USES THEREOF

BACKGROUND OF THE INVENTION

Technical Field of the Invention

[0001] The present invention relates to circular RNA molecules, vectors, method of producing circular RNA, and utilization methods.

Background

[0002] The success of introducing messenger RNA (mRNA) encoding SARS-CoV-2 spike protein as a novel vaccine strategy undoubtedly sparks the potential of RNA research in molecular therapy. In theory, the longer the half-life of delivered mRNA within the cell, the extended duration of its functionality, and the reduced amount of mRNA needed for delivery. Most mRNAs have 5'-Cap and 3' poly(A) in conjunction with various cellular proteins to reach each other and form a loop structure for efficient protein expression and avoiding exonuclease digestion. In response to elevating translation efficiency and longevity of the exogenous mRNA in cells, covalently circularizing single-stranded mRNA to generate the circular RNA (aka circRNA or oRNA) became a new choice.

[0003] Production of circular RNA (aka circRNA or oRNA) molecules has been attempted by various methods. However, the process is intricate and necessitates extensive purification. One popular oRNA formation strategy, the permuted intron-exon (PIE) splicing, involves fused partial exons flanked by intron and corresponding complementary sequences. When the RNA elements of the intron, splice site, and exon are permuted correctly, the RNA acts as an enzyme, the so-called ribozyme, catalyzes the splicing event to form oRNA via RNA breakdown and re-ligation. The PIE splicing strategy tolerates linear RNA in large and can be performed in vitro and in vivo but consumes quota in RNA sequence length for being the spliced wastes. Despite a particular condition with GTP and Mg ²⁺ accelerating PIE splicing in vitro, small byproduct RNAs after splicing required additional removal procedures to eliminate potential side effects.

[0004] Therefore, the present invention provides a method of oRNA synthesis by a U.s-acting ligase ribozyme (Rz ^L ). Unlike the splicing method of acquiring RNA breakdown and re-ligation, the present invention depends on ligase ribozyme RNA structure to ligate RNA directly. That said, neither RNA breakdown is needed, nor byproduct RNA is generated. Also, the present invention further provides a circular RNA and uses thereof.

SUMMARY OF INVENTION

[0005] One aspect of the present invention provides one or more plasmids having an arrangement of the region, wherein said arrangement is a 5’ polymerase promoter sequence, a nucleic acid sequence encoding a ligase ribozyme RNA, a nucleic acid sequence encoding an internal ribosome entry site (IRES), a nucleic acid sequence encoding an effector molecule, and a nucleic acid harboring a free OH group at the very 3’ end for conjugation to the ligase ribozyme RNA aforementioned.

[0006] Another aspect of the present invention relates to a method of producing a circular RNA molecule comprising the steps of: (a) providing a plasmid; (b) transcribing an RNA base on the plasmid and (c) allowing the RNA to self-ligation, thereby producing the circular RNA; wherein the plasmid comprises a 5’ polymerase promoter sequence, a ligase ribozyme RNA sequence, an IRES sequence, a nucleic acid sequence encoding an effector molecule, and a nucleic acid harboring a free OH group at the very 3’ end for conjugation to the ligase ribozyme RNA aforementioned.

[0007] In some embodiments of the present invention, the ligase ribozyme RNA sequence is a c/.s-acting ligase ribozyme sequence.

[0008] In some embodiments of the present invention, the 5’ polymerase promoter sequence is prokaryotic T7 promoter, T3 promoter, SP6 promoter, Lac promoter, or eukaryotic RNA polymerase I promoter.

[0009] In some embodiments of the present invention, the IRES sequence is selected from a virus, a cell, an artificial sequence, or a gene selected from the group consisting of CVB3 virus, EV71 virus, EMCV virus, PV virus, and CSFV virus.

[0010] In some embodiments of the present invention, the effector molecule sequence is microRNA sequence, microRNA sponge sequence, receptor protein sequence, target protein sequence, antigen sequence, protein enzyme sequence, functional protein sequence, or bicistronic protein expression sequence.

