Paul Terasak i
Apr 01, 2015
Paul
Terasaki
GENE THERAPY
Transplantation of thePancreas, Liver, Bone Marrow and Islets are all proceduresin which new functioning genesare transferred from one individual to another as treatments
GrowingaNew Pancreas?
We have all done it!But can it be possible to
repeat this miracle of naturein the laboratory?
Progenitor Cells
Are not fully differentiated
Committed to a special tissue, e.g. pancreas
Castaing et alDiabetologia (2001)
Human early foetal pancreas precursor tissue under renal capsule NOD/scid mice
After 6 months beta cell increase x 5000
There areThree Golden Rules
forMaking Surrogate -Cells
Unfortunately,
We Don’t Know
Any of Them
Shinya Yamanaka
VASCULARIZED FUNCTIONAL HUMAN LIVER from iSPC derived ORGAN BUD TRANSPLANT (Takebe and Taniguchi Nature 2013)
iPSCells from Type 1diabetics differentiatedto produce Insulin
PNAS 2009 Maehr et al. Melton’s Lab
Caution iPS Cells can become antigenic and
undergo rejection (Zhao et al Nature 2011 vol 474)
Can accumulate DNA Abnormalaties (Christine Mummary NEJM 2011)
Can Retain epigenitic memory of to Cell type of Origin (Kim et al Nature 2010
Expression of desired gene
• Coax with Differentiation factors
• Engineer by gene insertion
• Combined coaxing and engineering
Manet
Caravaggio
Goya
Goya
“SOUP” Hypothesis
Stem cells in a soup ofgrowth & differentiation
factors
Stem Cell Differentiating Cell
Genetic Engineering
Electroporation Viral vectors
– Adeno & adeno-associated virus
– Retrovirus e.g. Lenti modified HIV can unmask oncogenes
Autologus Cell Transplant
No immunosuppressive drugs
Requires suitable source of cells
? Autoimmune recurrence risk
unless target eliminated
In Vivo v Ex Vivo
In Vivo - Reduced hazards of infection, but transfection difficulties Ex Vivo - Problem of where and how to transplant manipulated cells
“TOO GOOD TO BE TRUE !”
CURIOUS !!
If it can be repeated in another laboratory, it would be
interesting.
Peter Medawar
The Literature Crescendo
Negotiating difficult and hazardous published traps
Human Insulin Gene transfection of Autologous Hepatocytes
Glucose Response Promoter EGR1 Plasmid ex-vivo electroporation Direct intra-hepatic cell injection Cured diabetic Pigs for up to 9 months
(Singapore, Nelson, Oi Lian Kon et al 2008 +1)
Vast literature scatteredin ever increasing numbers of journals
ASSESSMENT -;
1) TRUE and INTERESTING2) TRUE and of NO interest 3) FALSE due to
experimental or perceptual error4) FALSE due to FRAUD
3 and 4 confuse, lead others astray and can waste years of effort
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
Mohamed Ghoneim
Mansoura Egypt
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)
2 HUMAN Bone Marrow MSC’s to diabetic scid mice,(Gabr et al, Cell Transplantation, 2012)
Mesenchymalal Stromal cells
un
un
Undifferentiated passage 3
Islet like clusters after 17 days culture
Islet like clusters Ditizone after 17 days culture
Insulin +ve (GREEN) & glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom factor 2.5)
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom Factor 2.5)
Cpp +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom Factor 2.5)
Insulin & Cpp +ve (Yellow) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom Factor 2.5)
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Mansoura Culture Proliferation and Differentiation
Small numbers of insulin producing cells 1-5%
Probably Glucose Responsive 7 Days After transplantation
insulin producing cells rise to 15%
Data suggest
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
Outstanding Problems
Sufficient cells to provide adequate therapy
Persistent gene expression & protein synthesis for a long period
Encouraging in- vivo studies with the AAV Vector IM injections of the human insulin and Glucokinase genes into STZ diabetic rats (Mas et al,
Diabetes 2006) andSTZ diabetic dogs prolonged cure
(Callejas et al Diabetes 2013)
IV injection of factor IX in human Hemophilia B (Amit et al, NEJM 2011)
iPS Cells and Mesenchymal Stromal Cells only new runners in 10 years
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
Insulin And Cpp in cultutred differentiated MSCs using Trichostatintransplanted under renal capsule, received on 28- 8-2013
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Cpp +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Insulin & Cpp +ve (Yellow) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom Factor 3)
Cpp +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom Factor 3)
Insulin & Cpp +ve (Yellow) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom Factor 3)
Insulin And Glucagon in differentiated MSCs transplanted under renal capsule, Kidney received on 28- 8-2013
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Insulin +ve (GREEN) & glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom factor 2.5)
Glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom factor 2.5)
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Insulin +ve (GREEN) & glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL)
Insulin +ve (GREEN) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom factor 2.5)
Glucagon +ve (RED) differentiated MSCs transplanted under renal capsule (63X/1.40 OIL, Zoom factor 2.5)
Path to Beta Cell from iPS (Nichols, New and Annes
“Wild Speculation”Genetic engineering to remove
gene of diabetic targetSBlood, bone marrow, fat - stem cell of patient
Culture with growth &differentiation factors ofembryonic pancreas soupto produce autologous cells
Autotransplant of “doctored”culture cells No imunosuppression
No target for autoimmune attack
“Wild Speculation”Genetic engineering to remove
gene of diabetic targetSBlood, bone marrow, fat - stem cell of patient
Culture with growth &differentiation factors ofembryonic pancreas soupto produce autologous cells
Autotransplant of “doctored”culture cells No imunosuppression
No target for autoimmune attack