Methods of modulating circular RNA translation by RNA-binding proteins

Abstract

Provided herein are circular RNAs (circRNAs) and linear mRNAs bound to one or more RNA-binding proteins (RBPs), polynucleotides, vectors, and host cells comprising circRNAs or linear mRNAs linked to RBPs, and methods of use thereof.

Description

METHODS OF MODULATING CIRCULAR RNA TRANSLATION BY RNA- BINDING PROTEINSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No.: 63 / 702,063, filed October 1, 2024, and U.S. Provisional Patent Application No.: 63 / 724,872, filed November 25, 2024, the content of each of which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under MH126719, HG011864, and HG004659 awarded by National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification and attached Appendices are herein incorporated by reference to the same extent as if each individual publication, patent, patent application or appendix, was specifically and individually indicated to be incorporated by reference.BACKGROUND

[0004] Circular RNAs (circRNAs) are covalently closed RNA species that are predominantly generated in mammalian cells through a process called backsplicing in the nucleus, where a downstream 3’ splice site is joined to an upstream 5’ splice site to form a loop1,2. CircRNAs are exported to the cytoplasm, where they can function as microRNA sponges, protein decoys, and templates for translation3,4. CircRNAs can accumulate or are degraded via specific pathways including microRNA-5, RNase P-6mediated degradation of subsets of circRNAs and global RNase L-7mediated degradation upon virus infection, with additional mechanisms yet to be discovered8. Contemporaneously, the use of circRNAs as vectors for RNA vaccines and therapeutics has garnered significant attention due to their increased stability and reduced immunogenicity compared to mRNAs9,10. Translation efficiency remains a significant challenge, as current antigen-expressing RNA constructs often exhibitsuboptimal protein output11,12. Enhancing translation efficiency could reduce the required delivery dose, thereby lowering manufacturing costs which are estimated to account for up to 80% of total mRNA vaccine production expenses13.SUMMARY OF THE DISCLOSURE

[0005] Circular RNAs (circRNAs), formed by back-splicing in mammalian cells, are increasingly recognized for their roles in various biological processes and their potential in RNA therapeutics. However, the RNA-binding proteins (RBPs) that regulate circRNAs throughout their lifecycle remain largely unknown. Here, to the best of Applicant’s knowledge, this disclosure presents the most comprehensive evaluation to date, assessing 730 RBPs for their potential to regulate circRNAs. Using both luciferase-based and qPCR assays, Applicant uncovered a range of potential regulatory mechanisms.

[0006] While the functions of circRNAs in various biological contexts are being increasingly explored, the identities and roles of RBPs in circRNA biogenesis and metabolism remain largely unaddressed. This critical gap not only limits the understanding of how circRNAs’ functions are modulated but also precludes us from generating highly efficacious vectors that could be used to treat or prevent human disease. To date, only a handful of RBPs, for example QKI, FUS, MBNL1, SFPQ, etc., have been shown to bind the intronic regions flanking exons that ultimately generates circRNAs15 20. However, given the low evolutionary sequence conservation of non-coding intronic sequences across species, the existence of a conserved / ra / z.s-regulatory mechanism governing circRNA biogenesis remains uncertain.

[0007] While previous studies have explored cv.s-regulatory RNA elements that may promote circRNA or linear RNA translation, the underlying mechanisms remain unclear. One advantage of the system disclosed herein is that the system has a pre-defined mechanism of action by tethering an RNA Binding Protein (RBP) that is known to promote circRNA or linear RNA translation. In one aspect, the system promotes circRNA translation.

[0008] In one aspect, Applicant demonstrated that recruitment of specific RNA binding protein (RBP) by unmodified or modified RNA aptamers onto endogenous or exogenously delivered synthetic circular mRNAs enhances their eventual translation. Applicant therefore provides herein an RNA bound (linear or circRNA) to one or more RBPs. In one aspect, the RNA is linear. In one aspect the RNA is circular. Also provided are polynucleotides encodingthe circRNA or linear RNA bound to one more RBPs, as well as vectors, host cells and compositions comprising one or more of the same.

[0009] Specifically, provided herein are mRNA (circular and / or linear) translation enhancing systems, wherein a gene of interest is packaged into an RNA vector and polynucleotides encoding the circRNA or linear mRNA. In one aspect, the polynucleotide encoding the gene of interest is DNA or RNA. In one embodiment, the system may have two constructs applied consecutively or simultaneously, wherein one construct comprises the RNA encoding the gene of interest and the other construct comprises a polynucleotide encoding the RBP. In another embodiment, the system may have one construct, wherein the construct comprises the a polynucleotide encoding the RNA of interest, and wherein the construct comprises an aptamer which can recruit an endogenous RBP. The constructs may further comprise regulatory sequences necessary for the expression and / or replication of the construct or the gene of interest. Examples of such are known in the art and described herein.

[0010] As such in one embodiment, Applicant provides herein a circRNA bound to one or more RBPs as well as polynucleotides encoding the circRNA bound to the one or more RBPs. The circRNA or polynucleotide encoding such can be directly or indirectly bound to the one or more RBPs. In one aspect, the circRNA comprises at least one RNA hairpin, aptamer, or AU-rich sequence capable of recruiting an endogenous or exogenous RBP that enhances translation. Non-limiting examples of “exogenously expressed RBP” includes exogenously expressed LARP4B that can enhance the translation of the circRNA. LARP4B is shown to interact with endogenous EIF4G2 which can enhance circRNA translation.

[0011] In another embodiment, Applicant provides a linear RNA bound to one or more RBPs as well as polynucleotides encoding the linear RNA directly or indirectly bound to the one or more RBPs. Applicant also provides herein a vector comprising: a) a circRNA comprising one or more RNA hairpins / aptamers; and b) an RBP linked to the circRNA at the one or more RNA hairpins, or recruited by an AU-rich aptamer encoded in the circRNA or linear RNA to recruit endogenous RBPs, e.g. LARP4B. As such, Applicant also provides herein a linear mRNA bound to one or more RBPs. Applicant also provides herein a vector comprising: a) a linear mRNA comprising one or more RNA hairpins / aptamers; and b) an RBP linked to the linear mRNA at the one or more RNA hairpins, and optionally wherein the RPB is recruited via an aptamer or an AU-rich sequence.

[0012] As used herein, in one embodiment throughout, the term “circRNA” includes circular and linear mRNA. In another aspects, the circRNA or linear RNA encodes a gene of interest.

[0013] Applicant also provides herein a vector comprising circRNA or linear RNA or polynucleotides encoding the same, wherein the circRNA or linear RNA comprise one or more hairpins / aptamers, wherein the aptamer is specific for one or more endogenous RBPs in a cell.

[0014] In some aspects, provided herein are host cells and / or compositions comprising the circRNA, linear RNA, polynucleotides and / or vectors. In some aspects, provided herein are methods of use of the circRNA, linear RNA, polynucleotides, host cells and / or vectors. In some aspects the circRNA, linear RNA, and / or vectors regulate circRNA translation and / or biogenesis in cells or in subjects in need.

[0015] In some aspects, the circRNA, linear RNA, or vector comprises at least one RBP selected from those listed in Table 2, 3, 7, or 8, or a functional fragment or variant of each. In some aspects the circRNA, linear RNA, or vector comprise at least one RBP selected from LARP4, LARP4B, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, or DDX52 or a functional fragment or variant of each. In a further aspect the RBP comprising LARP4B and / or ANXA2.

[0016] In some aspects, the endogenous RBP specific for the aptamer is selected from at least one RBP listed in from Tables 2, 3, 7, or 8 or a functional fragment or variant of each. In some aspects, the endogenous RBP specific for the aptamer is selected from LARP4, LARP4B, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, or DDX52 or a functional fragment or variant of each. In a further aspect the RBP comprising LARP4B and / or ANXA2.

[0017] Also provided herein are methods to regulate endogenous circRNAs or linear RNAs in a cell, the method comprising contacting the cell with one or more RBPs and / or vector comprising a polynucleotide encoding the one or more RBPs as described herein. In some aspects, the vector comprises a circRNA or a linear RNA or a polynucleotide encoding the circRNA or linear RNA.

[0018] As exemplified herein, at least 296 RBPs were identified that encompass diverse components of circRNA metabolism including biogenesis, nuclear export, translation anddegradation. Without being bound by theory, it is shown that LARP4B universally enhances circRNA translation, while ANXA2 enhances circRNA translation in a cell type-specific manner, thus providing compositions and methods to advance circRNA therapeutic vector design. Moreover, RBM15 was identified as a multifaceted regulator of circRNAs, promoting their biogenesis via interactions with SRSF proteins and retaining circRNAs in the nucleus through RNA-dependent interactions with IGF2BP1. Notably, RBM15 binds directly to exonic regions near back-splicing junctions and is a conserved RBP regulator of circRNA biogenesis. These insights advance the application of / ra / / .s-regulators in circRNA biogenesis and metabolism and offers new avenues for RNA therapeutic development.

[0019] The discovery of cell type-specific RBPs that promote circRNA translation that could be recruited offers the potential for finely tuned, cell-specific regulation of circRNA translation, paving the way for precision medicine applications.BRIEF DESCRIPTION OF THE FIGURES

[0020] FIGS. 1A - II: A circRNA luciferase reporter system for RBP tethering screen.(FIG. 1A) Schematic design of the circFirefly and circRenilla reporters. Upon backsplicing, split Firefly and Renilla fragments form intact luciferase genes and are translated driven by EMCV IRES. circFirefly reporter carries 6x MS2 sequence for RBP tethering through MCP fusions. Created with Biorender.com (FIG. IB) Luciferase assays of the positive control (mean ± s.d., n = 3). Luciferase signals were normalized to the Flag control. Error bars indicate standard deviations of biological replicates. *: p<0.05 (FIG. 1C) qPCR of circFirefly RNA in total RNA extracts (mean ± s.d., n = 4). Relative expressions were normalized to the Flag control. Error bars indicate standard deviations of biological replicates, “ns” means not significant by student t-test (FIG. ID) qPCR of circFirefly RNA in cytoplasmic RNA fractions (mean ± s.d., n = 4). Relative expressions were normalized to the Flag control.Error bars indicate standard deviations of biological replicates. *: p<0.05 (FIG. IE) Luciferase assays of the negative controls (mean ± s.d., n = 3). Luciferase signals were normalized to the Flag control. Error bars indicate standard deviations of biological replicates. (FIG. IF) Volcano plot of RBP tethering screen results. Cutoff is set at |log2FC| > 0.5 and FDR < 0.05. Red dots represent RBP candidates that pass both cutoffs. Blue and green dots represent RBPs that only pass one of the cutoffs. Gray dots represent RBPs thatdoes not pass either cutoff. (FIG. 1G) Heatmap summary of secondary screen results. K- means clustered heatmap shows the log2FC of luciferase assays in two cell lines and qPCR results from HEK293T cells. PS = primary screen, SS = secondary screen (FIGS. 1H - II) Bar plots showing examples of RBPs from different clusters (mean ± s.d., n = 3). The first two bars in each group present luciferase results from the secondary screen. The third bar in each group presents qPCR results from separate experiments with biological replicates. Fold changes are all normalized to the corresponding Flag controls. Error bars represent standard deviations of biological replicates.

[0021] FIGS. 2A - 2H: Identification of RBPs promoting circRNA translation. (FIG. 2A, FIG. 2B) Luciferase assay showing effects of RBP tethering on protein production of exogenously delivered circRNAs (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. *: p<0.05, **: p<0.01, ***: p<0.001. (FIG. 2C, FIG. 2D) Relative RNA level by qPCR showing the effects of RBP tethering on circRNA stability of exogenously delivered circRNAs (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. (FIG. 2E, FIG. 2F) Luciferase assay showing effects of RBP tethering on protein production of exogenously delivered PV IRES driven circRNAs (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. **: p<0.01, ***: p<0.001, ****: p<0.0001. (FIG. 2G) Luciferase assay showing effects of RBP tethering on protein production of exogenously delivered CrPV IRES driven circRNAs (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. **: p<0.01. (FIG. 2H) Western blot showing interaction between LARP4B with eIF3 A and eIF4G2 with V5 pulldown.

[0022] FIGS. 3A - 3F: LARP4B is associated with endogenous mRNA and circRNA translation. (FIG. 3A) Bar plot showing the region distribution of LARP4B eCLIP binding sites. (FIG. 3B) Selective top motifs of LARP4B binding sites showing poly A and polyU tracts. (FIG. 3C, FIG. 3D) Translation efficiency analysis showing difference between transcript with high binding and low binding of LARP4B on 3 ' UTR or CDS. (FIG. 3E) Enrichment of TransCirc evidences for reproducibly enriched circRNAs in LARP4B RIP. (FIG. 3F) Base percentage of LARP4B RIP enriched circRNAs showing high AU content.

[0023] FIGS. 4A - 41: RBM15 is a multifaceted regulator of circRNAs. (FIG. 4A)RBM15 positively regulate the expression of endogenous circRNAs. Overexpression and knockdown of 13 RBP candidates followed by circRNA sequencing identifies RBM15 as a positive regulator of many endogenous circRNA expression. (FIG. 4B) RBM15 tethering on circFirefly reporter. Luciferase assay and qPCR results of RBM15 tethering on circFirefly reporter showing an increase of circFirefly biogenesis and a decrease in luciferase signal (mean ± s.d., n = 3). Dots represent individual biological replicates. Error bars represent standard deviations of biological replicates. **: p<0.01; ***: p<0.001 (FIG. 4C) Fractionation qPCR of circFirefly upon RBM15 tethering showing strong nuclear retention of circFirefly RNA (mean ± s.d., n = 4). Dots represent individual biological replicates. Error bars represent standard deviations of biological replicates. (FIG. 4D, FIG. 4E) Endogenous circRNA expression level upon RBM15 overexpression and knockdown by qPCR (mean ± s.d., n = 3). Dots represent individual biological replicates. Error bars represent standard deviations of biological replicates. *: p<0.05; ***: p<0.001 (FIG. 4F) Fractionation qPCR of endogenous circRNAs upon RBM15 overexpression showing nuclear retention of them. Dots represent individual biological replicates. Error bars represent standard deviations of biological replicates. *: p<0.05 (FIG. 4G) Schematic view of RBM15 domains and truncation constructs. (FIG. 4H) Luciferase assay results showing the effect of truncated RBM15 tethering on circFirefly reporter (mean ± s.d., n = 3). Dots represent individual biological replicates. Error bars represent standard deviations of biological replicates. *: p<0.05; **: p<0.01 (FIG. 41) Relative expression by qPCR showing the effect of truncated RBM15 tethering on circFirefly and linear Firefly (linFirefly) RNA level (mean ± s.d., n = 3). Dots represent individual biological replicates. Error bars represent standard deviations of biological replicates.

[0024] FIGS. 5A - 51: RBM15 interactome illustrates molecular mechanisms ofRBM15’s functions. (FIG. 5A, FIG. 5B) Venn diagram showing minimal overlap between the alternative spliced genes and RBM15 positively regulated circRNAs’ parental genes in RBM15 overexpression and knockdown samples. (FIG. 5C) RIP-qPCR showing enriched binding of RBM15 to the precursor mRNA where circSPECCl(4) originated from. (FIG. 5D) Schematics showing primer design for qPCR of precursor mRNA, mature mRNA and mature circRNA from SPECC1. (FIG. 5E) Integrative Genomics Viewer (IGV) tracks showingRBM15’s binding sites on YTHDF3 exon 3. (FIG. 5F) Schematic design of YTHDF3 minigene and RBM15 binding site mutants. (FIG. 5G) Bar plot showing qPCR detection of ci cYTHDF3(3') expression level upon RBM15 binding site deletion. (FIG. 5H) Volcano plot of RBM15 AP-MS result without RNase treatment. Purple, orange, green and yellow dots represent proteins passing both log2FC (>2) and p value (<0.05) cutoffs. Green dots represent well known RBM15 interacting partners, WTAP and SETD1B. Orange dots represent SRSF splicing factors. Yellow dot represents IGF2BP1. (FIG. 51) Heatmap of luciferase assay result from the primary tethering screen showing increased luciferase signals when SRSF splicing factors are tethered.

[0025] FIGS. 6A - 6E: RBM15 retains IGF2BP1 in the nucleus. (FIG. 6A) Scatter plot comparing RBM15 AP-MS data with and without RNase treatment. Dash lines represent the log2FC cutoff. RBM15 interacting partners that are only significant without RNase treatment are highlighted in purple. IGF2BP1 is highlighted in yellow. (FIG. 6B) RBM15 western blot showing successful pulldown of RBM15 by V5 tag or anti-RBM15 antibody. (FIG. 6C) IGF2BP1 western blot showing the pulldown of IGF2BP1 from RBM15 co-IP experiments. (FIG. 6D) Immunofluorescence showing nuclear retention of IGF2BP1 protein upon RBM15 overexpression. Green: IGF2BP1, Blue: DAPI, Magenta: RBM15-V5. Scale bar: 10pm. (FIG. 6E) Quantification of immunofluorescence images (mean ± s.d., n = 10). Error bars represent standard deviations of 10 imaging fields from each group. **: p<0.01.

[0026] FIGS. 7 A - 7F: RBM15 is a conserved circRNA regulator. (FIG. 7 A) Metadensity plot of RBM15 eCLIP binding on circRNAs showing enriched binding of RBM15 near the BSJs of the circRNAs that it positively regulates. (FIG. 7B) Integrative Genomics Viewer (IGV) tracks showing RBM15 binding on RTN4 exon2 and 3. (FIG. 7C) Comparison of the conserveness between RBM15 positively regulated circRNAs and all circRNAs expression in HEK293T cells. The line represents the median of the MCS from each group. **** Welch’s corrected p value < 0.0001 (FIG. 7D; FIG. 7E) Relative expression of mouse circRNAs upon RBM15 overexpression and knockdown in N2A cells (mean ± s.d., n = 3). Error bars represent standard deviations of 10 imaging fields from each group. ***: p<0.001; **: p<0.01 (FIG. 7F) Scatter plot showing the mean CPM of three biological replicates in RBM15 overexpression (OE) samples (y axis) vs Flag control samples (x axis). Each dot represents one circRNA.

[0027] FIGS. 8A - 8P: Validation of circRNA luciferase reporter system and tethering screen candidates. (FIG. 8A) Schematic view of divergent and convergent primer design. (FIG. 8B) Bar plot showing the RNA level comparison of circFirefly, circRenilla, linFirefly and linRenilla before and after RNase R treatment by qPCR (FIG. 8C) Sanger sequencing of the qPCR product detected successful backsplicing junctions. (FIG. 8D) Schematic view of truncated circFirefly and circRenilla mutants generated. The circularization deficient mutants are used to confirm that the upstream fragments do not generate luciferase signals. Created with Biorender.com (FIG. 8E, FIG. 8F) Bar plots showing raw Firefly and Renilla luciferase signals of circFirefly and circRenilla reporters and their mutants in HeLa and HEK293T cells (mean ± s.d., n = 2). Error bars represent standard deviations of biological replicates. (FIG. 8G) Immunofluorescence staining of YTHDF2-MCP and METTL3-MCP RBP fusions. Green: V5 stained fusion proteins. (FIG. 8H) Stacked bar plot showing the performance of cell fractionation (mean ± s.d., n = 3). Samples are normalized by RNA loading of 500ng. Error bars represent standard deviations of biological replicates. (FIG. 81) Gene Ontology analysis of positive effectors. Background list is set to all RBPs in the tethering library. Top 10 GO terms in Biological Processes are shown. (FIG. 8 J) Gene Ontology analysis of negative effectors. Background list is set to all RBPs in the tethering library. Top 10 GO terms in Biological Processes are shown. (FIG. 8K) Proposed mechanisms of different clusters. Cluster 1 is hypothesized to inhibit biogenesis or promote degradation. Cluster 2 is hypothesized to play multimodal functions, for example both promoting biogenesis and inhibiting nuclear export. Cluster 3 is hypothesized to promote biogenesis, stability and / or translation. Cluster 4 is hypothesized to promote nuclear export or translation. Created with Biorender.com (FIG. 8L) Venn diagram showing overlap between our tethering screen and a recent CRISPR screen to identify RBP regulators of circRNA biogenesis. (FIG. 8M) Scatter plot comparison between circRNA tethering screen with mRNA tethering screen. Solid line presents the linear regression. Top ten upregulators in circRNA screen are highlighted in red and top ten downregulators in circRNA screen are highlighted in blue. (FIG. 8N) Schematic design of exogenous circRNA luciferase reporter system. exo-circFirefly and exo-circRenilla circRNAs can be produced through in vitro transcription, circularization with the permuted intron and purification through RNase R digestion. RBP-MCP mRNAs can be made by in vitro transcription with capping and tailing. All three components are delivered together toassess the effect of an RBP on exogenously delivered circRNAs. Created with Biorender.com (FIG. 80) Tapestation result of exo-circFirefly and exo-circRenilla RNA reporters showing the purity of circRNAs after RNase R digestion. (FIG. 8P) Luciferase assay of tethering CN0T7 and mutant CN0T7 D40A and DCP1 A to the exo-circFirefly reporter system.

