Gene Editing: Swine in Biomedical Research (Angelika Schnieke)

Technische Universitt Mnchen
Gene Editing
Animals in biomedical research
Swine
Angelika Schnieke
30 years of genetic engineering of large animals

1985-2015
Brief history: Methods for genetic engineering

Genetically modified pigs for biomedical research
Why genome editing
Livestock Biotechnology
History of genetic modification
Selective breeding of plants and animals

First domestication 12000 BC (dog)
Unnatural' hybrids 3000 years ago

(Mules: male donkey and a mare)

1985-2015
1980 Gordon, Ruddle 1st transgenic mouse produced by DNA microinjection
1983 Palmiter, Brinster human GH transgenic mouse

The implicit possibility is to use this
technology to stimulate rapid growth
of commercially valuable animals
1985 Palmiter, Brinster Production of transgenic
rabbits, sheep and pigs by microinjection.
Beltville pig (GH)
1986 Pharming
Approved products
2006
Anti thrombin III
2010
C1 esterase inhibitor
Pharming: recombinant fibrinogen

- fibrin sealant, haemostat, anti-adhesive
Fibrinogen
chains
330 kDa hexamer
Multiple disulphides
Untreated control
at 14 days
extensive adhesions
Fibrin monomer
in acidic buffer
Neutralising
buffer
Fibrin I
polymer
Fibrin I treated
at 14 days
no adhesions
Drawbacks of pronuclear microinjection

DNA microinjection: only <1-5% transgenic animals
Transposons:
size limitation
Viral vectors:
safety issues, size limitation
No control over where the transgene integrates

Transgene analysis only possible after birth
No control over sex of founder - males allow faster flock expansion
Only gene addition possible - no gene deletion, modification, substitution
Microinjection: sheep, pig
Sheep zygote
Sheep zygote
Pig zygote
Pig zygote after centrifugation
Images: H. Nieman, Mariensee
Drawbacks of pronuclear microinjection

DNA microinjection: only <1-5% transgenic animals
Transposons:
size limitation
Viral vectors:
safety issues, size limitation
No control over where the transgene integrates

Transgene analysis only possible after birth
No control over sex of founder - males allow faster flock expansion
Only gene addition possible - no gene deletion, modification, substitution
What do we want?
We want: 100% transgenic animals
Analysis of transgenic status before generation of animals
Decide the sex of animals
Control over where the transgene integrates
Methods for precise manipulation of the animal genome
Homologous recombination/gene targeting
Cell mediated transgenesis
Targeted gene modification

Mouse ES cells (1981)
Blastocyst
Somatic cells
Livestock
ES cells
ES or iPS cells
Transfection
Targeting Vector
Porcine iPS ??
Homologous recombination
Selection
Chimeric animals
iPS cells
Targeted gene modification

Nuclear transfer
Oozyte
Slaughter house
Somatic cell
Refine
In vitro
In vivo
1996 Dolly
Enucleation
Genetic manipulation
DNA, RNA analysis
3R
Reduce
Refine
Replace
Cell transfer
Electro fusion
Replace
Embryo culture
Pregnancy
(150 days)
Embryo transfer
Reduce
History of genetic modification

1996 First animal cloned from an adult somatic cell (Dolly)
1997 First cloned transgenic lamb (Polly, Molly, Holly, Olly)
1999 First gene targeting in livestock (Diana and Cupid)
2000 First cloned pigs from an adult somatic cell (Millie, Christa,
Alexis, Carrel & Dotcom)

1985-2015

Why genome editing
Large animal models for biomedicine
Translation into the clinic

Success rate 1/5.000
Basic research
Preclinical tests
Establishing new models
Validation / Safety
Proof of principle
Similarity in physiology/anatomy
Bench to bedside
Clinical trials
Patients
Linear growth in life expectancy over last 170 years

Increased life expectancy increased cancer incidence
Half the babies born in Japan in 2007

will live to be 107
Why do we need large animal cancer models?

Mouse models
Until recently, genetically defined models only in mice
Mouse models do not always replicate human disease phenotype
Large animal models - pig

Similar size to human
Use of human sized equipment (surgery, radiation treatment, imaging)
Longer lifespan than mice
Longitudinal studies in single individual
Similar pharmacokinetics
Can be fed human diet
Disease phenotype closer to humans (?)
Porcine cancer model: Colorectal cancer
Gene targeting in somatic cells, selection
Nuclear transfer
mouse human = pig

Nature Reviews Cancer 8, 387-398 (May 2008)

Flisikowska et al. 2012; Gastroenterology 143, 1173-1175
Organ specific inducible oncogenic mutation of p53

Marker gene expressed
TP53R167H silent
*
TP53R167H expressed
*
Cre
recombinase
lox-sa-bs-polyA-polyA-polyA-lox
lox
Promoter
Cre recombinase
Gene targeting in somatic cells, selection
Transfection, selection
Nuclear transfer
Nuclear transfer
Orthologues
Human
R175H
Mouse
R172H
Pig
R167H
X
Leuchs et al., 2012 and Li et al., 2015

