Targeted genetic modification of the porcine genome using zinc-finger nucleases
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
at the University of Veterinary Medicine Hannover
by
Janet Hauschild-Quintern (Bremen)
Hannover 2012
Advisory Committee: Prof. Dr. Heiner Niemann Prof. Dr. Gerhard Breves Prof. Dr. Ulrich Martin 1st Evaluation: Prof. Dr. Heiner Niemann
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Mariensee, Germany
Prof. Dr. Gerhard Breves
Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany
Prof. Dr. Ulrich Martin
LEBAO, Hannover Medical School, Hannover, Germany
2nd Evaluation: Prof. Dr. Angelika Schnieke
Livestock Biotechnology, Center of Life and Food Sciences Weihenstephan, Freising, Germany
Technical University Munich
Date of oral exam: 5.11.2012
This PhD-Thesis was conducted at the Institute of Farm Animal Genetics, Friedrich-Loeffler- Institut (FLI) in Mariensee, Germany.
For my family and my husband.
Presentations of the thesis
These topics have been presented at national or international conferences as poster or oral presentations:
J. Hauschild, A.-L. Queisser, J.W. Carnwath, G. Cost, Y. Santiago, E. Rebar, P. Gregory, H.
Niemann, B. Petersen (2011) Genetic targeting of the porcine α1,3-galactosyltransferase gene in fetal fibroblast cells using zinc-finger nucleases. Poster presentation at the International Embryo Transfer Society (IETS) in Orlando, USA (Abstract #: 329). Abstract was published in Reproduction, Fertility and Development. 23(1): 260-1, December 2010
J. Hauschild, B. Petersen, Y. Santiago, A.-L. Queisser, J.W. Carnwath, A. Lucas-Hahn, L. Zhang, X. Meng, P. D. Gregory, R. Schwinzer, G. J. Cost, and H. Niemann (2011) Zinc-finger nuclease mediated knockout of the porcine α1,3-galactosyltransferase gene. Oral presentation at the International Xenotransplantation Association (IXA) held by B. Petersen in Miami, USA (Abstract #: 124)
J. Hauschild, B. Petersen, Y. Santiago, A.-L. Queisser, J.W. Carnwath, A. Lucas-Hahn, L. Zhang, X. Meng, P. D. Gregory, R. Schwinzer, G. J. Cost, and H. Niemann (2012) Gender-unspecific knockout of the GGTA1 gene in pigs using zinc-finger nucleases. Poster presentation at the International Embryo Transfer Society (IETS) in Phoenix, USA (Abstract #: 233). Abstract was published in Reproduction, Fertility and Development. 24(1): 229, December 2011
J. Hauschild, B. Petersen, Y. Santiago, A.-L. Queisser, J.W. Carnwath, A. Lucas-Hahn, L. Zhang, X. Meng, P.D. Gregory, R. Schwinzer, G.J. Cost, H. Niemann (2012) Knockout of the α1,3- galactosyltransferase gene in pigs using zinc-finger nucleases. Poster presentation at the 45th Annual Conference of Physiology and Pathology of Reproduction in Berlin, Germany (Abstract #: 54)
Content
Abbreviations ... I List of Figures ... III List of Tables ... IV Summary ... V Zusammenfassung ... VII
1. General Introduction ... 1
1.1. Transgenic animals ... 1
1.2. Xenotransplantation ... 5
1.3. Somatic cell nuclear transfer (SCNT) ... 10
1.4. Transgenic technologies ... 11
1.5. GGTA1 gene knockout ... 14
1.6. Zinc-finger nuclease (ZFN)-mediated gene targeting ... 15
1.7. Transcription activator like effector nucleases (TALENs) ... 16
1.8. Motivation and goal of this study ... 17
2. Publication 1 ... 19
Gene knockout and knockin by zinc-finger nucleases: Current status and perspectives 3. Publication 2 ... 21
Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases 4. Publication 3 ... 23
Gender non-specific efficacy of ZFN mediated gene targeting in pigs 5. General Discussion ... 25
5.1. Production and enrichment of GGTA1 negative cells via ZFN-mediated gene targeting ... 25
5.2. Transgenic fetuses and pigs produced by SCNT ... 26
5.3. Analysis of transgenic pigs... 28
5.4. Exclusion of negative side effects by ZFN-mediated targeting ... 29
5.5. Production of female and male ZFN-mediated GGTA1-KO animals ... 30
5.6. Importance of this PhD-Thesis ... 32
5.7. Conclusions and perspectives ... 33
6. References ... 35
Acknowledgments ... 61
Abbreviations
AAVS – adeno-associated virus site (safe harbor site in human genome) AD – Alzheimer’s Disease
AI – artificial insemination
ATryn® – commercially available heme-antithrombin III AVR – acute vascular rejection
bp – base pair
BSE – bovine spongiform encephalopathy BVDV – bovine viral diarrhea virus
CCR2 – human chemokine receptor 2 gene CCR5 – human chemokine receptor 5 gene CF – Cystic Fibrosis
CFTR – cystic fibrosis transmembrane conductance regulator CJD – Creutzfeldt-Jakob disease
CSF(V) – classical swine fever (virus) DAF – decay accelerating factor (hCD55) DNA – deoxyribonucleic acid
Dox – doxycycline
DSB – double-strand break
DXR – delayed xenograft rejection ESCs – embryonic stem cells
FACS – fluorescence-activated cell sorting
FokI – type IIS restriction endonuclease found in Flavobacterium okeanokoites FUT8 – α1,6-fucosyltransferase gene
GAL – product of α1,3-galactosyltransferase gene GFP – green fluorescent protein
GGTA1 – α1,3-galactosyltransferase gene HAE – Hereditary Angioedema
HAR – hyperacute rejection HD – Huntington’s Disease HDR – homology direct repair hEPO – human erythropoietin hFIX – human coagulation factor IX hHOI – human hemoxygenase-I hPC – human protein C
HR – homologous recombination
II HUS – hemolytic uremic syndrome ICAM-I – intercellular adhesion molecule 1 ICER Iɣ – inducible cAMP early repressor iGb3S – isoglobotriaosylceramide synthase iPSCs – induced pluripotent stem cells IRs – inverted repeats
KI – gene knockin KO – gene knockout LOH – loss of heterozygosity MAC – membrane attack complex
MCP – membrane cofactor protein (hCD46)
MIRL – membrane inhibitor of reactive lysis (hCD59) NHEJ – non homologous end joining
NPI – neonatal porcine islet
OCT4 – octamer-binding transcription factor 4 OTS – off-target site
PERV – porcine endogenous retrovirus PITX3 – pituitary homeobox 3 gene PrP – prion protein
rhC1INH – recombinant human C1 inhibitor RNA – ribonucleic acid
RP – Retinitis Pigmentosa RVD – repeat variable diresidue SB – sleeping beauty transposon SCNT – somatic cell nuclear transfer sh – short hairpin
SMGT – sperm-mediated gene transfer SNP – single nucleotide polymorphism
TALEN – transcription activator like effector nuclease TFPI – tissue factor pathway inhibitor
TNFAIP3 – tumor necrosis factor α-induced protein 3 gene (A20) TRE – tetracycline response element
VCAM-I – vascular cell adhesion protein 1 WT – wild-type
ZF – zinc-finger
ZFN – zinc-finger nuclease
List of Figures
Figures in General Introduction and Discussion
Figure 1: Waiting list and number of kidney transplantations in Germany ... 5
Figure 2: Hyperacute rejection (HAR) after anti-Gal antibodies bind to Gal-epitopes ... 7
Figure 3: Comparison between DNA binding of ZFNs and TALENs ... 16
Figure 4: Breeding of GGTA1-KO pigs... 31
Following figures are not included in this thesis
Figures in Publication 1
Figure P1.1: ZF and ZFN molecules binding to DNA
Figure P1.2: ZFN-mediated gene targeting: NHEJ and HDR
