Protocol: Design and Validation of Mirtron-Mediated RNA Interference for Gene Therapy
Publication details
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.
Part of the book series: Methods in Molecular Biology ((MIMB,volume 3023))
Keywords: DNA cloning; Gene therapy; Mirtron; RNAi.
Related publications
Personalized CRISPR therapies could soon reach thousands — here’s how
Authors: Fyodor D Urnov, Sadik H Kassim
CRISPR targeting of SNPs associated with age-related macular degeneration in ARPE-19 cells: a potential model for manipulating the complement system
This study explores a novel CRISPR targeting approach to manipulate the complement cascade in AMD using the reliable ARPE19 cell line as an in vitro model. Our results demonstrate efficient DNA editing with CRISPR in ARPE-19 cells, with notable allele specificity in the editing of various AMD-related SNPs in the complement system. The key factor influencing allele specificity is the distance of the target SNP from the PAM site.
Central Retinal Sensitivity Decline in RPGR-related Retinal Phenotypes
Purpose: To characterize the natural history of central retinal sensitivity decline in RPGR-associated retinopathy, and to evaluate and quantify phenotypic differences in disease progression using microperimetry-derived metrics.
Design: A retrospective cohort study SUBJECTS: 50 patients with genetically confirmed RPGR mutations (36 rod-cone, 14 cone-rod dystrophy [CORD]), contributing 357 microperimetry assessments.
