Vocalisation as a Viable Assessment for Phenotyping Minipigs Transgenic for the Huntington Gene?

BACKGROUND: Large animal models, such as the transgenic (tg) Huntington disease (HD) minipig, have been proposed to improve translational reliability and assessment of safety, efficacy and tolerability in preclinical studies. Minipigs are characterised by high genetic homology and comparable brain structures to humans. In addition, behavioural assessments successfully applied in humans could be explored in minipigs to establish similar endpoints in preclinical and clinical studies. Recently, analysis of voice and speech production was established to characterise HD patients. OBJECTIVE: The aim of this study was to investigate whether vocalisation could also serve as a viable marker for phenotyping minipigs transgenic for Huntington's disease (tgHD) and whether tgHD minipigs reveal changes in this domain compared to wildtype (wt) minipigs. METHODS: While conducting behavioural testing, incidence of vocalisation was assessed for a cohort of 14 tgHD and 18 wt minipigs. Statistical analyses were performed using Fisher's Exact Test for group comparisons and McNemar's Test for intra-visit differences between tgHD and wt minipigs. RESULTS: Vocalisation can easily be documented during phenotyping assessments of minipigs. Differences in vocalisation incidences across behavioural conditions were detected between tgHD and wt minipigs. Influence of the genotype on vocalisation was detectable during a period of 1.5 years. CONCLUSION: Vocalisation may be a viable marker for phenotyping minipigs transgenic for the Huntington gene. Documentation of vocalisation provides a non-invasive opportunity to capture potential disease signs and explore phenotypic development including the age of disease manifestation.

Minipigs as a Large-Brained Animal Model for Huntington's Disease: From Behavior and Imaging to Gene Therapy.

Large animal models offer novel opportunities in exploring safety, biology, and efficacy of novel therapeutic approaches for Huntington's disease (HD). Challenges in the development of, for example, gene therapy, such as delivery, distribution, and persistence of virus vectors or oligo sense nucleotides, can be explored in large brains and organisms approaching human size. We here introduce the transgenic Libechov minipig as a large animal model of HD. Methods developed to assess motor, cognitive, and behavioral features expected to manifest in an HD model are described. We also outline established protocols for magnetic resonance imaging (MRI) including magnetic resonance spectroscopy (MRS) for minipigs. The successful conduct of long-term follow-up studies over several years with repeated behavioral testing and imaging is reported. We discuss the advantages and limitations of using this model with regard to translational reliability, homology to humans and with respect to feasibility, breeding, housing, handling, and finally ethical considerations. It is concluded that minipigs can fulfill an important role in preclinical development to bridge the gap between rodents and nonhuman primate research in the translation to humans.

Behavioral Assessment of Stress Compensation in Minipigs Transgenic for the Huntington Gene Using Cortisol Levels: A Proof-of-Concept Study.

BACKGROUND: Huntington's disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder with motor, cognitive, behavioral and metabolic symptoms. HD patients exhibit an altered response to stress which is reflected in changes of cortisol levels. Large animal models of HD such as the Libechov minipig are currently explored in preclinical studies to improve translational reliability and assessing behavior is of interest. OBJECTIVE: This study aimed to investigate whether cortisol metabolism and response to stress are changed in minipigs transgenic for the Huntington gene (tgHD) compared to wildtype (wt) animals suggesting that cortisol may be used as a marker for stress in minipigs. METHODS: Thirty-two Libechov minipigs (14 tgHD and 18 wt) were tested before, during and after a stressor, i.e., a hoof trimming procedure, was applied at baseline and after one year. A total of six saliva samples were collected at each assessment and cortisol was measured. In addition, body temperature and respiratory rate were assessed at three pre-determined points during each hoof trimming procedure. RESULTS: All minipigs showed a rise of cortisol in response to the hoof trimming stressor similarly to cortisol changes induced by stress observed in humans. No relevant differences were detected between tgHD and wt minipigs. CONCLUSION: Cortisol testing for the assessment of stress compensation, e.g., during hoof trimming, is feasible and well tolerated in wt and tgHD minipigs. To elucidate the time profile of cortisol responses to stressors further studies with assessments at multiple time points and exploration of the diurnal profiles of cortisol in minipigs are recommended.