Figure P1.3: ZFN-mediated gene targeting: SCNT and microinjection Figure P1.4: Cel-I assay (Surveyor nuclease assay) scheme
Figure P1.5: Function of mutated FokI nucleases as obligate heterodimer
Figures in Publication 2
Figure P2.1: Design and characterization of zinc-finger nucleases Figure P2.2: FACS measurement of transfected cells
Figure P2.3: Live-born piglet
Figure P2.4: GGTA1 sequencing results of female fetuses and piglets Figure P2.5: Antibody/complement-mediated lysis assay
Figure P2.S1: FACS results of six cloned fetuses
Figure P2.S2: Surveyor Nuclease assay results of the GGTA1 gene Figure P2.S3: FokI PCR results
Figure P2.S4: Surveyor Nuclease result of off-target sites (OTS)
Figures in Publication 3
Figure P3.1: FACS result of male ZFN-GGTA1-KO piglets Figure P3.2: Sequences of cloned male piglets
IV
List of Tables
Tables in General Introduction and Discussion
Table 1: Porcine models for human diseases ... 2
Table 2: Overview of recombinant human proteins produced in transgenic livestock ... 3
Table 3: Summary of pig-to-baboon transplantation ... 8
Table 4: Somatic cloning and recloning results using male ZFN-mediated KO cells ... 27
Table 5: SCNT results of other GGTA1-KO studies ... 27
Following tables are not included in this thesis
Tables in Publication 1
Table P1.1: Listing of organisms treated with ZFNs against specific genes
Tables in Publication 2
Table P2.1: Somatic cloning and recloning results using female ZFN-mediated KO cells Table P2.S1: Off-target sites list
Summary
Janet Hauschild-Quintern:
Targeted genetic modification of the porcine genome using zinc-finger nucleases
Zinc-finger nucleases (ZFNs) are artificial molecular scissors that induce a double-strand break (DSB) at a specific site of the genome. After cleavage, error prone repair by non homologous end joining (NHEJ) can result into a reading frame shift causing a functional gene knockout (KO). This targeting method is much more efficient than conventional gene targeting, such as homologous recombination (HR).
ZFNs have successfully been applied in several species, including insects, amphibians, plants, nematodes, and several mammals, including humans. Within the scope of this thesis, the first biallelic gene knockout of an endogenous gene in the porcine genome via ZFN- mediated targeting is reported. The knockout of the α1,3-galactosyltransferase (GGTA1, Gal) gene is essential for the success of pig-to-human xenotransplantation to abolish Gal- epitopes, the major antigen. Transplantation of a xenograft leads to a hyperacute rejection response (HAR) due to pre-existing antibodies in human blood against Gal-epitopes which are expressed on the surface of porcine cells.
Organ survival was prolonged when porcine organs with a biallelic knockout of the GGTA1 gene had been transplanted into primate recipients. Gal-negative piglets were produced by conventional targeting. This method is very time consuming and critically depends on a knockout vector introducing the foreign DNA into the porcine genome. The biallelic GGTA1-KO mediated by ZFNs has several advantages and may lead to more viable piglets as many negative side effects of conventional gene targeting can be avoided. Due to the high targeting efficiency, a biallelic gene knockout can be achieved in only one experiment. Negative side effects by ZFN-mediated targeting, such as off-target site cleavage, only rarely occur. This thesis describes the path from wild-type fetal pig cell line to biallelic ZFN-mediated GGTA1 gene knockout animals.
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ZFN-mediated gene targeting in porcine cells led to a maximum of 1% biallelic GGTA1-KO cells in the cell population. Enrichment of Gal-negative cells with magnetic beads led to >95% pure cells. These Gal-negative cells were used for SCNT to produce fetuses for a first analysis and subsequently to produce live Gal-negative piglets. Cells from fetuses and piglets were analyzed by fluorescent activated cell sorting (FACS) and Gal-epitopes could not be detected. A complement lysis assay demonstrated protection of fetal ZFN-mediated GGTA1-KO cells that was similar to that of conventional HR-GGTA1-KO cells. Sequence analysis revealed that mutations mediated by ZFN binding and cleavage mostly consisted of small deletions (1 to 7 bp). Only a few insertions (4 bp) and one large deletion of 96 base pairs were observed. Most fetuses and pigs carried two different mutated GGTA1 alleles (heterozygous mutation) and three of eleven haplotyes showed the same mutation on both alleles (homozygous mutation). Unwanted side effects of ZFN-mediated gene targeting such as plasmid integration and off-target site (OTS) mutations could not be detected.
Female and male Gal-negative piglets were produced by using ZFN-mediated gene targeting and SCNT. This method was much more efficient than conventional gene targeting by HR, negative side effects and gender specific differences were not observed. This study demonstrates that this new targeting tool can be successfully applied in the domestic pig to produce transgenic animals for xenotransplantation or disease models.
Zusammenfassung
Janet Hauschild-Quintern:
Spezifische genetische Modifikation des Schweinegenoms mittels Zinkfinger- Nukleasen
Zinkfinger-Nukleasen (ZFNn) sind künstlich generierte “molekulare Scheren”, die permanente genetische Veränderungen an einer spezifischen Stelle im Genom induzieren.
Das Schneiden der DNA durch die Nukleasen führt zur Aktivierung eines fehleranfälligen Reparaturmechanismus der Zelle („nicht-homologe End-Verknüpfung“), wodurch eine Mutation in der Basenabfolge der DNA Sequenz entstehen kann. Ist die Mutation ausreichend groß oder führt sie zu einer Leseraster Verschiebung, kann dies zu einem funktionellen „Gen-Knockout“ (KO) führen. Für die Generierung gezielter Modifikationen im Genom ist die ZFN-Methode wesentlich effizienter als konventionelle Methoden, wie z.B.
homologe Rekombination (HR). ZFNn wurden bereits erfolgreich in verschiedenen Organismen angewendet, wie z.B. Insekten, Amphibien, Pflanzen, Nematoden, und verschiedene Säugetiere, wie der Mensch.
Im Rahmen dieser Studie wird der erste „biallelische Gen-Knockout“ eines endogenen Schweinegens mittels Zinkfinger-Nukleasen dargestellt. Der „Knockout“ des α1,3-Galaktosyltransferase (GGTA1, Gal) Gens ist Voraussetzung für die Verwendung von Schweinen im Rahmen der Xenotransplantation (Transplantation von Organen vom Schwein zum Menschen). Transplantationen zwischen verschiedenen Arten, wie Schwein und Pavian, führen zu einer hyperakuten Abstoßungsreaktion (HAR). Grund dafür sind die sogenannten Gal-Epitope, welche sich auf der Zelloberfläche von Schweinezellen und Gewebe befinden.
Dem Menschen und Altweltaffen fehlen evolutionsbedingt diese Zuckermoleküle auf der Zelloberfläche. Stattdessen weisen sie Antikörper gegen die Gal-Epitope auf, welche bei einer Xenotransplantation das Schweinegewebe angreifen und dessen Zerstörung induziert.
