Compositions and methods for cyclic RNA expression

Abstract

The present disclosure provides nucleic acid molecules encoding at least two circular RNAs (circRNAs), adeno-associated virus (AAV) particles comprising the nucleic acid molecules encoding at least two circRNAs, pharmaceutical compositions, and methods for delivering them to a subject. In some embodiments, the compositions herein may comprise nucleic acid molecules encoding at least two circular RNAs (circRNAs), wherein the nucleic acid molecules further comprise: (i) a first circRNA having a first circRNA coding sequence of interest; (ii) a second circRNA having a second circRNA coding sequence of interest, wherein the second circRNA is tandem with the first circRNA coding sequence of interest in the first circRNA, and the tandem first circRNA coding sequence of interest and the second circRNA are flanked by intron elements.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 969,758, filed on February 4, 2020, the content of which is hereby incorporated by reference in its entirety.

[0002] Description of Federal Government Support This invention was made with government support under Federal Grant No. R01NS099371 awarded by the National Institutes of Health. The federal government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Sequence Listing An electronic version of the sequence listing has been submitted together with this specification, the content of which is hereby incorporated by reference in its entirety. This electronic file is 23 kilobytes in size and has the title 21_2000_WO_Sequence_Listing_ST25.txt.

Background Art

[0004] Background of the Invention RNA modulation has emerged as a promising therapeutic approach for the treatment of several types of diseases. Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for the expression of therapeutic proteins and non - coding RNAs. CircRNAs introduce an additional level of control by modulating various cellular functions and pathways either directly or indirectly. Increasing evidence indicates that circRNAs play important roles in neurological disorders, atherosclerotic vascular diseases, and cancer. In addition to the long lifespan of circRNAs and their resistance to RNA decay mechanisms, there has been a growing interest in the development of circRNA - based therapeutics for at least these reasons.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Currently, there is a lack of designs for synthetic circRNAs for therapeutic use. This disclosure provides a rational design for a synthetic circRNA cassette comprising a composition encoding at least two circular RNAs (circRNAs) that can be packaged into a recombinant AAV vector for therapeutic delivery. [Means for solving the problem]

[0006] Summary of this disclosure This disclosure provides, in part, nucleic acid molecules encoding at least two circular RNAs (circRNAs), adeno-associated virus (AAV) particles comprising nucleic acid molecules encoding at least two circRNAs, pharmaceutical compositions, and methods for delivering them to a target.

[0007] One aspect of the present disclosure provides a nucleic acid molecule encoding at least two circular RNAs (circRNAs). In some embodiments, the compositions herein may comprise a nucleic acid molecule encoding at least two circular RNAs (circRNAs), the nucleic acid molecule further comprising (i) a first circRNA having a target first circRNA coding sequence, and (ii) a second circRNA having a target second circRNA coding sequence, the second circRNA being tandem with the target first circRNA coding sequence in the first circRNA, with intron elements adjacent to the tandem target first circRNA coding sequence and the second circRNA. In some embodiments, the first circRNA disclosed herein may further have an internal ribosome entry site (IRES), a promoter region in the 5' untranslated region (UTR) and outside the intron element adjacent to the tandem target first circRNA coding sequence and the second circRNA, a translational regulatory region in the 3'UTR and outside the intron element adjacent to the tandem target first circRNA coding sequence and the second circRNA, or any combination thereof. In some embodiments, the composition herein may have an intron element adjacent to the tandem target first circRNA coding sequence and the second circRNA, such intron element is back-spliced ​​to produce at least two circRNAs without forming a scar at the site where the circRNA is covalently ring-closed.

[0008] In some embodiments, the Disclosure provides an adeno-associated virus (AAV) genome having one of the nucleic acid molecules encoding at least two circRNAs disclosed herein.

[0009] Another aspect of this disclosure provides adeno-associated virus (AAV) particles. In some embodiments, the AAV particles disclosed herein include at least one AAV genome cassette having a nucleic acid molecule encoding at least one circular RNA (circRNA), at least one circRNA containing an internal ribosome entry site (IRES) element preceding an open reading frame (ORF) in a backsplicing cassette, the backsplicing cassette containing the at least one circRNA coding sequence of interest. In some embodiments, the AAV particles herein may further include an intron pair adjacent to the at least one circRNA coding sequence of interest, the

[0010] In some embodiments, the Disclosure provides a method for delivering a target circRNA coding sequence to cells, comprising the step of introducing the cells into either a composition or AAV particles disclosed herein. In some embodiments, the Disclosure provides a method for delivering a target circRNA coding sequence to tissue, comprising the step of introducing the tissue into either a composition or AAV particles disclosed herein.

[0011] In some embodiments, the Disclosure provides a method for treating a disease or condition in a subject. In some embodiments, the method for treating a disease or condition in a subject according to the Specified Method comprises the step of administering an effective amount of either a composition or AAV particles disclosed herein to a subject, the effective amount being an amount that reduces at least one symptom of the disease or condition in the subject.

[0012] In some embodiments, the methods herein include the step of administering either one of the compositions disclosed herein or AAV particles to tissue to cause expression of at least one circular RNA (circRNA) in the tissue, the expression of at least one circRNA may be substantially increased compared to baseline. In some embodiments, the expression of at least one circRNA increases by at least twofold compared to baseline. In some other embodiments, when at least one AAV particle is delivered to cardiac tissue, liver tissue, skeletal muscle tissue, or any combination thereof, the expression of at least one circRNA increases by at least fourfold compared to baseline. In some other embodiments, when at least one AAV particle is delivered to skeletal muscle tissue, the expression of at least one circRNA increases by at least fiftyfold compared to baseline.

[0013] In some embodiments, any of the compositions or AAV particles disclosed herein may be administered by intramuscular injection, intravenous injection, intra-coronary injection, intra-arterial injection, or any combination thereof.

[0014] Certain aspects of this disclosure provide a kit which may comprise either one of the compositions or AAV particles disclosed herein and at least one container.

[0015] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure, which can be better understood by referring to the drawings in conjunction with a detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0016] [Figure 1-1]Figures 1A–1Q are schematic diagrams, images, and graphs illustrating distance requirements between Alu elements and splice sites, with different upstream and downstream introns, according to embodiments of the present disclosure. Figure 1A shows a schematic diagram of a reporter constructed by extracting intronic portions from the HIPK3 gene and arranging them around a split GFP reporter exon. Inverted Alu repeats within the intron interacted to form circRNA, allowing backsplicing to occur. The presence of the IRES sequence drove translation, resulting in GFP protein expression. Figures 1B–1F are schematic diagrams, images, and graphs illustrating left-side sequence insertions into HIPK3 introns. Figure 1B shows a schematic diagram of a construct with a sequence ranging from 100nt to 1,500nt inserted into the left HIPK3 intron at the indicated location. Figures 1C and 1D show Western blot analysis for GFP expression, with Figure 1C showing a representative image of the blot and Figure 1D showing a graph of quantification of the blot using actin as a loading control. Figures 1E and 1F show Northern blot analysis for GFP RNA, with Figure 1E showing a representative image of the blot and Figure 1F showing a graph of blot quantification. Figures 1G-1K are schematic diagrams, images, and graphs showing sequence insertion into the right HIPK3 intron. Figure 1G shows a schematic diagram of a construct with a sequence in the range of 100nt-1,500nt inserted into the right HIPK3 intron at the indicated location. Figures 1H and 1I show Western blot analysis for GFP expression, with Figure 1H showing a representative image of the blot and Figure 1I showing a graph of blot quantification using actin as a loading control. Figures 1J and 1K show Northern blot analysis for GFP RNA, with Figure 1J showing a representative image of the blot and Figure 1K showing a graph of blot quantification. Figures 1L-1Q show schematic diagrams, images, and graphs showing tricRNA formation when tricRNA is inserted into the right HIPK3 intron. Figure 1L shows a schematic diagram of a construct containing a sequence that drives the formation of a circular tricRNA (tricY-Broccoli) with Broccoli inserted at the same position in the right HIPK3 intron.Figure 1M shows a representative image of a gel demonstrating the verification of tricY-Broccoli expression by gel electrophoresis followed by DFHBI-1T staining. Figures 1N and 1O show Western blot analysis for GFP expression, Figure 1N shows a representative image of the blot, and Figure 1O shows a graph of the quantification of the blot using actin as a loading control. Figures 1P and 1Q show Northern blot analysis probing for GFP RNA, Figure 1P shows a representative image of the blot, and Figure 1Q shows a graph of the quantification of the blot. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. <故

[0017] [Figure 2-1] Figures 2A - 2C are schematic diagrams showing sequence information for HIPK3 (Figure 2A), laccase 2 (Figure 2B), and ZKSCAN1 (Figure 2C) intron pairs according to embodiments of the present disclosure. The distance between the complementary region and the splice site is shown above the schematic diagram, and information on the complementary region and splice site is overlaid on the sequence. [Figure 2-2] Same as above.

[0018] [Figure 3] Note: There seems to be a typo in the original text where "故0000083" is likely a mistake and should be something else. I've translated it as it is for now. If it's an important tag that should be something specific, please correct it in the original text for a more accurate translation.Figures 3A - 3E are schematic diagrams, images, and graphs showing that an increase in the distance between the left Alu element and the splice acceptor site is harmful even when inserted distally to the splice site, according to an embodiment of the present disclosure. Figure 3A shows a schematic diagram of a construct having a sequence in the range of 100 nt to 1500 nt inserted into the left HIPK3 intron at a site distal to the splice site. Figures 3B and 3C show Western blot analysis for GFP expression, where Figure 3B shows a representative image of the blot and Figure 3C shows a graph of the quantification of the blot using actin as a loading control. Figures 3D and 3E show Northern blot analysis probing for the GFP sequence, where Figure 3D shows a representative image of the blot and Figure 3E shows a graph of the quantification of the blot.

[0019] [Figure 4-1]Figures 4A–4P are schematic diagrams, images, and graphs showing that partial shortening of intron sequences increased circRNA expression according to embodiments of this disclosure. Figure 4A shows schematic diagrams of constructs with deletions of portions of the left and right introns of HIPK3, with deletions numbered from the beginning of each intron. Figures 4B–4G show representative images of GFP fluorescence in HEK293 cells 4 days after transfection with the following constructs: HIPK3 split GFP (Figure 4B); LΔ42-267 (Figure 4C); RΔ10-654 (Figure 4D); RΔ10-497 (Figure 4E); RΔ161-654 (Figure 4F); and LΔRΔ (Figure 4G). Figures 4H and 4I show Western blot analysis for GFP expression, with Figure 4H showing a representative image of the blot and Figure 4I showing a graph of quantification of the blot using actin as a loading control. Figures 4J and 4K show Northern blot analyses probing GFP RNA, with Figure 4J showing a representative image of the blot and Figure 4K showing a graph of blot quantification. Figure 4L shows a schematic diagram of a construct in which either the left or right partial Alu element was deleted from the HIPK3 intron. Figures 4M and 4N show Western blot analyses for GFP expression, with Figure 4M showing a representative image of the blot and Figure 4N showing a graph of blot quantification using actin as a loading control. Figures 4O and 4P show Northern blot analyses probing GFP RNA, with Figure 4O showing a representative image of the blot and Figure 4P showing a graph of blot quantification. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0020] [Figure 5-1]Figures 5A–5H are schematic diagrams, images, and graphs showing that the effects of insertions and deletions according to embodiments of the present disclosure were conserved in glioblastoma and hepatocellular carcinoma cell lines. Figures 5A–5D show GFP expression in U87 glioblastoma cells transfected with the indicated constructs. Figures 5A and 5B show Western blot analysis for GFP expression, with Figure 5A showing a representative image of the blot and Figure 5B showing a graph of quantification of the blot using actin as a loading control. Figures 5C and 5D show Northern blot analysis probing for GFP RNA, with Figure 5C showing a representative image of the blot and Figure 5D showing a graph of quantification of the blot. Figures 5E–5H show GFP expression in Huh7 hepatocellular carcinoma cells transfected with the indicated constructs. Figures 5E and 5F show Western blot analysis for GFP expression; Figure 5E shows a representative image of the blot, and Figure 5F shows a graph of blot quantification using actin as a loading control. Figures 5G and 5H show Northern blot analysis for GFP RNA probing; Figure 5G shows a representative image of the blot, and Figure 5H shows a graph of blot quantification. [Figure 5-2] Same as above.

