Dr.Padmesh. VSTEPS IN GENE THERAPY:

1. Identification of the defective gene.

2. Cloning of normal healthy gene.

3. Identification of target cell / tissue / organ.

4. Insertion of the normal functional gene into the host DNA.

METHOD:

Introduction of FUNCTIONAL GENES into appropriate cells

Transferred gene (TRANSGENE) encodes & produces proteins

The Proteins encoded by Transgene corrects the disorder

Dr.Padmesh. V Gene Therapy:

Approaches: Two ways to deliver genes:

1. Ex vivo approach

2. In vivo approach

Dr.Padmesh. V 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.

Dr.Padmesh. V

Dr.Padmesh. V 2. In vivo approach: (Direct Gene

Transfer) -Cloned therapeutic gene is introduced

directly into the affected tissue, without removing cells from the body.

-Specially designed vehicles are needed. -Examples are: Lungs, Brain

Dr.Padmesh. V

Dr.Padmesh. V METHODS OF GENE DELIVERY:

1. PHYSICAL METHODS:

-Parenteral injection

-Microinjection

-Aerosol

-Gene gun

2. CHEMICAL METHODS:

-Calcium phosphate

-DEAE-Dextran

-Liposomes

3. BIOLOGICAL METHODS:

Viral Vectors like

-Retrovirus -Adenovirus -HSV

Dr.Padmesh. V

METHODS OF GENE DELIVERY: contd…

4. NEO-ORGAN IMPLANTS

5. TISSUE TRANSPLANTATION

6. HUMAN ARTIFICIAL CHROMOSOMES

7. OTHERS:

-Receptor mediated delivery

-Virally directed enzyme prodrug therapy

Dr.Padmesh. V TYPES OF GENE THERAPY:

1. SOMATIC CELL THERAPY 2. GERM LINE THERAPY

Dr.Padmesh. V 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

However,in somatic cell therapy, Transgene cannot be passed on to the siblings etc.

Dr.Padmesh. V 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.

Dr.Padmesh. V VARIOUS STRATEGIES FOR GENE

THERAPY: 1. GENE AUGMENTATION THERAPY

2. TARGETED MUTATION CORRECTION

3. INHIBITION OF GENE EXPRESSION

4. GENE THERAPY TO ACHIEVE PHARMACOLOGICAL EFFECTS

Dr.Padmesh. V 1. GENE AUGMENTATION THERAPY:

If a disease is caused by a mutation causing loss of function,

introduction of a FUNCTIONAL COPY OF THE GENE into the cell will restore the normal function of the gene.

Examples:

1. Deficiency of ADA

2. Haemophilia

Dr.Padmesh. V 2. TARGETED MUTATION

CORRECTION:

Correction of mutation,by changing the mutated nucleotide sequence to normal.

Practically difficult,but in principle can be done by homologous recombination.

Dr.Padmesh. V 3. INHIBITION OF GENE

EXPRESSION: In case of mutations that have a negative dominant

effect,

the expression of the mutated gene can be blocked at the

DNA / RNA / protein level.

Examples:

This strategy is useful in Cancers caused by inappropriate expression of a gene.

Dr.Padmesh. V 4. GENE THERAPY TO ACHIEVE

PHARMACOLOGICAL EFFECTS: Examples:

1. Introduction of a gene that makes cancer cells susceptible

to anticancer drugs.

2. Introduction of a toxic gene whose expression kills cancer

cells.

3. Genes of cytokines can be introduced into cells of immune

system to enhance their potential to kill diseased cells.

Dr.Padmesh. V

COMMON VECTORS USED FOR GENE THERAPY:

1.Retro viruses

2. Adeno viruses

3. Liposomes

Dr.Padmesh. V 1. RETRO VIRUSES:

Retroviruses used in gene therapy are made incapable of independent replication,to prevent side effects associated with infectivity.

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.

Dr.Padmesh. V

Dr.Padmesh. V 2. ADENO VIRUSES:

Second most commonly used delivery system in gene therapy.

Adenoviruses can be produced at high titres in cultures.

Advantages: Can infect non-dividing cells,thus suitable for gene

therapy of Cystic fibrosis, DMD. Non-integration to chromosome. Avoids the risks of

uncontrolled integration. Efficient gene transfer.

Disadvantages: Transient expression of gene due to episomal

integration. Provokes immune response.

Dr.Padmesh. V 2. ADENO VIRUSES:

Second most commonly used delivery system in gene therapy.

Adenoviruses can be produced at high titres in cultures.

Advantages: Can infect non-dividing cells,thus suitable for gene

therapy of Cystic fibrosis, DMD. Non-integration to chromosome. Avoids the risks of

uncontrolled integration. Efficient gene transfer.

Disadvantages: Transient expression of gene due to episomal

integration. Provokes immune response.

Dr.Padmesh. V

Dr.Padmesh. V 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.

Dr.Padmesh. V

Dr.Padmesh. V

Dr.Padmesh. V

Dr.Padmesh. V Some results of gene therapy:

1. Adenosine Deaminase deficiency: -First attempt at gene therapy.

2. Severe Combined Immuno Deficiency: -SCID-X1 successfully treated with gene therapy.

3. Hemophilia A & Hemophilia B: -Ex vivo method using fibroblasts

-Clinical improvement was present.

4. DMD: -Successful in mice,but human trials not yet.

5. Cystic Fibrosis: -In vivo trials with Transmembrane Conductance

Regulator CFTR.

Dr.Padmesh. V RISKS OF GENE THERAPY:

1.Adverse reactions due to the virus or new genes.

2.Activation of proto-oncogene leading to formation of oncogene.

3.Introduction of a mutation to the next generation.

Dr.Padmesh. VGENE THERAPY (Summary)

Steps in Gene therapy

Approaches: - Ex-vivo -In-vivo

Types:-Somatic cell therapy -Germ line

therapy

Methods of delivery:-Physical -Chemical -Biological

etc etc

Strategies.

Dr.Padmesh. V

You are no

more

Dominant!!!

Gene

Dr.Padmesh. V

Are we heading in the right direction?

?

Dr.Padmesh. V

Who are we , to play

God??

Dr.Padmesh. V

Thank you!