Disease
Genetic modification
Reference
TP53
GLI2
v-H-RAS
BRCA1
APC
Conditionally activated targeted mutation

Human transgene
MMTV directed v-H-ras transgene
Knockout
Targeted truncating mutations
Leuchs 2012
McCalla-Martin 2010
Yamakawa 1999
Luo 2011
Flisikowska 2012
LDLR
PCSK9
NOS3
Catalase
PPAR-G
APOC3
Knockout
Human mutant transgene
Porcine transgene
Human transgene
Knockout
Human transgene
Carlson 2012
Al-Mashhadi 2013
Hao 2006
Whyte 2011
Yang 2011
Wei 2011
GIPR
INS
HNF-1
Human dominant negative mutant transgene

Porcine mutant transgene
Human dominant negative mutant transgene
Renner 2010
Renner 2013
Umeyama 2009
APP
HTT
SMN1
Human mutant APP transgene

Human mutant transgenes
Knockout
Takeuchi 2000
Baxa 2013
Lorson 2011
RHO
ELOVL4
Porcine and human mutant transgenes

Human mutant transgenes
Ross 2012
Sommer 2011
CFTR
DMD
F8
IL2RG
Knockout
Knockout
Knockout
Knockout
Rogers 2008
Klymiuk 2013
Kashiwakura 2013
Suzuki 2012
Cancer
Various cancers
Basal cell carcinoma
Breast cancer
Colorectal cancer
Cardiovascular diseases
Atherosclerosis
Hypercholesterolemia
Nitric oxide regulation
Hydrogen peroxide regulation
Atherosclerosis
Hypertriglyceridemia
Diabetes mellitus
Diabetes type 2
PNDM
Diabetes type 3 (MODY)
Neurodegenerative diseases
Alzheimers disease
Huntingtons disease
Spinal muscular atrophy
Ophthalmic neurodegenerative diseases

Retinitis pigmentosa
Stargardt disease-3
Other diseases
Cystic fibrosis
Duchenne muscular dystrophy
Haemophilia A
SCID

1985-2015

Why genome editing
Why genome editing

ROSA 26
RAG1
IL2R
GGTA1
LDLR
DMD
P53
APC
KRAS
CFTR
BRCA1
SMN1
Ig heavy chain
Ig light chain
Gene targeting is inefficient requires cultured cells

Gene targeting in somatic cells is very inefficient
~100x less efficient than in mouse ES cells
Few genes targeted in swine in the last 15 years

Very few examples of conditional gene targeting
Complex manipulation very, very inefficient
Targeted modification only on 1 allele breeding to homozygosity
SA
bsr
Lox
*3
Lox
Why genome editing

Gene editing is very efficient (>40%)
Editing both alleles
Multiple target genes simultaneously
Can be done in cells or embryo
Necessary vectors are easy to construct
Applied as DNA, RNA, protein
* Lox
Lox
SA
bsr
*3
Fig. Nature Protocols 9, 810827 (2014)
Why genome editing

Gene editing is very efficient
Gene editing
Analysis of single cell clones

DNA, RNA, Protein
Nuclear
transfer
Gene editing
Piglet with predicted

modification
Analaysis post partum

Modification?
Mosaicism?
Gene editing: example xenotransplantation
Genome-wide inactivation of porcine endogenous retroviruses (PERVs).

Yang et al., Science. 2015 Nov 27;350(6264):1101-4.
62 PERVs
Why genome editing

Gene editing is very efficient
Can be done in all species (no requirement for ES cell or SCNT)
Human polyclonal antibodies

- more effective than mAbs for passive immunotherapy
- mimic natural immune response
Can only be produced in people or transgenic animals
Require animals larger than mice for production of adequate quantities of serum.
Human polyclonal antibodies

produced in rabbit
Rabbit nuclear transfer: difficult
Putative rabbit ES cells
Zakhartchenko et al., 2011 Biol Reprod 84
Immunoglobulin gene disruption using Zinc Finger Nucleases

2011 Flisikowska et al PLoS ONE 6(6): e21045
ZFN RNA
ZFN RNA
+ targeting vector DNA
pronucleus & cytoplasm
Targeted replacement in
up to 18% of fetuses.
Porcine cancer models

KRAS, TP53, APC involved in many different types of cancer
KRAS:
pancreas, large/small intestine, lung, testis, liver, thyroid
TP53:
most human tumours, small cell lung carcinoma
APC:
Colorectal cancer, gastric and duodenal malignancies, pancreas, biliary tract,

gall bladder, hepatoblastomas
Just add Cre
In vivo CRISPR/Cas9 application

Just add 1 or more guide RNAs
Genetically modified
large animals
Environment
Pharming
Disease
models
Health
Products
Improving
Regenerative
Medicin
Genome editing in swine

Beyond biomedicine
Edited genes
Targeted genes
Double-muscled pigs
Nature 523, 1314 (02 July 2015)
Mutation in the myostatin gene (MSTN).
Micro pigs as pets

18 | NATURE | VOL 526 | 1 OCTOBER 2015
TALENs inactivated 1 of 2 copies of the growth hormone receptor gene (GHR)
Fighting African Swine Fever

