Circular tough decoy RNAS and methods of use thereof

Abstract

The present disclosure provides circular RNAs comprising tough decoys for miRNA suppression and methods of use thereof. The circular RNAs described herein can be generated from an expression cassette or an adeno-associated virus (AAV) genome via backsplicing or tRNA splicing mechanisms following their transcription.

Description

ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CIRCULAR TOUGH DECOY RNAS AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS|0001] This application claims the benefit of and priority from U. S. Provisional No. 63 / 737,383 filed on December 20, 2024, the disclosure of which is incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to circular RN / Xs comprising tough decoys for miRNA suppression and methods of use thereof. The circular RN / Xs described herein can be generated from an expression cassette or an adeno-associated virus (AAV) genome via backsplicmg or tRN A splicing mechanisms following their transcription.INCORPORATION OF THE SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (TORQJ313 0I WO_SeqList_ST26.xml; Size: 430,888 bytes; and Date of Creation: December 19, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0004] MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcnptionally by binding to target messenger RNAs (mRN / Xs), resulting in their downregulation or inhibition of translation. Erroneous miRNA expression plays a significant role in the development and progression of various diseases and genetic disorders, including cancer, cardiovascular disease, and autoimmune disease.

[0005] The discovery of various pathological conditions associated with changes in miRNA expression levels has triggered an interest in the development of miRNA inhibitors as therapeutics. One such miRNA inhibitor is a tough decoy (TuD), which is designed to form a hairpin-shaped structure with stabilizing stems and an intervening unpaired region consisting of two miRN / X binding sites. Unfortunately, the use of I'uDs as a therapeutic modality has remained a challenge due to their complex structure and inefficient delivery to target cells and tissues.

[0006] Thus, there is a need in the art for stable, effective, and quantifiable TuDs for the treatment of disease.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069BRIEF SUMMARY

[0007] The present disclosure relates to circular RNAs encoding tough decoys for miRNA inhibition and methods of use thereof. In some embodiments, the present disclosure provides a recombinant nucleic acid molecule encoding a circular RNA, wherein the recombinant nucleic acid molecule comprises, from 5’ to 3’, (1) a 5’ backsplicing intronic element, a cargo, and a 3’ backsplicing intronic element; or (2) a tRNA 5’ leader, a tRNA 5’ exonic element, a cargo, a tRNA 3 ’ intronic element, a tRNA 3 ’ exonic element, and a tRNA 3 ’ trailer, wherein the cargo comprises a hairpin-shaped tough decoy, wherein the hairpin-shaped tough decoy comprises two single¬ stranded miRNA binding sites, and wherein the two single-stranded miRNA binding sites are flanked by a double-stranded stem region 1 and a double-stranded stem region 2.

[0008] In some embodiments, the present disclosure provides a vector comprising the recombinant nucleic acid molecule disclosed herein. In some embodiments, the present disclosure provides an rAAV particle comprising the recombinant nucleic acid molecule disclosed herein, or the vector disclosed herein, and a capsid of AAV1, AAV2, AAV218, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhSR, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or a variant thereof.

[0009] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the recombinant nucleic acid molecule disclosed herein, the vector disclosed herein, or the rAAV particle disclosed herein, and a pharmaceutically acceptable excipient.

[0010] In some embodiments, the present disclosure provides a method of expressing a circular RNA in a cell or tissue, comprising introducing to the cell or tissue the recombinant nucleic acid molecule disclosed herein, the vector disclosed herein, the rAAV particle disclosed herein, or the pharmaceutical composition disclosed herein.

[0011] In some embodiments, the present disclosure provides a method of expressing a circular RNA in a subject, comprising introducing to the tissue the recombinant nucleic acid molecule disclosed herein, the vector disclosed herein, the rAAV particle disclosed herein, or the pharmaceutical composition disclosed herein, under conditions wherein the circular RNA is transcribed.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0012] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutic effective amount of the recombinant nucleic acid molecule disclosed herein, the vector disclosed herein, the rAAV particle disclosed herein, or the pharmaceutical composition disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and / or features of the present invention. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0014] Figures 1A-1E depict schematic representations of expression cassettes for linear and circular tough decoys for miRNA suppression. Figure 1A shows an expression cassette for Linear TuDNheI, a linear tough decoy. The tough decoy-encoding sequence comprises two Nhel restriction enzyme (RE) sites in the Stem I region (miRNA TuDNheI). Figure IB shows an expression cassette for circTuD-spl, a circular tough decoy produced by tRNA splicing. The tough decoy-encoding sequence is an unmodified tough decoy (miRNA TuD). The tough decoy- encoding sequence is flanked by spacer elements (5’ Spacer and 3’ Spacer). tRNA splicing elements include tRNA leaders, trailers, exons, introns or partial introns, ligation motifs, and RE sites (5’ RE and 3’ RE).Figure 1C shows an expression cassette for circTuD-sp2, a circular tough decoy produced by backsplicing. The tough decoy-encoding sequence is an unmodified tough decoy (miRNA TuD). The tough decoy- encoding sequence is flanked by spacer elements (5’ Spacer and 3’ Spacer) and RE sites (5’ RE and 3’ RE). Backsplicing elements include backsplicing introns, splice acceptors, and splice donors. Figure ID shows an expression cassete for circTuDW, a modified circular tough decoy produced by tRN / X splicing. The tough decoy-encoding sequence incorporates tRNA splicing elements (e.g., RE sites and ligation motifs) into Stem 1 along with three additional mutations for detection (miRNA TuD1*). Figure IE shows an expression cassette for circTuDW2, a modified circular tough decoy produced by backsplicing. The tough decoy-encoding sequence incorporates RE sites into Stem I, an exonic splicing enhancer (ESE) into Stem II, and has three additional mutations for detection (miRNA TuDW2). All miRNA binding sites include a four-nucleotide bulge sequence. Dashed lines encompass elements that are within circRNAs after splicing.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0015] Figures 2A-2E show the predicted RNA structures of linear and circular tough decoys. Figure 2A shows the predicted RNA structure of the linear tough decoy (Linear TuDNheI). Figure 2B shows the predicted RNA structure of the circular tough decoy produced by tRNA splicing (circTuD-spl). Figure 2C shows the predicted RNA structure of the circular tough decoy (miRNA TuD-spl) produced by backsplicing (circTuD-sp2). Figure 2D shows the predicted RNA structure of the modified circular tough decoy produced by tRNA splicing (circTuDw). Figure 2E shows the predicted RNA structure of the modified circular tough decoy produced by backsplicing (circTuD^2). MBS, niiRNA binding site; RE, restriction enzyme site; ESE, exonic splicing enhancer.

[0016] Figure 3A shows the binding of miRNA to a tough decoy miRNA binding site (MBS) and the location of the four-nucleotide bulge. Figures 3B-3D show the sequences of the linear tough decoy (Linear TuDNheI) (Figure 3B), the modified circular tough decoy produced by tRNA splicing (circTuD^) (Figure 3C), and the modified circular tough decoy produced by backsplicing (circTuD^2) (Figure 3D). Key engineered elements like restriction enzyme (RE) sites, G-U wobble base pairs, ligation motif, and exonic components of splice sites are labeled in Figures 3C and 3D. Highlighted nucleotides show mutations in the modified tough decoy sequences compared to the unmodified tough decoy sequence.

[0017] Figure 4A shows luciferase activity m HeLa cells transfected with a miRNA dual luciferase reporter plasmid compared to cells transfected with a non-targeted reporter. The target miRNA is endogenously expressed in the cells. A dual luciferase assay was conducted 48 hours after transfection, and relative luminescence was normalized to cells transfected with the miRNA reporter, as indicated by the dotted line at fold-change of 1. Statistical analyses were performed using two-tailed t-test; ****p<0.0001. Figure 4B shows miRNA inhibition in HeLa cells co¬ transfected with the miRNA reporter plasmid and varying amounts of the linear tough decoy expression cassette (U6+27-Linear TuDNheI) or corresponding negative control (U6+27-Neg Ctrl TuDNheI). Total plasmid dose was held constant by addition of a stuffer plasmid. A dual luciferase assay was conducted 48 hours after transfection, and relative luminescence was normalized to cells transfected with the miRNA reporter along with the stuffer plasmid only, as indicated by the dotted line at fold-change of 1. Statistical analyses were performed using two-way ANOV A, and post-hoc comparisons to the negative control at each dose were conducted using Sidak’s multiple comparisons test; * * * *p<0.0001. Figure 4C shows niiRNA inhibition in HeLa cells co-transfectedATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069with the miRNA reporter plasmid and varying amounts of a mirVana™ miRNA inhibitor or corresponding negative control. A dual luciferase assay was conducted 48 hours after transfection, and relative luminescence was normalized to cells transfected with the miRNA reporter and vehicle control, as indicated by the dotted line at fold-change of 1. Statistical analyses were performed using two-way ANOVA, and post-hoc comparisons to the negative control at each dose were conducted using Sidak’s multiple comparisons test; ns=not significant, ****p<0.0001.

[0018] Figure 5A shows miRNA inhibition in HeLa cells co-transfected with a miRNA reporter plasmid and a linear tough decoy (U6+27-Linear TuDNi,el__PEX190) expression cassette, a circular tough decoy (U6+27-TricY-circTuD-spl__PEX332, CBA-HIPK3-circTuD-sp2_PEX334, or U6+27-TricY-circTuD*__PEX350) expression cassette, or a stuffer plasmid as a negative control (Neg Ctrl). Cells were harvested 24 hours after transfection, and miRNA inhibition was determined using a dual luciferase assay. Relative luminescence was normalized to Neg Ctrl cells. Data are represented as a percentage of activity of the positive control (Pos Ctrl), and the doted line indicates the relative activity level of the Neg Ctrl, Statistical analyses were performed using oneway ANOVA on In-transformed data, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test. Only relevant statistical comparisons are shown; ns=not significant, ***p<0.001, ****p<0,0001. Figures 5B-5D show expression levels of circRNA (Figure 5B) and total RNA (Figure 5C) in these cells, as well as the circularization efficiency for each vector (Figure 5D). Copies per nanogram (cp / ng) for circRNA and precursor RN / X were determined using reverse transcription followed by digital PCR (RT-dPCR). Total RNA was calculated as the sum of circRNA and precursor RNA, and circularization efficiency was calculated as the proportion of total RNA present as circRNA. Statistical analyses were performed using one-way ANOVA, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test; ns=not significant, *p<0.05, **p<0.01, ****p<0.0001.

[0019] Figure 6A shows the sequences of a modified circular tough decoy produced by tRNA splicing (circTuDw), a linear (i.e., non-circularizing) version of circTuDw, and the linear tough decoy (Linear TuDNhel). Highlighted nucleotides are mutations in circTuDwcompared to Linear TuDKIheI. The tough decoys shown have an AACA bulge (Bulgel) in each miRN A binding site. Other tough decoys not shown have a CCAA bulge (Bulge2) in each miRN A binding site. Figure 6B shows miRNA inhibition in HeLa cells co-transfected with the miRNA reporter plasmid and circular tough decoys (U6+27-TricY-circTuDw-Bulgel PEX350 or U6+27-TricY-circTuDw-ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Bulge2_PEX399) or linear tough decoys (U6+27-circTuDV1-Bulgel_PEX409, U6+27-circTuDw-Bulge2__PEX387, or U6+27-Linear TuDheI-Bulgel_PEX190). Cells were harvested 24 hours after transfection, and miRNA inhibition was determined using a dual luciferase assay. Relative luminescence was normalized to cells expressing the miRNA reporter along with a stuffer plasmid as a negative control (Neg Ctrl). Data are represented as a percentage of activity of the positive control (Pos Ctrl), and the dotted line indicates the relative activity level of the Neg Ctrl. Statistical analyses were performed using one-way ANOVA, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test. Only relevant statistical comparisons are shown; **p<0,01, ***p<0.001, ****p<0.0001. Figure 6C shows the expression levels of tough decoys in these cells, circRNA expression is shown for circular tough decoys, and mRNA expression is shown for linear tough decoys. Copies per nanogram (cp / ng) for circRNA and mRNA were determined using reverse transcription followed by digital PCR (RT-dPCR). Statistical analyses were performed using one-way ANOVA on In -transformed data; ns=not significant.

[0020] Figure 7A shows miRNA inhibition in HeLa cells co-transfected with the miRNA reporter plasmid and a modified circular tough decoy expression cassette comprising various tRNA splicing scaffolds (U6+27-TricY-circTuDw_PEX350, U6+27-TR Y-GT A5- 1 -circTuDw_PEX388, U6+27-TRL-CAA4-l-circTuDw_PEX389, or U6+27-TRR-TCT1 -1 -circTuDw_PEX390). Cells were harvested 24 hours after transfection, and miRNA inhibition was determined using a dual luciferase assay. Relative luminescence was normalized to cells expressing the miRNA reporter along with a stuffer plasmid as a negative control (Neg Ctrl). Data are represented as a percentage of activity of the positive control (Pos Ctrl), and the dotted line indicates the relative activity level of the Neg Ctrl. Statistical analyses were performed using one-way ANOVA; ns:::not significant. Figures 7B-7D show expression levels of circRNA (Figure 7B) and total RNA (Figure 7C) m these cells, as well as the circularization efficiency for each vector (Figure 7D). Copies per nanogram (cp / ng) for circRNA and precursor RNA were determined using reverse transcription followed by digital PCR (RT-dPCR), and total RNA was calculated as the sum of circRNA and precursor RNA. Circularization efficiency was calculated as the proportion of total RNA present as circRN A, and average values are shown within bars. Statistical analyses were performed using one-way ANOVA on In-transformed data (Figure 7B and Figure 7C) or on untransformed data (Figure 7D). Post-hoc comparisons to U6+27-TricY-circTuDw(PEX350) were conducted using Dunnett’s multiple comparisons test; ns=not significant, *p<0.05, **p<0.01, ****p<0.0001.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0021] Figure 8 A shows miRNA inhibition in HeLa cells co-transfected with the miRNA reporter plasmid and a circular tough decoy expression cassette (CBA-HIPK3-circTuD-sp2_PEX334, CBA-HIPK3-circTuDW2_PEX410, or CBA-HIPK3-AMP255-Alu-circTuDW2_PEX411). Cells were harvested 24 hours after transfection, and miRNA inhibition was determined using a dual luciferase assay. Relative luminescence was normalized to cells expressing the miRNA reporter along with a stuffer plasmid as a negative control (Neg Ctrl). Data are represented as a percentage of activity' of the positive control (Pos Ctrl), and the dotted line indicates the relative activity' level of the Neg Ctrl. Statistical analyses were performed using one-way ANOVA, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test; ns=not significant, **p<0.01. Figures 8B-8D show expression levels of circRNA (Figure SB) and total RNA (Figure SC) in these cells, as well as the circularization efficiency for each vector (Figure 8D). Copies per nanogram (cp / ng) for circRNA and precursor RNA were determined using reverse transcription followed by digital PCR (RT-dPCR). Total RNA was calculated as the sum of circRNA and precursor RNA, and circularization efficiency was calculated as the proportion of total RNA present as circRNA. Statistical analyses were performed using one-way ANOVA on In-transformed data (Figure 8B and Figure 8C) or on untransformed data (Figure 8D). Post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test; ns::::not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0022] Figure 9A shows miRNA inhibition in cells co-transfected with the miRNA reporter plasmid and a modified circular tough decoy (U6+27-TricY-circTuDw, CBA-HIPK3-AMP255-Alu-circTuDW2, or CBA-HIPK3-circTuDW2) or linear (i.e., non-circularizing) tough decoy (U6+27-circTuDw) expression cassette. Tough decoys with an AACA bulge (Bulgel) in each miRNA binding site were compared to versions with a CCAA bulge (Bulge2) in each miRNA binding site. Cells were harvested 24 hours after transfection, and miRNA inhibition was determined using a dual luciferase assay. Relative luminescence was normalized to cells expressing the miRN A reporter along with a stuffer plasmid as a negative control ( eg Ctrl). Data are represented as a percentage of activity of the positive control (Pos Ctrl), and the dotted line indicates the relative activity level of the N eg Ctrl. Figure 9B shows the expression levels of tough decoys in these cells. circRNA expression is shown for circular tough decoys, and mRNA expression is shown for linear versions. Copies per nanogram (cp / ng) for circRNA and mRNA were determined using reverse transcription followed by digital PCR (RT-dPCR). Figures 9C-9EATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069show, for circular tough decoys only, the expression levels of circRNA (Figure 9C) and total RNA (Figure 9D), as well as the circularization efficiency for each vector (Figure 9E). Expression levels for circRNA and precursor RNA ’ere determined using RT-dPCR. Total RNA was calculated as the sum of circRNA and precursor RNA, and circularization efficiency was calculated as the proportion of total RNA present as circRNA. All statistical analyses were performed using two-way ANOVA; ns=not significant, **p<0.01.

[0023] Figure 10A shows a schematic of an in vitro pharmacology study to measure the efficacy and potency of tough decoys delivered by AAV. Dose-response experiments were performed in a human kidney cell line engineered to stably express a miRNA-sensitive Renilla luciferase reporter. Assay-ready cells were plated, transduced at various multiplicity of infections (MOIs), and harvested 72 hours later to measure tough decoy activity and expression levels. Figure 10B shows miRNA inhibition in engineered cells transduced with AAV expressing a modified circular tough decoy (AAV186, AAV174, AAV146, or AAV182), linear tough decoy (AAV116), or negative control linear tough decoy (AAV117), miRNA inhibition was determined using a Renilla luciferase assay. Luminescence was normalized to vehicle (formulation buffer)-treated cells, as indicated by the dotted line at fold-change of 1. An increase in luciferase signal is indicative of miRNA inhibition. Statistical analyses were performed using two-way ANOVA, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test. Statistical comparisons to AAV116 are shown; ns:::not significant, ****p<0.0001. Figure 10C shows the miRNA Pharmacodynamic Signature (PD-Sig) for selected A / XVs at le7 MOI. PD-Sig is a reverse transcription-quantitative PCR (RT-qPCR) gene panel that reports changes in the expression of 13 miRNA target genes normalized across six housekeeping (non-miRNA target) genes, and it is reported as the log2 fold change compared to levels in vehicle-treated cells. An increase in PD-Sig is indicative of miRNA inhibition. Statistical analyses were performed using one-way ANOVA, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test. Statistical comparisons to AAV116 are shown; ns=not significant, **p<0.01. Figure 10D shows the expression levels of tough decoys in these cells. circRNA expression is shown for circular tough decoys, and mRNA expression is shown for linear tough decoys. Copies per nanogram (cp / ng) for circRNA and mRNA were determined using reverse transcription followed by digital PCR (RT-dPCR). Statistical analyses were performed using two- way ANOVA on In-transformed data, and post-hoc comparisons between all groups wereATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069conducted using Tukey’s multiple comparisons test. Statistical comparisons to AAV146 are shown; ns=not significant, **p<0.01, ***p<0.001. Figure 10E shows, for circular tough decoys only, the circularization efficiency for each vector. Expression levels for circRNA and precursor RNA were determined using RT-dPCR, and total RNA was calculated as the sum of circRNA and precursor RNA. Circularization efficiency was calculated as the proportion of total RNA present as circRNA, and average values across all MOIs are shown within bars. Statistical analyses were performed using one-way ANOVA, and post-hoc comparisons between all groups were conducted using Tukey’s multiple comparisons test. Statistical comparisons to AAV146 are shown; ns=not significant, **p<0.01. Figure 10F visualizes the relative potency of circular and linear tough decoy molecules by plotting expression levels from Figure 10D against miRNA inhibition levels from Figure 10B. No statistical analyses were performed.

[0024] Figure HA provides a description of the circular tough decoy vectors that were packaged into AAV for in vitro expression analysis in HEK293T cells. These vectors allow for direct comparison of scaffold, promoter, and AAV genome configuration fore expression of the circTuD* cargo. AAV146, AAV174, and AAV198 contain self-complementary (sc) genomes, and AAV199 contains a single-stranded (ss) genome. HEK293T cells were transduced with the AAVs at le6 multiplicity7of infection (MOI), RNA was harvested 72 hours post-transduction, and circRNA and precursor RNA levels were determined by reverse-transcription followed by digital PGR (RT-dPCR) As a negative control group, cells were incubated with formulation buffer only during the transduction experiment. Figures 11B-11D respectively show expression levels of circular RNA (Figure 11B), precursor RNA (Figure 11C), and total RNA (Figure 11D) in copies per ng input (cp / ng). Total RNA is calculated as the sum of circRNA and precursor RNA. Figure HE shows circularization efficiency, which is the proportion of total RNA present as circRNA; average values are shown within bars. All bars represent the group mean ± SEM; N=4. For statistical analyses, circRN A, precursor RN A, and total RNA data were log-transformed (Figures 11B-11D), and circularization efficiency data were logit-transformed (Figure HE) to conduct parametric statistical tests. To compare all groups to AAV146, ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was performed for circRNA and total RNA (Figures HB-11C), and Welch’s ANOVA followed by Dunnett’s T3 multiple comparison test was performed for precursor RN A and circularization efficiency due to variance heterogeneity, asATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069determined by the Brown-Forsythe test (Figures 11D-11E). ns=not significant, *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001.

[0025] Figure 12A shows a schematic of an in vivo study to measure the biodistribution and expression of circTuDwvectors delivered by AAV to mice. The AAVs described m Figure 11 A were injected intravenously via temporal facial vein into P0-P1 neonatal male mice at a dose of lei 4 vector genomes (vg) per kilogram (kg) (N=5). A group of mice were injected with formulation buffer only as a negative control (N=3). Mice were euthanized 14 days after injection, and the left kidneys were harvested and processed for biomolecular analysis by dPCR and RT-dPCR. Figure 12B shows biodistribution levels in the kidney, represented as vector genome (vg) copies per 1000 ng gDNA. Figure 12C shows expression levels of circRNA in the kidney, represented as copies per 100 ng input. Figure 12D shows circularization efficiency, which is the proportion of total RNA present as circRNA. Figure 12E shows circRNA copies expressed per vector genome. Bars represent the group mean ± SEM. Ordinary one-way ANOVA followed by Dunnett’s multiple comparison test was performed on non-transformed (Figure 12D) or log-transformed (Figures 12B-12C and 12E) data to compare all groups to AAV146. ns=not significant, *** p<0.001, **** p<0.0001. Figure 12F shows the average fold change in circRNA expression for each vector compared to AA V 146 in HEK293T cells and mouse kidneys (data from Figure 11B and Figure 12C). The dotted line represents a fold-change of 1.

[0026] Figure 13A shows a schematic of an in vivo study to measure the biodistribution and expression of AAV146 (U6+27-TricY-circTuDw) in mice. AAV146 was injected intravenously via temporal facial vein into P0-P1 neonatal mice at a dose of 3el4 vector genomes (vg) per kilogram (kg). Mice were euthanized 14 days after injection, and kidneys and livers were harvested for biomolecular analysis. Figure 13B shows biodistribution of vector genomes in the kidney and liver. Copies per 1000 ng of genomic DNA were determined using digital PCR (dPCR). Figures 13C-13E show' expression levels of circRNA (Figure 13C) and precursor RNA (Figure 13D) m these organs, as well as the circularization efficiency for each vector (Figure 13E). Copies per 100 ng of cDNA for circRNA and precursor RNA were determined using reverse transcription followed by digital PCR (RT-dPCR), and total RNA was calculated as the sum of circRNA and precursor RNA. Circularization efficiency was calculated as the proportion of total RNA present as circRNA, and average values are shown within bars. No statistical analyses were performed. Figure 13F shows a representative circRNA in situ hybridization image from formulation buffer-ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069treated or AAV146-treated animal kidneys. Probes were designed to detect only mature circRNA species. Arrows indicate circRNA-positive cells.

[0027] Figure 14A shows a schematic of an in vivo study to evaluate circTuDwexpression from various AAV-delivered tRNA splicing scaffolds in mice. AAV146 (U6+27-TricY-circTuDV1') or AAV174 (U6+27-TRY-GTA5-l-circTuDw) was injected intravenously via temporal facial vein into P0-P1 neonatal mice at a dose of 3el4 vector genomes (vg) per kilogram (kg). Mice were euthanized 21 days after injection, and kidneys, livers, and hearts were harvested for biomolecular analysis. Figure 14B shows biodistribution of vector genomes in the kidney, liver, and heart. Copies per 1000 ng of genomic DNA were determined using digital PCR (dPCR). Statistical analyses were performed using two-way ANOVA, and post-hoc comparisons between AAVs were conducted using Sidak’s multiple comparisons test; ns=not significant. Figures 14C-14E show expression levels of circRNA (Figure 14C) and precursor RNA (Figure 14D) in these organs, as well as the circularization efficiency for each vector (Figure 14E). Copies per 100 ng of cDNA for circRNA and precursor RNA were determined using reverse transcription followed by digital PCR (RT-dPCR), and total RNA was calculated as the sum of circRNA and precursor RNA. Circularization efficiency was calculated as the proportion of total RNA present as circRNA, and average values are shown within bars. Statistical analyses were performed using two-way ANOVA, and post-hoc comparisons between AAVs were conducted using Sidak’s multiple comparisons test; ns:::not significant, ****p<0.0001.

[0028] Figure 15A shows biodistribution of AAV146 in pig kidneys. Female Yorkshire-Landrace crossbreed pigs were dosed with formulation buffer or AAV146 at either I el4 or 3el4 total vector genomes per kidney, bilaterally, via retrograde ureteral administration. After a 28-day in-life period, kidneys were harvested and processed. 23 biopsies across the whole kidney were analyzed, and results can also be viewed in the cranial, mid, and caudal sections of the kidney. Copies per 1000 ng of genomic DNA were determined using digital PCR (dPCR). Figures 15B-15D show expression levels of circRNA (Figure 15B) and precursor RNA (Figure 15C), as well as the circularization efficiency (Figure 15D) at each dose. Copies per 100 ng of cDNA for circRN A and precursor RNA were determined using reverse transcription followed by digital PCR (RT-dPCR), and total RNA was calculated as the sum of circRNA and precursor RNA. Circularization efficiency was calculated as the proportion of total RNA present as circRN A, and average values are shown within bars. Figure 15E shows a representative circRNA in situ hybridization imageATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069from formulation buff er- treated or AAV146-treated pig kidneys. Probes were designed to detect only mature circRNA species. Arrows indicate circRNA-positive cells.DETAILED DESCRIPTION

[0029] All publications, patents and patent applications, including any drawings and appendices, are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0030] The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed disclosures, or that any publication specifically or implicitly referenced is prior art.Definitions

[0031] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa unless the content clearly dictates otherwise. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0032] The terms “a”, “an”, and “the”, as used herein, include plural references unless the context clearly dictates otherwise. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0033] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to theATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0034] The term “and / or”, as used herein, refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0035] The term “between”, as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0036] The terms “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0037] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0038] The term “coding sequence” or a polynucleotide which “encodes” a polypeptide, as used herein, is a nucleic acid molecule which is transcribed (in the case of DNA) and / or translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory’ sequences. The boundaries of the coding sequence are determined by a start codon atthe 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A transcription termination sequence may be located 3’ to the coding sequence. A Kozak sequence may be located 5’ to the coding sequence.

[0039] The term “flanked”, as used herein, with respect to a sequence that is flanked by other elements, indicates the presence of one or more of the flanking elements upstream and / or downstream, i.e., 5’ and / or 3’, relative to the sequence. The term “flanked” is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the gene of interest and a flanking element. A nucleic acid molecule that is “flanked” by two other elements indicates that one element is located 5’ to the sequence and the other is located 3’ to the sequence; however, there may be intervening sequences therebetween.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0040] The term “polynucleotide” or “nucleic acid”, as used herein, refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be single¬ stranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides can comprise modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction.

[0041] As used herein, the term “promoter” refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream coding or non-coding sequence.

[0042] As used herein, the term “terminator” refers to a DNA regulatory region that signals the end of transcription, causing the RNA polymerase to detach from the DNA template and release the newly synthesized RNA molecule,

[0043] The term “recombinant” means a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in the production of a construct that is distinct from a polynucleotide found in nature.

[0044] As used herein, the term “operably linked” refers to regulatory elements that are contiguous or function in trans with the nucleic acid molecule encoding the circular RNA in order to facilitate transcriptional or translational expression of the nucleic acid molecule in the target cell or tissue.

[0045] The term “tough decoy,” as used herein, refers to a type of miRNA inhibitor comprising a hairpin-shaped structure with stabilizing stems and an intervening unpaired region consisting of two miRNA binding sites. In some embodiments, the tough decoy binds to miRNA through complementary base pairing, which sequesters the miRNA into stable complexes, leading to its inhibition. In some embodiments, the tough decoy is a circular tough decoy.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0046] The term “regulatory' element”, as used herein, refers to transcriptional or translational control sequences that regulate transcription of a non-coding sequence (e.g., a circular RNA) or a coding sequence and / or regulate synthesis of an encoded polypeptide.

[0047] As used herein, the term “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. “Percent id entity” is the identity' fraction times 100. Comparison of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BL AST suite of sequence analysis programs. Unless noted otherwise, the term “sequence identity” in the claims refers to sequence identity as calculated by Clustal Omega® using default parameters.

[0048] The term “vector”, as used herein, refers to a molecule or moiety' which transports, transduces, or transfects a nucleic acid molecule of interest into a host cell or tissue. Vectors applicable for use include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA-derived cDNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce aATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0049] The term “viral vector”, as used herein, refers to a virus particle that functions as a nucleic acid delivery vehicle, which comprises a viral nucleic acid (i.e., a viral or vector genome) packaged within a virion. In some embodiments, the viral genome comprises a nucleic acid molecule comprising any one of the circRNAs described herein and the necessary engineered tRNA scaffolds required to produce such circRNA.

[0050] The term “recombinant AAV vector (rAAV vector)”, as used herein, refers to a polynucleotide vector comprising a nucleic acid sequence from an AAV and one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin). In some embodiments, the one or more heterologous sequences are flanked by at least one, in some embodiments two, AAV inverted terminal repeat sequences (ITRs). In some embodiments, such rAAV vectors can be replicated and packaged into infectious viral capsid particles, e.g., when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). An rAAV vector may be incorporated into a larger polynucleotide (e g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), and can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral capsid particle, particularly an AAV particle. An rAAV vector can be packaged into an A / XV capsid to generate a “recombinant adeno-associated viral capsid particle (rAAV particle).” The terms “recombinant AAV vector”, “rAAV vector” and “AAV vector” are used interchangeably herein.

[0051] The term “heterologous nucleotide sequence” refers to a nucleic acid sequence that is not naturally occurring in the virus. Generally, the heterologous nucleic acid comprises a sequence that encodes the circular RNA of interest (e.g., for delivery to a cell or tissue of a subject).

[0052] The term “inverted terminal repeat” or “ITR” sequence, as used herein, refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation. An “AAV inverted terminal repeat (ITR)” sequence is well known in the art, and is usually an approximatelyATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069145 -nucleotide sequence that is present at both termini of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contains several shorter regions of self¬ complementarity (designated A, A’, B, B’, C, C’ and D regions), allowing intra-strand base-pairing to occur within this portion of the ITR.

[0053] The term “capsid”, as used herein, refers to a capsid protein of a virus, such as an adeno- associated virus, wherein the capsid encapsulates a nucleic acid molecule or viral genome. In some embodiments, the capsid is a variant capsid. The term “variant capsid” refers to a capsid protein that has been modified (e.g., one or more amino acid substitutions) compared to a parental capsid protein. In some embodiments, the variant capsid protein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the native or parental capsid protein. The term “AAV capsid” or “AAV capsid protein” or “AAV cap”, as used herein, refers to a protein encoded by an AAV capsid (cap) gene (e.g., VPI, VP2, and XT’S) or a variant thereof. For example, the term includes but not limited to a capsid protein derived from any AAV serotype such as AAV1, AAV2, AAV218, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAVTO, AAVrhlO, AAVT1, AAV12, AAV13, AAV-DJ, AAV-2 / 1, AAV 2 / 6, AAV 2 / 7, AAV 2 / 8, AAV 2 / 9, AAV LK03, AAVrhlO, AAVrh74, AAV44-9, or a variant thereof. In some embodiments, the capsid is an AAV 9 capsid variant, such as? XAV.kl3 or AAV.k20. The term also includes a capsid protein expressed by or derived from a recombinant AAV such as a chimeric AAV. The term “? XAV capsid particle” or “AAV particle”, as used herein, includes at least one AAV capsid protein (e.g., a VPI protein, a VP2 protein, a VP3 protein, or variant thereof) and optionally encapsulates a nucleic acid from an AAV genome or a nucleic acid derived from an AAV genome. The term “serotype” used with respect to vector or virus capsid is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure.

[0054] The term “transduced”, as used herein, refers to a process by which a transgene is introduced into a host cell from a virus particle.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0055] The term “tropism”, as used herein, refers to preferential entry’ of a virus into a certain cell or tissue, optionally followed by expression of nucleic acid sequences (e.g., circular RNA) carried by the viral genome in the cell or tissue (e.g., expression of circular RNA).

