Reinventing the Wheel: Synthetic Circular RNAs for Mammalian Cell Engineering

TIBTEC 1820 No. of Pages 14

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Review

Reinventing the Wheel: Synthetic Circular RNAs for Mammalian Cell Engineering Alan Costello

,1,*,@ Nga T. Lao,2 Niall Barron,2,3 and Martin Clynes1

The circular RNA renaissance is upon us. Recent reports demonstrate applications of synthetic circular RNA molecules as gene therapies and in the production of biologics from cell-based expression systems. Circular RNAs are covalently closed loop RNA species that are formed naturally through noncolinear splicing of pre-mRNA. Although once thought to be noncoding artefacts from splicing errors, it is now accepted that circular RNAs are abundant and have diverse functions in gene regulation and protein coding in eukaryotes. Numerous reports have investigated circular RNAs in various diseases, but the promise of synthetic circular RNAs in the production of recombinant proteins and as RNA-based therapies is only now coming into focus. This review highlights reported uses of synthetic circular RNAs and describes methods for generating these molecules.

Highlights Both in vitro and in vivo methods to circularize RNA have been reported. Synthetic circular RNAs have been designed to dysregulate natural miRNA function. Circular RNAs can code for protein, and novel mechanisms of translation have been demonstrated. Perturbation of protein function and intracellular metabolite sensing are now possible with circular RNA aptamers.

The Circular RNA Renaissance Originally discovered in the early 1990s [1,2], eukaryotic circular RNAs (see Glossary; also known as circRNAs) were believed to be nothing more than an artefact of splicing errors because very few such transcripts had been identified. However, in recent years the advent of hybridization-free, next-generation RNA sequencing (RNA-seq) methods permitting the detection of novel, noncanonical transcripts revealed that ~10% of expressed genes produce circular RNA splice variants [3–5]. Independent studies demonstrating that levels of circular RNA isoforms can exceed those of associated linear RNA [6,7] have sparked renewed interest in the study of this distinct RNA species. Circular RNAs are formed through ‘back-splicing’ [3] (Box 1), can be composed of exons [8], introns [9], or both [10], and are highly stable compared with their linear counterparts because they lack free ends [3,11]. The classification of this RNA species is difficult because functional circular RNAs can be either noncoding [8,12] or coding [11,13–16]. Despite the still largely understudied and ill-defined role of eukaryotic circular RNAs, examples of endogenous functions and synthetic applications have been reported. These developments are generating new cell engineering opportunities to capitalize on the desirable attributes of these molecules to form synthetic circular RNA molecules with promising potential as gene therapies or in the mass production of recombinant proteins from cellular hosts. This review covers the methods available for generating synthetic circular RNAs and discusses current and future applications of these RNA species.

Generating Circular RNA Learning from Natural RNA Circles In forced expression studies of putative naturally occurring circular RNAs [8,10], criteria for the artificial circularization of any sequence in mammalian cells were established. In addition, functional endogenous circular RNAs have been a source of inspiration in the application of synthetic circular RNA molecules. Continued research into naturally occurring circular RNAs should elucidate future, but so far unidentified, functional motifs and applications of these molecules. To this end N10 computational methods for the discovery of circular RNAs from RNA-seq datasets have now been published [12,17–25]. A reference genome is essential for all circular Trends in Biotechnology, Month 2019, Vol. xx, No. xx

1

National Institute for Cellular Biotechnology, and Synthesis and Solid State Pharmaceutical Centre (SSPC)– Science Foundation Ireland (SFI) Centre for Pharmaceuticals, Dublin City University, Dublin 9, Ireland 2 National Institute for Bioprocessing Research and Training, Dublin, Ireland 3 School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, Ireland

*Correspondence: [email protected] (A. Costello). @ Twitter: @yestello (A. Costello).

https://doi.org/10.1016/j.tibtech.2019.07.008 © 2019 Elsevier Ltd. All rights reserved.

