1. GENE THERAPY
P. K. Choudhury (Ph. D, 1st year,
Dairy Microbiology)

2. Genes……......
• Are carried on a chromosome
• The basic unit of heredity
• Encode how to make a protein
DNARNA proteins
• Proteins carry out most of life’s function.
• When altered causes dysfunction of a protein
• When there is a mutation in the gene, then it will change the
codon, which will change which amino acid is called for which
will change the conformation of the protein which will change
the function of the protein. Genetic disorders result from
mutations in the genome.

3. What is Gene Therapy
• It is a technique for correcting defective genes that
are responsible for disease development
• There are four approaches:
1. A normal gene inserted to compensate for a nonfunctional
gene.
2. An abnormal gene traded for a normal gene
3. An abnormal gene repaired through selective reverse
mutation
4. Change the regulation of gene pairs

4. Background…………….
• 1970s Gene surgery proposal
• 1980s Great momentum to GT
• 1983 Gene therapy Lesch-Nyhan disease GT
• In 1988 (OTA)US Differentiate in somatic & germ LINE gene
therapy
• September 14, 1990 ADA deficiency GT(W. French Anderson
and colleagues)successful
• IN 1991 US govt.$58 million for gene therapy research
• Oct.1999.Jesse Jelsinger first fatality in human gene EXP
:Multiple organ failure.(Ornithine Trancarbamylase
Deficiency)
•2002 X-SCID: Fatal Leukemia

5. The First Case……….
• The first gene therapy was performed on
September 14th, 1990
• Ashanti DeSilva was treated for SCID
• Sever combined immunodeficiency
• Doctors removed her white blood cells,
inserted the missing gene into the WBC, and
then put them back into her blood stream.
• This strengthened her immune system
• Only worked for a few months 

6. How It Works………….
• A vector delivers the therapeutic gene into a
patient’s target cell
• The target cells become infected with the viral vector
• The vector’s genetic material is inserted into the
target cell
• Functional proteins are created from the therapeutic
gene causing the cell to return to a normal state

7. Types: Gene Therapy
 Somatic gene therapy:
a. Ex vivo gene therapy
b. In vivo gene therapy
 Germline & embryonic gene therapy:

8. a. Human ex-vivo gene therapy

9. b. Human in-vivo gene thepary

11. Germline and embryonic gene therapy
1. Germline gene therapy refers to the permanent
transfer of a gene into sperm or egg cells.
2. Embryonic gene therapy refers to the
permanent transfer of a gene into the cells of
an early embryo, just after the sperm and egg
unite.
3. In both cases, the delivered gene would
become a permanent part of cells in the
resulting adult.

12. Transfection Technology
• Calcium phosphate • Gold nanoparticles (1 to 3
• Electroporation micrometer) :800psi
• Microinjection • Chitosan
• Elevated temprature
• DEAE dextran
• Cationic liposome
• Activated dendrimer

13. Gene Transfer Vehicles
• Viral vector:
-Retroviruses
-Adenoviruses (dsDNA)
-Adeno-associated viruses(ssDNA)
-Herpes simplex virus(HSV)
• Non-viral methods:
-Naked DNA
-Anti sesne Oligonucleotides
-Lipoplexes
-Prodrug activation therapy
-Ribozymes
-Chimeroplasty
-Schistosoma egg Proteins

16. Ideal gene delivery system……
 Biocompatible
 Non-immunogenic
 Stable in blood stream
 Protect DNA during transport
 Small enough to extravagate
 Cell and tissue specific

17. The Ideal Vector for Gene Transfer
• High concentration of virus allowing many cells to be infected or
transduced
• Convenience and reproducibility of production
• Ability to transduce dividing and non-dividing cells
• Ability to integrate into a site-specific location in the host
chromosome, or to be successfully maintained as stable episome
• A transcriptional unit that can respond to manipulation of its
regulatory elements
• Ability to target the desired type of cell
• No components that elicit an immune response

18. Hallmarks of successful gene delivery
1. Targeting the right cells
2. Activating the gene
3. Integrating the gene in the cells
4. Avoiding harmful side effects

19. Retrovirus vector system

22. How one know whether a disorder is a
good candidate for gene therapy ?
 Does the condition result from mutations in one or more genes?
 Which genes are involved?
 What one know about the biology of the disorder?
 Which tissues are affected
 What role does the protein encoded by the gene play within
the cells of that tissue?
 Exactly how do mutations in the gene affect the protein's
function?
 Will adding a normal copy of the gene fix the problem in the
affected tissue?
 Can one deliver the gene to cells of the affected tissue?

23. Step 1: Learn about the disease
Is the disorder a good candidate for gene therapy?
To find out, study the disease
1) Get money for the project
2) Get approval for the project
3) Perform clinical research
4) Perform biological research
5) DECISION: Is the disorder a good candidate for gene
therapy?