[0011] In some embodiments of the present invention, the arrangement further comprises a substrate sequence of the ligase ribozyme RNA.

[0012] In some embodiments of the present invention, the substrate sequence of the ligase ribozyme is substrate sequence, substrate_2 sequence, or substrate_3 sequence.

[0013] A further aspect of the present invention is a circular RNA produced by the present method.

[0014] Still yet another aspect of the present invention relates to a protein expression method. This method involves using one or more plasmids from the present invention under conditions effective to express the effector molecules.

[0015] Described infra is a novel system for generating circular RNAs in virtually any metazoan or bacterial cell. In one embodiment, this can be done without the co-expression of any additional proteins or enzyme. This expression system takes advantage of the ligase ribozyme with related functions, which autonomously process the ligation of RNA 5’ and 3’ end.

BREIF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1 A illustrates a DNA template with elements for the PIE strategy carrying out the group I intron self-splicing as the conventional oRNA production method. Note the Xbal and Stul restriction enzyme recognition sites were applied in the DNA template linearization before RNA synthesis. IVT, in vitro transcription; T7, T7 promoter; homo, homology arm; IRES, internal ribosome entry site of coxsackievirus B3; GOI, gene of interest.

[0017] Fig. IB illustrates the precursor linear RNA produced by IVT, and the subsequent RNA circularization through PIE splicing generates target oRNA, which is resistant to digestion by RNase R but susceptible to RNase A.

[0018] Fig. 1C-1D depict the analysis of IVT products from either Xbal- or 5/Ml-linearized DNA templates, following treatment with the specified enzymes. Fig. 1C shows the results of agarose electrophoresis. Fig ID shows the quantification results of band intensity, which were measured using Image!

[0019] Fig. IE provides a schematic illustration of the RT-PCR primer sets used for detecting the target oRNA. Arrows indicate the primer orientation from 5'-end to 3'-end with specific regions of the oRNA targeted.

[0020] Fig. IF illustrates that IVT products from the Xba - or A7//I-li near) zed template were purified, circularized with indicated conditions, digested with RNase R, and then sampled for RT-PCR measuring oRNA. The PCR products were analyzed by agarose electrophoresis.

[0021] Fig 1G shows the quantitative PCR (qPCR) result using SYBR Green dye (data are mean+SD, n=3 per group, and were compared by two-tailed Student t test).

[0022] Fig 1H-J show that IVT products from the Slu\ -linearized template were incubated in the indicated condition (GTP, 2 mM; 55°C, 15 min) and subsequently analyzed. Fig 1H presents the analysis results of agarose electrophoresis. Fig. II presents the analysis results of RT-PCR. Fig. 1J shows the quantification results of RT-qPCR (data are mean ± SD, n=3 per group, and were compared by two-tailed Student t test).

[0023] Fig. 2A-2E exhibit the czs-acting ligase ribozyme (Rz ^L ) system in this invention. Fig. 2A is a schematic illustration of a pair of Rz ^L and substrate sequence (SUB) flanking the IRES-driven protein expression cassette. The RNA product could undergo circularization autonomously while in the IVT reaction. Fig. 2B exhibits the design of DNA template with different ends for IVT. Fig. 2C-2E show the analysis of IVT products from different DNA templates. Fig. 2C shows the results of agarose electrophoresis. Fig. 2D shows the quantification results of RT-qPCR (data are mean + SD, n=3 per group, and were compared by two-tailed Student t test). Fig. 2E shows the sequencing data as indicated.

[0024] Fig. 2F shows the RNA folding prediction of the Rz ^L and the designed endings.

[0025] Fig. 3A-3B illustrate the impact of N7-m ethyl -guanosine-5’ -ppp-5 ’-guanosine (5’-Cap) addition (0%, 80%, and 90%) in the IVT on the analyzed RNA products. Fig. 3A depicts the analysis of IVT products via agarose electrophoresis. Fig. 3B shows the quantification results of agarose electrophoresis. (Data are mean + SD (n=3 per group) and were compared by two-tailed Student t test).