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RNA detection algorithms. The unique back-splice junctions formed during RNA circularization aid in their detection in prediction pipelines. Comprehensive evaluation of circular RNA detection tools was conducted by Zeng and colleagues [26]. Most circular RNA discovery tools mine for noncanonical splice junctions but provide little information about the internal sequence, quantification, or embedded functional motifs. Additional computational tools have therefore been developed for such downstream analyses [21,27–31]. Depletion of polyadenylated RNA and enrichment for circular RNA with RNase R has proved useful in reducing the signal to noise ratio in circular RNA detection [32]. Comprehensive reviews of current computational strategies for circular RNA prediction and detection have recently been published [33,34]. Although some strategies have been heavily influenced by natural biogenesis pathways, other in vitro methods to generate synthetic circular RNAs have proved very successful. Intron-Mediated RNA Circularization In Vivo and In Vitro It is known that circularization of exon sequence requires canonical splice signals [35], and that flanking the sequence of interest with complementary introns dramatically improves backsplicing efficiency [10]. Transgene sequences can be circularized by bracketing the sequence of interest with a 5′ splice acceptor (SA) and 3′ splice donor (SD) (Figure 1A). This cassette is then further flanked with intronic sequences – a long intron at the 5′ end and a short b100 bp intron at the 3′ end. Downstream of the 3′ intron is a sequence of reverse complementarity to the 5′ intron. Upon transcription, the pre-mRNA generated undergoes hybridization of complementary introns, generating a large hairpin-like structure. This is an exogenous mechanism to mimic the natural biogenesis of circular RNA by intron pairing (Box 1). The splice signals made proximal by intron pairing facilitates a back-splice junction that produces nascent circular RNA. This method of in vivo circular RNA biogenesis has been utilized to mimic natural noncoding RNA circles [8] and to form coding synthetic circular RNAs [11,36]. Through absolute quantification of linear and circular isoforms, it has been shown that this system can produce circular RNA in living cells with an efficiency of N90% [36]. Competition between forward- and back-splicing was used to design a system for inducible circular RNA formation [37]. The 3′ intron of a circular RNA expression cassette was engineered to contain a hairpin targeted by the CRISPR endoribonuclease Csy4 [38] (Figure 1B). In the absence of Csy4, forward splicing is favored, yielding linear mRNA encoding two partial GFP sequences and an intact RFP sequence. Introduction of Csy4 leads to cleavage of the premRNA at the targeted hairpin, releasing the forward splice acceptor and promoting circularization of the upstream transcript. This yielded a circular RNA molecule containing an internal ribosome entry site (IRES) and an intact GFP open reading frame. Alternative strategies of intron-mediated RNA circularization have now been optimized for the production of long circular RNAs in vitro [39,40] and in vivo [41]. These methods iterate the first reports of autocatalytic group I intron ribozyme-mediated RNA circularization. To circularize long (up to 5 kb) RNA transcripts containing an encephalomyocarditis virus (EMCV) IRES and open reading frame, Wesselhoeft and colleagues [39] built on the permuted intron–exon (PIE) splicing method first described in the early 1990s [42]. This optimization included the addition of 5′ and 3′ homology arms flanking the autocatalytic split introns, additional short homology arms, and disruptive or pervasive spacer elements within the circular RNA molecule (Figure 1C). Using a group I catalytic intron from Anabaena pre-tRNA [42] and strong internal homology arms, RNA circularization of 95% was achieved in vitro [39]. Recently a new method for in vivo ribozymatic RNA circularization has been reported for the generation of small circular RNA aptamers, but is applicable to circularization of any sequence 2

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Glossary Cell factory: a cell used for the manufacture of a specific protein or chemical product. Circular RNA: a covalently closed RNA loop, lacking free ends. Cleavage peptide: a peptide sequence that is recognized by host cell proteases leading to cleavage of the nascent polypeptide chain. Internal ribosome entry site (IRES): a sequence within an mRNA that permits recruitment of ribosome subunits in a cap-independent manner. miRNA: small, ~22 nt, noncoding single-stranded (ss) RNAs that function in post-transcriptional regulation of coding mRNAs through complementary pairing with UTRs. miRNA sponge: synthetic decoy targets for specific miRNAs that divert miRNAs from their endogenous targets. Monocistronic: a mature mRNA encoding a single protein. Polycistronic: a mature mRNA encoding multiple proteins. Ribosome arrest peptide (RAP): a peptide sequence that pauses translation during protein synthesis. RNA aptamer: short RNA sequences/ structures that bind to intracellular molecules or proteins and can modulate their function. Untranslated regions (UTRs): segments of a mature mRNA that do not code for protein.

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Box 1. Endogenous Eukaryotic Circular RNA Biogenesis Eukaryotic circular RNAs are formed during pre-mRNA splicing and are dependent on the spliceosome machinery [35,79]. Circular RNA biogenesis competes with canonical colinear pre-mRNA splicing (Figure IA). It has been reported that the ‘back-splicing’ required to form circular RNA is much less efficient than canonical splicing [20]. Circular RNA formation may be facilitated by cis- [10,35,79] or trans-elements [79–82]. Exon circularization is associated with flanking complementary intron sequence (Figure IB) bringing noncolinear splice sites into close proximity. Approximately 38% and 9% of circular RNAs identified in C. elegans and human, respectively, are formed by the presence of complementary flanking intronic regions [82]. The splicing factors Quaking and Musclebind can induce circular RNA formation from exons with intronic flanks containing binding motifs for the respective splicing factors [79,80]. In this case interactions with RNA-binding proteins (RBPs) bring the exon terminal splice signals close together (Figure IC).

