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Paul Terasaki. GENE THERAPY Transplantation of the Pancreas, Liver, Bone Marrow and Islets are all procedures and Islets are all procedures in which new.

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Presentation on theme: "Paul Terasaki. GENE THERAPY Transplantation of the Pancreas, Liver, Bone Marrow and Islets are all procedures and Islets are all procedures in which new."— Presentation transcript:

1 Paul Terasaki

2 GENE THERAPY Transplantation of the Pancreas, Liver, Bone Marrow and Islets are all procedures and Islets are all procedures in which new functioning genes are transferred from one individual to another as treatments individual to another as treatments

3 Growing a New Pancreas?

4 We have all done it! But can it be possible to repeat this miracle of nature in the laboratory?

5 Progenitor Cells Are not fully differentiated Are not fully differentiated Committed to a special tissue, e.g. pancreas Committed to a special tissue, e.g. pancreas

6 Castaing et al Diabetologia (2001) Human early foetal pancreas precursor tissue under renal capsule NOD/scid mice After 6 months beta cell increase x 5000

7 There are Three Golden Rules for Making Surrogate  -Cells

8 Unfortunately, We Don’t Know Any of Them

9

10 Shinya Yamanaka

11

12 VASCULARIZED FUNCTIONAL HUMAN LIVER from iSPC derived ORGAN BUD TRANSPLANT (Takebe and Taniguchi Nature 2013)

13 iPSCells from Type 1 diabetics differentiated to produce Insulin PNAS 2009 Maehr et al. Melton’s Lab PNAS 2009 Maehr et al. Melton’s Lab

14 Caution iPS Cells can become antigenic and undergo rejection (Zhao et al Nature 2011 vol 474) iPS Cells can become antigenic and undergo rejection (Zhao et al Nature 2011 vol 474) Can accumulate DNA Abnormalaties (Christine Mummary NEJM 2011) Can accumulate DNA Abnormalaties (Christine Mummary NEJM 2011) Can Retain epigenitic memory of to Cell type of Origin (Kim et al Nature 2010 Can Retain epigenitic memory of to Cell type of Origin (Kim et al Nature 2010

15 Expression of desired gene Coax with Differentiation factorsCoax with Differentiation factors Engineer by gene insertionEngineer by gene insertion Combined coaxing and engineeringCombined coaxing and engineering

16 Manet

17 Caravaggio

18 GoyaGoya Goya

19 “SOUP” Hypothesis Stem cells in a soup of growth & differentiation factors Stem Cell Differentiating Cell

20 Genetic Engineering Electroporation Electroporation Viral vectors Viral vectors – Adeno & adeno-associated virus – Retrovirus e.g. Lenti modified HIV can unmask oncogenes

21 Autologus Cell Transplant No immunosuppressive drugs No immunosuppressive drugs Requires suitable source of cells Requires suitable source of cells ? Autoimmune recurrence risk ? Autoimmune recurrence risk unless target eliminated

22 In Vivo v Ex Vivo In Vivo v Ex Vivo In Vivo - Reduced hazards In Vivo - Reduced hazards of infection, but transfection of infection, but transfectiondifficulties Ex Vivo - Problem of where Ex Vivo - Problem of where and how to transplant manipulated cells

23 “TOO GOOD TO BE TRUE !” CURIOUS !! If it can be repeated in another laboratory, it would be interesting. Peter Medawar

24 The Literature Crescendo Negotiating difficult and hazardous published traps

25 Human Insulin Gene transfection of Autologous Hepatocytes Glucose Response Promoter EGR1 Glucose Response Promoter EGR1 Plasmid ex-vivo electroporation Plasmid ex-vivo electroporation Direct intra-hepatic cell injection Direct intra-hepatic cell injection Cured diabetic Pigs for up to 9 months Cured diabetic Pigs for up to 9 months (Singapore, Nelson, Oi Lian Kon et al ) (Singapore, Nelson, Oi Lian Kon et al )

26 Vast literature scattered in ever increasing numbers of journals ASSESSMENT -; 1) TRUE and INTERESTING 2) TRUE and of NO interest 3) FALSE due to experimental or perceptual error 3) FALSE due to experimental or perceptual error 4) FALSE due to FRAUD 3 and 4 confuse, lead others astray and can waste years of effort

27 To show that “A” is true, you don’t do “B”, you do “A” again (Brian Nosek,quoted Nature 2012 Ed Yong) Unfortunately doing “A” again is BORING so replication experiments are UNPOPULAR BORING so replication experiments are UNPOPULAR

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29 Mohamed Ghoneim Mansoura Egypt

