Human gene therapy

Gene Therapy, Somatic cell gene therapy, germ line gene therapy, classical gene therapy, non-classical gene therapy, targets of gene therapy, barriers of gene therapy, ex vivo gene therapy, in vivo gene therapy, vectors for gene delivery, antisense therapy

1. HUMAN GENE THERAPY PRADEEP SINGH M.Sc. Medical Biochemistry IIIrd Yr HIMSR, Jamia Hamdard New Delhi 04/11/2019 1

2. CONTENT What is Gene Therapy? Types of Gene Therapy Barriers of g ene delivery Strategies for transgene delivery Types of vectors for gene therapy Some diseases treated by gene therapy 2

3. Gene therapy Gene Therapy is the use of genetic material (DNA) as a pharmaceutical agent to prevent or cure a disease. Gene therapy typically aims to supplement a defective/mutant allele with a functional one. 3

4. impact of gene manipulation on the practice of medicine 4

5. What is gene therapy? It is a technique for correcting defective genes that are responsible for a disease. It is essentially an intervention that alters the instruction set of a cell. There are four approaches:- A normal gene is inserted to compensate for a non-functional/mutated gene. An abnormal gene expression is suppressed. An abnormal gene is repaired through selective reverse mutation. Change the regulation of gene pairs. 5

6. History of gene therapy 1930 ’ s “Genetic Engineering ” - plant/animal breeding 1960’s First idea of using genes therapeutically 1950’s-1970’s Gene transfer methods developed 1970’s-1980’s Recombinant DNA technology 1990 First gene therapy in human (ADA deficiency) Since then, more than 2,500 clinical studies have been initiated for a broad range of applications, from a variety of monogenic diseases to infectious diseases, complex neurodegenerative disorders, and cancer. 6

7. Gene Therapy vs conventional therapy Gene Therapy Convential Therapy Materials DNA, RNA; Cells, Tissues or organs Small molecules, Peptide, Proteins Delivery Usually required to be delivered into cells (Antisense Oligonucleotides) or Nucleus (genes) Effect on the cell membrane or diffuse into cells Mechanisms Usually cure the cause of the diseases Usually relieve the symptoms or signs of disease Duration of Effect Can be permanent and also can be passed down to next generation in germline gene therapy. Usually stop the effect after stop taking it. Ethics Major Issues Usually Not 7

8. Principle of gene therapy 8

9. How it works? 9

10. Methods of classifying gene therapy On the basis of class of disease: Genetic disease Vs complex acquired disorder By the characteristics of the gene delivery vehicle: Integrating Vs non-integrating vectors W hether the vector is administered in vivo (directly into the patient) or ex vivo (in cultured cells taken from the patient that are subsequently transplanted back ) 10

11. Purpose & Approch of gene therapy Monogenic gene therapy Provides genes for the production of a specific protein Cystic fibrosis, Muscular dystrophy, Sickle cell disease, hemophilia, SCID Suicide Gene Therapy Provide ‘suicide’ genes to target cancer cells for destruction Cancer Antisense Gene Provides a single stranded oligonucleotides in an ‘antisense’ (backward) orientation to block the production of harmful proteins AIDS/HIV 11

12. Types of gene therapy Somatic cell gene therapy Germ line gene therapy Current gene therapy is exclusively somatic cell gene therapy which involves the introduction of genes into somatic cells of an affected individual. The prospect of human germline gene therapy is currently not sanctioned. Germline gene therapy targets the reproductive cells, meaning any changes made to the DNA will be passed on to the next generation. The effects of gene therapy are too unpredictable. A ny additional mutations that are introduced as a result will be passed on to the next generation. 12

13. Classical V s Non-classical gene therapy Classical Gene Therapy : Genes are delivered to the appropriate target cells with the aim of obtaining the optimal expression of the introduced genes. Non-classical Gene Therapy : Genes are delivered to inhibit the expression of genes associated with the pathogenesis. 13

