A Transgenic Minipig Model of Huntington's Disease

Article type: Research Article

Authors: Baxa, Monika^; | Hruska-Plochan, Marian^{; ; ;} | Juhas, Stefan | Vodicka, Petr^; | Pavlok, Antonin | Juhasova, Jana | Miyanohara, Atsushi | Nejime, Tetsuya | Klima, Jiri | Macakova, Monika^; | Marsala, Silvia^; | Weiss, Andreas^; | Kubickova, Svatava | Musilova, Petra | Vrtel, Radek | Sontag, Emily M.^; | Thompson, Leslie M.^{; ;} | Schier, Jan | Hansikova, Hana | Howland, David S. | Cattaneo, Elena | DiFiglia, Marian | Marsala, Martin^{; ;} | Motlik, Jan

Affiliations: 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 in 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 | Vector Development Laboratory, Human Gene Therapy Program, Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA | Novartis Institutes for Biomedical Research, Neuroscience Discovery, Basel, Switzerland | Department of Genetics and Reproduction, Veterinary Research Institute, Brno, Czech Republic | Department of Clinical Genetics and Fetal Medicine, Palacky University, University Hospital Olomouc, Olomouc, Czech Republic | 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 | Institute of Information Theory and Automation v.v.i., AS CR, Prague, Czech Republic | Laboratory for Study of Mitochondrial Disorders, First Faculty of Medicine, Department of Pediatrics and Adolescent Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic | CHDI Foundation, Princeton, NY, USA | Department of Pharmacological Sciences and Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy | Department of Neurology, Massachusetts General Hospital, Boston, MA, USA | Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic | IRBM Promidis, Pomezia, Italy

Note: [] The first two authors contributed equally to this work.

Note: [] The first two authors contributed equally to this work.

Note: [] Correspondence to: Jan Motlik, Institute of Animal Physiology and Genetics, v.v.i, Laboratory of Cell Regeneration and Plasticity, Rumburska 89, 27721 Libechov, Czech Republic. Tel: +420 315639560; Fax: +420 315639510; E-mail: [email protected]

Abstract: 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.

Keywords: Huntington's disease, mutant huntingtin, minipigs, large animal model, lentiviral transgenesis, FISH analysis, mRNA and protein expression, immunohistochemistry, DARPP32, AGERA assay, TR-FRET assay, spermatozoa

DOI: 10.3233/JHD-130001

Journal: Journal of Huntington's Disease, vol. 2, no. 1, pp. 47-68, 2013