[0028] FIGS. 9A - 9D: Identification of translation promoting RBPs. (FIG. 9A) Immunofluorescence of RBP-MCP fusion proteins translated from the in vitro transcribed mRNAs. Green: V5 stained RBP-MCP fusion proteins; Blue: DAPI (FIG. 9B) Raw luciferase signal of different IRES driving circFirefly reporter (by backsplicing in the cells). (FIG. 9C) Raw luciferase signal of different IRES driving exo-circFirefly or exo-circRenilla reporters (by in vitro circularization with permuted introns). (FIG. 9D) Western blot showing not interaction between LARP4B with a number of other elFs proteins tested.

[0029] FIGS. 10A - 10E: LARP4B eCLIP and RIP-circRNA-seq. (FIG. 10A) Violin plot showing LARP4B eCLIP binding level difference between 3 ' UTR and CDS. Black dash lines indicate the media. ****: p<0.0001. (FIG. 10B) t-SNE plot showing LARP4B eCLIP clustering close to 3 ' UTR binding RBPs from the ENCODE project. (FIG. IOC) Western blot showing successful pulldown of LARP4B for the RIP experiment. (FIG. 10D) Bar plot showing factions of BSJ reads in input and LARP4B IP samples. (FIG. 10E) Venn diagram showing the overlap between three biological replicates of LARP4B RIP experiments. Purple labels the number of reproducible circRNAs been identified.

[0030] FIGS. 11A - 11G: RBM15 is a multifaceted regulator of circRNA. (FIG. 11A, FIG. 11B) Box plot of RBM15 read counts showing successful overexpression and knockdown. (FIG. 11C, FIG. HD) Volcano plot showing mRNA expression level changes upon RBM15 overexpression and knockdown. Red dots indicate the linear cognate of RBM15 positively regulated circRNAs. (FIG. 11E, FIG. 11F) Bar plots showing raw luciferase signal of the circFirefly and circRenilla reporters upon RBM15 tethering assay (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. (FIG. 11G) Bar plot showing relative expression of RBM15 upon siRNA knockdown of RBM15 (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. *: p<0.05

[0031] FIGS. 12A - 12H: RBM15 directly regulate circRNA biogenesis. (FIG. 12A) Bar plot showing relative expression of endogenous circRNAs upon RBM15 overexpression in METTL3 knockdown stable cell lines (mean ± s.d., n = 2). Error bars represent standard deviations of biological replicates. (FIG. 12B) Bar plot showing relative expression of METTL3 mMETTL3 knockdown stable cell lines (mean ± s.d., n = 2). Error bars represent standard deviations of biological replicates. (FIG. 12C) RIP-qPCR showing enriched binding of RBM15 to the precursor mRNA where circA7V- / (2,3) originated from. (FIG. 12D) Schematics showing primer design for qPCR of precursor mRNA, mature mRNA and mature circRNA from RTN4. (FIG. 12E) Bar plot showing similar level of \\nYTHI)F3 expression of the YTHDF3 minigene and mutants. (FIG. 12F) Bar plot showing luciferase results of SRSF splicing factors from secondary screen in HEK293T and HeLa cells (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. (FIG. 12G) Bar plot showing qPCR results of circFirefly upon SRSF splicing factors tethering from secondary screen in HEK293T cells (mean ± s.d., n = 4). Error bars represent standard deviations of biological replicates. (FIG. 12H) Heatmap showing enriched eCLIP co-binding of RBM15 and SRSF1 on RBM15 positively regulated circRNAs.

[0032] FIGS. 13A - 13B: RBM15’s interaction with IGF2BP1 is RNA-dependent. (FIG. 13A) Volcano plot of RBM15 AP-MS result with RNase treatment. Purple, orange and green dots represent proteins passing both log2FC (>2) and p value (<0.05) cutoffs. Green dots represent well known RBM15 interacting partners, WTAP and SETD1B. Orange dots represent SRSF splicing factors. (FIG. 13B) Volcano plot showing the overlap between RBM15 positively regulated circRNAs and all ENCODE eCLIP datasets. Each dot represents one eCLIP experiment. RBM15 is highlighted in orange and IGF2BP1 and IGF2BP2 proteins are highlighted in blue.

[0033] FIGS. 14A - 14D: RBM15 is a conserved regulator of circRNA biogenesis. (FIG. 14A) Bar plot showing region distribution of RBM15 eCLIP binding sites. (FIG. 14B) Bar plot showing relative expression of Rbml5 in N2A cells with Rbml5 knockdown by siRNA (mean ± s.d., n = 3). Error bars represent standard deviations of biological replicates. **: p<0.01 (FIG. 14C) Protein sequence alignment between human and mouse RBM15 proteins. Red color highlights the mismatches. (FIG. 14D) Box plot of circRNA expression in N2Acells showing conserved circRNAs positively regulated by RBM15. The mid line shows the mean of each group. Error bars represent standard deviations of all conserved RBM15 positively regulated circRNAs. **: p<0.01.

[0034] FIGS. 15A-G: Plasmid map (FIG. 15A) and sequence of the pTwist circren plasmid (FIGS. 15B-15G)

[0035] FIGS. 16A-H: Plasmid map (FIG. 16A) and sequence of the pTwist_circluc_MS2 plasmid (FIGS. 16B-16H)

[0036] FIG. 17: Results of exocircFirefly RBP tethering screen in HEK293T cells.Volcano plot showing the results of the screen. Red dots represent the significant RBPs passing the threshold of p<0.05 and log2(Fold Change)>0.5.

[0037] FIG. 18 Results of exocircFirefly RBP tethering screen in HeLa cells. Volcano plot showing the results of the screen. Red dots represent the significant RBPs passing the threshold of p<0.05 and log2(Fold Change)>0.5.DETAILED DESCRIPTION

[0038] Definitions

[0039] Throughout this application various technical and patent publications are referenced, the disclosure of which are incorporated herein to more fully describe the state of the art to which this disclosure pertains. Technical reference may be identified by an Arabic numeral wherein the complete bibliographic details of the publication are provided in the Reference section preceding the claims. The disclosures of these technical publications also are referenced herein to more fully describe the state of the art.

[0040] As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0041] As used herein, the term “comprising” is intended to mean that the compositions or methods include the recited steps or elements, but do not exclude others. “Consisting essentially of’ shall mean rendering the claims open only for the inclusion of steps or elements, which do not materially affect the basic and novel characteristics of the claimed compositions and methods. “Consisting of’ shall mean excluding any element or step notspecified in the claim. Embodiments defined by each of these transition terms are within the scope of this disclosure. For example, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the present disclosure.

[0042] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which can vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1 %.

[0043] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals.

[0044] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method, cell or composition described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. A mammal can be a pregnant female. In some embodiments a subject is a human.

[0045] “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is thenucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human.

[0046] “Prokaryotic cells” usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome.Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0047] A “composition” typically intends a combination of the active agent, and a naturally- occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches,and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0048] The compositions used in accordance with the disclosure, including cells, treatments, therapies, agents, drugs and pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0049] As used herein, the terms “nucleic acid sequence,” “oligonucleotide,” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, circRNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, synthetic, recombinantly produced, chemically or biochemically modified, nonnatural, or derivatized nucleotide bases. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any aspect of this technology that is a polynucleotideencompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0050] The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is the to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0051] As used herein, the term “isolated cell” generally refers to a cell that is substantially separated from other cells of a tissue. The term cell and isolated cell includes prokaryotic and eukaryotic cells. In one aspect the cell is a mammalian cell selected from a murine cell, a bovine cell, an equine cell, a canine cell, a feline cell, or a human cell. The cell can be a cultured cell that is reproduced in the laboratory or obtained from commercial sources. In some respects, the term “cell” intends a cell in vivo in a subject. In one aspect, the cell is a HeLa cell or a HEK293T cell that are commercially available from vendors such as the American Type Culture Collection (ATCC) (https: / / www.atcc.org / ).

[0052] As used herein, the term “vector” refers to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a viral vector, a cosmid, a phage, a BAC, a YAC, etc, and optionally where the viral vector is selected from a baculovirus, a retrovirus or an adenovirus. It can also be referred to as an “expression cassette.”

[0053] A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. In some embodiments, plasmid vectors can be prepared from commercially available vectors, examples of such are provided herein. In other embodiments, viral vectors can be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacture proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors,such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. Further details as to modem methods of vectors for use in gene transfer can be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.).

[0054] An “effective amount” or “efficacious amount” refers to the amount of an agent or combined amounts of two or more agents, that, when used for its intended purpose as disclosed herein or alternatively when administered for the treatment of a mammal or other subject, is sufficient to cause such treatment for the disease. The “effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated or the purpose of use

[0055] As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and can be used interchangeably with the term “tumor.”

[0056] The tumor is not limited and can be any kind of cancer, e.g., solid or blood cancer, e.g., carcinoma or sarcoma. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’ s sarcoma, multiple idiopathic hemorrhagic sarcoma);endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’ s adenocarinoma); Ewing’ s sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T- cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom’ s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T- cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms’ tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemicmastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (c.g, polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g.,bone cancer); ovarian cancer (c.g, cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (c.g, pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’ s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (c.g, squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (c.g, seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’ s disease of the vulva).

[0057] A “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. In some embodiments, a solid tumor comprises bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.

[0058] As used herein, the term “hematologic malignancy” refers to cancers with hematopoietic origin. In some instances, the hematologic malignancy is a B-cell malignancy.In some instances, the hematologic malignancy is a lymphoma, optionally a B-cell lymphoma. Exemplary hematologic malignancies include, but are not limited to, Diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantel cell lymphoma (MCL), marginal zone lymphomas, Burkitt lymphoma, Waldenstrom macroglobulinemia, hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma.

[0059] As used herein, the term “detectable marker” or “detectable label” refers to at least one marker capable of directly or indirectly, producing a detectable signal. A non-exhaustive list of markers includes enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, P-galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent, luminescent dyes, groups with electron density detected by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance, detectable groups, for example whose molecules are of sufficient size to induce detectable modifications in their physical and / or chemical properties, such detection can be accomplished by optical methods such as diffraction, surface plasmon resonance, surface variation , the contact angle change or physical methods such as atomic force spectroscopy, tunnel effect, or radioactive molecules such as32P,35S or1251. Also provided herein are detectably labeled circRNA, linear RNA, polynucleotides, vectors, and host cells.

[0060] As used herein, the term “purification marker” or “reporter protein” refer to at least one marker useful for purification or identification. A non-exhaustive list of purification markers includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten. Also provided herein are circRNA, linear RNA, polynucleotides, vectors, and host cells that are linked to a purification marker or a reporter protein.

[0061] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotideis derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of a compound, or the circRNA, linear RNA, polynucleotide, vector, cell, composition or plurality of each, as described herein.

[0062] As used herein, “homology” or “identical”, percent “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding the RBP described herein). Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds.1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. The terms “homology” or “identical,” percent “identity” or “similarity” also refers to, or can be applied to, the complement of a test sequence. The terms also include sequences that have deletions and / or additions, as well asthose that have substitutions. As described herein, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein.

[0063] The phrase “first line” or “second line” or “third line” refers to the order of treatment received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. The National Cancer Institute defines first line therapy as “the first treatment for a disease or condition. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008. Typically, a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.

[0064] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, polypeptide, or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any of the above also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and / or exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0065] The phrase “equivalent polypeptide” or “equivalent peptide fragment” refers to protein, polynucleotide, or peptide fragment encoded by a polynucleotide that hybridizes to a polynucleotide encoding the exemplified polypeptide or its complement of the polynucleotide encoding the exemplified polypeptide, under high stringency and / or which exhibit similar biological activity in vivo, e.g., approximately 100%, or alternatively, over 90% or alternatively over 85% or alternatively over 70%, as compared to the standard or control biological activity. Additional embodiments within the scope of this disclosure are identified by having more than 60%, or alternatively, more than 65%, or alternatively, more than 70%, or alternatively, more than 75%, or alternatively, more than 80%, or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98% or 99% sequence homology. Percentage homology can be determined by sequence comparison using programs such as BLAST run under appropriate conditions. In one aspect, the program is run under default parameters.

[0066] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0067] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can comprise two strands forming aduplex structure, three or more strands forming a multi -stranded complex, a single selfhybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0068] Examples of stringent hybridization conditions include: incubation temperatures of about 25 °C to about 37 °C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40 °C to about 50 °C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. A high stringency hybridization refers to a condition in which hybridization of an oligonucleotide to a target sequence comprises no mismatches (or perfect complementarity). Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O. lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[0069] The term “isolated” as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. Theterm “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.

[0070] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. In another aspect, the subunit can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which can comprise a protein’s or peptide’s sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0071] As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein, biological complexes or other active compound is one that is isolated in whole or in part from proteins or other contaminants. Generally, substantially purified peptides, proteins, biological complexes, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, biological complex or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical formulation for therapeutic administration. More typically, the peptide, protein, biological complex or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation can be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.

[0072] As used herein, the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. In one aspect, the term includes proteins that are chemically manufactured without the use of a host cell system.

[0073] The terms “fusion” or “chimeric” and grammatical variations thereof, when used in reference to a molecule, means that a portions or part of the molecule contains a different entity distinct (heterologous) from the molecule as they do not typically exist together in nature. That is, for example, one portion of the fusion or chimera includes or consists of a portion that does not exist together in nature, and is structurally distinct.

[0074] As used herein, “direct binding” refers to direct interaction with RNA with an interacting interface between the two molecules. For example, MCP directly binds MS2 sequence.

[0075] “Indirect binding” refers to interaction between a protein and a RNA mediated by a third molecule. For example, an RBP tethered to MCP does not directly interact with MS2 sequence but could forcibly binds to MS2 sequencing mediated by MCP-MS2 interaction.

[0076] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor. In one aspect, treatment excludes prophylaxis.

[0077] As used herein, the term "overexpress" with respect to a cell, a tissue, or an organ expresses a protein to an amount that is greater than the amount that is produced in a control cell, a control issue, or an organ. A protein that is overexpressed can be endogenous to the host cell or exogenous to the host cell.

[0078] A regulatory sequence intends a sequence that is necessary or preferred for the in vitro or in vivo expression or replication of a polynucleotide or vector, e.g., a promoter, an enhancer, or an ITR.

[0079] As used herein, the term “enhancer”, denotes sequence elements that augment, improve or ameliorate transcription of a nucleic acid sequence irrespective of its location and orientation in relation to the nucleic acid sequence to be expressed. An enhancer can enhance transcription from a single promoter or simultaneously from more than one promoter. As long as this functionality of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of wild-type activity, that is, activity of a full-length sequence), any truncated, mutated or otherwise modified variants of a wild-type enhancer sequence are also within the above definition.

[0080] The term “promoter” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters can be constitutive, inducible, repressible, or tissue-specific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It can contain genetic elements at which regulatory proteins and molecules can bind such as RNA polymerase and other transcription factors.

[0081] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0082] The term “introduce” as applied to methods of producing modified cells such as chimeric antigen receptor cells refers to the process whereby a foreign (i.e. extrinsic or extracellular) agent is introduced into a host cell thereby producing a cell comprising the foreign agent. Methods of introducing nucleic acids include but are not limited to transduction, retroviral gene transfer, transfection, electroporation, transformation, viral infection, and other recombinant DNA techniques known in the art. In some embodiments, transduction is done via a vector (e.g., a viral vector). In some embodiments, transfection isdone via a chemical carrier, DNA / liposome complex, or micelle (e.g., Lipofectamine (Invitrogen)). In some embodiments, viral infection is done via infecting the cells with a viral particle comprising the polynucleotide of interest (e.g., AAV). In some embodiments, introduction further comprises CRISPR mediated gene editing or Transcription activator-like effector nuclease (TALEN) mediated gene editing. Methods of introducing non-nucleic acid foreign agents (e.g., soluble factors, cytokines, proteins, peptides, enzymes, growth factors, signaling molecules, small molecule inhibitors) include but are not limited to culturing the cells in the presence of the foreign agent, contacting the cells with the agent, contacting the cells with a composition comprising the agent and an excipient, and contacting the cells with vesicles or viral particles comprising the agent.

[0083] The term “culturing” refers to growing cells in a culture medium under conditions that favor expansion and proliferation of the cell. The term “culture medium” or “medium” is recognized in the art and refers generally to any substance or preparation used for the cultivation of living cells. The term “medium”, as used in reference to a cell culture, includes the components of the environment surrounding the cells. Media can be solid, liquid, gaseous or a mixture of phases and materials. Media include liquid growth media as well as liquid media that do not sustain cell growth. Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices. Exemplary gaseous media include the gaseous phase to which cells growing on a petri dish or other solid or semisolid support are exposed. The term “medium” also refers to material that is intended for use in a cell culture, even if it has not yet been contacted with cells. In other words, a nutrient rich liquid prepared for culture is a medium. Similarly, a powder mixture that when mixed with water or other liquid becomes suitable for cell culture can be termed a “powdered medium.” “Defined medium” refers to media that are made of chemically defined (usually purified) components. “Defined media” do not contain poorly characterized biological extracts such as yeast extract and beef broth. “Rich medium” includes media that are designed to support growth of most or all viable forms of a particular species. Rich media often include complex biological extracts. A “medium suitable for growth of a high-density culture” is any medium that allows a cell culture to reach an OD600 of 3 or greater when other conditions (such as temperature and oxygen transfer rate) permit such growth. The term “basal medium” refers to a medium which promotes the growth of many types of microorganisms which do not require anyspecial nutrient supplements. Most basal media generally comprise of four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. A basal medium generally serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. In one aspect, the growth medium can be a complex medium with the necessary growth factors to support the growth and expansion of the cells of the disclosure while maintaining their self-renewal capability. Examples of basal media include, but are not limited to, Eagles Basal Medium, Minimum Essential Medium, Dulbecco’s Modified Eagle’s Medium, Medium 199, Nutrient Mixtures Ham’s F-10 and Ham’s F-12, McCoy’s 5A, Dulbecco’s MEM / F-12, RPMI 1640, and Iscove’s Modified Dulbecco’s Medium (IMDM).

[0084] “Cryoprotectants” are known in the art and include without limitation, e.g., sucrose, trehalose, and glycerol. A cryoprotectant exhibiting low toxicity in biological systems is generally used.

[0085] A non-coding RNA (ncRNA) is an RNA molecule that is not translated into a protein. Non-limiting examples of non-coding RNA include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), microRNAs, siRNAs etc.

[0086] Circular RNA (circRNA) intends a type of single-stranded RN A which, unlike linear RNA, forms a covalently closed continuous loop. In circular RNA, the 3’ and 5’ ends normally present in an RNA molecule are joined together. This feature confers numerous properties to circular RNA, many of which have only recently been identified. Studies have identified three main types of circRNA: ecircRN, circular intronic RNA (ciRNA), and exonintron circRNA (EIciRNA). As used herein and unless as specifically recited, the term circRNA includes ecircRN, circular intronic RNA (ciRNA), and exon-intron circRNA (EIciRNA).

[0087] “Linear RNA” intends a type of single-stranded RNA which is not covalently closed. In this disclosure, “Linear RNA” refers to linear mRNA that possesses 5’ cap and poly A tail and encodes proteins. Linear mRNA also includes in vitro synthesized mRNAs that often incorporates modified bases.

[0088] The term “RBP tethering,” “RBP tether,” or “tethered” as provided herein refers to a system comprised of an RNA and a RBP. The RNA (e.g. a circRNA or linear RNA) containsan RNA structural element that is recognized by an exogeneous RNA-binding moiety which is fused to the RBP. The RNA-binding moiety binds to the RNA structural element, thereby recruiting the RBP to the RNA. The RNA structural element in the RBP tethering system is an aptamer. An aptamer is a sequence that bind specific molecules. In one aspect, the RNA structural element is an RNA stem-loop structure (“hairpin”). The hairpin is type of aptamer structures. The number of hairpins / aptamers can be modified, and multiple RNA-binding moieties can be recruited to a hairpin. Common tethering systems include, but are not limited to, the hairpins and coat proteins derived from bacteriophages MS2, X, PP7, QP, GA, the bovine immunodeficiency virus, human U1 small nuclear ribonucleoprotein-specific protein U1A, and the iron response protein tethering system. Further details as to tethering can be found, for example, in Bos, T. J., et al. Tethered Function Assays as Tools to Elucidate the Molecular Roles of RNA-Binding Proteins. Advances in experimental medicine and biology, 907, 61-88 (2016). https: / / doi.org / 10.1007 / 978-3-319-29073-7_3 and Luo, E. C. et al. Large- scale tethered function assays identify factors that regulate mRNA stability and translation. Nat. Struct. Mol. Biol. 27, 989 (2020).

[0089] Modes of Carrying Out the Disclosure

[0090] Circular RNAs (circRNAs) are emerging as important regulators in mammalian cells and promising substrates for RNA therapeutics, yet the / ra / z.s-acting factors governing their metabolism remain poorly defined. Here, Applicant systematically evaluated 730 RNA- binding proteins (RBPs) using a tethered reporter assay and identify 281 RBPs that significantly modulate circRNA activity. Follow-up assays implicated these RBPs across key stages of the circRNA lifecycle including biogenesis, nuclear export, degradation, and translation. Applicant highlights LARP4B as a potent enhancer of endogenous and exogenous circRNA translation likely through recruitment of eIF3 A and eIF4G2. Applicant also uncovered a role for RBM15 in circRNA biogenesis and nuclear retention, supported by its binding near back-splice junctions and evolutionarily conserved function. Together, this disclosure provides compositions and methods for circRNA trans-regulation and practical entry points for therapeutic development.

[0091] CircRNA, and Vectors and Compositions comprising circRNA and / or linear RNA

[0092] In one aspect, provided herein is a circular RNA (circRNA) bound to one or more RNA-binding proteins (RBPs), as well as polynucleotides encoding same, e.g. DNA or RNA.

[0093] In another aspect, provided herein is a vector comprising, or alternatively consisting essentially of, or yet further consisting of a circRNA comprising one or more RNA hairpins / aptamers and one or more RBPs linked to the circRNA at the one or more RNA hairpins / aptamers. In one aspect, the RBP is linked to the circRNA at the one or more hairpins / aptamers by the process of tethering, wherein the RNA hairpin / aptamer is bound to an exogenous RNA-binding moiety, and the RNA-binding moiety is fused to the RBP.

[0094] In one aspect, the RBP is linked to the circRNA at the aptamer by the process of tethering, wherein the RNA aptamer is bound to an endogenous RBP.

[0095] In one aspect, provided herein is a linear RNA bound to one or more RNA-binding proteins (RBPs).

[0096] In another aspect, provided herein is a vector comprising, or alternatively consisting essentially of, or yet further consisting of a linear RNA comprising one or more RNA hairpins / aptamers and one or more RBPs linked to the linear RNA at the one or more RNA hairpins / aptamers. In one aspect, the RBP is linked to the linear RNA the one or more hairpins / aptamers by the process of tethering, wherein the RNA hairpin is bound to an exogenous RNA-binding moiety, and the RNA-binding moiety is fused to the RBP.

[0097] In one aspect, the RBP is linked to the linear RNA at the aptamer by the process of tethering, wherein the RNA aptamer is bound to an endogenous RBP.

[0098] In one aspect, the RNA hairpin / aptamer and RNA-binding moiety are derived from the same or different organism. In some embodiments, the RNA hairpin and RNA-binding moiety are derived from a bacteriophage selected from: MS2, R17, , PP7, Qp or GA; iron responsive protein (IRP); bovine immunodeficiency virus (BIV), or human UI small nuclear ribonucleoprotein A. In some embodiments, the RNA hairpin or RNA-binding moiety comprise a sequence as shown in any one of SEQ ID NOs: 1-19, as shown in Table 1 herein.

[0099] In some embodiments the RNA-binding moiety has at least 80%, 90%, 95%, 99%, or 100% sequence identity to at least one of SEQ ID NOs: 11-19.

[0100] The sequences of the RNA hairpin and / or RNA-binding moiety shown in Table 1 are representative, rather than exhaustive. In some embodiments, the RNA hairpin can bind to more than one RNA-binding moiety. In some embodiments, the aptamer / hairpin and / or RNA-binding moiety are different than what is shown in Table I .

[0101] In one embodiment the RNA hairpin and RNA binding moiety are derived from the bacteriophage MS2. In one embodiment, the RNA hairpin and RNA binding moiety are derived from the bacteriophage MS2, and the hairpin comprises an amino acid sequence according to SEQ ID NOs: 1 or 2, and the RNA-binding moiety comprises an amino acid sequence according to SEQ ID NO: 11.

[0102] In some embodiments, there are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 RNA hairpins in the circRNA or linear RNA. In one embodiment the circRNA or linear RNA comprises six hairpins. In one embodiment the circRNA or linear RNA comprises six hairpins derived from MS2, optionally wherein each hairpin has a sequence according to SEQ ID NOs: 1 or 2. In some aspects, each VI S2 hairpin can bind to trvo RNA-binding moieties. According to some embodiments embodiment, a circRNA or linear RNA comprising six hairpins binds at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 RNA-binding moieties, wherein at least one RNA-binding moiety is bound to a RBP, optionally wherein the each RBP are the same or different.

[0103] In some aspects, the disclosure provides a circular RNA (circRNA) bound to one or more RNA-binding proteins (RBPs), or a polynucleotide encoding the circRNA, optionally wherein the circRNA is directly or indirectly bound to the one or more RBPs.

[0104] In some aspects, the disclosure provides a circRNA, wherein the circRNA includes at least one RNA hairpin, aptamer, or AU-rich sequence capable of recruiting an endogenous or exogenous RPB that enhances translation.

[0105] In some aspects, the disclosure provides a vector comprises, or consists essentially of, consists: a) a circRNA including one or more RNA hairpins or a polynucleotide encoding the circRNA; and b) an RNA-binding protein (RBP) linked to the circRNA at the one or moreRNA hairpins, or recruited by an AU-rich aptamer encoded in the circRNA to recruit endogenous LARP4B.

[0106] In some aspects, the disclosure comprises, or consists essentially of, consists: a linear RNA bound to one or more RNA-binding proteins (RBPs), or a DNA or RNA polynucleotide encoding the linear RNA, optionally wherein the linear RNA is directly or indirectly bound to the one or more RBPs.

[0107] In some aspects, the disclosure provides a vector comprises, or consists essentially of, consists: a) a linear RNA including one or more RNA hairpin, or a polynucleotide encoding the linear RNA; and b) an RNA-binding protein (RBP) linked to the mRNA at one or more RNA hairpins, and optionally wherein the RPB is recruited via an apatamer or an AU-rich sequence.

[0108] In some aspects, the disclosure provides a vector, wherein the RNA hairpin is derived from a bacteriophage selected from: MS2, R17, X, AN, UlsnRNP aptamer, a boxB element, an AU-rich motif, PP7, QP or GA; iron responsive protein (IRP); bovine immunodeficiency virus (BIV), or human U1 small nuclear ribonucleoprotein A; a hairpin engineered to bind a specific RBP or an aptamer, or an AU-rich aptamer or element that recruits an endogenous RPB, optionally where in the RPB includes LARP4B or a functional fragment or variant of thereof.

[0109] In some aspects, the disclosure provides a circRNA, the linear RNA, the polynucleotide or the vector, wherein the RBP is selected from the group consisting of LARP4, LARP4B, HSPB1, NUFIP2, BCIN3D, NOB1, PKM, GNB2L1, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, DDX52, or a functional fragment or variant of each thereof, or wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8 or a functional fragment or variant of each thereof.

[0110] In some aspects, the disclosure provides a circRNA, the linear RNA, the polynucleotide or the vector, wherein the RBP is a translation enhancer by in vitro exocircFirefly luciferase reporter assay screening in a mammalian cell, optionally a mammalian cell that is an HEK293 cell or a HeLa cell.

[0111] In some aspects, the disclosure provides a vector, wherein the circRNA the linear RNA, or the polynucleotide further includes a sequence encoding a therapeutic polypeptide or protein, a vaccine antigen, a cytokine, a reporter polypeptide (optionally luciferase or GFP), an immunoregulatory protein, or a chimeric antigen receptor.

[0112] In some aspects, the disclosure provides a vector, wherein the RNA hairpin includes a sequence as shown in any one of SEQ ID NOs: 1-10.

[0113] In some aspects, the disclosure provides a vector, wherein the vector is selected from a plasmid, a viral vector, a cosmid, or a phage, optionally wherein the viral vector is selected from a baculovirus, a retrovirus, or an adenovirus.

[0114] In some aspects, the disclosure provides a plurality, wherein the circRNAs, linear RNAs, polynucleotides or vectors are the same or different from each other.

[0115] In some aspects, the disclosure provides an isolated host cell including or expressing one or more.

[0116] In some aspects, the disclosure provides an isolated host cell, wherein the cell is a procaryotic cell or a eukaryotic cell, and optionally a mammalian cell.

[0117] In some aspects, the disclosure provides a composition including a pharmaceutically acceptable carrier and one or more of: the circRNA, the linear RNA, the polynucleotide, or the vector, and / or the plurality, or the isolated host cell. In one aspect, the composition also comprises a carrier, such as a pharmaceutically acceptable carrier.

[0118] In some aspects, the disclosure provides a composition, further including a preservative, a lyophilization agent, or a stabilizer.

[0119] In some aspects, the disclosure provides a composition, further including an additional therapeutic agent.

[0120] In some aspects, the disclosure provides a composition, wherein the additional therapeutic agent is selected from an RBP, wherein the RBP is: a. the same or different as the one or more RBP; b. a positive or negative regulator of circRNA; or c. an RBP regulates RNA biogenesis, splicing, transport, translation, and / or degradation.

[0121] In some aspects, the disclosure provides a composition, wherein the RBP is selected from HSPB1, NUFIP2, BCIN3D, NOB1, PKM, GNB2L1, RBM15, ANXA2, DCP1A,DDX19A, DDX52, G3BP2, LARP4, LARP4B, RBM17, TH0C1, TOB2, U2AF2, or ZC3HAV1.

[0122] In some aspects, the disclosure provides a composition, wherein the PRPB is selected from LARP4, LARP4B, RBM4, EIF3C, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX55, GTF2E2, RDBP, or a functional fragment or variant of each thereof, or wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8 or a functional fragment or variant of each thereof.

[0123] Methods of Use

[0124] In some aspects, the disclosure provides a method to regulate circRNA translation in a cell, including contacting the cell with one or more.

[0125] In some aspects, the disclosure provides a method, wherein the method enhances circRNA translation.

[0126] In some aspects, the disclosure provides a method to regulate circRNA or linear biogenesis in a cell, including contacting the cell with one or more.

[0127] In some aspects, the disclosure provides a method, wherein the contacting is in vivo, in vitro, or ex vivo.

[0128] In some aspects, the disclosure provides a method, wherein the cell is a mammalian cell, optionally a HeLa cell or a HEK293T cell.

[0129] In some aspects, the disclosure provides a method to regulate circRNA or linear RNA translation in a subject in need, the method including administering to the subject one or more.

[0130] In some aspects, the disclosure provides a method to enhance circRNA or linear RNA translation in a cell, the method including contacting the cell with a vector including a circRNA or linear RNA bound to LARP4B.

[0131] In some aspects, the disclosure provides a method to enhance circRNA or linear RNA translation in a cell, the method including contacting the cell with a vector including a circRNA or linear RNA bound to ANXA2.

[0132] In some aspects, the disclosure provides a method to regulate endogenous circRNAs or linear RNAs in a cell, the method including contacting the cell with one or more RBPs and / or vector including a polynucleotide encoding the one or more RBPs, optionally where the vector includes a circRNA or linear RNA.

[0133] In some aspects, the disclosure provides a method to enhance circRNA or linear RNA biogenesis in a cell, the method including contacting the cell with a polynucleotide encoding RBM15.

[0134] In some aspects, the disclosure provides a method, wherein the contacting is in vitro or in vivo.

[0135] In some aspects, the disclosure provides a method to regulate RNA in a cell, the method including contacting the cell with the single construct system, wherein an endogenous RBP specific to the aptamer is recruited to the construct system and binds with the aptamer.

[0136] In some aspects, the disclosure provides a method, wherein the circRNA is bound to LARP4B, ANXA2, or an RBP identified as a translation enhancer.

[0137] In some aspects, the disclosure provides a method of recruiting an endogenous RBP to a circRNA or linear RNA, including introducing into a cell a vector or RNA including an aptamer, hairpin, or sequence specific for an endogenous RBP, thereby enhancing translation, stability, nuclear export, or degradation of the RNA.

[0138] In some aspects, the disclosure provides a method of enhancing translation of a circRNA in a cell, the method including co-delivering: (a) a circRNA including at least one RNA hairpin or aptamer, and (b) an RBP, or a linear RNA or DNA encoding an RBP that binds the RNA hairpin or aptamer.

[0139] In some aspects, the disclosure provides a method of enhancing translation of a circRNA in a cell, the method including delivering a single construct including a circRNA encoding a gene-of-interest and at least one aptamer moiety that recruits an endogenous translation enhancing RBP.

[0140] In some aspects, the disclosure provides a method of treating a disease or disorder in a subject, including administering an effective amount of the composition of any precedingcomposition claim, wherein the composition enhances translation of a therapeutic or prophylactic protein by recruitment or tethering of at least one RBP.

[0141] In some aspects, the disclosure provides a kit including: (a) a circRNA, linear RNA, or vector of any preceding circRNA, linear RNA or vector as described herein; (b) an RBP or encoding nucleic acid, or instructions for co-delivery or recruitment of a translation enhancing RBP.

[0142] In some aspects, the disclosure provides a method of screening candidate RBPs, hairpin, or aptamer sequences for their ability to enhance translation, stability, export, or degradation of a circRNA or linear RNA, including contacting a candidate RBP, hairpin, or aptamer with a reporter RNA in a cell and measuring a change in reporter expression or RNA abundance.

[0143] In some aspects, the disclosure provides a method, wherein the cell is a mammalian cell, optionally a HET293 cell or a HeLa cell.

[0144] In some aspects, the disclosure provides a composition for use in the manufacture of a medicament for the treatment or prevention of a disease or condition.

[0145] In some aspects, the disclosure provides a method for increasing translation of a protein from a circRNA or linear RNA in a mammalian cell, including introducing into the cell a circular RNA containing (a) an AU-rich aptamer sequence and (b) a coding sequence, such that the AU-rich aptamer recruits endogenous LARP4B and thereby enhances translation of the encoded protein.

[0146] In some aspects, the disclosure provides a method, further including the step of identifying an RNA-binding protein that increases translation of circRNA including the steps of: a) expressing a plurality of translation-enhancing RBPs, each fused to a specific tethering domain, b) screening for increased expression of circRNA-encoded reporter protein in a human cell line, and c) selecting RBPs which provide a statistically significant increase in translation (p < 0.05, and log2(fold-change) > 0.5) in two or more cell lines, preferably both HEK293T and HeLa cells.

[0147] In some aspects, the disclosure provides a method for enhancing circRNA or linear RNA translation in a cell in vitro or in vivo, including administering a circular RNA with anAU-rich aptamer and / or a tethering system for an RBP from the group of LARP4B, HSPB1, NUFIP2, BCDIN3D, N0B1, PKM, or GNB2L1, optionally together with a polynucleotide or protein corresponding to the RBP, such that translation from the circRNA is increased compared to a control lacking the recruited RBP or aptamer.

[0148] In some aspects, the disclosure provides a method wherein the cell is a mammalian cell.

[0149] In some aspects, the disclosure provides a method 34 or 35, wherein enhanced translation is caused by recruitment of LARP4B and subsequent interaction of LARP4B with cellular translation-initiation factors EIF3 A and EIF4G2.

[0150] In some aspects, the disclosure provides a composition, system, or method, wherein the composition or system is configured for use in multiple mammalian cell types, including but not limited to HEK293T and HeLa cells.

[0151] In some aspects, the disclosure provides a panel of engineered circular RNAs or RNA-binding protein effectors selected by exocircFirefly luciferase reporter screening in at least two cell types, wherein each RBP demonstrates a statistically significant increase in reporter translation, the panel including at least two, three, four, five, or all of HSPB1, NUFIP2, BCDIN3D, NOB1, PKM, and GNB2L1.

[0152] In some aspects, the disclosure provides a method of designing a circular RNA for therapeutic use, including the steps of: a) analyzing AU content of known LARP4B-bind sequences, b) encoding an AU-rich aptamer of at least 6 nucleotides in the 5' or 3' UTR or non-coding region of the circular RNA, and c) introducing the designed circRNA into a mammalian cell, wherein the AU-rich region recruits endogenous LARP4B and enhances translation efficiency.

[0153] Experimental Materials and Methods

[0154] Establishment of tethered circRNA reporter system

[0155] Previous screens to identify RBP regulators mostly rely on reporters with pre-existing cis regulatory elements in the flanking intronsl8,20, limiting comprehensive evaluation of RBPs with uncharacterized preferences and the identification of exonic regulators. A recent CRISPR screen utilized a single-exon derived circRNA reporter and identified ZC3H14regulates circRNA biogenesis by binding on the exon-intron boundary21. While their screen identified an exonic regulator, it still depended on the sequence context of the reporter design. Applicant reasoned that by forcibly tethering RBPs to a circRNA reporter, Applicant could more comprehensively evaluate their potential to alter circRNA metabolism.

[0156] Therefore, Applicant designed two luciferase minigene reporters containing minimal intronic sequences derived from ZKSCAN172that facilitate backsplicing, splitting the Firefly or Renilla luciferase gene into two exons. Intact Firefly or Renilla luciferase is only produced upon successful backsplicing. Translation was initiated using the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES). In the Firefly reporter (circFirefly), Applicant incorporated 6x MS2 hairpins to recruit MS2 coat protein (MCP) fused to individual RBPs, allowing us to assess the effects of specific RBPs on the circRNA reporter. The Renilla reporter (circRenilla), lacking MS2 hairpins, was co-delivered and served as an internal control (FIG. 1A, Methods). One goal was to identify RBPs that could affect circular RNA metabolism and Applicant selected a circular internal control instead of an mRNA control to avoid excluding RBPs that may influence both circRNAs and their linear counterparts.

[0157] To confirm successful circularization and rule out potential trans-splicing events, Applicant performed RNase R treatment and performed qPCR with divergent (that amplify circRNA or trans-splicing product) or convergent (that amplify linear RNA precursors) primers (FIG. 8A). Applicant observed almost no reduction of circRNA products upon RNase R treatment, suggesting successful circularization and minimal trans-splicing FIG. 8B. The backsplicing junction was further confirmed by Sanger sequencing of the qPCR product (FIG. 8C). To rule out the possibility that unspliced linear mRNA could generate luciferase signals from the upstream fragments of the Firefly or Renilla genes (Firefly up or Renilla up), Applicant excised the upstream intron and downstream fragments (Firefly down or Renilla down) to create mutants that only produce the linear RNA versions of Firefly up and Renilla up (Firefly Notl, Renilla Notl) (FIG. 8D). In luciferase assays, Applicant observed that only circFirefly and circRenilla produced luminescence signals in both HeLa and HEK293T cells, while none of the mutants showed any signal. This indicated that the reporter system specifically reflected the ultimate translation of circRNA-derived luciferase (FIG. 8E, FIG. 8F)

[0158] To validate the sensitivity of the our assay, Applicant selected YTHDF2, a known circRNA regulator previously shown to degrade circRNAs6. Applicant co-transfected the dual luciferase reporters with fusion YTHDF2-MCP plasmid, including a Flag-MCP fusion as a control. As expected, tethering of YTHDF2 led to decreased luciferase signal by 1.7-fold (FIG. IB). Successful expression of YTHDF2 was confirmed by immunofluorescence (FIG. 8G). At the RNA level, YTHDF2 tethering reduced circFirefly RNA levels, particularly in the cytoplasmic fraction, consistent with its predominantly cytoplasmic localization (FIG. 1C, FIG. ID, FIG. 8G, FIG. 8H) To assess the rate of potential false discovery, Applicant cloned eight zinc finger transcription factors that Applicant previously characterized to have minimal binding and regulation on RNA23, and found that they did not alter circRNA expression in the tethering assay, suggesting relatively low false discovery rate (FIG. IE). In summary, these results demonstrate that the reporter system effectively reflects the functional impact of RBPs on circRNAs.