14. Types of somatic cell gene therapy Ex vivo Approach R apidly dividing cells such as hematopoietic stem cells are used Integrating vectors are used (Retroviral/lentiviral vectors) In vivo approach Long lived post-mitotic cells are used (lymphocytes) Non-integrating vectors are used (Adenoviruses/AAV) 14

15. 15

16. Targets of gene therapy Central Nervous System (Huntington’s Disease) Respiratory Tract Other s olid o rgans (Liver) T cells and Hematopoeitic cells Muscles Vascular system (Lymphocytes) 16

17. Barriers of gene delivery 17

18. Barriers of gene delivery Extracellular Epithelial Barriers; Circulation and The components such as RBC, serum proteins, Enzymes, etc. Reticuloendothelial cleaning system, et c. Require inert surface and targeting molecules for specific transportation of complex in the blood. Intracellular Cell membrane Endosomal Membrane Nuclear Membrane DNA releasing at Right Site and Right Time Require cationic groups for successful gene delivery. 18

19. Strategies of transgene delivery 19

20. Strategies for Transgene Delivery 20 Ex Vivo In Vivo Cells are removed from the body Transgene delivered into cultures cells Cells returned to the body Transgene delivered directly into host

21. 21

22. Vectors for gene delivery 22

23. Types of vectors for gene delivery RNA viruses (Retroviruses) Murine leukemia virus ( MuLV ) Human immunodeficiency virus (HIV) Human T-cell lymphotropic viruses (HTLV) DNA viruses Adenoviruses Adeno-associated viruses (AAV) Herpes simplex virus (HSV) Pox viruses Foamy viruses Non-viral vectors Liposomes Naked DNA Liposomes- polycation complexes Peptides delivery systems. 23

24. Viral Vectors: gene + Protein coat Disabled viral vectors Pathogenicity is attenuated but infection ability is still maintained. Gene of interest is inserted Altered virus should transfer helpful genes to cells but should not multiply or produce disease Viruses bind to the cell surface receptors of cell membrane and deliver its genetic contents. The cell will use the inserted gene to produce a therapeutic protein 24

25. Retrovirus for gene delivery 25

26. Modified Retroviruses (RNA viruses) Advantages Good at inserting genes into host chromosome Used with partial success treating Gaucher’s disease Successfully cured 4 babies of SCID in early 2000 [ S evere C ombined I mmunodeficiency S yndrome (Bubble baby)] Disadvantages Insert genes randomly. Usually needs an actively dividing host cell to enter Therefore, not used for Cystic Fibrosis Modified virus may cause mutation and serious disease. 26

27. 3-D visualization of retrovirus structure 27

28. Life cycle of a retrovirus 28

29. Life cycle of a retrovirus 29

30. Retrovirus genome 30 Gag : processed to matrix and other core proteins that determine retroviral core. Pol : reverse transcriptase, RNase H and integrase functions. Env : envelope protein, resides in lipid layer; determine viral protein

31. 31

32. Reconstruction of retrovirus genome for gene therapy 32 5’ LTR Packaging Gene X Neo r 3’ LTR Insertion of new gene

33. Engineering a virus into a viral vector 33 Vector DNA Helper DNA wildtype virus Viral vector replication proteins structural proteins Packaging Therapeutic gene essential viral genes Packaging cell

34. Gene transfer 34 Y vector Vector uncoating Therapeutic mRNA and protein Episomal vector Integrated expression cassette Target cell

35. adenovirus 35

36. Adenovirus structure Non-enveloped virus Contains linear double stranded DNA Can enter both dividing as well as non-dividing cells. Does not integrate into the host genome Replicates as an episomal element in the nucleus 36

37. Generation of non-replicating Adenovirus expression vector 37

38. Adeno-associated virus 38

39. Adeno-associated virus vector AAV is a simple, non-pathogenic, single stranded DNA virus dependent on the helper virus (usually adenovirus) for replication. AAV enters the host cell, becomes double stranded and gets integrated into host genome. 39

40. Herpes Simplex Viruses 40

41. Herpes simplex virus Advantages Large insert size Could provide long-term CNS gene expression High titer Disadvantages System currently under development Current vectors provide transient expression Low transduction efficiency 41

