Exploring the potential of cell-derived vesicles for transient delivery of gene editing payloads

Abstract: Machado-Joseph disease (MJD) is the most common dominant autosomal inherited ataxia worldwide, caused by the over-repetition of the trinucleotide CAG in the ATXN3 gene. This leads to the accumulation of ataxin-3 protein and neurodegeneration. Currently, treatment remains symptomatic, though gene therapy has emerged as a promising approach. However, efficient and minimally invasive delivery to the brain remains a challenge. Extracellular vesicle-associated adeno-associated vectors (EV-AAVs) are a novel delivery system, combining AAVs' ability to deliver genes with extracellular vesicles' capacity to bypass the immune system and cross the blood-brain barrier (BBB). Previous studies, however, have only combined AAV serotypes known to efficiently cross the BBB with EVs as a non-invasive delivery system to the brain. Thus, the ability of EV-AAVs to cross the BBB remained inconclusive. In this study we evaluated whether AAV1/2 serotype, combined with rabies virus glycoprotein (RVg)-coated EVs, could effectively target the brain. Two isolation methods, differential ultracentrifugation and size exclusion chromatography (SEC) were compared, with SEC yielding higher EV recovery. Moreover, RVg-EV-AAV1/2 successfully crossed the BBB and transduced mouse brains, leading to motor and neuropathological improvements in an MJD mouse model. This study demonstrates that RVg-EV-AAVs are promising non-invasive delivery systems for MJD gene therapy.

TL;DR: Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown.