CE17 - Recherche translationnelle en santé

Investigating UNC45A dependent epithelial polarization in microvillus inclusion disease (MVID)-like disease: from mechanism to development of models for preclinical studies – INCEPTION

Submission summary

Background:
A main objective of Partner 1 is to improve mechanistic insight and care of monogenic intestinal disorders. A new syndrome combining congenital diarrhea, cholestasis, deafness and bone fragility was recently ascribed to mutations in UNC45A, a member of the UCS protein family (UNC-45/ CRO1/ She4p) that acts as co-chaperone for myosin folding. The mechanism of intestinal failure is not known and there is no curative therapy.

Preliminary results and working hypothesis:
We have identified novel biallelic UNC45A mutations in 4 unrelated patients with severe congenital diarrhea. Immunohistochemistry and electron microscopy showed brush border abnormalities and intracellular inclusions in enterocytes that are reminiscent of Microvillous Inclusion Disease, an untractable form of congenital diarrhea mainly caused by mutations in myosin Vb. This motor protein is indispensable for apical targeting of brush border proteins and enterocyte polarization. Our working hypothesis is that UNC45A orchestrates the folding of myosins, notably of myosin Vb, and is thereby indispensable for intestinal epithelial polarization and brush border differentiation. Accordingly, preliminary data obtained in the model enterocyte line Caco-2 indicate that: i) CRISPR-Cas9 inactivation of UNC45A prevents organization into well-formed cysts in 3D culture; ii) UNC45A interacts with myosin Vb and HSP90; iii) large protein aggregates accumulate when proteasome is blocked. Moreover, preliminary observations in unc45a-/- zebrafish mutants showed microvillous inclusions in enterocytes.

Objectives:
We intend now:
- In Aim 1: to characterize the consequences of UNC45A mutations on epithelial morphogenesis and polarization using UNC45A-deficient Caco2 cells complemented with UNC45A mutant alleles. We will i) study apical polarization in 3D culture, ii) use pull-down assays and proteomics to identify UNC45A partners and compare their interactions with wild type and mutated UNC45A, iii) assess expression and misfolding of myosin Vb and possibly of other myosins interacting with UNC45A, iv) validate mislocalization of apical markers and loss of myosin expression in patients’ biopsies.
- In Aim 2: to set up and characterize disease models of UNC45A deficiency. We intend: i) to recapitulate the developmental defect in intestinal organoids differentiated from patients’ blood-derived pluripotent cells; 2) to use the zebrafish model for in vivo analysis of the role of UNC45A and UNC45A partners in epithelial polarity and brush border development.
- In Aim3: to perform preclinical studies and identify possible therapeutic strategies. We will implement: i) gene editing of UNC45A in patients’ intestinal stem cells in order to provide proof-of-concept for gene correction; ii) drug screen for compounds that may rescue myosin folding and thereby the UNC45A mutant phenotype.
The project relies on the complementary expertise of 4 partners in the study of the human intestinal diseases (team 1), developmental studies in zebrafish (team 2), epithelial differentiation (teams 3 and team 4). It will benefit from of a combination of cutting-edge approaches including in vitro and in vivo genetic manipulations, proteomics and high-resolution imaging techniques that are mastered by the four partners.

Expected Outcomes:
We expect to establish the key role of UNC45A in intestinal epithelial polarization and to unravel the mechanism of intestinal failure in O2H2 syndrome. Our multi-organismal approach will provide a general view of the conserved and/or divergent functions of UNC45A in the intestine and will allow us to address the feasibility of new treatment options aiming at genetic correction of UNC45A mutations or pharmacological rescue of UNC45A chaperone activity in intestinal cells. The rescue, even partial, of UNC45A functions should have long-term implication for the treatment of this rare but extremely severe disease.

Project coordination

Nadine Cerf-Bensussan (IHU IMAGINE - INSTITUT DES MALADIES GÉNÉTIQUES)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IdV Institut de la vision
IGDR INSTITUT DE GENETIQUE ET DEVELOPPEMENT DE RENNES
The Enteric Nervous System in Gut and Brain Disorders - TENS INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
IMAGINE IHU IMAGINE - INSTITUT DES MALADIES GÉNÉTIQUES

Help of the ANR 592,463 euros
Beginning and duration of the scientific project: March 2021 - 36 Months

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