Technische Universität München

Gene Editing
Animals in biomedical research
Swine
Angelika Schnieke

Technische Universität München

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

Technische Universität München

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)

Livestock Biotechnology

Technische Universität München

30 years of genetic engineering of large animals
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)
Livestock Biotechnology

Technische Universität München

1986 Pharming

Approved products

Livestock Biotechnology

2006

Anti thrombin III

2010

C1 esterase inhibitor

Technische Universität München

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

Livestock Biotechnology

Fibrin I
polymer

Fibrin I treated
at 14 days
no adhesions

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

Microinjection: sheep, pig

Sheep zygote

Sheep zygote

Pig zygote

Pig zygote after centrifugation

Livestock Biotechnology

Images: H. Nieman, Mariensee

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

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

Technische Universität München

Oozyte
Slaughter house
Somatic cell

Refine

In vitro

In vivo
1996 Dolly
Livestock Biotechnology

Enucleation

Genetic manipulation
DNA, RNA analysis

3R
Reduce
Refine
Replace

Cell transfer

Electro fusion

Replace

Embryo culture

Pregnancy
(150 days)

Embryo transfer

Reduce

Technische Universität München

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)

Livestock Biotechnology

Technische Universität München

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

Technische Universität München

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
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Clinical trials
Patients

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

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 (?)
Livestock Biotechnology

Technische Universität München

Porcine cancer model: Colorectal cancer

Gene targeting in somatic cells, selection

Nuclear transfer

mouse ≠ human = pig
Livestock Biotechnology

Nature Reviews Cancer 8, 387-398 (May 2008)
Flisikowska et al. 2012; Gastroenterology 143, 1173-1175

Technische Universität München

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

Livestock Biotechnology

X
Leuchs et al., 2012 and Li et al., 2015

Technische Universität München
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-1α

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
Alzheimer’s disease
Huntington’s disease
Spinal muscular atrophy

Ophthalmic neurodegenerative diseases
Retinitis pigmentosa
Stargardt disease-3

Other diseases
Cystic fibrosis
Duchenne muscular dystrophy
Haemophilia A
SCID

Livestock Biotechnology

Technische Universität München

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

Technische Universität München

Why genome editing
ROSA 26
RAG1
IL2Rγ
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

1

SA

bsr
Lox

Livestock Biotechnology

2

3

2

*3

4

Lox

Technische Universität München

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

2

1

* Lox

Lox

SA

Livestock Biotechnology

bsr

3

2

*3
Fig. Nature Protocols 9, 810–827 (2014)

4

Technische Universität München

Why genome editing
Gene editing is very efficient
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

Gene editing

Analysis of single cell clones
DNA, RNA, Protein

Livestock Biotechnology

Nuclear
transfer

Gene editing

Piglet with predicted
modification

Analaysis post partum
Modification?
Mosaicism?

Fig. Nature Protocols 9, 810–827 (2014)

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

Why genome editing
Gene editing is very efficient
Editing both alleles
Multiple target genes simultaneously
Can be done in cells or embryo
Can be done in all species (no requirement for ES cell or SCNT)
Necessary vectors are easy to construct
Applied as DNA, RNA, protein

Livestock Biotechnology

Fig. Nature Protocols 9, 810–827 (2014)

Technische Universität München

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

Livestock Biotechnology

Putative rabbit ES cells

Zakhartchenko et al., 2011 Biol Reprod 84

Technische Universität München

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.

Livestock Biotechnology

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

Genetically modified
large animals
Environment

Pharming

Disease
models

Health
Products

Improving

Regenerative
Medicin

Livestock Biotechnology

Technische Universität München

Genome editing in swine
Beyond biomedicine
Edited genes

Targeted genes

‘Double-muscled’ pigs
Nature 523, 13–14 (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
Livestock Biotechnology

Technische Universität München

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

Livestock Biotechnology

Technische Universität München

Genetic modification: Cas9-based applications

Livestock Biotechnology

Sander & Joung, Nature Biotech April 14

Technische Universität München

Genetic engineering of large animals
State of the art

CRISPR/Cas9
Leading tool in the fields of synthetic biology and genome engineering.

Technische Universität München

COST Action BM1308

Sharing Advances on Large Animal Models

Livestock Biotechnology

http://www.salaam.genzentrum.lmu.de/

Technische Universität München

Genetic engineering of large animals
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
Livestock Biotechnology

Technische Universität München

Genetic engineering of large animals
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 ?

Livestock Biotechnology

Technische Universität München

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 Kühn
Wolfgang Wurst
Poznan University of Life Sciences
Marek Switonski,
Livestock Biotechnology

TUM Klinikum rechts der Isar
Dieter Saur
Günter Schneider
Stefan Eser
Roland Schmid
Klaus-Peter Janssen
LMU
Barbara Kessler
Mayuko Kurome
Valeri Zakhartchenko
Eckhard Wolf
Hannover
Björn Petersen
Heiner Niemann
Ralf Schwinzer
Roche
Josef Platzer
Antonio Iglesisas
Ulrich Certa

Livestock Biotechnology

Technische Universität München

Thank you for your attention

Livestock Biotechnology

Technische Universität München

Livestock Biotechnology

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