Tric-derived scaffolds for circular RNA biogenesis

Abstract

The present disclosure relates to circular RNAs generated from tric scaffolds via tRNA splicing and uses thereof.

Description

TRIC-DERIVED SCAFFOLDS FOR CIRCULAR RNA BIOGENESISFIELD OF THE DISCLOSURE

[0001] The present disclosure relates to circular RNAs generated from trie scaffolds via tRNA splicing and uses thereof.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from U.S. Provisional No. 63 / 667,403 filed on July 3, 2024 and U.S. Provisional Application No. 63 / 757,184 filed on February 11, 2025; the disclosure of each of which are incorporated by reference in their entireties.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (TORQ_009_02WO_SeqList_ST26.xml; Size: 296,952 bytes; and Date of Creation: July 3, 2025) are herein incorporated by reference in its entirely.BACKGROUND

[0004] tRNA intronic circular RNA (tricRNA) is a by-product that results from splicing pre- tRNA by the tRNA splicing endonuclease (TSEN) complex. This endogenous process can be co- opted and used for generation of therapeutic circRNA by flanking a sequence encoding a cargo of interest with tRNA splicing elements to facilitate circRNA production in vitro and in vivo. However, the particular tRNA splicing elements, including leader, trailer, intronic, and exonic elements, required to support efficient circRNA production remains to be explored. The present disclosure provides novel approaches for generating therapeutic circRNAs using tRNA splicing elements.BRIEF SUMMARY

[0005] In some embodiments, the present disclosure provides a recombinant nucleic acid molecule comprising an engineered trie scaffold that enables circular RNA formation via tRNA splicing.

[0006] In some embodiments, the present disclosure provides an engineered tRNA sequence, comprising: i) a tRNA 5’ leader and a tRNA 3’ trailer; ii) a tRNA 5’ exonic element and a tRNA 3’ exonic element; iii) a tRNA 5’ intronic element and / or a tRNA 3’ intronic element; iv) a 5’ restriction endonuclease (RE) site and 3’ RE site; v) a 5’ ligation motif and a 3’ ligation motif; and vi) a cargo. In some embodiments, the cargo is incorporated into a circular RNA following tRNA splicing.

[0007] In some embodiments, the engineered tRNA sequence is derived from a gene encoding a tyrosine tRNA, an isoleucine tRNA, a leucine tRNA, or an arginine tRNA. In some embodiments, the gene is a human gene. In some embodiments, the gene is a mouse gene.

[0008] In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 1, 29-54, and 139. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 2, 55-80, and 140.

[0009] In some embodiments, the 5’ RE site comprises a polynucleotide sequence with at least about 60%, at least about 75%, at least about 85%, or 100% identity to GCGGCCGC or SEQ ID NO: 142. In some embodiments, the 3’ RE site comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 4.

[0010] In some embodiments, the engineered tRNA sequence comprises the tRNA 3’ intronic element but does not comprise the tRNA 5’ intronic element In some embodiments, the tRNA 3’ intronic element comprises a polynucleotide sequence with at least about 75%, or 100% identity to any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC.

[0011] In some embodiments, the engineered tRNA sequence comprises the tRNA 5’ intronic element and the tRNA 3’ intronic element In some embodiments, the tRNA 5’ intronic element comprises a polynucleotide sequence of TG, CT, GA, or GC. In some embodiments, the tRNA 3’ intronic element comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs: 101-104 and 141.

[0012] In some embodiments, the engineered tRNA sequence is cleaved by tRNA splicing endonuclease (TSEN) complex during tRNA splicing.

[0013] In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence that interacts with an RNA ligase. In some embodiments, the 5’ ligation motif and the 3’ ligation motif comprise a binding site for an RNA ligase. In some embodiments, the RNA ligase is RtcB. In some embodiments, the 5’ ligation motif and the 3’ ligation motif stabilize the secondary structure formed by the linear engineered tRNA sequence prior to and during tRNA splicing. In some embodiments, the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 6, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 7. In some embodiments, the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 7, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO:6.

[0014] In some embodiments, the engineered tRNA sequence comprises an acceptor stem sequence. In some embodiments, the acceptor stem sequence comprises a polynucleotide sequence with at least about 70%, at least about 85%, or 100% identity to any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 25 and 81-86.

[0015] In some embodiments, the tRNA 3 ’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 26 and 87-100.

[0016] In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence with 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% identity to SEQ ID NO: 27, wherein the polynucleotide sequence is upstream of the cargo. In some embodiments, theengineered tRNA sequence comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 109-118, and 145-164, wherein the polynucleotide sequence is upstream of the cargo. In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 190-192, wherein the polynucleotide sequence is upstream of the cargo sequence.

[0017] In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence with 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% identity to SEQ ID NO:28, wherein the polynucleotide sequence is downstream of the cargo. In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 119-128, and 170-189, wherein the polynucleotide sequence is downstream of the cargo. In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 193-196, wherein the polynucleotide sequence is downstream of the cargo.

[0018] In some embodiments, the engineered tRNA sequence comprises: i) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:29, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81 , the tRNA 3’ intronic element comprises a polynucleotide sequence of AGCA, the tRNA 3’exonic element comprises a polynucleotide sequence of SEQ ID NO: 87, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 55; ii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:31, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGTG, the tRNA 3’exonic elementcomprises a polynucleotide sequence of SEQ ID NO: 88, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 57; iii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:35, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 82, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGA, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 90, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 61; iv) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:37, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGGC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 92, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:63; v) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:38, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGCT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 93, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 64; vi) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:41, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 84, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGTC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 96, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:67; vii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:43, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 97, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 69; viii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:45, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’intronic element comprises a polynucleotide sequence of AGGT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO:26, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 71; ix) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:47, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 85, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:73; or x) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:49, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 85, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:75.

[0019] In some embodiments, the cargo encodes a therapeutic protein. In some embodiments, the cargo encodes a therapeutic RNA. In some embodiments, the therapeutic RNA is an antisense oligonucleotide, a ribozyme, a siRNA, a shRNA, a miRNA, a tough decoy, a miRNA sponge, a self-amplifying RNA, a guide RNA, an activating RNA, or a repressive RNA. In some embodiments, the therapeutic RNA is a miRNA sponge. In some embodiments, the therapeutic RNA is a tough decoy.

[0020] In some embodiments, the engineered tRNA sequence comprises a 5’ spacer and / or a 3’ spacer. In some embodiments, the 5’ spacer and / or the 3’ spacer comprise a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs: 105-108.

[0021] In some embodiments, the present disclosure provides a recombinant nucleic acid molecule comprising the engineered tRNA sequence described herein.

[0022] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase III promoter or an RNA polymerase II promoter.

[0023] In some embodiments, the RNA polymerase III promoter is an external RNA polymerase III promoter selected from the group consisting of H1, H1. M11, 7SK, U6, U6+27, and U6+1. In some embodiments, the RNA polymerase III promoter is an internal tRNA promoter.

[0024] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase terminator. In some embodiments, the RNA polymerase terminator comprises a polyT sequence or a polyadenylation signal. In some embodiments, the RNA polymerase terminator comprises at least 4 T nucleotides. In some embodiments, the RNA polymerase terminator comprises 8 T nucleotides. In some embodiments, the RNA polymerase terminator comprises a polynucleotide sequence with 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% identity to SEQ ID NO: 15.

[0025] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase in promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises an H1 promoter and the polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises an H1. M11 promoter and the polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a U6 promoter and the polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyT sequence.

[0026] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase in promoter and a polyadenylation signal. In some embodiments, the recombinant nucleic acid molecule comprises an H1 promoter and the polyadenylation signal. In some embodiments, the recombinant nucleic acid molecule comprises an H1.M11 promoter and the polyadenylation signal. In some embodiments, the recombinant nucleic acid molecule comprises a U6 promoter and the polyadenylation signal. In some embodiments, the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyadenylation signal.

