Abstract Details

D - Oligonucleotide Therapeutics (including siRNAs, aptamers, antagomirs, miRNAs, shRNA, antisense, and splice switching oligos, plasmids)

1216: miX^on, a System for Tunable Control of miRNA Expression by Drug-Induced Splicing

Type: Poster Session

Poster Board Number: 1216

Presentation Details

Session Title: Thursday Posters: Oligonucleotide Therapeutics






MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression by blocking translation or inducing decay of target mRNAs through complementary base pairing. Mature miRNAs are processed from long primary miRNAs (pri-miRNAs) in a multi-step process in which the RNAseIII endonucleases, Drosha and Dicer, cleave a stable hairpin to generate functional 20-24 bp sequences. Ultimate strand-loading preference into the RNA-induced silencing complex (RISC) is determined by multiple factors, including the identity of the 5’ base and low 5’ thermodynamic stability within the RNA duplex. Endogenous pri-miRNA scaffolds have been successfully adapted to host artificial miRNAs that, when delivered using adeno-associated viruses (rAAVs), lead to sustained knockdown of disease-related transcripts and protein for therapeutic benefit. Although we previously demonstrated that artificial miRNAs mitigate the toxicity of short hairpin RNAs (shRNAs) (McBride et al. 2008), toxicity remains possible due to unintended silencing (Monteys et al. 2014). Thus, a system to fine-tune miRNA expression following AAV delivery would be a beneficial addition to the RNA interference (RNAi) toolkit. Previously, we developed X^on, a drug-induced splicing switch that controls translation initiation in response to the small molecule LMI070 (Monteys et al. 2021). Here, we modified X^on to provide drug-inducible control over miRNA expression, which we present as miX^on. The miX^on cassette was designed to control the expression of miS1, our well-characterized RNAi sequence targeting human ATXN1. Following modifications to optimize strand biasing and sequence engineering to minimize background splicing, we developed a construct that showed 47% knockdown in a luciferase reporter system in response to LMI070, with no background knockdown in untreated, transfected cells. Moreover, this construct demonstrated reduced silencing efficiency in Drosha knockout cells, supporting miRNA processing by Drosha. Next, we engineered miX^on to target an endogenous mRNA, PCSK9. Transfection of a constitutively spliced miX^on cassette into HuH-7 cells led to a 22% knockdown of PCSK9. Interestingly, we observed a 12-fold increase in miRNA processing from a canonical miR-30 scaffold compared to our miX^on construct, with a 60% knockdown in PCSK9. This disparity between miRNA processing and knockdown could indicate an upper threshold for miRNA silencing by transfection and supports future efforts to optimize strand biasing and scaffold design for robust silencing. Overall, miX^on displays significant promise as a novel system for drug-inducible control over miRNA expression.

Ryan C. Giovenco¹, Alejandro Mas Monteys², Ellie Carrell³, Beverly L. Davidson<sup>3

1</sup>Children's Hospital of Philadelphia, Philadephia, PA,²University of Pennsylvania, Philadelphia, PA,³Children's Hospital of Philadelphia, Philadelphia, PA"