3Rs in use of laboratory animals for testing and

developing regenerative medicine

Frank Staal

Stem Cell Biology, Dept. Immunohematology

November 2016

Replacement in regenerative medicine

3 Rs

= Replacement, Reduction, Refining

Replacement in general by:

-Human volunteers, tissues and cells

-Mathematical and computer models

-Established animal cell lines,

-Invertebrates, such as Drosophila

and nematode worms.

Regenerative medicine or severe immune disorders:

Gene therapy

SCID= Severe Combined Immune Deficiency.

Genetic disease of the immune system

Sometimes called Bubble Boy Disease

Treatment: bone

marrow

transplantation.

Or: gene therapy

with own stem

cells

The Immune system:

Defense aginst invaders

Macrofages Granulocytes B cells T cells

Development of B and T cells

What is gene therapy? Transfer of correct version of mutated gene with therapeutic intent

How? Virus as vector

Crippled virus with therapeutic gene

How does stem cell - gene therapy work?

Blood Stem Cell from Patient

plus

B cell making antibodies Mature B Immature B

precursor

THYMUS

BLOOD

T Pre T

Immature B

Stem cell

Pre T Mature T

Relief of developmental block

Mouse studies needed as proof of principle

Efficacy: specific disease models

Mice that miss the same gene as human patients

Heatlth cells Deficient cells Gene therapy treated cells

Restoration of T cells in gene therapy treated mice

Successful gene therapy in patients: example from Paris

Restoration of T cells,

thymus tissue and

immunity

in the first 5 treated

patients in the world

NEJM, 2000

Severe adverse effects in SCID gene

therapy trials

Leukemia after gene therapy in 5 patients:

4 in the Paris trial and 1 in the London trial

T-cell kinetics lymphoid blasts

Hacein-Bey-Abina Science, 2003

Safety studies in gene therapy (and other cellular

therapies !)

Traditional: transplanting hundreds of mice with gene therapy treated

cells. Waiting for leukemia to happen.

Not predictive for human disease

time and labor consuming

severe stress on lab animals

Required GMP testing of viral batches in suckling mice

In > 10,000 mice never positive, but required

Alternative: IVIM assay: in vitro immortalization assay (Hannover)

In vitro immortalization assay – FDA approved surrogate safety assay

IVIM assay is a better predictor for safety than

mouse experiments

Data from Axel Schambach,

Ute Modlich, Hannover

Needs inmprovement:

• human cells

• No primary cells

• lymphoid cells/leukemia

Urgent need for grant money

But mice are not humans: larger animals needed?

• Often proposed: studies in

dogs or monkeys

• However, seldom the type of

disease (SCID)

• Better: use transplantation of

human cells into a limited

number of mice that accept

human cells

• xenotransplantation

models

N.B. Testing cosmetics on animals not acceptable

Xenotransplantation: human stem cells into

deficient mice (NSG)

hCD45

SS

C-A

hCD45

CD

13

/33

CD

19

CD

3

CD

16

/56

Transplantation of healthy CD34+ BM

CD4

Myeloid B T NK

IgM

CD

8

IgD

Thymocytes

hCD45+

PB B cells

hCD45+/CD19+

0.50% 73.9%

7.50% 18.0%

3.6% 78.2%

2.9% 15.3%

GT GT

Healthy donor Treated SCID Healthy donor Treated SCID

Alternative for large animal studies !

Lab animals in gene therapy applications

Efficacy: use in proof of principle studies: disease models (e.g. KO

mice): Needed, limited numbers (dozens)

Alternative for large animals (dogs, monkeys): xenotransplantation

(human stem cells into mice)

Safety studies: to investigate severe adverse effects.

Required by legislation, but not informative. 100s of mice needed

IVIM or other in vitro assays better

Better legislation: requirements for safety testing need change. There

often are equivalent or better alternatives, or tests are not informative

Harmonized procedures across Europe/EU: repeat of same animal

studies (in various EU countries) useless

Acknowledgements

Immunohematology and

Blood Transfusion Karin Pike-Overzet

Miranda Baert

Marja van Eggermond

Edwin de Haas

Sandra Vloemans

Anna-Sophia Wiekmeijer

Frank Staal

Wim Fibbe

Jaap-Jan Zwaginga

Pediatrics Dagmar Berghuis

Arjan Lankester

Robbert Bredius

Pharmacy Pauline Meij

Maarten Zandvliet

Molecular Cell Biology Rob Hoeben

Department of Immunology, Rotterdam Mirjam van den Burg

Jacques van Dongen

Hannover Medical School Michael Rothe

Axel Schambach

UCL, London Fang Zhang

H. Bobby Gaspar

Adrian Thrasher

Necker Hospital, Paris Chantal Lagresle-Peyrou

Marina Cavazzana