[0027] In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 200-232, 236, 238-240, and 264-276. In some embodiments, the circularization efficiency of the recombinant nucleic acid molecule is at least about 10% higher compared to an recombinantnucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively. In some embodiments, the abundance of the resulting circular RNA is at least about 100% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo, respectively.

[0028] In some embodiments, the recombinant nucleic acid molecule comprises an external RNA polymerase III promoter, wherein a vector comprising the external RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an internal tRNA promoter when transcribed in a cell. In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase III promoter, wherein a vector comprising the RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an RNA polymerase II promoter when transcribed in a cell.

[0029] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) vector encoding the engineered tRNA sequence described herein or the recombinant nucleic acid molecule described herein. In some embodiments, the engineered tRNA sequence or recombinant nucleic acid molecule is flanked by AAV inverted terminal repeats (ITRs).

[0030] In some embodiments, the present disclosure provides an AAV particle comprising the AAV vector described herein and a capsid protein. In some embodiments, the capsid protein is a capsid protein of AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or a variant thereof. In some embodiments, the capsid protein is an AAV9 capsid variant In some embodiments, the capsid protein is a variant of a parental wild-type capsid protein, and wherein the capsid protein improves gene transfer and / or expression in one or more region(s) or part(s) of kidney when compared to the parental wild-type capsid protein.

[0031] In some embodiments, the capsid protein is an AAV.kl3 capsid protein. In some embodiments, the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 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: 136. In some embodiments, the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV.kl3 capsid protein consists of an amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of theAAV capsid protein are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV.kl3 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.

[0032] In some embodiments, the capsid protein is an AAV.k20 capsid protein. In some embodiments, the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 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: 136. In some embodiments, the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136. In some embodiments, 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 AAV9 capsid.

[0033] In some embodiments, the present disclosure provides a composition comprising the engineered tRNA sequence described herein, the recombinant nucleic acid molecule described herein, the AAV vector described herein, or the AAV particle described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0034] In some embodiments, the present disclosure provides a kit, comprising: the engineered tRNA sequence described herein, the recombinant nucleic acid molecule described herein, the AAV vector described herein, the AAV particle described herein, or the composition described herein. In some embodiments, the kit comprises an instruction for delivering the AAV vector, the AAV particle, or the composition to a target cell or tissue.

[0035] In some embodiments, the present disclosure provides a method of expressing a recombinant circular RNA in a cell, comprising introducing the engineered tRNA sequence described herein, the recombinant nucleic acid molecule described herein, the AAV vector described herein, the AAV particle described herein, or the composition described herein into the cell under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a myoblast. In some embodiments, the cell is a kidney cell.

[0036] In some embodiments, the present disclosure provides a method of expressing a recombinant circular RNA in a tissue, comprising introducing the engineered tRNA sequence described herein, the recombinant nucleic acid molecule described herein, the AAV vector described herein, the AAV particle described herein, or the composition described herein into the tissue under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

[0037] In some embodiments, the present disclosure provides a method of expressing a recombinant circular RNA in a subject, comprising administering an effective amount of the engineered tRNA sequence described herein, the recombinant nucleic acid molecule described herein, the AAV vector described herein, the AAV particle described herein, or the composition described herein to the subject, wherein the effective amount is an amount that reduces at least one symptom of a disease or condition in the subject

[0038] In some embodiments, the present disclosure provides a method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the engineered tRNA sequence described herein, the recombinant nucleic acid molecule described herein, the AAV vector described herein, the AAV particle described herein, or the composition described herein to the subject. In some embodiments, the subject is a human subject. In some embodiments, the subject has autosomal dominant polycystic kidney disease (ADPKD). In some embodiments, the subject has ADPKD1. In some embodiments, the subject has ADPKD2. In some embodiments, the method inhibits or ameliorates renal cyst development.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0040] FIGs. 1A-1D depict schematic representations of engineered trie scaffolds for circular RNA (circRNA) biogenesis. FIG 1A depicts a schematic representation of an engineered trie scaffold expression cassette. The expression cassette encodes a linear engineered pre-tRNA comprising a cargo that is flanked by tRNA expression and splicing elements — including leader, trailer, exonic, and intronic elements — RE sites, and ligation motifs. The 5’ and 3’ spacer sequences are engineered to be unstructured sequence elements surrounding the cargo and to create primer and probe binding sites for circRNA quantitation. The expression cassette also contains an RNA polymerase promoter and a terminator for optimal expression. FIG. 1B shows a schematic representation of the components of the linear engineered pre-tRNA that are transcribed from the expression cassette shown in FIG. 1A. FIG. 1C shows the predicted secondary structure that is formed from the linear engineered pre-tRNA prior to splicing. The BHB-like motif is marked by a box. FIG. 1D shows the components of circRNA that are formed following pre-tRNA splicing. The splice junction is between the 5’ RE site and the partial tRNA 3’ intronic element Elements in the schematic representations are not to scale. RE, restriction endonuclease; BHB, bulge-helix- bulge.

[0041] FIGs. 2A-2D depict schematic representations of engineered trie scaffolds for circRNA biogenesis. FIG. 2A depicts a schematic representation of an engineered trie scaffold expression cassette. The expression cassette encodes a linear engineered pre-tRNA comprising a cargo that is flanked by tRNA expression and splicing elements — including a leader, trailer, exonic, and intronic elements — RE sites, and ligation motifs. The 5’ and 3’ spacer sequences are engineered to be unstructured sequence elements surrounding the cargo and to create primer and probe binding sites for circRNA quantitation. The expression cassette also contains an RNA polymerase promoter and a terminator for optimal expression. FIG. 2B shows a schematic representation of the components of the linear engineered pre-tRNA that are transcribed from the expression cassette shown in FIG. 2A. FIG.2C shows the predicted secondary structure that is formed from the linearengineered pre-tRNA prior to splicing. The BHB-like motif is marked by a box. FIG. 2D shows the components of circRNA that are formed following pre-tRNA splicing. The splice junction is between the partial tRNA 5’ intronic element and the partial tRNA 3’ intronic element. Elements in the schematic representations are not to scale. RE, restriction endonuclease; BHB, bulge-helix- bulge.

[0042] FIGs. 3A-3C show the number of copies of precursor RNA per nanogram (cp / ng) (FIG. 3A), the number of copies of circRNA per nanogram (cp / ng) (FIG. 3B), and circularization efficiency (FIG. 3C) in HEK293T cells following transfection with a standard hTricY scaffold (PEX040), a hTricY scaffold with a disrupted ligation motif (PEX033), or a CMV-GFP control lacking the hTricY scaffold and circRNA sequences (PEX018). All test plasmids expressed GFP from a separate promoter for transfection normalization. All engineered tRNAs were expressed using a CMV / polyA expression system. Precursor and circular RNA abundance were determined by reverse-transcription followed by digital PCR (RT-dPCR) and normalized to GFP. Bars represent the group mean ± SEM. A two-tailed impaired t-test was performed to compare the scaffold with intact ligation motif and the scaffold with a disrupted ligation motif; * P≤0.05; **** P≤0.0001; ns, not significant. HEK293T, human embryonic kidney 293T; CMV-GFP, cytomegalovirus promoter-driven green fluorescent protein.

[0043] FIGs. 4A-4C show the number of copies of precursor RNA per nanogram (cp / ng) (FIG. 4A), the number of copies of circRNA per nanogram (cp / ng) (FIG. 4B), and circularization efficiency (FIG. 4C) in HEK293T cells following transfection with a standard hTricY scaffold (PEX040), a hTricY scaffold having no tRNA leader (PEX034), a hTricY scaffold having no tRNA trailer (PEX035), a hTricY scaffold having no tRNA leader and trailer (PEX036), or CMV- GFP control lacking the hTricY scaffold and circRNA sequences (PEX018). All test plasmids expressed GFP from a separate promoter for transfection normalization. All engineered tRNAs were expressed using a CMV / polyA expression system. Precursor and circular RNA abundance were determined by RT-dPCR and normalized to GFP. Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison was performed to compare the scaffolds (compared all means to each other); * P≤0.05; ** P≤0.01; **** P≤0.0001; ns, not significant. HEK293T, human embryonic kidney 293T; CMV-GFP, cytomegalovirus promoter-driven green fluorescent protein.

