GENE THERAPY P. K. Choudhury (Ph. D, 1st year,              Dairy Microbiology)
Genes……......• Are carried on a chromosome• The basic unit of heredity• Encode how to make a protein               DNARNA...
What is Gene Therapy•   It is a technique for correcting defective genes that    are responsible for disease development• ...
Background…………….• 1970s Gene surgery proposal• 1980s Great momentum to GT• 1983 Gene therapy Lesch-Nyhan disease GT• In 19...
The First Case……….• The first gene therapy was performed on  September 14th, 1990  • Ashanti DeSilva was treated for SCID ...
How It Works………….• A vector delivers the therapeutic gene into a  patient’s target cell• The target cells become infected ...
Types: Gene Therapy Somatic gene therapy:      a. Ex vivo gene therapy      b. In vivo gene therapy Germline & embryonic...
a. Human ex-vivo gene therapy
b. Human in-vivo gene thepary
Germline and embryonic gene therapy1. Germline gene therapy refers to the permanent   transfer of a gene into sperm or egg...
Transfection Technology• Calcium phosphate     • Gold nanoparticles (1 to 3• Electroporation        micrometer) :800psi• M...
Gene Transfer Vehicles• Viral vector:   -Retroviruses   -Adenoviruses (dsDNA)   -Adeno-associated viruses(ssDNA)    -Herpe...
Ideal gene delivery system…… Biocompatible Non-immunogenic Stable in blood stream Protect DNA during transport Small ...
The Ideal Vector for Gene Transfer• High concentration of virus allowing many cells to be infected or  transduced• Conveni...
Hallmarks of successful gene delivery   1. Targeting the right cells   2. Activating the gene   3. Integrating the gene in...
Retrovirus vector system
How one know whether a disorder is a   good candidate for gene therapy ? Does the condition result from mutations in one ...
Step 1: Learn about the diseaseIs the disorder a good candidate for gene therapy? To find out, study the disease    1) Get...
From Research to Trials……………..•   Understand the biology behind the disorder•   Develop the treatment approach•   Test its...
Step 2: design a gene therapy  1) Use your knowledge of the disorder to design a    gene therapy  2) Test the therapy in a...
STEP 4: Phase One clinical trial  1) Establish safety and dosage limits in a small group of people (20-     80)  2) DECISI...
Step 6: Phase Three clinical trial  1) Test the therapy in a large group of people (1,000-3,000)  2) DECISION: Is your tre...
Cystic Fibrosis Defective gene (called delta F508) protein  (cystic fibrosis transmembrane conductance  regulator - CFTR)...
Pedigree Analysis
Gene Therapy for Cystic Fibrosis• Cystic fibrosis should be an ideal candidate for  gene therapy, for four main reasons:• ...
Gene Therapy :Cystic Fibrosis1993 vector used: AdenovirusCFTR (cystic fibrosis transmembrane regulator)1995 liposome : cat...
Gene therapy in animals• Hemophilia A: Clotting factor VIII has introduced in Rat and  Dog                                ...
Problems with Gene Therapy………• Short Lived   – Hard to rapidly integrate therapeutic DNA into genome and     rapidly divid...
Social & Ethical issues• Privacy & Confidentiality• Psychological Impact& Stigmatization• Available to rich & powerful• Pl...
Recent Developments• Genes get into brain using liposomes coated in polymer call  polyethylene glycol   – potential for tr...
Conclusions………• Neurogenic disease may be cured e.g. Alzeimer & Parkinsons  Disease (life threatening diseases)• Differenc...
Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana   5th Dec, 2011
Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana   5th Dec, 2011
Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana   5th Dec, 2011
Gene therapy
Gene therapy
Gene therapy
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Gene therapy

  1. 1. GENE THERAPY P. K. Choudhury (Ph. D, 1st year, Dairy Microbiology)
  2. 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. 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. 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. 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. 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. 7. Types: Gene Therapy Somatic gene therapy: a. Ex vivo gene therapy b. In vivo gene therapy Germline & embryonic gene therapy:
  8. 8. a. Human ex-vivo gene therapy
  9. 9. b. Human in-vivo gene thepary
  10. 10. Germline and embryonic gene therapy1. 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.
  11. 11. Transfection Technology• Calcium phosphate • Gold nanoparticles (1 to 3• Electroporation micrometer) :800psi• Microinjection • Chitosan• Elevated temprature• DEAE dextran• Cationic liposome• Activated dendrimer
  12. 12. 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
  13. 13. Ideal gene delivery system…… Biocompatible Non-immunogenic Stable in blood stream Protect DNA during transport Small enough to extravagate Cell and tissue specific
  14. 14. 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
  15. 15. 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
  16. 16. Retrovirus vector system
  17. 17. 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 proteins 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?
  18. 18. Step 1: Learn about the diseaseIs 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?
  19. 19. 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
  20. 20. 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
  21. 21. 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?
  22. 22. 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 approvalStep 8: Phase Four clinical trial Further test the efficacy and optimal use of the treatment in general use
  23. 23. 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
  24. 24. Pedigree Analysis
  25. 25. 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.
  26. 26. Gene Therapy :Cystic Fibrosis1993 vector used: AdenovirusCFTR (cystic fibrosis transmembrane regulator)1995 liposome : cationic liposome-based systems1998 Adeno-associated virus
  27. 27. 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)
  28. 28. 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
  29. 29. 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.
  30. 30. 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
  31. 31. Conclusions………• Neurogenic disease may be cured e.g. Alzeimer & Parkinsons 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
  32. 32. Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana 5th Dec, 2011
  33. 33. Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana 5th Dec, 2011
  34. 34. Gene Therapy and Genetic Engineering, P. K. Choudhury, NDRI, Karnal, Haryana 5th Dec, 2011

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