Recombinant DNA Technology and Drug Discovery

RDNA technology
Gene therapy

1. t RECOMBINANT DNA TECHNOLOGY AND DRUG DISCOVERY PRESENTED BY DIVYA V 1 st MPHARM PHARMACEUTICAL CHEMISTRY

2. RECOMBINANT DNA TECHNOLOGY INTRODUCTION A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from different organism. Also called as chimeric gene. Achieved by cutting DNA(restriction enzymes)into suitable fragments and joining together the appropriate fragments(ligation). Proteins expressed by rDNA called as recombinant proteins. Cloning is the process to create rDNA

3. TOOLS OF GENETIC ENGINEERING A)RESTRICTION ENDONUCLEASES(RE) Bacterial enzyme that can cut DNA at specific sites Recognition sequences; site in DNA which is cut by RE. Cleavage pattern; Form sticky ends which can easily pair with other DNA having complementary sticky ends. Also called as molecular scissors

4. B)DNA LIGASE Cutted DNA fragments are covalently joined by this. Join the fragments by forming phosphodiester bond between phosphate group of 5’carbon of one deoxy ribose with hydroxyl group of 3’carbon of another deoxy ribose.

5. BASIC PRINCIPLES STEPS IN GENE CLONING 1)Identification and isolation of desired gene. 2)Insertion of isolated DNA into a suitable vector 3)Introduction of this vector into suitable organism 4)Selection of transformed host cells 5)Multiplication/Expression/Integration followed by expression of the gene in the host

6. 1)ISOLATON OF DESIRED GENE DNA fragment to be cloned is called as DNA insert. Desired fragments can be obtained from, a)Genomic libraries Libraries are collection of DNA clones in a certain vector. Genomic - made from RE DNA fragments of total genomic DNA cDNA (complementary DNA) – made from DNA synthesized from mRNA

7. b)Polymerase chain reaction Allows the isolation of a specific segment of DNA from a small DNA (or cell sample) using DNA primers. c)Chemical synthesis of gene Base sequence of protein is identified, a polynucleotide of same sequence can be synthesized chemically or enzymatically .

8. 2)INSERTION OF THE GENE INTO SUITABLE VECTORS Can carry foreign DNA fragment to be cloned and are self replicating in host cells

9. PLASMID They are extrachromosomal,circular,self replicating DNA molecules.

10. E.g. pBR322

11. BACTERIOPHAGE They are viruses that attack bacteria. Can accept short fragments of foreign DNA into their genome.

12. COSMID It posses the characteristics of both plasmid and bacteriophage.

13. ARTICIAL CHROMOSOME VECTORS HUMAN ARTIFIAL CHROMOSOME(HAF) Synthetically produced vector DNA possesing characteristics of human chromosome.

14. BACTERIAL ARTIFICIAL CHROMOSOME Bacterial artificial chromosomes (BACS) are bacterial plasmids derived from the F plasmid. They are capable of carrying up to 300 kb of DNA.

15. YEAST ARTIFICIAL CHROMOSOME(YAC) Behaves like yeast chromosome and can accept large pieces of foreign DNA.

16. PHAGEMID/PHASMID Contain several copies of plasmid but one copy of plasmid is retained in the DNA.

17. 3)INTRODUCTION OF rDNA INTO SUITABLE HOST rDNA is introduced in to suitable host. Host are the living cells in which carrier of rDNA/vector can be propagated. TYPES,

18. 4)SELECTION OF TRANSFORMED CELLS rDNA containing cells can be identified from non-transformed cells when a marker gene is present in it. Only the cells that posses such gene will survive.

19. 5)MULTIPLICATION/EXPRESSION OF GENE The multiplied copies of gene can be used in number of ways, Introduced to bacterium for production of protein. Introduced into eukaryotic host. Expression of gene.

20. APPLICATIONS Manufacture of proteins/hormones Interferon, plasminogen activating factor, blood clotting factors, insulin, growth hormone,several enzymes etc. Diagnosis of molecular diseases: sickle cell anaemia, thalassaemia, familial hypercholesterolaemia, cystic fibrosis. Prenatal diagnosis: DNA from cells collected from amniotic fluid, chorionic villi.

21. Gene Therapy: This is achieved by cloning a gene into a vector that will readily be taken up & incorporated into genome of a host cell. ADA deficiency has been successfully treated Application in Agriculture: Genetically engineered plants are developed to resist draught & diseases. Good quality of food & increased yield of crops is also possible.

22. HYBRIDOMA TECHNOLOGY

23. INTRODUCTION It is a hybridization technique which is used to produce antibody producing hybrid cell. Antibodies produced are called as Monoclonal antibodies.

24. APPLICATIONS DIAGNOSTIC A monoclonal antibody can be used to detect pregnancy in only 14 days after conception. Their selective binding property allow detection of low levels of human corionic gonadotropin (HCG) in urine and serum.

25. THERAPEUTIC Earlier horses were inoculated with Coryne bacterium diphtheriae,the resulting crude horse antiserum was used to treat diphtheria. Organ transplantation For the treatment of solid organ transplant rejection, several Mabs against T cell antigens have been evaluated. Bone marrow transplantation MAbs are being evaluated for graft versus host disease in bone marrow transplantation.

26. CANCER TREATMENT mAbs act directly when binding to cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth etc.

27. IMMUNOPURIFICATION Monoclonal antibodies can also be used to purify a substance with techniques called affinity chromatography.