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Das Überleben der Organe im Empfänger Organismus kann verlängert werden, indem das GGTA1-Gen ausgeschaltet wird, welches für die Generierung der Gal-Epitope verantwortlich ist. Gal-negative Schweine wurden bereits von anderen Gruppen produziert, allerdings mit der konventionellen HR Methode. Die ZFN-Methode hat mehrere Vorteile:
wegen ihrer hohen Effizienz ist es möglich innerhalb eines Experimentes beide Gene auszuschalten, zudem ist keine Antibiotika Selektion notwendig. Die zufällige Integration von fremder DNA in das Genom mittels HR kann zu schädlichen genetischen Veränderungen führen. Dieses Risiko ist im Falle der ZFN-vermittelten Modifikationsmethode drastisch minimiert. Ein eventueller negativer Nebeneffekt der ZFNn könnte die Induktion von unerwünschten Mutationen im Genom sein, die allerdings nur selten vorkommen. Die folgende Doktorarbeit beschreibt den Weg von fetalen „Wildtyp“ Schweinefibroblasten hin zu „biallelischen“ ZFN-vermittelten GGTA1 „Knockout“ Schweinen.
Die ZFN-vermittelte Genmodifikation von Schweinefibroblasten ergab eine maximale Effizienz von 1% im Falle des biallelischen GGTA1 „Knockouts“. Die Gal-negativen Zellen wurden mit Hilfe magnetischer „Beads“ angereichert, was mit >95%iger Reinheit gelang. Die Gal-negativen Zellen wurden für den somatischen Zellkerntransfer eingesetzt, um einerseits Feten für erste Analysen, und andererseits lebende Gal-negative Schweine zu produzieren.
Zellen der modifizierten Feten und Schweine wurden mittels Durchflusszytometrie analysiert, es konnten keine Gal-Epitope nachgewiesen werden. Eine „Komplement-Lysis- Prüfung“ wies einen Schutz der ZFN-vermittelten „Knockout“ Zellen vor der Lyse auf. Dieser war dem Schutze der konventionell erzeugten HR-GGTA1-KO Zellen ähnlich. Sequenz Analysen ergaben, dass die ZFN-vermittelten genetischen Veränderungen in der DNA Sequenz meist aus kleinen Deletionen (1 bis 7 bp), wenigen Insertionen (4 bp) und einer großen Deletion von 96 Basenpaaren bestanden. Die meisten Feten und Schweine hatten unterschiedliche Mutationen auf beiden Allelen (heterozygote Mutation), nur drei von elf
„Haplotypen“ wiesen die gleiche Mutation auf beiden Allelen (homozygote Mutation) auf.
Negative Nebeneffekte der ZFN-vermittelten Genmodifikation, wie z.B. Plasmid-DNA Integration in das Schweinegenom oder unerwünschte Mutationen an anderen Genorten, konnten nicht detektiert werden.
Weibliche und männliche Gal-negative Schweine wurden mit Hilfe der ZFN- vermittelten Genmodifikation und des somatischen Zellkerntransfers erstellt. Diese Methode ist effizienter als herkömmliche konventionelle genetische Modifikationsmethoden (HR). Negativen Effekte oder Geschlechtsspezifische Unterschiede wurden nicht festgestellt.
Die Ergebnisse dieser Studie zeigen, dass sich ZFNn als gezielte Modifikationsmethode im domestizierten Schwein eignen, um transgene Schweine zu erstellen, welche sich für Xenotransplantation oder als Krankheitsmodelle eignen.
1. General Introduction
1.1. Transgenic animals
Gene targeting is an important tool to understand biological systems and to alter the genomes for the production of transgenic animals. Several transgenic animals were produced in the last decade contributing to human welfare, agriculture and pharmaceutical production. Animal models mimicking human diseases can be used to study onset and progression of human diseases and might help to develop new therapies. Additionally, transgenic pigs are considered as organ donors for humans, called xenotransplantation.
Transgenic pigs as human disease models
Due to the high physiological similarity of pigs and human, the domestic pig has emerged as suitable model for human diseases. Transgenic pigs carrying a specific mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene developed human Cystic Fibrosis (CF) symptoms such as meconium ileus, exocrine pancreatic destruction, focal biliary cirrhosis and lung disease (including mucus accumulation and infection) with a timely onset comparable to humans (ROGERS et al. 2008a; ROGERS et al. 2008b; STOLTZ et al. 2010). In contrast, various transgenic mouse models for CF only partly reflected the ion-transport abnormalities, but failed to develop clinical manifestations typical of human CF (GRUBB and BOUCHER 1999).
Pigs are a suitable model for eye diseases, due to similar anatomy, size and retinal structure of human and porcine eyes. Retinitis Pigmentosa (RP) is an inherited degenerative retinal disease leading to night blindness due to rod photoreceptor degeneration followed by future blindness caused by slower cone photoreceptor degeneration (MILAM et al. 1998).
Production of transgenic pigs carrying a mutated rhodopsin gene (Pro347Leu is one of several mutations leading to human RP) resulted in similar disease manifestation as in human RT patients (PETTERS et al. 1997).
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Mimicking human neurodegenerative diseases such as Huntington’s Disease (HD) or Alzheimer’s Disease (AD) in transgenic pigs is favored, as the porcine brain is more similar to the human brain than that of rodents. Transgenic pigs with an extended polyglutamine tract in the huntingtin gene showed a similar phenotype (apoptotic neurons with DNA fragmentation in brain) as seen in HD patients (YANG et al. 2010). Table 1 summarizes successful porcine models for human diseases.
Table 1: Porcine models for human diseases leading to similar manifestation as in human patients
Disease Genetic modification Authors
Alzheimer’s Disease Human amyloid precursor protein gene with “Swedish mutation”
KRAGH et al. 2009 Cardio Vascular Disease Overexpression of human catalase gene WHYTE et al. 2011
Cystic Fibrosis CFTR+/- or CFTR ΔF508/+ ROGERS et al. 2008b
Diabetes Mutant human hepatocyte nuclear factor
1α gene
UMEYAMA et al. 2009 Huntington’s Disease Extended repeat part in huntigtin gene YANG et al. 2010 Retinitis Pigmentosa Pro347Leu mutation in rhodopsin gene PETTERS et al. 1997
(KRAGH et al. 2009) (WHYTE et al. 2011) (ROGERS et al. 2008b) (UMEYAMA et al. 2009) (YANG et al. 2010) (PETTERS et al. 1997)
Gene pharming
Using recombinant bacteria for the production of therapeutically human proteins has some drawbacks. Some proteins can not be synthesized, are aggregated and difficult to isolate or are not folded in the right manner (HOUDEBINE 2009). Gene pharming (combination of
"farming" and "pharmaceuticals”) in transgenic animals is well suited for a cost effective production of proteins that are required in large volumes or are difficult to be expressed in conventional recombinant production systems. Additionally, correct protein folding is assured. The mammary gland was chosen to be the optimal site for the production of recombinant human proteins, due to its high consistently milk yield. Expression in mammary gland requires a mammary gland specific promoter fused to the DNA coding for the human protein.
Cattle and goat are the preferred species for production of recombinant human proteins, because of the large amount of milk they produce every year. The rabbit is often used as “bioreactor”, because it is small, easy to reproduce and maintain and has a short
generation time with large litter sizes. It is an attractive alternative to large animals, due to the short time interval between production of transgenic livestock and first lactation. The following table (Table 2) gives a small overview on recombinant human proteins produced in transgenic livestock. These recombinant proteins can be used for blood coagulation (human coagulation factor IX (hFIX)), regulation of hemostasis (human protein C (hPC)), strengthening of human defense system (human lactoferrin (hLF)) or regulation of red blood cell production (human erythropoietin (hEPO)).