24. From Research to Trials……………..
• Understand the biology behind the disorder
• Develop the treatment approach
• Test its effectiveness in biological models of the
disease
• Establish its safety in humans

25. Step 2: design a gene therapy
1) Use your knowledge of the disorder to design a
gene therapy
2) Test the therapy in appropriate models of the
disease
3) DECISION: Does your therapy look promising?
Step 3: Get money and approval for clinical trials
1) Get money for the trials
2) Get approval for the trials

26. STEP 4: Phase One clinical trial
1) Establish safety and dosage limits in a small group of people (20-
80)
2) DECISION: Does your therapy still look promising?
STEP 5: Phase Two clinical trial
1)Test the efficacy and safety in a larger group of people (100-300)
2) DECISION: Is your therapy effective in a larger group of people?
• Use your knowledge of the disorder to design a gene therapy
• Test the therapy in appropriate models of the disease
• DECISION: Does your therapy look promising?

27. Step 6: Phase Three clinical trial
1) Test the therapy in a large group of people (1,000-3,000)
2) DECISION: Is your treatment successful?
Step 7: Get FDA&RAC(NIH) approval for general clinical
use
Write proposals, fill out paperwork, answer questions and wait for approval
Step 8: Phase Four clinical trial
Further test the efficacy and optimal use of the treatment in general use

31. Cystic Fibrosis
 Defective gene (called delta F508) protein
(cystic fibrosis transmembrane conductance
regulator - CFTR)
 Flow of salt (sodium)
 salt is trapped
 overproducing bodily secretions such as
water, sweat and mucus
 secretions then build up in the body

35. Pedigree Analysis

36. Gene Therapy for Cystic Fibrosis
• Cystic fibrosis should be an ideal candidate for
gene therapy, for four main reasons:
• (1) it is a single gene defect;
• (2) it is a recessive condition, with heterozygotes
being phenotypically normal (suggesting gene
dosage effects are not critical)
• (3) the main pathology is in the lung, which is
accessible for treatment;
• (4) it is a progressive disease with a virtually normal
phenotype at birth, offering a therapeutic window.

37. Gene Therapy :Cystic Fibrosis
1993 vector used: Adenovirus
CFTR (cystic fibrosis transmembrane regulator)
1995 liposome : cationic liposome-based systems
1998 Adeno-associated virus

39. Gene therapy in animals
• Hemophilia A: Clotting factor VIII has introduced in Rat and
Dog (Xu et al., 2005)
• Epilepsy: Type A receptors - In Rat
(Brooks et al., 2002 )
• Deafness: Atoh1 or Math1- Guinea pigs
(Izumikawa, 2003)
• Mastitis: lysostaphin gene in Jersey cattle
(Robert-J-Wall et al., 2007)
• Blindness: GC1 gene in incubating egg
(Semple Rowland et al., 2005)

40. Problems with Gene Therapy………
• Short Lived
– Hard to rapidly integrate therapeutic DNA into genome and
rapidly dividing nature of cells prevent gene therapy from long
time
– Would have to have multiple rounds of therapy
• Immune Response
– new things introduced leads to immune response
– increased response when a repeat offender enters
• Viral Vectors
– patient could have toxic, immune, inflammatory response
– also may cause disease once inside
• Multigene Disorders
– Heart disease, high blood pressure, Alzheimer’s, arthritis and
diabetes are hard to treat because you need to introduce more
than one gene
• May induce a tumor if integrated in a tumor suppressor gene
because insertional mutagenesis

41. Social & Ethical issues
• Privacy & Confidentiality
• Psychological Impact& Stigmatization
• Available to rich & powerful
• Playing with God
• Designer babies
• Accumulation of defective genes in future generation
• Mutation or defective arrangement of genes
• Probability of occurrence of new diseases.

42. Recent Developments
• Genes get into brain using liposomes coated in polymer call
polyethylene glycol
– potential for treating Parkinson’s disease
• RNA interference or gene silencing to treat Huntington’s
– siRNAs used to degrade RNA of particular sequence
– abnormal protein wont be produced
• Create tiny liposomes that can carry therapeutic DNA through
pores of nuclear membrane
• Sickle cell successfully treated in mice

43. Conclusions………
• Neurogenic disease may be cured e.g. Alzeimer & Parkinson's
Disease (life threatening diseases)
• Difference types of Cancer gene therapy trials may get
success in future
• Germ cell gene therapy trials may get success in curing
hereditary diseases
• Pharmacogenetics & Geneticular molecular biotechnology
• Applicable in livestock species.
• GT: cautiously & seriously evaluate

44. Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana 5th Dec, 2011

45. Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana 5th Dec, 2011

46. Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana 5th Dec, 2011