Abstract
Purpose :
Mutations leading to protein misfolding are associated with many retinal dystrophies. The cellular response to this can result in reduced expression or mislocalisation of the mutant protein. Gene therapy is expensive and can time consuming to develop, and is restricted to autosomal recessive disease. We sought to identify small molecules that could rescue the function of multiple mutant alleles associated with different modes of inheritance as a faster and more economic approach to developing therapeutic agents using bestrophin-1 as an exemplar.
Methods :
Expression and localization of wildtype and mutant bestrophin-1 was tested by western blot and immunofluorescence in stably-transfected MDCKII cell lines. Whole-cell patch-clamp was performed on transiently transfected HEK293T cells for functional analysis. Sodium phenylbutyrate (4PBA) and its analogue 2-Naphthoxyacetic acid sodium salt (2-NOAA) were tested for their ability to rescue the expression and function of mutant bestrophin-1.
Results :
Western blot result showed that the expression level of mutant bestrophhin-1 was significantly increased by 4PBA (p<0.05) or 2-NOAA (p<0.001) compared to the untreated groups. Immunofluorescence showed that the membrane-localized mutant bestrophin-1 was remarkably increased by 4PBA (p<0.05) or 2-NOAA (p<0.01) treatment. The impaired Cl- channel function of the dominant mutants was partially rescued by the treatment (p<0.05 compared to the untreated group and p<0.05 compared to the wild-type) and that of the recessive mutant was fully restored to the wild-type level.
Conclusions :
Our data demonstrates that small molecules can restore the expression, trafficking and channel function of autosomal dominant and recessive mutant bestrophin-1 in vitro, paving the way for using small molecules as a therapy for bestrophinopathies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.