Table 2: Overview of recombinant human proteins produced in transgenic livestock
Species Protein Expression level
(maximum)
Authors
Cattle Human lactoferrin (hLF) 3.4 mg/ml YANG et al. 2008
Goat Human lactoferrin (hLF) 2.8 mg/ml VAN BERKEL et al. 2002
Goat Human antithrombin III (hATIII, ATryn®) >1 mg/ml EDMUNDS et al. 1998
Pig Human protein C (hPC) 1 mg/ml VELANDER et al. 1992
Pig Human coagulation factor IX (hFIX) 3 mg/ml LINDSAY et al. 2004
Pig Human erythropoietin (hEPO) 878 IU/ml PARK et al. 2006
Rabbit Human erythropoietin (hEPO) 0.5 mg/ml KORHONEN et al. 1997 Rabbit Human C1 inhibitor (rhC1INH, Ruconest®) No information VAN DOORN et al. 2005 (YANG et al. 2008) (VAN BERKEL et al. 2002) (EDMUNDS et al. 1998) (VELANDER et al. 1992) (LINDSAY et al. 2004) (PARK et al. 2006) (KORHONEN et al. 1997) (VAN DOORN et al. 2005)
The first commercially established gene pharming product is the heme-antithrombin III (ATryn®), which is secreted into the milk of transgenic goats, collected and then purified.
Antithrombin III is a natural anticoagulant that plays an important role in controlling the formation of blood clots. ATryn® is a new therapeutic option to benefit patients with hereditary antithrombin deficiency, a clotting disorder that is associated with venous thromboembolic events. ATryn® was approved by the EMEA in 2006 and achieved approval by the FDA in 2009. Ruconest® provided by Pharming Group NV (Netherlands) is a recombinant human C1 inhibitor (rhC1INH) produced in rabbit milk and is used for treatment of acute attacks of angioedema in patients with Hereditary Angioedema (HAE) (VAN DOORN et al. 2005). Transgenic goats were also generated for producing malaria antigen for vaccination in the mammary gland which is still in clinical phase (BEHBOODI et al.
2005; HOUDEBINE 2009). There will be more pharmaceuticals on the market soon, since
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further human pharmaceutical recombinant milk proteins are under investigation in different phases of clinical trials.
Agriculture and benefits in human welfare
Transgenic pigs can also play an important role in protecting the environment. The EnviroPig™ is a transgenic pig with an additional gene coding for phytase. This enzyme helps decomposing phosphorus in saliva and leads to reduced fecal phosphorus output (up to 75%) and an increase in absorption rate of nutrients (GOLOVAN et al. 2001). A transgenic pig with expression of a fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach had a significantly altered ratio of fatty acid in body fat. This meat could be an alternative source of essential fatty acids with more polyunsaturated fatty acids, which can help to prevent lifestyle-related diseases, such as coronary heart disease and thrombotic diseases (SAEKI et al. 2004).
Disease resistance
In most cases, susceptibility to pathogens is polygenic in nature. Only very few loci are currently known to be responsible for a specific disease in farm animals. The production of transgenic cattle with bovine spongiform encephalopathy (BSE) resistance was achieved by knocking out the responsible prion protein (PrP) gene on both alleles (RICHT et al. 2007).
Cattle with an age of 20 months were clinically, physiologically, histopathologically, immunologically and reproductively normal. Analysis of brain tissue showed no propagation of misfolded variant of PrP, which would lead to BSE in cattle and Creutzfeldt-Jakob disease (CJD) in humans.
The membrane cofactor protein (MCP or CD46) is known to be a receptor for several viruses and bacteria, including the bovine viral diarrhea virus (BVDV). The pathogens recognize the different structures of the CD46 ectodomain, resulting in cell infection and diseases (CATTANEO 2004; MAURER et al. 2004). The classical swine fever virus (CSFV) belongs to the same genus (pestvirus) as the BVDV. Swine fever has a damaging impact on global pig production. During an outbreak of classical swine fever (CSF) in Europe 1997, a
total of 876.000 pigs were killed because they belonged to infected or contact herds (EDWARDS et al. 2000). A biallelic gene knockout (KO) of the CD46 in pig could lead to swine fever resistant pigs. However, mutations in the human CD46 gene can be a predisposition factor for the hemolytic uremic syndrome (HUS), but without developing the end-stage renal failure typical for HUS (FREMEAUX-BACCHI et al. 2006). If CD46-KO pigs would show predisposition for HUS needs to be analyzed.
1.2. Xenotransplantation
Transgenic pigs are considered to be a perfect organ donor for humans to reduce or even eliminate the growing shortage of suitable human organs (COOPER and AYARES 2011). Due to the lack of suitable organs for allotransplantation many people die while waiting for a human donor organ (an average of three per day in Germany). In Germany about 8,000 dialyze-patients are waiting for a kidney. In 2011, 2,850 patients were transplanted, while the number of required organs was three times higher, resulting in a waiting time for a kidney of 5-6 years (Figure 1) (DSO 2012).
Figure 1: Waiting list and number of kidney transplantations in Germany from 2002 to 2011 (DSO 2012)
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Quantity
Year
active waiting list
kidney
transplantation
living-donation
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The use of concordant xenografts (between closely related species) like primate-to- human transplantation would lead to an immunological response similar to allotransplantation, which can be controlled by an efficient immunosuppressive treatment.
High costs, the difficulty to breed and keep the animals under strict hygienic conditions, the risk of zoonosis and ethical concerns have prevented primates to be used as organ donors for human patients. Additionally, most of the potential primate species are already on the list of endangered species. After discordant xenotransplantation (between distantly related species) a more severe immune response appears which can not be prevented by immunosuppressive agents. The domestic pig is considered as perfect organ donor for humans, due to its physiological similarity, size-matching organs, low production costs, high reproductive capacity and the knowledge about maintaining pigs under hygienic specific- pathogen-free conditions (PETERSEN et al. 2009a).
Transplantation of discordant xenografts leads to hyperacute rejection (HAR) within minutes after transplantation and finally destroys the transplanted organ. The responsible major antigens are carbohydrate structures called Gal-epitopes on the surface of porcine cells and tissues (Figure 2). The α1,3-galactosyltransferase encoded by the GGTA1 gene synthesizes these epitopes on porcine cells. Gal-epitopes are attacked by the human immune system immediately after transplantations. About 1% of all circulating antibodies in the human blood are directed against Gal-epitopes. Binding of pre-formed antibodies activates the complement cascade, that ultimately leads to the formation of a membrane attack complex (MAC) which in turn causes cell lysis and xenograft rejection (PARKER et al.
1996).
The presence of Gal-epitopes in placental and marsupial mammals, but not in non- mammalian vertebrates indicates the evolutionary appearance of the GGTA1 gene about 80 million years ago (GALILI 1995). Human and old world monkeys lost the function of this gene about 28 million years ago. It might have been an evolutionary advantage to express anti-Gal antibodies for ancestral old world higher primates, which were exposed to endemic pathogens (viruses, bacteria and protozoa possess Gal-epitopes). One requirement for the production of anti-Gal antibodies was the suppression of Gal-epitopes by a naturally
occurring gene knockout in order to prevent autoimmune response (GALILI 1995; GALILI and SWANSON 1991).