[0021] [Figure 6-1]Figures 6A–6T are schematic diagrams, images, and graphs showing that the intron spacing effect on circRNA formation, according to embodiments of this disclosure, is conserved in laccase 2 and ZKSCAN1 intron pairs. Figures 6A–6E show GFP expression in HEK293 cells transfected with a construct containing a ZKSCAN1 intron pair with the indicated randomized sequence insertion. Figure 6A shows a schematic diagram of the ZKSCAN1 construct with the insertion site. Figures 6B and 6C show Western blot analysis for GFP expression, with Figure 6B showing a representative image of the blot and Figure 6C showing a graph of quantification of the blot using actin as a loading control. Figures 6D and 6E show Northern blot analysis probing GFP RNA, with Figure 6D showing a representative image of the blot and Figure 6E showing a graph of quantification of the blot. Figures 6F–6J show GFP expression in HEK293 cells transfected with a construct containing a laccase 2 intron pair with the indicated randomized sequence insertion. Figure 6F shows a schematic diagram of the laccase 2 construct with the location of the sequence insertion. Figures 6G and 6H show Western blot analysis for GFP expression; Figure 6G shows a representative image of the blot, and Figure 6H shows a graph of blot quantification using actin as a loading control. Figures 6I and 6J show Northern blot analysis probing GFP RNA; Figure 6I shows a representative image of the blot, and Figure 6J shows a graph of blot quantification. Figures 6K-6O show GFP expression in HEK293 cells transfected with a construct containing the ZKSCAN1 intron pair with the indicated sequence deletion. Figure 6K shows a schematic diagram of the ZKSCAN1 construct with the location of the sequence deletion. Figures 6L and 6M show Western blot analysis for GFP expression; Figure 6L shows a representative image of the blot, and Figure 6M shows a graph of blot quantification using actin as a loading control. Figures 6N and 6O show Northern blot analysis for GFP RNA, with Figure 6N showing a representative image of the blot and Figure 6O showing a graph of blot quantification.Figures 6P–6T show GFP expression in HEK293 cells transfected with a construct containing a laccase 2 intron pair with the indicated sequence deletion. Figure 6P shows a schematic diagram of the laccase 2 construct with the location of the sequence deletion. Figures 6Q and 6R show Western blot analysis for GFP expression; Figure 6Q shows a representative image of the blot, and Figure 6R shows a graph of blot quantification using actin as a loading control. Figures 6S and 6T show Northern blot analysis probing GFP RNA; Figure 6S shows a representative image of the blot, and Figure 6T shows a graph of blot quantification. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0022] [Figure 7-1]Figures 7A–7R are schematic diagrams, images, and graphs illustrating the effect of IRES elements on translation efficiency and circRNA expression levels according to embodiments of this disclosure. Figure 7A shows a schematic diagram of a HIPK3-splitter GFP construct created using one of the EMCV, polio, KSHV vFLIP, or HCV IRES elements. Figures 7B–7E show representative images of GFP fluorescence in HEK293 cells 4 days after transfection with HIPK3-splitter GFP constructs created using EMCV IRES (Figure 7B); poliovirus IRES (Figure 7C); KSHV IRES (Figure 7D); and HCV IRES (Figure 7E). Figures 7F and 7G show Western blot analysis for GFP expression, with Figure 7F showing a representative image of the blot and Figure 7G showing a graph of blot quantification using actin as a loading control. Figures 7H–7P show constructs transfected into HEK293 cells as shown, collected in cycloheximide, and then subjected to sucrose gradient and fractionation, with arrowheads marking the last fraction in which circRNA was detected. Figures 7H–7J show representative images of OD254 traces of gradients with the HIPK3 EMCV construct, with fractions marked by lines (Figure 7H); RNA extracted from the gradient, separated by gel electrophoresis, transferred to a membrane, and stained with methylene blue to visualize ribosomal RNA (Figure 7I); and the same membrane shown in Figure 7I (Figure 7J) probed for GFP RNA. Figures 7K–7M show representative images of OD254 traces of gradients with HIPK3 polio constructs, with fractions marked by lines (Figure 7K); RNA extracted from the gradient, separated by gel electrophoresis, transferred to a membrane, and stained with methylene blue to visualize ribosomal RNA (Figure 7L); and the same membrane shown in Figure 7L (Figure 7M) probed for GFP RNA.Figures 7N–7P show representative images of OD254 traces of a gradient containing the HIPK3 KSHV construct, with fractions marked by lines (Figure 7N); RNA extracted from the gradient, separated by gel electrophoresis, transferred to a membrane, and stained with methylene blue to visualize ribosomal RNA (Figure 7O); and the same membrane shown in Figure 7O (Figure 7P) probed for GFP RNA. Figures 7Q and 7R show Northern blot analyses probed for GFP sequences, with Figure 7Q showing a representative image of the blot and Figure 7R showing a graph of the blot quantification. Figure 7S shows representative Northern blots probed for GFP RNA in RNA isolated from the indicated construct, either digested (+) or undigested (-) by RNase R (RnR). [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above.

[0023] [Figure 8] Figures 8A–8F are schematic diagrams, images, and graphs showing translation efficiency using HCV IRES and control linear RNA according to embodiments of the present disclosure. Figures 8A–8C show representative images of OD254 traces of gradients containing a HIPK3 HCV construct with fractions marked by lines (Figure 8A); RNA extracted from the gradient, separated by gel electrophoresis, transferred to a membrane, and stained with methylene blue to visualize ribosomal RNA (Figure 8B); and the same membrane shown in Figure 8B (Figure 8C) probed for GFP RNA. Figures 8D–8F show representative images of OD254 traces of gradients containing cap-driven linear GFP mRNA (linGFP) with fractions marked by lines (Figure 8D); RNA extracted from the gradient, separated by gel electrophoresis, transferred to a membrane, and stained with methylene blue to visualize ribosomal RNA (Figure 8E); and the same membrane shown in Figure 8E (Figure 8F) probed for GFP RNA.

[0024] [Figure 9-1] Figures 9A–9H are schematic diagrams, images, and graphs showing how IRES-mediated translation efficiency and circRNA expression varied in glioblastoma and hepatocellular carcinoma cell lines according to embodiments of the present disclosure. Figures 9A–9D show GFP expression in U87 glioblastoma cells transfected with the indicated constructs. Figures 9A and 9B show Western blot analysis for GFP expression, with Figure 9A showing a representative image of the blot and Figure 9B showing a graph of quantification of the blot using actin as a loading control. Figures 9C and 9D show Northern blot analysis probing GFP RNA, with Figure 9C showing a representative image of the blot and Figure 9D showing a graph of quantification of the blot. Figures 9E–9H show GFP expression in Huh7 hepatocellular carcinoma cells transfected with the indicated constructs. Figures 9E and 9F show Western blot analysis for GFP expression; Figure 9E shows a representative image of the blot, and Figure 9F shows a graph of blot quantification using actin as a loading control. Figures 9G and 9H show Northern blot analysis for GFP RNA probing; Figure 9G shows a representative image of the blot, and Figure 9H shows a graph of blot quantification. [Figure 9-2] Same as above.

[0025] [Figure 10-1]Figures 10A–10G are schematic diagrams, images, and graphs illustrating how IRES elements influence splicing and further RNA species formation according to embodiments of this disclosure. Figure 10A shows a representative image of RNase H digestion performed using an oligonucleotide targeting the backsplice junction, with the sample analyzed by Northern blotting and probed for GFP RNA. Figure 10B shows a representative image of RNA from the indicated constructs, analyzed by Northern blotting and probed using an oligonucleotide extending to the backsplice junction. Figures 10C and 10D show representative images of RNA from the HIPK3 polio construct (Figure 10C) and the HIPK3 KSHV construct (Figure 10D), analyzed by Northern blotting and probed for GFP and specific IRES sequences. Figure 10E shows representative images of virtual Northern blots and RT-PCR using primers extending to either the backsplice junction (top) or the IRES sequence (center, poliovirus IRES; bottom, KSHV vFLIP IRES), confirming the presence of both backsplices and linear splices with small circRNA bands. Figures 10F and 10G show representative images of mutations to the splice donor sites identified in Figure 10E in the HIPK3 polio construct (Figure 10F) and HIPK3 KSHV construct (Figure 10G), analyzed by Northern blotting probed for GFP, with splicing modified to remove small circRNA bands. For all Northern blots, * indicates small circular species. [Figure 10-2] Same as above.

[0026] [Figure 11-1]Figures 11A–11P are schematic diagrams, images, and graphs showing that circRNA size could be increased without loss of expression according to embodiments of this disclosure. Figure 11A shows a schematic diagram of a construct having a self-cleaving P2A peptide attached to the exon at the end of a GFP fragment, either in the original HIPK3 construct or in an LΔRΔ intron pair, followed by a dsRed ORF. Figures 11B–11D show representative images of GFP fluorescence in HEK293 cells 4 days after transfection with the following constructs: HIPK3 split GFP (Figure 11B); PP2A dsRed (Figure 11C); and LΔRΔ PP2A dsRed (Figure 11D). Figures 11B–11D show representative images of red fluorescence in HEK293 4 days after transfection with the following constructs: PP2A dsRed (Figure 11E) and LΔRΔ PP2A dsRed (Figure 11F). Figures 11G and 11H show Western blot analysis for GFP expression, with Figure 11G showing a representative image of the blot and Figure 11H showing a graph of quantification of the blot using actin as a loading control. Figures 11J–11L show images of the HIPK3 polio P2AdsRed construct transfected into HEK293 cells, collected in cycloheximide, and then fractionated with a sucrose gradient, with Figure 11J showing the OD254 trace of the gradient with fractions marked by lines; Figure 11K shows RNA extracted from the gradient, separated by gel electrophoresis, transferred to a membrane, and stained with methylene blue to visualize ribosomal RNA; Figure 11L shows the same membrane from Figure 11K probed for GFP RNA (arrowheads mark the last fraction in which circRNA was detected). Figures 11M and 11I show Northern blot analysis for GFP RNA, with Figure 11M showing a representative image of the blot and Figure 11I showing a graph of blot quantification. Figure 11N shows RNA treated with RNase R and then analyzed by Northern blot for GFP RNA. Figure 11O shows RNase H (RnH) digestion performed using oligonucleotides targeting the back splice junction, and the samples were analyzed by Northern blot for GFP RNA.Figure 11P shows RNA probed using an oligonucleotide extending to the back splice junction, analyzed by Northern blotting. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0027] [Figure 12-1]Figures 12A–12L are schematic diagrams, images, and graphs demonstrating that LΔRΔ intron pairs, according to embodiments of this disclosure, increased circRNA expression in vivo in multiple mouse tissues. Figure 12A shows a schematic diagram of a construct intravenously injected into C57BL / 6 mice, packaged into a recombinant AAV9 vector. Figure 12B shows the AAV vector genome per cell quantified in each tissue by quantitative PCR (qPCR) for the CMV promoter, standardized against the mouse lamin B2 locus. Figures 12C and 12D show that results from quantitative RT-PCR performed using GFP-amplifying primers extending to the backsplice junction revealed the increase in circRNA expression when using LΔRΔ intron pairs. CircRNA expression is graphed in each tissue, standardized against GAPDH (Figure 12C) or against HIPK3 polio expression (Figure 12D). Figures 12E–12G show immunofluorescence staining of sectioned cardiac tissue collected from mice injected with HIPK3 polio (Figure 12F) and HIPK3 polio ΔΔ (Figure 12G), with Figure 12E being unstained with the primary antibody. Figure 12H shows a graph of GFP expression levels in cardiac tissue collected from mice injected with HIPK3 polio or HIPK3 polio ΔΔ, quantified by corrected total cell fluorescence (CTCF). Figures 12I–12K show immunofluorescence staining of sectioned skeletal muscle tissue collected from mice injected with HIPK3 polio (Figure 12J) and HIPK3 polio ΔΔ (Figure 12K), with Figure 12I being unstained with the primary antibody. Figure 12L shows a graph of GFP expression levels in skeletal muscle tissue collected from mice injected with HIPK3 polio or HIPK3 polio ΔΔ, quantified by corrected total cell fluorescence (CTCF). [Figure 12-2] Same as above. [Figure 12-3] Same as above.

[0028] [Figure 13]Figures 13A–13D are images showing that a circRNA reporter, according to embodiments of this disclosure, expressed low levels of GFP protein in the liver. Figures 13A and 13B show immunofluorescence staining of sectioned liver tissue for GFP expression in tissue collected from mice injected with HIPK3 polio, with Figure 13B being a magnified view of Figure 13A. Figures 13C and 13D show immunofluorescence staining of sectioned liver tissue for GFP expression in tissue collected from mice injected with HIPK3 polio ΔΔ, with Figure 13D being a magnified view of Figure 13C. [Modes for carrying out the invention]

[0029] Detailed explanation RNA modulation has become a promising therapeutic approach for treating several types of diseases, and circular RNA (circRNA) is rapidly becoming an attractive template for the expression of therapeutic proteins and non-coding RNAs. This disclosure is at least partly based on the finding that further insertions into nucleic acid molecules encoding at least two circular RNAs (circRNAs), tolerated within a right intron, can be utilized to create bifunctional RNA molecules from a single template. Specifically, this disclosure provides tRNA intron-derived circRNA that can be incorporated into an intron sequence without affecting back-spliced ​​circRNA formation. The findings herein provide rational design for synthetic circRNAs that enable the uptake of aptamers, guide RNAs, protein sponges, miRNA sponges, protein-binding RNAs, naturally occurring circRNAs, antisense RNAs, miRNA precursors, siRNA precursors, long non-coding RNAs (lncRNAs), small activating RNAs (saRNAs), functional non-coding RNAs such as transfer RNA (tRNAs), ribosomal RNAs (rRNAs), nucleolar small RNAs (snoRNAs), intranuclear small RNAs (snRNAs), piwi-interacting RNAs (piRNAs), Y-RNAs, 7SK RNAs, and 7S RNAs, which can be packaged into recombinant AAV vectors for therapeutic delivery. I. Definition

[0030] For the purpose of facilitating an understanding of the principles of this disclosure, references are made herein to preferred embodiments, and specific languages ​​are used to describe them. Nevertheless, it is understood that no limitation of the scope of this disclosure is intended therein, and that such modifications and further alterations of the disclosure illustrated herein are intended to be commonly recalled by those skilled in the art relating to this disclosure.

[0031] As used herein, the articles “a” and “an” are used to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means at least one element and may include more than one element.

[0032] "Approximately" is used to provide flexibility to the endpoints of a numerical range by defining that a given value can be "slightly above" or "slightly below" the endpoint without affecting the desired result. In relation to a number, the term "approximately" means that the number can vary by plus or minus 5% or less.

[0033] Throughout this specification, unless the context requires otherwise, the words “comprise” and “include” and their variations (e.g., “comprises,” “comprising,” “includes,” “including”) are understood to imply that they include the stated constituent elements, features, elements, or steps, or groups of constituent elements, features, elements, or steps, but do not exclude any other integers or steps, or groups of integers or steps.

[0034] As used herein, "and / or" encompasses any and all possible combinations of one or more of the enumerated items in question, and, when interpreted as an alternative ("or"), the absence of any combination.

[0035] As used herein, the transitional phrase “essentially from” (and its grammatical variants) should be interpreted as encompassing the enumerated materials or steps and “not substantially affecting the essential and novel features” of the claimed invention. Therefore, as used herein, the term “essentially from” should not be interpreted as equivalent to “comprising.”

[0036] Furthermore, this disclosure intends that in some embodiments, any feature or combination of features shown herein may be excluded or omitted. For example, where this specification states that a complex includes components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, may be excluded individually or in any combination.

[0037] The enumeration of value ranges in this specification is intended only as a simplified way of referring individually to each separate value that falls within that range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually cited herein. For example, if the concentration range is stated as 1% to 50%, values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be expressly enumerated herein. These are merely examples of what is specifically intended, and any possible combination of numerical values ​​between and including the enumerated minimum and maximum values ​​is considered expressly stated in this disclosure.

[0038] As used herein, “treatment,” “therapy,” and / or “treatment regimen” refer to clinical interventions made in response to a disease, disorder, or physiological condition presented by or to which a patient may be susceptible. The objectives of a treatment include reducing or preventing symptoms, slowing or halting the progression or worsening of a disease, disorder, or condition, and / or achieving remission of the disease, disorder, or condition.

[0039] As used herein, “prevent” or “prevention” means the exclusion or delay of the onset of a particular disease, disability or physiological condition, or a reduction in the degree of severity of a particular disease, disability or physiological condition, compared to the time and / or degree of onset or severity in the absence of the intervention.

[0040] The term "effective dose" or "therapeutic effective dose" refers to a quantity sufficient to produce a beneficial or desirable biological and / or clinical outcome.

[0041] As used herein, the terms “subject” and “patient” are interchangeable herein and refer to both humans and non-human animals. The term “non-human animal” in this disclosure includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. In some embodiments, the subject includes humans. In other embodiments, the subject includes humans who require treatment for a disease or illness.

[0042] Unless otherwise defined, all technical terms used herein have the same meanings as those generally understood by those skilled in the art of the field to which this disclosure pertains. I. Nucleic acid molecule composition

[0043] In certain embodiments, the Disclosure provides a nucleic acid molecule encoding at least one covalently ring-bound circular RNA (circRNA). In some embodiments, the nucleic acid molecule herein comprises one or more of the following: a) a circRNA coding sequence that can be transcribed into non-coding RNA or translatable mRNA; b) one or more intron elements adjacent to the circRNA coding sequence that are back-spliced ​​by a cellular splicing apparatus to produce a covalently ring-bound circular RNA; c) an internal ribosome entry site (IRES) that drives translation of the translatable mRNA transcribed from the circRNA coding sequence; d) a promoter upstream of the intron element adjacent to the circRNA coding sequence; and e) a translational regulatory region in the 3'UTR and outside the intron element adjacent to the circRNA coding sequence.

[0044] In certain embodiments, the Disclosure provides nucleic acid molecules encoding at least two circular RNAs (circRNAs). In some embodiments, compositions of the Specified having nucleic acid molecules encoding at least two circular RNAs (circRNAs) may optionally include a first circRNA having a target first circRNA coding sequence and a second circRNA having a target second circRNA coding sequence, the second circRNA being tandem with the target first circRNA coding sequence of the first circRNA, and in some embodiments, intron elements may be adjacent to the tandem target first circRNA coding sequence and the second circRNA.

[0045] In certain embodiments, the first circRNA having the desired first circRNA coding sequence may further include an internal ribosome entry site (IRES). Non-limiting examples of IRESs suitable for use herein include encephelomyocarditis virus (EMCV) IRES, poliovirus IRES, Kaposi's sarcoma-associated herpesvirus (KSHV) vFLIP IRES, hepatitis C virus (HCV) IRES, or any combination thereof. In some embodiments, IRESs suitable for use herein may share at least 85% (e.g., at least 85%, 90%, 95%, 99%, 100%) sequence similarity with any one of sequence numbers 26-29.

[0046] In certain embodiments, the first circRNA having the desired first circRNA coding sequence may further include a promoter region upstream of an intron element adjacent to the tandem first circRNA coding sequence and the second circRNA coding sequence. In some embodiments, the promoter may be a human cytomegalovirus (CMV) promoter, a truncated chimeric CMV-chicken β-actin (smCBA) promoter, a mammalian β-actin promoter, an albumin promoter, and the like. In some embodiments, the promoter may be a polymerase II-driven promoter. The promoters are generally well known in the art.

[0047] In certain embodiments, the nucleic acid molecules herein may include at least one promoter region capable of recruiting RNA polymerase. The promoter controls the binding of RNA polymerase to DNA for initiating gene transcription. Currently, there are three known types of RNA polymerase, each transcribing a different gene: RNA polymerase type I can transcribe genes encoding ribosomal RNA (rRNA); RNA polymerase type II can transcribe messenger RNA (mRNA); and RNA polymerase type III can transcribe genes encoding transfer RNA (tRNA). RNA polymerase type III can also transcribe small RNAs, such as shRNA and gRNA. In some embodiments, the nucleic acid molecules herein may include at least one promoter region capable of recruiting RNA polymerase type I, type II, type III, or any combination thereof. In some embodiments, the nucleic acid molecules herein may include a promoter region capable of recruiting RNA polymerase type II or type III.

[0048] In certain embodiments, the first circRNA having the target first circRNA coding sequence may further include a translational regulatory region in the 3'UTR and outside the intron elements adjacent to the tandem target first circRNA coding sequence and the second circRNA.

[0049] In certain embodiments, a first circRNA having a target first circRNA coding sequence may have one or more intron elements adjacent to the circRNA coding sequence, which are back-spliced ​​by the cell splicing apparatus to produce a covalently circumferentially closed circular RNA. In some embodiments, the intron elements adjacent to the tandem target first circRNA coding sequence and second circRNA of this Specification are back-spliced ​​to produce at least two circRNAs without forming a scar at the site where the circRNA is covalently circumferentially closed. As used herein, a “scar” in a circRNA may be generated at the site where the intron elements are joined to produce a circular RNA. In this disclosure, at least two circRNAs of this Specification may form a seamless circular RNA (i.e., a circRNA that does not have a scar at the site where the circRNA is covalently circumferentially closed).

[0050] In a particular embodiment, the first circRNA having the desired first circRNA coding sequence may have one or more intron elements adjacent to the circRNA coding sequence, and one or more intron elements may share about 85% (e.g., about 85%, about 90%, about 95%, about 99%, about 100%) sequence similarity with any one of sequence numbers 1, 2, 6, 7, 11, 12 and 20-25.

[0051] In certain embodiments, a first circRNA having a target first circRNA coding sequence may have one or more intron elements adjacent to the circRNA coding sequence, and one or more intron elements may have an Alu element (or complementary region) that shares about 85% (e.g., about 85%, about 90%, about 95%, about 99%, about 100%) sequence similarity with any one of sequence numbers 3, 4, 8, 9, 13, and / or 14. In certain embodiments, a first circRNA having a target first circRNA coding sequence may have one or more intron elements adjacent to the circRNA coding sequence, and one or more intron elements may have a polypyrimidine tract that shares about 85% (e.g., about 85%, about 90%, about 95%, about 99%, about 100%) sequence similarity with any one of sequence numbers 5, 10, and / or 15. In certain embodiments, a first circRNA having a target first circRNA coding sequence may have one or more intron elements adjacent to the circRNA coding sequence, and one or more intron elements may have a splice site that shares about 85% (e.g., about 85%, about 90%, about 95%, about 99%, about 100%) sequence similarity with any one of the following: CAGGTAGGT(HIPK3), CAGGTAAGT(laccase 2), and / or CAGGTAAGA(ZKSCAN1).

[0052] In some embodiments, the intron elements of this specification adjacent to at least one circRNA coding sequence of interest may have one or more insertions in the naturally occurring nucleic acid sequence. In some embodiments, one or more insertions in the naturally occurring nucleic acid sequence intron elements of this specification may be insertions of at least about 1 to about 1000 nucleic acids. In some embodiments, one or more insertions in the naturally occurring nucleic acid sequence intron elements of this specification may be insertions of at least about 1 to about 1000 nucleic acids in the left intron adjacent to the circRNA coding sequence. In some embodiments, one or more insertions in the naturally occurring nucleic acid sequence intron elements of this specification may be insertions of at least about 1 to about 1000 nucleic acids in the right intron adjacent to the circRNA coding sequence.

[0053] In some embodiments, the intron elements of this specification adjacent to at least one circRNA coding sequence of interest may have one or more deletions in the naturally occurring nucleic acid sequence. In some embodiments, one or more deletions in the naturally occurring nucleic acid sequence intron elements of this specification may be deletions of at least about 1 to about 2000 nucleic acids. In some embodiments, one or more deletions in the naturally occurring nucleic acid sequence intron elements of this specification may be deletions of at least about 1 to about 2000 nucleic acids in the left intron adjacent to the circRNA coding sequence. In some embodiments, one or more deletions in the naturally occurring nucleic acid sequence intron elements of this specification may be deletions of at least about 1 to about 2000 nucleic acids in the right intron adjacent to the circRNA coding sequence.

[0054] In some embodiments, the intron pairs of this specification adjacent to at least one circRNA coding sequence of interest may contain a deletion of at least about 500 nucleotides (e.g., about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1000 nucleotides, about 1500 nucleotides). In some embodiments, the intron pairs of this specification adjacent to at least one circRNA coding sequence of interest may have a left intron containing a deletion of about 100 to about 500 nucleotides (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500). In some embodiments, the intron pairs of this specification adjacent to at least one circRNA coding sequence of interest may have a left intron containing a deletion of about 250 nucleotides. In some embodiments, the intron pair of this specification adjacent to at least one circRNA coding sequence of interest may have a right intron containing a deletion of about 300 to about 900 nucleotides (e.g., about 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900). In some embodiments, the intron pair of this specification adjacent to at least one circRNA coding sequence of interest may have a right intron containing a deletion of about 500 nucleotides. In some embodiments, the intron pair of this specification adjacent to at least one circRNA coding sequence of interest may have a left intron containing a deletion of about 100 to about 500 nucleotides (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500) and a right intron containing a deletion of about 300 to about 900 nucleotides (e.g., about 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900). In some embodiments, the intron pair of this specification adjacent to at least one circRNA coding sequence of interest may include a left intron containing a deletion of about 250 nucleotides and a right intron containing a deletion of about 500 nucleotides.

[0055] In certain embodiments, a composition of this specification having at least two nucleic acid molecules encoding circular RNA (circRNA) can produce at least one circRNA via backsplicing and at least one circRNA via autosplicing. In some embodiments, the circRNA formed via autosplicing may be a tRNA intron circular RNA, i.e., a "tricRNA". In some embodiments, a composition of this specification having at least two nucleic acid molecules encoding circular RNA (circRNA) can produce one or more tricRNAs. In some embodiments, a composition of this specification having at least two nucleic acid molecules encoding circular RNA (circRNA) can produce about 1 to about 50 tricRNAs, about 1 to about 40 tricRNAs, 1 to about 30 tricRNAs, 1 to about 20 tricRNAs, 1 to about 10 tricRNAs, or 1 to about 5 tricRNAs. In some embodiments, compositions of this specification having nucleic acid molecules encoding at least two circular RNAs (circRNAs) can produce one or more tRNA introns having about 85% (e.g., about 85%, about 90%, about 95%, about 99%, about 100%) sequence similarity to any one of sequence numbers 17-18.

[0056] In some embodiments, the tRNA intron elements herein may include any known tRNA intron elements in any combination and in any multiple and / or ratio. Examples of tRNA intron elements suitable for use herein may include those described in Abelson et al., J Biol Chem. 273(21):12685-8 (1998) and / or Schmidt et al., Wiley Interdiscip Rev RNA. 11(3):e1583 (2020), the disclosures of which are incorporated herein by reference in their entirety.

[0057] In some embodiments, the compositions of this specification having nucleic acid molecules encoding at least two circular RNAs (circRNAs) may have a nucleic acid insertion in a right intron adjacent to the circRNA coding sequence of interest. In some embodiments, the compositions of this specification may have a nucleic acid insertion in a right intron adjacent to the circRNA coding sequence of interest, and the insertion may be about 0.1 kb to about 2.0 kb (e.g., about 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 kb). In some embodiments, the compositions of this specification may have a nucleic acid insertion in a right intron adjacent to the circRNA coding sequence of interest, and the insertion may be about 1.5 kb. In some embodiments, the compositions of this specification may have a nucleic acid insertion in a right intron adjacent to the circRNA coding sequence of interest, and the insertion may produce at least one circRNA via backsplicing. In some embodiments, the compositions herein may have a nucleic acid insertion in a right intron adjacent to the circRNA coding sequence of interest, the insertion being able to produce at least one circRNA via self-splicing. In some embodiments, the compositions herein may have a nucleic acid insertion in a right intron adjacent to the circRNA coding sequence of interest, the insertion being able to produce one or more tricRNAs.

