Work

Analysis of Processing, Post-Maturation, and By-Products of shRNA in Gene and Cell Therapy Applications

Year: 2025

Type: article

Abstract: Short hairpin RNAs (shRNAs) are potent tools for gene silencing, offering therapeutic potential for gene and cell therapy applications. However, their efficacy and safety depend on precise processing by the RNA interference machinery and the generation of minimal by-products. In this protocol, we describe how to systematically analyze the processing of therapeutic small RNAs by DROSHA and DICER1 and their incorporation into functional AGO complexes. Using standard small RNA sequencing and tailored bioinformatic analysis (QuagmiR), we evaluate the different steps of shRNA maturation that influence processing efficiency and specificity. We provide guidelines for troubleshooting common design pitfalls and off-target effects in transcriptome-wide profiling to identify unintended mRNA targeting via the miRNA-like effect. We provide examples of the bioinformatic analysis that can be performed to characterize therapeutic shRNA. Finally, we provide guidelines for troubleshooting shRNA designs that result in suboptimal processing or undesired off-target effects. This protocol underscores the importance of rational shRNA design to enhance specificity and reduce biogenesis by-products that can lead to off-target effects, providing a framework for optimizing the use of small RNAs in gene and cell therapies.

Source: Methods and Protocols

DOI: 10.3390/mps8020038

Authors: Zhenyi Hong, Nikola Tesic, Xavier Bofill-De Ros

Institutions: Aarhus University, Seven Bridges Genomics (United States)

Language: English

Topic: RNA Interference and Gene Delivery

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $2,172