GGTA1-KO pigs were produced by using conventional genetic modification tools (DAI et al. 2002; PHELPS et al. 2003). Transplantation of kidneys and hearts from pigs with a homozygous GGTA1-KO to baboons with support of immunosuppressive agents resulted in significantly improved organ survival up to 179 days (KUWAKI et al. 2005; TSENG et al. 2005;
YAMADA et al. 2005). The production of transgenic pigs is essential for studies in the field of xenotransplantation. Table 3 summarizes published achievements in pig-to-baboon Figure 2: Hyperacute rejection (HAR) after anti-Gal antibodies bind to Gal-epitopes. (A) Binding of anti-Gal antibodies to Gal-epitopes induces complement cascade activation, resulting in a membrane attack complex (MAC), which is responsible for movement of ions and water across the membrane and ultimately for cell lysis; Decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and membrane inhibitor of reactive lysis (MIRL, CD59) can prevent the formation of MAC; C3- C9 are transition proteins responsible for the formations of MACs (modified from TURNBERG and BOTTO 2003). (B) Humoral graft rejection; Binding of preformed anti-Gal antibodies to endothelial cells of a xenograft leads to complement activation and formation of MACs and finally to massive haemorrhage and thrombosis (modified from YANG and SYKES 2007).
(TURNBERG and BOTTO 2003) (YANG and SYKES 2007)
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xenotransplantation. To further prolong survival of xenografts, multi-transgenic pigs are required to overcome the hurdle of HAR.
Various attempts have been made to prevent rejection of porcine xenografts.
Complement regulatory proteins such as human decay accelerating factor (hDAF, or hCD55) work in a species specific manner. Organs from transgenic animals expressing hCD55 were compatible with reduced complement-mediated injury (Figure 2). Transgenic pig hearts expressing hCD55 were transplanted into baboons, and led to a survival for up to 99 days (BHATTI et al. 1999). The combination of Gal-KO and hCD55 transgenic pig in xenotransplantation did not improve graft survival, but hCD55 expression seemed to restrict local complement activation (McGREGOR et al. 2012). Expression of hCD59 (membrane inhibitor of reactive lysis, MIRL) on islet cells led to reduced complement-mediated lysis (SCHMIDT et al. 2003). HCD46 transgenic pigs were generated and increased number of MCP proteins lead to enhanced protection of host cells from damage by complement activation (McGREGOR et al. 2005). A combination of Gal-KO, hCD46-KI and B-cell depletion showed a maximum xenograft survival of 236 days in regard of entire organs (MOHIUDDIN et al. 2012).
(MCGREGOR et al. 2012)
Table 3: Summary of pig-to-baboon transplantation regarding genetically knockin (KI) or knockout (KO), kind of organ and maximum survival (days)
Type of genetic engineering
Organ Max. survival
(days)
Authors
CD59-KI and hCD55-KI Heart 30h McCURRY et al. 1995
Gal-KO Kidney 16 CHEN et al. 2005
Gal-KO and hCD55-KI Heart, heterotopic 28* McGREGOR et al. 2012
hCD55-KI Heart, heterotopic 39 VIAL et al. 2000
hCD55-KI Kidney 75 CHEN et al. 2006
Gal-KO Heart, heterotopic 78* KUWAKI et al. 2005
Gal-KO Kidney 83 YAMADA et al. 2005
hCD55-KI Heart, heterotopic 99 BHATTI et al. 1999
hCD46-KI Heart, heterotopic 137 McGREGOR et al. 2005
Gal-KO Heart, heterotopic 179 TSENG et al. 2005
Gal-KO and hCD46-KI Heart, heterotopic 236 MOHIUDDIN et al. 2012
* median (MCCURRY et al. 1995) (CHEN et al. 2005) (MCGREGOR et al. 2012) (VIAL et al. 2000) (CHEN et al. 2006) (KUWAKI et al. 2005) (YAMADA et al. 2005) (BHATTI et al. 1999) (MCGREGOR et al. 2005) (TSENG et al. 2005) (MOHIUDDIN et al. 2012)
The first porcine xenografts potentially to be used for clinical applications in human are pancreatic islet cells. Porcine insulin has been successfully used for years in human patients to treat diabetes. Transplantation of porcine islets cells in chemically induced diabetic monkeys made injection of insulin dispensable. Survival of functional hCD46 porcine islet cells in monkeys lasted over 1 year (VAN DER WINDT et al. 2009). Gal-expression on porcine islet cells is age dependent; while fetal porcine islet cells express high levels of GGTA1, Gal-epitopes on adult islet cells are only occasionally detectable (BENNET et al.
2000; RAYAT et al. 2003). Using Gal-KO neonatal porcine islet (NPI) cells for xenotransplantation resulted in lower rates of primary graft dysfunction compared to wild- type (WT) NPI cells (THOMPSON et al. 2011).
After overcoming HAR by knocking out the GGTA1 gene, the major hurdle for long- term xenograft survival remains the acute vascular rejection (AVR). It was shown, that thrombotic microangiopathy is the predominant histopathological feature of rejected porcine Gal-KO cardiac xenografts (TSENG et al. 2005). This coagulation might be due to the inability of porcine tissue factor pathway inhibitor (TFPI) to adequately neutralize human factor Xa which leads to activation of endothelial cells and thrombosis (PETERSEN et al.
2009b). Transgenic pigs expressing the human TFPI are considered to effectively suppress organ rejection by its anticoagulating effect (LEE et al. 2011). The endothelial cell activation during AVR up-regulates adhesion molecules (e.g. E-selectin, VCAM-I and ICAM-I) which lead to endothelial swelling, focal ischemia and diffuse microvascular thrombosis (LEVENTHAL et al. 1993; OSBORN 1990). The human A20 (encoded by the tumor necrosis factor α-induced protein 3 gene (TNFAIP3)) expressed in transgenic pigs provides protection against tumor necrosis factor α-mediated apoptosis, and partial protection against CD95(Fas)L- mediated cell death, revealing hA20 as a promising molecule for controlling AVR in xenotransplantation studies (OROPEZA et al. 2009).
The third hurdle is the delayed xenograft rejection (DXR). This rejection is characterized by endothelial cell activation and infiltration of the graft by host monocytes and natural killer cells leading to intra-graft inflammation and thrombosis (BACH et al. 1996).
This reaction is similar to the rejection observed after allotransplantation and might be controlled by appropriate immunosuppressive agents.
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When these hurdles are overcome, there still is the risk of potential cross-species infection with the porcine endogenous retrovirus (PERV). PERVs are encoded by the porcine genome and were demonstrated to infect human cells in vitro (PATIENCE et al. 2001; SPECKE et al. 2001). However, in 160 patients, that underwent different forms of exposure to porcine tissue, no PERV infection was observed ten years after exposure (EKSER et al. 2009).
Besides, significant reduction of PERV expression was achieved by short hairpin (sh) RNA (DIECKHOFF et al. 2008; SEMAAN et al. 2012). More than 50 copies of PERV have been identified in the porcine genome and it is conceivable that the efficient targeting by zinc- finger nucleases (ZFN) could lead to a systemic PERV knockout.
1.3. Somatic cell nuclear transfer (SCNT)
The first cloned mammal was reported by WILMUT et al. in 1997. Dolly the sheep was the first live mammalian offspring generated by somatic cell nuclear transfer (SCNT) using differentiated adult somatic cells. Due to the reproductive uniqueness of the pig, that four embryos need to implant into the uterus to maintain a pregnancy, it took three years until the first cloned pigs were reported by three different groups (BETTHAUSER et al. 2000;
ONISHI et al. 2000; POLEJAEVA et al. 2000). (WILMUT et al. 1997)
Using SCNT for the production of transgenic livestock is superior to microinjection of foreign DNA into the pronucleus of zygotes. Transgene integration efficiency is low and random integration of the foreign DNA is associated with variation in transgene expression (WALL 1996). In mammals, transgene integration into somatic cells (e.g. fibroblasts) by transfection is achieved via electroporation or nucleofection. Enrichment for targeting events can be accomplished prior the use of donor cells for SCNT. In case of microinjection of foreign DNA into 1-cell embryos, late targeting can lead to mosaicism, which is characterized by appearance of modification in only a subset of the cells (CARBERY et al.
2010; MEYER et al. 2010).
For SCNT, in vitro matured oocytes isolated from abattoir ovaries are enucleated by removal of the first polar body and methaphase plate. Donor cells (arrested at G0/G1 phase by serum starvation and/or contact inhibition) are placed in the perivitelline space in close
contact to the oocyte membrane. After fusion (electrically) and activation (electrically/chemically) reconstructed embryos are incubated for 3 h until they are transferred to synchronized recipients (PETERSEN et al. 2008). Alternatively, the reconstructed embryos can be cultured to the morulae/blastocyst stage prior to transfer (GALLI et al. 2008).
About 1-5% of transferred SCNT-derived porcine embryos survive to term (HARRISON et al. 2004; LAGUTINA et al. 2006; ROGERS et al. 2008a). Several factors influence cloning efficiency, e.g. reduced oocyte quality, embryo culture, inadequate reprogramming of the transplanted nucleus and treatment of donor cells prior to SCNT (BEAUJEAN et al. 2004;
HIIRAGI and SOLTER 2005). To enhance cloning efficiency, the use of good quality biological material is a necessary prerequisite. Usually about 80-100 reconstructed embryos are surgically transferred into a synchronized recipient sow. SCNT is successfully applied and essential for the production of transgenic pigs (PARK et al. 2001). The first gene knockout pigs were produced via SCNT and carried a heterozygous knockout of the GGTA1 gene achieved by classical homologous recombination (HR) approaches (LAI et al. 2002).
Unfortunately, it was shown that porcine SCNT derived pregnancies resulted in smaller litters (in average 6.2 piglets) compared to pregnancies induced by artificial insemination (AI) (in average 11.5 piglets) (ESTRADA et al. 2007). The average birth weight was significantly smaller in SCNT derived piglets and postnatal mortality and number of stillbirths were higher compared to the AI group, leading to the assumption that incomplete or false reprogramming occurs during SCNT.
1.4. Transgenic technologies
In nature, harmful DNA double-strand breaks (DSBs; induced by radiation or mutagenic chemicals) are repaired using the sister chromosome for gene correction or non homologous end joining (NHEJ) DNA repair. Conventional gene targeting for the production of genetically modified organisms uses HR to copy foreign DNA into the host genome (CAPECCHI 1989). In general, DNA coding for the exogenous gene is placed behind a target specific promoter (ubiquitous or tissue-specific) and is flanked by regions homologous to the targeting site.
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Usually, a selection cassette is incorporated carrying antibiotic resistance to enrich for targeting events. Unfortunately, targeting efficiency is extremely low (only 1 of 106-109 cells) because DSBs rarely appear at the specific site of the genome (DENNING et al. 2001;
DOETSCHMAN et al. 1988; JIN et al. 2003). In rare cases the targeted gene is broken, donor DNA is used as template for DNA repair and the transgene is integrated into the genome.
Another negative side effect of HR is the potential risk of random integration of foreign DNA at other genomic sites, which can lead to disruptions of genes.
Lentiviral transfection
Transgenic farm animals, such as cattle and pig, have successfully been produced by injection of lentiviruses into zygotes (HOFMANN et al. 2003; HOFMANN et al. 2004).
Lentiviral vectors containing the green fluorescent protein (GFP) reporter transgene were injected into porcine embryos. After SCNT, 70% of the piglets showed transgene integration (1 to 20 copies) in organs from the three germ layers and 90% of these animals expressed GFP (HOFMANN et al. 2003). Lentiviral transfection shows high integration efficiency in dividing and non-dividing cells and is compatible with long-term stable expression of transgenes. Additionally, it does not activate protooncogenes in host organisms or leads to gene silencing as other viral-mediated gene transfer methods (CHAN et al. 1998). Due to the viral background of this transfection method, transgenic animals would not be useful for xenotransplantation.
Sperm-mediated gene targeting
Sperm-mediated gene transfer (SMGT) uses the intrinsic ability of sperm to bind and internalize foreign DNA and to transfer it into the oocyte at fertilization (BRACKETT et al.
1971). This method does not require expensive and time consuming experimental protocols, such as SCNT (LAVITRANO et al. 2006). The production of multi-transgenic pigs using SMGT was reported (enhanced fluorescent proteins (eFP): eBFP (blue), eGFP (green) and DsRed2 (red) as donor DNA). All 18 born piglets carried at least one transgene (39% triple transgenic,
39% double transgenic and 22% single transgenic), and fluorescent protein expression was found in all analyzed tissues (WEBSTER et al. 2005). The same research group reported the generation of porcine blastocysts via SMGT carrying transgenes relevant for xenotransplantation leading to 21% blastocysts carrying all three transgenes (human hemoxygenase-I (hHOI), human CD39 and human CD73) (VARGIOLU et al. 2010).
Unfortunately, targeted genetic modification is not possible, offspring have to be analyzed for transgene integration and several studies reported a lack of reproducibility (BRINSTER et al. 1989; GARCIA-VAZQUEZ et al. 2011). Another negative side effect of SMGT is a 2-fold decrease in motility of sperm treated with transgenetic DNA and an increase of sperms with highly damaged DNA. This is associated with the removal of seminal plasma during the washing step (KANG et al. 2008).
Transposon technology
Transposable elements are mobile DNA sequences that can change their genomic position.
There are two types of transposons, retrotransposons transpose through an RNA intermediate and reverse transcription into DNA leading to a copy, which inserts at another place into the genome (copy-and-paste). DNA transposons move directly as DNA element (cut-and-paste) and are transposed to another genomic position (IZSVAK and IVICS 2004).
Only DNA transposons have been developed as tool for insertional mutagenesis in invertebrate model organisms (COOLEY et al. 1988). Sleeping Beauty (SB) transposons allow efficient transgene integrations in mammals avoiding drawbacks accompanied by viral targeting methods (GARRELS et al. 2011; IVICS et al. 2007; JAKOBSEN et al. 2011). The gene of interest is cloned between the inverted repeats (IRs) of SB and the transposase (encoded on a second plasmid) binds to the IRs and catalyzes transposition. Targeted integration at a specific site of the genome was successful in >10% of transfected cells, using an approach based on interactions between the transposase and a targeting protein (IVICS et al. 2007).