[0044] FIG. 5 shows the number of copies of circRNA per nanogram (cp / ng) in HEK293T cells following transfection with a standard hTricY scaffold (PEX040), hTricY-CCTT>GGCC (PEX037), hTricY-MENβ (PEX038), hTricY-Masc (PEX039), or CMV-GFP control lacking the hTricY scaffold and circRNA sequences (PEX018). All test plasmids expressed GFP from a separate promoter. All tRNA scaffolds were expressed using a CMV / polyA expression system. Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison was performed to compare the scaffolds (compared all means to each other); * P≤0.05; ** P≤0.01. HEK293T, human embryonic kidney 293T; CMV-GFP, cytomegalovirus promoter-driven green fluorescent protein.

[0045] FIG. 6 shows circularization efficiency of hTricY cassettes in HEK293T cells following transfection. The hTricY cassettes comprised various combinations of RNA polymerase II or III promoters (CMV, U6+27, U6+1, H1, H1.M11, or 7SK) and terminators (bGHpA and / or poly T sequence). The tmaY7 Internal cassette contained no external promoter or terminator. A CMV- GFP plasmid lacking the hTricY scaffold and circRNA sequences (PEX018) was used as a negative control (mock). Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was performed on logit-transformed data (compared all means to each other); ns, not significant. CMV-GFP, cytomegalovirus promoter-driven green fluorescent protein; bGHpA, bovine growth hormone polyadenylation signal.

[0046] FIG. 7 shows circularization efficiency of hTricY cassettes in HEK293T cells following AAV vector transduction at 1e4, 1e5, and 1e6 multiplicity of infection (MOI). The hTricY cassettes comprised various combinations of RNA polymerase II or III promoters (CMV, U6+27, U6+1, H1, H1. M11, or 7SK) and terminators (bGHpA and / or poly T sequence). The tmaY7 Internal cassette contained no external promoter or terminator. Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was performed on data from the 1e6 MOI group (compared all means to each other); ns, not significant.

[0047] FIG. 8 shows circularization efficiency of hTricY cassettes in HEK293T cells following either plasmid transfection or AAV vector transduction at 1e6 multiplicity of infection (MOI). The hTricY cassettes comprised various combinations of RNA polymerase II or III promoters (CMV, U6+27, U6+1, H1, H1.M11, or 7SK) and terminators (bGHpA and / or polyT sequence). ThetrnaY7 Internal cassette contained no external promoter or terminator. Bars represent the group mean ± SEM.

[0048] FIG.9 A and FIG. 9B show circularization efficiency in myoblasts following AAV vector transduction with hTricY cassettes comprising CMV / bGHpA, U6+27 / bGHpA, or U6+27 / polyT expression systems at 1e4, 1e5, or 1e6 multiplicity of infection (MOI). FIG. 9 A shows the data for each test article at an individual MOI, and FIG. 9B shows the plot of aggregated data for each test article across all MOIs. Bars represent the group mean ± SEM. One-way ANOVA was performed to compare the effect of dose on circRNA efficiency with Tukey’s multiple comparison test used for statistical analysis. ANOVA was significant (F = 355.9, p<0.001). Tukey’s multiple comparison test revealed significant differences between treatment groups where **, **** denotes statistically significant difference with p = 0.0066 or <0.0001, respectively.

[0049] FIG. 10A and FIG. 10B show the circularization efficiency of hTricY cassettes with a different cargo than FIGs. 6-9 following plasmid transfection (FIG. 10A) or AAV vector transduction at 1e4, 1e5, or 1e6 multiplicity of infection (MOI) (FIG. 10B) in HEK293T cells. The hTricY cassettes comprised various combinations of RNA polymerase II or III promoters (CMV, U6+27, U6+1, H1, H1.M11, or 7SK) and terminators (bGHpA and / or polyT sequence). The trnaY7 Internal cassette contained no external promoter or terminator. Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was performed on logit-transformed data (compared all means to each other). In FIG. 10B, only statistics for the 1e6 MOI group is shown.

[0050] FIGs. 11A-11E show expression of hTricY cassettes driven by CMV / bGHpA, U6+27 / bGHpA, or U6+27 / polyT expression systems in adult mice following AAV delivery. FIG. 11A shows vector genome levels in liver, FIG. 11B shows circRNA expression in liver, FIG. 11C shows precursor RNA expression in liver, FIG. 11D shows the ratio of total RNA (circRNA + precursor) to vector genomes in liver, and FIG. 11E shows circularization efficiency in liver. Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison was performed to compare the three scaffolds to each other; * P≤0.05; ** P≤0.01; ns, not significant.

[0051] FIG. 12 shows a schematic representation of the predicted secondary structure that is formed from the linear pre-tRNA prior to circularization for intron-containing tRNAs used as basescaffolds. The scaffolds consisted of four isotype / isodecoder families: tyrosine (TRY-GTA), isoleucine (TRI-TAT), leucine (TRL-CAA), and arginine (TRR-TCT).

[0052] FIGs. 13A-13D show the number of copies of circRNA per nanogram (cp / ng) (FIG. 13A), number of copies of precursor RNA per nanogram (cp / ng) (FIG. 13B), number of copies of total RNA per nanogram (cp / ng) (FIG. 13C), and circularization efficiency (FIG. 13D) yielded by the standard hTricY scaffold (PTR175) and hTricY scaffolds with extended leader and trailer sequences (PTR228 and PTR238) in HEK293T cells following transfection. PTR238 contains a single mutation that abrogates splicing. Bars represent the group mean ± SEM. One-way ANOVA with Tukey ’ s multiple comparison was performed to compare scaffold yields using log 10- or logit- transformed values (compared all means to each other); * P≤0.05; ** P≤0.01; **** P≤0.0001; ns, not significant Only significant changes compared to PTR175 are marked.

[0053] FIG. 14A and FIG. 14B show the number of copies of precursor RNA per nanogram (cp / ng) yielded by trie scaffolds derived from the tyrosine (TRY) family (FIG. 14A) and the isoleucine (TRI) and leucine (TRL) families (FIG. 14B) compared to the standard hTricY scaffold (PTR175) in HEK293T cells following transfection. FIG. 14C and 14D show the number of copies of circRNA per nanogram (cp / ng) yielded by the trie scaffolds derived from the tyrosine (TRY) family (FIG. 14C) and the isoleucine (TRI), leucine (TRL), and arginine (TRR) families (FIG. 14D) compared to the standard hTricY scaffold (PTR175). Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison was performed to compare scaffold yields using loglO-transformed values (compared all means to each other); * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001; ns, not significant. Only significant changes compared to PTR175 are marked. FIG. 14E shows the circRNA fold change yielded by the tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds compared to the standard hTricY scaffold (PTR175). One-way ANOVA with Dunnett’s multiple comparison was performed to compare scaffold yields using log2-transformed values (compared all means to the mean of PTR175); * P≤0.05; ** P≤0.01; **** P≤0.0001; ns, not significant. Only significant increases compared to PTR175 are marked. FIG. 14F and 14G show the circularization efficiency for the trie scaffolds derived from the tyrosine (TRY) family (FIG. 14F), isoleucine (TRI), and leucine (TRL) families (FIG. 14G) compared to the standard hTricY scaffold (PTR175). One-way ANOVA with Tukey’s multiple comparison was performed to compare scaffold yields using logit-transformed values(compared all means to each other); **** P≤0.0001; ns, not significant. Only significant increases compared to PTR175 are marked.