[0058] In some embodiments, compositions of this specification having nucleic acid molecules encoding at least two circular RNAs (circRNAs) can produce one or more circRNAs, each encoding an aptamer, guide RNA, protein sponge, miRNA sponge, protein-binding RNA, naturally occurring circRNA, antisense RNA, long non-coding RNA (lncRNA), small activating RNA (saRNA), functional non-coding RNA, such as transfer RNA (tRNA), ribosomal RNA (rRNA), nucleolar small RNA (snoRNA), intranuclear small RNA (snRNA), piwi-interacting RNA (piRNA), Y-RNA, 7SK RNA, 7S RNA, or any combination thereof. In some embodiments, the compositions herein can produce at least one circRNA via backsplicing, the circRNA may encode an aptamer, guide RNA, protein sponge, miRNA sponge, protein-binding RNA, naturally occurring circRNA, antisense RNA, long non-coding RNA (lncRNA), small activating RNA (saRNA), functional non-coding RNA, such as transfer RNA (tRNA), ribosomal RNA (rRNA), nucleolar small RNA (snoRNA), intranuclear small RNA (snRNA), piwi-interacting RNA (piRNA), Y-RNA, 7SK RNA, 7S RNA, or any combination thereof. In some embodiments, the compositions herein can produce at least one circRNA via self-splicing, which may encode aptamers, guide RNAs, protein sponges, miRNA sponges, protein-binding RNAs, naturally occurring circRNAs, antisense RNAs, long non-coding RNAs (lncRNAs), small activating RNAs (saRNAs), functional non-coding RNAs, such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), nucleolar small RNAs (snoRNAs), intranuclear small RNAs (snRNAs), piwi-interacting RNAs (piRNAs), Y-RNAs, 7SK RNAs, 7S RNAs, or any combination thereof.In some embodiments, the compositions herein can produce one or more tricRNAs capable of encoding aptamers, guide RNAs, protein sponges, miRNA sponges, protein-binding RNAs, naturally occurring circRNAs, antisense RNAs, long non-coding RNAs (lncRNAs), small activating RNAs (saRNAs), functional non-coding RNAs, such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), nucleolar small RNAs (snoRNAs), intranuclear small RNAs (snRNAs), piwi-interacting RNAs (piRNAs), Y-RNAs, 7SK RNAs, 7S RNAs, or any combination thereof.

[0059] In some embodiments, compositions of this specification having nucleic acid molecules encoding at least two circular RNAs (circRNAs) can be prepared using conventional molecular biology techniques known in the art, including recombinant techniques. Such techniques are fully described in the literature, for example, in *Molecular Cloning: A Laboratory Manual, second edition* (Sambrook, et al., 1989) Cold Spring Harbor Press; *Oligonucleotide Synthesis* (MJ Gait, ed. 1984); *Methods in Molecular Biology*, Humana Press; and *Cell Biology: A Laboratory Notebook* (JE Cellis, ed., 1989) Academic Press. II. AAV virus particles

[0060] In certain embodiments, the present disclosure provides AAV virus particles for use as a vehicle for delivery to any of the circRNAs disclosed herein.

[0061] Adeno-associated viruses (AAVs) are members of the Parvoviridae family and are small, non-enveloped viruses. AAV particles suitable for use herein may contain an AAV capsid composed of capsid protein subunits VP1, VP2, and VP3 that encapsulate a single-stranded DNA genome.

[0062] In some embodiments, the AAV virus particles disclosed herein may harbor a single-stranded AAV DNA vector which may contain any of the circRNAs disclosed herein. In some embodiments, the circRNA coding sequence may be in a ligated state that can operate with a suitable promoter that drives circRNA expression. In some examples, the AAV DNA vector herein may contain one or more regulatory elements that modulate circRNA expression, such as one or more miRNA binding sites, enhancers, transcription factor binding sites, poly(A) signaling elements, or a combination thereof. (A) AAV vector

[0063] In certain embodiments, the AAV virus particles disclosed herein may have an AAV vector for expressing one or more of the circRNAs disclosed herein. The AAV vector is derived from the wild-type genome of a virus, e.g., AAV, by using molecular methods to remove the wild-type genome from the virus and replacing it with non-native nucleic acids, e.g., heterologous polynucleotide sequences (e.g., coding sequences for circRNAs). Typically, for an AAV vector, one or both inverted end repeat (ITR) sequences of the wild-type AAV genome are retained in the AAV vector, while other parts of the wild-type viral genome are replaced between the retained ITRs with non-native sequences, such as heterologous polynucleotide sequences. The AAV vectors disclosed herein may comprise AAV genome-derived skeletal elements, coding sequences for the circRNAs disclosed herein, and appropriate promoters in a ligated state activating with the coding sequences. In some examples, the AAV vectors disclosed herein may further comprise regulatory sequences that modulate the expression and / or secretion of the encoded protein. Examples include, but are not limited to, sequences encoding enhancers, polyadenylation signaling sites, internal ribosome entry sites (IRESs), protein transduction domains (PTDs), microRNA target sites, or combinations thereof.

[0064] In some cases, the AAV vectors disclosed herein may be ordinary AAV vectors containing single-stranded nucleic acids. In other cases, the AAV vectors disclosed herein may be self-complementary AAV vectors capable of containing a double-stranded portion. (1) AAV skeletal elements

[0065] In some embodiments, the AAV vectors disclosed herein may have one or more AAV genome-derived skeletal elements, which refer to the minimum number of AAV genome elements required for the biological activity of the AAV vector. For example, the AAV genome-derived skeletal elements may include a packaging site for the AAV vector to be assembled into an AAV virus particle, and elements necessary for vector replication in a host cell and / or expression of the circRNA coding sequence contained therein.

[0066] In some examples, the AAV vector skeleton disclosed herein may include at least one inverted terminal repeat (ITR) sequence. In some examples, the AAV vector skeleton herein includes two ITR sequences. In some examples, one ITR sequence is located at the 5' end of the polynucleotide sequence encoding circRNA. In some examples, one ITR sequence is located at the 3' end of the polynucleotide sequence encoding circRNA. In some examples, the polynucleotide sequence encoding circRNA herein is adjacent to an ITR sequence from either side.

[0067] In some embodiments, the AAV vectors of this specification include sequences or components originating from at least one distinct AAV serotype. In some examples, the AAV vector skeletons disclosed herein may include at least one ITR sequence derived from one distinct AAV serotype. In some examples, the AAV vector skeletons disclosed herein may include at least one ITR sequence derived from one distinct human AAV serotype. Such human AAV may originate from any known serotype, e.g., any one of serotypes 1 to 11. In some examples, the AAV serotypes used herein have tropism to the central nervous system (CNS), cardiac tissue, skeletal muscle, and / or liver tissue. In some examples, the AAV vector skeletons disclosed herein may have ITR sequences of serotypes AAV1, AAV2, AAV4, AAV5, AAV8, or AAV9.

[0068] In some embodiments, the AAV vectors herein may be pseudotyped AAV vectors (i.e., containing sequences or components originating from at least two distinct AAV serotypes). In some embodiments, the pseudotyped AAV vectors herein contain an AAV genomic backbone derived from one AAV serotype and a capsid at least partially derived from a distinct AAV serotype. In some examples, the pseudotyped AAV vectors herein may have an AAV2 genomic backbone and a capsid derived from an AAV serotype having tropism to the CNS (e.g., AAV1, AAV2, AAV4, AAV5, AAV8, or AAV9). Specific examples of such pseudotyped AAV vectors include, but are not limited to, vectors containing an AAV2-derived genome in an AAV5-derived capsid; or vectors containing an AAV2-derived genome in an AAV8-derived capsid; or vectors containing an AAV2-derived genome in an AAV9-derived capsid; or vectors containing an AAV2-derived genome in an AAV1-derived capsid.

[0069] To analyze the success of viral vector-mediated gene transfer, it may be important to be able to monitor both vector distribution and the effectiveness of vector-mediated gene expression. This can be achieved by subcloning a reporter gene into the viral vector skeleton. In some examples, the AAV vector skeleton disclosed herein may include a reporter gene. Several reporter genes are commonly used for this purpose and include, but are not limited to, fluorescent proteins of various colors (including green fluorescent protein (GFP) and red fluorescent protein (RFP)), E. coli β-galactosidase (LacZ), and various forms of luciferase (Luc). In some examples, the AAV vector skeleton disclosed herein may include GFP.

[0070] The vector constructs disclosed herein may be prepared using known techniques (see, for example, Current Protocols in Molecular Biology, Ausubel., F. et al., eds, Wiley and Sons, New York 1995). Fragment lengths may be selected so as not to exceed the packaging capacity of the AAV particles. If necessary, “stuffer” DNA sequences are added to the construct to maintain a standard AAV genome size for comparison purposes. Such fragments may be derived from such non-viral sources, e.g., lacZ, or other genes known and available to those skilled in the art. (2) Self-complementary AAV virus vector

[0071] In some embodiments, the AAV vectors disclosed herein may be self-complementary AAV (scAAV) vectors. Self-complementary AAV (scAAV) vectors contain complementary sequences that can spontaneously anneal (fold back into themselves to form a double-stranded genome) upon entering an infected cell, thus avoiding the need to convert a single-stranded DNA vector using the cell's DNA replication machinery. The AAVs herein having a self-complementary genome can readily form a double-stranded DNA molecule thanks to their partially complementary sequences (e.g., complementary coding and non-coding strands of a circRNA coding sequence).

[0072] In some embodiments, the scAAV viral vectors disclosed herein may comprise a first heterogeneous polynucleotide sequence and a second heterogeneous polynucleotide sequence, which can form intrachain base pairs. In some examples, the first and second heterogeneous polynucleotide sequences are linked by sequences that promote intrachain base pairing, for example, to form a hairpin DNA structure. In some examples, the dimer structure of the scAAV vector upon entry into a cell may be stabilized by a mutation or deletion of one of two terminal resolution sites (trs). Since trs are Rep-binding sites contained within each ITR, such mutations or deletions of trs can prevent the cleavage of the scAAV vector's dimer structure by the AAV Rep protein for monomer formation. In some embodiments, the scAAV viral vectors disclosed herein may comprise a truncated 5' inverted terminal repeat (ITR), a truncated 3' ITR, or both. In some examples, the scAAV vectors disclosed herein may include a truncated 3'ITR, in which the D region or a portion thereof (e.g., a terminally separable sequence) may be deleted. Such a truncated 3'ITR may be located between the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence described above. (3) Promoter

[0073] In some embodiments, the AAV vectors disclosed herein include at least one suitable promoter that controls the expression of further elements necessary for expression, such as a circRNA coding sequence. Such promoters may be ubiquitous, tissue-specific, strong, weak, regulated, chimeric, etc., to enable efficient and appropriate production of the protein in the infected tissue. The promoter may be homologous to the encoded protein or heterogeneous, including cellular, viral, fungal, plant, or synthetic promoters. The most preferred promoter for use in the present invention must be functional in human cells. Non-limiting examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, RSV promoter, SV40 promoter, etc., and cellular promoters, such as the PGK (phosphoglycerin kinase) promoter. In some embodiments, the viral promoters herein may be the CMV promoter, the SV40 promoter, or any combination thereof. In some embodiments, the viral promoters herein may have one of the following sequences: [Table A]

[0074] In some embodiments, the viral promoters herein may share approximately 85% (e.g., approximately 85%, 90%, 95%, 99%, 100%) sequence similarity with any one of sequence numbers 44-45.

[0075] In some embodiments, the AAV vectors disclosed herein include additional elements necessary for expression, such as at least one suitable promoter that controls the expression of a circRNA coding sequence after infection of suitable cells. Suitable promoters for use herein include, in addition to the AAV promoter, the cytomegalovirus (CMV) promoter or the chicken beta-actin / cytomegalovirus hybrid promoter (CAG), endothelial cell-specific promoters such as the VE-cadherin promoter, and steroid promoters and metallothionein promoters. In some embodiments, the promoter used in the vectors disclosed herein may be the CAG promoter.

[0076] In some embodiments, the circRNA coding sequence according to the present invention includes a tissue-specific promoter functionally linked to the circRNA coding sequence to be expressed. Therefore, the specificity of the vector according to this disclosure to a tissue (e.g., brain, heart, muscle, liver) can be further increased. In some examples, the vector disclosed herein may have a tissue-specific promoter whose activity in a particular tissue is at least 2, 5, 10, 20, 50, or 100 times higher than its activity in a non-specific tissue. In some examples, the tissue-specific promoter herein is a human tissue-specific promoter. In some examples, the expression cassette may also include an enhancer element for increasing the expression level of the exogenous protein to be expressed. Furthermore, the expression cassette may further include a polyadenylated sequence, e.g., an SV40 polyadenylated sequence or a bovine growth hormone polyadenylated sequence. Generally, tissue-specific promoters are well known in the art. (4) Other regulatory elements for gene expression

[0077] In some embodiments, the AAV vectors disclosed herein may comprise one or more conventional regulatory elements operably ligated to a circRNA coding sequence in a manner that enables its transcription, translation, and / or expression in cells transfected with the plasmid vector produced by the present invention or in cells infected with the virus. As used herein, “operably ligated” sequence includes both expression regulatory sequences that are contiguous with the circRNA coding sequence and expression regulatory sequences that act trans or at a certain distance to control the circRNA coding sequence. Expression regulatory sequences may further comprise appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance the secretion of encoded products. Numerous expression regulatory sequences, including promoters that are native, constitutive, inducible, and / or tissue-specific, are known in the art and may be utilized.

[0078] In some embodiments, the AAV vectors disclosed herein may include a modified capsid containing a non-viral or structurally modified protein or peptide to alter the vector's tropism. For example, the capsid may contain a ligand for a specific receptor or a receptor for a specific ligand to target the vector toward a cell type(s) expressing that receptor or ligand, respectively. (B) Serotype of AAV virus particle

[0079] In some embodiments, the AAV vectors disclosed herein may be prepared or derived from various serotypes of AAV. The term “serotype” refers to a distinction relating to an AAV having a serologically distinct capsid from other AAV serotypes. Serological discriminability is determined based on the absence of cross-reactivity between antibodies against AAVs compared to other AAVs. Cross-reactivity can be measured using methods known in the art. For example, the cross-reactivity described herein may be measured using a neutralizing antibody assay. For this assay, polyclonal serum is generated against a specific AAV in a rabbit or other suitable animal model using adeno-associated virus. In this assay, the serum generated against a specific AAV is then tested for its ability to neutralize either the same (homologous) or heterologous AAV. The dilution that achieves 50% neutralization is considered the neutralizing antibody titer. For two AAVs, if the quotient of heterologous titers divided by homologous titers is less than 16 in reciprocal form, these two vectors are considered to be the same serotype. Conversely, if the ratio of heterologous titers to homologous titers is 16 or higher in reciprocal terms, the two AAVs are considered distinct serotypes.