[0056] The term “pharmaceutical composition” or “therapeutic composition”, as used herein, refers to a composition capable of being administered to a subject for the treatment of a particular disease, disorder, or condition.

[0057] The term “pharmaceutically acceptable excipient, carrier or diluent”, as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject’s immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by well-known conventional methods (see, e.g,, Remington, The Science and Practice of Pharmacy, 23rd edition, A. Adejare, ed., Academic Press, 2020).

[0058] As used herein, an “effective amount” or “therapeutically effective amount” is an amount or dose of a composition (e.g., a therapeutic composition, compound, or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition. The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type, disease stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069namely by monitoring a subject’s response to administration of a composition and adjusting the dosage accordingly (see e g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed., Mack Publishing Co., Easton, PA, U. S., 19th ed., 1995)).

[0059] As used herein, the terms “prevent,” “preventing,” “prevention” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease, or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures. In some embodiments, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g., a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” includes slowing the onset of disease relative to the absence of treatment and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “preventing” or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition or preventing or delaying the development of symptoms associated with the condition.

[0060] In some embodiments, the term “treating” refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. In some embodiments, treatment refers to a short¬ term (e.g., temporary and / or acute) and / or a long-term (e.g., sustained) improvement or remediation in one or more disease symptoms. In some embodiments, the improvement is an observable or measurable improvement. In some embodiments, the improvement is an improvement in the general feeling of well-being of the subject.

[0061] As used herein, the term “subject” refers to any subject, e.g., a human or a non-human mammal, for whom diagnosis, prognosis, or therapy is desired. The term “subject” may mean a human or non-human mammal affected, likely to be affected, or suspected to be affected with a disease. The terms “subject” and “patient” are used interchangeably herein. In some embodiments, a subject is a mammal. A mammal includes primates, such as humans, monkeys, chimpanzee, and apes, and non-primates such as domestic animals, including laboratory animals (such as rabbitsATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069and rodents, e.g., guinea pig, rat, or mouse) and household pets and farm animals (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife, birds, reptile, fish, or the like. As used herein, the term “a subject in need thereof’ includes subjects that could or would benefit from the methods described herein. Subjects in need of treatment include, without limitation, those already with the disease, disorder, or condition, those prone to having the disease, disorder, or condition, those in which the disease, disorder, or condition is suspected, as well as those in which the disease, disorder, or condition is to be prevented, ameliorated, or reversed. In some embodiments, the subject is human. In some embodiments, the subject is a neonate, a juvenile, or an adult.

[0062] The term “administer”, “administration”, or “administering”, as used herein refers to the act of injecting or otherwise physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject (e.g., human), such as by oral, mucosal, topical, intradermal, parenteral, intravenous, retrograde ureteral, mtravitreal, intraarticular, subretmal, intramuscular, intrathecal deli very and / or any other method of physical delivery described herein or known in the art. The delivery can be systemic or to a specific tissue. In some embodiments, the pharmaceutical composition is administered by intravenous infusion. In some embodiments, the pharmaceutical composition is administered by retrograde ureteral infusion. In some embodiments, the pharmaceutical composition is administered by using an intravenous catheter (e.g., by inserting an IV catheter into a suitable peripheral vein for intravenous infusion of the pharmaceutical composition).

[0063] The term “baseline” or “baseline expression”, as used herein, refers to the level against which expression of a particular gene, RNA, protein, or signaling pathway in a test sample (e.g., a transduced cell or tissue) is compared.

[0064] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic PressATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20691997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.Recombinant Nucleic Acid Molecules

[0065] The present disclosure provides a recombinant nucleic acid molecule encoding a circular RNA, wherein the circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy binds to at least one miRNA. In some embodiments, the binding of an miRNA by the hairpin-shaped tough decoy results in suppression of the miRNA. In some embodiments, the binding of an miRNA by the hairpin-shaped tough decoy prevents the miRNA from binding to its endogenous target(s).

[0066] In some embodiments, the recombinant nucleic acid molecule encodes a circular RNA, wherein the circular RNA comprises a hairpin-shaped tough decoy, wherein the hairpin-shaped tough decoy comprises two single-stranded miRNA binding sites. In some embodiments, at least one of the two single-stranded miRNA binding sites binds to an miRNA. In some embodiments, each of the two single-stranded miRNA binding sites binds to an miRNA. In some embodiments, each of the two single-stranded miRNA binding sites binds to the same miRNA. In some embodiments, each of the two single-stranded miRNA binding sites binds to a different miRNA.

[0067] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (1) a 5’ intromc element, a cargo, and a 3’ mtronic element, or (2) a tRNA 5’ leader, a tRNA 5’ exonic element, a cargo, a tRNA 3’ intromc element, a tRNA 3’ exonic element, and a tRNA 3’ trailer, wherein the cargo comprises a hairpin-shaped tough decoy, wherein the hairpin-shaped tough decoy comprises two single-stranded miRNA binding sites, and wherein the two single-stranded miRNA binding sites are flanked by a double-stranded stem region 1 and a double-stranded stem region 2.

[0068] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA, comprises, from 5’ to 3’, (a) a 5’ intromc element; (b) a cargo; and (c) a 3’ mtronic element, wherein the cargo comprises a hairpin-shaped tough decoy, wherein the hairpin-shaped tough decoy comprises two single-stranded miRNA binding sites, and wherein the two single-stranded miRNA binding sites are flanked by a double-stranded stem region 1 and a double-stranded stem region 2.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0069] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) a tRNA 5’ leader; (b) a tRNA 5’ exonic element; (c) a cargo; (d) a tRNA 3 ’ intronic element; (e) a tRNA 3 ’ exonic element; (f) and a tRNA 3 ’ trailer, wherein the cargo comprises a hairpin-shaped tough decoy, wherein the hairpin-shaped tough decoy comprises two single-stranded miRNA binding sites, and wherein the two single-stranded miRNA binding sites are flanked by a double-stranded stem region 1 and a double-stranded stem region 2.

[0070] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises two single-stranded miRNA binding sites. In some embodiments, the two single-stranded miRNA binding sites are flanked by a double-stranded stem region 1 and a double-stranded stem region 2.

[0071] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, at least one of the two singlestranded miRNA binding sites binds to at least one miRNA, In some embodiments, the miRNA is any miRNA known in the art, for example, any miRNA listed m miRBase (a public miRNA database). In some embodiments, at least one of the two single-stranded miRNA binding sites binds to a microRNA selected from any one of: miR-9, miR-10, miR-17, miR-19, miR-21, miR-22, miR-23, miR-24, miR-26, miR-27, miR-29, miR-34, miR-39, miR-92, miR-98, miR-100, miR-101, miR-103, miR-107, miR-122, miR-124, miR-125, miR-126, miR-129, miR-130, miR-132, miR-133, miR-135, miR-143, miR-145, miR-146, miR-148, miR-155, miR-182, miR-185, miR-195, miR-199, miR-200, miR-206, miR-208, miR-210, miR-212, miR-214, miR-219, miR-221, miR-326, miR-339, miR-375, miR-192, miR-320, miR-326, miR-337, miR-338, miR-369, miR-379, miR-384, miR-410, miR-425, miR-451, miR-516, miR-532, miR-638, and miR-663. In some embodiments, each of the two single-stranded miRNA binding sites binds to at least one miRNA.

[0072] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, each of the two single-stranded miRNA binding sites comprises at least one bulged nucleotide when bound to miRNA. As used herein, the term “bulged nucleotide” refers to an unpaired base in one strand of a nucleic acid complex (e.g., an unpaired base in the single-stranded miRNA binding site when bound to miRNA). In some embodiments, each of the two single-stranded miRNA binding sites comprisesATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069at least two continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises two to ten continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises two to eight continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises two to six continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises two to four continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises two continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises three continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises five continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises six continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRNA, and the four continuous bulged nucleotides consist of a polynucleotide sequence of AACA. In some embodiments, each of the two smgle- stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRNA, and the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0073] In some embodiments, each of the two single-stranded miRNA binding sites comprises at least two continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRN A binding sites comprises at least one bulged nucleotide in addition to the at least two continuous bulged nucleotides when bound to miRNA. In some embodiments, each of the two single-stranded miRNA binding sites comprises one bulged nucleotide, two bulged nucleotides, three bulged nucleotides, four bulged nucleotides, five bulged nucleotides, six bulged nucleotides, seven bulged nucleotides, eight bulged nucleotides, or more, in addition to the continuous bulged nucleotides when bound to miRN A. In some embodiments, each of the two single-stranded miRNA binding sites comprises one bulged nucleotide, two bulged nucleotides, three bulged nucleotides, four bulged nucleotides, five bulged nucleotides, six bulged nucleotides,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069seven bulged nucleotides, eight bulged nucleotides, or more, in addition to the four continuous bulged nucleotides when bound to miRNA. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of AACA. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0074] In some embodiments, each of the two single-stranded miRNA binding sites comprises at least two continuous bulged nucleotides, wherein each of the two single-stranded miRNA binding sites is otherwise 100% complementary to its corresponding miRNA target. In some embodiments, each of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides, wherein each of the two single-stranded miRNA binding sites is otherwise 100% complementary to its corresponding miRNA target. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of AACA, In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0075] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, each of the two single-stranded miRNA binding sites comprises at least one bulged nucleotide when bound to miRNA. In some embodiments, the at least one bulged nucleotide is located 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides downstream of the 5’ end of each of the two singlestranded miRNA binding sites. In some embodiments, the at least one bulged nucleotide is located 13 nucleotides downstream of the 5’ end of each of the two single-stranded miRNA binding sites. In some embodiments, the at least one bulged nucleotide is located 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides upstream of the 3’ end of each of the two single¬ stranded miRNA binding sites. In some embodiments, the at least one bulged nucleotide is located 10 nucleotides upstream of the 3’ end of each of the two single-stranded miRNA binding sites. In some embodiments, each of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides, and the continuous bulged nucleotides are located 13 nucleotides downstream of the 5’ end of the single-stranded miRN A binding site. In some embodiments, each of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides, and the continuous bulged nucleotides are located 10 nucleotides upstream of the 3’ end of the single-stranded miRNA binding site. In some embodiments, the four continuous bulgedATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069nucleotides consist of a polynucleotide sequence of AACA. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0076] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, at least one of the two single¬ stranded miRNA binding sites comprises at least one bulged nucleotide when bound to niiRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises at least two continuous bulged nucleotides when bound to niiRNA. In some embodiments, at least one of the two single-stranded niiRNA binding sites comprises two to ten continuous bulged nucleotides. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises two to eight continuous bulged nucleotides. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises two to six continuous bulged nucleotides. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises two to four continuous bulged nucleotides. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises two continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises three continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises five continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises six continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRNA, and the four continuous bulged nucleotides consist of a polynucleotide sequence of AACA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRN A, and the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0077] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, at least one of the two smgle- stranded miRNA binding sites comprises at least one bulged nucleotide when bound to miRNA. In some embodiments, the at least one bulged nucleotide is located 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069nucleotides, 19 nucleotides, or 20 nucleotides downstream of the 5’ end of at least one of the two single-stranded miRNA binding sites. In some embodiments, the at least one bulged nucleotide is located 16 nucleotides downstream of the 5’ end of at least one of the two single-stranded miRNA binding sites. In some embodiments, the at least one bulged nucleotide is located 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides upstream of the 3’ end of at least one of the two single-stranded miRNA binding sites. In some embodiments, the at least one bulged nucleotide is located 13 nucleotides upstream of the 3’ end of at least one of the two single-stranded miRNA binding sites. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides, and the continuous bulged nucleotides are located 13 nucleotides downstream of the 5’ end of the single-stranded miRNA binding site. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides, and the continuous bulged nucleotides are located 10 nucleotides upstream of the 3’ end of the single-stranded miRNA binding site. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of AACA. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0078] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, at least one of the two single¬ stranded miRNA binding sites comprises at least two continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises at least one bulged nucleotide in addition to the continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises one bulged nucleotide, two bulged nucleotides, three bulged nucleotides, four bulged nucleotides, five bulged nucleotides, six bulged nucleotides, seven bulged nucleotides, eight bulged nucleotides, or more, in addition to the continuous bulged nucleotides when bound to miRNA. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises one bulged nucleotide, two bulged nucleotides, three bulged nucleotides, four bulged nucleotides, five bulged nucleotides, six bulged nucleotides, seven bulged nucleotides, eight bulged nucleotides, or more, in addition to the four continuous bulged nucleotides when bound to miRNA. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotideATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069sequence of AACA. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0079] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites. In some embodiments, at least one of the two single¬ stranded miRNA binding sites comprises at least two continuous bulged nucleotides when bound to miRNA, wherein at least one of the two single-stranded miRNA binding sites is otherwise 100% complementary to its corresponding miRNA target. In some embodiments, at least one of the two single-stranded miRNA binding sites comprises four continuous bulged nucleotides when bound to miRNA, wherein at least one of the two single-stranded miRNA binding sites is otherwise 100% complementary to its corresponding miRNA target. In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of AACA, In some embodiments, the four continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.

[0080] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites flanked by a double-stranded stem region 1 and a doublestranded stem region 2. In some embodiments, the double-stranded stem region 1 is 10 base pairs to 30 base pairs in length. In some embodiments, the double-stranded stem region 1 is 10 base pairs to 25 base pairs in length. In some embodiments, the double-stranded stem region 1 is 10 base pairs to 20 base pairs in length. In some embodiments, the double-stranded stem region 1 is 15 base pairs to 20 base pairs in length. In some embodiments, the double-stranded stem region 1 is 16 base pairs to 20 base pairs in length. In some embodiments, the double-stranded stem region 1 is 16 base pairs in length. In some embodiments, the double-stranded stem region 1 is 17 base pairs in length. In some embodiments, the double-stranded stem region 1 is 18 base pairs in length. In some embodiments, the double-stranded stem region 1 is 19 base pairs in length. In some embodiments, the double-stranded stem region 1 is 20 base pairs in length.

[0081] In some embodiments, the hairpin-shaped tough decoys described herein comprise two single-stranded miRNA binding sites flanked by a double-stranded stem region 1 and a double¬ stranded stem region 2, In some embodiments, the double-stranded stem region 2 is 5 base pairs to 20 base pairs in length. In some embodiments, the double-stranded stem region 2 is 5 base pairs to 15 base pairs in length. In some embodiments, the double-stranded stem region 2 is 5 base pairs to 10 base pairs in length. In some embodiments, the double-stranded stem region 2 is 6 base pairsATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069to 8 base pairs in length. In some embodiments, the double-stranded stem region 2 is 5 base pairs in length. In some embodiments, the double-stranded stem region 2 is 6 base pairs in length. In some embodiments, the double-stranded stem region 2 is 7 base pairs in length. In some embodiments, the double-stranded stem region 2 is 8 base pairs in length. In some embodiments, the double-stranded stem region 2 is 9 base pairs in length. In some embodiments, the double¬ stranded stem region 2 is 10 base pairs in length.

[0082] In some embodiments, the double-stranded stem region 1 or double-stranded stem region 2 comprises at least one wobble base pair. The term “wobble base pair” as used herein, refers to a hydrogen-bonded pairing between two nucleotides in RNA that is not in the canonical Watson- Crick base-pairing configuration. Some examples of wobble base pairs include, but are not limited to, guanine- uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). In some embodiments, the wobble base pairs are G-U wobble base pairs.

[0083] In some embodiments, the double-stranded stem region 1 comprises at least one wobble base pair. In some embodiments, the double-stranded stem region 1 comprises one wobble base pair, two wobble base pairs, three wobble base pairs, four wobble base pairs, five wobble base pairs, six wobble base pairs, seven wobble base pairs, eight wobble base pairs, nine wobble base pairs, ten wobble base pairs, or more. In some embodiments, the double-stranded stem region 1 comprises three wobble base pairs. In some embodiments, the double-stranded stem region 1 comprises at least one wobble base pair and the wobble base pair is a G-U wobble base pair. In some embodiments, the double-stranded stem region 1 comprises three wobble base pairs and the wobble base pairs are G-U wobble base pairs.

[0084] In some embodiments, the double- stranded stem region 2 comprises at least one wobble base pair. In some embodiments, the double-stranded stem region 2 comprises one wobble base pair, two wobble base pairs, three wobble base pairs, four wobble base pairs, five wobble base pairs, or more. In some embodiments, the double-stranded stem region 2 comprises at least one wobble base pair and the wobble base pair is a G-U wobble base pair.

[0085] Exemplary hairpin-shaped tough decoy sequences are provided in Table 1 below. The RE sites in the hairpin-shaped tough decoy sequence are underlined.Table 1. Exemplary Hairpin-Shaped Tough Decoy SequencesATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Name Nucleic Acid Sequence S EQ ID NO Negative Control GCTAGCGCTAGGATCATCAACTGAACAGTGTAG 64 TuDNlieIATCCGTACGAACCCAAGTATTCTGGTCACAGAA TACAACTGAACAGTGTAGATCCGTACGAACCCA AGATGATCCTAGCGCTAGCmiRNA TuD GACGGCGCTAGGATCATCAACCTACCTGCACTG 3TAACAAAGCACTTTGCAAGTATTCTGGTCACAG AATACAACCTACCTGCACTGTAACAAAGCACTT TGCAAGATGATCCTAGCGCCGTCmiRNA TuDhJheIGCTAGCGCTAGGATCATCAACCTACCTGCACTG 4TAACAAAGCACTTTGCAAGTATTCTGGTCACAG AATACAACCTACCTGCACTGTAACAAAGCACTT TGCAAGATGATCCTAGCGCTAGCmiRNA TuDwGCGGCCGCTAGGATCATCAACCTACCTGCACTG 65TAACAAAGCACTTTGCAAGTATTCTGGTCACAG AATACAACCTACCTGCACTGTAACAAAGCACTT TGCAAGATGGTTCTAGTGGCCGCmiRNA TuD'' - GCGGCCGCTAGGATCATCAACCTACCTGCACTG 66 Bulge2 TCCAAAAGCACTTTGCAAGTATTCTGGTCACAG AATACAACCTACCTGCACTGTCCAAAAGCACTT(Genel 13) TGCAAGATGGTTCTAGTGGCCGCmiRNA TuDw 2AAGCTTGCAGGACGGTCATCAACCTACCTGCAC 67TGTAACAAAGCACTTTGCAAGACTGAAGAAGA CTTCAGTCAACCTACCTGCACTGTAACAAAGCA CTTTGCAAGATGGCTGTCCTGCAGGCmiRNA TuDW2- AAGCTTGCAGGACGGTCATCAACCTACCTGCAC 68 Bulge2 TGTCCAAAAGCACTTTGCAAGACTGAAGAAGACTTCAGTCAACCTACCTGCACTGTCCAAAAGCACTTTGCAAGATGGCTGTCCTGCAGGC

[0086] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 3, 4, and 64-68. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence of any one of SEQ ID NOs: 3, 4, and 64-68 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence of any one of SEQ ID NOs: 3, 4, and 64-68. In some embodiments, theATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069hairpin-shaped tough decoy consists of a polynucleotide sequence of any one of SEQ ID NOs: 3, 4, and 64-68.

[0087] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NO: 3 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NOs: 3. In some embodiments, the hairpin-shaped tough decoy consists of a polynucleotide sequence of SEQ ID NOs: 3.

[0088] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NO: 4 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NOs: 4. In some embodiments, the hairpin-shaped tough decoy consists of a polynucleotide sequence of SEQ ID NOs: 4.

[0089] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 65. In some embodiments, the hairpin-ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069shaped tough decoy comprises a polynucleotide sequence of SEQ ID NO: 65 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NOs: 65. In some embodiments, the hairpin-shaped tough decoy consists of a polynucleotide sequence of SEQ ID NOs: 65.

[0090] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 66. In some embodiments, the hairpinshaped tough decoy comprises a polynucleotide sequence of SEQ ID NO: 66 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpinshaped tough decoy comprises a polynucleotide sequence of SEQ ID NOs: 66. In some embodiments, the hairpin-shaped tough decoy consists of a polynucleotide sequence of SEQ ID NOs: 66.

[0091] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 67. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NO: 67 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NOs: 67. In some embodiments, the hairpin-shaped tough decoy consists of a polynucleotide sequence of SEQ ID NOs: 67.

[0092] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a hairpin-shaped tough decoy. In some embodiments, the hairpin-shaped tough decoy comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least aboutATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206980%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 68. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NO: 68 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the hairpin¬ shaped tough decoy comprises a polynucleotide sequence of SEQ ID NOs: 68. In some embodiments, the hairpin-shaped tough decoy consists of a polynucleotide sequence of SEQ ID NOs: 68.

[0093] Exemplary sequences of cargos and components thereof are provided in Table 2. The 5’ and 3’ RE sites are underlined in circTuD* sequences. The tRNA 3’ intronic element is italicized in circTuD-spl and circTuDv* sequences. The exonic elements for backspacing are bolded in circTuD-sp2 and circTuD*2sequences.Table 2. Exemplary Sequences for Cargos and Components ThereofName Nucleic Acid Sequence SEQ ID NO circTuDwCargo GCGGCCGCTAGGATCATCAACCT 1ACCTGCACTGTAACAAAGCACTT TGCAAGTATTCTGGTCACAGAAT ACAACCTACCTGCACTGTAACAA AGCACTTTGCAAGATGGTTCTAG TGGCCGCGGAGGTcircTuDW2Cargo GTTGAAGCTTGCAGGACGGTCAT 2CAACCTACCTGCACTGTAACAAA GCACTTTGCAAGACTGAAGAAGA CTTCAGTCAACCTACCTGCACTG TAACAAAGCACTTTGCAAGATGG CTGTCCTGCAGGCCAGcircTuD-spl Cargo GCGGCCGCAACCCCTACCACATA 69CTGTTGAAGAAATATACGGGTAG AGCTCGACGGCGCTAGGATCATC AACCTACCTGCACTGTAACAAAG CACTTTGCAAGTATTCTGGTCAC AGAA TACAACC TACCTGCACI GT AACAAAGCACTTTGCAAGATGAT CCTAGCGCCGTCTACGCGTTCCG ACTACACAAATCAGCGATTCCGG TTGGTCGGGGTTGTGGCCGCGGAGGTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069circTuD-sp2 Cargo GTTGAAGCTTAACCCCTACCACG 70CAGTAGTATAGGTAGACATGAAC TCTTAAGGACGGCGCTAGGATCA TCAACCTACCTGCACTGTAACAA AGCACTTTGCAAGTATTCTGGTC ACAGAATACAACCTACCTGCACT GTAACAAAGCACTTTGCAAGATG ATCCTAGCGCCGTCGACACCATC TGCCTGACACCACTGTAGATTTC GGATGGTCGGGGTTAG CGCTCAGcircTuDw-AGAC Cargo GCGGCCGCTAGGATCATCAACCT 71ACCTGCACTGTAACAAAGCACTT TGCAAGTATTCTGGTCACAGAAT ACAACCTACCTGCACTGTAACAA AGCACTTTGCAAGATGGTTCTAG TGGCCGCGGAGACcircTuDw-GGCT Cargo GCGGCCGCTAGGATCATCAACCT 79ACCTGCACTGTAACAAAGCACTT TGCAAGTATTCTGGTCACAGAAT ACAACCTACCTGCACTGTAACAA AGCACTTTGCAAGATGGTTCTAG TGGCCGCGGGGCTcircTuDw-AGGC Cargo GCGGCCGCTAGGATCATCAACCT 73ACCTGCACTGTAACAAAGCACTT TGCAAGTATTCTGGTCACAGAAT ACAACCTACC TGCACTGTAACAA AGCACTTTGCAAGATGGT TCTAG TGGCCGCGGAGGCcircTuDw-Bulge2 Cargo GCGGCCGCTAGGATCATCAACCT 74ACCTGCACTGTCCAAAAGCACTT TGCAAGTATTCTGGTCACAGAAT ACAACCTACCTGCACTGTCCAAA AGCACTTTGCAAGATGGTTCTAG TGGCCGCGGAGGTcircTuDW2-Bulge2 Cargo GTTGAAGCTTGCAGGACGGTCAT 75CAACCTACCTGCACTGTCCAAAA GCACT TTGCAAGACTGAAGAAGA CTTCAGTCAACCTACCTGCACTG TCCAAAAGCACTTTGCAAGATGG CTGTCCTGCAGGCCAG5’ or 3’ RE site (Nhel only) GCTAGC Not Applicable5’ RE site (Notl only) GCGGCCGC Not ApplicableATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20695’ RE site (Hindlll only) AAGCTT Not Applicable 5’ RE site (Hindlll with Sbfl AAGCTTgcagg 80 reverse complement)3’ RE site (Afel only) AGCGCT Not Applicable 3’ RE site (Sbfl only) CCTGCAGG Not Applicable 3’ RE site (SacII only) CCGCGG Not Applicable 3’ RE site (SacII withNotl reverse gtggCCGCGG 5 complement)3’ RE site (Sbfl with partial CCTGCAGGc Not Applicable Hindlll reverse complement)tRN A 5 ’ Ligation motif TAGGATCATC 6 tRNA 3’ Ligation motif GATGGTTCTA 7 tRN A 5’ Ligation Motif AACCCCTACCA 57 tRNA 3’ Ligation Motif TGGTCGGGGTT 58Exomc Splicing Enhancer GAAGAAGAC Not Applicable

[0094] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 1, 2, and 69-75. In some embodiments, the cargo comprises a polynucleotide sequence of any one of SEQ ID NOs: 1, 2, and 69-75 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of any one of SEQ ID NOs: 1, 2, and 69-75. In some embodiments, the cargo consists of a polynucleotide sequence of any one of SEQ ID NOs: 1, 2, and 69-75.

[0095] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 1 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 1.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0096] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 2 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 2. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 2.

[0097] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 69. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 69 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 69. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 69.

[0098] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 70. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 70 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 70. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 70.

[0099] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 71. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 71 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 71. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 71.

[0100] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 72. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 72 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 72. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 72.

[0101] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 73. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 73 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 73. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 73.

[0102] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 74. In some embodiments, the cargo comprises a polynucleotide sequence of SEQATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069ID NO: 74 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 74. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 74.

[0103] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo. In some embodiments, the cargo comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 75. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 75 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the cargo comprises a polynucleotide sequence of SEQ ID NO: 75. In some embodiments, the cargo consists of a polynucleotide sequence of SEQ ID NO: 75.

[0104] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one restriction enzyme (RE) site. As used herein, the term “restriction site” or “RE site” refers to a polynucleotide sequence that is recognized and cleaved by a restriction endonuclease. In some embodiments, the RE site is defined as a recognition sequence of the restriction endonuclease (e.g., Notl RE site: GCGGCCGC). In some embodiments, the RE site comprises two restriction endonuclease recognition sequences. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises two RE sites capable of base pairing. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises two RE sites that contain G-U wobble base pairing mutations (e.g, SacII with reverse complement Notl RE site: gtggCCGCGG (SEQ ID NO: 5)). Exemplary sequences for RE sites are provided in Table 2.

[0105] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo and the cargo comprises at least one RE site. In some embodiments, the cargo comprises two RE sites. In some embodiments, the cargo comprises an RE site on the 5’ end of the cargo and is referred to herein as the 5’ RE site. In some embodiments, the cargo comprises an RE site on the 3’ end of the cargo and is referred to herein as the 3’ RE site.

[0106] In some embodiments, the hairpin-shaped tough decoy comprises at least one RE site. In some embodiments, the hairpin-shaped tough decoy comprises two RE sites. In someATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069embodiments, a 5’ RE site is located at the 5’ end of the hairpin-shaped tough decoy. In some embodiments, a 3’ RE site is located at the 3’ end of the hairpin-shaped tough decoy. In some embodiments, the 5’ RE site and the 3’ RE site form part of the double-stranded stem region 1 of the hairpin-shaped tough decoy. In other words, in some embodiments, the hairpin-shaped tough decoy comprises a 5’ RE site and a 3’ RE site.

[0107] In some embodiments, the RE site comprises a polynucleotide sequence that is at least about 50% identical, about 55% identical, at least about 60% identical, at least about 62.5% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 87.5% identical, at least about 90% identical, or 100% identical to any one of GCGGCCGC, AAGCTT, AGCGCT, CCTGCAGG, CCTGCAGGc, SEQ ID NO: 5, and SEQ ID NO: 80, In some embodiments, the RE site comprises a polynucleotide sequence selected from any one of GCGGCCGC, AAGCTT, AGCGCT, CCTGCAGG, CCTGCAGGc, SEQ ID NO: 5, and SEQ ID NO: 80 with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the RE site comprises a polynucleotide sequence selected from any one of GCGGCCGC, AAGCTT, AGCGCT, CCTGCAGG, CCTGCAGGc, SEQ ID NO: 5, and SEQ ID NO: 80. In some embodiments, the RE site consists of a polynucleotide sequence selected from any one of GCGGCCGC, AAGCTT, AGCGCT, CCTGCAGG, CCTGCAGGc, SEQ ID NO: 5, and SEQ ID NO: 80.

[0108] In some embodiments, the 5’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 62.5% identical, at least about 75% identical, at least about 87.5% identical, or 100% identical to GCGGCCGC. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC. In some embodiments, the 5’ RE site consists of a polynucleotide sequence of GCGGCCGC. In some embodiments, the hairpin-shaped tough decoy comprises the 5’ RE site and the 5’ RE site forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

[0109] In some embodiments, the 5’ RE site comprises a polynucleotide sequence that is at least about 55% identical, at least about 64% identical, at least about 73% identical, at least about 82% identical, at least about 91% identical, or 100% identical to SEQ ID NO: 80. In some embodiments,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 80 with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 80. In some embodiments, the 5’ RE site consists of a polynucleotide sequence of SEQ ID NO: 80. In some embodiments, the hairpm-shaped tough decoy comprises the 5’ RE site and the 5’ RE site forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

[0110] In some embodiments, the 3’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 5. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 5 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 5. In some embodiments, the 3’ RE site consists of a polynucleotide sequence of SEQ ID NO: 5, In some embodiments, the hairpin-shaped tough decoy comprises the 3’ RE site and the 3’ RE site forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy,

[0111] In some embodiments, the 3’ RE site comprises a polynucleotide sequence that is at least about 55% identical, at least about 64% identical, at least about 73% identical, at least about 82% identical, at least about 91 % identical, or 100% identical to CCTGCAGGc. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of CCTGCAGGc with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of CCTGCAGGc. In some embodiments, the 3’ RE site consists of a polynucleotide sequence of CCTGCAGGc. In some embodiments, the hairpm-shaped tough decoy comprises the 3’ RE site and the 3’ RE site forms part of the double-stranded stem region 1 of the hairpm-shaped tough decoy.

[0112] In some embodiments, the cargo comprises a 5’ RE site and a 3’ RE site. In some embodiments, the cargo comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 62.5% identical, at least about 75% identical, at least about 87.5% identical, or 100% identical to GCGGCCGC. In some embodiments, the cargo comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC with one or more mutations, such as 1, 2, 3, 4, or more mutations. In someATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069embodiments, the cargo comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC. In some embodiments, the cargo comprises a 5’ RE site, wherein the 5’ RE site consists of a polynucleotide sequence of GCGGCCGC. In some embodiments, the cargo comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 5. In some embodiments, the cargo comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 5 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the cargo comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 5. In some embodiments, the cargo comprises a 3’ RE site, wherein the 3’ RE site consists of a polynucleotide sequence of SEQ ID NO: 5.

[0113] In some embodiments, the cargo comprises a 5’ RE site and a 3’ RE site. In some embodiments, the 5’ RE site comprises a polynucleotide sequence that is at least about 55% identical, at least about 64% identical, at least about 73% identical, at least about 82% identical, at least about 91% identical, or 100% identical to SEQ ID NO: 80. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 80 with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 80. In some embodiments, the 5’ RE site consists of a polynucleotide sequence of SEQ ID NO: 80. In some embodiments, the cargo comprises the 5’ RE site and the 5’ RE site forms part of the double-stranded stem region 1 of the cargo. In some embodiments, the 3’ RE site comprises a polynucleotide sequence that is at least about 55% identical, at least about 64% identical, at least about 73% identical, at least about 82% identical, at least about 91% identical, or 100% identical to CCTGCAGGc. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of CCTGCAGGc with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of CCTGCAGGc. In some embodiments, the 3’ RE site consists of a polynucleotide sequence of CCTGCAGGc.

