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