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Figure I. Circular RNA Biogenesis. (A) Canonical splicing of pre-mRNA to remove intronic sequence occurs in a colinear fashion, joining exons in sequential order. During this process ‘back-splicing’ can occur, joining the splice signals of downstream exons to an upstream sequence. Circular RNA (circRNA) arises from backsplicing of the pre-mRNA sequence. (B) Intron complementarity brings noncolinear splice junctions close together to create a back-splice junction (BSJ). (C) This can also result from RNA-binding proteins (RBPs) interacting with the intronic sequence. circRNA products can be purely exonic or can retain an intron.

[41]. The method combines a natural form of RNA circularization that is not dependent on back-splicing, and which occurs during tRNA splicing [43] via a newly discovered group of autocatalytic ribozymes known as ‘Twister’ ribozymes [44]. The tRNA endonuclease generates a 5′ hydroxyl and 2′,3′-cyclic phosphate at the 3′ end. These ends are recognized by the nearly ubiquitous endogenous RNA ligase, RtcB. Before this report, the use of ribozyme-mediated RNA circularization in vivo was hindered by the very slow cleavage rates under physiological conditions – tens of minutes to hours [45]. However, Twister ribozymes undergo selfcleavage several hundred-fold faster than their hammerhead ribozyme counterparts under physiological conditions [46]. In their study, Litke and Jaffrey [41] evaluated the circularization products of numerous ribozyme combinations. Flanking the sequence of interest with a 5′ P3 Twister U2A [41,44] and a 3′ P1 Twister [47] resulted in the highest expression of circular RNA in vivo (Figure 1D). To distinguish this method of circular RNA generation they termed this type of circular RNA a ‘ribozyme-assisted circular RNA’ or racRNA. They called the U6 promoter-driven expression cassette the ‘Tornado’ expression system because it uses multiple Twister ribozymes. Trends in Biotechnology, Month 2019, Vol. xx, No. xx

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(A)

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Figure 1. Intron-Mediated RNA Circularization. (A) Expression of circular RNA (circRNA) from plasmid DNA (pDNA). The gene or sequence of interest is directly flanked with a 5′ splice acceptor (SA) and a 3′ splice donor (SD). These splice motifs are further flanked by intronic sequence. Arrows (NNN/bbb) indicate the direction of the intron sequence. Upon transcription the complementary intronic sequences hybridize, mirroring the natural intron pairing pathway. This intron pairing brings the SA and SD into close proximity, facilitating a back-splice junction to produce a circRNA and rapidly degraded intron side-products. (B) A Csy4-targeting aptamer is placed in the 3′ intron and no intron complementarity is present. In the absence of Csy4 protein, forward splicing is favored, yielding a linear mRNA product. In the presence of Csy4, the intron is cleaved at the aptamer site, thereby promoting a back-splice to yield circular RNA. (C) A split catalytic group I intron is used to circularize an internal sequence. Additional 5′ and 3′ homology and spacer elements are necessary to efficiently circularize the internal sequence. (D) Twister ribozymes are used to circularize short RNA aptamers. Ligation sequences provide a substrate for intracellular RNA ligase-mediated circularization. (E) In vitro RNA circularization facilitated by an oligonucleotide bridge and T4 ligase. Abbreviations: E1/E2, exons 1 and 2; FP/G, inverted GFP; IRES, internal ribosome entry site; m 7 G, 7-methylguanosine cap; Pmin, mimimal promoter; pU6, U6 promoter; racRNA, ribozymeassisted circular RNA; RCI, reverse complementary intron.

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In Vitro Enzymatic Generation of Circular RNA RNA circularization can be facilitated in vitro by enzymatic, chemical, and metal chelation-mediated reactions [48,49]. Investigation of synthetic circular RNA translation and gene regulatory functions has used enzyme-mediated RNA circularization and cap-independent mechanisms of translation of synthetic circular RNAs in eukaryotes [50]. RNA molecules were transcribed by T7 RNA polymerase with the use of a linearized DNA template. Annealing a deoxyoligonucleotide bridge complementary to the terminal ends of the linear RNA was used to bring the 3′-phosphate (p) and the 5′-OH together (Figure 1E). Following incubation with T4 DNA ligase, the resulting circular RNA products could be separated and purified by denaturing polyacrylamide gel electrophoresis (PAGE). In addition, it has been reported that in vitro enzymatic circularization can also be achieved in a similar bridging or ‘splint’ approach by way of T4 RNA ligase I, using an RNA bridge, or with T4 RNA ligase II with a DNA or RNA bridge [49]. Enzyme-mediated RNA circularization has been achieved with b500 nt, single-stranded (ss) circular miRNA sponges, but the authors report that efficiency of the T4 RNA ligase I reaction may be reduced with larger molecules [51]. Circular ss miRNA sponges were generated in vitro using enzymatic ligation. Two ssDNA oligonucleotides composed of two or three concatenated sponge sequences with 4 nt spacers were designed with ~20 nt complementary overhangs at their respective 3′ ends, allowing hybridization after heat denaturation. This provided a template for a single cycle of overlap PCR, the products of which were blunt-end ligated into a TOPO cloning vector containing a T7 promoter. Transcription by T7 RNA polymerase was used to produce short RNAs. These RNAs were dephosphorylated with calf intestinal phosphatase, phosphorylated with T4 polynucleotide phosphatase, and finally circularized with T4 RNA ligase 1. This strategy yielded highly pure circular RNA miRNA sponges containing five repeated miR-21 binding sites [51]. There are potential size limitations with this strategy that may limit its use in generating coding scripts.

Validation of RNA Circularity and Stability Owing to the unique characteristics of circular RNA, several techniques are available to verify the circularity of an RNA molecule. RNase R degrades all RNA with a free end of 7 nt or greater [32]. RNase R treatment of total RNA has been used to enrich for circular RNAs in high-throughput RNA-seq studies [12]. It can also be used as a validation method for detecting endogenous or exogenous expression of circular RNA in vivo [12,36,41] and as an enrichment step when generating circular RNA in vitro [39,51] (Figure 2A). RNase H can also be used to distinguish between linear and circular RNAs. RNase H cleaves RNA at sites that are hybridized to complementary DNA oligonucleotides [41]. In this instance cleavage of a linear RNA will yield two bands when run on a denaturing PAGE gel, whereas circular RNA will be linearized and generate a single band (Figure 2B). RNase R treatment is advantageous when validating circular RNAs from total RNA because there is no need to purify linear or putative circular RNAs before treatment with RNase H. Divergent PCR is another popular validation method for detecting RNA circularization. This works by designing primers to amplify the unique junction formed during RNA circularization (Figure 2C). For linear transcripts the primers are divergent and will not yield an amplicon, and thus circular and linear forms can be discriminated. Sanger sequencing of the amplicon detected in circular products can then be performed to verify the junction. One of the most attractive qualities of circular RNA is its inherent increased stability, and the molecular half-life is regularly reported in RNA circularization tests as an additional form of validation [3,11,36,39–41]. Numerous studies have reported on the use of actinomycin D (ActD) treatment as a means to test RNA stability [3,11,36,41]. Linear RNAs have very short residence times in cells, typically less than 6 h [52]. ActD is a transcriptional inhibitor, therefore by treating cells with ActD and sampling RNA over a subsequent time-course it is possible to identify the relative stabilities of circular and linear variants of the same sequence. Liu and colleagues demonstrated Trends in Biotechnology, Month 2019, Vol. xx, No. xx

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(A)

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Total RNA

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circRNA

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circRNA with convergent primers 3′

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Figure 2. Validation of RNA Circularization. (A) RNase R is used to deplete all linear RNAs, enriching for circular RNAs. (B) Linear and circular RNAs can be distinguished by the use of a DNA probe and RNase H. Digestion of linear RNA with RNase H gives two products, whereas circular RNA is merely linearized. (C) Divergent PCR primers can be used to validate RNA circularity by amplification of the junction sequence that is only present in circular RNA.

an in vitro method for assessing the stability of circular RNA [51]. Several types of RNA endonucleases and exonucleases are enriched in serum. Incubation of linear or circular RNA for 30 minutes at 37°C in 0–9% fetal bovine serum (FBS) solutions was used to test their stability: 92% of linear RNA was degraded in a 4% FBS solution after 30 minutes compared with only 9% of a comparable in vitro generated circular RNA.