42. Non-viral vectors 42

43. Non-viral vectors Naked DNA Liposomes 43

44. Naked DNA 44

45. Naked DNA It is simplest method of injecting naked DNA into the human body. Relatively low level of expression and but sufficient for use in DNA vaccination. Techniques commonly used for naked DNA delivery: Biolistics (or gene gun ) now used routinely. DNA coated particles are literally blasted into cells by an explosive discharge. Electroporation Pronuclear microinjection 45

46. Biolistics / gene gun/ Particle gun 46

47. Gene Gun DNA coated on pellets ( gold particles ) is forced down to the barrel of a ‘Particle Gun’ by an explosive charge. The particles are forced through the cell wall where the DNA is released. 47

48. Nano particles for gene therapy The electrostatistically coated poly (beta-amino ester) nanoparticles can facilitate ligand-mediated gene delivery. The more promising polymers for gene delivery is degradable poly(beta-amino ester), 1,3-diaminopentane-terminated poly (5-amino-1-pentanol-co-1,4-butanediol diacrylate ) (C32-117) . This polymer function by binding to and protecting DNA from degradation, enabling efficient cellular uptake, and enabling subsequent intracellular endosomal escape. However, as with many nanoparticle formulations, its systemic use in vivo is limited due to poor bio-distribution and lack of tissue-specific targeting 48

49. 49

50. electroporation 50

51. What is electroporation ? A short controlled pulse of electricity to cell momentarily disrupting lipid bilayer. Steps:- Use of high-voltage electric shocks to introduce DNA into cells. Voltage results in temporary breakdown and formation of pores. Small pores (40-120nm) reseal quickly. Harvest cell and resuspend in electroporation buffer Add DNA to cell suspension for stable transfection DNA should be lineralized , for transient the DNA may be supercoiled Electroporate Selection process for transfectant 51

52. 52

53. 53

54. Ex vivo Electroporation 54

55. liposomes 55

56. liposomes 56 Jfgxdwjopfbwe2ok[ wfj

57. Liposomes Liposomes are made up of phospholipid bilayer which can carry both hydrophilic as well as hydrophobic substances. Therefore, commonly used in drug delivery. Lipofection (or transfection) is a technique used to inject genetic material into a cell by means of liposomes., which are vesicles that can easily fuse with the cell membrane ( endocytosis ). Advantages: Safer when compared to viral vectors Can carry large DNA molecules 57

58. Gene Therapy For Silencing Unwanted Gene Expression Antisense Technology 58

59. A single-stranded RNA or DNA molecule that is complementary to a target mRNA pairs with the mRNA and prevents translation. This strategy works well in the laboratory on cultured cells and on model organisms. Clinical Example: Treatment against cancers. The tumor sizes decreased but this was mainly due to production of interferons in response to high doses of foreign RNA. If the dose was lowered to prevent the interferon response, the clinical benefits largely disappeared as well. 59

60. 60

61. Inherited diseases 61

62. Inherited Disease A large number of disease are known to be inherited from the parents to the offsprings . Such diseases are known as I nherited Diseases. A large number of diseases are known to be inherited from the parents to the offspring. Such diseases are known as genetic diseases. Most of these diseases are caused by the expression of recessive genes. The genetic diseases can be broadly classified into two types: Autosomal disorders Allosomal disorders Autosomal Disorders: These are metabolic disorders caused by the expression of some genes present on somatic chromosomes. Such disorders express equally in both the sexes. Allosomal Disorders : These disorders are caused by genes present on the sex chromosomes. The abnormal disorders express more commonly in males than females. 62