[0080] In some examples, the AAV vectors herein may be a mixture of at least two serotypes of AAV to produce a chimeric (e.g., pseudotyped) AAV virus, or a mixture with other types of viruses. In certain embodiments, the AAV vector for use in the present invention is a human serotype AAV vector. Such human AAV may be derived from any known serotype, for example, one of serotypes 1 to 11, more preferably AAV1, AAV2, AAV4, AAV6, and AAV9. Specific examples of such AAV vectors include vectors containing an AAV1-derived genome (an AAV1-derived ITR and an AAV1-derived packaging sequence, preferably an AAV1-derived packaging sequence and two AAV1-derived ITRs adjacent to the nucleic acid encoding the therapeutic protein, operably linked to the nucleic acid encoding the therapeutic protein) in an AAV1-derived capsid; vectors containing an AAV2-derived genome in an AAV2-derived capsid; vectors containing an AAV4-derived genome in an AAV4-derived capsid; vectors containing an AAV6-derived genome in an AAV6-derived capsid; or vectors containing an AAV9-derived genome in an AAV9-derived capsid. (C) Method for producing AAV particles

[0081] In some embodiments, the AAV vectors disclosed herein may be packaged into viral particles that can be used to deliver a genome for circRNA coding sequence expression in target cells. In some embodiments, the AAV vectors disclosed herein may be packaged into particles by transient transfection, the use of a production cell system, the combination of viral features into an Ad-AAV hybrid, the use of a herpesvirus system, or production in insect cells using a baculovirus.

[0082] Methods for generating packaging cells for use herein may involve creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking the AAV rep and cap genes, a separate AAV rep and cap gene from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, is incorporated into the cell genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing, addition of a synthetic linker containing restriction endonuclease cleavage sites, or direct blunt-end ligation. The packaging cell line is then infected with a helper virus, such as an adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale rAAV production. An example of a suitable method herein uses an adenovirus or baculovirus instead of a plasmid to introduce the rAAV genome and / or rep and cap genes into packaging cells. Mai. Pharmaceutical composition

[0083] In some embodiments, either of the circRNAs and / or AAV virus particles disclosed herein can be formulated to form a pharmaceutical composition. In some examples, the pharmaceutical compositions herein may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. Any of the pharmaceutical compositions used in the methods of the present invention may comprise a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formation or an aqueous solution.

[0084] The carriers in a pharmaceutical composition must be “acceptable” in the sense that they are compatible with the active ingredient of the composition, preferably capable of stabilizing the active ingredient, and not harmful to the subject being treated. For example, “pharmaceutically acceptable” may refer to molecular entities and other components of the composition that are physiologically acceptable and do not typically produce undesirable reactions when administered to mammals (e.g., humans). In some examples, “pharmaceutically acceptable” carriers used in the pharmaceutical compositions disclosed herein may be those approved by federal or state regulatory agencies for use in mammals, more particularly humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0085] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphoric acid, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, e.g., serum albumin, gelatin or immunoglobulin; amino acids; hydrophobic polymers; monosaccharides; disaccharides and other carbohydrates; metal complexes; and / or nonionic surfactants. For example, Remington: The Science and Practice of Pharmacy 20 th See Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0086] In some embodiments, the pharmaceutical composition or formulation is intended for parenteral administration, such as intravenous, intraventricular injection, intracisional injection, intraparenchymal injection, or a combination thereof. Such pharmaceutically acceptable carriers may be sterile liquids, such as water, and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions disclosed herein may further include, for example, preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, viscosity enhancers, and the like. The pharmaceutical compositions described herein may be packaged in single unit dose or multi-dose forms.

[0087] Suitable formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that are isotonic with the blood of the recipient to whom the formulation is intended; as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Aqueous solutions can be adequately buffered (preferably to a pH of 3–9). The preparation of suitable parenteral formulations under sterile conditions is readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0088] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. Sterile injectable solutions are generally prepared by incorporating the required amount of the active ingredient (e.g., circRNA, AAV particles, compositions containing circRNA and / or AAV particles) into a suitable solvent, along with various other components listed above, and then sterilizing by filtration as needed. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient + any further desired components from a pre-sterilically filtered solution.

[0089] The pharmaceutical compositions disclosed herein may also include other components, such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to the recipient at the dosage and concentration used, and include buffers, such as phosphoric acid, citrate or other organic acids; antioxidants, such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as Tween, Pluronic or polyethylene glycol. IV. How to Use

[0090] Any of the compositions described herein (e.g., nucleic acid molecules encoding one, two or more circular RNAs, AAV particles, AAV genomes) may be used to alleviate and / or treat a disease or condition. Accordingly, in some embodiments, this disclosure provides methods for alleviating and / or treating a disease or condition in subjects requiring the treatment composition disclosed herein, as well as pharmaceutical compositions comprising the treatment composition. Exemplary diseases or conditions that can be treated using the methods disclosed herein include cystic fibrosis (cystic fibrosis transmembrane regulatory protein) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi for repetition removal), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor) and other neurological disorders, and cancer (cytokines including endostatin, angiostatin, TRAIL, FAS-ligand, and interferon).RNAi including RNAi against VEGF or multidrug resistance gene products, mir-26a (e.g., for hepatocellular carcinoma), diabetes (insulin), Duchenne type (dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycans (e.g., α, β, γ), RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides (e.g., Kappa B dominant mutant I, sarcospan, eutrophin, mini-eutrophin), antisense or RNAi against splice junctions in the dystrophin gene to induce exon skipping (see, for example, WO2003 / 095647), U7 to induce exon skipping Antisense antibodies against snRNA (see, e.g., WO2006 / 021724), and antibodies or antibody fragments or myostatin propeptides against myostatin; muscular dystrophy including Becker type; Gaucher disease (glucocerebrosidase); Hurler disease (α-L-idulonidase); adenosine deaminase deficiency (adenosine deaminase); glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]); and other metabolic disorders; congenital pulmonary emphysema (α1-antitrypsin); Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase); Niemann-Pick disease (sphingomyelinase); Tay-Sachs disease (lysosomal hexosaminidase A); maple syrup urine disease (branched-chain keto acid dehydrogenase); retinal degenerative diseases (as well as other diseases of the eye and retina;For example, PDGF and / or vasohibin or other VEGF inhibitors for macular degeneration, or other angiogenic inhibitors to treat / prevent retinal damage in type 1 diabetes, for example, diseases of solid organs, such as the brain (including Parkinson's disease [GDNF], astrocytoma [RNAi against endostatin, angiostatin and / or VEGF], glioblastoma [RNAi against endostatin, angiostatin and / or VEGF]), liver, kidney, congestive heart failure or peripheral artery disease (P The heart (including AD), for example, protein phosphatase inhibitors I(I) and their fragments (e.g., IIC), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, P2 adrenergic receptor, p2 adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that result in knockdown of G protein-coupled receptor kinase type 2, for example, a truncated constitutively active bARKct; calsarcin; RNAi against phospholamban; phospholamban inhibitor or dominant-negative molecule (by delivering phospholamban S16E, etc.); arthritis (insulin-like growth factor); joint damage (insulin-like growth factor 1 and / or 2); intimal hyperplasia (by delivering enos, inos, etc.); improved cardiac graft survival (superoxide dismutase); AIDS (soluble CD4); muscle wasting (insulin-like growth factor I); kidney This may include, but is not limited to, visceral deficiencies (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, e.g., IRAP and TNFα soluble receptors), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, spinocerebraxa including SCA1, SCA2, and SCA3, phenylketonuria (phenylalanine hydroxylase), and autoimmune diseases.

[0091] To carry out the methods disclosed herein, a therapeutically effective amount of a composition (e.g., circRNA, AAV particles) or a pharmaceutical composition containing the same may be administered in appropriate amounts disclosed herein to a subject in need of treatment via an appropriate route (e.g., intramuscular, intravenous, intraventricular injection, intracisional injection, intravitreous, subretinal, subconjunctival, retrobulbar, anterior chamber, superior choroid, intracoronary injection, intra-arterial injection, and / or intraparenchymal injection).

[0092] In certain embodiments, the disclosure also provides a method for introducing a nucleic acid molecule into cells, comprising the step of contacting the cells with a viral vector and / or composition disclosed herein. In some embodiments, the method herein may include the step of delivering the nucleic acid molecule herein to eye cells, which comprises contacting the eye cells or layers with a viral vector disclosed herein, the viral vector comprising the nucleic acid molecule of interest. In some embodiments of the method, the nucleic acid molecule of interest may encode a therapeutic protein or therapeutic RNA. In some embodiments, the therapeutic protein may be a monoclonal antibody or a fusion protein.

[0093] In certain embodiments, the Disclosure also provides a method for introducing nucleic acid molecules into cardiac tissue, liver tissue, skeletal muscle tissue, or any combination thereof, comprising the step of contacting cells with the viral vectors and / or compositions disclosed herein. In some embodiments, the nucleic acid molecules herein may be delivered to specific tissues by administering AAV particles having one or more nucleic acid molecules herein into cardiac tissue, liver tissue, skeletal muscle tissue, or any combination thereof.

[0094] In some embodiments, a method of administering at least one AAV particle having one or more nucleic acid molecules according to this specification to a tissue substantially increases the expression of at least one circRNA compared to baseline. As used herein, “baseline” refers to the expression of at least one circRNA (and its encoding product) before administration of the AAV particle having one or more nucleic acid molecules. As used herein, “substantially increases expression” refers to at least a 1-fold change in expression compared to baseline. In some embodiments, a method of administering at least one AAV particle having one or more nucleic acid molecules according to this specification to a tissue increases the expression of at least one circRNA by at least about 2-fold to about 50-fold (e.g., about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) compared to baseline. In some embodiments, a method of administering at least one AAV particle having one or more nucleic acid molecules according to this specification to a tissue increases the expression of at least one circRNA by at least about 2 to about 50 times (e.g., about 2, 4, 6, 8, 10, 20, 30, 40, 50 times) compared to baseline when at least one AAV particle is delivered to cardiac tissue, liver tissue, skeletal muscle tissue, or any combination thereof. In some embodiments, a method of administering at least one AAV particle having one or more nucleic acid molecules according to this specification to a tissue increases the expression of at least one circRNA by at least about 4 times compared to baseline when at least one AAV particle is delivered to cardiac tissue, liver tissue, or any combination thereof. In some embodiments, a method of administering at least one AAV particle having one or more nucleic acid molecules according to this specification to a tissue increases the expression of at least one circRNA by at least about 50 times compared to baseline when at least one AAV particle is delivered to skeletal muscle tissue.

[0095] In any of the methods disclosed herein, an effective amount of the composition described herein (e.g., a nucleic acid molecule encoding one, two or more circular RNAs, an AAV particle, or an AAV genome) may be given to a subject in need to alleviate one or more symptoms associated with a disease and / or condition. “Effective amount,” as used herein, means a dose of the disclosed composition sufficient to confer a therapeutic effect to a subject having the disease and / or condition. In some embodiments, the effective amount may be an amount that reduces at least one symptom of the disease or condition in the subject. V. Kit

[0096] This disclosure also provides kits for use in preparing any one of the compositions described herein (e.g., nucleic acid molecules encoding one, two or more circular RNAs, AAV particles, AAV genomes), and kits having one or more therapeutic uses described herein. The kits for use described herein may comprise one or more containers further containing the compositions described herein (e.g., nucleic acid molecules encoding one, two or more circular RNAs, AAV particles, AAV genomes) formulated in a pharmaceutical composition.

[0097] In some embodiments, the kit may further include instructions for use of the composition (e.g., nucleic acid molecules encoding one, two or more circular RNAs, AAV particles, AAV genomes) in any of the methods described herein. The included instructions may include instructions for administering the composition or a pharmaceutical composition containing it to a subject to achieve the intended activity in the subject. The kit may further include instructions for selecting a subject appropriate for the treatment, based on the identification of whether the subject requires the treatment. Instructions for use of the compositions described herein generally include information on the dosage, administration schedule and route of administration for the intended treatment.

[0098] The container may be a unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit dose. The instructions for use provided in the kit of this disclosure are typically written instructions for use on the label or in the accompanying leaflet. The label or accompanying leaflet indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0099] The kits provided herein are in appropriate packaging. Appropriate packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging. Packaging for use in combination with specific devices, such as inhalers, nasal administration devices, or infusion devices, is also intended. The kits may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). The containers may also have a sterile access port.

[0100] The kit may provide further components, such as buffers and explanatory information, as needed. Typically, the kit includes a container and labels or accompanying documents on or associated with the container. In some embodiments, the disclosure provides a product comprising the contents of the kit.

[0101] In certain embodiments, the disclosure provides a nucleic acid molecule encoding a covalently ring-bound circular RNA (circRNA), comprising: a) a gene of interest that can be transcribed into non-coding RNA or translatable mRNA; b) an intron element adjacent to the gene of interest that is back-spliced ​​by a cell splicing apparatus to produce a covalently ring-bound circular RNA; c) an internal ribosome entry site (IRES) that drives the translation of the translatable mRNA transcribed from the gene of interest; d) a promoter region in the 5' untranslated region (UTR) and outside the intron element adjacent to the gene of interest; and e) a translation regulatory region in the 3' UTR and outside the intron element adjacent to the gene of interest. In certain embodiments, the Disclosure provides a nucleic acid molecule encoding a covalently circulated circular RNA (circRNA), comprising: a) a circRNA coding sequence that can be transcribed into non-coding RNA or translatable mRNA; b) an intron element adjacent to the circRNA coding sequence, which is backspliced ​​by a cellular splicing apparatus to produce a covalently circulated circular RNA; c) an internal ribosome entry site (IRES) that drives translation of translatable mRNA transcribed from the circRNA coding sequence, optionally including a self-splicing mechanism for circRNA generation; d) a promoter region upstream of the intron element adjacent to the circRNA coding sequence; and e) a translational regulatory region in the 3'UTR and outside the intron element adjacent to the circRNA coding sequence. In some embodiments, the Disclosure provides a nucleic acid molecule in which the intron element of (b) (i.e., the intron element adjacent to the gene of interest) comprises any combination thereof, and any multiples and / or ratios, of any nucleotide sequences of SEQ ID NOs: 1, 2, 6, 7, 11 and / or 12.In some embodiments, the Disclosure provides a nucleic acid molecule in which the intron elements of (b) (i.e., intron elements adjacent to the circRNA coding sequence) comprise any combination thereof and any multiple and / or ratio of any nucleotide sequences of SEQ ID NOs. 20–25. In some embodiments, the Disclosure provides a nucleic acid molecule in which the IRES of (c) comprise any combination thereof and any multiple and / or ratio of any nucleotide sequences of SEQ ID NOs. 26–29. In some embodiments, the Disclosure provides a nucleic acid molecule in which the translational regulatory region of (e) is a polyadenylated (poly-A) sequence and / or structural element that stabilizes the circRNA.

