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.