[0159] CircRNA tethering screen proposes RBP functions throughout circRNA lifecycle

[0160] Applicant then systematically evaluated how 730 RBPs, represented by 846 unique isoforms and fused to MCP, affect circRNA expression24,25. Applicant co-transfected individual RBP-MCP plasmids with circFirefly and circRenilla reporter plasmids and conducted arrayed luciferase assays in triplicate in HeLa cells. To control batch variability and validate assay performance, each 96-well plate included triplicate Flag-MCP negative controls for normalization, and positive controls for consistency. The results were analyzed using multiple t-tests, followed by multiple hypothesis correction for false discovery rate (FDR). With a cutoff of |log2(fold change)| > 0.5 and FDR < 0.05, Applicant identified 296 candidate RBP isoforms (281 RBPs), including 267 positive effectors and 29 negative effectors of the relative luciferase signal (FIG. IE). Notably, YTHDF2 was retrieved as a candidate, underscoring the sensitivity and reproducibility of the assay (data not shown.)

[0161] To better understand the functions of the candidate RBPs, Applicant performed Gene Ontology (GO) analysis with the whole RBP tethering library as background. The positive effectors are enriched with factors involved in RNA splicing, transport and translation processes, while the negative effectors are enriched with factors involved in RNA degradation and splicing processes (FIG. 81, FIG. 8J). Among the positive effectors, Applicant recovered many translation initiation factors and ribosomal subunits, that might promote circRNAtranslation, and a number of splicing factors that might enhance circRNA biogenesis. Interestingly, Applicant identified both the protein nuclear export (RAN and XP01) and mRNA nuclear export (NXF2, THOCs, DDX39A / B) proteins enhancing the relative luciferase signal, in concordance with recent observations that both protein and mRNA nuclear export pathways play roles in circRNA nuclear export26,27(data not shown). Among the effectors that decreased circRNA signal, Applicant identified ZC3HAV1 (also known as ZAP) which was recently shown to degrade circRNAs as part of the exogenous RNA surveillance mechanism28. Surprisingly, Applicant also identified known mRNA degraders involved in de-capping and de-polyadenylation such as DCP1A, TOB2, CNOT proteins (CN0T2, CN0T4, CN0T7), which were anti-intuitive as circRNAs do not have a 5' cap or polyAtail. Notably, U2AF1 and U2AF2 emerged as negative effectors, which may reflect their dependence on specific binding sites for functional activity, as shown in our prior splicing regulator screen29. This underscores a limitation of this tethering assay in accurately capturing position-sensitive RBP functions.

[0162] To increase the confidence of the candidates and shed light on potential mechanisms, Applicant selected top candidates across GO terms for a secondary screen in both HeLa cells and HEK293T cells, incorporating RNA level measurements by qPCR (data not shown). The secondary screen showed strong reproducibility in HeLa cells relative to the primary screen (FIG. 1G). In HEK293T cells, the negative effectors were consistent with the HeLa screen, but the positive effectors exhibited more cell-type specificity (FIG. 1G). Applicant integrated the luciferase data across the two cell types together with the RNA level changes, performed hierarchical clustering to group RBPs which similar regulatory effects (FIG. 1G), and validated selected candidates by qPCR to include biological replicates (FIG. 1H, FIG. II). Cluster 1, with decreased RNA and luciferase levels, included factors likely promoting circRNA degradation (e.g., ZC3HAV130, TOB2, DCP1A31, which are known to degrade linear RNAs) and factors inhibiting biogenesis (e.g., U2AF232) (FIG. 1G, FIG. II). Cluster 2 showed increased RNA but decreased luciferase levels, indicating potential multifunctionality (e.g., RBM15 as demonstrated in the latter part of this article) (FIG. 1G, FIG. II). Cluster 3 exhibited increased RNA and luciferase levels, suggesting roles in enhancing circRNA biogenesis, stability, or translation (FIG. 1G, FIG. 1H). Cluster 4 showed modest change in RNA but increased luciferase levels, with candidate proteins having known roles in nuclearexport (e.g., THOC133) or promoting translation (e.g., LARP434) (FIG. 1G, FIG. 1H). Applicant uncovered several uncharacterized candidates from different functional clusters (e.g., RBM17, DDX52), whose roles remain poorly defined. Hypothesized mechanisms for these RBPs are summarized in Fig. Slk and provide a framework for future mechanistic studies.

[0163] Applicant also compared our candidates with the aforementioned CRISPR screen to identify regulators of circRNA biogenesis21. Out of 41 positive regulators that they identified, 8 of them were included in the tethering library and 4 out of these 8 were identified as positive effectors from our screen (FIG. 81). Notably, their top candidate ZC3H14 was also identified in our screen, highlighting the reliability and the sensitivity of our screen to identify exonic circRNA regulators. Applicant had previously conducted a tethering screen with the same RBP library and identified RBPs that impact RNA stability and translation of linear mRNAs25. When comparing the circRNA tethering results to our previous linear mRNA stability and translation tethering data, Applicant observed a modest correlation (R2= 0.1449). Stronger concordance between the two screens was observed among the top 10 circRNA negative effectors, whereas the top positive effectors were largely specific to circRNAs. This implies that degradation may rely on shared pathways, while biogenesis and translation are more circRNA-specific (FIG. 8M).

[0164] Given that the decapping and deadenylation complexes consistently reduced luciferase signal in two different cell lines, Applicant next sought to confirm that this effect was mediated through their action on circRNAs. There could be two possibilities. First, these factors might act on linear precursor RNAs in the nucleus prior to backsplicing. Second, they might be capable of recruiting endonucleases to degrade circRNAs independently of their canonical decapping or deadenylation activities. To delineate these two possibilities, Applicant redesigned the luciferase reporters (exo-circFirefly and exo-circRenilla) to flank permuted group I introns for in vitro production of circRNA (FIG. 8N). The reporters were transcribed in vitro into linear RNA, induced to circularize, and subjected to RNase R digestion to remove any residual linear RNA (FIG. 80). The RBP-MCP fusion genes were also transcribed in vitro into mRNAs with 5’ Caps and 3’ polyA tails and co-delivered to the cells with exo-circFirefly and exo-circRenilla circRNAs. Interestingly, tethering CNOT7 and DCP1 A to the in vitro synthesized circRNA reporter still decrease the luciferase signal by 12to 33-fold, ruling out the possibility of acting on the linear precursors (FIG. 8P). Additionally, tethering an enzymatically dead mutant35of CN0T7 (D40A) also decreased the luciferase signal (FIG. 8P). Furthermore, tethering DCP2 which is the enzymatic subunit in the decapping complex did not decrease the luciferase signal in the original screen (data not shown). These observations suggest that they might function through a mechanism independent of their canonical enzymatic functions, possibly by recruiting endonucleases that merits future investigation.

[0165] Together, this screen identifies both known and unexpected circRNA effector RBPs reproducibly and reliably. These candidate RBP regulators are hypothesized to act throughout the circRNA lifecycle. By integrating RNA and luciferase level data, Applicant can infer potential regulatory mechanisms that identify distinct classes of circRNA RBP regulators.

[0166] Identification of RBPs promoting circRNA translation

[0167] Applicant next focused on two goals: identifying candidates that enhance translation of exogenous circRNAs for improved therapeutic vector design, and uncovering regulators of endogenous circRNAs that reveal previously unappreciated biological mechanisms. Reasoning that RBPs promoting circRNA translation would localize to the cytoplasm, Applicant selected six top candidate effectors based on their cytoplasmic localization determined by immunofluorescence (data not shown). To eliminate confounding effects on splicing or nuclear export and to model a therapeutic scenario involving exogenous circRNA delivery, Applicant leveraged the previously described exo-circFirefly and exo-circRenilla reporter system. The expression of RBP-MCP fusion proteins was confirmed via immunofluorescence (FIG. 9A).

[0168] Applicant then performed luciferase assays and extracted RNA for qPCR in both HEK293T and HeLa cells. Notably, Applicant observed that LARP4B consistently upregulated the luciferase signal by more than two-fold in both cell types. In contrast, two isoforms of ANXA2 significantly upregulated the luciferase signal by around two-fold, but only in HeLa cells, indicating that circRNA translation may be regulated in a cell typespecific manner (FIG. 3C, FIG. 3D). None of the RBPs increased the RNA levels of the exo- circFirefly reporter, ruling out RNA stabilization as a mechanism (FIG. 3E, FIG. 3F). Therefore, the increased luciferase signal is likely attributable to enhanced translation.Notably, the other 4 RBPs tested only upregulated luciferase signal of endogenously produced circRNAs, suggesting that they may rely on early processing events to facilitate translation.

[0169] To assess the generalizability of the translation-enhancing effect, Applicant incorporated two additional IRES elements, PV (poliovirus) and CrPV (cricket paralysis virus), into the exo-circFirefly and exo-circRenilla reporter systems. These IRES elements represent mechanistically distinct modes of IRES-mediated translation compared to the original EMCV IRES36. Interestingly, when these IRES elements were introduced into the endogenously expressed circFirefly construct generated via backsplicing, Applicant observed comparable luciferase signals (FIG. 9B). In contrast, the exo-circFirefly and exo-circRenilla constructs showed a stepwise increase in luciferase signal as their structures were extended to scaffold more IRES trans-acting factors (ITAFs), consistent with previous reports9(FIG.9C). These findings support the notion that circRNA processing steps can predefine translational competency, distinguishing endogenously processed circRNAs from exogenously delivered ones. This underscores the value of using exogenous circRNA systems for optimizing therapeutic constructs. Applicant next applied tethering of LARP4B and ANXA2 to the exo-circFirefly reporters bearing different IRES elements. Consistent with prior observations, LARP4B consistently enhanced translation across all IRES types, while ANXA2 promoted translation specifically in HeLa cells (FIGS. 2E - 2G). Notably, CrPV IRES-driven exo-circFirefly expression was too low in HeLa cells to confidently assess this effect.

[0170] Given that LARP4B emerged as a broadly effective enhancer of circRNA translation, Applicant next sought to investigate the underlying mechanism. Applicant performed coimmunoprecipitation (co-IP) experiments and screened several eukaryotic translation initiation factors (elFs), identifying interactions between LARP4B and both eIF3A and eIF4G2 (FIG. 2H, FIG. 9D). Notably, eIF4G2 has previously been implicated in promoting IRES-dependent translation of mRNAs37and m6A-mediated translation of circRNAs38, which may explain the translational enhancement observed in our tethering assays. In addition, LARP4B has been reported to associate with the 40S ribosomal subunit and polysomes39, further supporting its role in facilitating translation.

[0171] In summary, using an exogenous circRNA luciferase reporter system, Applicant identified LARP4B as a universal enhancer of circRNA translation and ANXA2 as a cell type-specific enhancer. LARP4B’s effect is consistent across multiple IRES types and cell lines, and may be mediated through its interactions with translation initiation factors eIF3 A and eIF4G2.

[0172] LARP4B is associated with endogenous mRNA and circRNA translation

[0173] Applicant asked if LARP4B is also associated with endogenous mRNA and circRNA translation. To address this question, Applicant first conducted eCLIP (enhanced crosslinking and immunoprecipitation) and RIP (RNA immunoprecipitation) followed by circRNA sequencing to identify LARP4B’s endogenous mRNA and circRNA targets. LARP4B binding sites were predominantly enriched in the 3’ UTR and CDS regions of its target RNAs, with notably higher enrichment in the 3’ UTR (FIG. 3A, FIG. 10A). Using t-SNE clustering of all RBP eCLIP profiles from the ENCODE project, LARP4B was grouped with RBPs that predominantly bind the 3’ UTR (FIG. 10B). Applicant observed both polyA and polyU tracts in motif analysis (FIG. 3B), consistent with previous findings that LARP4B binds AU-rich 3’ UTR regions40. Applicant then categorized the LARP4B bound transcripts into high binding and low binding by an arbitrary mean eCLIP enrichment cutoff of 3 and compared their translation efficiency obtained from Ribo-seq analysis in HEK293T cells41. Applicant found that higher LARP4B binding at the 3’ UTR is significantly correlated with increased translation efficiency, consistent with its role in promoting mRNA translation39.While LARP4B binding to the CDS also correlates with translation efficiency, the association is weaker than that observed at the 3’ UTR, likely due to reduced binding levels in the CDS region (FIG. 3D, FIG. 10A)

[0174] In the LARP4B RIP-circRNA-seq experiment, Applicant included three biological replicates (FIG. 10C) to robustly identify enriched circRNAs and minimize false positives, given that the majority of circRNAs are not believed to undergo translation despite recent suggestions of low-level, pervasive circRNA translation42. Applicant observed lower fraction of backsplicing junction (BSJ) reads in LARP4B IPs compared to the inputs, indicating that fewer circRNAs might be associated with LARP4B compared to mRNA targets. By intersecting enriched circRNAs across all three IP replicates, Applicant identified 404 high- confidence circRNAs bound by LARP4B (FIG. 10E). To investigate whether thesecircRNAs are likely to be translated, Applicant examined supporting evidence from the TransCirc database43. Applicant observed enriched evidence of translation for LARP4B- bound circRNAs, particularly in ribosome profiling and polysome profiling datasets (FIG. 3E), supporting an association between LARP4B and endogenous circRNA translation. Additionally, these circRNAs exhibited a higher proportion of adenosine and uridine bases, suggesting that LARP4B’s preference for AU-rich sequences may underlie its selective promotion of circRNA translation (FIG. 3F).

[0175] RBM15 is a multifaceted regulator of endogenous circRNAs

[0176] A key objective of the tethering screen was to identify RBPs that regulate endogenous circRNAs. To evaluate this, Applicant performed gain- and loss-of-function studies on 13 selected RBPs using overexpression and siRNA-mediated knockdown, respectively. Endogenous circRNAs that were upregulated upon overexpression and downregulated upon knockdown were classified as positively regulated by the RBP, while those showing the opposite pattern were considered negatively regulated (FIG. 4A). Among these RBPs, candidates implicated in translation enhancement, such as ANXA2, LARP4, LARP4B, and G3BP2, did not broadly alter circRNA abundance, consistent with their proposed roles being primarily translational, as supported by earlier assays. Similarly, nuclear export factors including THOC1 and DDX19A did not significantly impact global circRNA levels, suggesting their functions may be more related to localization, a possibility warranting further investigation. In contrast, known or putative RNA degradation factors such as TOB2, DCP1A, and ZC3HAV1 regulated a limited subset of endogenous circRNAs, with a greater number showing negative regulation — consistent with their expected roles in RNA decay (FIG. 4A). Surprisingly, RBM15 emerged as a strong positive regulator, influencing over 700 endogenous circRNAs (FIG. 4A). To exclude the possibility that these circRNA changes might be due to the expression level changes of their linear cognates, Applicant conducted ribosomal-RNA (rRNA)-depleted total RNA sequencing in RBM15 overexpression and knockdown samples (overexpression and knockdown confirmed by RBM15 read counts, (FIG. 11 A, FIG. 11B) and found minimal expression changes of their linear cognates (FIG. 11C, FIG. 11D) This confirms RBM15’s regulatory role on circRNA expression.

[0177] To further explore the molecular mechanism underlying RBM15's effect on circRNAs, Applicant revisited the results from the tethering assay. RBM15 tethering caused a dramatic decrease in the relative luciferase signal while increasing circFirefly RNA expression (FIG. 4B). Given RBM15’s known role in alternative splicing, this suggested that RBM15 might promote circRNA biogenesis. Since RBM15 is exclusively nuclear, Applicant hypothesized that it might retain circRNAs in the nucleus, thereby reducing cytoplasmic circFirefly levels and causing the observed discrepancy in decreasing the relative luciferase signal. Indeed, RBM15 tethering led to a dramatic ~140-fold increase of circFirefly in the nuclear fraction, along with a modest 2.17-fold upregulation in the cytoplasmic fraction (FIG. 4C). Upon reviewing the raw luciferase data, Applicant noted that both Firefly (due to tethering) and Renilla (due to RBM15 overexpression) luciferase signals were upregulated (FIG. HE, FIG. HF). The stronger nuclear retention of circFirefly relative to circRenilla explained the reduced cytoplasmic ratio of circFirefly, thereby accounting for the luciferase assay results, which were normalized against the Renilla luciferase signal. These findings suggest that RBM15 has dual functions: promoting circRNA biogenesis and retaining circRNAs in the nucleus.

[0178] To determine whether RBM 15 similarly regulates endogenous circRNAs, Applicant performed qPCR analysis following RBM15 overexpression and knockdown (successful knockdown was confirmed by qPCR, FIG. 11G), using divergent primers for endogenously expressed circRNAs. Applicant observed a significant increase in circ / ?7N- / (2,3) and circ5PE'CC7(4) levels upon RBM15 overexpression, and a corresponding decrease upon knockdown (FIG. 4D, FIG. 4E). Fractionation analysis revealed a greater upregulation of circ7?77V¥(2,3) and circ5PE'CC7(4) in the nuclear fraction compared to the cytoplasmic fraction, suggesting that RBM15 also retains endogenous circRNAs in the nucleus. However, its effect was less pronounced than with the tethered circFirefly reporter (FIG. 4F). Applicant hypothesize that RBM15's nuclear retention function may be linked to its RNA-binding affinity.

[0179] In summary, RBM15 is a multifaceted circRNA regulator, promoting circRNA biogenesis while also retaining circRNAs in the nucleus, both for the circRNA reporter and for endogenous circRNAs.

[0180] Different domains of RBM15 are responsible for its multifaceted functions

[0181] Applicant next sought to dissect the functions of the various domains within RBM15. RBM15 contains three RNA recognition motif (RRM) domains, one SPOC domain and several disordered domains, with two longer ones, before RRM1 (Disorderedl) and between the third RRM and the SPOC domain (Disordered2), respectively. Applicant generated a series of plasmid constructs with different RBM15 truncations, as illustrated in FIG. 4G, namely, A RRM1, A all RRM, A SPOC, RRM2 / 3, disordered and SPOC. Each RBM15 fragment was fused to MCP and subjected to the tethering assay, where Applicant measured changes in both luciferase activity and RNA levels to infer the functional contributions of each domain.

[0182] As a positive control, RBM15 tethering decreased linFirefly RNA levels and increased circFirefly RNA levels, reaffirming its role in promoting circRNA biogenesis. RBM15 also reduced the relative luciferase signal, indicating its function in retaining circRNAs in the nucleus. The Disorderedl and RRM1 domain appeared to be dispensable for both functions, as the A RRM1 construct displayed similar changes to the full-length RBM15 (FIG. 4H, FIG. 41) The RRM2 and RRM3 domains were not essential for promoting circRNA biogenesis, as the A all RRM construct still decreased linFirefly RNA and increased circFirefly RNA expression. However, the partial recovery of luciferase activity suggested that RRM2 and RRM3 contribute to circRNA nuclear retention. Consistent with this, tethering RRM2 / 3 domains alone increased circFirefly RNA levels, likely by enhancing transcription, but still reduced luciferase signal compared to the Flag control, supporting their role in nuclear retention (FIG. 4H, FIG. 41). The Disordered2 and SPOC domains worked synergistically to promote circRNA biogenesis, as neither domain alone increased circFirefly RNA levels. Furthermore, the SPOC domain had an additional role in transcriptional regulation, as A SPOC significantly reduced linFirefly RNA levels, while tethering the SPOC domain increased linFirefly RNA expression (FIG. 4H, FIG. 41). These findings are consistent with previous reports showing that the SPOC domain interacts with SETD1B to regulate transcription44. In conclusion, distinct RBM15 domains play separate roles in promoting circRNA biogenesis and retaining circRNAs in the nucleus.