[0102] For example, in certain embodiments, the disclosed nucleic acid molecules may include viral IRESs, such as the viral IRESs listed in Table 1 below. [Table 1-1] [Table 1-2]

[0103] For example, in certain embodiments, the disclosed nucleic acid molecules may include cellular IRESs, such as those listed in Table 2 below. [Table 2-1] [Table 2-2]

[0104] In some embodiments, the disclosed nucleic acid molecule may comprise a combination of one or more IRESs, such as viral IRESs or cellular IRESs. In some embodiments, the combination of one or more IRESs comprises one or more viral IRESs. In some embodiments, the combination of one or more IRESs comprises one or more cellular IRESs. In some embodiments, the combination of one or more IRESs comprises both viral IRESs and cellular IRESs. In certain particular embodiments, the disclosure provides a nucleic acid molecule comprising IRESs in any combination thereof and in any multiple and / or ratio, wherein the IRESs are viral IRESs listed in Table 1 and cellular IRESs listed in Table 2.

[0105] In certain embodiments, the Disclosure provides an adeno-associated virus (AAV) genome comprising an AAV inverted terminal repeat (ITR) adjacent to any one of the nucleic acid molecules disclosed herein. In some embodiments, the Disclosure provides an AAV capsid or particle comprising an AAV genome disclosed herein. In some embodiments, the Disclosure provides an AAV capsid or particle comprising any of the nucleic acid molecules disclosed herein.

[0106] In certain embodiments, the Disclosure provides a composition comprising any of the nucleic acid molecules disclosed herein, any of the AAV genomes disclosed herein, and / or any of the AAV capsids or particles disclosed herein, in a pharmaceutically acceptable carrier.

[0107] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-bound circular RNA molecule in a cell, comprising the step of introducing one of the nucleic acid molecules disclosed herein into the cell under conditions in which the covalently ring-bound circular RNA molecule is transcribed and / or produced.

[0108] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-bound circular RNA molecule in a cell, comprising the step of introducing one of the AAV genomes disclosed herein into the cell under conditions in which the covalently ring-bound circular RNA molecule is transcribed.

[0109] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-closed circular RNA molecule in a cell, comprising the step of introducing either an AAV capsid or particle disclosed herein into the cell under conditions in which the covalently ring-closed circular RNA molecule is transcribed.

[0110] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-closed circular RNA molecule in a cell, comprising the step of introducing one of the compositions disclosed herein into the cell under conditions in which the covalently ring-closed circular RNA molecule is transcribed.

[0111] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-bound circular RNA molecule in a tissue-specific and / or cell-specific manner, comprising the step of contacting a tissue and / or cell with any of the nucleic acid molecules disclosed herein under conditions in which the covalently ring-bound circular RNA molecule is expressed.

[0112] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-closed circular RNA molecule in a tissue-specific and / or cell-specific manner, comprising the step of contacting a tissue and / or cell with one of the AAV genomes disclosed herein under conditions in which the covalently ring-closed circular RNA molecule is expressed.

[0113] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-closed circular RNA molecule in a tissue-specific and / or cell-specific manner, comprising the step of contacting a tissue and / or cell with any of the AAV capsids or particles disclosed herein under conditions in which the covalently ring-closed circular RNA molecule is expressed.

[0114] In certain embodiments, the Disclosure provides a method for expressing a covalently ring-closed circular RNA molecule in a tissue-specific and / or cell-specific manner, comprising the step of contacting the tissue and / or cell with any of the compositions disclosed herein under conditions in which the covalently ring-closed circular RNA molecule is expressed.

[0115] In some embodiments, the present disclosure provides any of the methods herein in which a covalently ring-closed circular RNA molecule is a therapeutic mRNA molecule encoding a protein, an RNA silencing molecule, a guide RNA molecule capable of targeting a genomic element, a guide RNA molecule capable of targeting an RNA transcript, a tRNA molecule, a long non-coding RNA molecule, an antisense RNA molecule, or any combination thereof.

[0116] In some embodiments, the Disclosure provides any of the methods herein in which the covalently ring-closed circular RNA molecule is a naturally occurring circRNA molecule, a functional non-coding RNA molecule, or any combination thereof.

[0117] In some embodiments, the present disclosure provides any of the methods herein in which the cells and / or tissues are of mammalian origin. [Examples]

[0118] While this disclosure has been described with reference to its specific embodiments, it will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of this disclosure. Furthermore, many modifications may be made to adapt specific circumstances, materials, compositions of substances, processes, or process steps(s) to the purposes, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of this disclosure. (Example 1) Backsplicing introns allow insertion and enable dual circRNA expression.

[0119] A common feature of introns that mediate backsplicing is the presence of an inverted Alu element or other complementary sequence. To investigate how the distance between these complementary regions and splice donor / acceptor sites affects circRNA backsplicing, we used the reporter system shown in Figure 1A, which includes an intron derived from the HIPK3 gene to drive the circularization of split GFP exons, as a template.

[0120] In short, a HIPK3 expression plasmid was first generated to produce the reporter system shown in Figure 1A. To generate the HIPK3 expression plasmid, a portion of the human HIPK3 gene sequence was inserted into a pcDNA3.1(+) vector. The pcDNA3.1(+) vector is a mammalian expression vector with a human cytomegalovirus (CMV) promoter, where the multicloning site (MCS) is forward-oriented (+). Novel vector cassettes using naturally occurring HIPK3 intron sequences were constructed by cloning them into a plasmid backbone isolated by the multicloning site. Figure 2A shows the HIPK3 intron sequences used in the novel vector cassettes. Cassettes containing IRESs (e.g., EMCV, polio, KSHV, or HCV) and split GFP were cloned between the intron sequences. The fragmented GFP cassettes used in the examples herein were derived from circGFP plasmids using a method similar to that disclosed in Wang and Wang, RNA. 2015;21(2):172-179, the entirety of which is incorporated herein by reference.

[0121] In addition to the reporter system using the naturally occurring HIPK3 intron sequence, other reporter systems were generated using a method similar to the one described above, except that they used the laccase 2 and ZKSCAN1 intron sequences. Figures 2B and 2C show the laccase 2 and ZKSCAN1 intron sequences used in these new vector cassettes, respectively. The sequences for the reporter systems using the HIPK3, laccase 2, and ZKSCAN1 intron sequences are provided in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0122] As shown in Figure 1A, the resulting construct contained an intronic region derived from the HIPK3 gene, positioned around a split GFP reporter exon. Inverted Alu repeats within the intron interacted, allowing backsplicing to occur and forming circRNA. The presence of the IRES sequence drove translation, resulting in GFP protein expression.

[0123] To probe the effects of spacing within introns, the distance between each Alu element and the splice site was artificially increased. Random inserts ranging from 100 to 1,500 nt were designed that had nucleotide content similar to that of the HIPK3 intron (e.g., G / C) but lacked any strongly predicted secondary structures. Once the insert designs met this criterion, 1.5 kb of randomized sequences were then synthesized for the left and right introns. The sequences were inserted into introns or exons using PCR and restriction cloning. Where necessary, site-directed mutagenesis was used to create 1-2 nt changes to generate restriction enzyme sites.

[0124] The first random insert was inserted into the “left” intron (upstream of the splice acceptor site) at a site approximately 100 nt from the splice acceptor (Figure 1B). The construct was transfected into HEK293 (human embryonic kidney) cells. Briefly, cells were seeded in 6-well plates and transfected at approximately 70% confluence. Transfection was performed using 2.5 μg of plasmid DNA (test constructs were equimolar, and mass was equalized using an empty vector) and 12.5 μL of 1 mg / mL PEI-MAX 40,000. Four days after transfection, cells were collected and assayed for GFP protein expression using Western blotting and for circRNA expression using Northern blotting.

[0125] As can be seen from the Western blots of GFP protein (Figures 1C and 1D) and the Northern blots of circRNA (Figures 1E and 1F), further insertions were highly detrimental. Insertions of only 100 nt reduced circRNA formation by approximately one-fifth, while greater distances effectively eliminated circRNA formation.

[0126] Considering that the HIPK3 splice acceptor has a large polypyrimidine tract (SEQ ID NO: 5), a second set of constructs was created in which additional sequences were inserted distal to the splice junction and proximal to the Alu element to control the possible impact on the branching environment (Figure 3A). This second set behaved virtually identically, with insertions of 100 nt or more significantly reducing GFP expression (Figures 3B and 3C) and circRNA formation (Figures 3D and 3E).

[0127] Insertions in the "right" intron (downstream of the splice donor site) were also generated, as shown in Figure 1G. Surprisingly, the effect of right insertions was distinct from that of left intron insertions, with sequences up to 1.5 kb in length showing no effect on GFP expression (Figures 1H and 1I) or circRNA formation (Figures 1J and 1K). RNA concatemers different from those formed in the case of the original HIPK3 intron were also observed in these constructs with inserts in the right intron. In particular, these additional band / molecular entities were later analyzed, as described in other examples herein, and were found to be dependent on other elements such as IRES.

[0128] Next, randomized inserts are given Drosophila tRNA:Tyr, which contains an intron containing the non-coding RNA-based fluorescent aptamer Broccoli, as provided in Table 4. GUA It was replaced with a sequence that codes for a gene. [Table 4]

[0129] The resulting constructs, shown in Figure 1L, independently produced both GFP circRNA and circular Broccoli tricRNA (tricY-Broccoli). Robust tricY-Broccoli expression was observed using an in-gel assay (Figure 1M). Briefly, 5 μg of RNA was resuspended in a denaturing buffer (67% deionized formamide, 6.7% formaldehyde, 1×MOPS running buffer), incubated at 65°C for 7 minutes, and cooled on ice. The denatured samples were separated on a 10% trisboric acid / EDTA (TBE)-urea gel electrophoresed at 300V. The gels were washed three times with water and then stained in DFHBI-1T fluorescent probe staining solution (40 mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl2, 10 μM DFHBI-1T) for 30 minutes. Stained gels were imaged using an Amersham Typhoon (GE Healthcare) with a 488 excitation / 526 emission setting.

[0130] Western and Northern blot analyses confirmed that tricRNA formation did not affect GFP protein levels (Figure 1N and 1O) or circRNA levels (Figure 1P and 1Q). Therefore, it is possible to increase the distance between the splice donor and the Alu element without further influence, and to insert additional functional RNA elements into the intron region. (Example 2) Synthetic intron sequences increase circRNA expression.

[0131] Next, the effect of reducing the distance between the Alu element and the splice site was tested by deleting the intron sequence of the construct described in Example 1. Examples of HIPK3, laccase 2, and ZKSCAN1 introns with deleted regions are provided in Table 5. [Table 5-1] [Table 5-2]

[0132] As disclosed in Example 1, the HIPK3 splice acceptor has a large polypyrimidine tract; therefore, an LΔ42-267 construct was created, preserving approximately 125 nt proximal to the splice junction. The right intron of HIPK3 had a complete deletion (RΔ10-654) and two smaller deletions (RΔ10-497 and RΔ161-654), but these together preserved a distance of approximately 150 nt (Figure 4A, deletions numbered from the beginning of their respective introns). The constructs were transfected into HEK293 cells, and expression was assayed 4 days after transfection to evaluate the ability of these modified introns to support circRNA formation and translation by GFP fluorescence (Figures 4B-4G), Western blotting (Figures 4H and 4I), and Northern blotting (Figures 4J and 4K).

[0133] In short, cells were imaged 4 days after transfection using an EVOS FL epifluorescence cell imaging system equipped with a GFP light cube (excitation 470 nm, emission 510 nm) or a Texas Red light cube (excitation 585 nm, emission 624 nm) to detect GFP fluorescence. Similar to the method in Example 1, cells were collected from plates, the resulting cell suspensions were separated into two tubes, and centrifuged at 300 × g at 4°C for 4 minutes. The cell pellets were suspended in either 1 × Passive Lysis Buffer or TRIzol Reagent, placed on a rocker at 4°C for 10 minutes, and then stored at -80°C before use. Lysates in Passive Lysis Buffer were centrifuged at 16,000 × g at 4°C for 5 minutes to remove cell debris before freezing. Lysates stored in Passive Lysis Buffer were processed for Western blotting analysis, and lysates stored in TRIzol Reagent were processed for Northern blotting analysis.

[0134] LΔ42-267 showed approximately twice as high GFP and circRNA expression compared to the original construct (Figures 4B-4K). A large RΔ10-654 deletion placing the Alu element within 20 nt of the splice junction dramatically reduced circularization efficiency. However, smaller deletions were tolerated without affecting circRNA biogenesis or GFP expression. It should be noted that both smaller deletions spliced ​​equally well, indicating a sequence-independent effect. Next, a combination of an upstream LΔ42-267 deletion and a downstream RΔ10-497 deletion was used to create a minimal LΔRΔ intron element. The deletion combination resulted in a synergistic effect, increasing GFP expression and circRNA levels by approximately fivefold (Figures 4B-4K). Deletion of either the left or right Alu region (Figure 4L) incapacitated GFP expression (Figures 4M and 4N) and circRNA formation (Figures 4O and 4P), confirming the necessity of the Alu element in this system.