[0114] In some embodiments, the hairpin-shaped tough decoy comprises a 5’ RE site and a 3’ RE site. In some embodiments, the hairp in-shaped tough decoy comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 62.5% identical, at least about 75% identical, at least about 87.5% identical, or 100% identical toATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GCGGCCGC. In some embodiments, the hairpin-shaped tough decoy comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the hairpin-shaped tough decoy comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC. In some embodiments, the hairpin-shaped tough decoy comprises a 5’ RE site, wherein the 5’ RE site consists of a polynucleotide sequence of GCGGCCGC. In some embodiments, the hairpin-shaped tough decoy comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 5. In some embodiments, the hairpin-shaped tough decoy comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 5 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the hairpin-shaped tough decoy comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 5, In some embodiments, the hairpin-shaped tough decoy comprises a 3’ RE site, wherein the 3’ RE site consists of a polynucleotide sequence of SEQ ID NO: 5. In some embodiments, the hairpin-shaped tough decoy comprises the 3’ RE site and the 3’ RE site forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

[0115] In some embodiments, the hairpin- shaped tough decoy comprises a 5’ RE site and a 3’ RE site. In some embodiments, the 5’ RE site comprises a polynucleotide sequence that is at least about 55% identical, at least about 64% identical, at least about 73% identical, at least about 82% identical, at least about 91% identical, or 100% identical to SEQ ID NO: 80. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 80 with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 80. In some embodiments, the 5’ RE site consists of a polynucleotide sequence of SEQ ID NO: 80. In some embodiments, the hairpin-shaped tough decoy comprises the 5’ RE site and the 5’ RE site forms part of the double-stranded stem region 1 of the hairpm-shaped tough decoy. In some embodiments, the 3’ RE site comprises a polynucleotide sequence that is at least about 55% identical, at least about 64% identical, at least about 73% identical, at least about 82% identical, at least about 91% identical, or 100% identical to CCTGCAGGc. In some embodiments, the 3’ RE site comprises a polynucleotide sequence ofATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CCTGCAGGc with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of CCTGCAGGc. In some embodiments, the 3’ RE site consists of a polynucleotide sequence of CCTGCAGGc. In some embodiments, the hairpin-shaped tough decoy comprises the 3 ’ RE site and the 3 ’ RE site forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

[0116] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one ligation motif. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises two ligation motifs. Exemplary sequences for ligation motifs are provided in Table 2.

[0117] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a cargo and the cargo comprises at least one ligation motif. In some embodiments, the cargo comprises two ligation motifs. In some embodiments, the cargo comprises a ligation motif on the 5’ end of the cargo and is referred to herein as the 5’ ligation motif. In some embodiments, the cargo comprises a ligation motif on the 3’ end of the cargo and is referred to herein as the 3’ ligation motif,

[0118] In some embodiments, the hairpin-shaped tough decoy comprises at least one ligation motif. In some embodiments, the hairpin -shaped tough decoy comprises two ligation motifs. In some embodiments, a 5’ ligation motif is l ocated at the 5’ end of the hairpin-shaped tough decoy. In some embodiments, a 3’ ligation motif is located at the 3’ end of the hairpm-shaped tough decoy. In some embodiments, the 5’ ligation motif and the 3’ ligation motif form part of the double-stranded stem region 1 of the hairpin-shaped tough decoy. In other words, in some embodiments, the hairpin-shaped tough decoy comprises a 5’ ligation motif and a 3 ’ ligation motif. In some embodiments, the 5’ ligation motif is complementary to the 3’ ligation motif and enables the formation of Watson-Crick base pairing to form suitable substrates for ligation by a ligase. In some embodiments, the ligation motif described herein interacts with an RNA ligase to promote tRNA splicing. In some embodiments, the 5’ ligation motif and 3’ ligation motif comprise a binding sequence for an RNA ligase. In some embodiments, the RNA ligase is RtcB. Exemplary ligation motifs are provided in Table 2.

[0119] In some embodiments, the ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least aboutATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206980% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 6. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6. In some embodiments, the ligation motif consists of a polynucleotide sequence of SEQ ID NO: 6.

[0120] In some embodiments, the ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 7. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7, In some embodiments, the ligation motif consists of a polynucleotide sequence of SEQ ID NO: 7.

[0121] In some embodiments, the 5’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 6. In some embodiments, the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5’ ligation motif forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

[0122] In some embodiments, the 3’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 7. In some embodiments, the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the 3’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the hairpin-shaped tough decoy comprises the 3 ’ ligation motif and the 3 ’ ligation motif forms part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0123] In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 6; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 7. In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 6; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 7, In some embodiments, the hairpin-shaped tough decoy comprises the 5’ and 3’ ligation motifs and the ligation motifs form part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

[0124] In some embodiments, the ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 57. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 57 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 57. In some embodiments, the ligation motif consists of a polynucleotide sequence of SEQ ID NO: 57.

[0125] In some embodiments, the ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 58. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 58 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the ligation motif comprises a polynucleotide sequence of SEQ ID NO: 58. In some embodiments, the ligation motif consists of a polynucleotide sequence of SEQ ID NO: 58.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0126] In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 57; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 58. In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 57; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 58. In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 57; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 58. In some embodiments, the 5’ circularizing element comprises the 5’ ligation motif In some embodiments, the 3’ circularizing element comprises the 3’ ligation motif.

[0127] In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 58; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence that is at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or 100% identical to SEQ ID NO: 57. In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 58; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 57. In some embodiments, the cargo comprises a 5’ ligation motif, wherein the 5’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 58; and the cargo comprises a 3’ ligation motif, wherein the 3’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 57. In some embodiments, the 5’ circularizing element comprises the 5’ ligation motif. In some embodiments, the 3’ circularizing element comprises the 3 ’ ligation motif.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0128] In some embodiments, the double-stranded stem region 2 of the hairpin-shaped tough decoy comprises an exonic splicing enhancer. In some embodiments, the exonic splicing enhancer increases backsplicing and improves circularization efficiency. In some embodiments, the exonic splicing enhancer comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to GAAGAAGAC. In some embodiments, the exonic splicing enhancer comprises a polynucleotide sequence of GAAGAAGAC with one or more mutations, such as 1, 2, 3, 4, 5, or more mutations. In some embodiments, the exonic splicing enhancer comprises a polynucleotide sequence of GAAGAAGAC, In some embodiments, the exonic splicing enhancer consists of a polynucleotide sequence of GAAGAAGAC.

[0129] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one intronic element for backsplicing. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises two intronic elements for backsplicing. Examples of intronic elements include those described in Rybak-Wolf et al. (2005) Mol, Cell 58(5):870-885 and the circBase circular RNA database (Glazar et al. (2014) RNA 20: 1666-1670; and http: / / www.circbase.org), all incorporated by reference herein in their entirety. In some embodiments, the intronic element for backsplicing is referred to herein as a circularizing element.

[0130] Backsplicing relies on a linear precursor RNA with introns arranged in an order such that the endogenous cellular splicing machinery produces a circular product (as opposed to a linear “forward spliced” product) comprised of the intervening exonic sequence. The backsplicing introns can be cloned into plasmid systems and the exonic sequence can be replaced by a sequence of interest, allowing for exogenous production of a desired circRNA. Introns can be truncated down to minimal sequences required for circRNA formation. Alu sequences are among the minimal sequences often required for backsplicing intron function. These repetitive sequences are typically found on both introns flanking the sequence to be circularized, and mediate circRNA formation by base-pairing across the introns, bringing the splice sites closer together. Importantly, only the portions of the Alu sequences that base-pair across introns are necessary for circularization of the intervening exon.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0131] In some embodiments, the intronic element for backsplicing comprises an Alu element. In some embodiments, the intronic element for backsplicing is derived from human ZKSCAN1, human HIPK3, human EPHB4, and / or fly (Drosophila) Laccase2 genes. In some embodiments, the intronic element for backsplicing is a truncated intronic element, wherein the truncated intronic element is shorter in length compared to a corresponding wild-type intronic element. In some embodiments, the intronic element for backsplicing is a truncated intronic element derived from human ZKSCAN1, human HIPK3, human EPHB4, and / or fly (Drosophila) Laccase2 genes, wherein the truncated intronic element is shorter in length compared to the corresponding endogenous intronic element (i.e., endogenous ZKSCAN1, HIPK3, EPHB4 or Laccase2). In some embodiments, the intronic element is shorter in length compared to the endogenous intronic element (i.e., endogenous ZKSCAN1, HIPK3, EPHB4 or Laccase2) due to one or more deletions within the endogenous intronic element.

[0132] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, a 5’ intronic element, a cargo, and a 3’ intronic element for backsplicing.

[0133] In some embodiments, the intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 8-23, 402, 410-423, 602, and 609-623. In some embodiments, the intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 8-23, 402, 410-423, 602, and 609-623 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 8-23, 402, 410-423, 602, and 609-623. In some embodiments, the intronic element consists of a polynucleotide sequence selected from any one of SEQ ID NOs: 8-23, 402, 410-423, 602, and 609-623.

[0134] In some embodiments, the 5’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423. In some embodiments,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069the 5’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423. In some embodiments, the 5’ intronic element consists of a polynucleotide sequence selected from any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423.

[0135] In some embodiments, the 3’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623. In some embodiments, the 3’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 3’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623. In some embodiments, the 3’ intronic element consists of a polynucleotide sequence selected from any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623.

[0136] In some embodiments, the recombinant nucleic acid molecule comprises two intronic elements for backsplicing. In some embodiments, the recombinant nucleic acid molecule comprises a 5’ intronic element and a 3’ intronic element. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423; and the 3’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423 with one or more mutations, suchATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations; and the 3’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423; and the 3’ intronic element comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623. In some embodiments, the 5’ intronic element consists of a polynucleotide sequence selected from any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 402 and 410-423; and the 3’ intronic element consists of a polynucleotide sequence selected from any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 602, and 609-623.

[0137] In some embodiments, the 5’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8, In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 8 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 8. In some embodiments, the 5’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 8.

[0138] In some embodiments, the 3’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the 3’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 9 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 3 ’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 9. In some embodiments, the 3’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 9.

[0139] In some embodiments, the 5’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8; and the 3’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 8 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations; and the 3’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 9 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 8; and the 3’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 9. In some embodiments, the 5’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 8; and the 3’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 9.

[0140] In some embodiments, the 5’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 410. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 410 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 410. In some embodiments, the 5’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 410.

[0141] In some embodiments, the 3’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 609. In some embodiments, the 3’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 609 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 3’ intronic element comprises a polynucleotideATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069sequence of SEQ ID NO: 609. In some embodiments, the 3’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 609.

[0142] In some embodiments, the 5’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 410; and the 3’ intronic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 609. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 410 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations; and the 3’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 609 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the 5’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 410; and the 3’ intronic element comprises a polynucleotide sequence of SEQ ID NO: 609. In some embodiments, the 5’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 410; and the 3’ intronic element consists of a polynucleotide sequence of SEQ ID NO: 609.

[0143] In some embodiments, the recombinant nucleic acid molecule comprises a splice donor site. In some embodiments, the splice donor site comprises a polynucleotide sequence of GU. In some embodiments, the splice donor site consists of a polynucleotide sequence of GU. In some embodiments, the recombinant nucleic acid molecule comprises a splice acceptor site. In some embodiments, the splice acceptor site comprises a polynucleotide sequence of AG. In some embodiments, the splice acceptor site consists of a polynucleotide sequence of AG.Table 3. Exemplary Sequences for BackspacingName Nucleic Acid Sequence SEQ ID NO5’ HIPK3 GCCTCAGCCTCTCAAAGTGCTAGGATTACAGGGATCTATACT 8TTTCTTTTGAGGGAAAATGTTGGCACCGTTTCTAGGGCATATT GGCCATTTCAGCTTCTCAGTAAATATTTGTTAAGTAATTAAAT GCACTTGATTCTTTATTCTTAGCCTTTTAACGCAATACTCAGA ATAGCTGAAGCACCAATTAACTGAAATGGAGATATTATAAAGATAGTTATCTTCTCCAAGGGAAAAAATCATCTTCATGGAAAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TTAATTACTTTTTTACAAATTGTGAATTTGACCCTTAAGAGTT TTCTTCCTGATATTTAAAATTGAAAAAAAAATTGTTGACATT AATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTT TTGCAG3’ HIPK3 GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 9ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAGATTCAGATATACAATAATCCCAGCAACTT GGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGATGG AGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA5’ HIPK3 A42- GCCTCAGCCTCTCAAAGTGCTAGGATTACAGGGATCTATACT 10 267 ACAAATTGTGAATTTGACCCTTAAGAGTTTTCTTCCTGATATT TAAAATTGAAAAAAAAATTGTTGACATTAATATTTCTTCTTTC CTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTTGCAG3’ IIIPK3 A 10- GTAGGTAACCCAACTAGTTAAGCACCCCCAAATTTGAAAATT 11 497 CGTTTCCTTTGAGTGTCATGTCAATGCCCAAAAAGTTTCAGA TATTTGGATTTGAGATGCTCAACCTGTATAAGGATTCAGAAA GTTATTCTGATTAATGATTTTAAGATTCAGATATACAATAATC CCAGCAACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACC CAGGAGATGGAGGTTGCAGTGAGCCGAGATCATGCCATTGC ACTCCA5’ ZKSCAN1 AGTGACAGTGGAGATTGTACAGTTTTTTCCTCGATTTGTCAG 12 (100-547) GATTTTTTTTTTTTTGACGGAGTTTAACTTCTTGTCTCCCAGGT AGGAAGTGCAGTGGCGTAATCTCGGCTCACTACAACCTCCAC CTCCTGGGTTCAAGCGTTTCTCCTGCCTCAGCTTTCCGAGTAGC'rGGGATTACAGGCGCCTGCCACCATGCCCTGCTGACTFTTG TATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTG GTCTTGAACTCCTGACCGCAGGCGATTGGCCTGCCTCGGCCT CCCAAAGTGCTGAGATTACAGGCGTGAGCCACCACCCCCGG CCTCAGGAGCGTTCTGATAGTGCCTCGATGTGCTGCCTCCTA TAAAGTGTTAGCAGCACAGATCACTTTTTGTAAAGGTACGTA CTAATGACTTTTTTTTTATACTTCAG3' ZKSCAN1 GTAAGAAGCAAGGTTTCATTTAGGGGAAGGGAAATGATTCA 13GGACGAGAGTC FTTG1 GCTGCTGAGFGCCTG1 GATGAAGAAG CATGTTAGTCCTGGGCAACGTAGCGAGACCCCATCTCTACAA AAAATAGAAAAATTAGCCAGGTATAGTGGCGCACACCTGTG ATTCCAGCTACGCAGGAGGCTGAGGTGGGAGGATTGCTTGAGCCCAGGAGGTTGAGGCTGCAGTGAGCTGTAATCATGCCACTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069ACTCCAACCTGGGCAACACAGCAAGGACCCTGTCTCAAAAG CTACTTACAGAAAAGAATTAGGCTCGGCACGGTAGCTCACAC CTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCAGATCAC TTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGA AACCTTGTCTCTACTAAAAATATGAAAATTAGCCAGGCATGG TGGCACATTCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCA GGAGAATCACTTGAACCCAGGAGGTGGAGGTTGCAGTAAGC CGAGATCGTACCACTGTGCTCTAGCCTTGGTGACAGAGCGAG ACTGTCTTAAAAAAAAAAAAAAAAAAAAAAGAATTAATTAA AAATTTAAAAAAAAATGAAAAAAAGCTGCATGCTTGTTTTTT GTTTTTAGTTATTCTACATTGTTGTCATTATTACCAAATATTG GGGAAAATACAACTTACAGACCAATCTCAGGAGTTAAATGTT ACTACGAAGGCAAATGAACTATGCGTAATGAACCTGGTAGG CATTA5’ ZKSCAN1 AGTGACAGTGGAGATTGTACAGTTTTTTCCTCGATTTGTCAG 14 A367-395 GATTTTTTTTTTTTTGACGGAGTTTAACTTCTTGTCTCCCAGGT AGGAAGTGCAGTGGCGTAATCTCGGCTCACTACAACCTCCAC CTCCTGGGTTCAAGCGTTTCTCCTGCCTCAGCTTTCCGAGTAG CTGGGATTACAGGCGCCTGCCACCATGCCCTGCTGACTTTTG TATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTG GTCTTGAACTCCTGACCGCAGGCGATTGGCCTGCCTCGGCCT CCCAAAGTGCTGAGATTACAGGCGTGAGCCACCACCCCCGG CCTCAGGAGCGTTCTGATAGTGCCTCGAACAGATCACTTTTT GTAAAGGTACGTACTAATGACTTTTTTTTTATACTTCAG 3' ZKSCAN1 GT AAGAAGCAGGAGGCT GAGGT GGGAGGATT GCTT GAGCCC 15 All-177 AGGAGGTTGAGGCTGCAGTGAGCTGTAATCATGCCACTACTC CAACCTGGGCAACACAGCAAGGACCCTGTCTCAAAAGCTAC TTACAGAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGT AATCCCAGCACTTTGGGAGGCTGAGGCGGGCAGATCACTTG AGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAAC CTTGTCTCTACTAAAAATATGAAAATTAGCCAGGCATGGTGG CACATTCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGA GAAT CACTTGAACCCAGGAGGrGGAGGTTGCAGT AAGCCGA GATCGTACCACTGTGCTCTAGCCTTGGTGACAGAGCGAGACT GTCTTAAAAAAAAAAAAAAAAAAAAAAGAATTAATTAAAAA TTTAAAAAAAAATGAAAAAAAGCTGCATGCTTGTTTTTTGTT TTTAGTTATTCTACATTGTTGTCATTATTACCAAATATTGGGG AAAATACAACTTACAGACCAATCTCAGGAGTTAAATGTTACT ACGAAGGCAAATGAACTATGCGTAATGAACCTGGTAGGCAT TA5" Laccase2 TCATTGAGAAATGACTGAGTTCCGGTGCTCTCAAGTCATTGA 16TCTTTGTCGACTTTTATTTGGTCTCTGTAATAACGACTTCAAA AACATTAAATTCTGTTGCGAAGCCAGTAAGCTACAAAAAGA AAAAACAAGAGAGAATGCTATAGTCGTATAGTATAGTTTCCC GACTATCTGATACCCATTACTTATCTAGGGGGAATGCGAACC CAAAATTTTATCAGTTTTCTCGGATATCGATAGATATTGGGG AATAAATTTAAATAAATAAATTTTGGGCGGGTTTAGGGCGTG GCAAAAAGTTTTTTGGCAAATCGCTAGAAATTTACAAGACTT ATAAAATTATGAAAAAATACAACAAAATTTTAAACACGTGG GCGTGACAGTTTTGGACGGTTTTAGGGCGTTAGAGTAGGCGAGGACAGGGTTACATCGACTAGGCTTTGATCCTGATCAAGAATATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069ATATATACTTTATACCGCTTCCTTCTACATGTTACCTATTTTTC AACGAATCTAGTATACCTTTTTACTGTACGATTTATGGGTATA ATAATAAGCTAAATCGAGACTAAGTTTTATTGTTATATATAT TTTTTTTATTTTATGCAG’ Laccase2 GTAAGTATTCAAAATTCCAAAATTTTTTACTAGAAATATTCG 17ATTTTTTAATAGGCAGTTTCTATACTATTGTATACTATTGTAG ATTCGTTGAAAAGTATGTAACAGGAAGAATAAAGCATTTCCG ACCATGTAAAGTATATATATTCTTAATAAGGATCAATAGCCG AGTCGATCTCGCCATGTCCGTCTGTCTTATTATTTTATTACCG CCGAGACATCAGGAACTATAAAAGCTAGAAGGATGAGTTTT AGCATACAGATTCTAGAGACAAGGACGCAGAGCAAGTTTGT TGATCCATGCTGCCACGCTTTAACTTTCTCAAATTGCCCAAA ACTGCCATGCCCACATTTTTGAACTATTTTCGAAATTTTTTCA TAATTGTATTACTCGTGTAAATTTCCATCAATTTGCCAAAAA ACTTTTTGTCACGCGTTAACGCCCTAAAGCCGCCAATTTGGT CACGCCCACACTATTGAACAATTATCAAATTTTTTCTCATTTT ATTCCCCAATATCTATCGATATCCCCGATTATGAAATTATTAA ATTTCGCGTTCGCATTCACACTAGCTGAGTAACGAGTATCTG ATAGTTGGGGAAATCGACTTATTTTTTATATACAATGAAAAT GAATTTAATCATATGAATATCGATTATAGCTTTTTATTTAATA TGAATATTTATTTGGGCTTAAGGTGTAACCTCCTCGACATAA GACTCACATGGCGCAGGCACATTGAAGACAAAAATACTCAT TGTCGGGTCTCGCACCCTCCAGCAGCACCTAAAATTATGTCT TCAATTATTGCCAACATTGGAGACACAATTAGTCTGTGGCAC CTCAG’ Laccase2 A TCATTGAGAAATGACTGAGTTCCGGTGCTCTCAAGTCATTGA 18TCTTTGTCGACTTTTATTTGGTCTCTGTAATAACGACTTCAAA AACATTAAATTCTGTTGCGAAGCCAGTAAGCTACAAAAAGA AAAAACAAGAGAGAATGCTATAGTCGTATAGTATAGTTTCCC GACTATCTGATACCCATTACTTATCTAGGGGGAATGCGAACC CAAAATTTTATCAGTTTTCTCGGATATCGATAGATATTGGGG AATAAATTTAAATAAATAAAI'TTTGGGCGGGTTTAGGGCGTG GCAAAAAGTTTTTTGGCAAATCGCTAGAAATTTACAAGACTT ATAAAATTATGAAAAAATACAACAAAATTTTAAACACGTGG GCGTGACAGTTTTGGACGGTTTTAGTATAATAATAAGCTAAA TCGAGACTAAGTTTTATTGTTATATATATTTTTTTTATTTTATG CAG’ Laccase2 A GTAAGTATTCAAAAGCATTTCCGACCATGTAAAGTATATATA 19TTCTTAATAAGGATCAATAGCCGAGTCGATCTCGCCATGTCC GTCTGTCTTATTATTTTATTACCGCCGAGACATCAGGAACTAT A A A A GCTAGAAGGAT GAG’l T I T AGCAT ACAGAT1 CT AGAGA CAAGGACGCAGAGCAAGTTTGTTGATCCATGCTGCCACGCTT TAACTTTCTCAAATTGCCCAAAACTGCCATGCCCACATTTTTG AACTATTTTCGAAATTTTTTCATAATTGTATTACTCGTGTAAA TTTCCATCAATTTGCCAAAAAACTTTTTGTCACGCGTTAACGC CCTAAAGCCGCCAATTTGGTCACGCCCACACTATTGAACAAT TATCAAATTTTTTCTCATTTTATTCCCCAATATCTATCGATAT CCCCGATTATGAAATTATTAAATTTCGCGTTCGCATTCACACT AGCTGAGTAACGAGTATCTGATAGTTGGGGAAATCGACTTAT TTTTTATATACAATGAAAATGAATTTAATCATATGAATATCGATTATAGCTTTTTATTTAATATGAATATTTATTTGGGCTTAAGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GTGTAACCTCCTCGACATAAGACTCACATGGCGCAGGCACAT TGAAGACAAAAATACTCATTGTCGGGTCTCGCACCCTCCAGC AGCACCTAAAATTATGTCTTCAATTATTGCCAACATTGGAGA CACAATTAGTCTGTGGCACCTCAG5’ EPHB4 CCAGCTACTCAGGAGGCTGAGGCAGAAGAATCATTTTAACCC 20GGGAGGCGGAGATTGCAGTGAGCCAAGATCGCGCCACTGCG CTCCAGGCCTGGGTGACACCACGGAGACAGGGGTTTGGGGC TAAAAGCTATGAGCCGAGCCTCCGAGTCCAGTGGGAGTTAAT TCCCAGCTGACGGGGCCCTGCCTGATTTCTCAG3’ EPHB4 GTGAGCACCCTCCCTGGCTTCTGCGGCCACCCGGAGTTCCCA 21CTTACACCCAGAGGCCACTTGGGTTAAGAAGCCAGGACAGA CAGTGGGTCCCAGGTCACCTCCTCCAGCCTTTTCCTCTTGGGC TAAGCCCTGGTCCTCTGCCTTTTCTTTTTTTTAAGACAGAGCC TCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCGATCTCGG CTCATTGCTGTCTCCACCTCCAGGGTTCAAGCGATTCTCCTGC CTCAGTCTCCCAAGTAGCTGGTACTATAGGCATGCACCACCA TGCTGACTAATTTTTGTATTTTTAGTAGACACAGGGTTTCACC ATGTAGGCCAGGCTGGTATCAAACTCCTGACCTCAAGTGATC TCCCCACCTCAGCCTCCCAAAGTGCTGGTATTACAGGTGTGA GGCACCACGCCTGGCCAGCCCTCTGCCTTTAATTTTCCCTCTG GGAAAGGCTGGGCTCCTGGGACCTTCCTTTCCCACTGCCCCA TACAGCTGAAGGTTGTC5’ EPHB4 AHO- CCAGCTACTCAGGAGGCTGAGGCAGAAGAATCATTTTAACCC 22 161 GGGAGGCGGAGATTGCAGTGAGCCAAGATCGCGCCACTGCG CTCCAGGCCTGGGTGACACCACGGAGTTAATTCCCAGCTGAC GGGGCCCTGCCTGATTTCTCAG3’ EPHB4 A J O- GTGAGCACCGTTCCCACTTACACCCAGAGGCCACTTGGGTTA 23 35 AGAAGCCAGGACAGACAGTGGGTCCCAGGTCACCTCCTCCA GCCTTTTCCTCTTGGGCTAAGCCCTGGTCCTCTGCCTTTTCTTT TTTTTAAGACAGAGCCTCGCTCTGTCGCCCAGGCTGGAGTGC AGTGGCGCGATCTCGGCTCATTGCTGTCTCCACCTCCAGGGT TCAAGCGATTCTCCTGCCTCAGTCTCCCAAGTAGCTGGTACT ATAGGCATGCACCACCATGCTGACTAATTTTTGTATTTTTAGT AGACACAGGGTTTCACCATGTAGGCCAGGCTGGTATCAAACT CCTGACCTCAAGTGATCTCCCCACCTCAGCCTCCCAAAGTGC TGGTATTACAGGTGTGAGGCACCACGCCTGGCCAGCCCTCTG CCTTTAATTTTCCCTCTGGGAAAGGCTGGGCTCCTGGGACCTT CCTTTCCCACTGCCCCATACAGCTGAAGGTTGTC5’ HIPK3- GCCTCAGCCTCTCAAAGTGCTAGGATTACAGGGATCTATACT 410 AMP255-Alu TTTCTT'rTGAGGGAAAA'rGTFGGCACCGTTTCTAGGGCATA'rT GGCCATTTCAGCTTCTCAGTAAATATTTGTTAAGTAATTAAAT GCACTTGATTCTTTATTCTTAGCCTTTTAACGCAATACTCAGA ATAGCTGAAGCACCAATTAACTGAAATGGAGATATTATAAA GATAGTTATCTTCTCCAAGGGAAAAAATCATCTTCATGGAAA TTAATTACTTTTTTACAAATTGTGAATTTGCCTCAGCCTCTCA AAGTGCTAGGATTAGACCCTTAAGAGTTTTCTTCCTGATATTT AAAATTGAAAAAAAAATTGTTGACATTAATATTTCTTCTTTC CTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTTGCAG5’ HIPK3- GCCTCAGCCTCTCAAAGTGCTAGGATTAGACCCTTAAGAGTT 411AMP255A TTCTTCCTGATATTTAAAATTGAAAAAAAAATTGTTGACATTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069AATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTT TTGCAG5’ HIPK3- GTGGAATTCAAAGAGCCGCTCGAGTCCGCCTCAGCCTCTCAA 412 ClipOn AGTGCTAGGATTACAGGGATCTATACTTTTCTTTTGAGGGAA AATGTTGGCACCGTTTCTAGGGCATATTGGCCATTTCAGCTTC TCAGTAAATATTTGTTAAGTAATTAAATGCACTTGATTCTTTA TTCTTAGCCTTTTAACGCAATACTCAGAATAGCTGAAGCACC AATTAACTGAAATGGAGATATTATAAAGATAGTTATCTTCTC CAAGGGAAAAAATCATCTTCATGGAAATTAATTACTTTTTTA CAAATTGTGAATTTGACCCTTAAGAGTTTTCTTCCTGATATTT AAAATTGAAAAAAAAATTGTTGACATTAATATTTCTTCTTTC CTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTTGCAG5’ HIPK3- TCAAGTGATTCTCCTAGCCTCAGCCTCTCAAAGTGCTAGGAT 413 AED15 TACAGGGATCTATACTTTTCTTTTGAGGGAAAATGTTGGCAC CGTTTCTAGGGCATATTGGCCATTTCAGCTTCTCAGTAAATAT TTGTTAAGTAATTAAATGCACTTGATTCTTTATTCTTAGCCTT TTAACGCAATACTCAGAATAGCTGAAGCACCAATTAACTGAA ATGGAGATATTATAAAGATAGTTATCTTCTCCAAGGGAAAAA ATCATCTTCATGGAAATTAATTACTTTTTTACAAATTGTGAAT TTGACCCTTAAGAGTTTTCTTCCTGATATTTAAAATTGAAAAA AAAATTGTTGACATTAATATTTCTTCTTTCCTTTTTTTTCTTTT CCTTTTTTTTTTTTTTTTTGCAG5’ HIPK3- GTGGAATTCAAAGAGCCGCTCGAGTCCGATCTATACTTTTCT 414 ClipOnAAlu TTTGAGGGAAAATGTTGGCACCGTTTCTAGGGCATATTGGCC ATTTCAGCTTCTCAGTAAATATTTGTTAAGTAATTAAATGCAC TTGATTCTTTATTCTTAGCCTTTTAACGCAATACTCAGAATAG CTGAAGCACCAATTAACTGAAATGGAGATATTATAAAGATA GTTATCTTCTCCAAGGGAAAAAATCATCTTCATGGAAATTAAT'TACTT'TT'rTACAAAT'rGTGAAT'TTGACCCTTAAGACTTTT'TCT TCCTGATATTTAAAATTGAAAAAAAAATTGTTGACATTAATA TTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTTGC AG5’ HIPK3- ■rTT'rTrTT'rGAGACAGAGTCTrCTC'rCTGTCTCACAGGCTGAAG 415 FullAlu TGCAGTGGTGCAATCTCGGCTCACTGCAACCTCCACCTCCCA GGTTCAAGCGATTCTCCTCCCTCAGCCTCCCGAGTAGCTGGG ACCACAGGCATGCACCACCATCCCCAGCTAATTTTTGCATTA TTAGTAGAGTTGGGATTTCTTCACCGTGTTGGCCAGGCTGGT CTTGGACTCCTGACCTCAAGTGATCCAACTGCCTCAGCCTCT CAAAGTGCTAGGATTACAGGGATCTATACTTTTCTTTTGAGG GAAAATGTTGGCACCGTTTCTAGGGCATATTGGCCATTTCAG CTTCTCAGTAAATATTTGTTAAGTAATTAAATGCACTTGATTC TTTATTCTTAGCCTTTTAACGCAATACTCAGAATAGCTGAAG CACCAATTAACTGAAATGGAGATATTATAAAGATAGTTATCT TCTCCAAGGGAAAAAATCATCTTCATGGAAATTAATTACTTT TTTACAAATTGTGAATTTGACCCTTAAGAGTTTTCTTCCTGAT ATTTAAAATTGAAAAAAAAATTGTTGACATTAATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTTGCAGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20695’ HIPK3- 416 AED65 TCCTGGCTTCAAGTGATTCTCCAGCCTCAGCCTCTCAAAGTG CTAGGATTACAGGGATCTATACTTTTCTTTTGAGGGAAAATG TTGGCACCGTTTCTAGGGCATATTGGCCATTTCAGCTTCTCAG TAAATATTTGTTAAGTAATTAAATGCACTTGATTCTTTATTCT TAGCCTTTTAACGCAATACTCAGAATAGCTGAAGCACCAATT AACTGAAATGGAGATATTATAAAGATAGTTATCTTCTCCAAG GGAAAAAATCATCTTCATGGAAATTAATTACTTTTTTACAAA TTGTGAATTTGACCCTTAAGAGTTTTCTTCCTGATATTTAAAA TTGAAAAAAAAATTGTTGACATTAATATTTCTTCTTTCCTTTT TTTTCTTTTCCTTTTTTTTTTTTTTTTTGCAG5’ HIPK3- CTCCATCTCCTGGGTATCAAGTGATTCTCCTAGCCTCAGCCTC 417 AED30 TCAAAGTGCTAGGATTACAGGGATCTATACTTTTCTTTTGAG GGAAAATGTTGGCACCGTTTCTAGGGCATATTGGCCATTTCA GCTTCTCAGTAAATATTTGTTAAGTAATTAAATGCACTTGATT CTTTATTCTTAGCCTTTTAACGCAATACTCAGAATAGCTGAA GCACCAATTAACTGAAATGGAGATATTATAAAGATAGTTATC TTCTCCAAGGGAAAAAATCATCTTCATGGAAATTAATTACTT■rTT'rACAAATTG'rGAATTTGACCCTTAAGAGrTTIC'rTCCTGA TATTTAAAATTGAAAAAAAAATTGTTGACATTAATATTTCTT CTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTTGCAG5’ HIPK3- TCGGCTCACTGCAACACTCCATCTCCTGGGTATCAAGTGATT 418 AED45 CTCCTAGCCTCAGCCTCTCAAAGTGCTAGGATTACAGGGATC TATACTTTTCTTTTGAGGGAAAATGTTGGCACCGTTTCTAGGG CATATTGGCCATTTCAGCTTCTCAGTAAATATTTGTTAAGTAA TTAAATGCACTTGATTCTTTATTCTTAGCCTTTTAACGCAATA CTCAGAATAGCTGAAGCACCAATTAACTGAAATGGAGATATT ATAAAGATAGTTATCTTCTCCAAGGGAAAAAATCATCTTCAT GGAAATTAATTACTTTTTTACAAATTGTGAATTTGACCCTTAA GAGTTTTCTTCCTGATATTTAAAATTGAAAAAAAAATTGTTG ACATTAATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTT TTTTTTTTGCAG5’HIPK3- CAGGGATCTATACTTTTCTTTTGAGGGAAAATGTTGGCACCG 419 AMP255 TTTCTAGGGCATATTGGCCATTTCAGCTTCTCAGTAAATATTT GTTAAGTAATTAAATGCACTTGATTCTTTATTCTTAGCCTTTT AACGCAATACTCAGAATAGCTGAAGCACCAATTAACTGAAA TGGAGATATTATAAAGATAGTTATCTTCTCCAAGGGAAAAAA TCATCTTCATGGAAATTAATTACTTTTTTACAAATTGTGAATT TGCCTCAGCCTCTCAAAGTGCTAGGATTAGACCCTTAAGAGTT'TTCT'TCrT'GATAT'TTAAAATTGAAAAAAAAA'rTGI'TGACATT'AATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTT TTGCAG5’ HIPK3- CAGGGATCTATACTTTTCTTTTGAGGGAAAATGTTGGCACCG 420 AMP85 TTTCTAGGGCATATTGGCCATTTCA. GCTTCTCAGTAAATATTT GCCTCA. GCCTCTCAAAGTGCTAGGATTAGTTAAGTAATTAAATGCACTTGATTCTTTATTCTTAGCCTTTTAACGCAATACTCAGAATA. GCTGAAGCACCAATTAACTGAAATGGAGATATTATAAAGATAGT'TATCT'TCTCCAACTCTCTAAAAAATCATCT,T'CA'rGGAAAT'TAATTAC;rTT'TT'TACAAATTDTGAAT'TTGACCCTTAAGAG'rTTTCTTCCTGATATTTAAAATTGAAAAAAAAATTGTTGACATTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069AATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTT TTGCAG5’ HIPK3AA1U CTATACTTTTCTTTTGAGGGAAAATGTTGGCACCGTTTCTAGG 421 GCATATTGGCCATTTCAGCTTCTCAGTAAATATTTGTTAAGTA ATTAAATGCACTTGATTCTTTATTCTTAGCCTTTTAACGCAAT ACTCAGAATAGCTGAAGCACCAATTAACTGAAATGGAGATA TTATAAAGATAGTTATCTTCTCCAAGGGAAAAAATCATCTTC ATGGAAATTAATTACTTTTTTACAAATTGTGAATTTGACCCTT AAGAGTTTTCTTCCTGATATTTAAAATTGAAAAAAAAATTGT TGACATTAATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTT TTTTTTTTTTGCAG5’ HIPK3-PHS GCCTCAGCCTCTCAAAGTGCTAGGATTACAGGGACCTAAATT 422 ACTCCTCTCTTGAGGGAAAATGTTGGCACCGTTTCTAGGGCA TATTGGCCATTTCAGCTTCTCAGTAAATATTTGTTAAGTAATT AAATGCACTTGATTCTTTATTCTTAGCCTTTTAACGCAATACT CAGAATAGCTGAAGCACCTTAGCAACGGAAATGGAGATATT ATAAAGATAGTTATCTTCTCCAAGGGAAAAAATCATCTTCAT GGAAATTAATTACTTTTTTACAAATTGTGAATTTGACCCTTAA GAGTTTTCTTCCTGATATTTAAAATTGAAAAAAAAATTGTTG ACATTAATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTT TTTTTTTTGGAG5’ HIPK3- CAGGGATCTATACTTTTCTTTTGAGGGAAAATGTTGGCACCG 423 AMP160 TTTCTAGGGCATATTGGCCATTTCAGCTTCTCAGTAAATATTT GTTAAGTAATTAAATGCACTTGATTCTTTATTCTTAGCCTTTT AACGCAATACTCAGAATAGCTGAAGCACCAATGCCTCAGCCT CTCAAAGTGCTAGGATTATAACTGAAATGGAGATATTATAAA GATAGTTATCTTCTCCAAGGGAAAAAATCATCTTCATGGAAA TTAATTACTTTTTTACAAATTGTGAATTTGACCCTTAAGAGTT TTCTTCCTGATATTTAAAATTGAAAAAAAAATTGTTGACATT AATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTTTTTTTTTTTT TTGCAG5’ HIPK3-A42- GCCTCAGCCTCTCAAAGTGCTAGGATTACAGGGATCTATACT 402 267-SG ACAAATTGTGAATTTGACCCTTAAGAGTTTTCTTCCTGATATT TAAAATTGAAAAAAAAATTGTTGACATTAATATTTCTTCTTTC CTTTTTTTTCTTTTCCTTTTTTTTTTTTTTTTGCAG3’ HIPK3-A10- GTAGGTAACGCCAACTAGTTAAGCACCCCCAAATCTGAAAAT 602 497-SG TCGTTTCCTTTGAGTGTCATGTCAATGCCCAAAAAGTTTCAG ATATTTGGATTTGAGATGCTCAACCTGTATAAGGATTCAGAA AGTTATTCTGATTAATGATTTTAAGATTCAGATATACAATAA TCCCAGCAACTTGGGAGGCTGAGGCAGGAGAATCACTTGAA CCCAGGAGATGGAGGTTGCAGTGAGCCGAGATCATGCCATT GCACTCCA3’ HIPK3- GrAGGTAACAACTCCA'rACTTITTGGTTG'rTrATTAA'rGTGAA 609 AMP255-Alu ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTTAAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GGCATT'T'TAGCACTTGAGAGAAATAGT'T'TATTAAAGATATAA TCAATCATATGTAACTGAACATTTATAATCCCAGCAACTTGG GAGGCTGAGGCGAAAAATTATATACAGGTTTGAGTAGCCCTT ATCTGAAACTTTTGGGGCCAGAAGTGTTTTGGATTCCAGATT TTTCCGGATTTTGGAATATTTGCACTGCCAACTAGTTAAGCA CCCCCAAATTTGAAAATTCGTTTCCTTTGAGTGTCATGTCAAT GCCCAAAAAGTTTCAGATATTTGGATTTGAGATGCTCAACCT GTATAAGGATTCAGAAAGTTATTCTGATTAATGATTTTAAGA TTCAGATATACAATAATCCCAGCAACTTGGGAGGCTGAGGCA GGAGAATCACTTGAACCCAGGAGATGGAGGTTGCAGTGAGC CGAGATCATGCCATTGCACTCCA3’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 610 AMP255dR ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTATAATCCCAGCAACTTGG GAGGCTGAGGC3’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 611 ClipOnl ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAGATTCAGATATACAATAATCCCAGCAACTT GGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGATGG AGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCAGGACT CGAGCGGCCCTTTGAATTCCAC3’ HIPK3 TAAGCAC, CCCCAAATTTGAAAATTCGTTTCCTTTGAGTGTCAT 612 Embedded GTCAATGCCCAAAAAGTTTCAGATATTTGGATTTGAGATGCT CAACCTGTATAAGGATTCAGAAAGTTATTCTGATTAATGATT TTAAGATTCAGATATACAATAATCCCAGCAACTTGGGAGGCT GAGGCAGGAGAATCACTTGAACCCAGGAGATGGAGGTTGCA GTGAGCCGAGATCATGCCATTGCACTCCAGTAGGTAACAACT CCATACTTTTTGGTTGTTTATTAATGTGAAATTTCTGCTAAAT GAAATACTTTTGTGTGTGTTTGTGGTAGAAGAGACCACTTCA GTTAAATAAGGAAATCAAGAGAGGATCAATTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAAAATCTACCCAAGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CAGGATAGAAATCTCCACTGCAAAGT'T'CCATGCCAAAGACAT CTGGTTATTTTTATTTTTAATGGAAGACTTGAAGGAATGATA GGTGATTAATAATGATCAAACAGAAGTCTTTAAATGTTGGAA AGTATTTACATTAATCTTTGTATATATCATTGGGCATTTTAGC ACTTGAGAGAAATAGTTTATTAAAGATATAATCAATCATATG TAACTGAACATTTAGAAAAATTATATACAGGTTTGAGTAGCC CTTATCTGAAACTTTTGGGGCCAGAAGTGTTTTGGATTCCAG ATTTTTCCGGATTTTGGAATATTTGCACTGCCAACTAGT3’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 613 AED45 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATAAACGGTGCCAACATATT TCCCTCAAAAGAACAAGTATAGATCCCTGATAATCCCAGCAA CTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGA TGGAGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA3’ HIPK3- GTAGGrAACAACTCCATACT'rTrTGGT'rGTITArTAATGrGAA 614 ClipOnlAAlu ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAGATTCAGATATACAGGACTCGAGCGGCCCT TTGAATTCCAC3’ H1PK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 615 FullAlu ArTTCrGCTAAATGAAArACTTT'rGTGIGTGrTTGrGGTAGAA GAGACCACTI CAG1 TAAA’l AAGGAAATCAAGAGAGGAFCAA TTTAGGTTCX1TTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTTAAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAGATTCAGATATACAGCCGGGTGCAGTGGCT CATGCCTGTAATCCCTGCACTTAGGGAGGCTGAGGCGGGTGG ATGACCTGAGGTTAGGAGTTCAAGACCAGCCTGGCCAACAT GGCGAAACCCCCATCTCTACTAAAAATAACAAAAATTAGCTG GGTGTGGTGGTGGGTGTCTATAATCCCAGCAACTTGGGAGGC TGAGGCAGGAGAATCACTTGAACCCAGGAGATGGAGGTTGC AGTGAGCCGAGATCATGCCATTGCACTCCAGCCTGGGCCACA AGAGCAAAACTCTGTCTCAAAAAAACAAAACAAA3’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 616 ClipOn2 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAGATTCAGATATACAATAATCCCAGCAACTT GGGAGGCTGAGGCAGGACTCGAGCGGCCCTTTGAATTCCAC3’ HIPK3- GrAGGTAACAACTCCA'rACTTITTGGTTG'rTrATTAA'rGTGAA 617 AED15 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAAAGTATAGATCCCTGATAATCCCAGCAACT TGGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGATGGAGGTTGCAGTGAGCCGAGATCATGCXATTGCACTCCAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20693’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 618 AMP255 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTATAATCCCAGCAACTTGG GA GGCTGA GGCGA A A A ATTAT AT AC AGGTTTGAGT A GCCCTT ATCTGAAACTTTTGGGGCCAGAAGTGTTTTGGATTCCAGATT TTTCCGGATTTTGGAATATTTGCACTGCCAACTAGTTAAGCA CCCCCAAATTTGAAAATTCGTTTCCTTTGAGTGTCATGTCAAT GCCCAAAAAGTTTCAGATATTTGGATTTGAGATGCTCAACCT GTATAAGGATTCAGAAAGTTATTCTGATTAATGATTTTAAGA TTCAGATATACAAAGGAGAATCACTTGAACCCAGGAGATGG AGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA3’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 619 AED30 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTTTT CCCTCAAAAGAACAAGTATAGATCCCTGATAATCCCAGCAAC TTGGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGAT GGAGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA3’ HIPK3- GTAGGrAACAACTCCATACT'rTrTGGT'rGTITArTAATG'rGAA 620 AED65 ArTTCrGCIAAATGAAArACTTT'rGTGTGTGrTTG'rGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTTGAGAAAATGAGCCAATATGCCCTAGAAAACGGTGCCAACATATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069ATTTCCCTCAAAAGAACAAGTATAGATCCCTGATAATCCCAG CAACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGG AGATGGAGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA3’ HIPK3- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 621 AMP85 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTAATCCCAGCAACTTG GGAGGCTGAGGCTCAGATATTTGGATTTGAGATGCTCAACCT GTATAAGGATTCAGAAAGTTATTCTGATTAATGATTTTAAGA TTCAGATATACAAAGGAGAATCACTTGAACCCAGGAGATGG AGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA3’ HIPK3-PHS- GTAGGTAACAACTCCATACTTTTTGGTTGTTTATTAATGTGAA 622 R2 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCCGTTGCATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGG TTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTT TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGCACTGCC AACTAGTTAAGCACCCCCAAATTTGAAAATTCGTTTCCTTTG AGTGTCATGTCAATGCCCAAAAAGTTTCAGATATTTGGATTT GAGATGCTCAACCTGTATAAGGATTCAGAAAGTTATTCTGAT TAATGATTTTAAGATTCAGATATACAATAATCCCAGCAACTT GGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGATGG AGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA3’ HIPK3- GTAGGrAACAACTCCATACT'rTFTGGT'rGTITArTAATG'rGAA 623 AMP160 ATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAA GAGACCACTTCAGTTAAATAAGGAAATCAAGAGAGGATCAA TTTAGGTTCGTTTTAAAGAGATTAAAAAAAATCAAGACATAA AATCTACCCAAGCAGGATAGAAATCTCCACTGCAAAGTTCCA TGCCAAAGACATCTGGTTATTTTTATTTTTAATGGAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACAGAAGTCTTT AAATGTTGGAAAGTATTTACATTAATCTTTGTATATATCATTG GGCATTTTAGCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTAGAAAAATTATATACAGGTTTGAGTAGCCCTTATCTGAAACTTTTGGGGCCAGAAGTGTTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TTGGATTCCAGATTTTTCCGGATTTTGGAATATTTGTAATCCC AGCAACTTGGGAGGCTGAGGCCACTGCCAACTAGTTAAGCA CCCCCAAATTTGAAAATTCGTTTCCTTTGAGTGTCATGTCAAT GCCCAAAAAGTTTCAGATATTTGGATTTGAGATGCTCAACCT GTATAAGGATTCAGAAAGTTATTCTGATTAATGATTTTAAGA TTCAGATATACAAAGGAGAATCACTTGAACCCAGGAGATGGAGGTTGCAGTGAGCCGAGATCATGCCATTGCACTCCA