[0054] FIGs. 15A-15D show the number of copies of circRNA per nanogram (cp / ng) yielded by trie scaffolds comprising 3’ intronic elements of either four or ten nucleotide length (“4-mer” and “10-mer,” respectively) derived from the isoleucine family (TRI-TATI-1, FIG. 1SA), leucine family (TRL-CAA2-1, FIG. 15B), arginine family (TRR-TCT3-2, FIG. 15C), and tyrosine family (TRY-GTA3-1.2, FIG. 15D) in HEK293T cells following transfection. Bars represent the group mean ± SEM. One-way ANOVA with Tukey’s multiple comparison was performed to compare 4- mer and 10-mer scaffold yields using log2-transformed values (compared all means to each other); ** P≤0.01; **** P≤0.0001; ns, not significant.

[0055] FIGs. 16A and 16B show the number of copies of circRNA per nanogram (cp / ng) yielded by the representative 4-mer and 10-mer tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds in HEK293T cells (FIG. 16 A) and HeLa cells (FIG. 16B) following transfection. FIG. 16C shows the microRNA inhibition activity yielded by the miRNA sponge cargo produced from the 4-mer and 10-mer tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds in HeLa cells following transfection. microRNA inhibition is defined by an increase in measured luciferase activity from a luciferase reporter containing two target miRNA binding sites.

[0056] FIG. 17 shows average circRNA and precursor RNA copies per nanogram (cp / ng) and circularization efficiency of cassettes comprising human-mouse hybrid trie scaffolds with a miRNA sponge cargo driven by a U6+27 / polyT expression system in HEK293T cells following transfection. One-way ANOVA with Tukey’s multiple comparison was used to compare trie scaffold yields using loglO-transformed values of all means. No statistically significant difference in circRNA abundance between PTR244, PTR245, and PTR246 was observed, although there was a statistically significant increase in circularization efficiency for PTR244 and PTR245 compared to PTR246.

[0057] FIG. 1SA shows the number of copies of circRNA (lighter bar) and precursor RNA (darker bar) per nanogram (cp / ng) yielded by trie scaffolds derived from the tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) families in HeLa cells following transfection; the top of each stacked bar represents the mean total RNA (circRNA + precursor RNA), with thetop error bars representing the SEM for total RNA, and the lower error bars representing the SEM for circRNA. Circularization efficiency for each scaffold is reported as text within each bar of the graph. FIG. 18B shows microRNA inhibition activity of the miRNA sponge cargo produced from tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds. microRNA inhibition is defined by an increase in measured luciferase activity from a luciferase reporter containing two target miRNA binding sites. Bars represent the group mean ± SEM. One-way ANOVA with Dunnett’s multiple comparison to PEX091 using In-transformed fold changes for activity assay was performed; * P≤0.05; **** P≤0.0001; ns, not significant. FIG. 18C shows the correlation between microRNA inhibition activity and circRNA abundance of tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds in HeLa cells. FIG. 18D shows the lack of correlation between miRNA inhibition activity and precursor RNA abundance of tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds in HeLa cells. The dotted line represents a linear regression fit to mean values (FIGs. 18C-18D).

[0058] PIG. 19 A shows a correlation between circRNA abundance yielded by tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds in transfected HEK293T cells versus HeLa cells. The dotted line represents a linear regression fit to mean values. FIG. 19B shows a comparison of circRNA abundance between tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) trie scaffolds in transfected HEK293T cells versus HeLa cells.

[0059] FIGs. 20A-20C show the number of copies of circRNA per nanogram (cp / ng) (FIG. 20A), number of copies of precursor RNA per nanogram (cp / ng) (FIG. 20B), and circularization efficiency (FIG. 20C) yielded by trie scaffolds derived from the tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) families following AAV vector transduction of HEK293T cells at 1e6 multiplicity of infection (MOI). Bars represent the group mean ± SEM. FIGs. 20A-20B show statistical analyses using one-way ANOVA with Tukey’s multiple comparison to compare scaffold circRNA and precursor yields using loglO-transformed values (compared all means to each other); * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001; ns, not significant. Only significant changes compared to hTricY_AAV136 are indicated. FIG. 20C shows statistical analyses using Kruskal-Wallis with Dunn’s multiple comparison to compare scaffold circularization efficiencies using logit-transformed values (compared all means to each other); ns, not significant.

[0060] FIGs. 21 A-21B show circRNA fold change of tyrosine (TRY), isoleucine (TRI), leucine (TRL), and arginine (TRR) scaffolds relative to the standard hTricY scaffold following transfection (FIG. 21A) or transduction (FIG. 21B). Bars represent the group mean ± SEM. Oneway ANOVA with Dunnett’s multiple comparison was performed to compare scaffold yields using log2-transformed values (compared all means to the standard hTricY scaffold); * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001; ns, not significant. FIG.21C shows the correlation between circRNA fold change resulting from transduction versus circRNA fold change resulting from transfection using the data from FIGs. 21A-21B. The dotted line represents a linear regression fit to mean values. FIG. 21D shows a schematic representation of TRY, TRI, TRL, and TRR-based scaffolds that resulted in a significant increase in circRNA expression over the standard hTricY scaffold using the data from FIGs. 21A-21B.

[0061] FIG. 22A shows average vector genome levels, circRNA, precursor RNA, and total RNA expression, and circularization efficiency of tyrosine (hTricY or TRY-GTA5-1) scaffolds comprising either a tough decoy cargo (circTuD) or a miRNA sponge cargo (12x12B) in mouse kidney and liver following AAV delivery. FIG. 22B shows in situ hybridization (ISH) performed on kidney sections from formulation buffer-treated mice or mice treated with AAV comprising the U6+27-hTricY-circTuD-polyT scaffold (AAV146). The ISH probe was designed to detect the circRNA splice junction and thus is specific for the circTuD versus the linear precursor RNA. Blue staining shows hematoxylin-labeled nuclei. The discrete red puncta in the right panel (black arrows) are representative of circTuD labeling in the kidney of AAV-treated mice.DETAILED DESCRIPTION

[0062] 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.

[0063] 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 artDefinitions

[0064] 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.

[0065] The term “a” or “an” refers to one or more of that entity, i.e., can refer to plural referents. 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.

[0066] 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”).

[0067] 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.

[0068] Throughout this application, the term “abouf ’ is used to indicate that a value includes the inherent variation of error for the composition 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 the endpoints 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.

[0069] 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 notpreclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0070] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0071] The terms “reduce” or “decrease”, as used herein, may indicate a reduction or decrease of at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or any value therebetween.

[0072] The terms “increase” or “enhance”, as used herein, may indicate an increase or enhancement of at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 300%, about 400%, about 500%, about 1000%, or any value therebetween.

[0073] The term “polynucleotide” or “nucleic acid”, as used herein, refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be singlestranded or double-stranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), transfer RNA (tRNA), pre-tRNA, 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.

[0074] 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 at the 5’ endand a translation stop codon at the 3 ’ end. A transcription termination sequence may be located 3 ’ to the coding sequence. A Kozak sequence may be located 5’ to the coding sequence. In some embodiments, the Kozak sequence overlaps with the start codon.

[0075] 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 identity” 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 BLAST 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.

[0076] The term “modulate”, as used herein, means to regulate expression of a particular gene, mRNA, protein, or signaling pathway. In some embodiments, the term “modulate” refers to upregulation or increased expression of a particular gene, mRNA, protein, or signaling pathway. In some embodiments, the term “modulate” refers to downregulation or decreased expression of a particular gene, mRNA, protein, or signaling pathway.

[0077] The term “baseline” or “baseline expression”, as used herein, refers to the level against which expression of a particular gene, mRNA, protein, or signaling pathway in a test sample (e.g., a transduced cell or tissue) is compared.

[0078] 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 theculture 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 a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art

[0079] 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.

[0080] 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 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 AAV capsid to generate a “recombinant adeno-associated viral capsid particle (rAAV particle).”