[0183] RBM15 directly regulates circRNA biogenesis

[0184] To further investigate how RBM15 regulates circRNA biogenesis, Applicant considered its known roles in alternative splicing45and m6A modification46. Applicanttherefore first examined whether its effect on circRNA biogenesis depends on either of these functions. Applicant analyzed the alternative splicing changes in the rRNA-depleted total RNA-seq data with RBM15 overexpression and knockdown. Applicant found that the majority of RBM 15 -regulated alternative splicing events involved exon skipping. However, there was minimal overlap between these splicing events and the parental genes of circRNAs that RBM15 positively regulates (FIG. 5A, FIG. 5B). This indicates that RBM15 regulates circRNA biogenesis through a mechanism independent of its alternative splicing activity. Furthermore, even in the absence of METTL3 (FIG. 12B), circRNA upregulation persisted at levels similar to control cells, ruling out m6A modification as a contributor to RBM15- mediated circRNA biogenesis (FIG. 12A).

[0185] To understand if RBM15 directly regulates circRNA biogenesis, Applicant conducted RBM15 RIP experiment followed up with qPCR analysis and found higher enrichment of RBM15 on the pre-mRNA precursors of circSPECCl(4) and circRTN4(2,3) compared to their mature circRNAs and mRNA cognates (FIG. 5C, FIG. 5D, FIGS. 12C-12D), demonstrating a direct occupancy on pre-mRNA to potentially mediate circRNA biogenesis. To further illustrate the necessity of RBM15 binding for circRNA biogenesis, Applicant focused on circYTHDF3(3), a representative circRNA with sparse RBM15 binding sites identified by eCLIP (FIG. 5E). Applicant generated ciwYTHDF3(3') minigene and mutants with these binding sites deleted, such as deleting binding sites 1 and 2 (A 12), 3 and 4 (A 34) or 1,2,3 and 4 (A 1234) (FIG. 5F). As binding site 4 spans the 3’ exon-intron junction, Applicant added the original 4 bases at the splice site (“GGgt”) to minimize the effect due to splice site removal. Applicant then transfected HEK293T cells with these constructs in comparison to the wild type (WT) control minigene and performed qPCR to measure c\vcYTHl)F3(3) and YmYTHDF3 level. Applicant detected close to 15-fold upregulation of circYTHDF3 by expressing the WT minigene normalized to the basal expression of cixcYTHDF3(3') in HEK293T cells, suggesting successful circRNA biogenesis from the minigene (FIG. 5G). Importantly, all three mutants markedly reduced circYTHDF3(3) production while leaving linYTHDF3 expression from the minigene unaffected. The milder effect observed upon deletion of the weaker binding sites (3 and 4) supports the conclusion that RBM15 binding is essential for promoting circRNA biogenesis (FIG. 5G, FIG. 12E).

[0186] Applicant then performed affinity pulldown followed by mass spectrometry (AP-MS) to identify RBM15's interacting partners (data not shown). RBM15 was identified, confirming successful immunoprecipitation. Additionally, Applicant detected well-known RBM15 interactors, including the m6A writer complex component WTAP47and the epigenetic modifier SETD1B44(FIG. 5H). Interestingly, RBM15 interacted with several SRSF splicing factors (FIG. 5H). Tethering SRSF4, SRSF6, and SRSF10 increased both luciferase signal (in the secondary screen) and circFirefly RNA levels (FIG. 12F, FIG. 12G), and many other SRSF proteins also enhanced luciferase signal in the primary screen (FIG. 51), suggesting that RBM15 may promote circRNA biogenesis through interactions with SRSF proteins. This potentially cooperative role between RBM15 and SRSF proteins was further supported by co-binding of SRSF1 on the circRNAs that RBM15 positively regulated (FIG. 12H). Due to the limited availability of IP-grade antibodies and a dense auto- and cross-regulation among SRSF proteins48, it is currently difficult to directly demonstrate their cooperative role and require future investigation into the potential redundancy or target selectivity of SRSF proteins.

[0187] RBM15 retains IGF2BP1 in the nucleus

[0188] To investigate RBM15’s additional role in retaining circRNAs in the nucleus, Applicant performed an AP-MS experiment with RNase A treatment, based on prior evidence suggesting that its nuclear retention function is RNA-dependent (FIG. 4C, FIG. 4F, FIG. 4H, FIG. 41). By comparing the AP-MS results with and without RNase A treatment, Applicant observed a marked reduction in the interaction between RBM15 and IGF2BP1, suggesting that this interaction is mediated through RNA (FIG. 6A). Applicant confirmed RBM15 and IGF2BPl’s interaction with co-IP both by overexpression of a V5-tagged RBM15 protein or immunoprecipitation with an antibody recognizing endogenous RBM15 (FIG. 6B, FIG. 6C). Notably, interactions with other partners such as WTAP, SETD1B, and SRSF proteins persisted following RNase A treatment, suggesting RNA-independent binding. This aligns with our observation that RBM15’s function in circRNA biogenesis is independent of its RRM domains (FIG. 13A).

[0189] Considering that IGF2BP1 had recently been shown to act as an adapter protein for circRNA nuclear export27, Applicant speculated that RBM15 might retain IGF2BP1, and thereby circRNAs, in the nucleus. Indeed, immunofluorescence revealed significantly highernuclear localization of IGF2BP1 when RBM15 was overexpressed (FIG, 5E, FIG. 5F). Furthermore, cells with elevated levels of RBM15 transgene expression displayed stronger nuclear retention of IGF2BP1 (FIG. 5E), supporting the idea that RBM15 retains circRNAs in the nucleus by sequestering IGF2BP1 there. Combinatorial analysis with the ENCODE eCLIP data revealed that the RBM 15 -regulated circRNAs were significantly enriched with both RBM15 and IGF2BP1 binding sites, potentially providing scaffolds to mediate this interaction and nuclear retention.

[0190] Taken together, RBM15 retains IGF2BP1 in the nucleus. Without being bound by theory, it is proposed that the shared binding on circRNAs might scaffold this interaction and in turn leads to the nuclear retention of circRNAs that are shuttled by IGF2BP1.

[0191] RBM15 binds directly to the exonic region near BSJs and is a conserved circRNA regulator

[0192] Re-examination of RBM15 eCLIP data revealed that RBM15 predominantly binds within coding sequence (CDS) regions (FIG. 14A). To further explore its role in circRNA regulation, Applicant generated a metadensity plot of RBM15 binding centered on backsplicing junctions (BSJs). Notably, RBM15 exhibited increased binding at exonic regions flanking the BSJs of circRNAs it positively regulates, compared to those it does not affect (FIG. 6A). This pattern strongly suggests that RBM15 facilitates circRNA biogenesis by binding proximal to BSJs. A representative example is c\xcRTN4 (2,3), which Applicant previously identified as an RBM 15 -regulated circRNA (FIG. 6B, FIG. 4D, FIG. 4E).

[0193] Given that exonic sequences are evolutionarily more conserved across closely-related species, Applicant hypothesized that RBM15’s regulatory role might also be conserved across orthologous target substrates. In contrast to ZC3H1421, the only other known exonic RBP regulator of circRNAs, RBM15 binds more extensively across exonic regions, which may enhance its ability to recognize conserved sequence elements. Protein sequence alignment revealed that human and mouse RBM15 proteins shared 94% sequence identity (FIG. 13C). The circRNAs positively regulated by RBM15 also appeared to be more conserved (FIG. 6C), as measured by a multiple conservation score (MCS) that takes into account the number of species, tissues and individuals sharing a given circRNA49. From the literature, Applicant identified the conserved circA7 / / - / (2,3) circRNA in the mouse genome50.Applicant then measured circRtn4 expression in the mouse neuronal cell line Neuro-2a (N2A) after overexpression or knockdown of Rbml5. Excitingly, Applicant observed upregulation of circAtw4(2,3) upon mouse RBM15 overexpression and downregulation upon Rbml5 knockdown, while another circRNA, c\vcRims2 (21-24), which is not regulated by Rbml5, showed no changes in expression (FIG. 6D, FIG. 6E). This provides compelling support for RBM15 as an evolutionarily conserved factor in circRNA biogenesis regulation. To assess the extent of RBM15’s regulation, Applicant performed circRNA sequencing in N2A cells after mouse RBM15 overexpression or knockdown. By lifting over RBM15- regulated circRNAs in HEK293T cells to the mouse genome, Applicant annotated 730 of 756 circRNAs using circPrimer2.051(data not shown). Of these, 357 circRNAs were identified in the N2A circRNA sequencing data. Applicant next calculated the average counts per million reads (CPM) of these circRNAs and observed a significant increase in circRNA expression in mouse RBM15 overexpression samples (mean of delta CPM = 0.05237, p = 0.0032, paired t- test; FIG. 6F, FIG. 13D) These findings define RBM15 as a conserved regulator of circRNA biogenesis, most likely via direct binding to conserved exonic sequences flanking backsplicing junctions.

[0194] I 'able 1. Sequences of RNA hairpins and RNA-bindhig moieties.

[0195] Hairpin sequences reproduced from Bos, T. J., et al. (2016) Advances in experimental medicine and biology, 907, 61-88. https: / / doi.org / 10. l007 / 978-3-3 l9-29073-7_3, the entire contents of which are incorporated herein.

[0196] In some embodiments, the aptamer is specific to the RBP.

[0197] Applicant also provides herein a vector comprising circRNA or linear RNA comprising one or more hairpins / aptamers, wherein the aptamer is specific for one or more endogenous RBPs in a cell.

[0198] In some aspects, the endogenous RBP specific for the aptamer is selected from at least one RBP listed in from Tables 2, 3, 7, or 8 or a functional fragment or variant of each thereof. In some aspects, the endogenous RBP specific for the aptamer is selected from LARP4, LARP4B, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, or DDX52.

[0199] In one aspect, the vector is selected from a plasmid, a viral vector, a cosmid, or a phage, optionally wherein the viral vector is selected from a baculovirus, a retrovirus, or an AAV, or an adenovirus. In one aspect, the vector may be selected from a different virus.

[0200] According to one aspect, the one or more RBPs is selected from RBM15, RBPMS, SRSF10, LARP4, LARP4B, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, or DDX52 or functional fragment or variant of each thereof. According to another aspect, the RBP is selected from an RBP set forth in Tables 2, 3 7, and / or 8 or a functional fragment or variant of each thereof. According to yet another aspect, the RBP is indicated as a top upregulator or top downregulator in the circRNA screen.

[0201] According to some aspects, described herein is a nucleic acid sequence encoding an RBP. In some aspects, the nucleic acid comprises a sequence having at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to at least one of the nucleotide sequences in an RBP selected from an RBP set forth in Tables 2, 3 7, and / or 8 or a functional fragment or variant of each thereof. In one aspect, the RBP is selected from: NOB1, NUFIP2, GAPDH, GNB2L1, BCDIN3D, PKM, HSPB1, GFM1, ILF3, RBPMS, DDX21, RPL35, FAM120A, SPATS2, LARP4B, ANXA2.

[0202] According to some aspects, described herein is an RBP having an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to at least one of the amino acid sequences in Tables 2, 3, 7 or 8 or a functional fragment or variant of each thereof. In one aspect, the RBP is selected from: NOB1, NUFIP2, GAPDH, GNB2L1, BCDIN3D, PKM, HSPB1, GFM1, ILF3, RBPMS, DDX21, RPL35, FAM120A, SPATS2, LARP4B, ANXA2.

[0203] According to some aspects, described herein is a nucleic acid sequence encoding a RBP. In some aspects, the nucleic acid comprises a sequence having at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to at least one of the nucleotide sequences in Table 8 or a functional fragment or variant of each thereof.

[0204] According to some aspects, described herein is an RBP having an amino acid sequence having at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to at least one of the amino acid sequences in Table 8 or a functional fragment or variant of each thereof.

[0205] In some aspects, the RBP enhances circRNA or linear RNA biogenesis, splicing, transport, translation, and / or degradation.

[0206] In some embodiments, the circRNA or linear RNA encodes a therapeutic polypeptide or reporter polypeptide. In one aspect the reporter polypeptide is luciferase or green fluorescent protein.

[0207] In some embodiments, the circRNA or linear RNA encodes a vaccine, such as a SARS-CoV-2, influenza, or Ebola virus vaccine. In some embodiments, the circRNA encodes a protein used as a protein replacement in protein replacement therapy (see, e.g. Vavilis, T. et al. (2023). mRNA in the Context of Protein ReplacementTherapy. Pharmaceutics, 75(1), 166. https: / / doi.org / 10.3390 / pharmaceuticsl5010166 and Rohner, E. et al. Unlocking the promise of mRNA therapeutics. Nat Biotechnol 40, 1586- 1600 (2022). https: / / doi.org / 10.1038 / s41587-022-01491-z, the contents of each are incorporated by reference herein). In still other embodiments, the circRNA or linear RNA is used in CAR T therapy.

[0208] In some embodiments, the circRNA or linear RNA encodes one or more of a cytokine, an immunomodulatory molecule, or a reporter protein, optionally wherein cytokine is selected from GM-CSF, TNFa, IFN-y, TGF-13, IL-2, IL-4, IL-10 and IL-13.

[0209] In some aspects, provided herein is a plurality of the circRNAs, linear RNAs, and / or vectors, wherein the circRNAs, linear RNAs, and / or vectors are the same or different from each other.

[0210] In some embodiments, provided herein are isolated host cells comprising one or more circRNAs and / or vectors as described herein. In some aspects the host cell is a prokaryotic or a eukaryotic cell.

[0211] Compositions

[0212] In one aspect, provided herein is a composition comprising, or alternatively consisting essentially of, or yet further consisting of a carrier and one or more circRNAs, linear RNAs, or vectors described herein.

[0213] Thus, this disclosure also provides compositions containing the circRNA, linear RNA, or vector and another substance, active or inert. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the disclosure. Those skilled in the art will know of other suitable carriers, or will be able to ascertain such, using routine experimentation.

[0214] In another aspect, the composition further comprises an additional therapeutic agent.

[0215] In some embodiments, the additional therapeutic agent is an RBP, wherein the RBP is the same or different as the RBP(s) in the circRNA, linear RNA, and / or vector. In some aspects the RBP is a positive regulator of circRNA or linear RNA. In some aspects the RBP is a negative regulator of circRNA or linear RNA. In some aspects the additional therapeutic RBP regulates RNA biogenesis, splicing, transport, translation, and / or degradation. In some aspects, the additional therapeutic RBP is selected from RBM15, ANXA2, DCP1 A, DDX19A, DDX52, G3BP2, LARP4, LARP4B, RBM17, THOC1, TOB2, U2AF2, or ZC3HAV1 or a functional fragment or variant of each thereof. In other aspects, the RBP is selected from an RBP as set forth in Tables 2, 3, 7 and / or 8 or a functional fragment or variant of each thereof.

[0216] In some embodiments the additional therapeutic agent is an anti-cancer therapy. In some embodiments, the additional therapeutic agent comprises chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof. Illustrative additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5 -fluorouracil (5-FU), 6-mercaptopurine (6-MP), capeci tabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors such as topotecan or irinotecan (CPT-11); topoisomerase II inhibitors such as etoposide (VP- 16), teniposide, or mitoxantrone; mitotic inhibitors such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or corticosteroids such as prednisone, methylprednisolone, or dexamethasone. In some cases, the additional therapeutic agent comprises pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

[0217] In some cases, the additional therapeutic agent comprises an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.

[0218] In some cases, the additional therapeutic agent comprises a cytokine. Exemplary cytokines include, but are not limited to, IL-ip, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNFa.

[0219] In some embodiments, the additional therapeutic agent comprises a receptor agonist. In some instances, the receptor agonist comprises a Toll-like receptor (TLR) ligand. In some cases, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases, the TLR ligand comprises a synthetic ligand such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2p, CFA, or Flagellin.

[0220] In some cases, the additional therapeutic agent comprises an adoptive T cell transfer (ACT) therapy. In one embodiment, ACT involves identification of autologous T lymphocytes in a subject with, e.g., anti-tumor activity, expansion of the autologous T lymphocytes in vitro, and subsequent reinfusion of the expanded T lymphocytes into the subject. In another embodiment, ACT comprises use of allogeneic T lymphocytes with, e.g., anti-tumor activity, expansion of the T lymphocytes in vitro, and subsequent infusion of the expanded allogeneic T lymphocytes into a subject in need thereof.

[0221] Pharmaceutical compositions of the present disclosure can be administered in a manner appropriate to the disease to be treated or prevented. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0222] In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal administration routes. In some cases, parenteral administration comprises intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intratechal administration. In some instances, the pharmaceuticalcomposition is formulated for local administration. In other instances, the pharmaceutical composition is formulated for systemic administration.

[0223] In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0224] In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999).

[0225] In some instances, the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0226] In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0227] In some embodiments, the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, sodium citrate, ammonium sulfate and / or other agents such as heparin to increase the solubility and in vivo stability of polypeptides.

[0228] In some instances, the pharmaceutical formulations further include diluent which are used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®, dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di- Pac® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.

[0229] In some cases, the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” include both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as com starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as awood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PHI 05, Elcema® Pl 00, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross- linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cationexchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

[0230] In some instances, the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0231] Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials.

[0232] Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like.

[0233] Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycolssuch as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.

[0234] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N- methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.

[0235] Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, poly sorb ate-20 or Tween® 20, or trometamol.

[0236] Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxy ethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[0237] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Additionalsurfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes.

[0238] Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

[0239] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

[0240] In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day or more. The pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0241] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the composition is given continuously, alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday is from 10%-100%, including,by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0242] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.

[0243] In some embodiments, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0244] The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages are altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0245] In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimaltoxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

[0246] Methods of Use

[0247] Also provided herein are methods to regulate circRNA or linear RNA translation and / or degradation in a cell, the method comprising, consisting of, or consisting essentially of, contacting the cell with one or more circRNA, linear RNA, vector, plurality, and / or composition described herein. In one aspect, the method enhances circRNA or linear RNA translation. In one aspect, the method enhances translation of exogenously applied circRNA or linear RNA. In one aspect, the method enhances degradation of circRNA or linear RNA.

[0248] In some aspects, the method comprises co-delivery of the RNA and RBP. In other aspects the method comprises delivery of the RNA in a vector comprising an aptamer that recruits an endogenous RBP.

[0249] In one aspect, provide herein is a method to enhance circRNA or linear RNA translation in a cell, the method comprising, consisting of, or consisting essentially of, contacting the cell with a vector comprising a circRNA or linear RNA bound to LARP4B or a functional fragment or variant of each thereof.

[0250] In one aspect, provide herein is a method to enhance circRNA or linear RNA translation in a cell, the method comprising, consisting of, or consisting essentially of, contacting the cell with a vector comprising a circRNA or linear RNA bound to ANXA2 or a functional fragment or variant of each thereof.

[0251] Also provided herein are methods to regulate circRNA biogenesis, and / or nuclear export in a cell, the method comprising, consisting of, or consisting essentially of, contacting the cell with one or more circRNA, vector, plurality, or composition described herein.. In one aspect, the method enhances biogenesis of circRNA endogenous to the cell. In one aspect, the method inhibits biogenesis of circRNA endogenous to the cell. In one aspect, the method inhibits export of circRNAs from the nucleus of the cell.

[0252] Further provided herein are methods to regulate endogenous circRNAs in a cell, the method comprising, consisting of, or consisting essentially of, contacting the cell with one or more RBPs and / or vector comprising a polynucleotide encoding the one or more RBPs,optionally where the vector comprises a circRNA.. In one aspect, the vector comprising the one or more RBPs is the circRNA vector described herein. In another aspect, the vector comprising the one or more RBPs is a vector comprised of linear RNA. In one aspect, the RBP positively regulates endogenous circRNAs in a cell. In one aspect, the RBP negatively regulates endogenous circRNAs in a cell.

