BACKGROUND: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. METHODS: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

CHDI Foundation Princeton NY USA

Department of Biological Chemistry University of California Irvine CA USA Department of Psychiatry and Human Behavior University of California Irvine CA USA

Department of Biological Chemistry University of California Irvine CA USA Department of Psychiatry and Human Behavior University of California Irvine CA USA Department of Neurobiology and Behavior University of California Irvine CA USA

Department of Clinical Genetics and Fetal Medicine Palacky University University Hospital Olomouc Olomouc Czech Republic

Department of Genetics and Reproduction Veterinary Research Institute Brno Czech Republic

Department of Neurology Massachusetts General Hospital Boston MA USA

Department of Pharmacological Sciences and Centre for Stem Cell Research Università degli Studi di Milano Milan Italy

Institute of Information Theory and Automation v v i AS CR Prague Czech Republic

Laboratory for Study of Mitochondrial Disorders 1st Faculty of Medicine Department of Pediatrics and Adolescent Medicine Charles University and General University Hospital Prague Prague Czech Republic

Laboratory of Cell Regeneration and Plasticity Institute of Animal Physiology and Genetics v v i AS CR Libechov Czech Republic

Laboratory of Cell Regeneration and Plasticity Institute of Animal Physiology and Genetics v v i AS CR Libechov Czech Republic Department of Neurology Massachusetts General Hospital Boston MA USA

Laboratory of Cell Regeneration and Plasticity Institute of Animal Physiology and Genetics v v i AS CR Libechov Czech Republic Faculty of Science Department of Cell Biology Charles University Prague Prague Czech Republic

Laboratory of Cell Regeneration and Plasticity Institute of Animal Physiology and Genetics v v i AS CR Libechov Czech Republic Faculty of Science Department of Cell Biology Charles University Prague Prague Czech Republic Neurodegeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA USA Sanford Consortium for Regenerative Medicine San Diego La Jolla CA USA

Neurodegeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA USA

Neurodegeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA USA Sanford Consortium for Regenerative Medicine San Diego La Jolla CA USA

Neurodegeneration Laboratory Department of Anesthesiology University of California San Diego La Jolla CA USA Sanford Consortium for Regenerative Medicine San Diego La Jolla CA USA Institute of Neurobiology Slovak Academy of Sciences Kosice Slovak Republic

Novartis Institutes for Biomedical Research Neuroscience Discovery Basel Switzerland IRBM Promidis Pomezia Italy

Vector Development Laboratory Human Gene Therapy Program Department of Pediatrics University of California San Diego La Jolla CA USA

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