[0026] Fig. 3C provides a schematic illustration of the DNA template that ended with SUB, which was either transfected into the BHK-21 cells stably expressing T7 RNA polymerase (BHK-T7) or subjected for IVT followed by RNA transfection into the parental BHK-21 cells.

[0027] Fig. 3D shows the analysis of oRNA using RT-PCR.

[0028] Fig. 3E shows the images of transfected cells using fluorescence microscopy.

[0029] Fig. 3F exhibits the intensity of RFP-encoded oRNA measured by flow cytometry.

[0030] Fig. 4A-4F show that RNA modification could reduce the Rz ^L -catalyzed oRNA formation. Fig. 4A-4B show that the Rz ^L -SUB pair was highlighted with dark circles to indicate possible N6-methyladenosine (m6A) (Fig. 4A) or 5-methylcytosine (m5C) (Fig. 4B) modifications. Note that the content of A in the pair is higher than C. Fig. 4C-4F shows that different ratios of m6A- or m5C-modified A or C were added in the IVT whose RNA products were analyzed for its RNase R resistance (Fig. 4C) and oRNA formation (Fig. 4C- Fig. 4D) as indicated. Data are mean + SD (n=3 per group). Fig. 4E-4F show that BHK-21 cells were transfected by the IVT products with the indicated ratio of m6A- or m5C-modified RNA, followed by analysis using fluorescence microscopy (FIG. 4E) and flow cytometry (Fig. 4F).

[0031] Fig. 5A-5E show that an extraneous IRES in cis, not trans, hampered the oRNA translation. Fig. 5A illustrates the strategy for designing mono- and bicistronic oRNA constructs to express the indicated reporter proteins. Fig. 5B shows that N18 cells were transfected with the indicated oRNA for 24 h and harvested for FLuc activity (data are mean + SD, n=3 per group). Fig. 5C illustrates the analysis of reporter protein expression using western blotting. Fig. 5D illustrates the analysis of reporter protein expression using flow cytometry. Fig. 5E illustrates the analysis of reporter protein expression using fluorescence microscopy.

[0032] Fig. 6A-6D show that an IRES-driven oRNA expressing erCasl3 could be antiviral against the recombinant RNA virus JEV-GFP. Fig. 6A shows the use of fluorescence microscopy to observe N18 cells infected with JEV-GFP virus (moi 10) or transfected with oRNA (1 ug) for the indicated time. Fig. 6B is a schematic illustration of timepoint of JEV-GFP virus (moi 10) infection and oRNA transfection. Fig. 6C-6D depict the results of flow cytometry analysis for JEV-GFP virus (moi 10) infection and oRNA transfection.

[0033] Fig. 6E shows that N18 cells were co-transfected with DNA plasmid encoding erGFP and oRNA encoding Flag-tagged Cast 3 as indicated.

[0034] Fig. 6F-6H shows that N18 cells were treated with oRNA, JEV-GFP, or oRNA at the time and with the dose as indicated (Fig 6F), which was analyzed by Western blotting (Fig. 6G) and flow cytometry (Fig. 6H). The design of ER-targeting Casl3 in the oRNA was illustrated (Fig 6G, lower panel). +, sgRNA targeting GFP; -, N18 cellular RNA.

[0035] Fig 7A-7B show that Reticulocyte lysate was incubated with control FLuc mRNA (5’-Capped, 3’-polyA-tailed) for translation in vitro and then with CHX to cease. Fig 7A shows that control conditions for CHX working concentrations were illustrated (upper panel) and tested (lower panel) as indicated (one of the represented results). Fig 7B shows that time intervals were illustrated (upper panel) and tested (lower panel) as indicated (one of the represented results).

[0036] Fig. 7C-7D show that IVT products from either Xha\- or S -linearized pORl DNA templates were added to the reticulocyte lysate (data are mean + SD, n=2 per group) or transfected to BHK-21 cells (data are mean + SD, n=3 per group) for the indicated time.

[0037] Fig. 8A-8B demonstrate that based on the designed substrate (SUB) sequence, the RZ ^L -SUB pair ends with an extra nucleotide could probably make overhanging at the 3’ end, except for additional U.

