Nucleic acid-and-cell-based-therapies

Nucleic acid-and-cell-based-therapies

1. Nucleic acid and cell based therapies

2. Gene Therapy 1980s and early 1990s Proteins of therapeutic use nucleic acid based therapeutics center around gene therapy and antisense Tehnology Early 2007 only three nucleic acid based therapeutics gained worldwide approval. antisense-based product ( Vitravene) aptamer ( Macugen ) gene therapy product( Gendicine )

3. In cell based, fully differentiated cells or groups of cells used organ or tissue transplantation

4. Principle of gene therapy

5. Some diseases for which gene based clinical trials are in process

6. Basic approach to gene therapy

7. Vector systems used to deliver genes into mammalin cells

8. P ractical approaches that may be pursued when undertaking gene therapy

9. Some additional questions

10. Vectors used in gene therapy

11. Types of vectors Vectors capable of introducing genes into recipient cells.

12. Retroviral vectors Enveloped viruses. Genome consists of ssRNA . RNA transcribed and yields dDNA . Integrates into host cell.

13. Structural genes in retroviral genome

14. Other components LTR harbours promoter and enhancers. promote integration ψ packaging sequence. viral RNA packaging

15. Retroviral genome LTR Ψ gene of interest LTR

16. Retrovirus life cycle

17. Packing cells

18. Properties of Retrovirus Integrate their proviral DNA into replicating cells. Efficiency of gene transfer in sensitive cells. Long term,high level expression. Integrates randomly. Retro virus are promiscuous. Complete copies are passed to daughter cells.

19. Continue .. Good,high level stocks of replication incompetent retroviral particles. Studies carried out in animal species.

20. Advantages Disadvantages Integrate into genome in a stable fashion Often damage during purification and concentration. Effectively enter into various cell types. Ability to infect only dividing cells. Transduction efficacy. Lack of selectivity. Fairly high level expression. Not infect all dividing cells. Easy to propagate. Integrate randomly into the chromosome of the recipient cells.

21. Drawback of retroviral gene therapy In 2002, Patients with SCID-X1 received with retroviral therapy developed leukemic like condition Due to the proviral integration at the site near the LM02 proto-oncogene promoter leading to gene activation. This resulted in initial ban on retro viral based gene therapy trials in some world regions.

22. Adenoviral and additional viral vectors

23. Adenoviruses Relatively large Non enveloped structure Double stranded DNA Large genome and complex

24. Adenoviruses have some advantages or disadvantages Advantages Disadvantges Capable of gene transfer to non dividing cells High immunogenic in man Easy to propagate Duration of tranferred gene can vary High gene expression Highly selective for cells

25. Adeno -associated virus Small Single stranded DNA Need coinfecting adenoviruses replicate

26. Small genes can be introduced into adenoviral vector. Facilitate long term gene expression

27. Herpes Simplex Virus Herpes simplex virus Neurotropic vector Can deliver genes to PNS and CNS. Upon infection Remains latent in non dividing neurons Genome is in unintegrated form

28. Difficult to generate viable herpes simplex particle

29. Sindbis Virus

30. Formation of engineered sindbis virus

31. Manufacture of viral vectors

32. Non-viral vectors

33. Use naked plasmid dna or dna complexes as non-viral vector. Advantages: their low/non-immunogenicity; non-occurrence of integration of the therapeutic gene into the host chromosome

34. Methods of non-viral gene delivery:

35. Naked DNA:   simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred . low expression.

36. Lipoplexes :   Plasmid DNA can be covered with lipids in an organized structure like a liposome complexed with DNA it is called a lipoplex 3 types of lipids: anionic (negatively charged ) neutral cationic (positively charged)

37. Polyplexes : Complexes of polymers with DNA are called polyplexes consist of cationic polymers and their production is regulated by ionic interactions polyplexes rapidly remove from circulation. PEG attachment

38. cellular entry of (non-viral) gene delivery : Target: appropriate cell surface Therapeutic plasmid must enter the cell and reach the nucleus intact. Cellular entry is generally achieved via endocytosis.

39. Routes by which plasmid can reach nucleus: direct nuclear entry transport through nuclear pores

40. manufacture of plasmid dna :

41. Gene Therapy and Genetic Disease

42. Genetic diseases? A genetic disease is any disease that is caused by an abnormality in an individual's genome. Over 4000 diseases are characterized Causes

43. Factors

44. Example EX VIVO GT 1990 – 4 year old Ashanti DaSilva had a genetic disorder called severe combined immunodeficiency (SCID) Defect in ADA gene results in an accumulation of dATP , which is toxic to certain types of T cells Takes down the entire immune system.

45. Example: IN-VIVO GT mutation on 7th chromosome. Defective cystic fibrosis transmembrane conductance regulator (CFTR) gene. Normally it serves as a pump at the cell membrane to move electrically charged chloride atoms out of the cells If cells can’t move chloride out, they absorb water trying to dilute the chloride in the cell This leads to the production of THICK sticky mucus

46. Vectors used to deliver CF gene to airway epithelial cells: 1993 vector used: Adenovirus 1995 liposome have potential to avoid critical problems immune response, limited packaging capacity, and random integration . Liposomes may be mildly effective, but their activity does not last .