28. NEW PHARMACEUTICALS DERIVED FROM BIOTECHNOLOGY

29. HORMONES INSULIN Used for treatment of diabetes.

30. VACCINES Hepatitis B vaccine. Myobloc vaccine. Menveo vaccine. Ixiaro vaccine. MONOCLONAL ANTIBODIES Used along with immunosuppressant's. E.g. Infliximab,Basiliximab,rituximab

31. ENZYMES Alteplase-Plasminogen activator. Recombinant dornase alpha-Cystic fibrosis Idursulphase-Hunter syndrome. GROWTH FACTORS Recombinant erythropoietin-Anemia. Palifermin-Oral mucositis in cancer patients.

32. ANTIBIOTICS Penicillin,Cephalosporin,Streptomycin BLOOD FACTORS Clotting factors 8,9-Hemophilia. Anti-thrombin recombinant-Prevention of thromboembolic events

33. OLIGONUCLEOTIDE THERAPY

34. They are short DNA or RNA molecules that has wide range of applications. Antisense oligonuleotides(ASO) are single strand of DNA or RNA that are complementary to a chosen sequence. They are chemically synthesized from protected phosphoramides or chemically modified nucleosides.

35. MECHANISM OF ACTION

36. MUSCULAR DYSTROPHY Group of diseases that cause weakening and breakdown of muscles. ASO therapy used to remove mutated exon. CANCER The high specificity of binding of ASO to their target mRNA make these compounds useful as therapeutic agents against human cancer. Suppresses malignant cells ASO AS THERAPEUTIC AGENT

37. THALASSEMIA Antisense 2'-O-methylribooligonucleotides were targeted against specific sequence elements in mutated human beta-globin and can repair thalassemia. ARTHRITIS Fibroblast-like cells obtained from RA synovium were stimulated with interleukin-1beta and treated with antisense or sense oligonucleotides targeting proliferating cell.

38. ASTHMA ASOs directed against chemokine receptor,granulocyte-macrophage colony stimulating factor are designed to inhibit allergic inflammation. AMYLOIDOSIS DIABETES LIMITATIONS High doses required. Half life in plasma is short Protected against nucleophilic attack.

39. GENE THERAPY

40. INTRODUCTION Gene therapy is a clinical procedure in which a gene or other DNA sequence used to treat a disease. TYPES EXVIVO INVIVO

41. VECTORS VIRAL VECTORS Viral DNA has been removed and is introduced into hosts. E.g. Adenoviruses,Adeno associated virus,retro virus,Lenti virus. NON-VIRAL VECTORS Pure DNA construct. DNA molecular conjugates. Lipoplexes. Human artificial chromosome.

42. METHODS OF GENE DELIVERY PHYSICAL METHODS CHEMICAL METHODS Using detergent mixtures Lipofection MICROINJECTION

43. RECENT ADVANCES & APPLICATION OF GENE THERAPY BLINDNESS Cure blindness of inherited condition. HOW IT WORKS; used harmless viruses enable access to the cells beneath the retina of patients. CANCER Used to treat various types of cancer.

44. HOW IT WORKS; Normal WBC taken from cancer patients infected with retrovirus that deliver genes to cells. PARKINSON’S DISEASE Improved the weakness of the symptoms such as tremors, motor skill problems,and rigidity. HIV Under clinical trials

45. CYSTIC FIBROSIS Adenovirus vector was used to deliver a normal ion channel protein(CFTR) to airway cells in a patient’s nose or lungs. SEVERE COMBINED IMMUNODEFICIENCY Due to defect of gene coding Adenosine deaminase.Gene of ADA is introduced for its treatment.

46. ORNITHINE TRANSCARBOXYLASE (OTC)DEFICIENCY Leads to accumulation of ammonia and can be corrected by gene therapy. THALASSEMIA It is an inherited autosomal recessive blood disease. Gene transfer of a regulated β-globin gene in would reduce the imbalance between a-and β-globin chains in erythroid cells.

47. CLINICAL TRIALS Alzheimers disease Hepatitis-B AIDS CANCER- Brain, Ovarian,Small cell lung, Prostrate, Breast cancer. Chronic granulomatous disease.

48. PRINCIPLES OF DNA AND RNA ESTIMATION. Important applications in PCR. DIPHENYLAMINE METHOD Diphenylamine + deoxy ribose Blue coloured complex(absorbs at 595nm) Concentration Vs Absorbance plotted.

49. SPECTROPHPTOMETRIC METHOD Sample is exposed to wavelength at 260nm and photo detectors measures the light that passes through the sample. AGAROSE GEL ELECTROPHORESIS Used to separate nucleic acid based on their size under the influence of electric field. Nucleic acids are negatively charged, on applying electric field they move to anode based on size and seperated.

50. ANALYSIS WITH FLUORESCENT DYE TAGGING Sample is tagged with fluorescent dye. Intensity of the dye that bind to nucleic acids is measured.

51. REFERENCES T.A.Brown.Gene cloning and DNA analysis.Blackwell publishers;2006(5);302-322. B.D.Singn.Text book of biotechnology. Kalyani Publishers.;2006(1);11-104. U.Satyanarayana and U.Chakrapani. Textbook of biochemistry.Allied Publishers.2006(3).578-618. Hugo Almeida, Maria Helena Amaral, Paulo Lobão. Drugs obtained by biotechnology processing. Brazilian Journal of Pharmaceutical Sciences;vol. 47;2011.

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