[0038] Fig. 8C shows the DNA template ended with either SUB or SUB ^EcoRI for IVT.

[0039] Fig. 8D shows the sequencing result of IVT products from each DNA template.

[0040] Fig. 8E shows the agarose electrophoresis and RT-PCR result of oRNA (data are mean ± SD, n=3 per group).

[0041] Fig. 8F shows the quantification results of RT-qPCR of oRNA (data are mean ± SD, n=3 per group).

[0042] Fig. 8G depicts the RNA folding prediction of oRNA transcribed from the DNA template designed with SUB_2 sequence and SUB_3 sequence at the ends.

[0043] Fig. 8H shows the RT-PCR result of oRNA transcribed from the DNA template designed with SUB_2 sequence and SUB_3 sequence at the ends.

[0044] Fig. 9Ais the schematic illustration of the experimental design. N18 cells were infected with JEV-GFP, transfected with oRNA harboring Vip or FLuc, and analyzed by flow cytometry.

[0045] Fig. 9B shows the IEV-GFP infection rate analyzed via flow cytometry.

[0046] Fig. 10A shows that N18 cells were co-transfected for 24 h with plasmid DNA encoding erGFP and oRNA expressing either Casl3 or erCasl3, followed by immunofluorescence microscopy.

[0047] Fig. 10B-10C show that cells were treated with oRNA, JEV-GFP, or oRNA at the time and with the dose indicated (Fig. 10B), which was analyzed by Western blotting with indicated antibodies (Fig. 10C). +, sgRNA targeting GFP; -, N18 cellular RNA.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Other features and advantages of the present invention will be further exemplified and described in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

[0049] The embodiments of the invention will be apparent from the following detailed description, which proceeds with reference to the accompanying drawings, wherein the same references relate to the same elements.

[0050] As used herein, the term “promoter sequence” refers to T7 promoter, T3 promoter, SP6 promoter, CMV promoter, SV40 promoter, Lac promoter, eukaryotic RNA polymerase I promoter.

[0051] As used herein, the term “ligase ribozyme” refers to Ligase Rl, Ribozyme L2, Ligate Z, Ribozyme XI, Ligase RibA, Circularize R, RibozymeLinker.

[0052] As used herein, the term “substrate sequence” refers to Fl substrate A sequence, Fl substrate 2 sequence, Fl substrate 3 sequence, Fl substrate B sequence, Fl substrate X sequence, Fl substrate Z sequence.

[0053] As used herein, the term “IRES sequence” refers to CVB3 virus sequence, EV71 virus sequence, EMC A virus sequence, PV virus sequence, CSFV virus sequence, cellular RNA sequence, and artificial RNA sequence.

[0054] Example 1. The PIE splicing strategy forms RNase R-resistant oRNA.

[0055] In this and following examples, the synthesized pORl vector comprises an IRES-driven protein expression cassette flanked by a sequence set of Anabaena catalytic intron/exon PIE system resided in a pair of homology arms under the control of T7 promoter (Fig. 1A). The restriction enzyme /Ml-linearized template contains all the elements forming oRNA, whereas the Xbal -lineari zed template generates RNA lacking pairing elements and staying in linear form (Fig. IB, left). RNase R, an exoribonuclease digesting linear RNA was applied to tell the oRNA formation because the RNA electrophoresis showed the Xbal- and 5/Ml-linearized templates generating a variety of RNA species (Fig. 1C). Because of the difficulty of oRNA quantification using photo intensity (Fig. ID), an RT-PCR method was designed with specific primers to measure oRNA (Fig. IE). The RT-PCR consistently showed the Stul but not Xbal group of RNA forms oRNA as expected (Fig. IF) despite low efficiency (Fig. 1G). RNA was heated in the presence of GTP for a better splicing activity generating oRNA (Fig. 1H-J). As expected, RNA from 57« I -linear! zed templates expressed more reporter proteins in vitro and the transfected cells (in vivo) than that from AAc/I -lineari zed ones (Fig. 7). Thus, oRNA production, qualification, and quantification by the conventional PIE splicing method were established.