30 Autologous Mesenchymal Stromal Cells (Mansoura) 1)Bone Marrow, Studies with MSC’s from rat to rat isologous (Gabr et al, exp. and clin. Transplantation, 2008) 1)Bone Marrow, Studies with MSC’s from rat to rat isologous (Gabr et al, exp. and clin. Transplantation, 2008) 2 HUMAN Bone Marrow MSC’s to diabetic scid mice,(Gabr et al, Cell Transplantation, 2012) 2 HUMAN Bone Marrow MSC’s to diabetic scid mice,(Gabr et al, Cell Transplantation, 2012)

31 Mesenchymalal Stromal cells

32 unun unun Undifferentiated passage 3

33 Islet like clusters after 17 days culture

34 Islet like clusters Ditizone after 17 days culture

35 Insulin +ve (GREEN) & glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom factor 2.5)

36 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom Factor 2.5)

37 Cpp +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom Factor 2.5)

38 Insulin & Cpp +ve (Yellow) differentiat ed MSCs transplante d under renal capsule (63X/1.40 OIL, Zoom Factor 2.5)

39 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

40 Mansoura Culture Proliferation and Differentiation Small numbers of insulin producing cells 1-5% Small numbers of insulin producing cells 1-5% Probably Glucose Responsive Probably Glucose Responsive 7 Days After transplantation insulin producing cells rise to 15% 7 Days After transplantation insulin producing cells rise to 15%

41 Data suggest Human bone marrow MSC’s can produce human insulin and C- peptide for at least 3 months in a xenogenic model Human bone marrow MSC’s can produce human insulin and C- peptide for at least 3 months in a xenogenic model Although small in amount there is sufficient to control ATZ diabetes in the diabetic SCID mouse Although small in amount there is sufficient to control ATZ diabetes in the diabetic SCID mouse

42 Outstanding Problems Sufficient cells to provide adequate therapy Sufficient cells to provide adequate therapy Persistent gene expression & protein synthesis for a long period Persistent gene expression & protein synthesis for a long period

43 Encouraging in- vivo studies with the AAV Vector IM injections of the IM injections of the human insulin and Glucokinase human insulin and Glucokinase genes into STZ diabetic rats (Mas et al, Diabetes 2006) and STZ diabetic dogs prolonged cure (Callejas et al Diabetes 2013) IV injection of factor IX in human Hemophilia B (Amit et al, NEJM 2011)

44 iPS Cells and Mesenchymal Stromal Cells only new runners in 10 years

45 BELIEVE NOTHING ! no matter where you read it or who has said it, not even if I have said it, unless it agrees with your own reason and your own common sense. Buddha

46 Insulin And Cpp in cultutred differentiated MSCs using Trichostatintransplanted under renal capsule, received on

47 Insulin +ve (GREEN) differentiat ed MSCs transplante d under renal capsule (63X/1.40 OIL)

48 Cpp +ve (RED) differentiat ed MSCs transplante d under renal capsule (63X/1.40 OIL)

49 Insulin & Cpp +ve (Yellow) differentiat ed MSCs transplante d under renal capsule (63X/1.40 OIL)

50 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom Factor 3)

51 Cpp +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom Factor 3)

52 Insulin & Cpp +ve (Yellow) differentiat ed MSCs transplante d under renal capsule (63X/1.40 OIL, Zoom Factor 3)

53 Insulin And Glucagon in differentiated MSCs transplanted under renal capsule, Kidney received on

54 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

55 Glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

56 Insulin +ve (GREEN) & glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

57 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom factor 2.5)

58 Glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom factor 2.5)

59 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

60 Glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

61 Insulin +ve (GREEN) & glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL)

62 Insulin +ve (GREEN) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom factor 2.5)

63 Glucagon +ve (RED) differentiat ed MSCs transplant ed under renal capsule (63X/1.40 OIL, Zoom factor 2.5)

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65 Path to Beta Cell from iPS (Nichols, New and Annes

66 “Wild Speculation” Genetic engineering to remove gene of diabetic target S Blood, bone marrow, fat - stem cell of patient of patient Culture with growth & differentiation factors of embryonic pancreas soup to produce autologous  cells Autotransplant of “doctored” culture  cells No imunosuppression No target for autoimmune attack

67 “Wild Speculation” Genetic engineering to remove gene of diabetic target S Blood, bone marrow, fat - stem cell of patient of patient Culture with growth & differentiation factors of embryonic pancreas soup to produce autologous  cells Autotransplant of “doctored” culture  cells No imunosuppression No target for autoimmune attack

68


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