Applications of Synthetic Circular RNA Reported applications of synthetic circular RNA molecules include exogenous protein expression [36,39,40], use as miRNA sponges [51], and as RNA aptamers [41]. Although this is a new area, 6

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and more publications regarding each of these functions will surely follow, the examples reported to date highlight a technology that is broadly applicable to enhance recombinant protein production from cellular and cell-free expression systems, as well as providing indications of a new generation of gene therapies (Figure 3, Key Figure). Circular RNA Gene Therapies Circular mRNAs comprise any circular RNA molecule with an intact open reading frame. The first evidence of eukaryotic translation of circular mRNA containing a viral IRES site was observed in the mid-1990s, but proved to be less efficient than for a linear RNA control in rabbit leukocyte lysate. In fact, the circular RNA yielded no detectable translated products [50]. However, until recently no comparative study evaluating the effectiveness of different viral and eukaryotic IRES sequences in circular RNA translation had been undertaken. Of the IRES sequences tested, that from EMCV was found to be the most efficient [15]. Although the initiation of circular

Key Figure

Applications of Synthetic Circular RNA Improving therapeutic production

DNA vector

Production host

Therapeutic functions

Diseased cell

circRNA

Cell engineering with miRNA sponges

Sequester onco-miRs

Transgene expression

Transient gene therapy

Metabolite sensing

Inhibit protein function

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Figure 3. Circular RNAs can be produced in vitro for delivery to cell systems, and DNA vector based-approaches have been used for their in vivo expression. Synthetic circular RNA molecules have been reported with rationally designed functions. These include miRNA sponge activity, translation of circular mRNA, and circular RNA aptamers. All known functions of circular RNAs described here have applications in both improving therapeutic protein cell factories and as therapeutic agents.

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mRNA translation, still largely undefined, differs from that of cap-dependent linear mRNA, the mechanisms by which elongation and translational termination occur should in theory be unchanged (Figure 4B). Knowing that IRES-mediated circular RNA translation was possible in

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Cleavage mechanism Long polyprotein Cleavage of continuously translating circular mRNA products

Continuous polyribosome translation of ‘infinite’ circular mRNA

eIF4A

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Figure 4. Mechanisms of Circular mRNA Translation. (A) The canonical pathway of eukaryotic translation initiation is a highly regulated process of ribosome assembly on the 5′ untranslated region of an mRNA. The mRNA substrate is bound by poly(A)-binding proteins (PABPs) and the eukaryotic initiation factor 4F (eIF4F) complex that comprises eIF4G, eIF4A, and the m7GpppG (m7G) cap-dependent initiation factor eIF4E. Together with eIF4B, this complex activates the mRNA and recruits the components of the ribosome 48S complex. (B) The optimal method of circular mRNA translation initiation is not well defined. Two distinct mechanisms have been reported. First, internal ribosome entry sites (IRESs) are RNA elements that recruit eukaryotic initiation factors in a cap-independent manner. The secondary structure of an IRES can vary greatly depending on the source (viral or eukaryotic). The localization of initiation factors can be stabilized by IRES trans-acting factors (ITAFs). Second, N6-methyladenosine (m6A) can directly bind to eIF3, a component of the 48S complex, to direct translation at internal locations in mRNA. (C) By removing the stop codon from the gene open reading frame in a circular mRNA, the message becomes infinite. This facilitates indefinite circling of the mRNA by the ribosome, known as rolling circle translation (RCT). RCT produces long concatemeric spools of repeating polyprotein. These polyproteins contain unique peptides encoded by translation of the back-splice junction (BSJ). (D) By coding for a cleavage motif in the absence of a termination site, the repeating polyprotein can be co- or post-translationally processed by intracellular mediators to yield monomeric proteins with repeat ribosome cycling of the circular RNA molecule. Abbreviation: m7G, 7-methylguanosine cap. 8