[0056] Example 2. A ligase ribozyme (Rz ^L ) resided at RNA 5’-end autonomously catalyzes RNA circularization in virto.

[0057] To generate oRNA without the splicing byproducts, the synthesized pOR2 vector contains the same protein translation cassette but is flanked by a pair of Rz ^L and Fl substrate sequences (Fig. 2A). The Hindlll linearization leaves the DNA template 5’-protruding ended with an imperfect but acceptable substrate sequence. For ending with the corresponding substrate RNA (SUB) fitting the Rz ^L , PCR was used to generate a blunted-ended template that should end as designed (Fig. 2B). Both Hindlll- and SUB-end templates seemed to generate equivalent RNA products (Fig. 2C, lanes 1 and 2) sensitive to RNase A digestion (Fig. 2C, lanes 3 and 4). However, the SUB-end group RNA was more RNase R-resistant than the Hindlll group (Fig. 2C, lanes 5 and 6). oRNA-specific RT-qPCR consistently showed that the SUB-end group automatically formed more oRNA than the Hindlll group in vitro transcription (IVT) (Fig. 2D). The PCR products were sequenced (Fig. 2E) to confirm details of the RNA covalent circularization design (Fig. 2F). Thus, RNA ending with the corresponding SUB sequence fitting the Rz ^L yielded oRNA efficiently, despite a minor presence of an extraneous nucleotide (Fig. 2E, star marked) that probably resulted from the none-5’ end-protruding IVT template.

[0058] Since T is relatively higher than others in the extraneous nucleotide site (Fig. 2E, star), the RNA folding of each Rz ^L -SUB pair which ends with a different extraneous nucleotide were analyzed (Fig. 8A, Fig. 8B). Because the extra U following SUB made the RNA end without an overhang that differs from the other three, the SUB template was ended with an EcoRI cutting site (SUB ^EcoRI ) to create a 5’ protruding end of AATT (Fig. 8C) for mimicking the circumstances with extra U (Fig. 8B). The SUB ^EcoRI showed a precise ligation as expected (Fig. 8D) but with lower efficiency than SUB in RNA circularization (Fig. 8E, F). Thus, certain RNA substrate sequence tolerances existed beyond perfect matches in the RZ ^L -SUB paired RNA ligation.

[0059] Optionally, the RNA molecules can have an ending that corresponds to the SUB sequence, SUB 2 sequence and SUB 3 sequence. In this optional configuration, the SUB sequence, the SUB_2 sequence and SUB_3 sequence are designed to form a conjunction ring with a 5 '-end-protruding overhang end (Fig. 8G). The conjunction ring consists of three nucleotides for bridging at the middle and two overhang nucleotides at the 5' end. This specific design has been found to efficiently yield oRNA (Fig. 8H).

[0060] Example 3. RNA modifications affect the Rz ^L -SUB oRNA formation.

[0061] N7-methyl-guanosine-5’ -ppp-5 ’-guanosine (m7G cap) was applied to mask the 5’-ppp and remove its circularization ability (Fig. 3A, B). Consistent with this, the design of RZ ^L -SUB failed to form oRNA by T7 RNA polymerase in vivo because the transcribed RNA was 5’-Capped immediately in the transfected cell (Fig. 3C, D). Consequently, the RNA in circular form has more translation capability than linear in the cell (Fig. 3E, F) despite bearing IRES in both formats. Thus, the Rz ^L -SUB method is capable of circularizing an RNA long enough in length for protein expression.

[0062] Since modifications in the Rz ^L and SUB regions could likely affect the circularization and the contents of A and C were not equivalent within the region (Fig.4A, B). Two common eukaryotic RNA modifications, N6-methyladenosine

(m6A) and 5 -methylcytosine (m5C) were enrolled to verify whether modified RNA is tolerated in the Rz ^L -mediated oRNA formation. As low as 1% A or C modification barely affecting the oRNA formation, which was perfectly blocked by replacing all the A with m6A (Fig. 4C, D). Thus, the RNA with 100% m6A modification remained linear and was inert in protein expression (Fig. 4E, F). In the case of replacing all the C with m5C, the oRNA formation was partially prohibited (Fig. 4C, D, lane 5). However, the m5C-modified oRNA remained incapable of protein expression (Fig. 4E, F), probably because of the m5C-mediated translation inhibition.