[0135] HIPK3 and modified HIPK3 constructs (e.g., L100, R1500, LΔ42-267, RΔ10-497, and LΔRΔ) were transfected into U87 glioblastoma and Huh7 hepatocellular carcinoma cell lines. Four days after transfection, U87 and Huh7 cells were collected and processed for Western and Northern blotting analysis as described above. GFP expression (Figures 5A and 5B) and circRNA expression (Figures 5C and 5D) in U87 cells, as well as GFP expression (Figures 5E and 5F) and circRNA expression (Figures 5G and 5H) in Huh7 cells, showed results similar to those observed in HEK293 cells detailed above. Similar results were observed in HEK293, U87 glioblastoma, and Huh7 hepatocellular carcinoma cell lines, indicating that the regulation of backsplicing was conserved across diverse cell types. (Example 3) The intron spacing effect on circRNA formation is conserved.

[0136] To confirm whether the conclusions drawn from HIPK3-derived introns are more generally applicable, the experiments described in Examples 1 and 2 were repeated using reporters driven by two different intron pairs derived from the human ZKSCAN1 gene or the Drosophila melanogaster laccase 2 gene (Figures 2B and 2C; Table 3). Three insertions were created in each intron pair (L100, L500, and R1500) (Figures 6A (ZKSCAN1) and 6F (laccase 2)). In both cases, insertions in the left intron reduced circRNA expression (Figures 6D-6E (ZKSCAN1) and 6I-6J (laccase 2)) and GFP expression (Figures 6B-6C (ZKSCAN1) and 6G-6H (laccase 2)). Insertions in the right laccase 2 intron did not alter GFP expression (Figures 6G-6H) or circRNA expression (Figures 6I-6J). However, insertion into the right ZKSCAN1 intron slightly reduced GFP expression (Figure 6B-6C) and circRNA expression (Figure 6D-6E).

[0137] For both laccase 2 (Figure 6P) and ZKSCAN1 (Figure 6K), deletion constructs were created that removed the largest possible sequences, based on the results observed in the HIPK3 intron pair in Example 2. Examples of laccase 2 and ZKSCAN1 intron sequences with deletion regions are provided in Table 3. For ZKSCAN1, it was not possible to design a deletion in the left intron due to space constraints between the Alu element and the splice site (Figure 6K). For laccase 2, LΔ403-507 and RΔ15-118 constructs, as well as combined deletions, were created (Figure 6P).

[0138] All ZKSCAN1 and laccase 2 deletions were tolerated, and GFP (Figure 6L-6M (ZKSCAN1) and 6Q-6R (laccase 2)) and circRNA (Figure 6N-6O (ZKSCAN1) and 6S-6T (laccase 2)) were expressed at levels equivalent to the original levels. Therefore, results from further intron pairs confirmed that the intron spacing effect on splicing efficiency appears to be generally conserved. (Example 4) IRES elements regulate circRNA levels and translation.

[0139] In the systems described in the above examples, GFP expression was a product of both circRNA level and IRES activity. Since circRNA does not contain a 5' end, canonical cap-dependent translation cannot occur, and therefore it must rely on the IRES element to initiate cap-independent translation and protein synthesis. Versions of the HIPK3 split GFP construct were created containing encephalomyocarditis virus (EMCV) IRES, poliovirus IRES, Kaposi's sarcoma-associated herpesvirus (KSHV) vFLIP IRES, or hepatitis C virus (HCV) IRES (Figure 7A). The sequences of these different IRES elements used in this example are provided in Table 6. [Table 6-1] [Table 6-2]

[0140] The constructs were transfected into HEK293 cells, and expression was assayed 4 days after transfection by GFP fluorescence (Figures 7B-7E) and Western blot analysis (Figures 7F-7G). These circRNAs containing different IRES elements showed fluctuating levels of GFP expression, with the poliovirus IRES-containing circRNA construct exhibiting approximately 5-fold higher expression compared to the EMCV and KSHV IRES elements. In contrast, no GFP expression was observed with the HCV IRES (Figures 7B-7G).

[0141] To directly evaluate translation efficiency, the polyribosome fraction was isolated, and the bound RNA was visualized by Northern blotting using a GFP-specific probe (Figure 7H-7P). Briefly, HEK293 cells were seeded overnight in 10 cm plates and transfected at approximately 70% confluence using PEI Max with 8 μg of the indicated plasmid. 24 hours after transfection, the cells were divided and seeded in a 1:2 ratio in 10 cm plates. The cells were grown to 60-70% confluence, then incubated at 37°C for 10 minutes in medium containing cycloheximide (CHX, 100 μg / mL), followed by two washes with ice-cold PBS containing CHX. Cells were lysed (20 mM Tris-HCl pH 7.4, 140 mM KCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100), and the cell membranes were disrupted by passing a 27 1 / 2 gauge needle through the cells five times. The lysate was spun to remove the nuclei, and then spun again to remove any remaining cellular debris. The clarified lysate was loaded onto a linear 10–50% sucrose gradient prepared in polysome gradient buffer (20 mM Tris-HCl pH 7.4, 140 mM KCl, 5 mM MgCl2) and spun uninterrupted at 32,000 rpm for 2 hours in an SW41 swinging bucket rotor. The gradient was fractionated into 750 μL fractions using a Brandel gradient fractionator system, while continuously monitoring absorbance at 254 nm. RNA was extracted from each gradient fraction using TRIzol reagent. The RNA was visualized by Northern blotting.

[0142] RNA associated with multiple ribosomes (polyribosomes) was translated more efficiently, while RNA associated with two ribosomes (disomes) or a single ribosome (monosomes) was not translated as efficiently. CircGFP RNA containing EMCV IRES was present on both monosomes and disomes, but most of the circRNAs were not translated. KSHV IRES-containing circRNAs were also present in fractions corresponding to monosomes and disomes, but, similar to EMCV circRNAs, the majority of these circRNAs were not translated, suggesting that low GFP protein levels were attributable to the low translation efficiency of these circRNAs. In contrast, poliovirus IRES-containing circGFP RNA was detected in higher fractions corresponding to multiple bound ribosomes, which correlated with the observation of higher levels of GFP expression (Figure 7H-7P).

[0143] When using HCV IRES, the absence of protein expression was confirmed by the fact that the majority of circRNAs were in the unbound fraction; in contrast, control linear GFP mRNA was detected in the high-weight polysome fraction (Figure 8A–8F). Overall, the RNA and protein levels of these constructs in U87 and Huh7 cell lines were similar to those observed in 293T cells (Figure 9A–9H), suggesting that the IRES-mediated translation efficiency of these circRNA vectors is similar in vitro.

[0144] When the total RNA produced from these constructs was visualized via Northern blotting, some differences were noted. Firstly, the total amount of circRNA produced varied among the four constructs. The HCV IRES construct expressed the highest level of circRNA, followed by KSHV, poliovirus, and finally EMCV IRES (Figures 7Q and 7R). While the sizes of the four IRES elements varied, there was no strong correlation between IRES (and therefore circRNA) size and circRNA levels. Secondly, there were dramatic differences in the types of RNA species produced other than circRNA. In particular, smaller RNA species than circRNA were observed in both the poliovirus and KSHV constructs. Furthermore, the KSHV construct produced several larger RNA species (Figure 7Q). Interestingly, these higher bands were detected in the polyribosome fraction, while the smaller RNA species were not (Figures 7H-7P). Both the smaller RNA species and the larger KSHV RNA species are RNase R resistant, suggesting that they may also be circular (Figure 7S). (Example 5) IRES elements significantly influence circRNA splicing patterns.

[0145] To further investigate the identity of the observed RNA species, RNase H digestion was performed using oligonucleotides that bind to the backsplice junction, confirming the circular nature of these RNA species. The band corresponding to the main circRNA remained. Interestingly, while many of the higher KSHV bands completely disappeared or decreased in intensity, the bands corresponding to the main and smaller circRNAs increased in intensity, suggesting that these larger species may be concatemers (Figure 10A). Total RNA was then probed using a probe specific to the backsplice junction. The RNA band formation pattern was similar to that observed with the probe for GFP exons, suggesting that these RNA species represent spliced ​​products (Figure 10B).

[0146] To elucidate the identity of further spliced ​​products produced by poliovirus and KSHV circRNAs, total RNA was probed against each IRES element of the construct. This probe detected most RNA species but not small circRNAs, indicating that this species lacks IRESs and suggests that the IRES elements were spliced ​​out from the RNA product (Figures 10C-10D). Next, a virtual Northern blot was performed using extracted RNA corresponding to the small circRNAs. Briefly, 5 μg of RNA was isolated on denatured agarose. The gel was stained with ethidium bromide to visualize ribosomal RNA. Gel fragments corresponding to larger and smaller circRNAs were cut based on the distance from 18S rRNA (distance measured from the Northern blot). RNA was extracted from the gel. The RNA was treated with DNase using the Turbo DNA-free kit (Ambion). Equal nanogram amounts of DNase-treated RNA were converted to cDNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems). The reverse transcription product was used as a template for PCR using backsplice-specific primers (5'-CTGCTTGTCGGCCATGATATAGACGTTGTGGC-3' (SEQ ID NO: 30); 5'-CAAGCTGACCCTGAAGTTCATCTGCACCACC-3' (SEQ ID NO: 31)) or primers extending to the IRES element (5'-GGCCGACAAGCAGAAGAACGGCATCAAG-3' (SEQ ID NO: 32); 5'-GGTGGTGCAGATGAACTTCAGGGTCAGCTTG-3' (SEQ ID NO: 33)), and the purified PCR product was subsequently sequenced.

[0147] After cDNA synthesis, PCR products were obtained using primers that amplified across the IRES and sequenced to confirm that the IRES was indeed spliced ​​out from these circRNA products. In both cases, weak splice donors in the last two codons of GFP were spliced ​​to weak acceptors at the end of the IRES (Figure 10E). Having identified the splice sites involved, alternative donor sites were deactivated by introducing silent mutations into the GFP coding sequence, thereby eliminating the formation of small circRNAs (Figures 10F-10G). These results highlight the importance of identifying weak donor / acceptor splice sites in circRNA vector design. (Example 6) The production of larger circRNAs does not affect expression.

[0148] The size of endogenous circRNA exons can range from approximately 100 nt to >1 kb, with an average of approximately 700 nt. The circGFP RNA in the examples herein is in the range of 1.1–1.5 kb in length, which is considerably larger than endogenous circRNA, but is expressed at high levels. To investigate whether the exon length could be further increased, a P2A autocleavable sequence and a subsequent dsRed ORF were added downstream of the terminal of a GFP ORF fragment (-FP), increasing the exon length by approximately 750 nt to a total of 2.2 kb (Figure 11A). When this larger exon was paired with both the original and LΔRΔ HIPK3 intron pairs, GFP expression (Figures 11B–11H) was obtained in addition to dsRed expression (Figures 11E and 11F), in addition to comparable GFP expression (Figures 11B–11H) to previous results (see Figures 4A–4I). Polyribosome analysis revealed that GFP-dsRed circRNA was present in fractions containing multiple ribosomes undergoing translation, indicating that GFP-dsRed circRNA is efficiently translated (Figures 11J-11L). Addition of the P2A sequence resulted in the removal of the GFP stop codon, mutating previously identified weak splice donor sites. Therefore, analysis of total RNA species by Northern blotting detected only two species: circGFP RNA and a larger RNA species (Figures 11M and 11I). RNase R and RNase H analysis confirmed the cyclic nature of the putative circRNA bands, revealing that the larger species was linear (Figures 11N and 11O). Probing for backsplice junctions revealed that both bands contained backsplice junctions, indicating that the larger linear species underwent a splicing event (Figure 11P). These results demonstrate that both endogenous and synthetic HIPK3 backsplicing introns can mediate the splicing of the larger circRNA product. (Example 7) CircRNAs can be highly expressed in cardiac and muscle tissue using AAV vectors.

[0149] To gather further insights into circRNA backsplicing and IRES-mediated translation efficiency in vivo, DNA constructs containing (1) the original HIPK3 intron and poliovirus IRES or (2) a cytomegalovirus (CMV) promoter driving either an LΔRΔ intron and poliovirus IRES, with a split GFP exon and an inverted terminal repeat flanked, were packaged into AAV vectors (Figure 12A). Briefly, recombinant AAV vectors were generated using a triple plasmid transfection protocol with modifications. Briefly, the transfection mixture contained (1) a pXR helper plasmid; (2) an adenovirus helper plasmid pXX6-80; and (3) the indicated circRNA coding sequence and SV40 polyA driven by a CMV promoter with an AAV2 inverted terminal repeat (ITR) flanked. Vector purification was performed by polyethylene glycol (PEG) precipitation (8% w / v) from the supernatant of the culture medium using iodixanol gradient ultracentrifugation, followed by desalting using a ZebaSpin desalting column (40K MWCO, Thermo Scientific). Vector genome (vg) titers were obtained by quantitative PCR (Lightcycler 480, Roche Applied Sciences) using SYBR Green (Roche Applied Sciences) and primers designed to selectively bind to the AAV2 inverted terminal repeat (forward: 5'-AACATGCTACGCAGAGAGGGAGTGG-3' (SEQ ID NO: 34); reverse: 5'-CATGAGACAAGGAACCCCTAGTGATGGAG-3' (SEQ ID NO: 35)).

[0150] In each cohort, mice (strain company: C57BL / 6) were given 3 × 10 11The animals were intravenously injected via the tail vein. Tissue samples were collected 4 weeks after injection. 4 weeks after injection, the mice were overdosed with tribromoethanol (Avertin) (1.25% solution at 0.2 mL / 10 g) via the intraperitoneal route. This was followed by transcardiac perfusion with phosphate-buffered saline. Parts of the collected organs (heart, liver, skeletal muscle) were dissected and stored in RNAlater solution (Invitrogen); the remainder was postfixed in 4% paraformaldehyde.