[0253] In one aspect, provided herein is a method to regulate endogenous circRNA in a cell, the method comprising, consisting of, or consisting essentially of, contacting the cell with a polynucleotide encoding the RBP RBM15 and / or the RBP RBM15. In one aspect, the method enhances biogenesis of endogenous circRNAs. In one aspect, the method regulates the circRNAs circRTN4 and / or circSPECCl.circRTN4 and circSPECCl each have therapeutic potential. Additional information about circRTN4 can be found, by non-limiting example, in Qi, Y., et al. (2021). CircRtn4 Acts as the Sponge of miR-24-3p to Promote Neurite Growth by Regulating CHD5. Frontiers in molecular neuroscience, 14, 660429. https: / / doi.org / 10.3389 / fnmol.2021.660429 (circRTN4 acts as a miRNA sponge for miR-24-3 to promote neurite growth). Additional information about circSPECCl can be found, by nonlimiting example, in Chen X, et al. m6A modification of circSPECCl suppresses RPE oxidative damage and maintains retinal homeostasis. Cell Rep. 2022 Nov 15;41(7): 111671. doi: 10.1016 / j.celrep.2022.111671. PMID: 36384115 (circSPECCl plays a role in resisting sub-retinal pigment epithelium oxidative damage and maintaining retinal homeostasis).

[0254] In some aspects, the contacting of the cell is in vivo, in vitro, or ex vivo. In some aspects the cell is a mammalian cell, optionally a HeLa cell or a HEK293 cell.

[0255] Also provided herein are methods to regulate circRNA or linear RNA translation in a subject in need, the method comprising, consisting of, or consisting essentially of, administering to the subject one or more circRNA, linear RNA, vector, plurality, composition, and / or isolated host cell described herein.

[0256] Also provided herein are methods to regulate circRNA or linear RNA biogenesis in a subject in need, the method comprising, consisting of, or consisting essentially of, administering to the subject one or more circRNA, linear RNA, vector, plurality, composition, and / or isolated host cell described herein. In one aspect, the method enhancesbiogenesis of circRNA or linear RNA endogenous in the subject. In one aspect, the method inhibits biogenesis of circRNA or linear RNA endogenous in the subject.

[0257] In some embodiments, the methods are used to administer a vaccine, such as a SARS- CoV-2, influenza, or Ebola virus vaccine to a subject in need. In some embodiments, the methods are used in protein replacement therapy. In still other embodiments, the methods are used in CAR T therapy. In still other embodiments, the methods are used in immune- oncology to treat cancer.

[0258] The methods are useful to treat subjects such as humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In certain embodiments the subject has or is suspected of having a neoplastic disorder, neoplasia, tumor, malignancy or cancer.

[0259] For the above methods, an effective amount is administered, and administration of the cell or population serves to attenuate any symptom or prevent additional symptoms from arising. When administration is for the purposes of preventing or reducing the likelihood of cancer recurrence or metastasis, the cell or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal.

[0260] The methods provide one or more of: (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression or relapse of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening)state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. Treatments containing the disclosed compositions and methods can be first line, second line, third line, fourth line, fifth line therapy and are intended to be used as a sole therapy or in combination with other appropriate therapies e.g., surgical recession, chemotherapy, radiation. In one aspect, treatment excludes prophylaxis.

[0261] Experimental

[0262] The following examples are intended to illustrate, and not limit the scope of the claims or this disclosure.

[0263] Example 1

[0264] Discussion

[0265] Applicant developed a tethering-based circRNA luciferase reporter system to evaluate the regulatory potential of 730 RBPs, comprising 846 unique isoforms, on circRNA expression. Our screen identified 267 isoforms that increased the luciferase signal and 29 isoforms that decreased it. A secondary screen in two cell lines validated the robustness of our results, allowing us to infer different regulatory modalities through which various RBPs control circRNA expression and translation. Our focus centered on RBPs that enhance circRNA translation, which holds great promise for applications in circRNA-based vaccines and therapeutics, as well as RBPs that regulate endogenous circRNAs, which delineates the biological mechanism underlying circRNA biogenesis.

[0266] By developing an exogenous circRNA luciferase reporter system, Applicant identified that two RBPs, LARP4B and ANXA2, enhanced circRNA translation. LARP4B increased circRNA translation universally, while ANXA2 promoted translation in a cell type-dependent manner. Interestingly, the translation promoting function of LARP4B also seems to stay true for its endogenous target, highlighting the capability of our screen to identify both effector for engineering purposes and regulators for understanding the circRNA biology in cells. Many previous studies have relied on plasmid-based reporter systems to identify regulatory elements that enhance circRNA translation, including m6A modification38and structured elements52that can drive translation of endogenous circRNAs. To assess the relevance of ourfindings to therapeutic formats, Applicant compared translation outcomes between plasmid- derived and in vitro synthesized circRNAs, the latter representing the format used in therapeutic applications. Applicant found both shared and distinct regulatory features, underscoring the importance of using synthesized circRNAs in future screens aimed at improving translation efficiency. This insight can better inform the design and interpretation of translational enhancement screens for therapeutic development. Our exogenous circRNA tethering assay provides a versatile platform for systematic screening of RNA-binding proteins (RBPs) with potential to enhance circRNA translation. Additionally, Applicant also demonstrated a proof-of-concept strategy to tether RNA-binding proteins (RBPs) to circRNAs, significantly enhancing their translational output. This lays the foundation for two future directions: (i) co-delivery of RBP-expressing constructs alongside therapeutic circRNAs, or (ii) engineering RNA aptamers into therapeutic circRNAs to recruit endogenous RBPs that enhance circRNA translation.

[0267] In exploring RBPs that regulate endogenous circRNAs, Applicant discovered an unexpected role of RBM15 separate from its known functions, serving as a multifaceted regulator that both promotes circRNA biogenesis and retains circRNAs in the nucleus. Domain mapping of RBM15 revealed that the RRM2 / 3 domains are responsible for nuclear retention of circRNAs, while the disordered and SPOC domains together enhance circRNA biogenesis. Using AP-MS, Applicant identified RBM15’s interaction with SRSF splicing factors, which likely promote circRNA biogenesis, and its RNA-dependent interaction with IGF2BP1, a circRNA adapter protein that RBM15 retains in the nucleus to mediate circRNA retention. Despite that SRSF proteins were previously shown to inhibit circRNA biogenesis in fly cells19, recently both this study and another CRISPR screen identified some of them as positive regulators of circRNA biogenesis in mammalian system, suggesting differences of circRNA regulation across species. Interestingly, the previously studied role of RBM15 in mediating m6A modification did not appear to contribute to its circRNA regulatory functions. Our analysis with eCLIP data revealed direct binding of RBM15 specifically near the BSJs of the circRNAs it regulates. This makes RBM15 one of the first identified exonic regulators of circRNA biogenesis, with its regulatory role conserved across species, as demonstrated in both human and mouse cells.

[0268] Although a varied percentage (13-40%) of circRNAs were shown to be conserved comparing different species and tissues49,50, a key unresolved question is whether and how these orthologous circRNAs are regulated in a conserved manner. Intronic repetitive elements have been previously proposed as conserved features that may regulate circRNA biogenesis through convergent evolution50, despite their very low evolutionary sequence conservation. In this study, Applicant identify RBM15 as one of the first examples of a circRNA regulator that binds evolutionary conserved exonic sequences to modulate circRNA biogenesis, helping to bridge this knowledge gap.

[0269] Materials and Methods

[0270] Generation of plasmid constructs

[0271] The circFirefly and circRenilla reporter sequences were designed by splitting Firefly and Renilla luciferase gene at an AG|GA sequence (to mimic original circZKSCANl backsplicing junction) and flanking EMCV IRES sequence. Additional multiple cloning sites were introduced before IRES for cloning 6xMS2 sequence into the circFirefly reporter. Both reporters without MS2 sequences were ordered from Twist Bioscience and cloned into the pTwist CMV vector.

[0272] The RBP-MCP tethering library were generated as previously described25. The RBM15 fragments and the negative zinc finger protein controls were cloned into the same vector by gateway cloning. The primers used to amplify RBM15 fragments were listed in Tables 4 and 6.

[0273] The exo-circFirefly and exo-circRenilla reporters were generated by amplifying from circFirefly and circRenilla reporter with overlap PCR and subcloned into circRNA-synlRES- R25-mNeonGreen plasmid9replacing synIRES and mNeonGreen by Gibson assembly. circRNA-synIRES-R25-mNeonGreen was a gift from Howard Chang (Addgene plasmid # 188115; http: / / n2t.net / addgene: 188115; RRID: Addgene_188115).

[0274] The overexpression plasmids were generated by cloning RBP ORFs into pEF5 / FRT / V5-DEST vector (Thermo Fisher, V602020) using gateway cloning. For knockdown, shRNA plasmids were derived from the Mission lentiviral shRNA library (Sigma-Aldrich).

[0275] All plasmids were verified by Sanger (Azenta) or Primordium sequencing. The reporter plasmid maps are available in Table 5, FIG. 15 and FIG. 16.

[0276] Cell lines

[0277] HEK293T, HeLa and Neuro-2a cells were originally purchased from the American Type Culture Collection and were not further authenticated. HeLa cells were a gift from Jamieson lab. Cells were routinely tested for mycoplasma contamination with a MycoAlert mycoplasma test kit (Lonza) and were found negative for mycoplasma.

[0278] Luciferase reporter assay

[0279] HeLa or HEK293T cells were seeded in 96 well plates precoated with poly D-lysine (PDL, Sigma-Aldrich) at 20K cells per well. After 24 hours, RBP-MCP, circFirefly, circRenilla plasmids were co-transfected in a ratio of 6: 1 : 1 (lOOng in total) with TransIT-LTl (Minis Bio) following manufacturers’ instructions. Cells were lysed and luciferase activity was measured with Dual-Luciferase Reporter Assay System (Promega) in a Spark Multimode Microplate Reader (Tecan), following the manufacturer’s instruction, 72 hours post transfection. RBM15 fragments tethering was carried out in HEK293T cells following the same procedures.

[0280] For exogenous luciferase reporters, HeLa or HEK293T cells were seeded in 96 well plates precoated with poly D-lysine at 20K cells per well. After 24 hours, RBP-MCP mRNA, exo-circFirefly, exo-circRenilla circRNAs were co-transfected in a ratio of 6: 1 : 1 (lOOng in total) with jetMESSENGER mRNA transfection reagent (Polyplus) following the manufacturer’s instructions. Cells were lysed and luciferase activity was measured with DualLuciferase Reporter Assay System (Promega) in a Spark Multimode Microplate Reader (Tecan), following the manufacturer’ s instruction, 48 hours post transfection.

[0281] Statistical analysis

[0282] Luciferase data was first normalized to the renilla internal control by calculating the ratio of firefly signal over renilla signal. The ratios were further normalized to the average ratio of Flag control to calculate fold change. Two-tailed Student’ s t-test was performed to calculate p value and multiple test correction was implemented to control FDR.

[0283] Gene Ontology analysis

[0284] Gene ontology (GO) analysis of the candidates from the screen was performed using DAVID online tools53,54. Background genes were set as the full list of the RBP-MCP library. Top GO terms in the Biological Processes were shown in the enriched GO term dot plot.

[0285] Protein Network analysis

[0286] Protein Network of the candidates from the screen were generated from the STRING database55. Only experimental and database evidences were used to build the network.

[0287] mRNA and circRNA synthesis

[0288] The RBP-MCP DNA template were linearized by PCR amplification and gel extraction. RBP-MCP mRNAs were produced with Hi Scribe® T7 ARC A mRNA Kit (with tailing, NEB) with the addition of 5mCTP and Pseudo-UTP, following the manufacturer’s instructions. The concentration was measured by nanodrop and size was measure by RNA Tapestation (Agilent).

[0289] For exo-circFirefly and exo-circRenilla circRNA production, the protocol was described previously27with slight modifications. Briefly, DNA templates were linearized by PCR amplification and gel extraction. circRNA precursors were synthesized from 100-200ng of linearized DNA template in a 20ul reaction using the HiScribe T7 High Yield RNA Synthesis Kit (NEB), following the manufacturer’s instructions. After transcription and DNase I digestion, 77ul RNase-free water and 2ul addition GTP (lOmM) were added to the reaction (total volume lOOul), and the reaction was heated to 55°C for 30 min. The RNA was purified with RNA Clean & Concentrator-25 (Zymo Research). To remove linear RNA, 20pg RNA was resuspended into final volume of 86pl RNase-free water and heated at 65°C for 3 min and immediately placed on ice for 3 min. Linear RNA was removed by digestion with 20U RNase R (Lucigen) for 15 min at 37°C, with addition of further 10U RNase R and 15 min incubation. RNA was then purified with RNA Clean & Concentrator-5. The concentration was measured by nanodrop and purity were assessed by RNA Tapestation.

[0290] RNA fractionation

[0291] Fractionation of cytoplasmic and nuclear RNA was performed as previously described56with slight modifications. In brief, 1 million cells were washed in IxPBS and subsequently permeabilized in 50pl of cold lysis buffer (lOmM Tris-HCl, pH 7.4, lOmMNaCl, 3mM MgC12, 0.1% IGEPAL CA-630) by gentle pipetting for 5 times. The nuclei were centrifuged at 500g for 5 min at 4°C. The supernatant was harvested as cytoplasmic faction and mixed with 150pl TRIzol LS and stored in -80°C until RNA extraction. The nuclei were washed in 200pl lysis buffer once, lysed in 200pl TRIzol Regent as the nuclear fractions, and stored in -80°C until RNA extraction. The performance of fractionation was tested by RT- qPCR to measure the relative levels of MALAT1 (nuclear) and ACTB mature mRNA (predominantly cytoplasmic).

[0292] RNA extraction, reverse transcription and qPCR

[0293] RNA samples were harvested in TRIzol Reagent or TRIzol LS Reagent and extracted with Direct-Zol RNA miniprep kit (Zymo Research), following the manufacturer’s instructions. 500pg RNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad) with the modification of scaling down to lOul reaction volume following the manufacturer’s instructions. cDNA were diluted 10 times and 1 pl of the diluted cDNA was loaded as template for qPCR reactions with Power SYBR™ Green PCR Master Mix (Thermo Fisher) following manufacturer’s instructions. qPCR primers for linear mRNAs were designed by NCBI Primer Blast. qPCR primers for circRNAs were either designed by circPrimer2.0 or acquired from literature. All qPCR primers (together with circRNA chromosome coordinate) used are listed in Supplementary Table 6. For human cells, HPRT (total and cytoplasmic fraction) and MALAT1 (nuclear fraction) were used as reference genes for normalization. For mouse cells, 18s rRNA was used as the reference gene for normalization.

[0294] Statistical Analysis

[0295] Relative expression was calculated with the delta delta Ct method. Statistical analysis of the qPCR results was performed on at least 3 biological replicates. One-tailed Student’s t- test was carried out to calculate p values.

[0296] Immunofluorescence

[0297] Cells were fixed with cold 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with 7% FBS in PBS for 30 min at room temperature. After blocking, cells were incubated with anti-V5 antibodies (CST), or IGF2BP1 antibodies (CST) for 1 h at room temperature, followed by washing 3 times with PBS and incubation with Alexa 488 or Alexa 555 conjugated secondary antibodies(Invitrogen) for another 1 h. The cells were counterstained with DAPI for 5 min, washed 3 times with PBS and subjected to imaging. Images were acquired with either a Keyence BZ- X800 microscope, or a Zeiss LSM780 confocal microscope (for IGF2BP1 localization quantification).

[0298] Imaging data analysis

[0299] Confocal images were processed and analyzed with Fiji (NIH)57. The “Threshold” function was used to determine the average intensity of antibody or nanobody staining for the corresponding fluorescence channel; the threshold value was set to capture all cellular intensity of the antibody or nanobody. To determine the fraction of nuclear signal in each image, Applicant created masks of the whole cell using the antibody or nanobody channel and masks of the nucleus using the DAPI channel. The DAPI mask was subtracted from the whole-cell mask to create a cytoplasm-only mask of each image. The masks were subtracted from the original image to remove extraneous intensity values, and the “Threshold” function was used to determine the intensity of the remaining pixels. The total intensity (i.e. area x intensity) of the nuclear antibody signal was divided by the total intensity of the whole cell to calculate the nuclear fraction. At least 400 cells were analyzed per condition. Student’s t-test was performed on the average nuclear faction in 10 imaging fields of both groups.

[0300] siRNA knockdown

[0301] ON-TARGETplus siRNAs were purchased from Horizon Discovery as pools of 4 siRNAs for each gene knockdown. siRNAs were transfected with DF1 transfection reagent (Horizon Discovery) following the manufacturer’s instructions using reverse transfection protocol. Cells were harvested 72 hours post transfection for RNA extraction. Knockdown efficiency was measured by qPCR.

[0302] shRNA lentivirus production and shMETTL3 stable cell line generation

[0303] To produce lentiviruses for generating METTL3 knockdown stable lines, HEK293T cells were seeded at 500K cells per well in antibiotic-free growth media (DMEM + 10% HI FBS) in 6-well tissue culture plates. After 24 hours, the cells were transfected with psPAX2, pMD2.G and pLKO.l shMETTL3 plasmids (500ng, 50ng, 500ng, respectively per well) with TransIT-LTl transfection reagent (3 pl per well). 18 hours post transfection, media was changed to high-FBS media (DMEM + 30% HI FBS). Lentiviruses were harvested twice at48 hours and 72 hours post transfection and combined. The lentiviruses were concentrated using PEG Virus Precipitation Kit (Abeam) following the manufacturer’s instructions.

[0304] For lentiviral transduction, 400,000 HEK293T cells were seeded in each well of a 6 well plate. After 24 hours, media were replaced with culture media supplemented with lOpl concentrated lentivirus and polybrene at the final concentration of 8pg / ml. The cells were selected in puromycin containing media (Ipg / ml) from 24 hours post infection for one week. The knockdown efficiency was measured by qPCR.

[0305] circRNA sequencing library preparation

[0306] circRNA sequencing libraries were prepared following a homebrewed protocol. Briefly, Ipg of total RNA from overexpression or knockdown samples were subjected to ribosomal RNA (rRNA) removal using RiboMinus kit (Thermo Fisher). The rRNA-depleted RNA were poly-A tailed with E.coli poly A polymerase (Thermo Fisher) and digested with 10U RNase R (Lucigen) at 37°C for 30 min. Reverse transcription was carried out with random hexamer with PCR handle 1. First strand cDNA was poly-A tailed by terminal deoxynucleotidyl transferase (Thermo Fisher), followed by second strand synthesis with NEBNext Hifi 2x PCR master mix (NEB) using oligo dT with PCR handle 2. The double stranded cDNA was pre-amplified with NEBNext Hifi 2x PCR master mix with PCR handle 1 and 2. Pre-amplified cDNA was then sized selected by 0.5x Ampure XP beads (Beckman Coulter) cleanup and subjected to Nextera XT DNA library preparation (Illumina), following the manufacturer’s instructions.

[0307] circRNA sequencing analysis

[0308] Reads were processed with ciri quant software58. First, reads with adapter-trimmed with skewer with parameter “-z -r 0.2 -d 0.2 -q 13 -1 20”, and then use ciri quant software with default parameters without RNase R correction. The output quantifies the number of BSJ and FSJ reads detected per circular RNA, as well as genomic annotations forming the circular RNA (exon, intron, antisense, intergenic or unknown.)

[0309] To quantify expression of each circular RNA, Applicant calculated counts per million (CPM) per each BSJ:CPMi = 106x BSJi / total sequencing depth

[0310] Applicant performed the differential expression analysis followed ciri quant instruction, “Study without biological replicates” section for the knockdown and overexpression circRNA sequencing screen; and “Study with biological replicates” section for Rbml5 overexpression and knockdown in N2A cells.

[0311] rRNA-depleted total RNA sequencing and analysis

[0312] rRNA-depleted total RNA libraries were prepared with Illumina Stranded Total RNA Prep with Ribo-Zero Plus following the manufacturers’ instruction.