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1.5. GGTA1 gene knockout
The catalytic domain of the α1,3-galactosyltransferase gene, which is responsible for the Gal-epitopes on porcine cell surfaces, is located in exon 9 of the GGTA1 gene (KATAYAMA et al. 1998). Genetic mutations in exon 9 have shown to result in a functional knockout of the GGTA1 gene (PETERSEN 2004; PHELPS et al. 2003). Heterozygous and homozygous GGTA1- KO pigs have been produced by conventional gene targeting such as HR (DAI et al. 2002;
FUJIMURA et al. 2008; LAI et al. 2002; PHELPS et al. 2003). Toxin A (toxin from Clostridium difficile) can be used to select for Gal-negative cells. The toxin binds to Gal-epitopes and inserts a cytotoxic effect on Gal-positive cells (CLARK et al. 1987). This selection method was used to enrich for HR events on the second GGTA1 allele of primal GGTA1+/- cells. However, sequencing revealed that a spontaneous mutation in exon 9 on the second GGTA1 allele occurred (loss of heterozygosity, LOH). Only one base pair was substituted (from T to G) which led to one amino acid change and a functional gene knockout (PHELPS et al. 2003).
The use of kidneys and hearts from GGTA1-KO pigs in a pig-to-baboon xenotransplantation model led to significantly improved organ survival (KUWAKI et al. 2005;
TSENG et al. 2005; YAMADA et al. 2005). Current view is that multi-transgenic pigs are required to improve organ survival after transfer into immune-suppressed monkeys.
Unfortunately, pigs carrying a GGTA1-KO suffer from low birth weights and health problems, and the cells have low cloning efficiency (DAI et al. 2002). Homozygous knockout of the GGTA1 gene in mice led to a complete elimination of Gal-epitopes and did not affect development of the organism (THALL et al. 1995). However, expression of Gal-epitopes in porcine organs is up to 500-fold (kidney) higher than in mouse organs, which may suggest that the Gal-epitopes play an essential role in porcine development (TANEMURA et al. 2000).
An alternative approach to abolish the effect of Gal-epitopes is the expression of H- transferase (α1,2-fucosyltransgferase gene, FUT8), which uses the same acceptor (N-acetyl lactosamine) as GGTA1 and competes very effectively with GGTA1 for the same substrate (PARKER et al. 1996). Transgenic FUT8 mice had decreased number of Gal-epitopes on cell surfaces (COHNEY et al. 1997). Unfortunately, down-regulation of porcine GGTA1 is not as effective as in mice, due to elevated expression of GGTA1 in porcine tissue (COWAN et al.
2000; MILLAND et al. 2006; TANEMURA and GALILI 2000; TANEMURA et al. 2000). Even when the GGTA1 gene is knocked out on both alleles (GGTA1−/−), not all cells are completely Gal-negative. About 1 to 2% of the cells still exhibit Gal-epitopes (SHARMA et al. 2003). In the laboratory rat, an additional gene is responsible for the formation of Gal-epitopes, the isoglobotriaosylceramide synthase (iGb3S) (KEUSCH et al. 2000; TAYLOR et al. 2003). This gene is also present in pigs, but it is not clear if this gene is responsible for the remaining Gal-epitopes in GGTA1-KO pigs (KEUSCH et al. 2000; MILLAND et al. 2006; SHARMA et al.
2003; TAYLOR et al. 2003). The production of multi-transgenic pigs with a biallelic GGTA1-KO is the best solution for preventing the HAR after xenotransplantation.
Breeding of heterozygous GGTA1-KO pigs produced normal sized litters. From 25 live born piglets (4 litters) 12 (44%) pigs were Gal-negative (NOTTLE et al. 2007). For an effective production of biallelic GGTA1-KO animals, breeding of two GGTA1+/- animals is preferred over cloning. Breeding of GGTA1−/− pigs might result in small litters with low birth weights but pure Gal-negative litter. Own observations in Mariensee lately showed a negative influence of the biallelic GGTA1 gene knockout on piglet development, which suggests that Gal-epitopes may play a role in the reproduction of pigs.
1.6. Zinc-finger nuclease (ZFN)-mediated gene targeting
ZFNs are genetic scissors that mediate highly efficient and permanent genetic mutations by inducing a DSB at a specific genomic site which is repaired by error prone NHEJ. ZFNs can be designed to bind at almost any target site with high specificity (Figure 3A). The applicability covers a broad range of species, such as fruit flies, mouse, human and pig. ZFN-mediated targeting could not only serve as tool for the production of transgenic animals, also transgenic cell lines can be produced for possible treatment of diseases (LI et al. 2011a).
For the production of transgenic animals, SCNT has emerged as the method of choice. Because cloning efficiency is quite low, good quality transgenic donor cells used for SCNT could improve results. ZFN-mediated targeting has several advantages. The targeting efficiency of ZFNs is much higher than that of conventional targeting methods. Putative negative side effects of ZFN-mediated targeting, such as cleavage at off-target sites, can be
16
verified by analyzing sequences that are highly similar to the specific binding site of the ZFN pair. As basis for breeding experiments or the production of multi-transgenic pigs, knockout animals lacking random integrated transgenes and antibiotic resistance cassettes would be beneficial.
The review paper in chapter 2 (Publication 1) provides an overview about ZFN- mediated gene targeting. It describes the invention and mode of function of ZFNs, aspects that have to be considered for the design of ZFNs and the state of the art in various biological systems.
Figure 3: Comparison between DNA binding of ZFNs and TALENs. (A) A pair of zinc-finger nucleases (ZFNs) bound to its target DNA; ZFNs contain three zinc-fingers (ZFs) each binding to one DNA triplet; The FokI endonuclease is ready to cleave the DNA in the spacer region between two ZFN binding sites on opposing strands. (B) A pair of transcription activator like effector nucleases (TALENs) bound to its target, ready to cleave the DNA; Each base pair is bound by one TALE with different variable diresidues (RVDs): A – NI, G – NN, C – HD, T – NG.
1.7. Transcription activator like effector nucleases (TALENs)
Recently, a new type of engineered nuclease called transcription activator like effector (TALEs) nucleases was described. TALEs are produced by plant pathogens in the genus Xanthomonas and bind to their host DNA to act as transcription factors. The TALEs consist of
repeats, each of about 33-35 amino acids long, with two polymorphic positions in the middle called the repeat variable diresidue (RVD). One RVD binds to one nucleotide of genomic DNA (Figure 3B) (BOCH et al. 2009; MOSCOU and BOGDANOVE 2009).
It is possible to join individual TALEs to engineer DNA binding domains capable of recognizing endogenous sequences in mammalian cells. By linking the binding domain to the non-specific cleavage domain from the type IIs restriction endonuclease FokI, TALENs can be used as tool for stimulating NHEJ and HDR (CERMAK et al. 2011; CHRISTIAN et al. 2010;
HOCKEMEYER et al. 2011; LI et al. 2011b; LI et al. 2011c; MAHFOUZ et al. 2011; MILLER et al.
2011). A comparative study in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and three different target genes AAVS1 (save harbor site), OCT4 (octamer- binding transcription factor 4) and PITX3 (pituitary homeobox 3 gene) revealed that TALENs and ZFNs had a similar targeting efficiency (HOCKEMEYER et al. 2011). Similar to ZFNs, TALENs have been used to subsequently knockout genes in rats and zebrafish (HUANG et al.
2011; SANDER et al. 2011; TESSON et al. 2011).
ZFNs and TALENs differ in some main aspects: (i) TALE repeats are 3 to 4 times longer than ZFNs, when recognized per base pair of the targeted DNA; (ii) the spacer length (the gap between two binding sites) is variable in TALEN and not restricted to a specific length, which complicates TALEN design and will lead to greater off-target activity relative to an identical nuclease with a fixed spacer length; (iii) ZFNs assembly requires an archive of high- quality, well characterized modules to achieve specific gene targeting, because cross-talk between the individual fingers can lead to imperfect DNA recognition (DEFRANCESCO 2011).
