Protocol: Design and Validation of Mirtron-Mediated RNA Interference for Gene Therapy

Abstract

Mirtrons, a subclass of intronic microRNAs, offer an alternative RNA interference (RNAi) mechanism by bypassing Drosha processing. This protocol details the design and production of mirtron-based constructs aimed at achieving efficient gene knockdown. By embedding mirtrons in the 5' untranslated region (UTR) of a transgene, we evaluated processing efficiency and RNAi activity. The compact size of mirtrons facilitates concurrent gene knockdown and supplementation within a single adeno-associated virus (AAV) vector. Our findings present a novel strategy for gene regulation in therapeutic and research applications.

Protocol: Design and Validation of Mirtron-Mediated RNA Interference for Gene Therapy

Qingyun Zheng, Harry O Orlans, Federica Staurenghi, Michelle E McClements, Robert E MacLaren

Part of the book series: Methods in Molecular Biology ((MIMB,volume 3023))

Keywords: DNA cloning; Gene therapy; Mirtron; RNAi.

Summary

This book gives researchers clear steps for developing gene therapies for inherited eye conditions. It covers how treatments are made, tested in the laboratory and animals, and studied in patients.

Topics include AAV delivery, CRISPR, lab-grown retina models and tests of eyesight.

Keywords: retinal gene therapy, inherited eye conditions, AAV delivery, CRISPR, retina models, animal testing, patient trials, eyesight tests, research methods