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mammalian cell systems, coupled with the persistent stability of circular RNA molecules over linear forms, Wesselhoeft and colleagues [39] investigated the potential of circular mRNA as an alternative to linear mRNA for the production of recombinant proteins. Indeed, the prolonged half-life of a circular mRNA versus a linear control resulted in greater quantities of bioactive protein. It has also been shown that delivery of circular mRNA in vivo has significantly diminished immunogenicity over linear mRNA [40]. Because of the transient nature of RNA, it has been proposed as an attractive alternative to DNA as a vector for gene delivery [53]. These works illustrate the potential of circular mRNAs as an alternative to DNA expression systems in the delivery of gene therapies, and highlight the benefits of circular RNA over linear RNA-based expression systems. The first reports of circular RNA function in mammalian cells [8,12,54] described miRNA sponge activity [55]. Direct argonaut II (AGO II)-mediated cleavage of endogenous circular RNA from the human CDR1 locus via binding of miR-671 was found to regulate the levels of CDR1 protein [54]. This was the first report of post-transcriptional regulation of a gene by an antisense transcript. It was later reported that this circular RNA, referred to as ciRS-7, harbored N60 miRNA binding sites for a second miRNA, miR-7 [8]. A second circular RNA sponge was found to come from the mouse Sry gene which regulated miR-138 levels [12]. Since these early discoveries, many more naturally occurring circular RNAs have been found to have miRNA sponge activity [56–62]. miRNA sponges have the potential to become a powerful therapeutic in and of themselves. A recent study investigated the potential for producing and utilizing a synthetic circular RNA – a miRNA sponge targeting the oncomiR, miR-21, in three gastric carcinoma cell lines [51]. miRNA sponge design followed the same strategy outlined for conventional linear sponge design [55]. The synthetic in vitro generated circular RNA miR-21 sponge elicited a greater effect than the linear sponge control. The authors attributed this to the increased stability of the circular RNA and not to any difference in the mechanism of miRNA sequestration [51]. Akin to miRNA sponge-mediated disruption of intracellular functional molecules, a recent study has highlighted the use of short circular RNA aptamers as functional therapeutics [41]. Litke and Jaffery demonstrated the ability of Tornado-derived circular RNA aptamers to significantly improve inhibition of NF-κB function versus linear RNA aptamers [41]. This is an exciting development because it adds a new tool for modulating intracellular protein function. Knowing that circular RNAs are susceptible to miRNA-mediated RNA decay, it could be possible to control the expression or operation of functional therapeutic circular RNA aptamers in a cell typedependent manner. Improving Cell Factories The undeniable market growth of high-value biotherapeutics [63] makes continued research into host cell engineering pertinent. Of yet there has been no report to our knowledge of engineering production cell systems by exogenous expression or repression of naturally occurring circular RNAs. Synthetic circular miRNA sponges could be a viable engineering strategy, given the successes in host cell engineering with linear miRNA sponges to date [64–66]. There has, however, been success in the design and expression of recombinant protein therapeutics from synthetic circular mRNA molecules. The closed loop structure of circular RNA offers a unique characteristic over linear mRNA. By encoding an open reading frame lacking a stop codon on a circular RNA molecule, the message becomes infinite. Should the number of nucleotides in the molecule be perfectly divisible by three, the circular mRNA acts as a polycistronic message, repeating the same message indefinitely, yielding large polyprotein chains (Figure 4C). The first investigation of this phenomenon was

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reported in the late 1990s [67]. Removal of the terminator sequence from a GFP open reading frame provided a model for producing long repeating polyprotein sequences in Escherichia coli. The objectives of this study were twofold. First, to evaluate the potential of circular RNAs to improve the translation of long repeating polyproteins [68] and, second, as a model for studying IRES-mediated cap-independent translation initiation in bacteria [50]. Although this system was able to generate multimeric forms of GFP exceeding 300 kDa in size, the levels of expression from the circular RNAs were poor and generated very heterogeneous products, with only monomeric forms being fluorescent. Postulating that the low-level protein expression from circular mRNAs seen by Perriman and Ares [67] was the result of poor translation initiation alone, Abe and colleagues [69] looked to solve this issue. By introducing in vitro generated circular RNA into a cell-free E. coli lysate, ‘rolling circle’ – continuous translation of small repeating circular mRNAs – improved translation by N100-fold, yielding heterogenous protein over a linear control. In this study, however, no gene was translated. The circular mRNAs contained multiple repeats of FLAG-tag sequence [69]. To evaluate the expression of protein from circular mRNAs, Abe and colleagues [13] expressed repeating FLAG-tag sequences from a synthetically circularized mRNA in cell-free and live human cells. Production in mammalian cell-free systems gave similarly large N250 kDa proteins, as seen in previous efforts with bacterial cell-free lysates [67]. Translation of infinite open reading frames in mammalian cells has produced long repeating polyproteins N250 kDa [13]. These synthetic circular mRNAs were all short fragments of genes of ≤300 nt. The circular RNAs lacked an IRES, poly(A), and cap structure, suggesting that capindependent translation initiation does not require any of these. In fact, incorporation of poly-A or poly-T sequences in circular RNAs reduced translation in one case [15]. Continuous translation was used in this case to prove the translation of circular mRNAs in human cells. However, as with the work by Perriman and Ares [67] in E. coli, the spooling polyprotein products were heterogeneous and unpredictable. Although the improved translational yield was promising in these reports, the homogeneity and functionality of the resulting proteins were still lacking. For a mammalian expression system there are two strategies. Thinking of infinite circular mRNA not as an endless loop but rather as a concatemeric series of genes or polycistronic mRNA, there are existing solutions to this issue that nature has kindly provided. Unlike polycistrons, however, in translation of circular RNA (rolling circle translation) the goal is to avoid the rate-limiting step of translational initiation at each start codon and instead have the ribosome traverse the message unabated and indefinitely. Cotranslational cleavage of the nascent polypeptide chain was the mechanism investigated by Costello and colleagues as a means to overcome the generation of a nonfunctional, heterogeneous polyprotein product – in this case the secreted therapeutic protein, erythropoietin (EPO) [36] (Figure 4D). To avoid nonsense translation of a long viral or eukaryotic IRES sequence, the system used a short ‘IRES-like’ sequence that was previously reported to be sufficient for translational initiation on circular RNA [15] (Box 2). Because the model therapeutic protein being expressed in their study, EPO, is a secreted protein, posttranslational cleavage was also prevalent – leading to a reduction in size of the multimeric polyprotein variant in comparison with prior findings in prokaryotes. Cotranslational cleavage via the addition of a 2A peptide motif [70] (Box 3) improved the specific productivity Nthreefold during transient transfection of Chinese hamster ovary cell (CHO-K1) host [36]. Rationale design of RNA aptamers has been used to develop biosensors for fluorescent imaging of metabolites and signaling molecules in bacteria [71–74], but their use in eukaryotes has been stifled by an inability to express these molecules at sufficient levels to evoke function. Litke and Jaffrey [41] developed the Tornado expression system, for the rapid expression of highly stable circular RNA aptamers in vivo. Using their racRNA they demonstrated the ability to monitor intracellular S-adenosyl methionine (SAM) levels in mammalian cells by fluorescent imaging. 10