[0151] Next, the vector genome copy number in the tissues was quantified. Briefly, genomic DNA was extracted from fixed tissue sections using the QiaAmp DNA FFPE Tissue Kit (QIAGEN, Germantown, MD, USA). Quantitative PCR was performed to calculate the viral genome copy number using primers specific to the CMV promoter (5'-CAAGTACGCCCCCTATTGAC-3' (SEQ ID NO: 36); and 5'-AAGTCCCGTTGATTTTGGTG-3' (SEQ ID NO: 37)). The vector genome copy number was standardized relative to the mouse lamin B2 locus as a housekeeping gene (primers 5'-GGACCCAAGGACTACCTCAAGGG-3' (SEQ ID NO: 38); and 5'-AGGGCACCTCCATCTCGGAAAC-3' (SEQ ID NO: 39)). The AAV vector genome copy numbers were statistically indistinguishable between cohorts, confirming equivalent drug delivery (Figure 12B).

[0152] Next, circRNA expression in heart, liver, and skeletal muscle tissue was evaluated using RT-PCR. Briefly, 5 µg of RNA was treated with DNase using the Turbo DNA-free kit (Ambion). Equal nanogram amounts of DNase-treated RNA were converted to cDNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). The reverse transcription products were used as templates for PCR (or quantitative PCR) using gene-specific primers for GFP (5'-CTGCTTGTCGGCCATGATATAGACGTTGTGGC-3' (SEQ ID NO: 40); 5'-CAAGCTGACCCTGAAGTTCATCTGCACCACC-3' (SEQ ID NO: 41)) and gene-specific primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5'-CCACTCCTCCACCTTTGAC-3' (SEQ ID NO: 42); 5'-ACCCTGTTGCTGTAGCC-3' (SEQ ID NO: 43)). Quantitative PCR was performed using a Roche Light-Cycler 480 and SYBR Green Mastermix (Roche Applied Sciences). Quantitative RT-PCR using primer pairs extending to the circRNA backsplice junction revealed that the LΔRΔ intron pair resulted in significantly higher circRNA expression compared to the original HIPK3 intron: approximately 4 times higher expression in the heart and liver, and approximately 50 times higher expression in muscle (Figures 12C-12D). In particular, circRNA expression in skeletal muscle was detected at very low levels with the original HIPK3 intron, but expression using the LΔRΔ intron pair dramatically increased to levels comparable to those in the heart and liver (Figures 12C-12D).

[0153] Next, immunofluorescence staining was performed on the collected tissues. Briefly, 50 μm thick sections were obtained from fixed tissue using a vibrating blade microtome for the heart and liver. Immunohistochemical analysis of GFP expression was performed using antibodies against GFP and Alexa Fluor goat anti-rabbit 488 secondary antibody. Sections were mounted on slides in ProLong Gold Antifade Mountant containing DAPI (4',6-diamidino-2-phenylindole), a fluorescent dye that strongly binds to adenine-thymine-rich regions in DNA. Imaging was performed at 20× magnification on Aperio ScanScope XT (bright-field) or Aperio ScanScope FL (fluorescence). Skeletal muscle samples were mounted and sectioned, then immunofluorescence and slide scanning were performed. Fluorescence was quantified in ImageJ for all tissues.

[0154] Immunofluorescence staining of sectioned tissue for GFP confirmed that the LΔRΔ construct demonstrated significantly higher GFP expression in the heart (Figure 12E-12H) and skeletal muscle (Figure 12I-12L). GFP expression in the liver was low in both constructs, despite differences in circRNA expression, indicating that poliovirus IRES did not result in efficient translation in the liver (Figure 13A-13D). These results demonstrate that altering intron distances can be utilized to regulate synthetic circRNA levels in vivo in different tissues.

[0155] Those skilled in the art will readily understand that this disclosure is well adaptable to achieve its purpose and to obtain the outcomes and benefits mentioned and those inherent thereto. The disclosure described herein is, for the time being, representative and illustrative of preferred embodiments and is not intended as a limitation on the scope of this disclosure. Variations and other uses therein, encompassing the spirit of this disclosure as defined by the claims, will be recalled to those skilled in the art.

[0156] No authorization is made that any reference, including any non-patent or patent document, cited herein constitutes prior art. In particular, unless otherwise stated, any reference to any document herein is understood not to constitute an authorization that any of these documents form part of the common general knowledge of the art in the United States or any other country. Any consideration of a reference represents the claims of its authors, and the applicant has the right to object to the accuracy and appropriateness of any of the documents cited herein. All references cited herein are incorporated entirely by reference unless expressly indicated otherwise.

[0157] If there are any discrepancies between any definitions and / or descriptions found in the cited references This disclosure shall govern the matter. The present invention provides, for example, the following items: (Item 1) A composition comprising nucleic acid molecules encoding at least two circular RNAs (circRNAs) in tandem. (Item 2) The composition comprises at least two nucleic acid molecules encoding circular RNA (circRNA), and the nucleic acid molecules are (i) a first circRNA containing the first circRNA coding sequence, and (ii) A second circRNA containing a second circRNA coding sequence The second circRNA is in tandem with the first circRNA coding sequence in the first circRNA. The composition according to item 1, wherein the tandem first circRNA coding sequence and the second circRNA are adjacent to an intron element. (Item 3) The nucleic acid molecule further comprises at least one promoter region, The composition according to either item 1 or item 2, wherein the at least one promoter region can recruit RNA polymerase type II, RNA polymerase type III, or any combination thereof. (Item 4) The nucleic acid molecule further comprises a left backsplicing intron element and a right backsplicing intron element, The first circRNA coding sequence is adjacent to the left backsplicing intron element and the right backsplicing intron element, The aforementioned right backsplicing intron includes at least one tRNA-derived intron, The composition according to any one of items 1 to 3, wherein the at least one tRNA-derived intron comprises a tRNA sequence that encodes a second circRNA during splicing. (Item 5) The first circRNA coding sequence is The 5' untranslated region (5'UTR) inside the intron element adjacent to the first circRNA coding sequence, which is the internal ribosome entry site (IRES), and Translational regulatory region within the 3'UTR, inside the intron element adjacent to the first circRNA coding sequence and the second circRNA. A composition according to any one of items 1 to 4, further comprising: (Item 6) The composition according to any one of items 1 to 5, wherein the intron elements adjacent to the tandem first circRNA coding sequence and the second circRNA are back-spliced ​​to produce the at least two circRNAs without forming a scar at the site where the circRNA is covalently ring-closed. (Item 7) An adeno-associated virus (AAV) genome comprising the nucleic acid molecules encoding at least two circRNAs from any of items 1 to 6. (Item 8) At least one AAV genome cassette containing at least one nucleic acid molecule encoding circular RNA (circRNA) At least one circRNA containing an internal ribosome entry site (IRES) element preceding the open reading frame (ORF) in the backsplicing cassette Adeno-associated virus (AAV) particles containing, The back-splicing cassette comprises an adeno-associated virus (AAV) particle containing at least one circRNA coding sequence of interest. (Item 9) The nucleic acid molecule encoding at least one circular RNA (circRNA) is adjacent to a tRNA-derived intron element, The AAV particle according to item 8, wherein the tRNA-derived intron element comprises at least one tRNA splicing cassette for at least one circRNA coding sequence. (Item 10) The AAV particle according to either item 8 or item 9, further comprising an intron pair adjacent to the at least one circRNA coding sequence of interest, wherein the intron pair adjacent to the at least one circRNA coding sequence of interest comprises a deletion of approximately 750 nucleotides. (Item 11) The AAV particle according to item 10, wherein the intron pair adjacent to at least one circRNA coding sequence of the aforementioned purpose comprises a left intron containing a deletion of about 250 nucleotides and a right intron containing a deletion of about 500 nucleotides. (Item 12) A method for delivering a target circRNA coding sequence to a cell, comprising the step of introducing the cell into a composition according to any one of items 1 to 7 or AAV particles according to items 8 to 11. (Item 13) A method for delivering a target circRNA coding sequence to a tissue, comprising the step of introducing the tissue into one of the compositions described in items 1 to 7 or AAV particles described in items 8 to 11. (Item 14) A method for treating a disease or condition in a subject, comprising the step of administering to the subject an effective amount of any of the compositions described in items 1 to 7 or the AAV particles described in items 8 to 11, wherein the effective amount is an amount that reduces at least one symptom of the disease or condition in the subject. (Item 15) A method for expressing at least one circular RNA (circRNA) in a tissue, comprising the step of administering at least one AAV particle described in any one of items 8 to 11 to the tissue, wherein the expression of the at least one circRNA is substantially increased compared to baseline. (Item 16) The method according to item 15, wherein the expression of at least one circRNA is increased by at least twofold compared to baseline. (Item 17) The method according to either item 15 or item 16, wherein when the at least one AAV particle is delivered to cardiac tissue, liver tissue, skeletal muscle tissue, or any combination thereof, the expression of the at least one circRNA increases by at least four times compared to baseline. (Item 18) The method according to any one of items 15 to 17, wherein, when the at least one AAV particle is delivered to skeletal muscle tissue, the expression of the at least one circRNA increases by at least 50 times compared to baseline. (Item 19) The method according to any one of items 12 to 18, wherein the at least one AAV particle is administered by intramuscular injection, intravenous injection, intracoronary injection, intra-arterial injection, or any combination thereof.

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence, wherein the nucleic acid sequence comprises a first circRNA coding sequence and a second circRNA coding sequence. The first circRNA coding sequence is adjacent to the left backsplicing intron element and the right backsplicing intron element, The second circRNA coding sequence is adjacent to the left tRNA splicing element and the right tRNA splicing element, The right backsplicing intron element includes the left tRNA splicing element, the second circRNA coding sequence, and the right tRNA splicing element. Nucleic acid molecule.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence further comprises at least one promoter region, the at least one promoter region capable of recruiting RNA polymerase type II, RNA polymerase type III, or any combination thereof.

3. The nucleic acid molecule according to claim 1 or 2, wherein the left backsplicing intron element includes an Alu element, and the right backsplicing intron element includes an Alu element.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the left backsplicing intron element comprises a nucleic acid sequence selected from any one of sequence numbers 1, 6, 11, 21, 23, and 25.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein the right backsplicing intron element comprises a nucleic acid sequence selected from any one of sequence numbers 2, 7, 12, 20, 22, and 24.

6. The nucleic acid molecule according to any one of claims 1 to 5, wherein the left backsplicing intron element includes a deletion of 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides compared to SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO:

11.

7. The nucleic acid molecule according to any one of claims 1 to 6, wherein the right backsplicing intron element includes a deletion of 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 nucleotides compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO:

12.

8. The nucleic acid molecule according to any one of claims 1 to 7, wherein the left tRNA splicing element comprises the nucleic acid sequence of sequence number 17.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the right tRNA splicing element comprises the nucleic acid sequence of sequence number 18.

10. The nucleic acid molecule according to any one of claims 1 to 9, wherein the first circRNA coding sequence includes a 5' untranslated region (5'UTR) and a 3'UTR, and the 5'UTR includes an internal ribosome entry site (IRES).

11. The nucleic acid molecule according to any one of claims 1 to 10, wherein the first circRNA coding sequence codes for a therapeutic protein or therapeutic RNA.

12. A nucleic acid molecule according to any one of claims 1 to 11, wherein the second circRNA coding sequence codes for an aptamer, guide RNA, protein sponge, miRNA sponge, protein-binding RNA, naturally occurring circRNA, antisense RNA, long non-coding RNA (lncRNA), small activating RNA (saRNA), functional non-coding RNA, ribosomal RNA (rRNA), nucleolar small RNA (snoRNA), intranuclear small RNA (snRNA), piwi-interacting RNA (piRNA), Y-RNA, 7SK RNA, or 7S RNA.

13. An adeno-associated virus (AAV) vector comprising a nucleic acid molecule according to any one of claims 1 to 12.

14. AAV particle comprising the AAV vector described in claim 13.

15. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 12, an AAV vector according to claim 13, or an AAV particle according to claim 14, and a pharmaceutically acceptable carrier or excipient.

16. For use in a method of delivering at least two circRNAs to cells or tissues, A composition comprising a nucleic acid molecule according to any one of claims 1 to 12, an AAV vector according to claim 13, or an AAV particle according to claim 14, A pharmaceutical composition according to claim 15, A composition or pharmaceutical composition comprising the step of introducing the composition or pharmaceutical composition into the cells or tissues.

17. For use in methods of treating diseases or conditions in subjects, A composition comprising a nucleic acid molecule according to any one of claims 1 to 12, an AAV vector according to claim 13, or an AAV particle according to claim 14, A pharmaceutical composition according to claim 15, The method comprises the step of administering an effective amount of the composition or pharmaceutical composition to a subject, wherein the effective amount is an amount that reduces at least one symptom of a disease or condition in the subject.

18. The composition for use or pharmaceutical composition according to claim 17, wherein the method comprises the step of administering at least one AAV particle to a tissue, wherein at least two circular RNAs are expressed in the tissue, and the expression of the at least two circular RNAs is substantially increased compared to baseline expression before administration of the at least one AAV particle.

19. The composition for use or pharmaceutical composition according to claim 18, wherein the expression of the at least two circRNAs is increased by at least twofold compared to the baseline expression before administration of the at least one AAV particle.

20. The composition for use or pharmaceutical composition according to claim 18, wherein the expression of the at least two circRNAs is increased by at least four times compared to baseline expression in cardiac tissue, liver tissue, skeletal muscle tissue, or any combination thereof, before administration of the at least one AAV particle.

21. The composition for use or pharmaceutical composition according to claim 18, wherein the expression of the at least two circRNAs is increased by at least 50 times compared to baseline expression in skeletal muscle tissue before administration of the at least one AAV particle.

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