63. Some diseases treated by gene therapy Disease Gene Therapy Severe Combined Immunodeficiency (SCID) Retroviral transfer of enzyme Adenosine deaminase gene into lymphocyte Familial Hypercholestrolemia Retroviral transfer of LDL receptor gene into hepatocytes Hemophilia Retroviral transfer of gene of factor VIII & IX into fibroblasts Cystic Fibrosis CFTR gene transfer into bronchial epithelium by Adenovirus Duchenne Muscular Dystrophy Retroviral transfer of dystrophin gene Leber’s Hereditary Optic Neuropathy Transfer of gene of the enzyme, isomer hydrolase, by adenovirus into the retina Lesch-Nyhan Syndrome Retroviral transfer of HGPRT into lymphocytes Sickle Cell Anemia Transfer of beta globin gene by adenoviral vector Thallasemia Transfer of α & β by an adenoviral vector Gaucher’s Disease Transfer of β -glucosidase by liposome Cancer ( Wilm’s Tumor) Insertion of p53 tumor suppressor gene by liposome 63

64. Severe Combined Immunodeficiency Disease ( SCid ) SCID is caused by deficiency of the enzyme Adenosine Deaminase (ADA) Gene is located on chromosome 22 Deficiency results in failure to develop function B & T lymphocytes ADA is involved in Adenine degradation Lack of ADA leads to a 100-fold increase in the cellular concentration of dATP , a strong inhibitor of ribonucleotide reductase. High levels of dATP produce a general deficiency of other dNTP S in T lymphocytes B cells doesn’t mature as they require help of T cells Patients cannot withstand infection  Die if untreated 64

65. 65

66. 66

67. 67

68. SCID September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial Ashanti (4 year old girl) Her lymphocytes were gene-altered (~10 9 ) ex vivo  used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used) Cynthia (9 year old girl) treated in same year Problem: WBC are short-lived, therefore treatment must be repeated regularly 68

69. Ornithine transcarbamylase deficiency (OTC) Ornithine transcarbamylase (OTC) deficiency Urea cycle disorder (1/10,000 births) Encoded on X chromosome Females usually carriers, sons have disease Urea cycle = series of 5 liver enzymes that rid the body of ammonia (toxic breakdown product of protein) If enzymes are missing or deficient, ammonia accumulates in the blood and travels to the brain (coma, brain damage or death ) 69

70. 70

71. Severe OTC deficiency Newborns  coma within 72 hours Most suffer severe brain damage ½ patients die in first month ½ of survivors die by age 5 Early treatment Low-protein formula called “keto-acid” Modern day treatment Sodium benzoate and another sodium derivative Bind ammonia  helps eliminate it from the body 71

72. Gene therapy – A failure Inability to produce ornithine transcarbamylase (OTC) is often lethal, but moderate deficiencies may be controlled by strict control of diet. Jess Gelsinger , a young volunteer in a gene therapy trial who had a moderate OTC deficiency, died on 17 Sept 1999 He had been injected in the liver with high concentrations of adenovirus that expressed OTC He apparently died of a massive immune response to the adenovirus vector 72

73. Familial hypercholestrolemia 73

74. Familial hypercholesterolemia This commonly results from an autosomal dominant defect in a gene for the LDL receptor or receptor function. At least 900 mutations have been identified affecting different aspects of LDL uptake, metabolism and regulation. De-novo cholesterol synthesis synthesis is normally suppressed by exogenous cholesterol intake; with receptor processing defects this function is lost and markedly elevated cholesterol levels result. Cholesterol levels are elevated to such an extent atherosclerotic disease resulting in fatal cardiovascular events beginning in the second & third decades. 74

75. There are five major classes of FH due to LDLR mutations: Class I : LDL receptor (LDLR) is not synthesized at all. Class II : LDLR is not properly transported from the endoplamic reticulum to the Golgi apparatus for expression on the cell surface. Class III : LDLR does not properly bind LDL on the cell surface (this may be caused by a defect in either Apolipoprotein B 100 or a defect in LDLR. Class IV : LDLR bound to LDL does not properly cluster in clathrin -coated pits for receptor-mediated endocytosis Class V : The LDLR is not recycled back to the cell surface 75