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Box 2. Cap-Dependent versus Cap-Independent Translation Initiation

Outstanding Questions

In 1979, Marilyn Kozak proposed a theory that eukaryotic translation initiates by ribosomes threading onto an mRNA like beads on a string [83]. Circularization of a message would abolish its ability to interact with a ribosome should this threading mechanism hold, and would explain the monocistronic character of eukaryotic mRNAs. It has since been shown that eukaryotic translation initiation occurs by two distinct mechanisms – either cap-dependent, or capindependent via an IRES [84]. The canonical model of eukaryotic translation initiation describes a process of assembly of the 48S ribosome complex at the 5′ untranslated region (5′-UTR) of an mRNA. The process is highly regulated, involving at least nine eukaryotic initiation factors before 48S recruitment and scanning [75]. This culminating in the formation of the elongation-competent 80S ribosome complex when a Met-tRNAi anticodon loop is paired to the P-site of a ribosome 40S subunit. The umbrella term ‘IRES-mediated translation initiation’ is less well defined owing to the lack of consensus RNA/protein binding motifs in viral and eukaryotic IRES variants. Viral IRES sequences are large, ranging from ~200 to 450 nt in length [75]. The first evidence of eukaryotic translation of circular RNA containing a viral IRES site was observed in the mid-1990s, but proved to be less efficient than a linear RNA control in a rabbit leukocyte lysate: the absence of an IRES in the circular RNA resulted in undetectable translated products [50]. A recent study by Yang and colleagues [15] demonstrated circular RNA translation in the absence of a viral or eukaryotic IRES. The unexpected translation of ‘negative control’ circular RNAs with no deliberate IRES sequences led them to identify a common 6 nt motif. The hexamer closely resembled the most abundant internal RNA modification [85], N6-methyladenosine (m6A) [86]. Replacing the IRES structure, and in the absence of a consensus Kozak sequence, a single m6A motif permitted translation of a reporter gene from a circular mRNA, whereas undetectable protein was produced in the m6A-free control. Furthermore, the discovery that m6A motifs are enriched in endogenous circular RNAs suggests a natural IRES-like function of this motif in eukaryotes [15]. The presence of a single m6A motif in the 5′-UTR of an mRNA can directly bind eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex and initiate translation in the absence of the cap-binding factor eIF4E [87]. Although the presence of an m6A motif in the 5′-UTR of an mRNA significantly improved translation versus a nonmethylated control in all cases, the position of the m6A relative to the start codon also has an effect [87].

This new method could open the floodgates for RNA aptamer technology. These racRNAs could be used in cell line development for heterologous protein production. The example of intracellular SAM detection demonstrated by Litke and Jaffrey indicates the potential for monitoring metabolite use and feed optimization by substitution of SAM by other metabolites [41].

Concluding Remarks and Future Perspectives It is still early days in the design and implementation of synthetic circular RNA molecules as either tools or therapies. Several methods have been recently developed to enable the generation of circular RNA in vitro with great efficiency. Expression of circular RNA at sufficient levels to modulate cellular function is also possible. The efficiency of in vivo RNA circularization remains a question (see Outstanding Questions). Measuring the relative expression efficiency of circular or linear variants of an RNA may be confounded by the differences in molecule stability. The observed levels of exogenous circular RNA could result from highly stable transcript accumulation with

Will circular RNAs be optimally delivered using lipid-based or exosome-mediated delivery? ActD treatment is commonly used to evaluate circular RNA stability, but its toxicity to cells limits long exposure studies. Could alternative methods of evaluating RNA stability aid in understanding the limits of circular RNA stability? How difficult would it be to translate circular mRNA therapeutics in a targeted cell-specific manner? Does this involve the use of multiple input regulatory molecules as well as endogenous regulators? To date all proteins generated from exogenous circular RNAs have been translated from a single open reading frame. For more complex molecules such as antibodies the best strategy for producing multiprotein complexes is not yet known: will it be a single circular mRNA with multiple open reading frames or multiple independent circles? A comparative study evaluating the performance of in vitro versus in vivo generated circular RNAs is lacking. For example, what are the challenges in scaling up the production of synthetic circular RNAs?