[0313] Reads were processed using NF-core RNAseq (available at https: / / github.com / nf- core / rnaseq) version 3.13.259. Briefly described, reads were trimmed using trimmomatic (cutadapt v3.4) and mapped using STAR (v2.7.9a). Mapped reads were quantified using Salmon (vl.10.1). Differential expression and splicing analysis compared triplicates of siRNA knockdown of RBM15 against a non-targeting control (HEK_siRBM15 / HEK siNT) as well as triplicates of RBM15 overexpression against a FLAG control (RBM15 OE / FLAG). For these analyses, DESeq260(vl.38.3) and rMATS61(v4.3) were used.

[0314] AP-MS

[0315] HEK293T cells were lysed and affinity purified using 10 pg per sample of anti- RBM15 antibody (Bethyl). In brief, the cell lysates with antibody were incubated with magnetic beads overnight in the cold room. Then, 5 pl of 10 mg mF1RNase A was added to ribonuclease-positive conditions at this step. Supernatants were removed, and beads were washed with NP-40 buffer, twice in Buffer 2 (50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgCh, 0.05% NP-40 and 5% glycerol) and twice in Buffer 3 (50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgCh and 5% glycerol). After the last wash, the wash buffer was spun down and aspirated completely, and the beads were resuspended in 80 pl of trypsin buffer (2 M urea, 50 mM Tris (pH 7.5), 5 pg mF1trypsin) to digest the bound proteins at 37 °C for 1 h with agitation. The beads were centrifuged at 100 g for 30 s, and the partially digested proteins (the supernatant) were collected. The beads were then washed twice with 60 pl of urea buffer (2 M urea, 50 mM Tris (pH 7.5)). The supernatant of both washes was collected and combined with the partially digested proteins (final volume, 200 pl). After brief centrifugation, the combined partially digested proteins were cleared from residual beads. Then, 80 pl of these partially digested proteins was used; disulfide bonds were reduced with5 mM dithiothreitol (DTT); and cysteines were subsequently alkylated with 10 mM iodoacetamide. Samples were further digested by adding 0.5 pg of sequencing-grade modified trypsin (Promega) at 25 °C. After 16 h of digestion, samples were acidified with 1% formic acid (final concentration). Tryptic peptides were desalted on C18 StageTips according to Protocol for micro-purification enrichment pre-fractionation and storage of peptides for proteomics using StageTips62, and evaporated to dryness in a vacuum concentrator and reconstituted in 15 pl of 3% acetonitrile / 2% formic acid for liquid chromatography with tandem mass spectrometry (LC-MS / MS).

[0316] LC-MS / MS analysis was performed on a Q Exactive HF. Five microliters of total peptides was analyzed on a Waters M-Class UPLC using a 15-cm lonOpticks column (1.7 pm, 120 A, 75 pm x 15 cm) coupled to a benchtop Thermo Fisher Scientific Orbitrap Q Exactive HF mass spectrometer. Peptides were separated at a flow rate of 400 nl / min with a 90-min gradient, including sample loading and column equilibration times. Data were acquired in data-dependent (DDA) mode. MSI spectra were measured with a resolution of 120,000, an AGC target of 3e6 and a mass range from 300 m / z to 1,800 m / z; MS2 spectra were measured at a resolution of 15,000, an AGC target of le5, a TopN of 12, an isolation window of 1.6 m / z and a mass range from 200 m / z to 2,000 m / z.

[0317] AP-MS data analysis

[0318] Proteomics raw data was analyzed by MaxQuant v2.0.3.063using a UniProt database (Homo sapiens, UP000005640), and MS / MS searches were performed under default settings with LFQ quantification and match between the runs. Data was further analyzed in R v3.6.3. Contaminants, and proteins only identified by site or reverse were removed, LFQ intensity values were transformed to parts per million, a pseudocount of 1 was added, and then the values were log2 transformed. Proteins with a mean MS / MS count value for each IP condition below 5 were removed from subsequent analysis and missing values were imputed with values from the bottom of the signal distribution for measured protein intensities. Interacting proteins were identified as those that passed a log2FC sample IP over control IP cutoff of 1 and a p-value of 0.05. Sample and control IPs were matched by RNase treatment.

[0319] eCLIP and eCLIP data analysis

[0320] eCLIP was performed as previously described64

[0321] Computational analysis of eCLIP data was performed using the default settings of Skipper65available on Github (https: / / github.com / YeoLab / skipper). Reads were mapped to human genome assembly GRCh38.

[0322] RNA immunoprecipitation (RIP) and RIP-circRNA-seq analysis

[0323] RIP experiments were performed as previously described66with slight modifications. Briefly, for each replicate, around 20 million HEK293T cells were harvested, washed with cold PBS and lysed on ice with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8, 5 mM MgC12, 1 mM CaC12, 0.5 mM EDTA, 0.5% NP-40, 1 :200 proteinase inhibitor, 1 :500 RNaseOUT). The lysates were centrifuged at 18,000 ref for 10 minutes in 4 °C to remove cell debris. The supernatant was first incubated with lysis buffer washed Protein A beads in 4 °C for 30 minutes to preclear non-specific binding of the beads. Meanwhile, additional Protein A Dynabeads (Invitrogen, 10002D) were washed and incubated with respective antibodies (anti-LARP4B, anti-RBM15, control rabbit IgG) in lysis buffer at room temperature for 15 minutes. Protein A beads coated with antibodies were then washed twice with lysis buffer. Then, 30 pl of the lysate supernatant were kept as input; while 450pl of the lysate supernatant was added to either anti-LARP4B / anti-RBM15 coated or control IgG coated Protein A beads and incubated at 4 °C for overnight. After the incubation, the beads were washed three times with lysis buffer. The IP RNA was released by proteinase K digestion, and both the IP and input RNA was cleaned up by the RNA Clean & Concentrator -5 kit following manufacturers’ instructions. The resulting RNA was subjected to circRNA library preparation or reverse transcription followed by qPCR quantifications.

[0324] The statistical procedure is similar as presented in Skipper65. Let there be two libraries: foreground library (IP), and background library (Input). Applicant use betabinomial distribution as the null distribution to model the BSJ counts between foreground and background. The overdispersion term is obtained by pairwise comparing the replicates. The probability of success is set to the fraction of total BSJs in the foreground library.

[0325] Integrated analysis of eCLIP and circRNA sequencing

[0326] RBM15 positively regulated circRNAs were converted to bed format using bedtools67. eCLIP bed files of reproducible binding sites were generated as Skipper output. The overlap between circRNAs and eCLIP bed files were identified by the intersect functionfrom bedtools. Odds ratios were calculated and Fisher’s exact test or chi-square test was used to calculate p value.

[0327] Metadensity plot

[0328] To visualize RBM15 eCLIP signals around circular RNAs, Applicant utilize metadensity68, a package that aggregates eCLIP raw signals and plot against selected genomic annotations. Circular RNAs are stratified into three groups, up-regulated upon knock-down (n = 614), downregulated upon knock-down (n = 4) and those without change. Applicant randomly sampled 500 circular RNA without change as comparison. Each circular BSJ was built into the gene annotation, and ENCODE HepG2 eCLIP signals was plotted around the BSJ-forming interval. Specifically, Applicant utilize 'relative information' method to quantify the difference between IP and SM-Input.

[0329] Table 2. Candidate RBP isoforms. RBP position and number correspond to the RBP position and number in Table 7 (accession numbers of the RBPs). Down-regulators have a negative log2foldchange and up-regulators have a positive log2foldchange.

[0330] Table 3. Localization of RBP. In table, localization number indicates: 1 cytoplasmic; 0 both; -1 nuclear.

[0331] Table 4. RBM15 primers.

[0332] Table 5 - Reporter Plasmid Maps

[0333] See also FIG. 15 and FIG. 16. The sequence map information at the benchling.com pages are incorporated by reference herein.

[0334] Cell lines

[0335] Table 6. qPCR primers.

[0336] RBP accession numbers

[0337] Table 7 shows the accession numbers for the RBPs tested in several embodiments of the screen. The RBP tested in the assays may be a functional fragment of the sequence under the accession number. These 296 RBPs were identified in the luciferase screen and / or qPCR assays as circRNA regulators. As seen in corresponding Table 2, the RBPs are involved in both up-regulation and down-regulation of circRNAs, and play a role in mechanisms including circRNA translation, circRNA biogenesis, and circRNA nuclear export. The sequences and / or accession numbers are intended to be representative, rather than exhaustive, of the RBPs.

[0338] Table 7 RBP accession numbers. Select RBPs have multiple isoforms. RBPs were identified as circRNA regulators in luciferase and qPCR assays. The number and position of RBPs in Table 5 corresponds to the position of RBPs in Table 2.

[0339] The RBPs

[0340] Table 8 shows the nucleotide and amino acid sequences for the RBPs tested in the secondary screen. The sequences for the RBPs in Table 8 are intended to be representative, rather than exhaustive, of the RBPs.

[0341] Table 8 RBP sequence information. Included in the table are symbol (gene ID), nucleotide length, NCBI accession number, nucleotide sequence, and amino acid sequence. Select RBPs have multiple isoforms.Example 2

[0342] In Experiment No. 1, Applicant established a mechanism supporting how circRNA translation can be enhanced by tethering LARP4B. Several embodiments include: 1. LARP4B universally enhances circRNA translation across different IRES elements. (See FIGS. 2E - 2G); 2. LARP4B may enhance circRNA translation through recruiting EIF3 A and EIF4G2. See FIG. 2H; and 3. LARP4B preferentially binds AU-rich sequence endogenously which can potentially be used to design aptamers to recruit endogenous LARP4B to therapeutic circRNA construct. See FIG. 3.

[0343] In addition, Applicant also performed an extended RBP tethering screen using the in vitro synthesized system (as illustrated as exocircFirefly, exocircRenilla and RBP-MCP mRNA system as disclosed in Experiment No. 1) to expand the RBP choices to enhance circRNA translation in a therapeutic relevant setting (exocircFirefly RBP tethering screen). This screen was performed in both HEK293T cells and HeLa cells.

[0344] In HEK293T cells, 13 RBPs significantly increased the luciferase signal upon tethering to the exocircFirefly reporter, namely, N0B1, NUFIP2, GAPDH, GNB2L1, BCDIN3D, PKM, HSPB1, GFM1, ILF3, RBPMS, DDX21, RPL35, FAM120A. See FIG.17.

[0345] In HeLa cells, 7 RBPs significantly increased the luciferase signal, namely, HSPB1, NUFIP2, SPATS2, BCDIN3D, NOB1, PKM, GNB2L1. (FIG. 18)

[0346] Among these RBPs, 6 of them, including HSPB1, NUFIP2, BCDIN3D, NOB1, PKM, GNB2L1, appeared to ubiquitously enhance circRNA translation in both cell lines, thus highlighting their utility to promote translation of therapeutic circRNA vectors in more cell types.

[0347] Linear mRNAs

[0348] Enhancing mRNA translation through tethering of specific RBPs can apply to linear mRNAs. The methods described herein for circRNAs can be applied to linear mRNAs.

[0349] Equivalents

[0350] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0351] The present technology illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0352] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0353] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0354] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0355] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0356] Embodiments

[0357] Clause 1. A circular RNA (circRNA) bound to one or more RNA-binding proteins (RBPs).

[0358] Clause 2. A vector comprising: a) a circRNA comprising one or more RNA hairpins; and b) an RNA-binding protein (RBP) linked to the circRNA at the one or more RNA hairpins.

[0359] Clause 3. A linear RNA bound to one or more RNA-binding proteins (RBPs).

[0360] Clause 4. A vector comprising: a) a linear RNA comprising one or more RNA hairpin; and b) an RNA-binding protein (RBP) linked to the mRNA at the one or more RNA aptamer.

[0361] Clause 5. The vector of any of clause 2-4, wherein the RNA hairpin is derived from a bacteriophage selected from: MS2, R17, X, PP7, QP or GA; iron responsive protein (IRP); bovine immunodeficiency virus (BIV), or human U1 small nuclear ribonucleoprotein A.

[0362] Clause 6. The vector of clause 5, wherein the RNA hairpin comprises a sequence as shown in any one of SEQ ID NOs: 1-10.

[0363] Clause 7. The circRNA of clause 1, the linear RNA of claim 3 or the vector of any one of clauses 2, or 4-6, wherein the circRNA encodes a therapeutic polypeptide or reporter polypeptide.

[0364] Clause 8. The vector of any one of clauses 2, 4-7, wherein the vector is selected from a plasmid, a viral vector, a cosmid, or a phage, optionally wherein the viral vector is selected from a baculovirus, a retrovirus, or an adenovirus.

[0365] Clause 9. The circRNA of clause 1, the linear RNA of claim 3 or the vector of any one of clauses 2, or 4-8, wherein the RBP is selected from: LARP4, LARP4B, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, or DDX52 or a functional fragment or variant of each thereof.

[0366] Clause 10. The circRNA of clause 1, the linear RNA of claim 3 or the vector of any one of clauses 2, or 4-8, wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8 or a functional fragment or variant of each thereof.

[0367] Clause 11. A plurality of circRNA of any of clauses 1, the linear RNA of claim 3 or the vector of any one of clauses 2, or 4-10, wherein the circRNAs and / or the vectors are the same or different from each other.

[0368] Clause 12. An isolated host cell comprising one or more of the circRNA of any of clauses 1, the linear RNA of clauses 3 or the vector of any one of clauses 2, 4-10.

[0369] Clause 13. The isolated host cell of clause 12, wherein the cell is a procaryotic cell or a eukaryotic cell.

[0370] Clause 14. A composition comprising a pharmaceutically acceptable carrier and one or more of: the circRNA of any of clauses 1, the linear RNA of claim 3 or the vector of any one of clauses 2, or 4-10; and / or the plurality of claim 11; and / or the isolated host cell of clause 12 or 13.

[0371] Clause 15. The composition of clause 14, further comprising a preservative or a lyophilization agent.

[0372] Clause 16. The composition of clause 14 or 15, further comprising an additional therapeutic agent.

[0373] Clause 17. The composition of clause 16, wherein the additional therapeutic agent is an RBP, wherein the RBP is: a. the same or different as the one or more RBP of clause 1 or 2; b. a positive or negative regulator of circRNA; or c. an RBP regulates RNA biogenesis, splicing, transport, translation, and / or degradation.

[0374] Clause 18. The composition of clause 17, wherein the RBP is selected from RBM15, ANXA2, DCP1A, DDX19A, DDX52, G3BP2, LARP4, LARP4B, RBM17, THOC1, TOB2, U2AF2, or ZC3HAV1 or a functional fragment or variant of each thereof.

[0375] Clause 19. A method to regulate circRNA translation in a cell, comprising contacting the cell with one or more of: circRNA of any of clauses l,the linear RNA of claim 3 or the vector of any one of claims 2, 4-10; and / or the plurality of clause 11; and / or the isolated host cell of clause 12 or 13; and / or the composition of clause 14-18.

[0376] Clause 20. The method of clause 19, wherein the method enhances circRNA translation.

[0377] Clause 21. A method to regulate circRNA biogenesis in a cell, comprising contacting the cell with one or more of: circRNA of any of clauses l,the linear RNA of clause 3 or the vector of any one of clause 2, or 4-10; and / or the plurality of clause 11; and / or the isolated host cell of clause 12 or 13; and / or the composition of clause 14-18.

[0378] Clause 22. The method of any of clauses 19-21, wherein the contacting is in vivo, in vitro, or ex vivo.

[0379] Clause 23. The method of any of clauses 19-22, wherein the cell is a mammalian cell, optionally a HeLa cell or a HEK293T cell.

[0380] Clause 24. A method to regulate circRNA translation in a subject in need, the method comprising administering to the subject one or more of: circRNA of any of clause 1, the linear RNA of clause 3 or the vector of any one or more of clause 2, 4-10; and / or the pluralityof clause 11; and / or the isolated host cell of clause 12 or 13; and / or the composition of clause 14-18.

[0381] Clause 25. A method to enhance circRNA translation in a cell, the method comprising contacting the cell with a vector comprising a circRNA bound to LARP4B or a functional fragment or variant of each thereof.

[0382] Clause 26. A method to enhance circRNA translation in a cell, the method comprising contacting the cell with a vector comprising a circRNA bound to ANXA2 or a functional fragment or variant of each thereof.

[0383] Clause 27. A method to regulate endogenous circRNAs in a cell, the method comprising contacting the cell with one or more RBPs and / or vector comprising a polynucleotide encoding the one or more RBPs, optionally where the vector comprises a circRNA.

[0384] Clause 28. A method to enhance circRNA biogenesis in a cell, the method comprising contacting the cell with a polynucleotide encoding RBM15 or a functional fragment or variant thereof.

[0385] Clause 29. The method of any one of clauses 26-28, wherein the contacting is in vitro or in vivo.

[0386] Clause 30. The single construct system of clause 32, wherein the aptamer is RBP specific.

[0387] Clause 31. A circular RNA (circRNA) bound to one or more RNA-binding proteins (RBPs), or a polynucleotide encoding the circRNA, optionally wherein the circRNA is directly or indirectly bound to the one or more RBPs.

[0388] Clause 32. The circRNA of clause 31, wherein the circRNA comprises at least one RNA hairpin, aptamer, or AU-rich sequence capable of recruiting an endogenous or exogenous RPB that enhances translation.

[0389] Clause 33. A vector comprising: a) a circRNA comprising one or more RNA hairpins or a polynucleotide encoding the circRNA; and b) an RNA-binding protein (RBP) linked to the circRNA at the one or more RNA hairpins, or recruited by an AU-rich aptamer encoded in the circRNA to recruit endogenous LARP4B or a functional fragment or variant thereof.

[0390] Clause 34. A linear RNA bound to one or more RNA-binding proteins (RBPs), or a DNA or RNA polynucleotide encoding the linear RNA, optionally wherein the linear RNA is directly or indirectly bound to the one or more RBPs.

[0391] Clause 35. A vector comprising: a) a linear RNA comprising one or more RNA hairpin, or a polynucleotide encoding the linear RNA; and b) an RNA-binding protein (RBP) linked to the mRNA at one or more RNA hairpins, and optionally wherein the RPB is recruited via an apatamer or an AU-rich sequence.

[0392] Clause 36. The vector of clause 33 or 35, wherein the RNA hairpin is derived from a bacteriophage selected from: MS2, R17, X, AN, UlsnRNP aptamer, a boxB element, an AU- rich motif, PP7, QP or GA; iron responsive protein (IRP); bovine immunodeficiency virus (BIV), or human U1 small nuclear ribonucleoprotein A; a hairpin engineered to bind a specific RBP or an aptamer, or an AU-rich aptamer or element that recruits an endogenous RPB, optionally where in the RPB comprises LARP4B or a functional fragment or variant thereof.

[0393] Clause 37. The circRNA, the linear RNA, the polynucleotide or the vector of any preceding clause, wherein the RBP is selected from the group consisting of LARP4, LARP4B, HSPB1, NUFIP2, BCIN3D, NOB1, PKM, GNB2L1, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, THOC1, DDX55, GTF2E2, RDBP, DDX52, or a functional fragment or variant of each thereof, or wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8 or a functional fragment or variant of each thereof.

[0394] Clause 38. The circRNA, the linear RNA, the polynucleotide or the vector of any preceding clause, wherein the RBP is a translation enhancer by in vitro exocircFirefly luciferase reporter assay screening in a mammalian cell, optionally a mammalian cell that is an HEK293 cell or a HeLa cell.

[0395] Clause 39. The vector of any of clauses 33 or 35-38, wherein the circRNA, the linear RNA, or the polynucleotide further comprises a sequence encoding a therapeutic polypeptide or protein, a vaccine antigen, a cytokine, a reporter polypeptide (optionally luciferase or GFP), an immunoregulatory protein, or a chimeric antigen receptor.

[0396] Clause 40. The vector of any one of clauses 33 or 35-37, wherein the RNA hairpin comprises a sequence as shown in any one of SEQ ID NOs: 1-10.

[0397] Clause 41. The vector of any one of clauses 33 or 35-39, wherein the vector is selected from a plasmid, a viral vector, a cosmid, or a phage, optionally wherein the viral vector is selected from a baculovirus, a retrovirus, or an adenovirus.

[0398] Clause 42. A plurality of circRNAs, linear RNAs, polynucleotides or vectors of any preceding clauses, wherein the circRNAs, linear RNAs, polynucleotides or vectors are the same or different from each other.

