AAVrh10-CAG-GFP

Spinocerebellar ataxia (SCA) is a devastating neurodegenerative disease for which there is currently no cure or preventive treatment. Dysregulated brain cholesterol metabolism and impaired brain cholesterol turnover are associated with a variety of neurodegenerative diseases. SCA3 or Machado-Joseph disease (MJD) is the most common ataxia worldwide. Cholesterol 24-hydroxylase (CYP46A1), a key enzyme that allows brain cholesterol efflux and activates brain cholesterol turnover, is reduced in cerebellar extracts from SCA3 patients and SCA3 mice. Here, researchers found that administration of an adeno-associated viral vector encoding CYP46A1 to a lentivirus-based mouse model of SCA3 reduced the accumulation of mutant ataxin 3, a hallmark of SCA3, and preserved neuronal markers. In a transgenic SCA3 model with a severe motor phenotype, cerebellar delivery of AAVrh10-CYP46A1 was potently neuroprotective in adult mice with established pathology. CYP46A1 significantly reduces ataxin 3 protein aggregation, alleviates motor impairments, and improves SCA3-related neuropathology. Specifically, improvement in Purkinje cell number and reduction of cerebellar atrophy are observed in AAVrh10-CYP46A1-treated mice.

Researchers co-overexpressed lentiviral vectors encoding human full-length mutant ataxin-3 (LV-based SCA3 mouse) and AAVs encoding CYP46A1 or control GFP (AAVrh10-CAG-GFP) in mice striatum (Figure 1a, b) and sacrifced the animals 2 months post-injection. As expected, HA-directed immunohistochemistry showed that the HA tag was present in LV-SCA3 mice injected with AAVrh10-CYP46A1 but absent in control mice injected with AAVrh10-CAG-GFP (AAVrh10-GFP) (Figure 1c). In contrast, control mice showed strong and extensive brain immunoreactivity for GFP, whereas no GFP-positive signal was detected in mice injected with AAVrh10-CYP46A1 (Figure 1d). In the LV-based SCA3 mouse model, immunohistochemistry (1H9 antibody) and quantitative analysis of mutant ataxin3-positive inclusions revealed a statistically significant decrease (59%) in the number of ataxin-3-positive inclusions present in animals expressing CYP46A1, compared to the control group (AAVrh10-GFP) (Figure 1e, f, i). Furthermore, the size of inclusion bodies was significantly reduced (47%) in LV-SCA3 mice treated with AAVrh10-CYP46A1 compared with LV-SCA3 mice injected with the control AAVrh10-GFP vector (Figure 1j). These results were complemented with an anti-ubiquitin immunostaining that demonstrated a reduction (46%) in the number of ubiquitinated inclusions in LV-SCA3 mice injected with AAVrh10-CYP46A1 relatively to control mice (Figure 1g, k).

Figure 1. CYP46A1 clears mutant human ataxin-3 and confers neuroprotection in a lentiviral-based SCA3 mouse model. (Nóbrega C, et al., 2019)