Box 3. Co- versus Post-Translational Cleavage of Polypeptide Chains The cleavage of polypeptide chains can occur either co- or post-translationally. Post-translational cleavage is the recognition and cleavage of a specific peptide motif post-synthesis. A limiting step in the production of many bioactive therapeutic proteins in mammalian cells is the proteolytic cleavage of precursor molecules. This was overcome by bridging the precursor and gene sequences with a furin cleavage motif [88]. Inclusion of a cleavage motif in between the start and end of a transgene open reading frame on a circular RNA would allow post-translational processing of the polyprotein strand. The second solution is cotranslational cleavage in the form of ribosome arrest peptides (RAPs). RAPs are present in a diverse range or organisms [89]. The best-known comes from foot and mouth disease virus. The ss positive-sense genome of this virus encodes a single polyprotein which is cotranslationally processed to produce multiple proteins. Two proteins ‘2A’ and ‘2B’ are processed by a process of ribosome ‘skipping’ [70] or ‘stop–carry on’ [90]. At the C-terminus of the 2A protein is a highly conserved proline–glycine–proline motif. Steric hindrance of the final proline sees it egress from the ribosome. The growing polypeptide chain is dissociated by release factors and translation continues from the proline to translate the downstream chain [91]. 2As work broadly in eukaryotes [70,92,93] but not in prokaryotes [94]. The ‘pause and carry-on’ manner of 2A-self cleavage peptides may introduce a rate-limiting step themselves, creating a polyribosome traffic jam.

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relatively low expression levels. Low-level detection of pre-circular RNA suggests this may not be the case [36], but this warrants further investigation. The stability of circular mRNA [39] and its ability to be continuously translated [36] have shown promise in protein coding systems. Cap-independent translation initiation is said to be less efficient in eukaryotes than cap-dependent initiation, which could be the result of a low intracellular concentration of relevant initiation factors [75]. Highly elevated levels of eIF4G are associated with many advanced breast cancers and lead to efficient IRES-mediated translation of p120-catenin (CTNND1) and VEGF mRNAs [76]. Translation of exogenous circular mRNA could perhaps be greatly enhanced by finding a host with highly abundant relevant cap-independent initiation factors or exogenous upregulation of such factors in an otherwise suitable host. In addition, evaluation of the continuous translation of circular RNA with co- or post-translational cleavage in cell-free expression systems could significantly improve yields from such systems. Another application of circular mRNAs to improve stable cell lines could be achieved by designing circular mRNAs with poor translational outputs, as seen in early evaluations of circular RNA translation [50]. Expression of a poorly translated selection marker gene could then be used for transgene amplification in the generation of stable cell lines. Engineering RNA-based gene therapies to express recombinant protein in a cell-specific manner could come from knowledge of distinct miRNA signatures between healthy and diseased states [77]. The use of miRNA response ‘elements’, ‘sequences’, or ‘sponges’ to regulate cell-specific replication of gene therapies was reported earlier [78]. This same strategy could be applied to circular RNA-based gene therapies. It is known that circular RNA can be turned over in an RNA-like manner through perfect complementarity to endogenous miRNAs [54]. Relying on a single miRNA regulator is likely to be too unspecific, given that miRNA signatures are highly dynamic in many cell types. Additional elements to control translation would need to be included. In conclusion, versatility coupled with the desirable stability and reduced immunogenicity of circular RNA molecules makes this RNA species a promising candidate for future cell engineering strategies and gene therapy applications. There has not yet been much in the way of cross-pollination between fields of the ideas discussed in this review. All the methods and applications reviewed are broadly applicable in both cell engineering for recombinant protein production and as therapeutics. Some outstanding questions remain regarding the best means of circular RNA expression or delivery for each of these applications. What we do know is that these molecules are uniquely stable, nonimmunogenic, and customizable with diverse functions and applications. Acknowledgments This work was conducted with financial support of Science Foundation Ireland (SFI; grants 13/IA/1963 and 13/IA/1841) and the European Regional Development Fund (ERDF; grant 12/RC/2275_P2).

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