[0144] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one circularizing element for tRNA splicing. In some embodiments, the circularizing element is a tRNA 5’ leader. In some embodiments, the circularizing element is a tRNA 3’ trailer. In some embodiments, the circularizing element is a tRNA intronic element. In some embodiments, the circularizing element is a tRNA exonic element.

[0145] In some embodiments, following transcription, the 5’ circularizing element and 3’ circularizing element facilitate tRNA splicing to yield the circular RNA, In some embodiments, the 5’ circularizing element and 3’ circularizing element are derived from a human gene encoding a tyrosine tRNA, an isoleucine tRNA, a leucine tRNA, or an arginine tRNA.

[0146] Transfer RNAs (tRNAs) are transcribed by RNA polymerase III as precursor molecules known as pre-tRNAs that undergo a series of processing steps, including splicing by the TSEN complex in the case of intron-containing transcripts. In metazoans, splicing of pre-tRNA results in ligated exons to produce a mature, functional tRN / X molecule and end-to-end ligated introns, forming circular RNA by-products referred to as tRNA intronic circular RNAs or tricRNAs. This cellular process can be co-opted and used for generation of a therapeutic circRN / X from a viral vector by flanking an RNA sequence of interest with pre-tRNA elements, such as leader, trailer, and exons. In other words, the pre-tRNA components serve as a scaffold for synthetic circRNA production in vivo. In some embodiments, the circular tough decoys of the present disclosure are generated using pre-tRNA splicing elements.

[0147] In some embodiments, the tricY system uses genes encoding tyrosine pre-tRNA, or fragments thereof, as scaffolds to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA1-1 (TRNAY10) gene to produce a circular RN A via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA2-1 (TRNAY4) gene to produce a circular RNA via tRNAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA3-1 (TRNAY7) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA4-1 (TRNAYl 3) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA5-1 (TRNAYl 5) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA5-2 (TRNAY6) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA5-3 (TRNAY14) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA5-4 (TRNAY2) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA5-5 (TRNAY3) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA6-1 (TRNAY8) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRY-GTA7-1 (TRNAYl) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRN / X sequence derived from the human TRY-GTA8-1 (TRNAYl 2) gene to produce a circular RN A via tRN A splicing.

[0148] In some embodiments, the tricl system, uses genes encoding isoleucine pre-tRNA, or fragments thereof, as scaffolds to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRI-TATI-1 (TRNAI18) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRI-TAT2-1 (TRNAI13) gene to produce a circular RNA via tRN A splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRI-TAT2-2 (TRNAI2) gene to produce a circular RNA via tRN A splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre- tRNA sequence derived from the human TRI-TAT2-3 (TRNAI6) gene to produce a circular RNAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRI-TAT3-1 (TRNAI21) gene to produce a circular RNA via tRNA splicing.

[0149] In some embodiments, the tricL system uses genes encoding leucine pre-tRNA, or fragments thereof, as scaffolds to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRL-CAAl-1 (TRNAL49) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRL-CAA1-2 (TRNAL22) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRL-CAA2-1 (TRNAL28) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRL-CAA3-1 (TRNAL8) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRL-CAA4-1 (TRNAL19) gene to produce a circular RNA via tRNA splicing.

[0150] In some embodiments, the tricR system uses genes encoding arginine pre-tRNA, or fragments thereof, as scaffolds to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRR-TCT1-1 (TRNARl 5) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRR-TCT2-1 (TRNARl) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre-tRNA sequence derived from the human TRR-TCT3-1 (TRNARl 2) gene to produce a circular RNA via tRNA splicing. In some embodiments, the recombinant nucleic acid molecule comprises a pre- tRNA sequence derived from the human TRR-TCT3-2 (TRNAR14) gene to produce a circular RN A via tRN A splicing.

[0151] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, a tRNA 5’ leader, a tRNA 5’ exonic element, a cargo, a tRNA 3’ mtronic element, a tRNA 3’ exonic element, and a tRNA 3’ trailer for tRNA splicing.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0152] In some embodiments, the recombinant nucleic acid molecule comprises a tRNA leader. In some embodiments, the tRNA leader comprises a complete or partial sequence derived from tyrosine, leucine, isoleucine, or arginine tRNA. In some embodiments, the tRNA leader is located 5’ to the cargo and is referred to herein as the “tRNA 5’ leader” or “tRNA 5’ leader sequence”. Non-limiting examples of sequences for tRNA 5’ leaders are provided in Table 4 below.Table 4. Exemplary tRNA Leader SequencesSEQ IDDescription Nucleic Acid SequenceNO. TRY-GTA3-1.1ACGTGTCTAAAAAGGACAGCGTTCCGTGT 24 (TricY)CGTGGGGTTATGGAGCCOTTCCGTCAACCGGGTTGCCmTRY-GTA3-l 302GCTC GAACCACTGAAGAAGACCCCGGCGCAGGGGTGCGGC TRI-TATI-1 303CGGA AGCGCACCCTGATAGAGCCATCACGAGGCCCATTCA TRI-TAT2-1 304GCAGA GTGTAGGCTGTGTAAGGAGTGACCGGAAGTAGAAAC TRI-TAT2-2 305TGGGT ATGGCTCTGTAGAGTGCGCATGGCCAAGCAAAGGAA TRI-TAT2-3 306AGCAT CATCTTCTCTTATACCTTTCCAAATCCTTATTATTTCT TRI-TAT3-1 307GTG TC TCTATTT TAGTGCAAAAATCAGGGCI GTTAT GCAG TRL-CAAl-l 308TGT ATCGAAGATTATATTAATTTGCGATGGTAAGTGTCCC TRL-CAA1-2 309AGGT ATTAC TCATCATGTATAGTTTC AAGGA TTTTI GTCACA TRL-CAA2-1 310GT CTACGAGTTACATAATGAAACTTAAGGAGTAACAGT TRL-CAA3-1 311GCGT CACAAGTATGAGGCTAAGTGAGAAGCGCTCTGCTTCC TRL-CAA4-1 312GAGT CTCTGCJCTTAACATAGCAGATGCGCTGAGACTCCAAC TRR-TCT1-1 314AGGT TCTAGTACCCGTAAGCTACAAGACGCCGCCGTTCGTC TRR-TCT2-1 313GGGT TTTCAGGCAGAGGTGAGCGGGCiCGGCCGGGTTCCGCTRR-TCT3-1 315AGCC CCTCCCCTGATGTGTTCATCGCGGAGAGGTCGCACGT TRR-TCT3-2 316GTCT CAGAGATGGCATCCCCAGCGGGAAGTGAAAGTCGTG TRY-GTA1-1 317TGAATATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069AAGCATTAGAGGGCTATCAGCAGCATCTTATCGCAGC TRY-GTA2-1 318GGAG GAACGCAGAACTACGTGTCTAAAAAGGACAGCGTTC TRY-GTA3-1.2 319CGTGT TTTGCAGAAAGTCCAATGAACCAGCTTTGATAGCATG TRY-GTA4-1 320CAT TCTGTGCTGAACCTCAGGGGACGCCGACACACGTAC TRY-GTA5-1 321ACGTC GTTATGGAGACAAGCICGGCACCCGGGAAGCTGTGCC TRY-GTA5-2 322CGCTC TTTGCTGAAAGATCAATGACCCAACCCCAGAAACGT TRY-GTA5-3 323GCGC TTTGCGGAAAGTCCAGTGATCCAGCTCTTGCAGCGTG TRY-GTA5-4 324CAC TGTCTCGAGTAGGGATTTTTAGGAGAGTTGTTTCCGT TRY-GTA5-5 325GCTT ATAGGTAAATCCTAAAGAATCCAAAACGAGGGTAAT TRY-GTA6-1 326GACAT TTTGCAGAAAGTCCAGTGACCCAGCCTTAACAGTGTG TRY-GTA7-1 327CAT ACATTATTTTAGGAATTCTAGAAGATGAAGGGCTAGG TRY-GTA8-1 328GGTC

[0153] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA leader. In some embodiments, the tRNA leader is located 5’ to the cargo and is referred to herein as the tRNA 5’ leader. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 24 and 302-328. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of any one of SEQ ID NOs: 24 and 302-328 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of any one of SEQ ID NOs: 24 and 302-328. In some embodiments, the tRNA 5’ leader consists of a polynucleotide sequence of any one of SEQ ID NOs: 24 and 302-328.

[0154] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA leader. In some embodiments, the tRN A leader is located 5’ to the cargo and is referred to herein as the tRNA 5’ leader. In some embodiments, the tRNA 5’ leader comprises aATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 24. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 24 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 24. In some embodiments, the tRNA 5’ leader consists of a polynucleotide sequence of SEQ ID NO: 24.

[0155] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA leader. In some embodiments, the tRNA leader is located 5’ to the cargo and is referred to herein as the tRNA 5’ leader. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 312. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 312 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 312. In some embodiments, the tRNA 5’ leader consists of a polynucleotide sequence of SEQ ID NO: 312.

[0156] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA leader. In some embodiments, the tRNA leader is located 5’ to the cargo and is referred to herein as the tRNA 5’ leader. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 314. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 314 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 314. In some embodiments, the tRNA 5’ leader consists of a polynucleotide sequence of SEQ ID NO: 314.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0157] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA leader. In some embodiments, the tRNA leader is located 5’ to the cargo and is referred to herein as the tRNA 5’ leader. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 321. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 321 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO: 321. In some embodiments, the tRNA 5’ leader consists of a polynucleotide sequence of SEQ ID NO: 321.

[0158] In some embodiments, the recombinant nucleic acid molecule comprises a tRNA trailer. In some embodiments, the tRNA trailer comprises a complete or partial sequence derived from tyrosine, leucine, isoleucine, or arginine tRNA. In some embodiments, the tRNA trailer is located 3’ to the cargo and is referred to herein as the “tRNA 3’ trailer” or “tRNA 3’ trailer sequence”. Non-limiting examples of sequences for tRNA 3’ trailers are provided in Table 5 below.Table 5. Exemplary tRNA Trailer SequencesSequence Name Nucleic Acid Sequence SEQ ID NO. TRY-GTA3-1.1 26CGGTAGTTTTGACCTACCAAG(TricY)crrTT.rTTTCCCCATCAGrrTTTTAI.AAACTTACACATC 553 TRI-TATI-1TAAT AGACCCTTTTGGATGTTCGCAACCCCTTTTGTGTAT 554 TRI-TAT2-1CTCG TTGTTTTCCAACCCAAAATAGCATTACTTGAAGTGC 555 TRI-TAT2-2CTTT TGTTTTCTTCCAATTGCGATTTCAGACATTTCACGAT 556 TRI-TAT2-3AAT GTTTTACTATTCTCCGACCTGTAGAAATTCAAAAAC 557 TRI-TAT3-1AGGG CATACTTTTCTTTTTCCTCTCCTTACTTTAGAGATAT 558 TRL-CAA1-1OCT CAACTATCTTATTCTCCTTTTACTCTACTTTCTCAGC 559 TRL-CAA1-2CATATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CAGCCTGATCTTTTGTTTTTCCCTACCAGATTTGTAC 560 TRL-CAA2-1CGAA CAGCTATTTTGCCCCTTTAATCCTAGGATTGGGGGA 561 TRL-CAA3-1CTGGG CATTCGTTTTATTAGCATTTTATTCCCCGGAAGAAA 562 TRL-CAA4-1CCCC TAACGCTTTTTTCCCTCCCCCCTACAATTTATTTTCT 564 TRR-TCTl-1GCC ATTTTATTTCAATTTTTCCTTTTTATCCGTTTTTTTCT 563 TRR-TCT2-1TA AATTTTGATGGCTCCTACTCCCTGACTTTCACCTGC 565 TRR-TCT3-1GTGTT CT TTTTGT TTGAAGAAGAGCAAACGGAAAGTTAGG 566 TRR-TCT3-2CGGAG GTTCACAATTTTTTTTTTTTGCGTAAGTTTTAAAATT 567 TRY-GTA1-1CAT CTTCGTCTGTAA TTTTTAACCTC AA TTTAATTCAATT 568 TRY-GTA2-1ATC CGGTAGTTTTGACCTACCAAGCTAATAATTCCAGAC 569 TRY-GTA3-1.2AATA AGTGCGTGATGCTTTGGTTAAAAGCCCTGCAGCTT 570 TRY-GTA4-1CCAAG GACAAGTGCGGTTTTTTTCTCCAGCTCCCGATGACT 571 TRY-GTA5-1TATG GAGACACCCCCCCCCCCATTATTTTGTTGCTTTGAA 572 TRY-GTA5-2CCAA GCTGCCGTATTCTTTTGCACACGCACGCACCAAAAC 573 TRY-GTA5-3TACGT AGTGCCCGATGCTTTTGCATGCAATGCCACCTGGTG 574 TRY-GTA5-4CCTGG GACGCTGCTGTTTTGGGGTTTACGGTTGCTCTTCGTT 575 TRY-GTA5-5TGT GTTCCATTTTTAAAAGTGTCTCTTCTGGGCCTGAAA 576 TRY-GTA6-1TAAG AGCGCCTGACTCTTTTGCGCACAATGCTGCCTGGCT 577 TRY-GTA7-1GCACC CGAAAGTTTTGAACTACGGGCTAATAATTCCAGAA 578 TRY-GTA8-1ATAACTyr-GTA-3-1 AAGAGCT rGC’TATITTCTITTACCTTrTAA TCCTA TC 552(Mouse) TAT

[0159] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA trailer. In some embodiments, the tRNA trailer is located 3’ to the cargo and is referred to herein as the tRNA 3’ trailer. In some embodiments, the tRNA 3’ trailer comprises aATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 26 and 552-578. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of any one of SEQ ID NOs: 26 and 552-578 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of any one of SEQ ID NOs: 26 and 552-578. In some embodiments, the tRNA 3’ trailer consists of a polynucleotide sequence of any one of SEQ ID NOs: 26 and 552-578.

[0160] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA trailer. In some embodiments, the tRNA trailer is located 3’ to the cargo and is referred to herein as the tRNA 3’ trailer. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 26, In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 26 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 26. In some embodiments, the tRNA 3’ trailer consists of a polynucleotide sequence of SEQ ID NO: 26.

[0161] n some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA trailer. In some embodiments, the tRNA trailer is located 3’ to the cargo and is referred to herein as the tRNA 3’ trailer. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 562. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 562 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 562. In some embodiments, the tRNA 3’ trailer consists of a polynucleotide sequence of SEQ ID NO: 562.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0162] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA trailer. In some embodiments, the tRNA trailer is located 3’ to the cargo and is referred to herein as the tRNA 3 ’ trailer. In some embodiments, the tRNA 3 ’ trailer comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 564. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 564 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 564. In some embodiments, the tRNA 3’ trailer consists of a polynucleotide sequence of SEQ ID NO: 564.

[0163] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA trailer. In some embodiments, the tRNA trailer is located 3’ to the cargo and is referred to herein as the tRNA 3’ trailer. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 571. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 571 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 571. In some embodiments, the tRNA 3’ trailer consists of a polynucleotide sequence of SEQ ID NO: 571.

[0164] In some embodiments, the recombinant nucleic acid molecule comprises a tRNA exonic element. In some embodiments, the tRNA exonic element comprises a sequence derived from tyrosine, leucine, isoleucine, or arginine tRNA. In some embodiments, the tRNA exonic element is located 5’ to the cargo and is referred to herein as the tR A 5’ exonic element. In some embodiments, the tRN A 5’ exonic element is referred to as the “tRN A 5’ exon” or “tRNA 5’ exon sequence”. N on-limiting examples of sequences for tRNA 5’ exonic elements are provided in Table 6 below.Table 6. Exemplary tRNA 5’ Exonic SequencesATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Description Nucleic Acid Sequence SEQ ID NO.5’ tncl Exon GCTCCAGTGGCGCAATCGGTTAGCGCGCGGTACTT 353ATA5’ tricL Exon GTCAGGATGGCCGAGTGGTOTAAGGCGCCAGACTC 354AAG5’ tricR Exon GGCTCCGTGGCGCAATGGATAGCGCATTGGACTTC 355TA5’ tricR Exon GGCTCTGTGGCGCAATGGATAGCGCATTGGACTTC 356TA5’ tricY Exon CCTTCGATAGCTCAGCTGGTAGAGCGGAGGACTGT 357AG5’ tricY Exon CTTTCGATAGCTCAGTTGGTAGAGCGGAGGACTGT 358AG5 ’ tricY Exon CCTTCGATAGCTCAGTTGGTAGAGCGGAGGACTGT 25AG

[0165] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 5’ to the cargo and is referred to herein as the tRNA 5’ exonic element. In some embodiments, the tRNA 5’ exonic element comprises a tRNA 5’ acceptor stem sequence. In some embodiments, the tRN A 5’ exonic element comprises a polynucleotide sequence that at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 25 and 353-358. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 25 and 353-358 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 25 and 353-358. In some embodiments, the tRNA 5’ exonic element consists of a polynucleotide sequence of any one of SEQ ID NOs: 25 and 353-358.

[0166] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 5’ to the cargo and is referred to herein as the tRNA 5’ exonic element. In some embodiments, the tRNA 5’ exonic element comprises a tRNA 5’ acceptor stem sequence. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence that at least about 70%, at leastATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 25. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 25 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 25. In some embodiments, the tRNA 5’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 25.

[0167] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 5’ to the cargo and is referred to herein as the tRNA 5’ exonic element. In some embodiments, the tRNA 5’ exonic element comprises a tRNA 5’ acceptor stem sequence. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence that at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 354. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 354 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 354. In some embodiments, the tRNA 5’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 354.

[0168] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 5’ to the cargo and is referred to herein as the tRNA 5’ exonic element. In some embodiments, the tRNA 5’ exonic element comprises a tRNA 5’ acceptor stem sequence. In some embodiments, the tRN A 5’ exonic element comprises a polynucleotide sequence that at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 355. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 355 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In someATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 355. In some embodiments, the tRNA 5’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 355.