[0081] 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.

[0082] 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).

[0083] 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 approximately 145-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 contain several shorter regions of selfcomplementarity (designated A, A’, B, B’, C, C’ and D regions), allowing intra-strand base-pairing to occur within this portion of the ITR.

[0084] 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 VP3) 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, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 , AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, A AV-2 / 1, AAV 2 / 6, AAV 2 / 7, AAV 2 / 8, AAV 2 / 9, AAV LK03, AAVrh10, AAVrh74, AAV44-9, or a variant thereof.The term also includes a capsid protein expressed by or derived from a recombinant AAV such as a chimeric AAV. The term “AAV capsid particle” or “AAV particle”, as used herein, includes at least one AAV capsid protein (e.g., a VP1 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.

[0085] 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).

[0086] The term “transduced”, as used herein, refers to a process by which a transgene is introduced into a host cell from a virus particle.

[0087] 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.

[0088] 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, post-transcriptional, or translational expression of the nucleic acid molecule in the target cell or tissue.

[0089] The term “regulatory elemenf ’, 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.

[0090] 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.

[0091] 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 or disorder.

[0092] 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).

[0093] 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 rabbits and 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.

[0094] As used herein, “treat,” “treating,” or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results. The terms may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. In some embodiments, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treat,” “treating,” or “treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g. a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The terms “treat,” “treating,” or “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment and is not necessarily meant to imply complete cessation of the relevant disease, disorder, or condition.

[0095] 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.

[0096] 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, intravitreal, intraarticular, subretinal, intramuscular, intrathecal delivery 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 isadministered 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).

[0097] 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. In some embodiments, the therapeutically effective dose is an amount of viral particles comprising the circular RNA that reduces at least one symptom of a disease or disorder in a subject. In some embodiments, the therapeutically effective dose is an amount of circular RNA that reduces at least one symptom of a disease or disorder in a subject. 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, namely 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)).

[0098] 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., Cold Spring 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 (L Lefkovits ed., AcademicPress 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. tRNA Intronic Circular (tric)RNA System

[0099] In some embodiments, the synthetic circular RNA described herein is processed and produced by a tRNA splicing mechanism.

[0100] 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 tRNA 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 circRNA 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 synthetic circular RNAs of the present disclosure are generated using pre-tRNA splicing elements. In some embodiments, the present disclosure provides a synthetic circular RNA (i.e., circRNA) generating system using pre-tRNA splicing elements.

[0101] 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from a human gene. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRY-GTA1-1 (TRNAY10) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRY-GTA2-1 (TRNAY4) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRY-GTA4-1 (TRNAY13) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRY-GTA5-1 (TRNAY15) gene to produce a circular RNA viatRNA splicing. In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence 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 engineered tRNA sequence 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 engineered tRNA sequence 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 engineered tRNA sequence 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRY-GTA7-1 (TRNAY1) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRY-GTA8-1 (TRNAY12) gene to produce a circular RNA via tRNA splicing.

[0102] 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from a mouse gene. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the mouse Tyr-GTA-3-1 (n-Tygta2) gene to produce a circular RNA via tRNA splicing.

[0103] 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from a mouse gene and a human gene. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the mouse Tyr-GTA-3-1 (n-Tygta2) gene and the human TRY- GTA3-1 (TRNAY7) gene to produce a circular RNA via tRNA splicing.

[0104] 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from ahuman gene. In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRI-TAT2-1 (TRNAI13) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRI-TAT2-2 (TRNAI2) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRI-TAT2-3 (TRNAI6) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRI-TAT3-1 (TRNAI21) gene to produce a circular RNA via tRNA splicing.

[0105] 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from a human gene. In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRL-CAAl-2 (TRNAL22) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRL-CAA4-1 (TRNAL19) gene to produce a circular RNA via tRNA splicing.

[0106] 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 engineered tRNA sequence comprises a pre-tRNA sequence derived from a human gene. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRR-TCT1-1 (TRNAR15) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNAsequence derived from the human TRR-TCT2-1 (TRNAR1) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRR-TCT3-1 (TRNAR12) gene to produce a circular RNA via tRNA splicing. In some embodiments, the engineered tRNA sequence comprises a pre-tRNA sequence derived from the human TRR-TCT3-2 (TRNAR14) gene to produce a circular RNA via tRNA splicing.

[0107] In some embodiments, the engineered tRNA sequence 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”. Exemplary sequences for tRNA 5’ leaders are provided in Table 1 below.Table 1. Exemplary tRNA Leader Sequences

[0108] 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: 1, 29-54, and 139. In some embodiments, the tRNA 5’ leader comprises a polynucleotide sequence of any one of SEQ ID NOs: 1, 29-54, and 139 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA5’ leader comprises a polynucleotide sequence of any one of SEQ ID NOs: 1, 29-54, and 139. Insome embodiments, the tRNA 5’ leader consists of a polynucleotide sequence of any one of SEQID NOs: 1, 29-54, and 139.

[0109] In some embodiments, the engineered tRNA sequence 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”. Exemplary sequences for tRNA 3’ trailers are provided in Table 2 below.Table 2. Exemplary tRNA Trailer Sequences

[0110] 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 any one of SEQ ID NOs: 2, 55-80, and 140. In some embodiments, the tRNA 3’ trailer comprises a polynucleotide sequence of any one of SEQ ID NOs: 2, 55-80, and 140 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the tRNA3’ trailer comprises a polynucleotide sequence of any one of SEQ ID NOs: 2, 55-80, and 140. In some embodiments, the tRNA 3’ trailer consists of a polynucleotide sequence of any one of SEQID NOs: 2, 55-80, and 140.

[0111] In some embodiments, the engineered tRNA sequence comprises a tRNA exonic elementIn 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 tRNA 5’ exonic element. In some embodiments, the tRNA 5’ exonic element is referred to as the “tRNA 5’ exon” or “tRNA 5’ exon sequence”. Exemplary sequences for tRNA 5’ exonic elements are provided in Table 3 below.Table 3. Exemplary tRNA 5’ Exonic Sequences

[0112] 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 any one of SEQ ID NOs: 25 and 81-86. In some embodiments, the tRNA 5’ exonic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 25 and 81-86 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 81-86. In some embodiments, the tRNA 5’ exonic element consists of a polynucleotide sequence of any one of SEQ ID NOs: 25 and 81-86.

[0113] In some embodiments, the engineered tRNA sequence 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 located3’ 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 “tRNA 3’ exon” or “tRNA 3’ exon sequence”.Exemplary sequences for tRNA 3’ exonic elements are provided in Table 4 below.Table 4. Exemplary tRNA 3’ Exonic Sequences

[0114] 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 about90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about95%, 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 87-100. In some embodiments, the tRNA 3’ exonic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 26 and 87-100 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 NOs: 26and 87-100. In some embodiments, the tRNA 3’ exonic element consists of a polynucleotide sequence of any one of SEQ ID NOs: 26 and 87-100.

[0115] In some embodiments, the engineered tRNA sequence comprises a tRNA intronic element. In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence 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, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, or more. In some embodiments, the engineered tRNA sequence comprises a partial tRNA intronic element and the partial tRNA intronic element comprises 2 nucleotides (2-mer). In some embodiments, the engineered tRNA sequence comprises a partial tRNA intronic element and the partial tRNA intronic element comprises 4 nucleotides (4-mer). In some embodiments, the engineered tRNA sequence 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. Exemplary tRNA intronic elements are provided in Table 5 below.Table 5. Exemplary tRNA Intronic Sequences

[0116] In some embodiments, the tRNA intronic element is located 3 ’ to the cargo and is referred to herein as the “tRNA 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 engineered tRNA sequence 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 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, 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.

