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Recombinant DNA (r-DNA) technology


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Therapeutic Agents Developed From Recombinant DNA Technology

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Recombinant DNA (r-DNA) technology

  1. 1. RECOMBINANT DNA (r-DNA) TECHNOLOGY (Therapeutic Agents Developed From Recombinant DNA Technology) S.SEETARAM SWAMY, M.Pharm., Asst. professor, Dept. of Pharmaceutical Chemistry, Chilkur Balaji College of Pharmacy.
  2. 2.  DNA is the keeper of the all information needed to recreate an organism Nucleotides are the building blocks of the DNA. All DNA is made up of a base consisting of sugar phosphates and nitrogen bases.  “Double helix"  The sugar used in DNA is deoxyribose.  DNA contain a anti-parallel strands.  DNA contains 4 nitrogen bases they are: Purines: Adenine, Guanine Pyrimidines: Thymine, Cytosine They are found in pairs, A&T and G&C DNA – THE GENETIC SECRET DNA is the keeper of the all the information needed to recreate an organism.
  3. 3.  Prokaryotic (bacteria) or eukaryotic (yeast, mammalian cell culture) systems are generally used as a host for the production of usable quantities of the desired r-DNA product.  Most of the r-DNA products approved by FDA are being produced using these systems. Bacteria such as Escherichia coli are widely used for the expression of r-DNA products.  They offer several advantages due to high level of recombinant protein expression, rapid growth of cell and simple media requirement. Yeast such as Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris are among the simplest eukaryotic organisms.  They grow relatively quickly and are highly adaptable to large-scale production.  These organisms do not produce endotoxin. Mammalian systems such as Chinese hamster ovary (CHO) cell and Baby hamster kidney (BHK) cell systems are often the choice for production of human therapeutic proteins.
  4. 4. A gene is a stretch of DNA that codes for a type of protein that has a function in the organism. It is a unit of heredity in a living organism. All living things depend on genes. Genes hold the information to build and maintain an organism’s cell and pass genetics traits to off spring. DNA does not actually make the organism, it only makes proteins. The DNA is transcribed into mRNA and mRNA is translated into protein, and the protein then forms the organism. GENE
  5. 5. 1970: Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Herbert Boyer Stanley Cohen 1978: Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology. 1972: Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. History of Recombinant DNA Technology
  6. 6. DNA that has been created artificially (not natural). DNA from two or more sources is incorporated into a single recombinant molecule Recombinant DNA(rDNA) is a form of artificial DNA that is created by combining two or more sequences. It is made possible by two important enzymes. Restriction enzymes and DNA Ligase are the two principal tools, first used by Paul Berg in 1972,employed to alter DNA.  METHODS BY WHICH RECOMBINANT DNA IS MADE ARE: TRANSFORMATION TRANSDUCTION CONJUGATION RECOMBINANT DNA (r DNA)
  7. 7. Recombinant DNA technology r-DNA involves using microorganisms 1. To create new pharmaceuticals 2. To create safer/ more effective version therapeutic agents Recombinant DNA (r-DNA) technology has made a revolutionary impact in the area of human healthcare by enabling mass production of safe, pure and effective r-DNA expression products. •Recombinant DNA is specifically from two or more DNA incorporated into a single molecule. • Genetic engineering, recombinant DNA technology, genetic modification and gene splicing are terms are applied to the direct manipulation of an organisms gene. • The development of these new technologies have resulted into production of large amount of biochemically products
  8. 8. Production of a unique DNA molecule by joining together or more DNA fragments not normally associated with each other, which can replicate in the living cell. Recombinant DNA is also called chimeric DNA. 3 different methods of DNA recombination… A. Transformation B. Non-bacterial transformation C. Phage induction
  9. 9. RESTRICTION ENDONUCLEASE: Cleaves DNA at a specific base sequence. DNA LIGASE: Binds two DNA molecules or fragments. DNA POLYMERASE: Fills single – stranded gaps in duplex DNA by stepwise addition of nucleotides to 3’ ends (removes RNA polymer). REVERSE TRANSCRIPTASE: Makes a DNA polymer of an RNA polymer POLYNUCLEOTIDE KINASE: Adds a phosphate to the 5’ OH end of a polynucleotide, to label it or permit ligation ALKALINE PHOSPHATASE: Removes terminal phosphates from the 5’end, the 3’ end, or both. ROLE OF ENZYMES IN r-DNA TECHNOLOGY