47. Gene therapy and cancer

48. 1.4 million cases reported. 50% survival rate using surgery, chemo/radiotherapy

49. Low success rate due to: requirement for improved, more target-specific vector systems. A requirement for a better understanding of how cancer cells evade the normal immune response. suffering from advanced and widespread terminal cancer (i.e. little/no hope of survival if treated using conventional therapies). Cancers at earlier stages of development will probably prove to be more responsive to gene therapy.

50. Boosting the immune response: aim to boost the body's natural ability to attack cancer cells. Our immune system has cells that recognize and kill harmful things that can cause disease, such as cancer cells. Involved introduction of TNF…

51. Steps:

52. Anti-cancer strategy:

53. Pro-drug gene therapy:

54. GENE THERAPY AND AIDS

55. INTRODUCTION U seful in treating medical condition inherited disease,cancer and infectious disease. AIDS is a viral disease caused by intracellular pathogens. D ifferent approaches used to treat AIDS

56. Introduction of a gene into pathogen susceptible cells is termed as intracellular immunization such as introduction of a gene into viral sensitive cell coding for an altered HIV protein such as gag tat env Mutant form of gag is capable of inhibiting viral replication

57. The transfer to sensitive cell of a gene coding for antibody fragments capable of binding to the HIV enveolpe proteins this interfere with viral assembly Recombinant cell have also been generated which are capable of secreting soluble form of HIV cell surface receptor like CD4 antigen These soluble viral receptor would bind with viriones

58. GENE BASED VACCINE Administration of a DNA vector housing the gene coding for a surface antigen protein from the target pathogen Any body cell could be targeted.The target cell export the resultant antigenic protein Gene expression need only be transient to facilitate the induction of an immune response Gene based vaccine have entered for clinical trials include malaria, hepatitis B and AIDS

59. Gene based vaccine have entered for clinical trials include malaria, hepatitis B and AIDS

60. Causes Of Disease Occurrence Hypertension

61. Antisense Technology Definition The specific sequence of nucleotide bind to mRNA or DNA when the sequence is known that will cause a specific disease. This leads to gene turning off

62. Antisense technology

63. Antisense oligonucleotides and their mode of action

64. Antisense oligonucleotides and their mode of action

65. Advantages of Oligos It acts as therapeutic agent in Cancer Viral diseases such as HIV,hepatitis B Herpes and Papilloma Infection

66. Example Cancer

67. Advantages

68. Disadvantages

69. Oligonucleotide pharmacokinetics and delivery Intravenous administration Sub cutaneous administration Intra dermal administration Charged Oligo's enter in cell by receptor mediated endocytosis UnCharge Oligos enter in the cell through Passive diffusion and endocytosis

70. Continue

71. Phosphodiester linkage

72. Manufacture of oligos

73. Oligonucleotides Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis.

74. Oligonucleotides “ Oligo ” – Prefix meaning few (~ 2-10 ). Nucleosides can be obtained from either natural sources (i.e. salmon sperm) or chemically synthesized . We use phosphoramidite method for manufacturing oligos.

75. Phosphoramidite Method Developed in 1980. The principle of this method was developed by McBride and Caruthers in 1983 . Currently considered as the standard synthesis . Used in most automated synthesizers today .

76. Cycle The cycle consists of four steps: D e-protection Coupling Oxidation Capping

77. De-protection Trityl group attached to the 5’ carbon of the pentose sugar of the recipient nucleotide is removed by trichloroacetic acid (TCA) leaving a reactive hydroxyl group .

78. Coupling The phosphoramidite monomer is added in the presence of an activator such as a tetrazole This structure then reacts with the hydroxyl group of the recipient and the 5’ to 3’ linkage is formed. Tetrazole = Class of synthetic organic heterocyclic compound, consisting of a 5-member ring of four nitrogen atoms and one carbon atom. The simplest is tetrazole itself, CH₂N₄. They are unknown in nature.

79. Oxidation The oxidation step stabilizes the phosphate linkage in the growing oligonucleotide. The traditional method of achieving this is by treatment with iodine in water.

80. Capping Any remaining free 5’-hydroxyl groups are blocked at the capping step in an irreversible process.

81. After having synthesized the full length sequence, the oligonucleotide is then released from the solid support using a base, such as aqueous ammonia or a mixture of ammonia and methylamine. This will also remove protection groups from the nucleobases. The oligonucleotide is now ready for purification. oligonucleotide is purified with RP-HPLC where the retention time is to a large.

82. RNA interference and ribozymes RNAi represents a sequence-specific post-translational inhibition mechanism of gene expression, Induced ultimately by dsRNA Entry of dsRNA triggers its cleavage into short (21–23 nucleotide long) sequences called short interfering RNAs

83. This cleavage is catalyzed by a Enzyme known as Dicer RNA inducedsilencing complex ( RISC) antisense ’ siRNA strand then facilitates RISC binding to a specifi c mRNA via Watson–Crick base complementarity, which is then degraded by RISC nuclease activity.

84. Ribozyme RNA sequences can function as catalysts. This is known as Ribozymes Many ribozymes will cleave Target mRNA where there exists a particular triplet nucleotide sequence G–U–C

85. Ribozyme

86. Aptamers Aptamers are single-stranded DNA or RNA-based sequences allowing them to bind a specific target molecule . Identification of specific aptamers binding the target molecule SELEX (systematic evolution of ligands by exponential enrichment)

87. affinity-based purifi cation, target validation drug discovery, diagnostics and therapeutics .

88. low immunogenicity In order to prevent renal removal, aptamers are usually conjugated to PEG. Their half-lives can most effectively be extended via chemical modification