[0169] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 5’ to the cargo and is referred to herein as the tRNA 5’ exonic element. In some embodiments, the tRNA 5’ exonic element comprises a tRNA 5’ acceptor stem sequence. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence that at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 357. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 357 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 357. In some embodiments, the tRNA 5’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 357

[0170] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element comprises a sequence derived from tyrosine, leucine, isoleucine, or arginine tRNA. In some embodiments, the tRNA exonic element is located 3’ to the cargo and is referred to herein as the tRNA 3’ exonic element. In some embodiments, the tRNA 3’ exonic element is referred to as the “tRN / X 3’ exon” or “tRNA 3’ exon sequence”. Non-limiting examples of tRNA 3’ exonic elements are provided in Table 7 below.Table 7. Exemplary tRNA 3’ Exonic SequencesDescription Nucleic Acid Sequence SEQ ID NO.3 ’ tricl Exon ATGGCGAGGTTGTGAGTTCGATCCTCACGTGGAGC 503A3 ’ tncl Exon ATGCCGAGGTTGTGAGTTCGAGCCTCACCTGGAGC 504A3 ’ tricl Exon ATGCCGAGGTTGTGAGTTCAAGCCTCACCTGGAGC 505AATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20693 ’ tricL Exon TTCTG<1TCTCCAATGGAG<1CGTG<1GTTCGAATCCC 506ACTTCTGACA3’ tricL Exon TTCTGGTCTCCGTATGGAGGCGTGGGTTCGAATCC 507CACTTCTGACA3 ’ tricL Exon TTCTGGTCTCCGCATGGAGGCGTGGGTTCGAATCC 508CACTTCTGACA3’ tricL Exon TTCTGGTCTCCGGATGGAGGCGTGGGTTCGAATCC 509CACTTCTGACA3 ’ tricR Exon ATTCAAAGGTTCCGGGTTCGAGTCCCGGCGGAGTC 510G3’ tricR Exon ATTCAAAGGTTGTGGGTTCGAATCCCACCAGAGTC 511G3 ’ tricR Exon ATTCAAAGGTTGTGGGTTCGAGTCCCACCAGAGTC 512G3 ’ tricY Exon ATCCTTAGGTCGCTGGTTCGAATCCGGCTCGAAGG 513A3 ’ tricY Exon ATCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGG 514A3 ’ tricY Exon ATCCTTAGGTCGCTGGTTCGATTCCAGCTCGAAGG 515A3’ tricY Exon ATCC1TAGGTCGCTGGTTCAAITCCGGCTCGAAGG 516A3 ’ tricY Exon ATCCTTAGGTCGCTGGTTCGAATCCGGCTCGGAGG 27A

[0171] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 3’ to the cargo and is referred to herein as the tRNA 3’ exonic element. In some embodiments, the tRNA 3 ’ exonic element comprises a tRNA 3 ’ acceptor stem sequence. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NO: 27 and 503-516. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of any one of SEQ ID NO: 27 and 503-516 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of any one of SEQ ID NO: 27 and 503-516. In some embodiments, the tRNA 3’ exonic element consists of a polynucleotide sequence of any one of SEQ ID NO: 27 and 503-516.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0172] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 3’ to the cargo and is referred to herein as the tRNA 3’ exonic element. In some embodiments, the tRNA 3’ exonic element comprises a tRNA 3’ acceptor stem sequence. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 27 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 27, In some embodiments, the tRNA 3’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 27.

[0173] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 3’ to the cargo and is referred to herein as the tRNA 3’ exonic element. In some embodiments, the tRNA 3’ exonic element comprises a tRNA 3’ acceptor stem sequence. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 509. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 509 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 509. In some embodiments, the tRNA 3’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 509.

[0174] In some embodiments, the recombinant nucleic acid molecule encoding a circular RN A comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 3’ to the cargo and is referred to herein as the tRNA 3’ exonic element. In some embodiments, the tRNA 3’ exonic element comprises a tRNA 3’ acceptor stem sequence. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence that is at least about 70%, at leastATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 510. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 510 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 510. In some embodiments, the tRNA 3’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 510.

[0175] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a tRNA exonic element. In some embodiments, the tRNA exonic element is located 3’ to the cargo and is referred to herein as the tRNA 3’ exonic element. In some embodiments, the tRNA 3’ exonic element comprises a tRNA 3’ acceptor stem sequence. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 514. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 514 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 514. In some embodiments, the tRNA 3’ exonic element consists of a polynucleotide sequence of SEQ ID NO: 514.

[0176] In some embodiments, the cargo of the recombinant nucleic acid molecule comprises a tRNA intronic element. In some embodiments, the tRNA intronic element is located 3’ to the tough decoy and is referred to herein as the tRN / X 3’ intronic element. In some embodiments, the tRNA 3’ intronic element is referred to herein as the “tRNA 3’ intron” or “tRNA 3’ intron sequence.” In some embodiments, the cargo comprises a partial or a complete tRNA intronic element derived from tyrosine, leucine, isoleucine, or arginine tRNA. In some embodiments, the tRNA intronic element is referred to herein as the “partial tRNA intron” or “partial tRNA intronic sequence”. In some embodiments, the cargo comprises a partial tRNA intronic element and the partial tRNA intronic element comprises 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, or more. In some embodiments, the cargo comprises a partial tRNA intronic element and the partial tRNA intronic element comprises 2 nucleotides (2- mer). In some embodiments, the cargo comprises a partial tRNA intronic element and the partial tRNA intronic element comprises 4 nucleotides (4-mer). In some embodiments, the cargo comprises a partial tRNA intronic element and the partial tRNA intronic element comprises 10 nucleotides (10-mer). In some embodiments, the partial intronic element is located 5’ and / or 3’ to the cargo. In some embodiments, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGT. In some embodiments, the tRNA 3’ intronic element consists of a polynucleotide sequence of AGGT, Exemplary tRNA intronic elements are provided m Table 8 below.Table 8. tRNA Intronic SequencesDescription Nucleic Acid Sequence SEQ ID NO.5’ Intron (2-mer)- TG Not Applicable5’ Intron (2-mer) CT Not Applicable5’ Intron (2-mer) GA Not Applicable5 ’ Intron (2-mer) Not Applicable3’ Intron (4-mer) AGGT Not Applicable3 ’ Intron (4-mer) AGCA Not Applicable3 ’ Intron (4-mer) AGTG Not Applicable3 ’ Intron (4-mer) GGTG Not Applicable3’ Intron (4-mer) GGGA Not Applicable3 ’ Intron (4-mer) AGGA Not Applicable3 ’ Intron (4-mer) GGTC Not Applicable3 ’ Intron (4-mer) GGGC Not Applicable3’ Intron (4-mer) GGCT Not Applicable3 ’ Intron (4-mer) AGGC Not Applicable3 ’ Intron (4-mer) AGAA Not Applicable3’ Intron (4-mer) AGAC Not Applicable3 ’ Intron (4-mer) GGCA Not ApplicableATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20693 ’ Intron (4-mer) GGAC Not Applicable3 ’ Intron (4-mer) GGCC Not Applicable3’ Intron (10-mer) GAGCGGAGCA 4943’ Intron (10-mer) TGTTCGGGTC 4953’ Intron (10-mer) AGTTAGAGAA 4963 ’ Intron ( 10-mer) TAAGCAAGGT 4973’ Intron (10-mer) GGCTGTGGAC 498

[0177] In some embodiments, the cargo of the recombinant nucleic acid molecule comprises a tRNA 3’ intronic element, but does not comprise a tRNA 5’ intronic element. In some embodiments, the tRNA 3’ intronic element comprises a polynucleotide sequence of any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC. In some embodiments, the tRN / X 3’ intronic element comprises a polynucleotide sequence of any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGA / X, AG / XC, GGCA, GG / XC, and GGCC, with one or more mutations. In some embodiments, the tRNA 3’ intronic element consists of a polynucleotide sequence of any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC.

[0178] In some embodiments, the tRNA intronic element is located 5’ to the tough decoy and is referred to herein as the “tRNA 5’ intronic element.” In some embodiments, the tRN A 5’ intronic element is referred to herein as the “tRNA 5’ intron” or “tRNA 5’ intron sequence.” In some embodiments, the tRN A 5’ intronic element comprises a polynucleotide sequence of any one of TG, CT, GA, or GC. In some embodiments, the tRNA 5’ intronic element consists of a polynucleotide sequence of any one of TG, CT, GA, or GC.

[0179] In some embodiments, the cargo of the recombinant nucleic acid molecule comprises a tRNA 5 ’ intronic element and a tRNA 3 ’ intronic element. In some embodiments, the tRNA 3 ’ intronic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 494-498. In some embodiments, the tRN A 3 ’ intronic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 494-498, with one or more mutations, such as 1, 2, 3, 4, 5, or more mutations. In some embodiments, the tRNA 3’ intronic element consists of a polynucleotide sequence of any one of SEQ ID NOs: 494-498.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0180] In some embodiments, the cargo of the recombinant nucleic acid molecule comprises a tRNA 5’ intronic element and a tRNA 3’ intronic element. In some embodiments, the tRNA 5’ intronic element comprises a polynucleotide sequence of any one of TG, CT, GA, or GC; and the tRNA 3’ intronic element comprises a polynucleotide sequence of any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC, and SEQ ID NOs: 494-498. In some embodiments, the tRNA 5’ intronic element consists of a polynucleotide sequence of any one of TG, CT, GA, or GC; and the tRNA 3’ intronic element consists of a polynucleotide sequence of any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC, and SEQ ID NOs: 494-498.

[0181] In some embodiments, the cargo of the recombinant nucleic acid molecule comprises a tRNA 5’ intronic element and a tRNA 3’ intronic element. In some embodiments, the tRNA 5’ intronic element comprises a polynucleotide sequence of any one of TG, CT, GA, or GC; and the tRNA 3’ intronic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 494-498. In some embodiments, the tRNA 5’ intronic element consists of a polynucleotide sequence of any one of TG, CT, GA, or GC; and the tRNA 3’ intronic element consists of a polynucleotide sequence of any one of SEQ ID NOs: 494-498.

[0182] In some embodiments, the recombinant nucleic acid molecule comprises a tRNA 3’ intronic element, but does not comprise a tRNA 5’ intronic element.

[0183] Additional exemplary sequences for tRNA splicing are provided in Table 9 below.Table 9. Additional Exemplary Sequences for tRNA SplicingName Nucleic Acid SequencetricY 5’ Acceptor Stem CCUUCGAtricY 3 ’ Acceptor Stem UCGGAGGAtricYCCTT> GGCC 5’ Acceptor Stem GGCCCGAtricYCCTT> GGCC 3’ Acceptor Stem UCGGACCGtricYMENp 5’ Acceptor Stem GGCACUGtncYMENP 3’ Acceptor Stem CAGUACCGtricYMasc 5’ Acceptor Stem GACGCUGTricYMasc 3 ’ Acceptor Stem CGGUGUCUATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0184] In some embodiments, the recombinant nucleic acid molecule comprises: (i) a tRNA 5’ leader; (ii) a tRNA 5’ exonic element; (iii) a 5’ RE site; and (iv) a 5’ ligation motif upstream of the cargo. In some embodiments, the recombinant nucleic acid molecule comprises a tRNA 5’ leader and a tRNA 5’ exonic element upstream of the cargo. In some embodiments, the 5’ engineered tRNA sequences shown in Table 10 below does not comprise the 5’ RE site and 5’ ligation motif.

[0185] In some embodiments, the recombinant nucleic acid molecule comprises: (i) a tRNA 3’ ligation motif; (ii) a 3’ RE site; (iii) a tRNA 3’ intronic element; (iv) a tRNA 3’ exonic element; and (v) a tRN A 3’ trailer downstream of the cargo. In some embodiments, the recombinant nucleic acid molecule comprises (i) a tRNA 3’ intronic element; (ii) a tRNA 3’ exonic element; and (iii) a tRNA 3’ trailer downstream of the cargo. In some embodiments, the 3’ engineered tRNA sequences shown in Table 11 below does not comprise the 3’ RE site and 3’ ligation motif.

[0186] Non-limiting examples of engineered tRNA sequences are listed in Table 10 and Table 11 below with the intron in underlined text, the RE in bold text, and the ligation motif in italicized text.Table 10. 5’ Engineered tRNA SequencesDescription Nucleic Acid Sequence SEQ ID NO.ACGTGTCTAAAAAGGACAGCGTTCCGTGTC 450TRY-GTA3-1.1 (TricY) CTTCGATAGCTCAGTTGGTAGAGCGGAGGA CTGTAGGCGGCCGCAACCCCTACCATncY (TRY-GTA3-1.1) ACGTGTCTAAAAAGGACAGCGTTCCGTGTC 285’ leader / exonic element CTTCGATAGCTCAGTTGGTAGAGCGGAGGACTGTAG TRY-GTA1-1 CAGAGATGGCATCCCCAGCGGGAAGTGAA 451AGTCGTGTGAATCCTTCGATAGCTCAGTTG GTAGAGCGGAGGACTGTAGGCGGCCGCA4 CCCCTACCA TRY-GTA2-1 AAGCATTAGAGGGCTATCAGCAGCATCTTA 452TCGCAGCGGAGCCTTCGATAGCTCAGTTGG TAGAGCGGAGGACTGTAGGCGGCCGCAACCCCTACCA TRY-GTA3-1.2 GAACGCAGAACTACGTGTCTAAAAAGGAC 453AGCGTTCCGTGTCCTTCGATAGCTCAGTTGG TAGAGCGGAGGACTGTAGGCGGCCGCA4CCCCTACCAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TRY-GTA3-1.2 (10- GAACGCAGAACTACGTGTCTAAAAAGGAC 454AGCGTTCCGTGTCCTTCGATAGCTCAGTTGGmer)TAGAGCGGAGGACTGTAGGCCTGCGGGCG CAACCCCTACCA TRY-GTA4-1 TTTGCAGAAAGTCCAATGAACCAGCTTTGA 455TAGCATGCATCCTTCGATAGCTCAGCTGGT AGAGCGGAGGACTGTAGGCGGCCGCA4CCCCTACCA TRY-GTA5-1 TCTGTGCTGAACCTCAGGGGACGCCGACAC 456ACGTACACGTCCCTTCGATAGCTCAGCTGG TAGAGCGGAGGACTGTAGGCGGCCGCAACCCCTACCA TRY-GTA5-1 TCTGTGCTGAACCTCAGGGGACGCCGACAC 4495’ leader / exonic element ACGTACACGTCCCTTCGATAGCTCAGCTGG TAGAGCGGAGGACTGTAG TRY-GTA5-2 GTTATGGAGACAAGGCGGCACCCGGGAAG 457CTGTGCCCGCTCCCTTCGATAGCTCAGCTGG TAGAGCGGAGGACTGTAGGCGGCCGCAACCCCTACCA TRY-GTA5-3 TTTGCTGAAAGATCAATGACCCAACCCCAG 458AAACGTGCGCCCTTCGATAGCTCAGCTGGT AGA GCGGAGGACTGTA GGCGGCCGC44CCCCTACCA TRY-GTA5-4 TTTGCGGAAAGTCCAGTGATCCAGCTCTTG 459CAGCGTGCACCCTTCGATAGCTCAGCTGGT AGAGCGGAGGACTGTAGGCGGCCGCA4CCCCTACCA TRY-GTA5-5 TGTCTCGAGTAGGGATTTTTAGGAGAGTTG 460TTTCCGTGCTTCCTTCGATAGCTCAGCTGGT AGAGCG(jAGGA( / TGTAGGlC(xGCCGG4y4CCCCTACCA TRY-GTA6-1 ATAGGTAAATCCTAAAGAATCCAAAACGAG 461GGTAATGACATCCTTCGATAGCTCAGCTGG TAGAGCGGAGGACTGTAGGCGGCCGCAACCCCTACCA TRY-GTA7-1 TITGCAGAAAGrCCAGTGACCCAGC’CTIAA 462CAGTGTGCATCCTTCGATAGCTCAGCTGGT AGAGCGGAGGACTGTAGGCGGCCGGA4CCCCTACCA TRY-GTA8-1 ACATTATTTTAGGAATTCTAGAAGATGAAG 463GGCTAGGGGTCCTTTCGATAGCTCAGTTGG TAGAGCGGAGGACTGTAGGCGGCCGCAACCCCTACCA TR1-TAT1-1 GAACC ACT GAAGAAGAC CCCGGC GC AGGG 464GTGCGGCCGGAGCTCCAGTGGCGCAATCGGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TTAGCGCGCGGTACTTATAGCGGCCGCAACCCCTACCA TRI-TATI-1 (10-mer) GAACCACTGAAGAAGACCCCGGCGCAGGG 465GTGCGGCCGGAGCTCCAGTGGCGCAATCGG TTAGCGCGCGGTACTTATATGCTGCGGCCG CAACCCCTACCA TRI-TAT2-1 AGCGCACCCTGATAGAGCCATCACGACGGCC 466CATTCAGCAGAGCTCCAGTGGCGCAATCGG TTAGCGCGCGGTACTTATAGCGGCCGCAACCCCTACCA TRI-TAT2-2 GTGTAGGCTGTGTAAGGAGTGACCGGAAGT 467AGAAACTGGGTGCTCCAGTGGCGCAATCGG TTAGCGCGCGGTACTTATAGCGGCCGCAACCCCTACCA TRI-TAT2-3 ATGGCTCTGTAGAGTGCGCATGGCCAAGCA 468AAGGAAAGCATGCTCCAGTGGCGCAATCGG TTAGCGCGCGGTACTTATAGCGGCCGCAACCCCTACCA TRI-TAT3-1 CATCTTCTCTTATACCTTTCCAAATCCTTAT 469TATTTCTGTGGCTCCAGTGGCGCAATCGGTT AGCGCGCGGTACTTATAGCGGCCGCA4CCCCTACCA TRL-CAA1-1 TCTCTATTTTAGTGCAAAAATCAGGGCTGTT 470ATGCAGTGTGTCAGGATGGCCGAGTGGTCT AAGGCGCCAGACTCAAGGCGGCCGC14CCCCTACCA TRL-CAA1-2 ATCGAAGATTATATTAATTTGCGATGGTAA 471GTGTCCCAGGTGTCAGGATGGCCGAGTGGT CTAAGGCGCCAGACTCAAGGCGGCCGCAACCCCTACCA TRL-CAA2-1 ATTACTCATCATGTATAGTTTCAAGGATTTT 472TGTCACAGTGTCAGGATGGCCGAGTGGTCT AAGGCGCCAGACTCAAGGCGGCCGCX4CCCCTACCA TRL-CAA2-1 (10-mer) ATTACTCATCATGTATAGTTTCAAGGATTTT 473TGTCACAGTGTCAGGATGGCCGAGTGGTCT AAGGCGCCAGACTCAAGCTCTGCGGCCGC AACCCCTACCA TRL-CAA3-1 CTACGAGTTACATAATGAAACTTAAGGAGT 474AACAGTGCGTGTCAGGATGGCCGAGTGGTC TA AGGCGCCAGACTCA AGGCGGCCGCA4C CCCTACCA TRL-CAA4-1 CACAAGTATGAGGCTAAGTGAGAAGCGC’TC 475TGCTTCCGAGTGTCAGGATGGCCGAGTGGT CTAAGGCGCCAGACTCAAGGCGGCCGCA4CCCCTACCAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TRL-CAA4-1 CACAAGTATGAGGCTAAGTGAGAAGCGCTC 4485’ leader / exonic element TGCTTCCGAGTGTCAGGATGGCCGAGTGGT CTAAGGCGCCAGACTCAAG TRR-TCTl-1 CTCTGGCTTAACATAGCAGATGCGCTGAGA 476CTCCAACAGGTGGCTCCGTGGCGCAATGGA TAGCGCATTGGACTTCTAGCGGCCGCA4CCCCTACCA TRR-TCTl-1 CTCTGGCTTAACATAGCAGATGCGCTGAGA 4475’ leader / exonic element CTCCAACAGGTGGCTCCGTGGCGCAATGGA TAGCGCATTGGACTTCTA TRR-TCT2-1 TCTAGTACCCGTAAGCTACAAGACGCCGCC 477GTTCGTCGGGTGGCTCTGTGGCGCAATGGA TAGCGCATTGGACTTCTAGCGGCCGC4ACCCCTACCA TRR-TCT3-1 TTTCAGGCAGAGGTGAGCGGGGCGGCCGG 478GTTCCGCAGCCGGCTCTGTGGCGCAATGGA TAGCGCATTGGACTTCTAGCGGCCGCA4CCCCTACCA TRR-TCT3-2 CCTCCCCTGATGTGTTCATCGCGGAGAGGT 479CGCACGTGTCTGGCTCTGTGGCGCAATGGA TAGCGCATTGGACTTCTAGCGGCCGC4.4CCCCTACCA TRR-TCT3-2 (10-mer) CCTCCCCTGATGTGTTCATCGCGGAGAGGT 480CGCACGTGTCTGGCTCTGTGGCGCAATGGA TAGCGCATTGGACTTCTAGACTGCGGCCG CAACCCCTACCATyrosine GTA3-1 ACGTGTCTAAAAAGGACAGCGTTCCGTGTC 481 Mouse-Human Hybrid CTTCGATAGCTCAGCTGGTAGAGCGGAGG / X CTGTAGGCCTGCGGCCGCAACCCCTACCATyrosine GTA3-1 GAACGCAGAACTACGTGTCTAAAAAGGAC 482 Mouse-Human Hybrid AGCGTTCCGTGTCCTTCGATAGCTCAGCTG GTAGAGCGGAGGACTGTAGGCCTGCGGCC GCAACCCCTACCATyrosine GTA3-1 CGTGGCGTTATGGAGCCCTTCCGTCAACCG 483 Mouse GCTTGCCGCTCCCTTCGATAGCTCAGCTGGTAGAGCGGAGGACTGTAGGCCTGCGGCCGC.4ACCCCTACCATable 11. 3’ Engineered tRNA SequencesDescription Nucleic Acid Sequence SEQ ID NO.TGGTCGGGGTTGTGGCCGCGGAGGTATCCTTA 650TRY-GTA3-1.1GGTCGCTGGTTCGAATCCGGCTCGGAGGACGGT(TncY)AGTTTTGACCT ACCAAGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TricY (TRY- ATCCTTAGGTCGCTGGTTCGAATCCGGCTCGGA 29 GTA3-I.1) 3’ GGACGGTAGTTTTGACCTACCAAGtrailer / exonicelementTRY-GTA1-1 TGGTCGGGGTTGTGGCCGCGGAGACATCCTTA 651GGTCGCTGGTTCGAATCCGGCTCGAAGGAGTTC ACAATTTTTTTTTTTTGCGTAAGTTTTAAAATTC AT TRY-GTA2-1 TGGTCGGGGTTGTGGCCGCGGGGCAATCCTTA 652GGTCGCTGGTTCGATTCCGGCTCGAAGGACTTC GTCTGTAATTTTTAACCTCAATTTAATTCAATTA TC TRY-GTA3-1.2 TGGTCGGGGTTGTGGCCGCGGAGGTATCCTTA 653GGTCGCTGGTTCGAATCCGGCTCGGAGGACGGT AGTTTTGACCTACCAAGCTAATAATTCCAGACA ATA TRY-GTA3-1.2 TGGTCGGGGTTGTGGCCGCGGTAAGCAAGGTA 654( 10-mer) TCCTTAGGTCGCTGGTTCGAATCCGGCTCGGAG GACGGTAGTTT TGACCTACCAAGC TAATAA TTC CAGACAATA TRY-GTA4-1 TGGTCGGGGTTGTGGCCGCGGGGACATCCTT'A 655GGTCGCTGGTTCGATTCCAGCTCGAAGGAAGTG CGTGATGCTTTTGGTTAAAAGCCCTGCAGCTTC CAAG TRY-GTA5-1 TGGTCGGGGTTGTGGCCGCGGAGACATCCTTA 656GGTCGCTGGTTCGATTCCGGCTCGAAGGAGACA AGTGCGGTTTTTTTCTCCAGCTCCCGATGACTTA TG TRY-GTA5-1 ATCCTTAGGTCGCTGGTTCGATTCCGGCTCGAA 6493’ leader / exonic GGAGAGAAGTGCGGTTTTTTTGTCCAGCTCCCGelement ATGACTTATGTRY-GTA5-2 ZGGZCGGGGZTGTGGCCGCGGGGCCATCCTTA 657GGTCGCTGGTTCGATTCCGGCTCGAAGGAGAGA CACCCCCCCCCCCATTATTTTGTTGCTTTGAACC AA TRY-GTA5-3 TGGTCGGGGTTGTGGCCGCGGGGACATCCTTA 658GGTCGCTGGTTCGATTCCGGCTCGAAGGAGCTGCCGTATTCTTTGCACACGCACGCACCAAAACTACGT TRY-GTA5-4 TGGTCGGGGTTGTGGCCGCGGGGACATCCTT'A 659GGTCGCTGGTTCGATTCCGGCTCGAAGGAAGTG CCCGATGCTTTTGCATGCAATGCCACCTGGTGC CTGG TRY-GTA5-5 TGGTCGGGGTTGTGGCCGCGGGGTCATCCTTA 660GGTCGCTGGTTCGATTCCGGCTCGAAGGAGACGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CTGCTGTTTTGGGGTTTACGGTTGGTCTTCGTTT GT TRY-GTA6-1 TGGTCGGGGTTGTGGCCGCGGGGTCATCCTTA 661GGTCGCTGGTTCGAATCCGGCTCGGAGGAGTTC CATTTTTAAAAGTGTCTCTTCTGGGCCTGAAAT AAG TRY-GTA7-1 TGGTCGGGG'nGTGGCCGCGGGGACAT'CCTTA 662GGTCGCTCKITTCAATTCCGGGTCGAAGGAAGCG CCTGACTCTTTTGCGCACAATGCTGCCTGGCTG CACC TRY-GTA8-1 TGGTCGGGGTTGTGGCCGCGGAGGCATCCTTA 663GGTCGCTGGTTCGAATCCGGCTCGAAGGACGAA AGTTTTGAACTACGGGCTAATAATTCCAGAAAT AAC TRI-TATI-1 TGGTCGGGGTTGTGGCCGCGGAGCAATGCCGA 664GGTTGTGAGriCGATCCTCACCTGGAGCACTIT TTTICCCCATCAGTTTITATAAACTTACACATCrAAT TRI-TATI-1 TGGTCGGGGTTGTGGCCGCGGGAGCGGAGCAA 665(10-mer) TGCCGAGGTTGTGAGTTCGATCCTCACCTGGAG CACTTTTTTTCCCCATCAGTTTTTATAAACTTAC ACATCTAAT TRI-TAT2-1 TGGTCGGGGTTGTGGCCGCGGAGCAATGCCGA 666GGTTGTGAGTTCGAGCCTCACCTGGAGCAAGAC CCTTTTGGATGTTCGCAACCCCTTTTGTGTATCT CG TRI-TAT2-2 TGGTCGGGGTTGTGGCCGCGGAGTGATGCCGA 667GGTTGTGAGTTCGAGCCTCACCTGGAGCATTGT TTTCCAACCCAAAATAGCATTACTTGAAGTGCC TTT TRI-TAT2-3 TGGTCGGGGTTGTGGCCGCGGGGAGATGCCGA 668GGTTGTGAGTTCGAGCCTCACCTGGAGCATGTT TTCTTCCAATTGCGATTTCAGACATTTCACGATA AT TRI-TAT3-1 TGGTCGGGGTTGTGGCCGCGGAGCAATGCCGA 669GGTTGTGAGTTCAAGCC TCACCTGGAGCAGT TT TACTATTCTCCGACCTGTAGAAATTCAAAAACA GGG TRL-CAA1-1 TGGTCGGGGTTGTGGCCGCGGGGGATTCTGGT 670CTCCAATGGAGGCGTGGGTTCGAATCCCACTTC TGA CAC ATA CTTTTCTTTTTCCTCTCCTTA CTTTA GAGATATCCT TRL-CAA1-2 TGGTCGGGGTTGTGGCCGCGGAGGATTCTGGT 671CTC’CAAIGGAGGCGrGGGI'TCGAATCCCACTTCTGACACAACTATCTTATTCTCCTTTTACTCTACTTTCTCAGCCATATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TRL-CAA2-1 TGGTCGGGGTTGTGGCCGCGGGGTCTTCTGGTC 672TCCGTATGGAGGCGTGGGTTCGAATCCCACTTC TGACACAGCCTGATCTTTTGTTTTTCCCTACCAG ATTTGTACCGAA TRL-CAA2-1 TGGTCGGGGTTGTGGCCGCGGTGTTCGGGTCTT 673CTGGTCTCCGTATGGAGGCGTGGGTTCGAATCC(10-mer) CACTTCTGACACAGCCTGATCTTTTGTTTTTCCC TACCAGATTTGTACCGAA TRL-CAA3-1 TGGTCGGGGTTGTGGCCGCGGGGGCTTTCTGGT 674GTCCGCATGGAGGCGTGGGTTCGAATCCCACTT CTGACACAGCTATTTTGCCCCTTTAATCCTAGG ATTGGGGGACTGGG TRL-CAA4-1 TGGTCGGGGTTGTGGCCGCGGGGCTTTCTGGTC 675TCCGGATGGAGGCGTGGGTTCGAATCCCACTTC TGACACATTCGTTTTATTAGCATTTTATTCCCCG GAAGAAACCCC TRL-CAA4-1 TTCTGGTCTCCGGATGGAGGCGTGGGTTCGAAT 6483’ leader / exonic CCCACTTCI GACACATTCGTTTTAT TAGCATTT Telement ATICCCCGGAAGAAACCCCTRR-TCT1-1 TGGTCGGGGTTGTGGCCGCGGAGGCATTCAAA 676GGTTCCGGGTTCGAGTCCCGGCGGAGTCGTAAC GCTTTTTTCCCTCCCCCCTACAATTTATTTTCTGccTRR-TCTl-1 ATTCAAAGGTTCCGGGTTCGAGTCCCGGCGGAG 6473’ leader / exonic TCGTAACGCTTTTTTCCCTCCCCCCTACAATTTAelement TTTTCTGCCTRR-TCT2-1 TGGTCGGGGTTGTGGCCGCGGAGCAATTCAAA 677GGTTGTGGGTTCGAATCCCACCAGAGTCGATTT TATTTCAATTTTTCCTTTTTATCCGTTTTTTTCTT A TRR-TCT3-1 TGGTCGGGGTTGTGGCCGCGGGGTCATTCAAA 678GGTTGTGGGTTCGAGTCCCACCAGAGTCGAATT TTGATGGCTCCTACTCCCTGACTTTCACCTGCGT GTT TRR-TCT3-2 7GC77’CC?(7GC777GTGGGCGCGGAGAAATTCAAA 679GGTTGTGGGTrCGAGrCCCAC'CAGAGTCGC’rTT TTGTi rGAAGAAGAGC'AAACGGAAAGTTAGGCGGAG TRR-TCT3-2 (10- TGGTCGGGGTTGTGGCCGCGGAGTTAGAGAAA 680 mer) TTCAAAGGTTGTGGGTTCGAGTCCCACCAGAGT CGCTTTTTGTTTGAAGAAGAGCAAACGGAAAGT TAGGCGGAGTyrosine GTA3-1 TGGTCGGGGTTGTGGCCGCGGGGCTGTGGACA 681 Mouse-Human TCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGHybrid (PTR244) GACGGTAGTTTTGACCTACCAAGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Tyrosine GTA3-1 TGGTCGGGGTTGTGGCCGCGGGGCTGTGGACA 682 Mouse-Human TCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGHybrid (PTR245) GACGGTAGTTTTGACCTACCAAGCTAATAATTC CAGACAATATyrosine GTA3-1 TGGTCGGGGTTGTGGCCGCGGGGCTGTGGACA 683 Mouse (PTR246) TCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAG GAAAGAGCTTGCTATTTTCTTTTACCTTTTAATC CTATCTATTyrosine GTA3-1 TGGTCGGGGTTGTGGCCGCGGGGCTGTGCACA 684 Mouse (PTR247) TCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGGAAAGAGCTTGCTATTTTCTTTTACCTTTTAATCCTATCTAT

[0187] In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element of Table 10, In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and the 5’ circularizing element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 28 and 447-483, In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and the 5’ circularizing element comprises a polynucleotide sequence of any one of SEQ ID NOs: 28 and 447-483 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and the 5’ circularizing element comprises a polynucleotide sequence of any one of SEQ ID NOs: 28 and 447-483. In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and the 5’ circularizing element consists of a polynucleotide sequence of any one of SEQ ID NOs: 28 and 447-483.

[0188] In some embodiments, the recombinant nucleic acid molecule comprises a 3’ circularizing element of Table 11. In some embodiments, the recombinant nucleic acid molecule comprises a 3’ circularizing element and the 3’ circularizing element comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069identical to any one of SEQ ID NOs: 29 and 647-684. In some embodiments, the recombinant nucleic acid molecule comprises a 3’ circularizing element and the 3’ circularizing element comprises a polynucleotide sequence of any one of SEQ ID NOs: 29 and 647-684 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a 3’ circularizing element and the 3’ circularizing element comprises a polynucleotide sequence of any one of SEQ ID NOs: 29 and 647-684. In some embodiments, the recombinant nucleic acid molecule comprises a 3’ circularizing element and the 3’ circularizing element consists of a polynucleotide sequence of any one of SEQ ID NOs: 29 and 647-684.

[0189] In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and a 3’ circularizing element, wherein the 5’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 28; and the 3’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 29.

[0190] In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and a 3’ circularizing element, wherein the 5’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 447; and the 3’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 647.

[0191] In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and a 3’ circularizing element, wherein the 5’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 448; and the 3’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 648.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0192] In some embodiments, the recombinant nucleic acid molecule comprises a 5’ circularizing element and a 3’ circularizing element, wherein the 5’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 449; and the 3’ circularizing element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 649.