[0117] In some embodiments, the tRNA intronic element is located 5’ to the cargo and is referred to herein as the “tRNA 5’ intronic element.” In some embodiments, the tRNA 5’ intronic elementis referred to herein as the “tRNA 5’ intron” or “tRNA 5’ intron sequence”. In some embodiments, the tRNA 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.

[0118] In some embodiments, the engineered tRNA sequence 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: 101-104 and 141. In some embodiments, the tRNA 3’ intronic element comprises a polynucleotide sequence of any one of SEQ ID NOs: 101-104 and 141, 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: 101-104 and 141.

[0119] In some embodiments, the engineered tRNA sequence 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: 101-104 and 141. 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: 101-104 and 141.

[0120] In some embodiments, the engineered tRNA sequence 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: 101-104 and 141. 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: 101-104 and 141.

[0121] In some embodiments, the engineered tRNA sequence comprises a tRNA 3’ intronic element, but does not comprise a tRNA 5’ intronic element.

[0122] In some embodiments, the engineered tRNA sequence comprises at least one restriction enzyme (RE) site. In some embodiments, the engineered tRNA sequence comprises two RE sites. In some embodiments, the RE site is located 5’ to the cargo and is referred to herein as the “5’ RE site,” “5’ RE sequence,” “first RE site,” or “first RE sequence.” In some embodiments, the RE site is located 3’ to the cargo and is referred to herein as the “3’ RE site,” “3’ RE sequence,” “second RE site,” or “second RE sequence.” In some embodiments, the engineered tRNA sequence comprises a 5’ RE site and a 3’ RE site. Exemplary sequences for RE sites are provided in Table 6 below where the lower-case letters denote filler nucleotides and the uppercase letters denote the restriction site.Table 6. Exemplary RE Site Sequences

[0123] In some embodiments, the RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 60%, at least about 62.5% identical, at least about 75% identical, at least about 85%, at least about 87.5% identical, or 100% identical to GCGGCCGC. In some embodiments, the 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 RE site comprises a polynucleotide sequence of GCGGCCGC. In some embodiments, the RE site consists of a polynucleotide sequence of GCGGCCGC.

[0124] In some embodiments, the 5’ RE site comprises a polynucleotide sequence that is at least about 50% identical, at least about 60%, at least about 62.5% identical, at least about 75% identical, at least about 85%, 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.

[0125] In some embodiments, the 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: 142. In some embodiments, the RE site comprises a polynucleotide sequence of SEQ ID NO: 142 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the RE site comprises a polynucleotide sequence of SEQ ID NO: 142. In some embodiments, the RE site consists of a polynucleotide sequence of SEQ ID NO: 142.

[0126] In some embodiments, the 5’ 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: 142. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 142 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the 5’ RE site comprises a polynucleotide sequence of SEQ ID NO: 142. In some embodiments, the 5’ RE site consists of a polynucleotide sequence of SEQ ID NO: 142.

[0127] In some embodiments, the 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: 4. In some embodiments, the RE site comprises a polynucleotide sequence of SEQ ID NO: 4 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the RE site comprises a polynucleotide sequence of SEQ ID NO: 4. In some embodiments, the RE site consists of a polynucleotide sequence of SEQ ID NO: 4.

[0128] 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: 4. In some embodiments, the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 4 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: 4. In some embodiments, the 3’ RE site consists of a polynucleotide sequence of SEQ ID NO: 4.

[0129] In some embodiments, the engineered tRNA sequence comprises a 5’ RE site and a 3’ RE site. In some embodiments, the engineered tRNA sequence 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 or SEQ ID NO: 142. In some embodiments, the engineered tRNA sequence comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC or SEQ ID NO: 142 with one or more mutations, such as 1, 2, 3, 4, or more mutations. In some embodiments, the engineered tRNA sequence comprises a 5’ RE site, wherein the 5’ RE site comprises a polynucleotide sequence of GCGGCCGC or SEQ ID NO: 142. In some embodiments, the engineered tRNA sequence comprises a 5’ RE site, wherein the 5’ RE site consists of a polynucleotide sequence of GCGGCCGC or SEQ ID NO: 142. In some embodiments, the engineered tRNA sequence 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: 4. In some embodiments, the engineered tRNA sequence comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 4 with one or more mutations, such as 1, 2, 3, 4, 5, 6, or more mutations. In some embodiments, the engineered tRNA sequence comprises a 3’ RE site, wherein the 3’ RE site comprises a polynucleotide sequence of SEQ ID NO: 4. In some embodiments, the engineered tRNA sequence comprises a 3’ RE site, wherein the 3’ RE site consists of a polynucleotide sequence of SEQ ID NO: 4.

[0130] In some embodiments, the engineered tRNA sequence comprises at least one ligation motif. In some embodiments, the engineered tRNA sequence comprises two ligation motifs. In some embodiments, the engineered tRNA sequence comprises a 5’ ligation motif and a 3’ ligation motif.

[0131] In some embodiments, the engineered tRNA sequence comprises a ligation motif 5’ to the cargo and is referred to herein as the “5’ ligation motif,” “5’ ligation sequence,” “first ligation motif,” or “first ligation sequence.” In some embodiments, the engineered tRNA sequence comprises a ligation motif 3’ to the cargo and is referred to herein as the “3’ ligation motif,” “3’ ligation sequence,” “second ligation motif,” or “second ligation sequence.”

[0132] 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 aligase. In some embodiments, the 5’ ligation motif and 3’ ligation motif stabilize the secondary structure formed by the linear engineered tRNA sequence prior to and during tRNA splicing.

[0133] In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence that interacts with an RNA ligase to promote tRNA splicing. In some embodiments, a 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 site for an RNA ligase. In some embodiments, the RNA ligase is RtcB. Exemplary ligation motifs are provided in Table 7 below.Table 7. Exemplary Ligation Motif Sequences

[0134] 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: 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.

[0135] 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.

[0136] In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence 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 engineered tRNA sequence comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6; and the engineered tRNA sequence comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the engineered tRNA sequence comprises a 5’ ligation motif, wherein the 5’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 6; and the engineered tRNA sequence comprises a 3’ ligation motif, wherein the 3’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 7.

[0137] In some embodiments, the engineered tRNA sequence 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: 7; and the engineered tRNA sequence 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: 6. In some embodiments, the engineered tRNA sequence comprises a 5’ ligation motif, wherein the 5’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 7; and the engineered tRNA sequence comprises a 3’ ligation motif, wherein the 3’ ligation motif comprises a polynucleotide sequence of SEQ ID NO: 6. In some embodiments, the engineered tRNA sequence comprises a 5’ ligation motif, wherein the 5’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 7; and the engineered tRNA sequence comprises a 3’ ligation motif, wherein the 3’ ligation motif consists of a polynucleotide sequence of SEQ ID NO: 6.

[0138] In some embodiments, the engineered tRNA sequence comprises an acceptor stem sequence. In some embodiments, the engineered tRNA sequence encodes an acceptor stem sequence comprising cytosines at the 5’ first and second positions. Exemplary acceptor stem sequences are provided in Table 8 below.Table 8. Exemplary tRNA Acceptor Stem Sequences

[0139] In some embodiments, the acceptor stem sequence comprises a polynucleotide sequence that is at least about 50% 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 85% identical, at least about 87.5% identical, or 100% identical to any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU. In some embodiments, the acceptor stem sequence comprises a polynucleotide sequence of any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU with one or more mutations, such as 1, 2, 3, 4, 5, or more mutations. In some embodiments, the acceptor stem sequence comprises a polynucleotide sequence of any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU. In some embodiments, the acceptor stem sequence consists of a polynucleotide sequence of any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU.