76. 76

77. Major issue is LDL receptor mutation. For Familial hypercholesterolemia there are 806 mutations. Out of which 457 mutations are missense and nonsense. Substitution mutations GGG-AGG Gly-Arg Hypercholesterolemia GCG-GAG Ala- Glu Hypercholesterolemia CTC-CCC Leu -Pro Hypercholesterolemia Gene Therapy for Familial Hypercholesterolemia 1993  First attempt Retroviral vector used to infect 3.2 x 10 9 liver cells (~15% of patients liver) ex vivo Has been used in many trials since then 77

78. Cystic fibrosis 78

79. Gene therapy for cystic fibrosis Cystic fibrosis (CF) is an inherited autosomal recessive disorder CF affects the epithelial cells lining air passage to the lungs CF affects the epithelial cells lining air passages to the lungs CF causes a buildup of thick mucus in the airways 79

80. Gene therapy for cystic fibrosis In CF, there is a defective ion channel protein i.e., Cystic fibrosis transmembrane conductance regulator (CFTR) protein CFTR regulates the balance of chloride ions in epithelial cell membranes Patients with cystic fibrosis make an altered version of this protein Protein is misfolded What types of proteins are involved in helping other proteins fold properly ? Adenovirus vector was used to deliver a normal ion channel protein to airway cells in a patient’s nose or lungs. 80

81. Thalassemia 81

82. Gene therapy for thalassemia Thalassemia is an inherited autosomal recessive blood disease. In thalassemia, the genetic defect which could be either mutations or deletion results in reduced rate of synthesis or no synthesis of one of the globin α or β -chains that make up hemoglobin . Reduced synthesis or no synthesis of one of the globin chains can cause the formation of abnormal hemoglobin molecules, thus causing anemia, the characteristic presenting symptom of the thalassemias . Gene transfer of a regulated β -globin gene in Hemopoietic stem cells (HSCs) would reduce the imbalance between α or β -globin chains in erythroid cells. Transplantation of autologous, genetically corrected HSCs would represent an alternative therapy for thalassemic patients lacking a suitable bone marrow donor. 82

83. 83

84. Lesch-nyhan syndrome 84

85. Lesch-nyhan syndrome It is an X-linked recessive disorder. It mostly affects male as they contain a single copy of X-chromosome. In females, a mutation must usually be present in both copies of the gene to cause the disorder. There is deficiency of the enzyme Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or Kelly Seegmiller syndrome that affects the levels of uric acid in the body. Gene therapy involves retroviral transfer of HGPRT gene in lymphocytes. 85

86. Sickel cell anaemia 86

87. The genetics of sickle cell anaemia The shape of the haemoglobin molecule is controlled by two alleles Normal Haemoglobin allele Sickle Cell Haemoglobin allele There are t hree phenotypes Normal – Normal individuals have two normal haemoglobin alleles Sickle cell anaemia – A severe form where all the red blood cells are affected. Sickle cell anaemia patients have two sickle cell alleles in their genotype – homozygous. Sickle cell trait – A mild condition where 50% of the red blood cells are affected. Sickle cell trait individuals are heterozygous, having one of each allele 87

88. Codominant Genotypes Genotypes Phenotypes Hb N Hb N Normal haemoglobin Hb N Hb S Sickle cell trait Hb S Hb S Sickle cell trait 88

89. Problems with gene therapy Efficient gene transfer into target cells (Insertional mutagenesis) Adequate level of transgene expression Persistence of gene expression Regulation of gene expression Tolerance to transgene product Safety 89

90. 90

91. 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 Patients 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. 91

92. Problems doing gene therapy Inefficient gene delivery – not suitable for all genetic diseases Most effective if stem cells are involved Only to correct a few cells with the gene E.g. Blood stem cells: SCIDS and Gaucher’s Disease Less effective or Ineffective if many cells must be corrected Brain cells ( Tay -Sachs disease, Huntington’s disease) Cystic fibrosis Insertion of gene is not always permanent E.g. Gaucher Disease: temporary cure until β -glucosidase gene “popped” out of chromosome Insertion of gene into genome could disrupt other genes Possible consequences? Some viruses elicit immune response or may cause disease E.g. Jesse Gelsinger died in 1999 92

93. 93