INTRODUCTION
The ability to manipulate RNA expression and splicing is one of the most powerful tools in research and holds great promise for therapeutic applications. In basic science, the ability to target and knockdown a specific gene of interest using tools such as siRNA and CRISPRi has allowed us to unravel the intricacies of biological pathways, leading to many new discoveries (Cong et al., 2013; Gilbert et al., 2014; Kampmann, 2018; Mullenders et al., 2009). In medicine, targeted manipulation of expression and splicing through antisense oligonucleotides (ASOs) has led to several promising therapeutics. For instance, the FDA has approved ASO-based treatments for both Duchenne muscular dystrophy, a child-onset disease in which patients progressively lose muscle function resulting in wheelchair dependency, the need for ventilation assistance and ultimately premature death (Aartsma-Rus & Krieg, 2017), and spinal muscular atrophy in infants, a disease caused by mutations in SNM1 leading to musculoskeletal wasting and respiratory failure (Stein & Castanotto, 2017).
While a variety of technologies exist to knockdown or modify pre-mRNA in the cell, few options are available that bridge the gap between research and clinical settings. Tools such as siRNA, lentivirus, and plasmid expression are frequently employed as screening tools to find therapeutic targets (Koike-Yusa et al., 2014). However, the end products of these methods often run into roadblocks as therapeutics themselves. siRNA therapies are challenged by double-stranded RNA degradation by the immune system, off-target effects due to improper strand loading, and toxicity due to oversaturation of the RNAi machinery (Nogrady, 2019). CRISPR-based technologies require the introduction of the exogenous Cas9 protein, which poses a significant barrier for many applications, and their off-target effects are still being evaluated for safety (Tycko et al., 2019). These limitations result in the identification and characterization of gene targets with technologies that are either clinically unviable or suboptimal, thus requiring their re-evaluation with small molecule screens or other therapeutic methods, resulting in significant delays in translating bench observations to the clinical setting.
While ASOs have found purchase as therapeutics, they are heavily reliant on a variety of nucleotide chemistries and backbone modifications to achieve the desired results (Roberts et al., 2020). ASOs that degrade their targets are often composed of RNA with central DNA “gap” sequences that hybridize with homologous regions of RNAs and trigger RNase H mediated degradation, whereas sterically hindering ASOs, such as morpholinos, incorporate morpholine moieties instead of ribose backbones and modified phosphonodiamidite linkages to evade degradation (Ekker, 2006). Reliance on these chemistries and modifications makes it difficult to perform large ASO based screens to identify genes that can be targeted with this modality. For example, this methodology is incompatible with the production of lentiviral libraries, which infect cells clonally and thereby can greatly parallelize the search for candidate molecules through a combination of cell culture and next-generation sequencing.
A technology that could seamlessly transition between construct-based screening and the synthesis of biologically functional molecules would greatly benefit both the research and clinical communities. The ideal technology would require minimal chemical modifications to transition from screening construct to lead compound, be compatible with modern molecular biology techniques, such as plasmid or viral expression, and would not require exogenous proteins to function. To address this need, we created hnRNPA1 recruiting oligonucleotides (AROs) to serve as both a standalone technology and paradigm for using endogenous RNA binding proteins to manipulate pre-mRNA.
hnRNPA1 is a ubiquitously expressed RNA binding protein with multiple roles that are still being uncovered; however, the protein is best known for the role it plays in alternative splicing and RNA processing (Jean-Philippe et al., 2013). Splice sites are defined by short 5’ (GU) and 3’ (AG) sequences flanking introns. While these dinucleotides are required for splicing, they do not contain enough information to correctly specify splice junctions. As a result, regulatory proteins such as SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) must bind to exonic splice enhancers (ESEs) and exonic splice silencers (ESSs) respectively to properly define exon-intron boundaries (Lee & Rio, 2015) . Binding of hnRNPA1 to an ESS on pre-mRNA blocks the binding of SR proteins and splicing machinery leading to cooperative recruitment of additional hnRNPA1 and suppression of the splice site (Jean-Philippe et al., 2013).
We sought to harness the ability of hnRNPA1 to suppress splice site selection by recruiting it to targeted pre-mRNA molecules. To do so, we constructed single-strand RNA molecules herein referred to as hnRNPA1 recruiting oligonucleotides or “AROs” (for A 1 r ecruitingo ligonucleotide). AROs consist of two parts, a short (20 – 25 bp) RNA oligonucleotide targeting domain which is complementary to the target pre-mRNA, and an hnRNPA1 recruiting loop derived from the HIV ESS 3, which binds to the RNA binding domain RRM1 of hnRNPA1 and recruits the protein (Jain et al., 2017) (Figure 1A). We hypothesized that, upon recruitment to the target pre-mRNA, hnRNPA1 would displace local SR proteins and splicing machinery (Figure 1B) resulting in the suppression of regional splice sites, leading to frameshifts caused by aberrant exon skipping or intron inclusion.
Due to the ubiquitous nature of endogenous hnRNPA1 across cell types, AROs do not require any exogenous proteins to function. Further, as AROs do not rely on DNA-RNA hybrids to trigger RNAase H degradation, AROs can be produced entirely as RNAs within the cell. Finally, their single-stranded nature and simple mechanism of action allow them to be transcribed in vivo using a standard Pol II promotor, so they are compatible with standard lentiviral screening methodologies. In the following experiments, we demonstrate that AROs can suppress target mRNA transcripts, are biologically functional, and can be expressed using standard molecular biology constructs or delivered directly as single-strand RNA oligonucleotides.