[0140] In some embodiments, the engineered tRNA sequence comprises at least one spacer. In some embodiments, the engineered tRNA sequence comprises two spacers. In some embodiments, the spacer is located 5’ to the cargo and is referred to herein as the “5’ spacer,”, “5’ spacer sequence,” “first spacer,” or “first spacer sequence.” In some embodiments, the spacer is located 3’ to the cargo and is referred to herein as the “3’ spacer,” “3’ spacer sequence,” “second spacer,”or “second spacer sequence.” In some embodiments, the engineered tRNA sequence comprises a 5’ spacer and a 3’ spacer. Exemplary sequences for spacers are provided in Table 9 below.Table 9. Exemplary Spacer Sequences

[0141] In some embodiments, the engineered tRNA sequence comprises at least one spacer. In some embodiments, the engineered tRNA sequence comprises at least one spacer, wherein the spacer 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: 105-108. In some embodiments, the engineered tRNA sequence comprises at least one spacer, wherein the spacer comprises a polynucleotide sequence of any one of SEQ ID NOs: 105-108 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the engineered tRNA sequence comprises at least one spacer, wherein the spacer comprises a polynucleotide sequence of any one of SEQ ID NOs: 105-108. In some embodiments, the engineered tRNA sequence at least one spacer, wherein the spacer consists of a polynucleotide sequence of any one of SEQ ID NOs: 105-108.

[0142] In some embodiments, the engineered tRNA sequence comprises two spacers. In some embodiments, the engineered tRNA sequence comprises a 5’ spacer, wherein the 5’ spacer 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: 105-108; and a 3’ spacer wherein the 3 ’ spacer 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 about92%, 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: 105- 108. In some embodiments, the engineered tRNA sequence comprises a 5’ spacer, wherein the 5’ spacer comprises a polynucleotide sequence of any one of SEQ ID NOs: 105-108 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations; and a 3’ spacer, wherein the 3’ spacer comprises a polynucleotide sequence of any one of SEQ ID NOs: 105-108 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the engineered tRNA sequence comprises a 5’ spacer, wherein the 5’ spacer comprises a polynucleotide sequence of any one of SEQ ID NOs: 105-108; and a 3’ spacer wherein the 3’ spacer comprises a polynucleotide sequence of any one of SEQ ID NOs: 105-108. In some embodiments, the engineered tRNA sequence comprises a 5’ spacer, wherein the 5’ spacer consists of a polynucleotide sequence of any one of SEQ ID NOs: 105-108; and a 3’ spacer, wherein the 3’ spacer consists of a polynucleotide sequence of any one of SEQ ID NOs: 105-108.

[0143] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 29; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 81; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of AGCA (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 87; and (v) a tRNA 3’ trailer comprising a polynucleotide sequence of SEQ ID NO: 55.

[0144] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 31; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 81; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of AGTG; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 88; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 57.

[0145] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 35; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 82; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of AGGA; (iv) a tRNA 3’ exonic element comprising apolynucleotide sequence of SEQ ID NO: 90; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 61.

[0146] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 37; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 82; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of GGGC; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 92; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 63.

[0147] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 38; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 82; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of GGCT; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 93; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 64.

[0148] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 41; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 84; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of GGTC; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 96; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 67.

[0149] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 43; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 25; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of AGAC; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 97; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 69.

[0150] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 45; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 25; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of AGGT; (iv) a tRNA 3’ exonic element comprising apolynucleotide sequence of SEQ ID NO: 26; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 71.

[0151] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 47; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 85; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of AGAC; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 98; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 73.

[0152] In some embodiments, the engineered tRNA sequence comprises: (i) a tRNA 5’ leader comprising a polynucleotide sequence of SEQ ID NO: 49; (ii) a tRNA 5’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 85; (iii) a tRNA 3’ intronic element comprising a polynucleotide sequence of GGAC; (iv) a tRNA 3’ exonic element comprising a polynucleotide sequence of SEQ ID NO: 98; and (v) a tRNA 3 ’ trailer comprising a polynucleotide sequence of SEQ ID NO: 75.

[0153] In some embodiments, the engineered pre-tRNA comprises a bulge-helix-bulge (BHB)- like motif. In some embodiments, the BHB-like motif comprises a tRNA 5’ exonic element. In some embodiments, the BHB-like motif comprises a tRNA 3’ exonic element In some embodiments, the BHB-like motif comprises a tRNA intronic element In some embodiments, the BHB-like motif comprises a tRNA 3’ intronic element. In some embodiments, the BHB-like motif comprises an RE site. In some embodiments, the BHB-like motif comprises a ligation motif. In some embodiments, the BHB-like motif comprises a 3’ RE site. In some embodiments, the BHB- like motif comprises: (i) a 5’ tRNA exonic element; (ii) a 3’ RE site; (iii) a tRNA 3’ intronic element; and (iv) a tRNA 3’ exonic element In some embodiments, the ligation motif(s) and RE site(s) support the formation of the BHB-like motif.

[0154] In some embodiments, the engineered tRNA sequence 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 engineered tRNA sequence 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 tRNA 3’ trailer downstream of the cargo. Exemplary 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 textTable 10. Exemplary 5’ Engineered tRNA SequencesTable 11. Exemplary 3’ Engineered tRNA Sequences

[0155] In some embodiments, the engineered tRNA sequence 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%, or100% identical to any one of SEQ ID NOs: 27, 109-118, 145-164, and 190-192. In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence of any one ofSEQ ID NOs: 27, 109-118, 145-164, and 190-192 with one or more mutations, such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the engineered tRNA sequencecomprises a polynucleotide sequence of any one of SEQ ID NOs: 27, 109-118, 145-164, and 190- 192. In some embodiments, the engineered tRNA sequence consists of a polynucleotide sequence of any one of SEQ ID NOs: 27, 109-118, 145-164, and 190-192.

[0156] In some embodiments, the engineered tRNA sequence 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, 119-128, 170-189, and 193-196. In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence of any one of SEQ ID NO: 28, 119-128, 170-189, and 193-196 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the engineered tRNA sequence comprises a polynucleotide sequence of any one of SEQ ID NOs: 28, 119-128, 170-189, and 193-196. In some embodiments, the engineered tRNA sequence consists of a polynucleotide sequence of any one of SEQ ID NOs: 28, 119-128, 170-189, and 193-196.

[0157] In some embodiments, the abundance of the resulting circular RNA from an engineered tRNA sequence disclosed herein is at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, or at least about 500% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the abundance of the resulting circular RNA from an engineered tRNA sequence comprising any one of SEQ ID NOs: 109-118, 145-164, and 190-192 in combination with any one of SEQ ID NOs: 119-128, 170-189, and 193-196 is at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, or at least about 500% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the abundance of the resulting circular RNA from an engineered tRNA sequence disclosed herein is at least about 10% higher compared to anengineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the abundance of the resulting circular RNA from an engineered tRNA sequence disclosed herein is at least about 100% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the abundance of the resulting circular RNA from an engineered tRNA sequence comprising any one of SEQ ID NOs: 109-118, 145-164, and 190-192 in combination with any one of SEQ ID NOs: 119-128, 170-189, and 193-196 is at least about 10% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the abundance of the resulting circular RNA from an engineered tRNA sequence comprising any one of SEQ ID NOs: 109-118, 145-164, and 190- 192 in combination with any one of SEQ ID NOs: 119-128, 170-189, and 193-196 is at least about 100% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively.

[0158] In some embodiments, the circularization efficiency of an engineered tRNA sequence disclosed herein is at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, or at least about 500% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the circularization efficiency of an engineered tRNA sequence comprising any one of SEQ ID NOs: 109-118, 145-164, and 190-192 in combination with any one of SEQ ID NOs: 119-128, 170-189, and 193-196 is at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, or at least about 500% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the circularization efficiency of an engineered tRNA sequence disclosed herein is at least about 10% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO:28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the circularization efficiency of an engineered tRNA sequence disclosed herein is at least about 100% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the circularization efficiency of an engineered tRNA sequence comprising any one of SEQ ID NOs: 109-118, 145-164, and 190-192 in combination with any one of SEQ ID NOs: 119-128,170-189, and 193-196 is at least about 10% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively. In some embodiments, the circularization efficiency of an engineered tRNA sequence comprising any one of SEQ ID NOs: 109-118, 145-164, and 190-192 in combination with any one of SEQ ID NOs: 119-128,170-189, and 193-196 is at least about 100% higher compared to an engineered tRNA sequence comprising SEQ ID NO: 27 and SEQ ID NO: 28 upstream and downstream of the cargo sequence, respectively.