  10. 10. © The term ‘clone’ means, exact copy of the parent. A duplicate or a look alike carrying the same genetic signature or genetic map. © Cloning is the best application of recombinant DNA technology and could be applied to something as simple as DNA fragment or a larger, sophisticated mammalian specie such as humans. Molecular cloning is carried out in‐vitro where a specific fragment of DNA is isolated from an organism ‘donor’ and introduced into a ‘plasmid’ that replicates in a ‘host’ cell making multiple copies of that DNA fragment. CLONING
  11. 11.  These are double stranded DNA that are usually circular and mostly found inside certain bacterial specie e.g. E.coli.  However most plasmids are now commercially available, ready to be used, providing specific fragment insertion sites.  Plasmids in genetic engineering are also known as ‘vectors’.  Vectors also include viruses known as bacterio‐phage that use bacteria as their host to replicate.  Hence a bacterio‐phage can be used to transfect and create several copies of the DNA fragment of interest by replicating several times in a bacteria. PLASMIDS
  12. 12. 1. This "sticky ends" from two different DNA molecules can hybridize together; then the nicks are sealed using ligase. 2. The result is recombinant DNA. 3. When this recombinant vector is inserted into E. coli, the cell processes the instructions and by translation & transcription, it assembles the amino acids forming the protein product of interest. 4. More importantly, the new instructions are passed along to the next generation of E. coli cells forming ‘recombinant clones’ on the culture agar.
  13. 13. Recombinant r-DNA technology Isolation of the gene of interest Preparation of vector DNA and DNA to be cloned Insertion of the gene to the vector molecule and ligation Introduction of the vector DNA to the appropriate host cell Amplification of the recombinant DNA molecule in the host cell
  14. 14. Recombinant DNA technology for production of human therapeutics.
  15. 15. Provide substantial quantity No need for natural or organic factors Unlimited utilizations Cheap Resistant to natural inhibitors Advantages of Recombinant Technology
  16. 16. Commercialized and became big source of income for businessman. Effects natural immune system of the body. Can destroy natural ecosystem that relies on organic cycle. Prone to cause mutation that could have harmful effects. Major international concern : manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype. Concerns of creating super – human race Disadvantages of recombinant technology
  17. 17. 1. Insulin for diabetics. 2. Factor VIII for males suffering from haemophilia A. 3. Factor IX for haemophilia B. 4. Human growth hormone (HGH). 5. Erythropoietin (EPO) for treating anemia. 6. Several types of interferon. 7. Granulocyte –macrophage colony-stimulating factor(GSM-CSF) for stimulating the bone marrow after a bone marrow transplant. 8. Many monoclonal antibodies 9. Pharmaceutical and therapeutic applications 10. Gene therapy 11. Medical diagnosis 12. Xenotransplants Applications of recombinant DNA technology
  18. 18.  Since Banting and Best discovered the hormone, insulin in 1921 diabetic patients, whose elevated sugar levels are due to impaired insulin production.  The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the use and storage of food, particularly carbohydrates.  Chemically, insulin is a small, simple protein. It consists of 51 amino acid, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain.  The two chains are linked by a disulfide bond. HUMULIN (INSULIN)
  19. 19.  Hepatitis B vaccine (rDNA) is produced by the expression of the viral gene coding for HBsAg in yeast (Saccharomyces cerevisiae) or mammalian cells (Chinese hamster ovary (CHO) cells or other suitable cell lines). HEPATITIS B VACCINE - (HB) PREPARATION OF HEPATITIS B VACCINE BY r-DNA HEPATITIS B is an infectious inflammatory illness of the liver caused by the hepatitis B virus (HBV). HEPATITIS B VACCINE:  It is a sterile solution of Immunoglobin containing antibody to hepatitis-B surface antigen.  Kept at p H 6.2  Formulated in 0.075M NaCl, 0.15M glycine, 0.01% polysorbate 80.  Used by intramuscular route as a vaccine for hepatitis B.