[0193] In some embodiments, the recombinant nucleic acid molecule comprises at least one regulatory element, such as circularizing elements (e.g., backsplicing or tRNA splicing elements), promoters, terminators, enhancers, internal ribosome entry sites, polyadenylation signals, capping elements, transcription control elements, translational control elements, cellular localization signals, stabilizing sequences, destabilizing sequences, introns, or any combination thereof. In some embodiments, the recombinant nucleic acid molecule comprises at least one backsplicing element. In some embodiments, the recombinant nucleic acid molecule comprises at least one tRNA splicing element. In some embodiments, the recombinant nucleic acid molecule comprises at least one promoter. In some embodiments, the recombinant nucleic acid molecule comprises at least one terminator,

[0194] In some embodiments, the recombinant nucleic acid molecule comprises an enhancer. Enhancers are nucleotide sequences that enhance promoter activity. In some embodiments, the enhancer is located upstream or downstream of the gene region to be transcribed, and / or located within the gene, to activate transcription.

[0195] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one promoter. In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase promoter located 5’ of the 5’ circularizing element. In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase promoter located 5’ of the tRNA 5’ leader or 5’ of the 5’ intronic element.

[0196] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase II (Pol II) promoter. In some embodiments, the RNA polymerase II promoter is located 5’ of the 5’ intronic element. In some embodiments, the RNA polymerase II promoter comprises a polynucleotide sequence derived from a chicken beta actin (CBA) promoter, a humanATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069phosphoglycerate kinase (hPGK) promoter, a human glucose-6-phosphatase (hG6Pase) promoter, a human elongation factor- 1 alpha (EF-la) promoter, a mouse E-cadherin (mECAD) promoter, a mouse or human polycystic kidney disease 2 (PKD2) promoter, or a mouse kidney-specific cadherin (mKSP-C) promoter. In some embodiments, the RNA polymerase II promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the RNA polymerase II promoter is a CBA promoter. In some embodiments, the RNA polymerase II promoter is a mouse Na⁺-K⁺-Cl⁻ cotransporter 2 (NKCC2) promoter, or a derivative thereof. In some embodiments, the RNA polymerase II promoter is a mouse uromodulin (UMOD) promoter, or a derivative thereof. In some embodiments, the RNA polymerase II promoter is a goat UMOD promoter, or a derivative thereof. In some embodiments, the RNA polymerase II promoter is a human UMOD promoter, or a derivative thereof.

[0197] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase III (Pol III) promoter. In some embodiments, the RNA polymerase III promoter is located 5’ of the tRNA 5’ leader. In some embodiments, the RNA polymerase III promoter comprises a polynucleotide sequence of a Hl promoter, a H1.M11 promoter, a 7SK promoter, a U6 promoter, a U6+1 promoter, or a U6+27 promoter. In some embodiments, the RNA polymerase III promoter is a U6+27 promoter.

[0198] In some embodiments, the recombinant nucleic acid molecule comprises a promoter selected from a ubiquitous promoter, constitutively active promoter (e.g., CMV), an inducible promoter (e.g., tetracycline-regulated promoter), a tissue- or cell-type specific promoter, or a synthetic promoter (e.g., a chimeric promoter). Examples of suitable promoters include, but are not limited to, cytomegalovirus (CMV) promoters, rous sarcoma virus (RSV) promoters, herpes simplex virus (HSV) promoters, SV40 promoters, chicken beta actin / cytomegalovirus hybrid (CAG) promoters, steroid-regulated promoters, and metal-regulated promoters.

[0199] In some embodiments, the promoter increases transcription of the recombinant nucleic acid molecule encoding the circular RNA by at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more above basal level in a target cell or tissue. In some embodiments, the promoter increases transcription of the recombinant nucleic acid molecule encoding the circular RNA by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069about 70%, about 80%, about 90%, or about 100% compared to a recombinant nucleic acid molecule encoding a circular RNA without the promoter.

[0200] In some embodiments, the promoter is a tissue- or cell-specific promoter that induces transcription of a recombinant nucleic acid sequence encoding a circular RNA in a specific tissue or cell. In some embodiments, the tissue- or cell-specific promoter increases transcription of the recombinant nucleic acid molecule encoding the circular RNA by at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more above basal level in the target tissue or cell.

[0201] Exemplary promoters of the present disclosure are provided in Table 12 below.Table 12. Exemplary Promoter SequencesPromoter Name Nucleic Acid Sequence SEQ ID NO CBA CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTG 30ACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT GACGTATGTTCCCATAGTAACGCCAATAGGGACTTT CCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC TGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT GGGACTTTCCTACTTGGCAGTACATCTACTCGAGGC CACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCC CCACCCCCAATTTTGTATTTATTTATTTTTTAATTAT TTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGG CGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGC GGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTAT GGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGC GAAGCGCGGGGCGGGCGGGAGCGGGATCAGCCACC GCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAA ACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTC TTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAA TCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACT TCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAA ACACTGCGGAATTGTACCCGCGGCCGATCCACCGGT CCGGAATTCCCGCGGATATCGTCGACCCACGCGTCCGG<1CCCCACGCTGCGCACCCGCGGGTTTGCTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069EF-la GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCG 31 CCCACAGTCCCCGAGAAGTTGCJGGGGAGGCIGTCGG CAATTGAACCGGTGCCTAGAGAAGGTGGGGCCCGG TAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGAGAACCGTATATAAGT GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCT TGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCA GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAG TGGGTGGGAGAGTTCGAGGCCTTGCCiCTTAAGGAGCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTG GGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCAC CTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTA GCCATTTAAAA1TTTTGATGACCTGCTGCGACGCIT TTTITCTGGC’AAGATAGICTTGTAAATGCGGGCCAA GATCTGCACACTGGTATTTCGGTT1TTGGGGCCGCG GGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATG TTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGA GAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCT GCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCC CCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACC AGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCC CTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGC TCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAG GAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATG TGACTCCACGGAGTACCGGGCGCCGTCCAGGCACC TCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTT AGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCA GCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCC CTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTC AGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGG TGTCGTGAhPGK GGGTTGCGCCTTCCAAGGCAGCCCTGGGTTTGCG 32CAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTT CACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCGGCGACGCTTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGC GTGCCGGACGTGACAAACGGAAGCCGCACGTCTCA CTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCA ATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAA TAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCG GCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCCTCTCCCTCGTTGACCGAATCACCGACCTCTC TCCCCAGGrnECAD ACCTGACATCCTCACACCAATACACATAAACAAAA 33TAAATTATTAAAAAATAAAACCCAACAGAACAACT GATGAGGAGGGCTGGAGAGATGGCTCAGTGGTTGA GAGCACTGGCTGCTTCCAAAGGACCCAGGTTCAAA TCCCAGCAACCACATGGTAGTTACAGCTGTCTGTAA TTCCAGTTCCAGGAGATCTGATACCCTCACATAGAC ATACACGGAGGGAGAACAATGTAAGTAAAAGATTT AAAATAAAAAGCAACAACAACAAAACAAAACAAA ACAAAAAACCTGATGGATGTGGGATGCATAGGATG CTGCTACTTGGGGGTCCCAAGGTCAAAGCCAGTCTG GGTTACCTAGTCCTGAGAACTGAAGTTAAGAGAAC AGATTTTAGCCGGGCAGTAAGGTAGTGGGTACCTG TAGCTCCATCCCAGAAGTGAGAAGGCTGAGTTTGA ACAATGATGAGTTTAAAGCCATGCTGGGCTACATA GCAAGGCTATGTCTCAAAAGGGAGCCGGTCAGGTG TTAGAAAATTAGATTAGCCTGGTCTGGTACCCCACT TGTGCAATCCCAGCATTCGGGAGACTGAAACAGGA GGATGGCTAAGACTAAGACAATTCCAGGCCCACCT GAGTTGGAAAACAAACAAACAAAAAAGCTACCAA ACAAAATAAAACCGTCGGAGAAATAGCTCAGTCAG TAAAGGCCAATGGCGGGAATGCAATCCCAAGACCC TCTTGGTGGAAGAAGAGAATTGACTCTTGAAGGCT GTCGTCTTATCTCCACAATCGGTCT(]TGGCACGTGC GTTTGGGAGCACAG<}ATCGCGCTC: TCGCCCGCGCAC GCACCCCTCCCCCCATGTTAAAATGTCATTTAAAAT CCCTAAGCAAACAAACTCATCCAACCAAAGAAAAT AAAAACATAAGAAACAAAACGGAAACCTAGATGAT GAATAAAGTCCTTTGTAACTCCATGTCTCCGTGGGT CAGAGCACAGCTAGGCTAGGATTCGAACGACCGTG GAATAGGAAGCTGGGAAGTCTTCTAAGGCCGGCCC CATGCCACCAACTACAGACAGGGGTGGAGGAAGIT GAGGGCCCTGCAGTTCC1TGGCTGCCACCTGCAGGTGCGTCCCCAGCCAATCAGCGGCGCCGGGGGCGGTGCCTGCGGGCTCACCTGGCGGCCGCAGCCTCTGCGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069mKSP-C AGCTTGCTCTGCCATGGGAAGGTCCCCAAACCTGA 34AAAAGAAACCCAGGCGCCTTGGGCAAACGGCTTAG CCTCTCTGTACCCCAGAGGG<1TATGACAAG<1G<1AC A<1TTCCCTCTTCAGAATGAGTG< K1TGG<1ATAGCAG CGATATGTGCTCACAGAAGTTCTGGGTGCTGGTTAGTAGGCACTCAGTATGTGTGAGACCTCCTTCAACAACCCCACATATAGCACTAACCTAGGCTCAGTGCCTCAGTGATCCTAAAACAGACACTCAGCTTGTCCCAGTCCCGCTTCCATCTGCAATGCTCACCACATATACCATGAGCAGTCCTCCGCGAGACCCCAAAACATGCAAGGAAAGCTGACAGCGAGACCAGGCTCTTCATACTCTG TACCCTCATCTGAGATTCCCAGAAAACAAGGTTTCT GCTGGGTCTGCCTTTCAGGCCTTACAGGGGGAGGTG AGGAGGCCCTGGTCAGCCCTGTAACTCCCTCAAAGT CACCTGAGCTGGCTCGGTCTICTCT TTTCTGCT TAGA GACA TGAGACAGGTC AGAGCCTCACCCTTA TCTGI G TTCAGGAGACCATGTGAGGCTAGGAGAGCTTCAGG GGGACCTCTAGGCTTCTGTCCCACCCACTGGTCTCA CI GATGAATGT TCTAGAGC TCCGAAC ACACACTTGG GGTCAGC'TGCCCTTTGGGTTACACrCCCAGTCCTTr CCITAGCTCCACGGGCCACrTGGGGC’AGGACAAGG TCATAGCCTACTCAGAGGAACTCCGAAGCTAATAC GTGAGAAACAAGCTGGTGTCTTCTGGGCATCCGTA GAGGCAGTTCATGGTATGGAGTGGGGGAGCCTAAT CCAGCCTGTGAATGTAAGGGCTTTCCTGAAGAAGG GGTTATTAGTGCTTGTTGCTAAAGCGTAAGCAGGTA CTTACTGTCCTGTAGAAAGGTGGGAAGAGCATTCC AGGCACAAGGAACAATATCTGGAGAGATCTGGCTA CAGATGCCACTGCACAGAGGAGAAGTGGGAGCCAA GTCTGAACACACACACACACACACACACACACACA CACAGAAGTTATGTCTAAGCCCAGAGGGCCAGCAA GTGCTCATTGGGCTGTGTCAAGGGGGCAGTGACAG ACCAAGAGCTGCCCACCTCCTAGGGCTGGCAGTCA CGGATGCTGAGCAGATCTGGCTCTCCAAAGTCAAT AAGTAACTTGGGGGGACTAGGCGGGGCCAGGCCTG CTCCTGTGGGCCCCGGTGGCATTTTCCACTCCTGAG CAAGCACGGCCAGACCGCCTACCTGCTCAAGTGTC CACCTTGCCTCGCCCCACCTAAGCCAAATTTGCU6+27 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCAT 35ATACGATACAAGGCTGTTAGAGAGATAATTAGAAT TAATTTGACTGTAAACACAAAGATATTAGTACAAA ATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGT TTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTGCTCGCTTCGGCAGCACATATACTAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069U6+1 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCAT 36ATACGATACAAGGCTGTTAGAGAGATAATTAGAAT TAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGHl GAACGCTGACGTCATCAACCCGCTCCAAGGAATCG 37CGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTTCTGTATGAGACCACH1.M11 ATATTTAGCATGTCGCTATGTGTTCTGGGAAACTTG 38ACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGTTCTGTATGAGACCACTCTTTCCC7SK CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGC 39ATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGT CCGTTTATCTCAAACTTTAGCATTTTC3GGAATAAAT GATATTTGC’TATGCTGGrTAAATTAGATITTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACCTCU6 GAGGGCCTATTTCCCATGATTCCTTCATATTTGCAT 40ATACGATACAAGGCTGTTAGAGAGATAATTAGAAT TAATTTGACTGTAAACACAAAGATATTAGTACAAA ATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGT TTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CMV TAGTTATTAATAGTAATCAATTACGGGGTCATTAGT 41TCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTC CCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG CAGTACATCAAGTGTATCATATGGCAAGTACGCCCC CTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATT / X CCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC GTGGA TAGCGGT TTGACTCACGGGGATTTCCAAGTC TCCACCCCATTGACGTCAATGGGAGTI’rGITlTGGC ACCAAAATCAACGGGACTTTCCAAAATGTCGTAAC AACTCCGCCCCATTGACGCAAATGGGCGGTAGGCG TGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTT AGTGAACCGTCAGATCGoat UMOD GACATTCCCACCTAGGATTGAGAAAAAGAATATTA 93 Promoter- 1473 AGAACTTTTATTTTCTTCTGAAGTTATAGCAAAGAA AGGGGAAAAAAAAAAACATTCTTATGGGGGATAAA CGGGCAAAGGATACAAACAGTTCAGAAAAGAATAA ATAGTAAGCAAATGAAAAGATAACTTCCTTTTTCAT CAAAGAACTGCAAAAGTAAATAATGATAAGATGTT TCTCACTTTTCCACAAAGATGAAAGTTAATGCCCAG GGTGGCTGAGTACTGTGCTGGGATTGTGAACTAACTGTTATAGATCTCTCTGGGGTGCTGTTTGGGAAGAAA CATCGCTGAAAACTGAGCTACCTCTTTTCCTATGAAATTCCCCTGAGGAGGTGAGTGAGCCGCTGCTGATCGTCACCCGAGGACTAGGCCAGAGAGAAGGAGAAAG CCCTCAAAGAGGCAATGCTGTGGATCACTGTCATATTTTCCTGCTCAGCCTGAGTTCACATGTGCCTGATTTTT CTCAATATGGCATTGCCATTAACGTGGAATTAGGTC AGGAGACCTAAGGCTGAACCAAGCCCTGTCATTCT CTGCCCCATGAC TGCGCA TCACCAAAACAGCATCG GCAGTGACTTCCACAGATGGTACCATTGCTATATGC CITAACTTGCATCATCTCCTTTAATGGCCATAACAA TTCTAGGACACGGGTATICTTGTTITACAGAIGATG AAAAT TACCTC TGGAAGGAAAATTACTGGCACACA AAAAACGCTGACCAGGAT TCAGA TAGACI GACTCC AAAGTCAGICTGTTCATCTACAAAAriAICTACTTC TCAAGGACCTTCCTICA TGGGAATTC AAAT TTCTTG ATTCACAGAGCATCTGGTCCAATGATGTCTGAATTATCTGCTGTCTCTGACCTTCAGCCATTCTCAGCTCCTTTCCTGATCACATTGGGACCCCAGGGGAGCTGGCTGAATCTGTGAGGATGACATTTGCTTTGGAATTAAGTGGCCACAAGTACACATCCTGGTGGGGACAATGAGCAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CCCCTTTTCTCCTGGAGCAGCCTGGCTTCAGATTCTGGCCTCTGCTTGGCTCCACTTTGTGCTTTTCAATGACCAAGAAAATCCCAGGCCCTTGGAATTGTTTACTCAG TTAATTTCTAACTAAAGAACCTCTTGTTGCCAAAAG GTATAAAACAGAGCCCTTGTAACTGTGGCTCACAGC TGTGACCCCCATGTCAATCATTTGGGGTCTCTACCT ATTAGGGAAAAGAACAACAACCACCTCACAGCCTA GAAAAGGAAAACACTGTGTCAAAAGGGAAAAATAT TCCACCCCCATTAAAATAATTAAGAAACAGAACCA GAGGATCATTGGAGGAGAGATTGCCAGTGGGGGAC AGATGTATATATATAGATATGAAAGTCACCTACTTG TAAAAGGATTAGTTCTACCTTTCTGGTTTCAGGAAGGCTATCTGCAGGoat UMOD TGATAGCGAGGTATATCTCCAGTGCTACCTAACTCC 94 Promoter- 1752 AAGGCATTAGTTCACCTTCAGTGGGGCAGTCCCTCC AGAATCACCAGTATCAAGATGTAAAAGCATCAAAC ACAAAAACTTGAATAATGTC3GTTGGTTGTTAGGCTTAAGATAGTCTCAGCCCAAACTCCCTATTTTCCTAC CCAAGATCTCTGCCCAGACAACTTCAGGAGCTACCT GGAGCICCATCITTAATCCTTIAAAGACAC'CCAGACT TAGGTTT TGACAGAGCCTCATGTTCACCAACCAGA AA TGAC A ITCACCACCTAGGATTGAGAAAAAGAAT ATTAAGAACTTITATITTCTICTGAAGrTATAGCAA AGAAAGGGGAAAAAAAAAAACATTCTTATGGGGG ATAAACGGGCAAAGGATACAAACAGTTCAGAAAAG AA TAAATAGTAAGC AAATGAAAAGATAACTTCC TT T TTCATCAAAGAAC TGCAAAAGTAAATAATGATAA GA TGTHCTC ACTT TTCCACAAAGAI GAAAGTTAAT GCCCAGGGTGGCTGAGTACTGTGCTGGGATTGTGA ACTAACTGTTATAGATCTCTCTGGGGTGCTGTTTGG GAAGAAACATCGCTGAAAACTGAGCTACCTCTTTTC CTATGAAATTCCCCTGAGGAGGTGAGTGAGCCGCT GCTGATCGTCACCCGAGCACTAGGCCAGACAGAAG GAGAAAGCCCTCAAAGAGGCAATGCTGTGGATCAC TGTCATATTTCCTGCTCAGCCTGAGTTCACATGTGC CTGATTTTTCTCAATATGGCATTGCCATTAACGTGG AATTAGGTCAGGAGACCTAAGGCTGAACCAAGCCC TGTCATTCTCTGCCCCATGACTGCGCATCACCAAAA CAGCATCGGCAGTGACTTCCACAGATGGTACCATTG CTATATGCCTTAACTTGCATCATCTCCTTTAATGGCC ATAACAATTCTAGGACACGGGTATTCTTGTTTTACA GATGATGAAAATTACCTCTGGAAGGAAAATTACTG GCACACAAAAAACGCTGACCAGGATTCAGATAGAC TGACTCCAAAGTCAGTCTGTTCATCTACAAAATTAT CTACTTCTCAAGGACCTTCCTTCATGGGAATTCAAATTTCTTGATTCACAGAGCATCTGGTCCAATGATGTCATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TGAATTATCTGCTGTCTCTGACCTTCAGCCATTCTCA GCTCCTTTCC: TGATCACATTG<}GACCCCAGGGGAGC TGGCTGAATCTGTGAGGATGACATTTGCTTTGGAAT TAAGTGGCCACAAGTACACATCCTGGTGGGGACAA TGAGGACCCCTTTTCTCCTGGAGCAGCCTGGGTTCA GATTCTGGCCTCTGCTTGGCTCCACTTTGTGCTTTTC AATGACCAAGAAAATCCCAGGCCCTTGGAATTGTTT ACTCAGTTAATTTCTAACTAAAGAACCTCTTGTTGC CAAAAGGTATAAAACAGAGCCCTTGTAACTGTGGG CACAGCTGTGACCCCCATGTCAATCATTTGGGGTCT CTACCTATTAGCIGAAAAGAACAACAACCACCTCAC AGCCTAGAAAAGGAAAACACTGTGTCAAAAGGGAA AAATATTCCACCCCCATTAAAATAATTAAGAAACA GAACCAGAGGATCATTGGAGGAGAGATTGCCAGTG GGGGAC AGAI G T ATATAT AT AGAT ATGAAAG TC AC CTACITGTAAAAGGATrAATTCTACCI’rTCTGGTnr AGGTAHuman UMOD cctgaaaagagcaattcacaatagcaaagacttggAaccaactcaaatgtccatcaa 95 Promoter tgatagactggataagaaaatgtggcacatatacaccgtggaatactatgcagccata aaaaatatgagttcatgtccttgtagggacatggatgaagatggaaaccatcattctca gcaaactatcgcaaggacaaaaaaccaaacaccgcatgtctcactcacaggtggga attgaacaatgagagcacttggacacaggaaggggaacatcacacccaggggccta ttgtgggatgcggggagcggggagggatagcattaggagttatacctaatgtaaatg acaagttaatgggtgcagcacatcaacatggcacatgtatacatatgtaacaaacctg cacgttgtgcacttgtaccctaaaacttaaagtataaaaaaaaaaaagatcaatgcagt gatcatggtgatatttcctgctcagcccaagttcacacatattttattttctcaacatgat gacagccactctcacactgacttttggaatgtcatgtatgttgaactgggtctgaagaca tggttttaactcaggctctgtcattttctacctcagtgattgcacaacagcaaagcagaat tttcactacttccatgaatataatcattactatatgactttacttgcatcatctcctttggttac tattactactgtgggagatgggtattctcattttatagacaaggaaattgacctctggacc tcaggaaggttaagaaatgagcccactgccacacaataaacaccagataaaggagg cagactgactccaaagtcagtctatttaagtgcaaatttatttcgcctccaaagggacct cccagtcatcagacctgattctttgttgtacagagtgggtcaggtccagtgatgtctgaa ctaccttctggttctgactttcagccattctcagctcctctcttgcttgtgtctggattctaa ggctgatctcatgagaatgggtgtttcagaagggtgccctctccaagacaggtgcacc tcccatctggggcagtgaatatcccttttgtccttatgcagcctggcttcagatactggct tctgcctggctccttgatcccaccctgcccttgtcagtgaccaagaagaagcccagca ccttggcactgctttcccagttaatttctaactatggaatctcttgctgttagaaggtgcga aacagtgaccttgtatttccgggcacaggtgtgaccccccaatgtcaatcatttggggt ctctagctattaggaaaaagaacaacaacaacctcacagcttggacaaggcaaacatt atgccaggaggaaaaaatattccacccccaagaaaacaatatcaaaaaacagaacta gagactaattggaggagagattgccagcctggggcaaatgtgtatatataagtatgag gcacatcatgaccAGACTAACTCTACCTTTCTGGCTTCAGG ACACCAGACATCAGAGACAGAGAGAAAAATTCAAA GGGCCAACCCGTCTTTCCTTTGGGCAGGTGCTATCTAGACCTGAAGTAGCGGGAAGAGCAGAAAGGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Mouse NKCC2 A AT A T GT G AGGCCCTGGGTT CGattctta ctgagagagagagag 96 Minimal Promoter agagagagagagagagagagagagagagagagagagagaaacagaaatagaga cagagacagaatggcagaccatagaactaaatttatctagtcccaaacaactgaagc aatattttttgaaggatttgttgttgtgcaatctgtaaattaaagaaactttattttgtttactg cctaaaaggattaatgatttactactagttactagttgatgtggtacttacagttggttcct cagggctaatgactggtgtggggcttttcccactattccaggccaatgactgaaaagtc atcgctttcaagaaagtttttaaccaatgagcagaagcctaaggttgtagcttaaactct aaggccccatatatgcgtgtgcgacgctaatgtgggttactgttGCTCAGAAG AAGGCTGCTGGATMouse UMOD CCAAGAGATCCAAGTCTCCTtcaaaaccaagtgtgtacagaacatt 97 Promoter gtctgaggagtaagattgcatttggcaacatgcatgtctttaatggtgtggaggaatttc agtggagttggcacgtcagaaagcacactggtgaaaaatggagagaatagatatatc ctttgagaaatttggtctcaaaaagtagggtatcaaattacttggtgtctgtgagatcaat tggttgtctctgtaggttagcttacataggagacaggaataagtgaaggagagaaggg aggacattggagcacccaaggagagagggaccttcctcctaaaagtgaatgaggtg gccttcattccaaggagaagagattcaggtcgcccgggaagatgagggaccaacat ccacaaggaatggcaggaagtcatcctgtgtgcataaatggagagagggggtcaaa gatggagcaaagaaggatgagcaagaaaatggtggatgtggatactctgaggatgg cctggctgtggtgagcaaaatgtgggcaaagtggcactccatgaacaagacagcttg ctctgtttgcagatccttaaataaaggcacatggcatgccatggaggctaggggagtg gaggggaaaggtatatagatagatgcagaagtaccagaggagccaggaaggacag gagtaggagggacaggtttgcacaaggctttgtcctctccccaccagctctctctccct tctgtatatgcacatacacagtgagctagtgtgcatatgtgtgcacatatgcatgtgatg aacagaggccagtcttgggtgtcagtcttcaggccctatctaccttgtttttgagacaat ctcacttgagtgagttgagtgactctcctagtattctacagaggtttcctcaggtgggga ggaatgggtgggagaagcaaatttaagactggttgatttcttgaatttcagtgggcttg ggaaatagcagctatatattcagtttcctcgttcctggctggcttcctggggtgatcaga gcagagtatagtagccctgtgtggcagtcacaccaagcagacaggagatagggcat ggctctggtgtggctggtagacataggaaaggatccttgtagcaagatgtttgccatct ccagagactagacagcccaggaaagtttgtcctcccaggaccagccagcactgaga ctggaatgcatcaaatccagagaccagaaagcacggtgctagcacttaggaagaga cactagcccaaagtctccttgctcctgcctaaagctttgccaattctgcaaaccttgaaaaatta gcatctttaaattcagaagggatacaagaagagaacttacatgggaccttgta a aaaagcatagggcatcagtaactaaagttacaaagataacaatcagtggtgagtgaa caaaggacatggccatgtttttttgttatgaaacacacgcacaggcacaggcactcac gtgtgcgcacgcgcgcacacacacacacgcgcacacacacacacacgcatgcaca catgcaccacacacaaactgcaaaagtgaataaaaagatatttctcactttggcaaagt ggatggaaagtgatcaaaatgaaagttatactcagaactattttgtactagagggagg ttataaattattgttattgttatattctatttactgttgtggcagcctaagttggtcttgaact cactatgaagctagcaatgaccttgagcttctgatccttatatctacactctcaagtgccc agattataagtgtgcaccactatactcagtttatgctgtgctaaggactaagcccaattat acaaacacacacacatatatacacacatacacacacacacacacgtatatatatgtata tatatatacatacatacacacacacacacacatatatgtaaaatttgggaagatatatca atcttctttaaagtacatgctactttggtccaaaactttcacttttaggaagttaagaaggaagagacagaataagagatgtcccaagaaagtcagtgtggttgtcttagttatgctcctgctcagtcaatgtttcagatttttctcagcacaatgacatctattctatcaagtttttgataaATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069ctctttacatgggactgggtgtggctgtggctctagctatttctatttgtgactgcctatc agcaaagcatccacttcagactttgactcaaacatcaccaagtattcccacttgcattgt ctctgttaaccagcatcactgttcacagggcagggcatcacatctcacaaagggaaa gggaaagggaagagttaaattccctgggatactagtcacggtggactcaggcaaac agcctcttcaattgtaagatgattccctagtccaaggaccctctactgtttggactccagt cttgtctgacagaggtccagttcaggagtgtccagatggtctgataacctgatgccatt ctcagagactctttcctgtctggaatctagtgaggaggacttatctggtgaagctgtcct ttagaacaggagtgtgttccagtcttcaaagcaaacattccttttatcctaacacagtctg acttcagatatactgtctttttcctggctccttgggcttaggtctaccttgtccttgcccag gtccaagaaaaggcccagaaccttggcactgttttgccagttaatgtctaactgagga atgtcttgctgccaaaaggtgaaaacagagaccttgtatttccaggcacaggtgtgac cccaatgtcaatcattttgtgtctaactcccaggggaaaaactaacaacaacagactca tggcttggaaaaggtgaattctatgccaaaagggaaggaaagttctacccccacaga aacaatctcagagggcagaagcagagaataatctgagggagagggccagccaagggcaggcaagtatatattgatcacaggcaCTTACTTGTGACI'GGACCAG

[0202] In some embodiments, the promoter comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 30-41 and 93-97. In some embodiments, the promoter comprises a polynucleotide sequence of any one of SEQ ID NOs: 30-41 and 93-97 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the promoter comprises a polynucleotide sequence of any one of SEQ ID NOs: 30-41 and 93-97. In some embodiments, the promoter consists of a polynucleotide sequence of any one of SEQ ID NOs: 30-41 and 93-97.

[0203] In some embodiments, the promoter comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the RNA polymerase II promoter comprises a polynucleotide sequence of SEQ ID NO: 30 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the RNA polymerase II promoter comprises a polynucleotide sequence of SEQ ID NO: 30. In some embodiments, the RNA polymerase II promoter consists of a polynucleotide sequence of SEQ ID NO: 30.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0204] In some embodiments, the promoter comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, the RNA polymerase III promoter comprises a polynucleotide sequence of SEQ ID NO: 35 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the RNA polymerase III promoter comprises a polynucleotide sequence of SEQ ID NO: 35. In some embodiments, the RNA polymerase III promoter consists of a polynucleotide sequence of SEQ ID NO: 35.

[0205] In some embodiments, the promoter comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 36, In some embodiments, the RNA polymerase III promoter comprises a polynucleotide sequence of SEQ ID NO: 36 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the RNA polymerase III promoter comprises a polynucleotide sequence of SEQ ID NO: 36, In some embodiments, the RNA polymerase III promoter consists of a polynucleotide sequence of SEQ ID NO: 36.

[0206] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one RNA polymerase terminator. In some embodiments, the recombinant nucleic acid molecule comprises at least one RNA polymerase terminator downstream of the 3’ circularizing element (a tRNA 3’ trailer or a 3’ intronic element). In some embodiments, the recombinant nucleic acid molecule comprises at least one RNA polymerase II or RNA polymerase III terminator. In some embodiments, the RNA polymerase terminator comprises a polyadenylation (polyA) signal. In some embodiments, the RNA polymerase terminator comprises a polyT sequence.

[0001] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one RNA polymerase terminator, wherein the RN A polymerase terminator is a polyadenylation (polyA) signal sequence. The term “polyA signal” or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of theATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069nascent RNA transcript by RNA polymerase II. In some embodiments, poly A signals promote mRNA stability by addition of a poly A tail to the 3’ end of the coding sequence. In some embodiments, the poly A signal is selected from any one of: AATAAA, ATTAAA, and AGTAAA. In some embodiments, the poly A signal is an SV40 poly A, a human growth hormone poly A signal (hGHpA), a bovine growth hormone poly A signal (bGHpA), a rabbit P-globin poly A signal (rPgpA), variants thereof, or another suitable heterologous or endogenous poly A signal known in the art. In some embodiments, the polyA signal is a bovine growth hormone polyA signal (bGHpA).

[0207] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises at least one RNA polymerase terminator, wherein the RNA polymerase terminator sequence is a polyT sequence. The term “polyT sequence” as used herein denotes a DNA sequence which directs the termination of the nascent RNA transcript by RNA polymerase III. In some embodiments, the polyT comprises at least 4, 5, 6, 7, or 8 thymine (T) nucleotides. In some embodiments, the polyT comprises at least 4 T nucleotides. In some embodiments, the polyT comprises 4 T nucleotides (polyT4), 5 T nucleotides (polyT.5), 6 T nucleotides (polyT6), 7 T nucleotides (polyT7), 8 T nucleotides (polyT8), 9 T nucleotides (polyT9), 10 T nucleotides (polyTIO), 11 T nucleotides (polyTl 1), or 12 T nucleotides (polyT12). In some embodiments, the polyT comprises 8 T nucleotides (polyT8).

[0208] Exemplary RNA polymerase terminator sequences are provided in Table 13 below.Table 13. Exemplary RNA Polymerase Terminator SequencesTerminator Name Nucleic Acid Sequence SEQID NO CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC 42 CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCAC TCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCbGHpA ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG G<1GTGGGGTGGGGCAGGACAGCAAG<}GGGAGGAT TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGT GGGCTCTATGGpolyT8 TTTTTTTT

[0209] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase terminator, wherein the RNA polymerase terminator is a polyA signal. In someATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069embodiments, the poly A signal is located 3’ of a 3’ circularizing element (e.g., a 3’ HIPK3 intronic element or a tRNA 3’ trailer). In some embodiments, the poly A signal comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, the poly A signal comprises a polynucleotide sequence of SEQ ID NO: 42 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the poly A signal comprises a polynucleotide sequence of SEQ ID NO: 42. In some embodiments, the poly A signal consists of a polynucleotide sequence of SEQ ID NO: 42.

