A Transgenic Minipig Model of Huntington\u27s Disease

Background: Some promising treatments for Huntington\u27s 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

The Generation of Transgenic Huntington's Disease Miniature Pig

Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt...

The Generation of Transgenic Huntington's Disease Miniature Pig

Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt...

The Generation of Transgenic Huntington's Disease Miniature Pig

Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt...

Tvorba transgenního miniaturního prasete s Huntingtonovou nemocí

Huntingtonova choroba (HCH) je devastující neurodegenerativní onemocnění, projevující se poruchami motorických i kognitivních funkcí, a také osobnostními změnami. Obrovské úsilí najít léčbu této nemoci přineslo na scénu několik slibných léčebných procedur. Každý z perspektivních terapeutických přístupů však musí být prozkoumán z hlediska bezpečnosti, snášenlivosti a účinnosti. Myší a potkaní modely byly velmi užitečné při zkoumání patologických mechanismů HCH, ale v předklinickém testování nejsou dostačující. Proto, aby se překlenula biologická vzdálenost mezi hlodavci a lidmi, zaměřili jsme se na vytvoření transgenního miniaturního prasete s HCH. Transgenní miniaturní prase kódující prvních 548 aminokyselin lidského mutovaného huntingtinu (TgHD) pod kontrolou lidského promotoru pro huntingtin vzniklo po lentivirové transdukci prasečích jednobuněčných embryí. V současné době je k dispozici šest generací miniprasat exprimujících jednu kopii N-terminální části lidského mutovaného proteinu huntingtin (mtHtt) s prodlouženou repeticí 124 glutaminů. Čím více model simuluje příznaky nemoci, tím lepší je pro translační výzkum, protože účinnost léčby lze vyhodnotit přesněji. Druhým cílem proto bylo demonstrovat v našem modelu fenotyp HCH. V důsledku exprese mtHtt byla pozorována testikulární degenerace,...Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

A transgenic minipig model of Huntington’s disease shows early signs of behavioral and molecular pathologies

Huntington's disease (HD) is a monogenic, progressive, neurodegenerative disorder with currently no available treatment. The Libechov transgenic minipig model for HD (TgHD) displays neuroanatomical similarities to humans and exhibits slow disease progression, and is therefore more powerful than available mouse models for the development of therapy. The phenotypic characterization of this model is still ongoing, and it is essential to validate biomarkers to monitor disease progression and intervention. In this study, the behavioral phenotype (cognitive, motor and behavior) of the TgHD model was assessed, along with biomarkers for mitochondrial capacity, oxidative stress, DNA integrity and DNA repair at different ages (24, 36 and 48 months), and compared with age-matched controls. The TgHD minipigs showed progressive accumulation of the mutant huntingtin (mHTT) fragment in brain tissue and exhibited locomotor functional decline at 48 months. Interestingly, this neuropathology progressed without any significant age-dependent changes in any of the other biomarkers assessed. Rather, we observed genotype-specific effects on mitochondrial DNA (mtDNA) damage, mtDNA copy number, 8-oxoguanine DNA glycosylase activity and global level of the epigenetic marker 5-methylcytosine that we believe is indicative of a metabolic alteration that manifests in progressive neuropathology. Peripheral blood mononuclear cells (PBMCs) were relatively spared in the TgHD minipig, probably due to the lack of detectable mHTT. Our data demonstrate that neuropathology in the TgHD model has an age of onset of 48 months, and that oxidative damage and electron transport chain impairment represent later states of the disease that are not optimal for assessing interventions