  20. 20. HUMAN GROWTH HORMONE(SOMATOTROPIN/ HUMATOTROPIN)  Polypeptide hormone of rdna origin,with 191 amino acids, molecular weight-2-115 dalton.It is a pituitary hormone.  Humatroph,a sterilised lauphilised powder sub-cutaneous or i.v. injection.  Phosphoric acid or NaOH added to adjust pH upto 7.5 and oxygen sensitive.  Recombinant human growth hormone is generally produced by inserting the human growth hormone gene into plasmids of E.coli bacteria.  Recombinant bacteria cells are cultured and human growth hormones produced by these bacteria are extracted from the extracellular media.  During the production of the human growth hormone in the body it is attached with signal peptides with 26 amino acids. This signal peptide is cleaved in the body before releasing the hGH to do their biological function  During the production of recombinant human growth hormone the signal peptides interrupts their production.First cDNA is produced using m RNA encoding for hGH. Then cDNA are integrated into the plasmid and are inserted into the E.coli bacteria,then they are cultured and HGH are extracted along with the signal peptide chains. PREPARATION OF HGH by rDNA
  21. 21. LIST OF SOME OTHER NEWER DRUGS PREPARED BY R-DNA TECHNOLOGY DRUGS APPLICATIONS Epogen/Procrit For patients with anemia due to Dialysis/ Chronic Kidney Disease / Renal Failure / Chemo / HIV Neulasta For Neutropenia: low WBC count febrile neutropenia (low WBC count with fever/ infection) due to chemo, BMT, AML Infergen For patients with Chronic, non-responding, or relapsing hepatitis C viral (HCV) infection. Avonex Treatment of relapsing forms of MS. Slows the progression of MS by regulating the body's immune response against myelin. Betaseron Multiple Sclerosis: Significantly delays the progression of secondary MS, including relapsing-remitting MS Forteo Treatment of osteoporosis in women and men IntronA treat different types of leukemia,malignant melanoma, multiple myeloma,basal cell carcinoma.
  22. 22. DRUGS APPLICATIONS HepatitisB surface antigen Vaccination (now in market) Interleukin-2 Treatment of cancer Tissue Plasminogen Activator Thrombosis Erythropoetin Treatment of Anemia LIST OF SOME OTHER NEWER DRUGS PREPARED BY R-DNA TECHNOLOGY
  23. 23. Some recombinant proteins that are used therapeutically
  24. 24. Applications of recombinant DNA technology
  25. 25. Recombinant DNA has been gaining in importance over the last few years, and recombinant DNA will only become more important in the 21st century as genetic diseases become more prevalent and agricultural area is reduced. Below are some of the areas where Recombinant DNA will have an impact.  Better Crops (drought & heat resistance) Recombinant Vaccines (ie. Hepatitis B)  Prevention and cure of sickle cell anemia  Prevention and cure of cystic fibrosis  Production of clotting factors  Production of insulin  Production of recombinant pharmaceuticals  Plants that produce their own insecticides Germ line and somatic gene therapy
  26. 26. REFERENCES • Pharmaceutical Biotechnology – Fundamentals And Applications • Biochemistry –U. Satyanarayana • A Text Book of Biotechnology – R .C Dubey • A Text book of intermediate second year BOTANY •
  27. 27. “What the public needs to understand is that these new technologies, especially in recombinant DNA technology, allow scientists to bypass biological boundaries altogether.” ―Jeremy Rifkin