[0210] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase terminator, wherein the RNA polymerase terminator is polyT, In some embodiments, the polyT is located 3’ of the tRNA 3’ trailer. In some embodiments, the polyT comprises a polynucleotide sequence of TTTTTTTT with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the polyT comprises a polynucleotide sequence of TTTTTTTT, In some embodiments, the polyT consists of a polynucleotide sequence of TTTTTTTT, In some embodiments, the recombinant nucleic acid molecule comprises an Hl. Ml 1 promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises an Hl promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a 7SK promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a U6 promoter, or any variant thereof, and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a U6+ 1 promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a U6+27 promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a 06+27 promoter and a poly A signal.

[0211] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) a promoter; (b) a tR A 5’ leader; (c) a tRNA 5’ exonic element; (d) a cargo comprising a tough decoy; (e) a tRNA 3’ intronic element; (f) a tRNA 3’ exonic element; (g) a tRNA 3’ trailer; and (h) a RNA polymerase terminator. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3 ’, (a) an RNA polymerase III promoter; (b) a tRNA 5’ leader; (c) a tRNA 5’ exonic element; (d) a cargoATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069comprising a tough decoy; (e) a tRNA 3’ intronic element; (f) a tRNA 3’ exonic element; (g) a tRNA 3’ trailer; and (h) a RNA polymerase III terminator. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) a U6+27 promoter; (b) a tRNA 5’ leader; (c) a tRNA 5’ exonic element; (d) a cargo comprising a tough decoy; (e) a tRNA 3 ’ intronic element; (f) a tRNA 3 ’ exonic element; (g) a tRNA 3 ’ trailer; and (h) a polyT. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) an RNA polymerase III promoter; (b) a tRNA 5’ leader; (c) a tRNA 5’ exonic element; (d) a cargo comprising a tough decoy; (e) a tRNA 3’ intronic element; (f) a tRNA 3’ exonic element; (g) a tRNA 3’ trailer; and (h) a RNA polymerase II terminator. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) a U6+27 promoter; (b) a tRNA 5’ leader; (c) a tRNA 5’ exonic element; (d) a cargo comprising a tough decoy; (e) a tRNA 3’ intronic element; (f) a tRNA 3’ exonic element; (g) a tRNA 3’ trailer; and (h) a poly A signal.

[0212] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) a promoter; (b) a 5’ intronic element; (c) a cargo comprising a tough decoy; (d) a 3’ intronic element; and (e) an RNA polymerase terminator. In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises, from 5’ to 3’, (a) an RN / X polymerase II promoter; (b) a 5’ intronic element; (c) a cargo comprising a tough decoy; (d) a 3’ intronic element; and (e) an RNA polymerase II terminator. In some embodiments, the recombinant nucleic acid molecule encoding a circular RN / X comprises, from 5’ to 3’, (a) a CB / X promoter; (b) a 5’ HIPK3 intronic element; (c) a cargo comprising a tough decoy; (d) a 3’ IIIPK3 intronic element; and (e) a bGUpA signal.

[0213] Exemplary linear and circular tough decoy expression cassettes are provided in Table 14 below.Table 14. Exemplary DNA Sequences for Tough Decoy Expression Cassettes Description Nucleotide Sequence SEQIDNOATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069U6-i-27-Linear TuDNhsIGagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga 43 gagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtg acgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatgg actatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtg gaaaggacgaaacaccgtgctcgcttcggcagcacatatactaGCTAGC GCTAGGATCATC: AACCTACCTGCACTGTAACAA AGCACTTTGCAAGTATTCTGGTCACAGAATACA ACCTACCTGCACTGTAACAAAGCACTTTGCAAG ATGATCCTAGCGCTAGCTTTTTTTT CBA-HIPK3-circTuD-sp2 cgtacataacttacggtaaatggcccgcctggctgaccgcccaacgaccccc 44 gcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttc cattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtaca tctactcgaggccacgttctgctcactctccccatctcccccccctccccacccc caatttgtatttattattttttaattatttgtgcagcgatgggggcggggggggg gggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcg gggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccga aagttcctttatggcgaggcggcggcggcggcggccctataaaaagcgaag cgcgcggcgggcgggagcgggatcagccaccgcggtggcggcctagagtc gacgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttat ttcagg tcccggatccggtggtggtgcaaatcaaagaactgctcctcag tggat gttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcgga attgtacccgcggccgatccaccggtccggaattcccgggatatcgtcgaccca cgcgtccgggccccacgctgcgcacccgcgggtttgctACCGGTTTA ATTAAGCCTCAGCCTCTCAAAGTGCTACICJATTAC AGGGATCTATACTTTTCTTTTGAGGGAAAATGTT GGCACCGTTTCTAGGGCATATTGGCCATTTCAGC TTCTCAGTAAATATTTGTTAAGTAATTAAATGCA CTTGATTCTTTATTCTTAGCCTTTTAACGCAATAC TCAGAATAGCTGAAGCACCAATTAACTGAAATG GAGATATTATAAAGATAGTTATC1TCTCCAAGG GAAAAAAICATCITCAIGGAAATrAATTACTTIT TTACAAAT TGTGAATTTGACCC TTAAGAGT TTTCTiCCTGATATriAAAAriGAAAAAAAAAi rGTIG ACA TTAATAT TTCTTCI TTCCT rITTlTTC IITTCC TITT rriTiTT TTHTTGC AGGTTGAAGCTTAACC CCTACCACGCAGTAGTATAGGTAGACATGAACT CT TAAGGACGGCGC TAGGATC ATC AACC TACCT GCACTGTAACAAAGCACTTTGCAAGTATTCrGGT CACAGAATACAACCTACCTGCACTGTAACAAAG CACTTTGCAAGAIGATCCTAGCGCCGTCGACAC CATCTGCCTGACACCACTGTAGATTTCGGATGGT CGGGGTTAGCGCTCAGGTAGGTAACAACTCCAT ACTTTTTGGTTGTTTATTAATGTGAAATTTCTGCTAAATGAAATACTTTTGTGTGTGTTTGTGGTAGAAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GAGACCACTTCA(]TTAAATAAGGAAATCAAGAG AGCJATCAATTTAGGTTCGTTTTAAAGAGATTAA AAAAAATCAAGACATAAAATCTACCCAAGCAGG ATAGAAATCTCCACTGCAAAGTTCCATGCCAAA GACATCTGGTTATTTTTATTTTTAATGGAAGACT TGAAGGAATGATAGGTGATTAATAATGATCAAA CAGAAGTCTTTAAATGTTGGAAAGTATTTACATT AATCTTTGTATATATCATTGGGCATTTTAGCACT TGAGAGAAATAGTTTATTAAAGATATAATCAAT CATATGTAACTGAACATTTAGAAAAATTATATA CAGGTTTGAGTAGCCCTTATCTGAAACTTTTGGG GCC AGAAGTGT TTTGGAT TCCAGATTT TTCCGGA TTTIGGAATATITGCACTGCCAACTAGTTAAGCA CCCCCAAATITGAAAATrCGTTTCCTTTGAGTGT CATGTCAATGCCCAAAAAG1TTCAGATATTTGG ATHGAGATGCTCAACCTGTATAAGGATTCAGA AAGITATTCTGATTAAIGATITTAAGATTCAGAT ATACAA TAATCCC AGC AAC TTGGGAGGCTGAGG CAGGAGAATCACTTGAACCCAGGAGATGGAGGT TGCAGTGAGCCGAGATCATGCCATTGCACTCCA ATCGATCTCGAGGCTAGCctgtgccttctagttgccagccatct gttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgt cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctatt ctggggggtggggtggggcaggacagcaagggggaggattgggaagacaa tagcaggcatgctggggatgcggtgggctctatggU6-i-27-TncY-circTuD- gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga 45 spl gagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtg acgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatgg actatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtg gaaaggacgaaacaccgtgctcgcttcggcagcacatatactaACCGGT TTAATTAACACGTGTCTAAAAAGGACAGCGTTC CGTGTCCTTCGATAGCTCAGTTGGTA(]AGCGGA GGACIGIAGGC’GGCCGCAACC'CCTACCACATAC TGrTGAAGAAAIArACGGGTAGAGCTCGACGGC1GCTAGGATCATCAACCTACCTGCACTGTAACAA AGCACrTTGCAAGTATTCTGGTCACAGAATACA ACCTACCTGCACTGTAACAAAGCACTTTGCAAG ATGATCCTAGCGCCGTCTACGCGTTCCGACTACA CAAAICAGCGATrCCGGTTGGTCGGGGTTGIGG CCGCGGAGGTATCCrrAGGTCGCTGGTTCGAATC CGGCTCGGAGGACGGTAGrTTTGACCTACCAAGGAATTCATCGATCTCGAGGCTAGCITTrnrrATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069U6-i-27-TricY-circTuDwGagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga 46 gagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtg acgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatgg actatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtg gaaaggacgaaacaccgtgctcgcttcggcagcacatatactaACCGGT TTAATTAACACGTGTCTAAAAAGGACAGCGTTC CGTGTCCTTCGATAGCTCAGTTGGTAGAGCGGA GGACTGTAGGCGGCCGCTAGGATCATCAACCTA CCTGCACTGTAACAAAGCACTTTGCAAGTATTCT GGTCACAGAATACAACCTACCTGCACTGTAACA AAGCACTTTGCAAGATGGTTCTAGTGGCCGCGG AGGTATCCTTAGGTCGCTGGTTCGAATCCGGCTC GGAGGACGGTAGTITTGACCTACCAAGGAATTC ATCGATCTCGAGGCTAGCi rriTITTCB A HIPK3 - AMP255- cgtacataacttacggtaaatggcccgcctggctgaccgcccaacgaccccc 109 Alu_circTuDW2_bGHpA gcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttc cattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtaca tctactcgaggccacgttctgcttcactctccccatctcccccccctccccacccc caatttgtattatttatttttaattattttgtgcagcgatgggggcggggggggg gggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcg gggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccga aagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaag cgcgcggcgggcgggagcgggatcagccaccgcggtggcggcctagagtc gacgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttat ttcagg tcccggatccggtggtggtgcaaatcaaagaactgctcctcag tggat gttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcgga attgtacccgcggccgatccaccggtccggaattcccgggatatcgtcgaccca cgcgtccgggccccacgctgcgcacccgcgggtttgctACCGGITTA ATTAAGCCTCAGCCTCTCAAAGTGCTAGGATTAC AGGGATCTA TACTTTTC ITTTGAGGGAAAATGTT GGC’ACCGITTCTAGGGCATAITGGC'CATTrCAGC TICTCAGTAAATATITGTTAAGTAATTAAATGCA CTTGAnriTTAnrrTAGCCrrTTAACGCAATAC TCAGAATAGCTGAAGCACCAATTAACIGAAATG GAGATATTATAAAGATAGTTATC1TCTCCAAGG GAAAAAATCATCITCATGGAAA1TAATTACTTIT TTACAAAT TGTGAATTTGCCTCAGCCTC TCAAAG TGCTAGGATTAGACCCTTAAGAGTTTI'CTTCCTG ATATTTAAAATTGAAAAAAAAATTGTTGACATT AATATTTCTTCTTTCCTTTTTTTTCTTTTCCTTTTT TTTTTTTTTTTTGCAGGTTGAAGCTTGCAGGACG GTCATCAACCTACCTGCACTGTAACAAAGCACTT TGCAAGACTGAAGAAGACTTCAGTCAACCTACCTGCACTGTAACAAAGCACTTTGCAAGATGGCTGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069TCCTGCAGGCCAGGTAGGTAACAACTCCATACT TTTTGGTTGTTTATTAATGTGAAATTTCTGCTAA ATGAAATACTTTTGTGTGTGTTTGTGGTAGAAGA GACCACTTCAGTTAAATAAGGAAATCAAGAGAG GATCAATTTAGGTTCGTTTTAAAGAGATTAAAA AAAATCAAGACATAAAATCTACCCAAGCAGGAT AGAAATCTCCACTGCAAAGTTCCATGCCAAAGA CATCTGGTTATTTTTATTTTTAATCiCjAAGACTTG AAGGAATGATAGGTGATTAATAATGATCAAACA GAAGTCTTTAAATGTTGGAAAGTATTTACATTAA TCTTTGTATATATCATTGCKJCATTTTAGCACTTG AGAGAAATAGTTTATTAAAGATATAATCAATCA TATGTAACTGAACATTTATAATCCCAGCAACTTG GGAGGCTGAGGCGAAAAAT TATATACAGGTT TG AGTAGC’CCTTATCTGAAACTTTTGGGGCCAGAA GTGlYTTGGATICCAGAITlTTCCGGArrTTGGA ATATITGCACTGCCAACTAGTTAAGCACCCCCAA ATHGAAAATTCGTTTCCTITGAGTGTCATGTCA ATGCCCAAAAAGTTTCAGATATTTGGATTTGAG ATGCTC AACCTG TATAAGGATTC AGAAAGTTAT T CTGAT TAATGATTT TAAGAT TCAGA TA TAC AA TA ATCCCAGCAACTTGGGAGGCTGAGGCAGGAGAA TCACTTGAACCCAGGAGATGGAGGTTGCAGTGA GCCGAGATCATGCCATTGCACTCCAATCGATCTC GAGGCTAGCctgtgccttctagttgccagccatctgttgtttgcccctccc ccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaat gaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt ggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctg gggatgcggtgggctctatggAAV146 TTGCiCCACTCCCTCTCTGCGCGCTCGCTCGCTCA 122 CTGAGGCCGGGCGACCAAAGGTCGCCCGAGGCC(U6-t-27__TncY_circTuDwC GGGC TTTGCCCC jGGCGGCC TO AGTGAGC G A GC _polyT8) GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCC rCAATTGcAATAAAAGATCTTIA TTTTCA TTAGATCTGTGTGTTGGHT TTTGTGTGG GATCCctagcttttctcttTAGtcaaccccacaGCcctttggcaccTAG aagtggTAGgTGAgTAACTAcTCAcTTActctctttagctGAg cttattccaggggtgtgtttGCtGCagTGAcacacaagagtgaggttgctT CAGAgttgTGAgTAGgTAATCAcTTAcCTAccaaagccttg gtgttgattgcctttgctcagtatcttcagcagtgtcCTAttgaagatcTGAta aaattagtgaTAGaagtaaTAGaatttgcaaaaacTAGtgttgTAGT GAagtcagTAGaaaattgtgacaaatcacttTCAaccctttttggagaTT AattTGATTAagttgcaactcttGCtgaaacctTGAgtgaaTAGgT AGaTAGT AGtgcaaaacaagaacT AGagagaaTAGaT AGcttc ttgcaacacaaagTGAacaacccaaacctccccGAattggtgagaccagaggttgTAGtgT AGtgccT AGcttttcT GAaccT AGaagaga CT AATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069ttttgaaaaaatacttatT G AaaatgcTT AaagaT C AccttacttttT GA ccc / \AgaactcctttctttgctaaaaggtataaagTAGctttacagaTAG ttgcTTAgTAGTAGataaagTAGcTAGgTAGttgGGATCC tgagagacacaaaaaattccaacacactattgcaatgaaaataaattcctttattA TTT A A AT gagggcctatttcccatgattccttcatatttgcatatacgatac aaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagta caaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat gtttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgattcttggctt tatatatcttgtggaaaggacgaaacaccgtgctcgcttcggcagcacatatact aACCGGTTTAATTAACACGTGTCTAAAAAGGACA GCGTTCCGTGTCCTTCGATAGCTCAGTTGGTAGA GCGGAGGACTGTAGGCGGCCGCTAGGATCATCA ACCTACCTGCAC TGTAACAAAGC ACTT TGC AAG TATTCTGGTCACAGAATACAACCTACCTGCACTG TAACAAAGCACTTTGCAAGATGGTTCTAGTGGC CGCGGAGGTATCC TTAGGTCGCTGGT TCGAATCC GGCTCGGAGGACGGTAGTTTTGACCTACCAAGG AATTCATCGATCTCGAGGC'TAGCTTTrniTGTTT AAACATGAGGTACCCCACTCCCTCTCTGCGCGCT CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGA AAV174 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA 123 CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC(U6+27 TRY-GTA5- CGGGCTTTGCCCGGGCGCICCTCAGTGAGCGAGC1 circT uDw_polyT8) GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCTCAATTGcAATAAAAGATCTTT / X TTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGG GATCCctagcttttctcttTAGtcaaccccacaGCcctttggcaccTAG aagtggTAGgTGAgTAACTAcTCAcTTActctctttagctGAg cttattccagggg tgtgtttGC tGCagT G Acacacaagag tgaggttgctT C / XGAgttgTGAgTAGgTAATCAcTTAcCTAccaaagccttg gtgttgattgcctttgctcagtatcttcagcagtgtcCTAttgaagatcTGAta aaattagtgaTAGaagtaaTAGaatttgcaaaaacTAGtgttgTAGT GAagtcagTAGaaaattgtgacaaatcacttTCAaccctttttggagaTT AattTGATTAagttgcaactcttGCtgaaacctTGAgtgaaTAGgT AGaTAGT AGtgcaaaacaagaacT AGagagaal AGaT AGcttc ttgcaacacaaagTGAacaacccaaacctccccGAattggtgagaccaga ggttgTAGtgTAGtgccTAGcttttcTGAaccTAGaagagaCTA ttttgaaaaaatacttatTGAaaattgcTTAaagaTCAccttacttttTGA cccAAgaactccttttctttgctaaaaggtataaagTAGcttttacagaTAG ttgcTTAgTAGTAGataaagTAGcTAGgTAGttgGGATCC tgagagacacaaaaaattccaacacactatgcaatgaaaataaatttcctttatt ATTTAAATgagggcctatttcccatgattccttcatatttgcatatacgatac aaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069gtttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgattcttggctt tatatatcttgtggaaaggacgaaacaccgtgctcgcttcggcagcacatatact aACCGCiTTTAATTAACACGTGTCTGTGCTGAACC TCAGCIGGACGCCGACACACGTACACGTCCCTTC GATAGCTCAGCTGGTAGAGCGGAGGACTGTAGG CGGCCGCTAGGATCATCAACCTACCTGCACTGT AACAAAGCACTTTGCAAGTATTCTGGTCACAGA ATACAACCTACCTGCACTGTAACAAAGCACTTT GCAAGATGGTTCTAGTGGGCGCGGagacATCC: TT / X GGTCGCTGGTTCGATTCCGGCTCGAAGGAGACA AGTGCGGTTTTTTTCTCCAGCTCCCGATGACTT / \ TGGAAITCATCGATCTCGAGGCTAGC'Trn nTT GTHAAACATGAGGTACCCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA GGTCGCCCGACGCCCGGGC1TTGCCCGGGCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA A A VI 82 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA 124 CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC(CBAJHPK3-AMP255- CGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC Alu circT uDw 2_bGHp A) GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCTCAATTGcAATAAAAGATCTTTA TTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGG GATCCctagcttttctcttTAGtcaaccccacaGCcctttggcaccTAG aagtggTAGgTGAgTAACTAcTCAcTTActctctttagctGAg cttattccagggg tgtgtttGC tGCagT G Acacacaagag tgaggttgctT C / XGAgttgTGAgTAGgTAATCAcTTAcCTAccaaagccttg gtgttgattgccttgctcagtatctcagcagtgtcCTAttgaagatcTGAta aaattagtgaTAGaagtaaTAGaatttgcaaaaacTAGtgttgTAGT GAagtcagTAGaaaattgtgacaaatcacttTCAaccctttttggagaTT / XattTGATTAagtgcaactcttCICtgaaacctTGAgtgaaTAGgTA GaTAGT AGtgcaaaacaagaacT AGagagaaT A GaT A Gcttc ttgcaacacaaagTGAacaacccaaacctccccGAattggtgagaccaga ggttglAGtgTAGtgccTAGcttttcTGAaccTAGaagagaCTA ttttgaaaaaatacttatTGAaaattgcTIAaagaTCAccttacttttTGA cccAAgaactccttttctttgctaaaaggtataaagTAGcttttacagaTAG ttgcI'TAgTAGTAGataaagTAGcTAGgl'AGtgGGAICC tgagagacacaaaaaattccaacacactatgcaatgaaaataaatttcctttatt ATI'TAAATcgttacataacttacggtaaatggcccgcctggctgaccgcc caacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcca atagggactttccattgacgtcaatgggtggagtattacggtaaactgcccactt ggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgac ggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac ttggcagtacatctactcgaggccacgttctgcttcactctccccatctccccccc ctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggc ggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069cgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctata aaaagcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggc ggcctagagtcgacgaggaactgaaaaaccagaaagttaactggtaagtttagt ctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgct cctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaa aagctgcggaattgtacccgcggccgatccaccggtccggaattcccgggata tcgtcgacccacgcgtccgggccccacgctgcgcacccgcgggtttgctAC CGGTTTAATTAAGCCTCAGCC'TCTCAAAGTGCT / \ GGATTACAGGGATCTATACTTTTCTTTTGAGGGA AAATGTTGGCACCGTTTCTAGGGCATATTGGCCA TTTCAGCTTCTCAGTAAATATTTGTTAAGTAATT AAAIGt’ACTTGATTCTITATTCTIAGCCTTITAACGC AA TACTCAGAATAGC TGAAGCACCAATTAA CTGAAA TGGAGATATTATAAAGATAGTTATCT TC TCCAAGGGAAAAAATCArCTTCATGGAAATIAA TTACTTrTTTACAAATTGTGAATITGCCTCAGCC TCTCAAAGTGCTAGGATTAGACCCTTAAGAGTIT TCTTCC IGATATT IAAAAT IGAAAAAAAAATTGT TGACATTAAI ATTTCTTCTTTCCTTTTTTTrCTTTTCCITT ITTI’rTTrTTrrTTGCAGGTTGAAGCTTGC AGGACGGTCATCAACCTACCTGCACTGTAACAA AGCACTTTGCAAGACTGAAGAAGACTTCAGTCA ACCTACCTGCACTGTAACAAAGCACTTTGCAAG ATGGCTGTCCTGCAGGCCAGGTAGGTAACAACT CCATACTTTTTGGTTGTTTATTAATGTGAAATTTC TGCTAAATGAAATACTTTTGTGTGTGTTTGTGGT AGAAGAGACCACTTCAGTTAAATAAGGAAATCA AGAGAGGATCAATTTAGGTTCGTTTTAAAGAGA TTAAAAAAAATCAAGACATAAAATCTACCCAAG CAGGATAGAAATCTCCACTGCAAAGTTCCATGC CAAAGACATCTGGTTATTTTTATTTTTAATGGAA GACTTGAAGGAATGATAGGTGATTAATAATGAT CAAACAGAAGTCTTTAAATGTTGGAAAGTATTT ACATTAATCTTTGTATATATCATTGGGCATTTTA GCACTTGAGAGAAATAGTTTATTAAAGATATAA TCAATCATATGTAACTGAACATTTATAATCCCAG CAACTTGGGAGGCTGAGGCGAAAAATTATATAC AGGTTTGAGTAGCCCTTATCTGAAACTTTTGGGG CCAGAAGTGTTTTGGATTCCAGATTTTTCCGGAT TTTGGAATATTTGCACTGCCAACTAGTTAAGCAC CCCCAAATTTGAAAATTCGTTTCCTTTGAGTGTC ATGTCAATGCCCAAAAAGTTTCAGATATTTGGAT TTGAGATGCTCAACCTGTATAAGGATTCAGAA / X GTTATTCTGATTAATGATTTTAAGATTCAGATAT ACAATAATCCCAGCAACTTGCIGAGGCTGAGGCAGGAGAATC: ACTTGAACCCAGGAGATGGAGGTTGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CA<1T<1A< KXX1A<1ATCATGCCATT<1CACTCCAAT CGATCTCGACKK / TAGCctgtgccttctagttgccagccatctgttg tttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctt cctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctgg ggggtggggtggggcaggacagcaagggggaggattgggaagacaatagc aggcatgctggggatgcggtgggctctatggGTTTAAACATGAGG TACCA<1GAACCCCTAGTGATG<1AGTTG<1CCACT CCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG< KX1ACCAAAGGTC< KXXX1ACGCCC< K1GCTTTG CCCGGGC < K1CCTCA<1T<1A< KX1A<1CGAGC GCGC / XG AG A G( JGAG T GGC C A AAAV186 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA 125 CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC(U6+27 TRY-GTA5- CGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC1 circT uDw_bGHp A) GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCTCAATTGcAATAAAAGATCTTTA TTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGG GATCCctagcttttctcttTA GtcaaccccacaGC ccttggcaccT A G aagtggT AGgTGAgT A ACTAcT CAcTT ActctctttagctGAg cttattccaggggtgtgtttGCtGCagTG A cacacaagagtgaggttgctT CAGAgttgTGAgTAGgTAATCAcTTAcCTAccaaagcctg gtgttgattgcctttgctcagtatcttcagcagtgtcCTAttgaagatcTGAta aaattagtgaTAGaagtaaTAGaatttgcaaaaacTAGtgttgTAGT GAagtcagTAGaaaattgtgacaaatcacttTCAaccctttttggagaTT / XattTGATTAagttgcaactctt< KXgaaacctTGAgtgaaTAGgTA <laTAGT AGtgcaaaacaagaacT AGagagaaT A <laT AGcttc ttgcaacacaaagT<lAacaacccaaacctccccGAattggtgagaccaga ggttgTAGtgT AGtgccT A GcttttcT G / XaccT AGaagagaCT A tttgaaaaaatacttatT <1 AaaatgcTT AaagaT C AccttactttT GA ccc / XAgaactcctttctttgctaaaaggtataaagTAGctttacagaTAG ttgcTT AgT AGT AGataaagT AGcTAGgT AGttgGGATCC tgagagacacaaaaaattccaacacactattgcaatgaaaataaattccttatt ATI'TAAATgagggcctatttcccatgattccttcatatttgcatatacgatac aaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagta caaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat gttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctt tatatatcttgtggaaaggacgaaacaccgtgctcgcttcggcagcacatatact aACCGGTTTAATTAACACGTGTCTGTGCTGAACC TCAGGGGACGCCGACACACGTACACGTCCCTTC GATAGCTCAGCTGGTAGAGCGGAGGACTGTAGG CGGCCGCTAGGATCATCAACCTACCTGCACTGT AACAAAGCACTTTGCAAGTATTCTGGTCACAGA ATACAACCTACCTGCACTGTAACAAAGCACTTT GCAAGATGGTTCTAGTGGCCGCGGagacATCCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGGAGACAAGTGCGGTTTTTTTCTCCAGCTCCCGATGACTTAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069T GGA ATT C ATCG AT CT CGAGGCT AGCctgtgccttctag t tgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgc cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtag gtgtcattctattctggggggtggggtggggcaggacagcaagggggaggatt gggaagacaatagcaggcatgctggggatgcggtgggctctatggGTTTA AACATGAGGTACCCCACTCCCTCTCTGCGCGCTC GCTCGCTCACTGAGGCCGGGCGACCAAAGGTCG CCCGACGCCC: GGGCTTTGCCCGGGCGGCCTCAG TGAGCGAGCGAGCGCGCAGAGAGGGA AAVi98 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA 126 CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC(U 6+ 1 _T ricY_circT uDw__ CGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC polyT8) GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCTCAATTGcAATAAAAGATCTTTA TTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGG GATCCctagctttctcttTAGtcaaccccacaGCccttggcaccTAG aagtggT AGgTGAgT A ACTAcT CAcTT ActctctttagctGAg cttattccaggggtgtgtttGCtGCagTGAcacacaagagtgaggttgctT CAGAgttgTGAgTAGgTAATCAcTTAcCTAccaaagcctg gtgtgattgcctttgctcagtatcttcagcagtgtcCT AttgaagatcT G Ata aaattagtgaTAGaagtaaTAGaattgcaaaaacTAGtgttgTAGT GAagtcagTAGaaaattgtgacaaatcacttTCAaccctttttggagaTT / XattTGATTAagttgcaactcttClCtgaaacctTGAgtgaaTAGgTA GaTAGT AG tgcaaaacaagaacT AGagagaaT A GaT A Gcttc ttgcaacacaaagTGAacaacccaaacctccccGAattggtgagaccaga ggttgTA GtgT AGtgccT A GcttttcT G / XaccT AGaagagaCT A ttttgaaaaaatacttatT G AaaatgcTT AaagaT CAccttacttttT GA ccc / XAgaactcctttctttgctaaaaggtataaagTAGctttacagaTAG ttgcTT AgT AGT AGataaagT AGcTAGgT AGttgGGATCC tgagagacacaaaaaattccaacacactattgcaatgaaaataaatttccttattA TTT A A AT gagggcctatttcccatgattccttcatatttgcatatacgatac aaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagta caaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattat gttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctt tatatatcttgtggaaaggacgaaacaccgACCGGTTTAATTAAC ACGTGTCTAAAAAGGACAGC'GTTCCGTGTCCITC GATAGC TCAGTTGGT AGAGCGGAGGACTGTAGG CGGCCGC'TAGGATCATCAACCTACCTGCACTGT AACAAAGCACTTTGCAAGTATTCTGGTCACAGA ATACAACCTACCTGCACTGTAACAAAGCACTTT GC'AAGATGGTICTAGTGGCCGC'GGAGGTATCCr TAGGTCGCTGGTTCGAATCCGGCTCGGAGGACG GTAGTT TTGACCTACCAAGGAATTCATCGATC TC GAGGCTAGCTTTTTTTTGTTTAAACATGAGGTAC CCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GCTTTGCCCGCIGCGGCCTCAGTGAGCGAGCGAG C GC GCA GAG AGGG / XAAVi99 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA 127 CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCC(U6+27 TncY circTuDwCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC polyT8) GAGCGCGCAGAGAGGGAGTGGCCAACTCCATCA CTAGGGGTTCCTCAATTGcAATAAAAGATCTTTA TTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGG GATCCctagcttttctcttTAGtcaaccccacaGCcctttggcaccTAG aagtggTAGgTGAgTAACTAcTCAcTTActctctttagctGAg cttattccaggggtgtgtttGCtGCagTGAcaca caagagtgaggttgctTC A G AgttgT G AgT AGggT C Actca gcctaccT G Aagagtgttga ttgccttgctcagtatctcagcagtgtcCTAttgaagatcTGAtaaaattag tgaT A GaagtaaT A GaatttgcaaaaacT A GtgtgT AGTGAagtc agTAGaaaattgtgacaaatcacttTCAacccttttggagaTTAattTG ATTAagttgcaactcttGCtgaaacctTGAgtgaaTAGgTAGaTAGT A GtgcaaaacaagaacT A Ga gagaaT AGaT AGcttcttgcaaca caaagT G Aacaacccaaacctcccc A AatggtgagaccagaggtgT A GtgTAGtgccTAGctttcTGAaccTAGaagagaCTAtttgaaaa aatacttatTGAaaattgcTTAaagaTCAccttacttttTGAcccAAg aactccttttcttgctaaaaggtataaagT AGctttacagaT AGttgcTT A gT AGT A GataaagT AGcT AGgT AGttgccaaa gctGCT GAa acttGAggTGAaagggaaggcttGCtT / XGccaaacagagactcaagt gtgcTTAtctccaaaaatttggagaaagagctttcaaagcTGAggcagtag ctGCcT AGagccagagatttcccaaagT AGagtttgcagaagtttcTT A gttagtgacagatcttaccaaagtccacaC jAgaT A GT AC IccTGAgag atT AGcttgaT AGtgT AGT GAacagggG Agaccttgccaagtatat TAGtgaaaatcaagattAAatctccagtaaaT / XGaaggaTAGTAGt gaaaaacctTAGttggaaaaatccccT AGCT Aggc A Aaagtggaaa TAGT GAagT GAcT AGT A GacttgccttCTA tagT AGT A Ga ttttgttgaaagtaaggTGAtttgcaaaaactTGATAGaggcaaaggT GAtcttcTAGggcTGAtttttgtTGAaatTGAcaagaaggTCAcT / XGattactTAGtGCtgTAGTAGTAGagacttgccaagaCTAT / XGaaaccactctagagaagtgT / XGtgcACTAGcagatcctcTGAa TAGctTGAcTTAagtgttGCTGAaatttaaacctcttgtggaagagc ctcagaatttaatcaaal'TATTTtgtgagctttttgagcagcttggagagtac aaattcTTAaTAGGCctattagttGCttacaccaagaaagtaccccaagt gtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaa TAGttgtaaaTCAcTAGaagcaaaaagaTAGccTAGtgcagaag actatctatcACtggtcTAGaaccagttTAGtgtgttgcTGAagaaaa GCccagtaagtgacagagtcaccaaTAGTAGcacagaatccttggtgaa caggACaccTGActtttcagctTAGgaagtGCTGAaaaTCAaG GttcccaaagagtttaTAGTAGaaaCTAtTTActtccTGATTAat atTGATTAactttTAGagaaggagagacaaatcaagaaaTTAaTAGcacttgttgagctGCtgaaacacaagccTTAggcaacaaaagagcaaTAGaaagTAGttTGAgTGAatttGCTTActtttgtagagaagtgTAATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GcaaggT AGaG AataaggagacT AGcttgc ACaggagggtaaaaa acttgttgTAGcaagtcaagTAGccttaggcttataaTCAcaCTAttaa aaAT cgC ActAaacCcT cTTAgAcTatataagagaaagaaaT AG aagatcaaaaggttattCTATAGtttttctttttGCttggtgtaaagccaaca ccTAGtctaaaaaaTC / AaaatttctttaatCT / XtttgcctcttttctTAGtg cttcaattaataaaaaTAGgaaagaatctaatagagtggtacagccTAGtta tttttcaaagT / XGtgttgctatcT / XGaaaattTAGtaggttT / XGtggaag ttccagtgttctctcttattccacttGAgtagaggattctagttctgtgccactat TgtggatAggGggaggAAataaGAGatA / XGGTgtTaTAGaata GcA aA Gc Aag AGAggaaga A AtagTG tg A AGCC TtttaGga aTAgaGCcTtactTGagG / WtattagGgctGatta / UACtttTT AAcCTaGC’agaTAGttaTtatcaggggtgctTgtgaaAactacttttT TgaTAAatacaacagtTGAtTtatAAatttTAGtgtGTGAgaTC AagGaaaAGCaAtaaGtAtACaaTAGtGtgaAtagAaacTaT AGaACACtagGCtatTTTGAacTaaAtaGgAGGttaagaaGa GtaaGCaGGataaAGtaGcccAGGTGAaacaaagtTCAGCtT cTTTtgAtagTfgAccttgtagGaAGC'TAAaTcaaggtGGGtG caAaaaaagcAaTAGttAATAGtaagaaattgagTGAGAatTata TAGggtaTAGAaGcAgGGACTAGtAttTGtaTAGGCcca aAaGcAgcTACAaagttTTGAgAcaAaattTTGAgaaGCtct TCAatctAcAagacTCAactTAAAttagtttctACTCAcTCAag aGAgTgCTAttCTAtttaTCAcAgAcactttaaaaaTaGCCAA gagtct AgacAaGC AAca AaT a caacTtaGAAT attT ggtAacT a gGtt Aa AT cAgagtttT GCttT AAaacT C AGCtt AattAgagttta TtctaTtTAGtgGtcAAgCtAgaaTAGTGATtgtgTggCaaA GGcaacagTggTtgAGcaaatTGAgccagacTtAggaATAGAT C Acactcta ctTaTaataT aatt AT AGGgGaa AGC AgtTgtT CA AGttcactcTtT ttag GgTT GAGa Tea ATC AcT A Atca aTac aAcTAcaaTCAGtGCtatTagAgtcTaAttctattttaTAGTagG GaGC cAcagtcTaggaataT AGa GgGaaccctT aAggGGgcTA GatTGAaTcTAGtAAgacctaTAGGAGaGtccttaAaaAgTt GGtgTAAcCTAtTtgcTAGTtgctaggagagaatcAacAGTAG tgGcaaaagT agtaTaagaattatT GAaaTtaT c AgeettettT C AAG Tgaa T C ActGCaat AaggttT aT etT gcaGc AC A A AacctttT G AcTTGAATctctatTtgtGGAgGttaCTAGtAtcagAGagtG tccCaagtgTaaGTtaagtGCaaT a AtT G Aat Agcct A CtAG A T CTTAGataaAagaGCaagttgcACTtaGttGAaTAtACcTcctT AAcTcagCTAATAAgTatcTATaggTaagTGAgGCtataaaa aGGATCCtgagagacacaaaaaattccaacacactattgcaatgaaaataa atttcctttattATTTAAATgagggcctattcccatgatccttcatattgca tatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaa agatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcag ttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcg atttcttggctttatatatcttgtggaaaggacgaaacaccgtgctcgcttcggcag cacatatactaACCGGTTTAATTA / XCACGTGTCTAAA / XAGGACAGCGTTCCGTGTCCTTC: GATAGCTCAGTTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069GGTAGAGCGGAGGACTGTAGGCGGCCGCTAGGA TCATCAACCTACCTGCACTGTAACAAAGCACTTT GCAAGTATTCTGGTCACAGAATACAACCTACCT GCACTGTAACAAAGCACTTTGCAAGATGGTTCT AGTGGCCGCGGAGGTATGGTTAGGTCGCTGGTT CGAATCCGGCTCGGAGGACGGTAGTTTTGACCT ACCAAGGAATTCATCGATCTCGAGGCTAGCTTTT TTTTGTTTAAACATGAGGTACCAGGAACCCCTAG TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTGG CTCGCTCACTGAGGCCGGCJCGACCAAAGGTCGC CCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA

[0214] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 43-47, 101-107, and 109-112. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of any one of SEQ ID NOs: 43-47, 101-107, and 109-112 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of any one of SEQ ID NOs: 43-47 and 101-112. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of any one of SEQ ID NOs: 43-47, 101-107, and 109-112. In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0215] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 46 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 46.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 46. In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0216] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 102 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 102, In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 102, In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0217] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 103. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 103 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 103. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 103. In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0218] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 105. In some embodiments, theATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 105 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 105. In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0219] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 109 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 109. In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0220] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 111. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 111 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 111. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 111. In some embodiments, a vector comprises the recombinant nucleic acid molecule. In some embodiments, the vector is a viral vector such as an AAV vector.

[0221] Exemplary RNA sequences for the tough decoys described herein are provided in Table 15 below.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Table 15. Exemplary RNA Sequences of Tough DecoysDescription Nucleotide Sequence SEQ ID NO Linear TuDNheIgugcucgcuucggcagcacauauacuaGCUAGCGCUAGGAU 48CAUCAACCUACCUGCACUGUAACAAAGCACUU UGCAAGUAUUCUGGUCACAGAAUACAACCUAC CUGCACUGUAACAAAGCACUUUGCAAGAUGAU CCUAGCGCUAGCUUUUUUUUcircTuD-sp2 GUUGAAGCUUAACCCCUACCACCUCAGUAGUAU 49AGGUAGACAUGAACUCUUAAGGACGGCGCUAG GAUCAUGAACCUACCUGCAGUGUAAGAAAGCA GUUUGCAAGUAUUCUGGUCACAGAAUACAACC UACCUGCACUGUAACAAAGCACUUUGCAAGAU GAUCCUAGGGCCGUCGACAGCAAUCUGCCUGAG ACCACUGUAGAUUUCGCUAUGGUCGGGGGUAGC GCUCAGcircTuD-spl GCGGCCGCAACCCCUACCACAUACUGUUGAAG 50AAAUAUACGGGUAGAGCUCGACGGCGCUAGGA UCAUCAACCUACCUGCACUGUAACAAAGCACU UUGCAAGUAUUCUGGUCACAGAAUACAACCUA CCUGCACUGUAACAAAGCACUUUGCAAGAUGA UCCUAGCGCCGUCUACGCGUUCCGACUACACA AAUCAGCGAUUCCGGUUGGUCGGGGUUGUGGC CGCGGAGGUcircTuDwGCGGCCGCUAGGAUCAUCAACCUACCUGCACU 51GUAACAAAGCACUUUGCAAGUAUUCUGGUCAC AGAAUACAACCUACCUGCACUGUAACAAAGCA CUUUGCAAGAUGGUUCUAGUGGCCGCGGAGGUcircTuDw-AGAC GCGGCCGCUAGGAUCAUCAACCUACCUGCACU 90GUAACAAAGCACUUUGCAAGUAUUCUGGUCAC AGAAUACAACCUACCUGCACUGUAACAAAGCA CUUUGCAAGAUGGUUCUAGUGGCCGCGGAGACcircTuDw-GGCT GCGGCCGCUAGGAUCAUCAACCUACCUGCACU 91GUAACAAAGCACUUUGCAAGUAUUCUGGUCAC AGAAUACAACCUACCUGCACUGUAACAAAGCA CUUUGCAAGAUGGUUCUAGUGGCCGCGGGGCTcircTuDW2GUUGAAGCUUGCAGGACGGUCAUCAACCUACC 52UGCACUGUAACAAAGCACUUUGCAAGACUGAA GAAGACUUCAGUCAACCUACCUGCACUGUAAC AAAGCACUUUGCAAGAUGGCUGUCCUGCAGGCCAGATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0222] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA is transcribed in a target cell or target tissue. In some embodiments, the circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 48-52, 90, and 91. In some embodiments, the circular RNA comprises a polynucleotide sequence of any one of SEQ ID NOs: 48-52, 90, and 91 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the circular RNA comprises a polynucleotide sequence of any one of SEQ ID NOs: 48-52, 90, and 91. In some embodiments, the circular RNA consists of a polynucleotide sequence of any one of SEQ ID NOs: 48-52, 90, and 91.

[0223] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 51. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 51 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 51. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 51.

[0224] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 52. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 52 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 52. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 52.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0225] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 90. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 90 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 90. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 90.

[0226] In some embodiments, the recombinant nucleic acid molecule encoding a circular RNA comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 91 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of SEQ ID NO: 91.

[0227] Exemplary recombinant nucleic acid molecules, and their corresponding elements, are provided in Table 16 below.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069Table 16. CassettesCassette SEQ ID NOs of Components Plasmid SEQ 5’ 3’Name Scaffold Promoter Cargo Terminator ID ID NO Scaffold ScaffoldPEX190 U6+27 Linear TuDNheT(Positive Control) Linear 43 35 NA 4 NA TTTTTTTT PEX191 U6+27 TuDM,eT(Negative Control) Linear 100 35 NA 64 NA TTTTTTTT PEX332 U6+27 TricY circTuD-spl polyT8 tricRNA 45 35 28 69 29 TTTTTTTT PEX334 CBA_HIPK3_circTuD-sp2_bGHpA Backsplice 44 30 8 70 9 42 PEX350 U6+27_TricY_circTuDw_polyT8 tricRNA 46 35 28 1 29 TTTTTTTT PEX387 U6+27 circTuD'A'-Bulge2 polyT8 Linear 101 35 NA 74 NA TTTTTTTT PEX388 U6+27 TRY-GTA5 - l__circTuDw_polyT8 tricRNA 102 35 449 71 649 TTTTTTTT PEX389 U6+27_TRL-CAA4-l_circTuDw_polyT8 tricRNA 103 35 448 72 648 TTTTTTTT PEX390 U6+27 TRR-TCT 1 - 1 _.circTuDw_polyT8 tricRNA 104 35 447 73 647 TTTTTTTT PEX399 U6+27__TricY__circTuDw-Bulge2_polyT8 tricRNA 105 35 28 74 29 TTTTTTTT CBA_HIPK3-AMP255-Alu_circTuDW2- PEX400 Backsplice 106 30 410 75 609 42 Bulge2 bGHpAPEX409 U6+27__circTuDw_polyT8 Linear 107 35 NA 1 NA TTTTTTTT PEX410 CBA_HIPK3_circTuDW2_bGHpA Backsplice 47 30 8 2 9 42 PEX411 CBA HIPK3-AMP255-Alu circTuDW2bGHpA Backsplice 109 30 410 2 609 42 PEX421 CBA_HIPK3_circTuDW2-Bulge2_bGHpA Backsplice 110 30 8 75 9 42 PTR283 U6+27 TRY-GTA5 - l__circTuD'vV_bGHp A tricRNA 111 35 449 71 649 42 PTR381 U6+1_TricY_circTuD_polyT8 tricRNA 112 36 28 1 29 TTTTTTTTPTR383 U6+27_TricY_circTuDw_polyT8 tricRNA 46 35 28 1 29 TTTTTTTTATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0228] Exemplary AAV vectors, and their corresponding capsids and cassettes, are provided in Table 17 below.Table 17. AAVsPlasmid SEQ ID AAV ID Capsid Name ScaffoldID NOU6+27-Linear TuDNheI-polyT8AAV116 k20 PTR178 Linear 120 (Positive Control)U6+27-Neg Ctrl TuDNheI-polyT8AAV117 k20 PTR180 Linear 121 (Negative Control)AAV146 k20 PTR251 U6 +27_TricY_circTuDw_polyT8 tricRNA 122 U6+27 TRY-GTA5- AAV 174 k20 PTR349 tricRNA 1231_circTuD^w_polyT8CBA_HIPK3-AMP255- AAV 182 k20 PTR361 Backsplice 124 Alu_circTuDW2_bGHpAPTR283 U6+27_TRY-GTA5- AAV186 k20 tricRNA 1251_circTuD^w_bGHpAAAV198 k20 PTR381 U6+l_TricY_circTuDw_polyT8 tricRNA 126AAV 199 k20 PTR383 U 6+27_Tric Y__circTuDw_polyT8 tricRNA 127

[0229] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 120-127. In some embodiments, the rAAV comprises a polynucleotide sequence of any one of SEQ ID NOs: 120-127 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the rAAV comprises a polynucleotide sequence of any one of SEQ ID NOs: 120-127. In some embodiments, the rAAV consists of a polynucleotide sequence of any one of SEQ ID NOs: 120-127.

[0230] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 122. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 122 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 122, In some embodiments, the rAAV consists of a polynucleotide sequence of SEQ ID NO: 122.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069

[0231] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 123. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 123 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 123. In some embodiments, the rAAV consists of a polynucleotide sequence of SEQ ID NO: 123.

[0232] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 124. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 124 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 124, In some embodiments, the rAAV consists of a polynucleotide sequence of SEQ ID NO: 124.

[0233] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 125. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 125 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 125. In some embodiments, the rAAV consists of a polynucleotide sequence of SEQ ID NO: 125.

[0234] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 126. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 126 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some1ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 126. In some embodiments, the rAAV consists of a polynucleotide sequence of SEQ ID NO: 126.

[0235] In some embodiments, the rAAV comprises a polynucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 127. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 127 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the rAAV comprises a polynucleotide sequence of SEQ ID NO: 127. In some embodiments, the rAAV consists of a polynucleotide sequence of SEQ ID NO: 127.Non- Viral and Viral Vectors

[0236] In some embodiments, a vector comprises the recombinant nucleic acid molecule encoding a circular RNA described herein. In some embodiments, the vector is double-stranded. In some embodiments, the vector ...

Claims

ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069CLAIMS1. A recombinant nucleic acid molecule encoding a circular RNA,wherein the recombinant nucleic acid molecule comprises, from 5’ to 3’, (1) a 5’ intronic element, a cargo, and a 3’ intronic element; or (2) a tRNA 5’ leader, a tRNA 5’ exonic element, a cargo, a tRNA 3 ’ intronic element, a tRNA 3 ’ exonic element, and a tRNA 3’ trailer, andwherein the cargo comprises a hairpin-shaped tough decoy, wherein the hairpin¬ shaped tough decoy comprises two single-stranded miRNA binding sites, and wherein the two single-stranded miRNA binding sites are flanked by a double-stranded stem region 1 and a double-stranded stem region 2.

2. The recombinant nucleic acid molecule of claim 1, wherein the 5’ end of the cargo comprises a 5’ restriction endonuclease (RE) site, and the 3’ end of the cargo comprises a 3’ RE site.

3. The recombinant nucleic acid molecule of claim 2, wherein the 5’ RE site and the 3’ RE site form part of the double-stranded stem region 1.

4. The recombinant nucleic acid molecule of any one of claims 1-3, wherein the double¬ stranded stem region 1 comprises at least one wobble base pair.

5. The recombinant nucleic acid molecule of claim 4, wherein the double-stranded stem region 1 comprises three wobble base pairs.

6. The recombinant nucleic acid molecule of claim 4 or 5, wherein the wobble base pair is a G-U wobble base pair.

7. The recombinant nucleic acid molecule of any one of claims 1 -6, wherein each of the two single-stranded miRNA binding sites comprises at least one bulged nucleotide.

8. The recombinant nucleic acid molecule of claim 7, wherein each of the two single¬ stranded miRNA binding sites comprises 2 to 6 continuous bulged nucleotides.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-20699. The recombinant nucleic acid molecule of claim 8, wherein each of the two single¬ stranded miRNA binding sites comprises 4 continuous bulged nucleotides.

10. The recombinant nucleic acid molecule of claim 8 or 9, wherein the continuous bulged nucleotides are located 10 nucleotides upstream of the 3 ’ end of each of the two singlestranded miRN A binding sites.

11. The recombinant nucleic acid molecule of any one of claims 8-10, wherein at least one of the two single-stranded miRNA binding sites comprises at least one bulged nucleotide in addition to the continuous bulged nucleotides.

12. The recombinant nucleic acid molecule of any one of claims 7-11, wherein each of the two single-stranded miRNA binding sites is otherwise 100% complementary to its corresponding miRNA target.

13. The recombinant nucleic acid molecule of any one of claims 1-12, wherein the double¬ stranded stem region 1 is 16 base pairs to 20 base pairs in length.

14. The recombinant nucleic acid molecule of claim 13, wherein the double-stranded stem region 1 is 18 base pairs in length.

15. The recombinant nucleic acid molecule of any one of claims 1-14, wherein at least one of the two single-stranded miRNA binding sites binds to an miRNA.

16. The recombinant nucleic acid molecule of claim 15, wherein at least one of the two single-stranded miRNA binding sites comprises 4 continuous bulged nucleotides, wherein the 4 continuous bulged nucleotides consist of a polynucleotide sequence of AACA.

17. The recombinant nucleic acid molecule of claim 15, wherein at least one of the two single-stranded miRNA binding sites comprises 4 continuous bulged nucleotides, wherein the 4 continuous bulged nucleotides consist of a polynucleotide sequence of CCAA.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206918. The recombinant nucleic acid molecule of any one of claims 1 to 14, wherein each of the two single-stranded miRNA binding sites binds to a miRNA, and the two miRNAs are the same miRNA or two different miRNAs.

19. The recombinant nucleic acid molecule of any one of claims 1-18, wherein the hairpinshaped tough decoy comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 3, 4, and 64-68.

20. The recombinant nucleic acid molecule of any one of claims 1-18, wherein the hairpinshaped tough decoy comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 65.

21. The recombinant nucleic acid molecule of any one of claims 1-18, wherein the hairpinshaped tough decoy comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 66.

22. The recombinant nucleic acid molecule of any one of claims 1-18, wherein the hairpinshaped tough decoy comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 67.

23. The recombinant nucleic acid molecule of any one of claims 1-18, wherein the hairpinshaped tough decoy comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 68.

24. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 1, 2, and 69-75.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206925. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1.

26. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2.

27. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 71.

28. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 72.

29. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 73.

30. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 74.

31. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the cargo comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 75.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206932. The recombinant nucleic acid molecule of any one of claims 1-31, wherein the recombinant nucleic acid molecule comprises, from 5’ to 3’, the 5’ backsplicing intronic element, the cargo, and the 3 ’ backsplicing intronic element.

33. The recombinant nucleic acid molecule of claim 32, wherein the double-stranded stem region 2 comprises an exonic splicing enhancer.

34. The recombinant nucleic acid molecule of claims 32 or 33, wherein the 5’ end of the cargo comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence that is at least about 85%, or 100% identical to SEQ ID NO: 80.

35. The recombinant nucleic acid molecule of any one of claims 32-34, wherein the 3’ end of the cargo comprises a 3 ’ RE site, wherein the 3 ’ RE site comprises a polynucleotide sequence that is at least about 85%, or 100% identical to CCTGCAGGc.

36. The recombinant nucleic acid molecule of any one of claims 32-35, wherein the 5’ intronic element comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 8.

37. The recombinant nucleic acid molecule of any one of claims 32-36, wherein the 3’ intronic element comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9.

38. The recombinant nucleic acid molecule of any one of claims 32-35, wherein the 5’ intronic element comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 410.

39. The recombinant nucleic acid molecule of any one of claims 32-36, wherein the 3’ intronic element comprises a polynucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 609.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206940. The recombinant nucleic acid molecule of any one of claims 1-31, wherein the recombinant nucleic acid molecule comprises, from 5’ to 3’, the tRNA 5’ leader, the tRNA 5’ exonic element, the cargo, the tRNA 3’ intronic element, the tRNA 3’ exonic element, and the tRNA 3 ’ trailer.

41. The recombinant nucleic acid molecule of claim 40, wherein the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 24.

42. The recombinant nucleic acid molecule of claim 40, wherein the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 312.

43. The recombinant nucleic acid molecule of claim 40, wherein the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 314.

44. The recombinant nucleic acid molecule of claim 40, wherein the tRNA 5’ leader comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 321.

45. The recombinant nucleic acid molecule of any one of claims 40-44, wherein the tRN A 3’ trailer comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 26.

46. The recombinant nucleic acid molecule of any one of claims 40-44, wherein the tRN A 3 ’ trailer comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 562.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206947. The recombinant nucleic acid molecule of any one of claims 40-44, wherein the tRNA 3’ trailer comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 564.

48. The recombinant nucleic acid molecule of any one of claims 40-44, wherein the tRN A 3’ trailer comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 571.

49. The recombinant nucleic acid molecule of any one of claims 40-48, wherein the tRN A 5’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 25.

50. The recombinant nucleic acid molecule of any one of claims 40-48, wherein the tRN A 5’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 354.

51. The recombinant nucleic acid molecule of any one of claims 40-48, wherein the tRN A 5’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 355.

52. The recombinant nucleic acid molecule of any one of claims 40-48, wherein the tRN A 5’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 357.

53. The recombinant nucleic acid molecule of any one of claims 40-52, wherein the tRN A 3’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 27.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206954. The recombinant nucleic acid molecule of any one of claims 40-52, wherein the tRNA 3’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 509.

55. The recombinant nucleic acid molecule of any one of claims 40-52, wherein the tRN A 3’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 510.

56. The recombinant nucleic acid molecule of any one of claims 40-52, wherein the tRN A 3’ exonic element comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 514.

57. The recombinant nucleic acid molecule of any one of claims 40-56, wherein the tRN A 3’ intronic element comprises a polynucleotide sequence of AGGT.

58. The recombinant nucleic acid molecule of any one of claims 40-56, wherein the tRN A 3’ intronic element comprises a polynucleotide sequence of AGAC.

59. The recombinant nucleic acid molecule of any one of claims 40-56, wherein the tRN A 3’ intronic element comprises a polynucleotide sequence of GGCT.

60. The recombinant nucleic acid molecule of any one of claims 40-59, wherein the 5’ end of the cargo comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence that is at least about 85%, or 100% identical to GCGGCCGC.

61. The recombinant nucleic acid molecule of any one of claims 40-60, wherein the 3’ end of the cargo comprises a 3 ’ RE site, wherein the 3 ’ RE site comprises a polynucleotide sequence that is at least about 85%, or 100% identical to SEQ ID NO: 5.

62. The recombinant nucleic acid molecule of any one of claims 40-61, wherein the cargo comprises, from 5’ to 3’, a 5’ RE site, a 5’ ligation motif, a 3’ ligation motif, and 3’ RE site.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206963. The recombinant nucleic acid molecule of claim 62, wherein the 5’ ligation motif and the 3’ ligation motif form part of the double-stranded stem region 1 of the hairpin-shaped tough decoy.

64. The recombinant nucleic acid molecule of claim 62 or 63, wherein the 5’ ligation motif comprises a polynucleotide sequence that is at least about 80%, at least about 90%, or 100% identical to SEQ ID NO: 6 and the 3’ ligation motif comprises a polynucleotide sequence that is at least about 80%, at least about 90%, or 100% identical to SEQ ID NO: 7.

65. The recombinant nucleic acid molecule of claim 62 or 63, wherein the 5’ ligation motif comprises a polynucleotide sequence that is at least about 80%, at least about 90%, or 100% identical to SEQ ID NO: 7 and the 3’ ligation motif comprises a polynucleotide sequence that is at least about 80%, at least about 90%, or 100% identical to SEQ ID NO: 6.

66. The recombinant nucleic acid molecule of any one of claims 1-65, wherein the recombinant nucleic acid molecule further comprises an RNA polymerase promoter located 5 ’ to the 5 ’ intronic element or the tRNA 5 ’ leader.

67. The recombinant nucleic acid molecule of claim 66, wherein the RNA polymerase promoter is an RNA polymerase II promoter or an RNA polymerase III promoter.

68. The recombinant nucleic acid molecule of claim 67, wherein the RNA polymerase II promoter is a chicken beta actin (CBA) promoter, a human phosphoglycerate kinase (hPGK) promoter, a human glucose-6-phosphatase (hG6Pase) promoter, a human elongation factor-1 alpha (EF-1α) promoter, a mouse E-cadherin (mECAD) promoter, or a mouse kidney-specific cadherin (mKSP-C) promoter, a mouse Na+-K+-Cl-cotransporter 2 (NKCC2) promoter, a mouse uromodulin (UMOD) promoter, a goat UMOD promoter, or a human UMOD promoter.

69. The recombinant nucleic acid molecule of claim 67 or 68, wherein the RNA polymerase II promoter is a CBA promoter.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206970. The recombinant nucleic acid molecule of any one of claims 67-69, wherein the RNA polymerase II promoter is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 30-34.

71. The recombinant nucleic acid molecule of any one of claims 67-69, wherein the RNA polymerase II promoter is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 30.

72. The recombinant nucleic acid molecule of claim 67, wherein the RNA polymerase III promoter is a U6 promoter, a U6+27 promoter, a Hl promoter, a H1.M11 promoter, a 7SK promoter, or a U6+1 promoter.

73. The recombinant nucleic acid molecule of claim 67 or 72, wherein the RNA polymerase III promoter is a U6+1 promoter.

74. The recombinant nucleic acid molecule of claim 67 or 72, wherein the RNA polymerase III promoter is a U6+27 promoter.

75. The recombinant nucleic acid molecule of any one of claims 67 and 72-74, wherein the RNA polymerase III promoter is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 35-40.

76. The recombinant nucleic acid molecule of claim 67, 72, or 74, wherein the RN A polymerase III promoter is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 35.

77. The recombinant nucleic acid molecule of claim 67, 72, or 73, wherein the RN A polymerase III promoter is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 36.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206978. The recombinant nucleic acid molecule of any one of claims 1 to 77, wherein the recombinant nucleic acid molecule further comprises an RNA polymerase terminator at the 3 ’ end of the 3 ’ intronic element or the tRNA 3 ’ trailer.

79. The recombinant nucleic acid molecule of claim 78, wherein the RNA polymerase terminator comprises a poly A signal or a polyT sequence.

80. The recombinant nucleic acid molecule of claim 79, wherein the poly A signal is a bovine growth hormone poly-adenylation (bGHpA), an SV40 poly A, a human GHpA (hGHpA), or a rabbit P-globin poly A (rflgpA) signal.

81. The recombinant nucleic acid molecule of claim 79 or 80, wherein the poly A signal is a bGHpA.

82. The recombinant nucleic acid molecule of any one of claims 79-81, wherein the poly A signal is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 42.

83. The recombinant nucleic acid molecule of claim 79, wherein the polyT sequence is polyT8 (TTTTTTTT).

84. The recombinant nucleic acid molecule of any one of claims 1-39, 66-71, and 78-82 wherein the recombinant nucleic acid molecule comprises, from 5’ to 3’, the 5’ intronic element, the cargo, and the 3 ’ intronic element for backsplicing, wherein the recombinant nucleic acid molecule further comprises an RNA polymerase II promoter located 5’ to the 5’ intronic element, and wherein the recombinant nucleic acid molecule further comprises a poly A signal located 3’ to the 3’ intronic element.

85. The recombinant nucleic acid molecule of any one of claims 1-31 and 40-83, wherein the recombinant nucleic acid molecule comprises, from 5’ to 3’, the tRNA 5’ leader, the tRN A 5’ exonic element, the cargo, the tRN A 3’ intronic element, the tR A 3’ exonic element, and the tRN A 3 ’ trailer for tR A splicing, wherein the recombinant nucleic acid molecule further comprises an RNA polymerase II or RNA polymerase III promoterATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069located 5’ to the tRNA 5’ leader, and wherein the recombinant nucleic acid molecule further comprises a poly A signal or a polyT sequence located 3’ to the tRNA 3’ trailer.

86. The recombinant nucleic acid molecule of any one of claims 1-85, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 43- 47, 101-107, and 109-112.

87. The recombinant nucleic acid molecule of any one of claims 1-85, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 46.

88. The recombinant nucleic acid molecule of any one of claims 1-85, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 109.

89. The recombinant nucleic acid molecule of any one of claims 1-85, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 102.

90. The recombinant nucleic acid molecule of any one of claims 1-85, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 105.

91. The recombinant nucleic acid molecule of any one of claims 1-85, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 111.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-206992. A vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 91.

93. The vector of claim 92, wherein the vector is a viral vector.

94. The vector of claim 93, wherein the viral vector is a recombinant adeno-associated virus (rAAV) vector.

95. The vector of claim 94, wherein the rAAV vector comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR flanks at the 5’ end of the recombinant nucleic acid molecule and the 3’ ITR flanks at the 3’ end of the recombinant nucleic acid molecule.

96. The vector of claim 94 or 95, wherein the rAAV vector is derived from AAV1, AAV2, AAV218, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAVI2, AAVT3, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

97. The vector of any one of claims 94-96, wherein the rAAV vector comprises a polynucleotide sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 120-12798. A rAAV particle comprising:(a) the recombinant nucleic acid molecule of any one of claims 1-91, or the vector of any one of claims 92 to 97; and(b) a capsid of AAV1, AAV2, AAV218, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV 13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

99. The rAAV particle of claim 98, wherein the capsid protein is an AAV9 capsid variant.

100. The AAV particle of claim 98 or 99, wherein the capsid protein is an AAV.k13 capsid protein.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069101. The AAV particle of claim 100, wherein the AAV.k13 capsid protein comprises an amino acid sequence of SEQ ID NO: 716 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 715.

102. The AAV particle of claim 100 or 101, wherein the AAV.k13 capsid protein comprises an amino acid sequence of SEQ ID NO: 716 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 715.

103. The AAV particle of any one of claims 100-102, wherein the AAV.k13 capsid protein consists of an amino acid sequence of SEQ ID NO: 716 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 715.

104. The AAV particle of any one of claims 100-103, wherein the AAV.k13 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wild-type AAV9 capsid.

105. The AAV particle of claims 98 or 99, wherein the capsid protein is an AAVk20 capsid protein.

106. The AAV particle of claim 105, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 717 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 715.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069107. The AAV particle of claim 105 or 106, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 717 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 715.

108. The AAV particle of any one of claims 105-107, wherein the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 717 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 715.

109. The AAV particle of any one of claims 105-108, wherein the AAV.k20 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wild-type AA V9 capsid.

110. The rAAV particle of claim 98, wherein the capsid is a variant of a parental wild-type capsid, and wherein the capsid improves transfer and / or expression of the recombinant nucleic acid molecule in one or more region(s) or part(s) of kidney when compared to the parental wild-type capsid.

111. A pharmaceutical composition, comprising the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, or the rAAV particle of any one of claims 98-110, and a pharmaceutically acceptable excipient.

112. A method of expressing a circular RNA in a cell, comprising introducing to the cell the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, the rAAV particle of any one of claims 98-110, or the pharmaceutical composition of claim 111, under conditions wherein the circular RNA is transcribed.ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069113. A method of expressing a circular RNA in a tissue, comprising introducing to the tissue the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, the rAAV particle of any one of claims 98-110, or the pharmaceutical composition of claim 111, under conditions wherein the circular RNA is transcribed.

114. A method of expressing a circular RNA in a subject, comprising introducing to the tissue the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, the rAAV particle of any one of claims 98-110, or the pharmaceutical composition of claim 111, under conditions wherein the circular RNA is transcribed.

115. A method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutic effective amount of the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, the rAAV particle of any one of claims 98-110, or the pharmaceutical composition of claim 111.

116. The method of claim 115, wherein the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, the rAAV particle of any one of claims 98-110, or the pharmaceutical composition of claim 111 is administered to the subject via retrograde ureteral infusion.

117. The method of claim 115, wherein the recombinant nucleic acid molecule of any one of claims 1 to 91, the vector of any one of claims 92-97, the rAAV particle of any one of claims 98-110, or the pharmaceutical composition of claim 111 is administered intravenously to the subject.

118. The method of any one of claims 115-117, wherein the disease or disorder is autosomal dominant polycystic kidney disease (ADPKD)119. The method of any one of claims 115-117, wherein the disease or disorder is ADPKD type 1 (ADPKD 1).

120. The method of any one of claims 115-117, wherein the disease or disorder is ADPKD type 2 (ADPKD2).ATTORNEY DOCKET NO.: TORQ-013 / 01 WO 339010-2069121. The method of any one of claims 115-117, wherein the disease or disorder is autosomal dominant tubulointerstitial kidney disease (ADTKD).

122. The method of any of claims 115-117, wherein the disease or disorder is ADTKD- UMOD.

123. The method of any of claims 115-117, wherein the disease or disorder is ADTKD-MUC1.

124. The method of any one of claims 115-123, wherein the method inhibits, ameliorates, or reverses renal cyst development.

125. The method of any one of claims 115-124, wherein the method inhibits, ameliorates, or reverses decline in kidney function.

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