Pig 26 Roslin Inst.
Selective transfer of naturally occurring gene variants

Alternative method to breeding
Genetic engineering
Mammalian Genome
3.3 x 109 bp
Genome engineering / Genome editing

Allows precise exchange of single or multiple base pairs in the genome
Prerequisite: genome sequence
Control:
genome sequencing
Genetic modification: Cas9-based applications
Sander & Joung, Nature Biotech April 14
Genetic engineering of large animals

State of the art
CRISPR/Cas9
Leading tool in the fields of synthetic biology and genome engineering.
COST Action BM1308
Sharing Advances on Large Animal Models
http://www.salaam.genzentrum.lmu.de/

Technologies for genetic manipulations are available and continue to
improve
In itself not a sufficient reason to produce genetically modified large
animals
For each project risk and benefits have to be evaluated
Animal wellbeing has to have a high priority
If it provides and advantage for the environment, the health or wellbeing of
human or animal, than genetic modification (incl. gene editing) is a
powerful and precise tool.
Few laboratories have capacity to carry out large animal experiments
Time and cost intensive need to coordinate efforts
Ethical and public concerns need to be addressed

Animal models
Work with animals is strictly regulated
Animal experiments require approval by governmental body
Biomedicine
Clinical and regulatory need for development of large animal models
Various models of human diseases are being developed
Genome editing: one more tool in the genetic toolbox Reduce, Refine
Agriculture
A tool to improve animal health (disease resistance/protection), traits
Reduce environmental impact
Replicate natural variants (not transgenic)
genetically modified ?
genetically improved ?
labelling ?
TUM WZW
Benedikt Baumer
Marlene Edlinger
Daniela Fellner
Konrad Fischer
Tatiana Flisikowska
Krzysztof Flisikowski
Alexander Kind
Simone Kraner
Shun Li
Denise Nguyen
Beate Rieblinger
Anja Saalfrank
Erica Schulz
Carolin Wander
Helmholz
Ralf Khn
Wolfgang Wurst
Poznan University of Life Sciences
Marek Switonski,
TUM Klinikum rechts der Isar

Dieter Saur
Gnter Schneider
Stefan Eser
Roland Schmid
Klaus-Peter Janssen
LMU
Barbara Kessler
Mayuko Kurome
Valeri Zakhartchenko
Eckhard Wolf
Hannover
Bjrn Petersen
Heiner Niemann
Ralf Schwinzer
Roche
Josef Platzer
Antonio Iglesisas
Ulrich Certa
Thank you for your attention

Gene Editing: Swine in Biomedical Research (Angelika Schnieke)

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Gene Editing: Swine in Biomedical Research (Angelika Schnieke)

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Technische Universitt Mnchen

Technische Universitt Mnchen

30 years of genetic engineering of large animals

Brief history: Methods for genetic engineering

Technische Universitt Mnchen

History of genetic modification

Selective breeding of plants and animals

Unnatural' hybrids 3000 years ago

Technische Universitt Mnchen

30 years of genetic engineering of large animals

1983 Palmiter, Brinster human GH transgenic mouse

Technische Universitt Mnchen

Anti thrombin III

Technische Universitt Mnchen

Pharming: recombinant fibrinogen

Technische Universitt Mnchen

Drawbacks of pronuclear microinjection

safety issues, size limitation

No control over where the transgene integrates

Technische Universitt Mnchen

Microinjection: sheep, pig

Pig zygote after centrifugation

Images: H. Nieman, Mariensee

Technische Universitt Mnchen

Drawbacks of pronuclear microinjection

safety issues, size limitation

No control over where the transgene integrates

Technische Universitt Mnchen

Cell mediated transgenesis

Technische Universitt Mnchen

Targeted gene modification

Targeted gene modification

Technische Universitt Mnchen

Technische Universitt Mnchen

History of genetic modification

1997 First cloned transgenic lamb (Polly, Molly, Holly, Olly)

1999 First gene targeting in livestock (Diana and Cupid)

Technische Universitt Mnchen

30 years of genetic engineering of large animals

Brief history: Methods for genetic engineering

Technische Universitt Mnchen

Large animal models for biomedicine

Translation into the clinic

Establishing new models

Technische Universitt Mnchen

Linear growth in life expectancy over last 170 years

Half the babies born in Japan in 2007

Technische Universitt Mnchen

Why do we need large animal cancer models?

Large animal models - pig

Technische Universitt Mnchen

Porcine cancer model: Colorectal cancer

Gene targeting in somatic cells, selection

mouse human = pig

Nature Reviews Cancer 8, 387-398 (May 2008)

Technische Universitt Mnchen

Organ specific inducible oncogenic mutation of p53

Gene targeting in somatic cells, selection

Technische Universitt Mnchen

Conditionally activated targeted mutation

Human dominant negative mutant transgene

Human mutant APP transgene

Porcine and human mutant transgenes

Ophthalmic neurodegenerative diseases

Technische Universitt Mnchen

30 years of genetic engineering of large animals

Brief history: Methods for genetic engineering