Generierung ALV-J resistenter Hühner mittels Gen-Editierung

ALV-J ist ein onkogenes, immunsuppressives Retrovirus, welches in Hühnern die myeloische Leukose und andere Tumore, Wachstums- und Legedepression verursachen kann. Die Eindämmung des Virus ist aufgrund fehlender Präventionsmaßnahmen, vor allem adäquater Impfstoffe, stark erschwert. Daher verursacht ALV-J trotz globaler Eradikationsmaßnahmen weiterhin enorme wirtschaftliche Einbußen, die vor allem asiatische Länder betreffen. Konventionelle Zuchtverfahren im Hinblick auf Selektion resistenter Hühnerrassen stehen nicht als Kontrollstrategie zur Verfügung, da keine natürlichen Resistenz-vermittelnden Genvarianten in der Hühnerpopulation vorkommen. Gen-Editierung ermöglicht die Erzeugung einer solchen Genvariante und kann so zur Eindämmung von ALV-J genutzt werden. Gegenstand dieser Studie ist die Etablierung und anschließende Beurteilung einer gentechnisch erworbenen Resistenz gegenüber des aviären Leukosevirus J (ALV-J) in einer kommerziellen Hühnerlinie. Dazu wurde die Bindestelle des Wirtszellrezeptors chNHE1 entsprechend modifiziert, sodass die Rezeptorbindung des ALV-J an die Zielzelle und eine Infektion daher nicht stattfinden kann. Die Modifikation des chNHE1 beinhaltet die Deletion der Aminosäure W38, die sich als entscheidendes Schlüsselelement für den Viruseintritt herausgestellt hat. Die W38-kodierenden Sequenz ist unter Verwendung des CRISPR/Cas9-Systems in Kombination mit HDR deletiert worden. Anhand eines etablierten Testsystems konnte die vollständige ALV-J Resistenz in der erstellten Hühnerlinie bestätigt werden. Die erzeugte Genvariante hatte keine unerwünschten Effekte auf den Phänotyp der Tiere. In dieser Arbeit konnte am Beispiel des relevanten Geflügelpathogens ALV-J gezeigt werden, dass die Gen-Editierung effektiv zur Krankheitsprävention im Huhn eingesetzt werden kann.ALV-J is an oncogenic, immunosuppressive retrovirus which infects chickens. It causes myeloid leucosis and leads to reduced growth and laying performance. The virus is difficult to control due to a lack of adequate prevention measures, such as an effective vaccine. Despite global efforts eradicating ALV-J it still causes severe economic losses, especially in Asian countries. Conventional breeding methods are not suitable to generate disease resistant chicken lines as a naturally resistant gene variant does not exist within the chicken population. Gene editing can be used to generate a resistant gene variant and thus contributing to the control of ALV-J. The objective of this work is the establishment and subsequent characterisation of genetically introduced ALV-J resistance in a commercial chicken line. For this purpose the binding site of the host cell receptor chNHE1 was modified to prevent ALV-J infection. The modification of chNHE1 implies the deletion of amino acid W38, which has been proven to be a key element for virus binding. The W38 coding sequence was deleted using CRISPR/Cas9 technology in combination with HDR. Complete ALV-J resistance in the generated chicken line was confirmed using an established testing system. No adverse phenotypic effects were observed. In this work we show that gene editing is an effective alternative breeding method to generate disease resistance against viral pathogens such as ALV-J in a commercial chicken line

Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons

A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na+/H+ exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field

A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na+/H+ exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field

Genetic determinants of risk in pulmonary arterial hypertension: international genome-wide association studies and meta-analysis.

BACKGROUND: Rare genetic variants cause pulmonary arterial hypertension, but the contribution of common genetic variation to disease risk and natural history is poorly characterised. We tested for genome-wide association for pulmonary arterial hypertension in large international cohorts and assessed the contribution of associated regions to outcomes. METHODS: We did two separate genome-wide association studies (GWAS) and a meta-analysis of pulmonary arterial hypertension. These GWAS used data from four international case-control studies across 11 744 individuals with European ancestry (including 2085 patients). One GWAS used genotypes from 5895 whole-genome sequences and the other GWAS used genotyping array data from an additional 5849 individuals. Cross-validation of loci reaching genome-wide significance was sought by meta-analysis. Conditional analysis corrected for the most significant variants at each locus was used to resolve signals for multiple associations. We functionally annotated associated variants and tested associations with duration of survival. All-cause mortality was the primary endpoint in survival analyses. FINDINGS: A locus near SOX17 (rs10103692, odds ratio 1·80 [95% CI 1·55-2·08], p=5·13 × 10-15) and a second locus in HLA-DPA1 and HLA-DPB1 (collectively referred to as HLA-DPA1/DPB1 here; rs2856830, 1·56 [1·42-1·71], p=7·65 × 10-20) within the class II MHC region were associated with pulmonary arterial hypertension. The SOX17 locus had two independent signals associated with pulmonary arterial hypertension (rs13266183, 1·36 [1·25-1·48], p=1·69 × 10-12; and rs10103692). Functional and epigenomic data indicate that the risk variants near SOX17 alter gene regulation via an enhancer active in endothelial cells. Pulmonary arterial hypertension risk variants determined haplotype-specific enhancer activity, and CRISPR-mediated inhibition of the enhancer reduced SOX17 expression. The HLA-DPA1/DPB1 rs2856830 genotype was strongly associated with survival. Median survival from diagnosis in patients with pulmonary arterial hypertension with the C/C homozygous genotype was double (13·50 years [95% CI 12·07 to >13·50]) that of those with the T/T genotype (6·97 years [6·02-8·05]), despite similar baseline disease severity. INTERPRETATION: This is the first study to report that common genetic variation at loci in an enhancer near SOX17 and in HLA-DPA1/DPB1 is associated with pulmonary arterial hypertension. Impairment of SOX17 function might be more common in pulmonary arterial hypertension than suggested by rare mutations in SOX17. Further studies are needed to confirm the association between HLA typing or rs2856830 genotyping and survival, and to determine whether HLA typing or rs2856830 genotyping improves risk stratification in clinical practice or trials. FUNDING: UK NIHR, BHF, UK MRC, Dinosaur Trust, NIH/NHLBI, ERS, EMBO, Wellcome Trust, EU, AHA, ACClinPharm, Netherlands CVRI, Dutch Heart Foundation, Dutch Federation of UMC, Netherlands OHRD and RNAS, German DFG, German BMBF, APH Paris, INSERM, Université Paris-Sud, and French ANR