Abstract
Purpose :
Usher Syndrome (USH), an inherited disorder, is the leading cause of deaf-blindness. USH2A, one of the genes that causes Usher syndrome, encodes protein Usherin with an open reading frame of 15.6kb. Our goal is to engineer minigenes with reduced size and yet sufficient function, to meet the AAV volumetric constraints (<~5kb) for gene augmentation to rescue vision.
Methods :
Full length Usherin consists of many motif repeats. We hypothesize that some of the repeats are redundant and aim to miniaturize USH2A through rational design of several exploratory minigenes and then high-throughput screening of combinatorial libraries with variant minigenes. We chose Oc-k1 cell line as our in vitro model as it displays the characteristic periciliary localization of Usherin and is originated from mouse organ of Corti. Our immunocytochemistry data showed that upon transfection, full length human USH2A expressed and localized at the periciliary region of Oc-k1 cell line (USH2A -/-). We observed that wild type Oc-k1 cells proliferate significantly faster than USH2A -/- cells, and full length USH2A can rescue cell proliferation. This yet to be explained feature is currently used as our in vitro screening platform.
Results :
We designed 7 minigenes to interrogate the motif repeats and screened them by proliferation rate. Three minigenes achieved >60% rescue effect of the full USH2A, two displayed >30% increase and two had no effect compared to control group (vehicle vector). Four of the positive minigenes also demonstrated periciliary localization. Our result showed that deletion among the FN3 repeats affects cell proliferation less than the other motifs.
Based on above results, we designed 3 independent libraries: Lib614 targets FN3 repeats #6~14, Lib1625 for #16~25, Lib2734 for #27~34. Each variant contains a unique barcode for batched screening through NGS. We have completed the assembly of Lib614, with Lib2734 assembly in progress. These libraries will be subject to cell proliferation assay and NGS for functionality screening.
Conclusions :
We conclude that the FN3 repeats region, occupying almost 2/3 of the entire USH2A gene, bears redundant motifs that may be deleted for USH2A miniaturization. The proliferation assay with Oc-k1 cells revealed potential physiological role of USH2A, though it calls for further investigation on the mechanism to justify the correlation between in vitro proliferation and in vivo photoreceptor rescue.
This is a 2021 ARVO Annual Meeting abstract.