Due to lower production costs, TALENs could be a good alternative to ZFNs.
1.8. Motivation and goal of this study
At the starting point of this project in 2009, ZFN-mediated gene targeting in pig had not been reported. To evaluate if ZFN-mediated targeting is feasible in the domestic pig, the endogenous α1,3-galactosyltransferase (GGTA1, Gal) gene was selected as target. The host laboratory already had great experience with the genetic modification of the GGTA1 locus by conventional gene targeting (HR) in combination with SCNT (PETERSEN 2004). There was a
18
tremendous interest in Gal-negative pigs for use in xenotransplantation studies (pig-to- baboon transplantation). Though Gal-negative pigs existed at that time, improvement of the technology was urgently needed for more routine use of viable GGTA1-KO pigs in xenotransplantation. The goal of the study was to establish a protocol for the use of ZFNs to knockout a genetic locus in the porcine system, preferably on both alleles. This protocol could also be used to knockout other genetic loci or to introduce transgenes or specific point mutations at a particular site in the porcine genome.
For this purpose, the host laboratory at the Institute of Farm Animal Genetics established a cooperation with Sangamo BioSciences (USA), who produced specific ZFNs targeting the catalytic domain of the α1,3-galactosyltransferase located in exon 9 of the GGTA1 gene. The production of ZFN-mediated biallelic knockout of the GGTA1 was to be achieved in cells and live knockout animals were to be generated via SCNT. Detailed analysis was meant to show that ZFN treatment was not accompanied with negative side effects (e.g.
off-target mutations or plasmid integration) and that the ZFN-mediated gene knockout was functionally similar to conventionally produced gene knockout. The production of male and female ZFN-mediated biallelic GGTA1-KO cells and pigs would provide evidence for potential gender specific differences in targeting frequency or other negative side effects. Moreover, breeding of two ZFN-mediated biallelic GGTA1-KO animals would be timesaving and cost effective for production of multiple biallelic GGTA1-KO pigs.
2. Publication 1
Gene knockout and knockin by zinc-finger nucleases:
Current status and perspectives
J. Hauschild-Quintern1, B. Petersen1, G. J. Cost2, and H. Niemann1,3 #
1Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, Neustadt a. Rbge., Germany
2Sangamo BioSciences, 501 Canal Blvd., Richmond, CA 94804, USA
3Rebirth, Cluster of Excellence, Hannover Medical School
#Corresponding author
Accepted by Cellular and Molecular Life Sciences (22.10.2012) DOI: 10.1007/s00018-012-1204-1
The extent of contribution from Janet Hauschild-Quintern to this article: ~70%.
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Abstract
Zinc-finger nucleases (ZFNs) are engineered site-specific DNA cleavage enzymes that may be designed to recognize long target sites and thus cut DNA with high specificity. ZFNs mediate permanent and targeted genetic alteration via induction of a double-strand break at a specific genomic site. Compared to conventional homology-based gene targeting, ZFNs can increase the targeting rate by up to 100,000-fold; gene disruption via mutagenic DNA repair is similarly efficient. The utility of ZFNs has been shown in many organisms, including insects, amphibians, plants, nematodes, and several mammals, including humans. This broad range of tractable species renders ZFNs a useful tool for improving the understanding of complex physiological systems, to produce transgenic animals, cell lines, and plants, and to treat human disease.
3. Publication 2
Efficient generation of a biallelic knockout in pigs using zinc- finger nucleases
Janet Hauschilda, Bjoern Petersena, Yolanda Santiagob, Anna-Lisa Queissera, Joseph W.
Carnwatha,c, Andrea Lucas-Hahna, Lei Zhangb, Xiangdong Mengb, Philip D. Gregoryb, Reinhard Schwinzerd, Gregory J. Costb, and Heiner Niemanna,c
aInstitute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt a.
Rbge., Germany
bSangamo BioSciences, Richmond, CA 94804, USA
cRebirth, Cluster of Excellence, and dTransplantation Laboratory Hannover Medical School, 30625 Hannover, Germany
Proceedings of the National Academy of Sciences U S A (PNAS) 108(29):12013-7 (July 2011)
Author contributions: J.W.C., P.D.G., and H.N. designed research; J.H., B.P., A.-L.Q., A.L.-H., X.M., R.S., and H.N. performed research; Y.S., L.Z., X.M., and G.J.C. analyzed data; and J.H., B.P., P.D.G., G.J.C., and H.N. wrote the paper.
The extent of contribution from Janet Hauschild-Quintern to this article:
A) Design of the project including the design and production of ZFNs: 0%
B) Performance of experiments (except SCNT): 90%
C) Analysis of experiments: 80%
D) Writing of the paper: 80%
22
Abstract
Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ~1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.
4. Publication 3
Gender non-specific efficacy of ZFN mediated gene targeting in pigs
Janet Hauschild-Quinterna, Bjoern Petersena, Anna-Lisa Queissera, Andrea Lucas-Hahna, Reinhard Schwinzerb, and Heiner Niemanna
aInstitute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt a.
Rbge., Germany
bTransplantation Laboratory Hannover Medical School, 30625 Hannover, Germany
Transgenic Research (in press) DOI: 10.1007/s11248-012-9647-6
The extent of contribution from Janet Hauschild-Quintern to this article:
A) Performance of experiments (except SCNT): 90%
B) Analysis of experiments: 80%
C) Writing of the paper: 80%
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5. General Discussion
5.1. Production and enrichment of GGTA1 negative cells via ZFN-mediated gene targeting
The ZFN pair targeting exon 9 of the GGTA1 gene was readily designed, produced and tested prior to delivery by Sangamo BioSciences. Transfection of porcine fetal fibroblasts with both ZFN plasmids led to a maximum of 1% biallelic (1% female and 0.8% male) targeting frequency as revealed by fluorescence-activated cell sorting (FACS) measurement (HAUSCHILD-QUINTERN et al. 2012; HAUSCHILD et al. 2011). This is in the range of biallelic targeting efficiencies previously achieved with ZFNs (LIU et al. 2010; SANTIAGO et al. 2008;
YU et al. 2011) and about 10,000-fold higher than conventional (HR) targeting efficiency of the GGTA1 gene (DENNING et al. 2001). The proportion of cells with a biallelic knockout mediated via ZFNs can be up to 1/3 of mutated cells (PEREZ et al. 2008). The NHEJ frequencies of ZFN-treated cell cultures were about 3% and 6% for female and male porcine fibroblasts, respectively. In case of targeting female cells, this correlates to the ratio of
“biallelic knockout rate/mutated alleles in cell population = 1/3”. However, not every mutation in the GGTA1 exon 9 might lead to a functional gene knockout.
The transfected cell population containing ~1% Gal-negative cells was selected with the aid of magnetic beads. A purity >95% was achieved and Gal-negative cells could easily be expanded. Using FACS for selection of Gal-negative cells was less successful. From the same amount of cells used for magnetic bead selection, fewer Gal-negative cells were enriched by FACS. In subsequent culture these cells only proliferated slowly and could not be used for further experiments, such as FACS measurement or SCNT. FACS selection was not further used in this study, because increasing shear stress reduced proliferation rate (PAPADAKI et al. 1996).
Only one experiment was necessary to obtain cells with a biallelic GGTA1 gene knockout for each gender. The advantage of this high targeting efficiency is that the cells do not have to be expanded to obtain sufficient amounts of donor cells for SCNT. In cell culture,
