Gene therapy (DNB Qn solved)

1. GENE
THERAPY
Dept of Pediatrics,
Dr.SMCSI Medical College, DR.PADMESH.V
Karakonam, India

2. 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”

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Alright,tell me
how you want to
change…!

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STEPS 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

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 Gene Therapy:
 Approaches:
Two ways to deliver genes:
1. Ex vivo approach
2. In vivo approach

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 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.

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 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

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 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

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 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

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

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 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.

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 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.

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 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

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 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

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 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.

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 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.

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 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.

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COMMON VECTORS USED FOR GENE THERAPY:
 1.Retro viruses
 2. Adeno viruses
 3. Liposomes

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 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.

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 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.

24. 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.

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 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.

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 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.

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 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.

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GENE 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.

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