The optimal window of RNAa activity was delayed by ~24-48 hours in comparison to RNAi. Perhaps, the delay in RNAa activity reflects a more complicated mechanism with additional rate-limiting steps. In nematode, a special ribonucleoprotein is required to shuttle small siRNAs into nuclei in order to facilitate nuclear RNAi [26
]. Although this protein is not conserved in humans, cytoplasmic miRNA has been shown to actively migrate into the nuclear fraction of living human cells [27
]. Because RNAa is a nuclear process acting on gene transcription, acquiring access to the nucleus may serve as an additional rate-limiting step for RNAa. Changes in chromatin structure are also associated with RNAa [4
], which may further contribute to the delayed kinetics. Regardless, identifying the delay and defining the optimal window of RNAa activity allows for proper assessment for gene induction and functional analysis of saRNAs. Assessing the rate of RNAa activity in cell culture also gives insight into the anticipated in vivo
pharmacological properties of RNAa. For instance, RNAa-based drugs may require several days before target gene induction or beneficial changes in phenotype are evident. Moreover, the longer-lasting effect of RNAa may result in less frequent administration of saRNA; a potential benefit as duplex RNA in excess can have toxic consequences [29
Identifying features and key factors involved in the RNAa pathway can influence saRNA design. As such, defining Ago2 as an important mechanistic component implicated that chemically-modified saRNAs may function to manipulate RNAa activity in a manner similar to RNAi [21
]. Utilizing dsP21-322 and dsEcad-215 as functional examples of saRNA molecules revealed that blocking the 5’-end or incorporating intentional mismatches can determine strand function. Studies have revealed an abundance of sense and antisense transcription within the promoters and flanking regions of active genes [30
]. Furthermore, overlapping noncoding RNAs and upstream cryptic transcripts have been shown to play substantial roles in regulating gene expression [33
]. As such, models for RNAa have included saRNAs targeting antisense transcripts and/or promoter-derived sequences to facilitate gene activation [5
]. RNAs transcribed in sense and antisense orientations have already been shown to serve as docking sites for transcriptional gene silencing (TGS) mediated by small duplex RNAs [38
]. Likewise, nascent sense and antisense transcripts may both serve as the targets for RNAa, as well. Utilizing modified saRNAs can not only improve mechanistic studies by defining strand activity, but also assist in determining orientation of such putative target transcripts.
Identifying functional modifications is also necessary for therapeutic development in order to improve the medicinal properties of saRNAs. In the case dsP21-322 and/or dsEcad-215 (i) blocking the 5’-end of the sense strand completely inhibited its potential off-target effects; (ii) incorporating an intentional mismatch opposite the 5’ most nucleotide in the antisense strand enhanced target gene induction, as well as reduced the off-target activity generated by the sense strand; (iii) 2’Ome modification to the sense strand inhibited RNAa activity, while the same modification to the antisense strand did not interfere with gene induction; (iv) modifying the 3’-end of either the sense or antisense strand had minimal effects on RNAa activity. Although the preferred guide strand may vary between the sense or antisense strand in different saRNAs, each modification may still be applied to manipulate saRNA activity or define strand function. As such, extrapolating these modifications to fit other saRNAs based on strand activity will also improve their medicinal properties.
Development of saRNAs for therapeutic application may also require multiple modifications to optimize medicinal benefits. For instance, we were able to enhance dsEcad-215 activity by blocking both the 5’-end of the sense strand and incorporating a mismatch opposite the 5’ most nucleotide of the antisense strand. The combination of both modifications alleviated any potential off-target effects that would arise from improper use of the sense strand and enhanced gene induction; features needed to manipulate in order to develop RNAa therapeutics. Modification to the sense and antisense backbones (i.e. 2’-OMe, 2’-flouro, etc.) in saRNA duplexes may also improve therapeutic application by increasing endonuclease resistance and serum stability, much as they are utilized to stabilize siRNAs, as long the passenger strand is devoid of inhibitory modifications. Tethering small molecules (i.e. cholesterol) to the 3’-ends of saRNAs could also be used to improve systemic delivery of RNAa-based drugs. Conjugation of other compounds (i.e. flurogenic labels) to the 3’-termini may be effective at providing visual confirmation of saRNA uptake into target cells or tissue, as well.
RNAi is rapidly developing into a promising new approach for combating disease at the genetic level; however, it can only provide antagonism of specific molecular targets. By utilizing saRNAs as therapeutic compounds, RNAa offers similar benefits as RNAi, while facilitating the exact opposite response – gene activation. This approach addresses a missing void in RNA-based gene therapies and offers a novel solution to provide greater efficacy in disease control. RNAa has already been shown to activate genes capable of suppressing cancer cell growth (e.g. p21, E-cadherin, p53, NKX3.1, ect.), triggering angiogenesis (e.g. VEGF), or influencing stem cell maintenance (e.g. CXCR4) [5
]. As such, the ability to selectively up-regulate genes acting against a disease state can have far-reaching impacts in almost every therapeutic realm. However, application of RNAa is not limited to only cancer therapeutics. RNAa also has potential to function as a surrogate tool for vector-based gene overexpression systems. RNAa offers a new approach to enhance endogenous gene expression that may be manipulated to target a variety of genes. As momentum within the biological sciences increases, RNAa may become an important technique to augment gene expression for therapeutics and functional gene studies.