Translational readthrough of ciliopathy genes BBS2 and ALMS1 restores protein, ciliogenesis and function in patient fibroblasts

Abstract

Background Ciliary dysfunction underlies a range of genetic disorders collectively termed ciliopathies, for which there are no treatments available. Bardet-Biedl syndrome (BBS) is characterised by multisystemic involvement, including rod-cone dystrophy and renal abnormalities. Together with Alström syndrome (AS), they are known as the ‘obesity ciliopathies’ due to their common phenotype. Nonsense mutations are responsible for approximately 11% and 40% of BBS and AS cases, respectively. Translational readthrough inducing drugs (TRIDs) can restore full-length protein bypassing in-frame premature termination codons, and are a potential therapeutic approach for nonsense-mediated ciliopathies. Methods Patient fibroblasts harbouring nonsense mutations from two different ciliopathies (Bardet-Biedl Syndrome and Alström Syndrome) were treated with PTC124 (ataluren) or amlexanox. Following treatment, gene expression, protein levels and ciliogenesis were evaluated. The expression of intraflagellar transport protein IFT88 and G-protein coupled receptor SSTR3 was investigated as a readout of ciliary function. Findings mRNA expression was significantly increased in amlexanox-treated patient fibroblasts, and full-length BBS2 or ALMS1 protein expression was restored in PTC124- and amlexanox-treated fibroblasts. Treatment with TRIDs significantly improved ciliogenesis defects in BBS2 Y24*/R275* fibroblasts. Treatment recovered IFT88 expression and corrected SSTR3 mislocalisation in BBS2 Y24*/R275* and ALMS1 S1645*/S1645* fibroblasts, suggesting rescue of ciliary function. Interpretation The recovery of full-length BBS2 and ALMS1 expression and correction of anatomical and functional ciliary defects in BBS2 Y24*/R275* and ALMS1 S1645*/S1645* fibroblasts suggest TRIDs are a potential therapeutic option for the treatment of nonsense-mediated ciliopathies.

Mutation profile of Bardet‐Biedl syndrome patients from India: Implicative role of multiallelic rare variants and oligogenic inheritance pattern

“…ARL6 and BBS12 gene were reported to be causative in ~0.4% and ~2%–6% of the cases respectively in other populations, 29 while 7.4% of our patients harbored ARL6 and BBS12 gene variations each. BBS2 variants have previously been reported in 8%–18% of the BBS cases, 30 contrary to 6% observed in the current study. Higher incidence of BBS4 and BBS5 are reported in patients from Middle Eastern and North African regions 23 while we observed BBS4 variations in 2.7% and BBS5 variations in 4.6% of our patients.…”

Emerging principles of primary cilia dynamics in controlling tissue organization and function

“…In turn, the loss of BBSome function results in defects of the stimulus-dependent control of GPCR localization in the cilium, whereby downstream GPCR signaling is severely affected (Schou et al, 2015;Wingfield et al, 2018;Anvarian et al, 2019). This has been shown for ciliary GPCRs, such as the somatostatin 3 receptor (SSTR3), the melanin-hormone concentrating receptor 1 (MCHR1) (Berbari et al, 2008;Nager et al, 2017;Eintracht et al, 2021), the neuropeptide Y receptor 2 (NPY2R) , and the dopamine receptor 1 (DR1) (Stubbs et al, 2022).…”

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs

“…Samples were analysed by western blotting as described previously 24,49 . Cells were washed with ice-cold PBS and total protein extract was prepared with RIPA buffer with 1x Halt ™ protease inhibitor cocktail and Halt ™ phosphatase inhibitor (ThermoFisher Scientific, MA, USA) at a ratio of 5x106 cells/mL.…”

“…We used these models to assess the potential of TRIDs amlexanox and 2,6-diaminopurine (DAP) to treat aniridia. Amlexanox is a FDAapproved drug used for the treatment of asthma and aphthous mouth ulcers , that was found to have both readthrough and NMD-inhibition properties [23][24][25] ; DAP is an antileukemia compound with recently identified strong readthrough capacity for UGA-type PTCs…”

“…Dosing concentrations of amlexanox (Abcam) and 2,6-diaminopurine (DAP, Sigma-Aldrich) were based on previous publications 23,25,26 . Known TRIDs ataluren/ PTC124 (ApexBio Tech LLC) and G418 (Life Technologies) were used as positive readthrough controls at 40µM and 100µg/mL, respectively, according to previous publications from our group 24,47,48 . 3D optic cups were dosed with amlexanox 250µM or ataluren 40µM in NIM+10%KOSR from day 15 to 35 of differentiation, with media refreshed every other day.…”