[0399] Clause 43. An isolated host cell comprising or expressing one or more of the circRNA, the linear RNA, the polynucleotide, or the vector of any preceding clause.

[0400] Clause 44. The isolated host cell of clause 43, wherein the cell is a procaryotic cell or a eukaryotic cell, and optionally a mammalian cell.

[0401] Clause 45. A composition comprising a pharmaceutically acceptable carrier and one or more of: the circRNA, the linear RNA, the polynucleotide, or the vector, and / or the plurality, or the isolated host cell of any preceding clause.

[0402] Clause 46. The composition of clause 45, further comprising a preservative, a lyophilization agent, or a stabilizer.

[0403] Clause 47. The composition of clause 45 or 46, further comprising an additional therapeutic agent.

[0404] Clause 48. The composition of clause 47, wherein the additional therapeutic agent is selected from an RBP, wherein the RBP is: a. the same or different as the one or more RBP of any preceding clause; b. a positive or negative regulator of circRNA; or c. an RBP regulates RNA biogenesis, splicing, transport, translation, and / or degradation.

[0405] Clause 49. The composition of clause 48, wherein the RBP is selected from HSPB1, NUFIP2, BCIN3D, NOB1, PKM, GNB2L1, RBM15, ANXA2, DCP1A, DDX19A, DDX52, G3BP2, LARP4, LARP4B, RBM17, THOC1, TOB2, U2AF2, or ZC3HAV1 or a functional fragment or variant thereof.

[0406] Clause 50. The composition of clause 48, wherein the RPB is selected from LARP4, LARP4B, RBM4, EIF3C, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX55, GTF2E2, RDBP, or a functional fragment or variant of each thereof, or wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8 or a functional fragment or variant thereof.

[0407] Clause 51. A method to regulate circRNA translation in a cell, comprising contacting the cell with one or more of the circRNA, the linear RNA, the polynucleotide, the vector, the plurality, the isolated host cell, or the composition of any preceding clause.

[0408] Clause 52. The method of clause 51, wherein the method enhances circRNA or linear RNA translation.

[0409] Clause 53. A method to regulate circRNA or linear biogenesis in a cell, comprising contacting the cell with one or more of the circRNA, the linear RNA, the polynucleotide, the vector, the plurality, the isolated host cell, or the composition of any preceding clause.

[0410] Clause 54. The method of any of clauses 51-53, wherein the contacting is in vivo, in vitro, or ex vivo.

[0411] Clause 55. The method of any of clauses 51-54, wherein the cell is a mammalian cell, optionally a HeLa cell or a HEK293T cell.

[0412] Clause 56. A method to regulate circRNA or linear RNA translation in a subject in need, the method comprising administering to the subject one or more of the circRNA, the linear RNA, the polynucleotide, the vector, the plurality, the isolated host cell, or the composition of any preceding clause.

[0413] Clause 57. A method to enhance circRNA or linear RNA translation in a cell, the method comprising contacting the cell with a vector comprising a circRNA or linear RNA bound to LARP4B or a functional fragment or variant thereof.

[0414] Clause 58. A method to enhance circRNA or linear RNA translation in a cell, the method comprising contacting the cell with a vector comprising a circRNA or linear RNA bound to ANXA2 or a functional fragment or variant thereof.

[0415] Clause 59. A method to regulate endogenous circRNAs or linear RNAs in a cell, the method comprising contacting the cell with one or more RBPs and / or vector comprising a polynucleotide encoding the one or more RBPs, optionally where the vector comprises a circRNA or linear RNA.

[0416] Clause 60. A method to enhance circRNA or linear RNA biogenesis in a cell, the method comprising contacting the cell with a polynucleotide encoding RBM15 or a functional fragment or variant thereof.

[0417] Clause 61. The method of any one of clauses 56-60, wherein the contacting is in vitro or in vivo.

[0418] Clause 62. A method to regulate RNA in a cell, the method comprising contacting the cell with the single construct system as described herein, wherein an endogenous RBP specific to the aptamer is recruited to the construct system and binds with the aptamer.

[0419] Clause 63. A method of enhancing translation of a circRNA or linear RNA in a cell, comprising contacting the cell with a circRNA or linear RNA respectively, or a vector of any preceding clause, wherein the circRNA or linear RNA is bound to LARP4B, ANXA2, or an RBP or a functional fragment or variant of each thereof, identified as a translation enhancer.

[0420] Clause 64. A method of recruiting an endogenous RBP to a circRNA or linear RNA, comprising introducing into a cell a vector or RNA comprising an aptamer, hairpin, or sequence specific for an endogenous RBP or a functional fragment or variant of each thereof, thereby enhancing translation, stability, nuclear export, or degradation of the RNA.

[0421] Clause 65. A method of enhancing translation of a circRNA or linear RNA in a cell, the method comprising co-delivering: (a) a circRNA comprising at least one RNA hairpin or aptamer, and (b) an RBP, or a linear RNA or DNA encoding an RBP that binds the RNA hairpin or aptamer or a functional fragment or variant of each thereof.

[0422] Clause 66. A method of enhancing translation of a circRNA or linear in a cell, the method comprising delivering a single construct comprising a circRNA or linear RNA respectively encoding a gene-of-interest and at least one aptamer moiety that recruits an endogenous translation enhancing RBP.

[0423] Clause 67. A method of treating a disease or disorder in a subject, comprising administering an effective amount of the composition of any preceding composition claim, wherein the composition enhances translation of a therapeutic or prophylactic protein by recruitment or tethering of at least one RBP.

[0424] Clause 68. A kit comprising: (a) a circRNA, linear RNA, or vector of any preceding circRNA, linear RNA or vector of any preceding clause; (b) an RBP or encoding nucleic acid, or instructions for co-delivery or recruitment of a translation enhancing RBP.

[0425] Clause 69. A method of screening candidate RBPs, hairpin, or aptamer sequences for their ability to enhance translation, stability, export, or degradation of a circRNA or linear RNA, comprising contacting a candidate RBP, hairpin, or aptamer with a reporter RNA in a cell and measuring a change in reporter expression or RNA abundance.

[0426] Clause 70. The method of clause 69, wherein the cell is a mammalian cell, optionally a HET293 cell or a HeLa cell.

[0427] Clause 71. The use of any circRNA, linear RNA, vector, or composition of any of the preceding clauses in the manufacture of a medicament for the treatment or prevention of a disease or condition.

[0428] Clause 72. A method for increasing translation of a protein from a circRNA or linear RNA in a mammalian cell, comprising introducing into the cell a circular RNA containing (a) an AU-rich aptamer sequence and (b) a coding sequence, such that the AU-rich aptamer recruits endogenous LARP4B and thereby enhances translation of the encoded protein.

[0429] Clause 73. The method of clause 72, further comprising the step of identifying an RNA-binding protein that increases translation of circRNA comprising the steps of: a) expressing a plurality of translation-enhancing RBPs, each fused to a specific tethering domain, b) screening for increased expression of circRNA-encoded reporter protein in a human cell line, and c) selecting RBPs which provide a statistically significant increase in translation (p < 0.05, and log2(fold-change) > 0.5) in two or more cell lines, preferably both HEK293T and HeLa cells.

[0430] Clause 74. A method for enhancing circRNA or linear RNA translation in a cell in vitro or in vivo, comprising administering a circular RNA with an AU-rich aptamer and / or a tethering system for an RBP from the group of LARP4B, HSPB1, NUFIP2, BCDIN3D, NOB1, PKM, or GNB2L1, or a functional fragment or variant of each thereof, optionally together with a polynucleotide or protein corresponding to the RBP, such that translation from the circRNA is increased compared to a control lacking the recruited RBP or aptamer.

[0431] Clause 75. The method of clause 74 wherein the cell is a mammalian cell.

[0432] Clause 76. The method of any clause 74 or 75, wherein enhanced translation is caused by recruitment of LARP4B and subsequent interaction of LARP4B with cellular translationinitiation factors EIF3 A and EIF4G2 or a functional fragment or variant of each thereof.

[0433] Clause 77. A composition, system, or method of any one of the preceding clauses, wherein the composition or system is configured for use in multiple mammalian cell types, including but not limited to HEK293T and HeLa cells.

[0434] Clause 78. A panel of engineered circular RNAs or RNA-binding protein effectors selected by exocircFirefly luciferase reporter screening in at least two cell types, wherein each RBP demonstrates a statistically significant increase in reporter translation, the panel comprising at least two, three, four, five, or all of HSPB1, NUFIP2, BCDIN3D, NOB1, PKM, and GNB2L1. or a functional fragment or variant of each thereof.

[0435] Clause 79. A method of designing a circular RNA for therapeutic use, comprising the steps of: a) analyzing AU content of known LARP4B-bind sequences, b) encoding an AU- rich aptamer of at least 6 nucleotides in the 5' or 3' UTR or non-coding region of the circular RNA, and c) introducing the designed circRNA into a mammalian cell, wherein the AU-rich region recruits endogenous LARP4B and enhances translation efficiency.

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Claims

WHAT IS CLAIMED IS:

1. A circular RNA (circRNA) bound to one or more RNA-binding proteins (RBPs), or a polynucleotide encoding the circRNA, optionally wherein the circRNA is directly or indirectly bound to the one or more RBPs.

2. The circRNA of claim 1, wherein the circRNA comprises at least one RNA hairpin, aptamer, or AU-rich sequence capable of recruiting an endogenous or exogenous RPB that enhances translation.

3. A vector compri sing : a) a circRNA comprising one or more RNA hairpins or a polynucleotide encoding the circRNA; and - b) an RNA-binding protein (RBP) linked to the circRNA at the one or more RNA hairpins, or recruited by an AU-rich aptamer encoded in the circRNA to recruit endogenous LARP4B.

4. A linear RNA bound to one or more RNA-binding proteins (RBPs), or a DNA or RNA polynucleotide encoding the linear RNA, optionally wherein the linear RNA is directly or indirectly bound to the one or more RBPs.

5. A vector compri sing : a) a linear RNA comprising one or more RNA hairpin, or a polynucleotide encoding the linear RNA; and b) an RNA-binding protein (RBP) linked to the mRNA at one or more RNA hairpins, and optionally wherein the RPB is recruited via an apatamer or an AU-rich sequence.

6. The vector of claim 3 or 5, wherein the RNA hairpin is derived from a bacteriophage selected from: MS2, R17, , kN, UlsnRNP aptamer, a boxB element, an AU-rich motif, PP7, QP or GA; iron responsive protein (IRP); bovine immunodeficiency virus (BIV), or human U1 small nuclear ribonucleoprotein A; a hairpin engineered to bind a specific RBP or an aptamer, or an AU-rich aptamer or element that recruits an endogenous RPB, optionally where in the RPB comprises LARP4B or a functional fragment or variant thereof.

7. The circRNA, the linear RNA, the polynucleotide or the vector of any preceding claim, wherein the RBP is selected from the group consisting of LARP4, LARP4B, HSPB1, NUFIP2, BCIN3D, N0B1, PKM, GNB2L1, RBM4, EIF3C, ANXA2, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX19A, TH0C1, DDX55, GTF2E2, RDBP, DDX52, or a functional fragment or variant of each thereof, or wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8 or a functional fragment or variant of each thereof.

8. The circRNA, the linear RNA, the polynucleotide or the vector of any preceding claim, wherein the RBP is a translation enhancer by in vitro exocircFirefly luciferase reporter assay screening in a mammalian cell, optionally a mammalian cell that is an HEK293 cell or a HeLa cell.

9. The vector of any of claims 3 or 5-8, wherein the circRNA the linear RNA, or the polynucleotide further comprises a sequence encoding a therapeutic polypeptide or protein, a vaccine antigen, a cytokine, a reporter polypeptide (optionally luciferase or GFP), an immunoregulatory protein, or a chimeric antigen receptor.

8. The vector of any one of claims 3 or 5-7, wherein the RNA hairpin comprises a sequence as shown in any one of SEQ ID NOs: 1-10.

10. The vector of any one of claims 3 or 5-9, wherein the vector is selected from a plasmid, a viral vector, a cosmid, or a phage, optionally wherein the viral vector is selected from a baculovirus, a retrovirus, or an adenovirus.

11. A plurality of circRNAs, linear RNAs, polynucleotides or vectors of any preceding claims, wherein the circRNAs, linear RNAs, polynucleotides or vectors are the same or different from each other.

12. An isolated host cell comprising or expressing one or more of the circRNA, the linear RNA, the polynucleotide, or the vector of any preceding claim.

13. The isolated host cell of claim 12, wherein the cell is a procaryotic cell or a eukaryotic cell, and optionally a mammalian cell.

14. A composition comprising a pharmaceutically acceptable carrier and one or more of: the circRNA, the linear RNA, the polynucleotide, or the vector, and / or the plurality, or the isolated host cell of any preceding claim.

15. The composition of claim 14, further comprising a preservative, a lyophilization agent, or a stabilizer.

16. The composition of claim 14 or 15, further comprising an additional therapeutic agent.

17. The composition of claim 16, wherein the additional therapeutic agent is selected from an RBP, wherein the RBP is: a. the same or different as the one or more RBP of any preceding claim; b. a positive or negative regulator of circRNA; or c. an RBP regulates RNA biogenesis, splicing, transport, translation, and / or degradation.

18. The composition of claim 17, wherein the RBP is selected from HSPB1, NUFIP2, BCIN3D, N0B1, PKM, GNB2L1, RBM15, ANXA2, DCP1A, DDX19A, DDX52, G3BP2, LARP4, LARP4B, RBM17, TH0C1, TOB2, U2AF2, or ZC3HAV1, or a functional fragment or variant of each thereof.

19. The composition of claim 17, wherein the PRPB is selected from LARP4, LARP4B, RBM4, EIF3C, ASS1, G3BP2, SRSF6, DDX39A, PINX1, DDX55, GTF2E2, RDBP, or a functional fragment or variant of each thereof, or wherein the RBP is selected from an RBP listed in Tables 2, 3, 7, or 8, or a functional fragment or variant of each thereof.

20. A method to regulate circRNA translation in a cell, comprising contacting the cell with one or more of the circRNA, the linear RNA, the polynucleotide, the vector, the plurality, the isolated host cell, or the composition of any preceding claim.

21. The method of claim 20, wherein the method enhances circRNA translation.

22. A method to regulate circRNA or linear biogenesis in a cell, comprising contacting the cell with one or more of the circRNA, the linear RNA, the polynucleotide, the vector, the plurality, the isolated host cell, or the composition of any preceding claim.

23. The method of any of claims 20-22, wherein the contacting is in vivo, in vitro, or ex vivo.

24. The method of any of claims 20-23, wherein the cell is a mammalian cell, optionally a HeLa cell or a HEK293T cell.

25. A method to regulate circRNA or linear RNA translation in a subject in need, the method comprising administering to the subject one or more of the circRNA, the linear RNA, the polynucleotide, the vector, the plurality, the isolated host cell, or the composition of any preceding claim.

26. A method to enhance circRNA or linear RNA translation in a cell, the method comprising contacting the cell with a vector comprising a circRNA or linear RNA bound to LARP4B, or a functional fragment or variant thereof.

27. A method to enhance circRNA or linear RNA translation in a cell, the method comprising contacting the cell with a vector comprising a circRNA or linear RNA bound to ANXA2, or a functional fragment or variant thereof.

28. A method to regulate endogenous circRNAs or linear RNAs in a cell, the method comprising contacting the cell with one or more RBPs and / or vector comprising a polynucleotide encoding the one or more RBPs, optionally where the vector comprises a circRNA or linear RNA.

29. A method to enhance circRNA or linear RNA biogenesis in a cell, the method comprising contacting the cell with a polynucleotide encoding RBM15.

30. The method of any one of claims 27-29, wherein the contacting is in vitro or in vivo.

31. A method to regulate RNA in a cell, the method comprising contacting the cell with the single construct system of claim 32 or 33, wherein an endogenous RBP specific to the aptamer is recruited to the construct system and binds with the aptamer.

32. A method of enhancing translation of a circRNA in a cell, comprising contacting the cell with a circRNA or a vector of any preceding claim, wherein the circRNA is bound to LARP4B, ANXA2, or an RBP identified as a translation enhancer, or a functional fragment or variant of each thereof.

33. A method of recruiting an endogenous RBP to a circRNA or linear RNA, comprising introducing into a cell a vector or RNA comprising an aptamer, hairpin, or sequence specificfor an endogenous RBP, thereby enhancing translation, stability, nuclear export, or degradation of the RNA.

34. A method of enhancing translation of a circRNA in a cell, the method comprising codelivering: (a) a circRNA comprising at least one RNA hairpin or aptamer, and (b) an RBP, or a linear RNA or DNA encoding an RBP that binds the RNA hairpin or aptamer.

35. A method of enhancing translation of a circRNA in a cell, the method comprising delivering a single construct comprising a circRNA encoding a gene-of-interest and at least one aptamer moiety that recruits an endogenous translation enhancing RBP.

36. A method of treating a disease or disorder in a subject, comprising administering an effective amount of the composition of any preceding composition claim, wherein the composition enhances translation of a therapeutic or prophylactic protein by recruitment or tethering of at least one RBP.

37. A kit comprising:(a) a circRNA, linear RNA, or vector of any preceding circRNA, linear RNA or vector of any preceding claim;(b) an RBP or encoding nucleic acid, or instructions for co-delivery or recruitment of a translation enhancing RBP.

38. A method of screening candidate RBPs, hairpin, or aptamer sequences for their ability to enhance translation, stability, export, or degradation of a circRNA or linear RNA, comprising contacting a candidate RBP, hairpin, or aptamer with a reporter RNA in a cell and measuring a change in reporter expression or RNA abundance.

39. The method of claim 41, wherein the cell is a mammalian cell, optionally a HEK293T cell or a HeLa cell.

40. The use of any circRNA, linear RNA, vector, or composition of the preceding claims in the manufacture of a medicament for the treatment or prevention of a disease or condition.

41. A method for increasing translation of a protein from a circRNA or linear RNA in a mammalian cell, comprising introducing into the cell a circular RNA containing (a) an AU- rich aptamer sequence and (b) a coding sequence, such that the AU-rich aptamer recruits endogenous LARP4B and thereby enhances translation of the encoded protein.

42. The method of claim 44, further comprising the step of identifying an RNA-binding protein that increases translation of circRNA comprising the steps of: a) expressing a plurality of translation-enhancing RBPs, each fused to a specific tethering domain, b) screening for increased expression of circRNA-encoded reporter protein in a human cell line, and c) selecting RBPs which provide a statistically significant increase in translation (p < 0.05, and log2(fold-change) > 0.5) in two or more cell lines, preferably both HEK293T and HeLa cells.

43. A method for enhancing circRNA or linear RNA translation in a cell in vitro or in vivo, comprising administering a circular RNA with an AU-rich aptamer and / or a tethering system for an RBP from the group of LARP4B, HSPB1, NUFIP2, BCDIN3D, NOB1, PKM, or GNB2L1, optionally together with a polynucleotide or protein corresponding to the RBP, such that translation from the circRNA is increased compared to a control lacking the recruited RBP or aptamer.

44. The method of claim 34 wherein the cell is a mammalian cell, optionally a human cell.

45. The method of any claim 34 or 35, wherein enhanced translation is caused by recruitment of LARP4B and subsequent interaction of LARP4B with cellular translationinitiation factors EIF3 A and EIF4G2.

46. A composition, system, or method of any one of the preceding claims, wherein the composition or system is configured for use in multiple mammalian cell types, including but not limited to HEK293T and HeLa cells.

47. A panel of engineered circular RNAs or RNA-binding protein effectors selected by exocircFirefly luciferase reporter screening in at least two cell types, wherein each RBP demonstrates a statistically significant increase in reporter translation, the panel comprising at least two, three, four, five, or all of HSPB1, NUFIP2, BCDIN3D, NOB1, PKM, and GNB2L1, or a functional fragment or variant of each thereof.

48. A method of designing a circular RNA for therapeutic use, comprising the steps of:a) analyzing AU content of known LARP4B-bind sequences, b) encoding an AU-rich aptamer of at least 6 nucleotides in the 5’ or 3’ UTR or noncoding region of the circular RNA, and c) introducing the designed circRNA into a mammalian cell, wherein the AU-rich region recruits endogenous LARP4B and enhances translation efficiency.

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