Pre-clinical evaluation of the brain-targeting AAV-PHP.eB encoding cholesterol hydroxylase CYP46A1 as a therapeutic strategy for spinocerebellar ataxia type 3 – towards the understanding of the neuroprotective mechanism

Overview

Project Summary

Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is the most prevalent autosomal dominant SCA in the world leading to severe clinical manifestations and premature death. SCA3 is caused by a CAG-repeat expansion >52 in the MJD1 gene, resulting in an expanded polyQ tract in the coding region of the ataxin-3 (atx3) protein that gains a toxic function, accumulates in neurons, and promotes the formation of insoluble intranuclear inclusions. SCA3 is a multisystem disorder with degeneration of the spinocerebellar tracts, brainstem nuclei, striatum, substantia nigra and subthalamic nucleus. Ataxia starts in midlife, with prominent cerebellar ataxia, supranuclear gaze palsy and peripheral neuropathy, evolving to severe disability and death.
Cholesterol, an essential component of the brain, and its local metabolism are involved in many neurodegenerative diseases. The blood-brain barrier (BBB) is impermeable to cholesterol; hence, cholesterol homeostasis in the central nervous system (CNS) represents a balance between in situ biosynthesis and elimination. CYP46A1, a CNS-specific enzyme, converts cholesterol to 24- hydroxycholesterol, which can freely cross the BBB and be degraded in the liver. By the dual action of initiating cholesterol efflux and activating the cholesterol synthesis pathway, CYP46A1 is the key enzyme that ensures brain cholesterol turnover.
In humans and mouse models, CYP46A1 activity is altered in several neurodegenerative disorders, including SCAs. In mice, modulation of CYP46A1 activity was shown to mitigate the manifestations of Alzheimer’s, Huntington’s, Nieman-Pick type C diseases and SCA3. Animal studies revealed that CYP46A1 activity effects are not limited to cholesterol maintenance but also involve critical cellular pathways, like gene transcription, endocytosis, misfolded protein clearance, vesicular transport and synaptic transmission. How CYP46A1 can exert central control of such essential brain functions is an unanswered question under investigation.
Intra-cerebellar injection of AAVs encoding CYP46A1 into SCA3 mice models, reduced mutant atx3 accumulation and was neuroprotective, alleviating motor impairments associated to the disease. Although AAVs have been extensively used as vehicles for gene transfer to the CNS, this approach is an invasive route of delivery, involving surgical risks and with considerable limitations from a clinical translational point of view.

We will take advantage of an AAV vector with the capacity to overcome the BBB and tropism for the CNS - the PHP.eB-AAV variant. In particular, we will investigate the expression of CYP46A1 in specific regions of the brain of these mice and analyse the reduction of mutant atx3 protein aggregates, the effectiveness in alleviating motor impairments and the amelioration in neuropathology of treated Tg mice. In parallel, we will use a human cell model of SCA3 (iPSC-derived neurons from MJD patients) that will be infected with the same vector, in order to complement and corroborate the beneficial role for CYP46A1 in this disorder. We will particularly focus on lipid modifications and on the transcriptomic signature of both SCA3 models, with special interest for the role of cholesterol metabolism impairment and the (de)regulation of genes involved in CYP46A1 pathways. This will be crucial to understand the mechanism whether delivery of CYP46A1 may impact on gene up- or down-regulation induced by mutant atx3 and strengthen its action towards a good candidate to treat MJD. Furthermore, we believe that the novelty in the use of a non-invasive strategy of CYP46A1 delivery will bring great promises as a relevant therapeutic approach not only for MJD but also for other SCAs.
Finally, as researchers and citizens, we believe we have the duty to show the relevance and applicability of our work.  Here, we designed a SciCom task, where we will be promoting society-targeted activities in the course of the project. These will include not only the engagement of MJD patients but also schools and the general public. 

Main Goals

This project focus on two main aims: i) investigating whether a non-invasive delivery of CYP46A1 will improve the disease phenotype in a SCA3 mouse model with established pathology and ii) to disclose the mechanism(s) behind the neuroprotective effect of this enzyme in SCA3 models.

Project Details