GENE THERAPY: TYPES, METHODS, FACTORS AND STANDARDS AND ITS APPLICATION IN HEALTHCARE FIELD INVIVO THERAPY AND EXVIVO THERAPY CHEMICAL AND PHYSICAL METHODS TO CARRY ON GENE THERAPY DEFECTIVE GENE IDENTIFICATION IN GENE THERAPY AND TREATMENT OF GENETICALLY AFFECTED GENE BY GENE THERAPY

1. GENE THERAPY
RVS Chaitanya Koppala
Assistant Professor
Vignan Institute of Pharmaceutical Technology
Visakhapatnam

2. GENE THERAPY
DEFINITION OF
“Novel approach to treat, cure or ultimately prevent a disease by hanging the expression of a
person’s genes” V
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:
1. Introduction of FUNCTIONAL GENES into appropriate cells
2. Transferred gene (TRANSGENE) encodes & produces proteins
3. The Proteins encoded by Transgene corrects the disorder
Gene Therapy:
Approaches:
Two ways to deliver genes:
1. Ex vivo approach
2. In vivo approach
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.

3. 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
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
4. NEO-ORGAN IMPLANTS
5. TISSUE TRANSPLANTATION

4. 6. HUMAN ARTIFICIAL CHROMOSOMES
7. OTHERS:
 Receptor mediated delivery
 Virally directed enzyme prodrug therapy
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 However,in somatic cell therapy,
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 somatiwell 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
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

5. 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.
COMMON VECTORS USED FOR GENE THERAPY:
1.Retro viruses
2. Adeno viruses
3. Liposomes

6. 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.
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.

7.  Provokes immune response.
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.

8. Some results of gene therapy:
1. Adenosine Deaminase deficiency:
First attempt at gene therapy.
2. Severe Combined Immune 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.
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.