[0063] Example 4. Three strategies for bicistronic expression of Flue and GFP.

[0064] In this and following examples, three strategies were enrolled for the bicistronic expression of FLuc and GFP: fuse the two in a single protein (FG), separate the two by 2a-mediated ribosome skipping (F2aG), or introduce another IRES between the two (FiresG) (Fig. 5A). FLuc activity was attenuated by fusion with GFP (Fig. 5B, FLuc vs. FG) that could be slightly rescued by separating the FLuc from GFP with a 2a peptide (Fig.5B, C). Unexpectedly, the dual-IRES strategy rendered FLuc the lowest level of protein expression among the three strategies (Fig. 5B, C). Consistently, flow cytometry (Fig.5D) and fluorescence microscopy (Fig. 5E) analysis of the GFP expression showed that it was not because of variations in transfection efficiency but because of different expression capabilities. To overcome this, two separated oRNA were co-transfected in which either FLuc or GFP was driven by a single IRES (FLuc + GFP) and found they worked well for each other in the transfected cells (Fig. 5). Thus, the IRES-driven oRNA translation could be interfered with by another identical IRES in cis, not trans. [0065] Example 5: Viral RNA comparable translation could bestow oRNA antiviral potentials.

[0066] A recombinant JEV harboring GFP (JEV-GFP) whose 5’-capped RNA genome undergoes continuous replication and efficient translation. The GFP was readily detected at 24 h of the infection with or without oRNA transfection (Fig. 6A, B). Flow cytometry consistently showed marginal effects of JEV-GFP infection on oRNA translation in the experimental model of infection-transfection and vice versa (Fig. 6C, D). Thus, IRES-driven oRNA translation is comparable to a cap-dependent self-replicating RNA in trans, which is the prerequisite against the virus successfully via expressing antiviral proteins.

[0067] Next, an oRNA overexpressing the known broad-spectrum antiviral protein viperin for virus inhibition (Fig. 9A). The GFP positive rate reached approximately 76% at 48 h after JEV-GFP infection with low moi (Fig. 9B left panel). Introducing control oRNA (FLuc) reduced a certain level of GFP, which was further attenuated by introducing the oRNA encoding viperin (Fig. 9B right panel). Thus, the delivery of a natural antiviral protein by oRNA could effectively inhibit the virus.

[0068] To have a specific-targeting antiviral, oRNA was used to express the RNA-guided RNA nucleases Casl3 (Fig. 6E and Fig. 10A left panel) first and then introduced the GFP-targeting single guide RNA (sgRNA) (Fig. 6F, upper panel). While GFP and JEV NS3 were readily detected after JEV-GFP infection, a certain level of antiviral activity resulting from Casl3 oRNA delivery was noticed (Fig. 6G and Fig. 10C lane 2 vs. 3). Although Casl3 oRNA with sgRNA lowered the NS3 level more than the control RNA (Fig. 10C lane 3 vs. 4), the difference was marginal by blotting GFP (Fig. 6G, lane 3 vs. 4). Regarding the orthoflavivirus RNA that may replicate and be concealed in the ER-associated membrane structures, the Casl3 was further modified to target ER (erCasl3) (Fig. 6G, lower panel). When the infection rate reached -81% in the group without any RNA delivery (Fig. 6H, left panel), delivery of the erCasl3 oRNA alone showed a basal antiviral activity suppressing the virus infection rate to -63% (Fig. 6H, right panel). Applied the sgRNA under such circumstances, erCasl3 could further suppress the infection rate toward as low as -45%, which worked better than the parental Cast 3 in the cytosol for a functional antiviral activity against the JEV-GFP virus (Fig. 6G and Fig. IOC).

[0069] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.