Restoring brain cholesterol metabolism : Development of clinical Gene therapy for Huntington's disease – HDcyp

Increasing evidences suggest that dysfunction of brain cholesterol metabolism plays a major role in neurodegenerative diseases particularly in Huntington disease (HD). Increased membrane cholesterol together with decreased activity of the cholesterol biosynthesis pathway (mevalonate pathway) are observed in the brain of HD patients and mice and strongly contribute to the pathology. Neuronal cholesterol 24 hydroxylase (CYP46A1) is the key enzyme allowing the clearance of excess cholesterol from the brain, after conversion into 24OH-cholesterol (24OH-Chol). CYP46A1 expression in striatum and blood 24OH-Chol are decreased in HD patients. Restoring CYP46A1 in striatum using AAV vector restores the mevalonate pathway, decreases cholesterol, improves behaviour and neuropathological defects and decreases aggregates of mutated Huntingtin (Htt*) in R6/2 and ZQ175 HD mice. AAV-CYP46A1 delivery corrects synaptic dysfunction (increasing synaptic plasticity, vesicular transport and neuronal survival pathways). Conversely, inhibiting CYP46A1 in the striatum of normal mice induces cholesterol accumulation, motor deficits and neuronal death. Altogether, these results support that CYP46A1 is a relevant therapeutic target for HD. In the absence of pharmacological activator, we propose AAV-CYP46A1 gene therapy to restore CYP46A1 enzyme in the striatum at an early stage of disease progression.

The objective of the proposal is, working with BrainVectis, a company devoted to brain gene therapy, to combine our efforts and expertise to 1) develop the production of the clinical AAV-CYP46A1 vector; 2) validate delivery protocol in Non-Human Primates (NPH); 3) study cholesterol metabolism in a cohort of HD patients to correlate 24OH-Chol biomarker to clinical and MRI progression.

WP1: AAV-CYP46A1 delivery in Non-Human Primates (NPH)
Our current results with a research vector (human CYP46A1 HA-tagged) demonstrate significant diffusion without toxicity into the striatum of a large size brain (NPH). In WP1, primates will receive the clinical vector developed in WP2 to document diffusion and tolerance of AAV and of CYP46A1 with the defined protocol (dose/volume/number of injections in caudate and putamen) and evaluate consequences on 24OH-Chol and cholesterol precursors in brain tissue, blood and cerebrospinal fluid.

WP2: Biomanufacturing of the clinical grade vector
Since Q4 2016, Nantes University/CPV is developing the large-scale GMP-compatible process for AAV2/rh10.CAG.hCYP46A1 manufacturing. Batches will be produced for bridging studies in mice (WP3) and validation in NPH (WP1) and for further regulatory toxicology studies requested by national (ANSM) and European (AMEA) agencies.

WP3: Bridging studies in mouse with the “clinical” vector
Proof-of-efficacy in HD mice have been performed with the research grade vector containing a Hemagglutinin (HA) tag to facilitate detection. Before engaging in primate studies with the clinical vector, we will need to validate its in vivo potency in wild-type mice then in R6/2 mice.

WP4: Evaluation of 24OH-Chol in Huntington patients
Converging results show that plasma 24OH-Chol is decreased in HD patients and correlated with striatal atrophy, suggesting that 24OH-Chol could represent a relevant biomarker of HD progression. To further correlate the link between 24OH-Chol decrease and disease progression (Clinic, MRI), we will perform a longitudinal retrospective and an observational prospective study with HD patients from the National Reference Center for Rare Diseases at Pitié Salpêtrière hospital (A Durr, partner 4).

Results already obtained and complementarity of the partner teams: N Cartier ( brain gene therapy), P Hantraye (primate studies), A Durr (Huntington clinical reference center) and BrainVectis gene therapy company (development, vector production) will ensure the feasibility of this program that will allow us to prepare clinical trial agreement (CTA) application for ANSM and EMEA submission.