[0159] In some embodiments, the engineered tRNA sequence comprises at least one cargo to be incorporated into the circular RNA after tRNA splicing. In some embodiments, the engineered tRNA sequence comprises one cargo. In some embodiments, the engineered tRNA sequence comprises two cargos. In some embodiments, the engineered tRNA sequence comprises three cargos. In some embodiments, the engineered tRNA sequence comprises four cargos. In some embodiments, the engineered tRNA sequence comprises five cargos.

[0160] In some embodiments, the engineered tRNA sequence comprises a cargo and the cargo encodes a therapeutic protein that can alleviate, reduce, prevent, and / or stabilize symptoms that result from an absence or defect in a protein in a cell or tissue of a subject In some embodiments, the engineered tRNA sequence comprises an IRES regulatory element that drives translation of the cargo into the therapeutic protein. In some embodiments, the therapeutic protein is wildtype or functional polycystin-1 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional polycystin-2 or a fragment thereof.

[0161] In some embodiments, the engineered tRNA sequence comprises a cargo, and the cargo encodes a therapeutic RNA. In some embodiments, the engineered tRNA sequence comprises a cargo, and the cargo encodes an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that affects spliceosome-mediated trans-splicing (see, Puttaraju et al.,Nature, 1999; U.S. Pat No. 6,013,487, and U.S. Pat. No. 6,083,702), an interfering RNA (RNAi) including siRNA, shRNA, or miRNA, a long noncoding RNA molecule, which mediates gene silencing (see, Sharp et al., Science, 2000), and any other non-translated RNA, such as a “guide” RNA (Gorman et al., PNAS, 1998; U.S. Pat No. 5,869,248), an aptamer, a naturally occurring circular RNA, a protein sponge, a RNA sponge (e.g., a miRNA sponge), a tough decoy, a proteinbinding RNA, an antisense RNA, a small activating RNA (saRNA), a self-amplifying RNA, a repressive RNA, a functional non-coding transfer RNA (tRNA), a ribosomal RNA (rRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a piwi-interacting RNA (piRNA), a Y- RNA, a 7SK RNA, a 7S RNA and the like as recognized by the art.

[0162] In some embodiments, the engineered tRNA sequence comprises a cargo, and the cargo encodes a miRNA inhibitor. In some embodiments, the miRNA inhibitor is a miRNA sponge. In some embodiments, the miRNA inhibitor is a tough decoy.

[0163] In some embodiments, the engineered tRNA sequence comprises a cargo, and the cargo encodes a miRNA sponge. As used herein, the term “miRNA sponge” refers to a nucleic acid molecule comprising one or more miKNA binding sites that are complementary to the targeted miRNA. In some embodiments, the miRNA sponge binds one or more miRNAs. In some embodiments, the miRNA sponge inhibits one or more miRNAs. In some embodiments, the miRNA sponge binds and inhibits one or more miRNAs.

[0164] In some embodiments, the engineered tRNA sequence comprises a cargo and the cargo encodes a miRNA sponge. In some embodiments, the miRNA sponge is specific for any miRNA known in the art, for example, any miRNA listed in miRBase (a public miRNA database). In some embodiments, the miRNA sponge is specific for 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-192, 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-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, the miRNA sponge is specific for miR-17, miR-21, miR-182, or miR-200.

[0165] In some embodiments, the engineered tRNA sequence comprises a cargo, and the cargo encodes a tough decoy. As used herein, the term “tough decoy” refers to 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.

[0166] In some embodiments, the engineered tRNA sequence comprises a cargo and the cargo encodes a tough decoy. In some embodiments, the tough decoy is specific for any miRNA known in the art, for example, any miRNA listed in miRBase (a public miRNA database). In some embodiments, the tough decoy is specific for 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-192, 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-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, the tough decoy is specific for miR-17, miR-21, miR-182, or miR- 200.

[0167] In some embodiments, the engineered tRNA sequence comprises a cargo, and the cargo encodes an RNA or protein that modulates alternative splicing of a pre-mRNA. In some embodiments, the alternative splicing results in exon inclusion or skipping, alternative splice-site selection, mutually exclusive exons, or intron retention. In some embodiments, the cargo encodes an RNA or protein that binds to an exon of the pre-mRNA. In some embodiments, the cargo encodes an RNA or protein that binds to an intron of the pre-mRNA. In some embodiments, the cargo encodes an RNA or protein that binds to an exon: intron junction of the pre-mRNA. In some embodiments, the cargo encodes an RNA or protein that binds to one or more components of a spliceosome. In some embodiments, the cargo encodes an RNA or protein that reduces expression of a pathogenic mRNA and / or protein. In some embodiments, the cargo encodes an RNA or protein that increases expression of a non-pathogenic mRNA and / or protein. In some embodiments, the cargo encodes an RNA or protein that provides a miRNA sponging effect.

[0168] In some embodiments, the cargo encodes an RNA or protein that binds to a polyadenylation signal sequence or complement thereof. In some embodiments, the cargo encodes an RNA or protein that reduces expression of a pathogenic mRNA and / or protein. In some embodiments, the cargo encodes an RNA or protein that increases expression of a non-pathogenic mRNA and / or protein.

[0169] In some embodiments, the recombinant nucleic acid molecule comprising the engineered tRNA sequence described herein further comprises one or more regulatory elements that regulate the expression of the circular RNA. In some embodiments, the regulatory element is a promoter, enhancer, RNA processing signal (e.g., polyadenylation or splicing signal), transcription initiation or termination signal, or internal ribosome entry site. In some embodiments, the regulatory element is a sequence that enhances translation efficiency (e.g., a Kozak consensus sequence), circular RNA stability, or circular RNA secretion. In some embodiments, the regulatory element is a sequence that enhances protein stability or protein secretion. In some embodiments, the recombinant nucleic acid molecule is operably linked to one or more regulatory elements. In some embodiments, the recombinant nucleic acid molecule is operably linked to a promoter or enhancer. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0170] In some embodiments, the recombinant nucleic acid molecule comprising the engineered tRNA sequence described herein further comprises at least one regulatory element, such as tRNA splicing elements, promoters, 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 tRNA splicing element. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0171] In some embodiments, the recombinant nucleic acid molecule comprising the engineered tRNA sequence described herein further 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 activatetranscription. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0172] In some embodiments, the recombinant nucleic acid molecule comprising the engineered tRNA sequence described herein further comprises at least one promoter. In some embodiments, the recombinant nucleic acid molecule comprises a promoter located 5’ of the tRNA 5’ leader. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0173] 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 tRNA 5’ leader. In some embodiments, the RNA polymerase II promoter comprises a polynucleotide sequence derived from 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 (ECAD) promoter, a mouse or human polycystic kidney disease 2 (PKD2) promoter, or a mouse kidney-specific cadherin (KSP-C) promoter. In some embodiments, the RNA polymerase II promoter is a CBA promoter. In some embodiments, the RNA polymerase II promoter is a CMV promoter. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0174] 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’ to the tRNA 5’ leader. In some embodiments, the RNA polymerase III promoter comprises a polynucleotide sequence of a H1 promoter, a H1.M11 promoter, a 7SK promoter, a U6 promoter, a U6+27 promoter, a U6+1 promoter, or an internal tRNA promoter. In some embodiments, the RNA polymerase III promoter is an internal tRNA promoter. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0175] In some embodiments, the recombinant nucleic acid molecule comprises a promoter selected from the group consisting of: 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 promotersinclude, 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. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0176] Exemplary promoters are provided in Table 12 below.Table 12. Exemplary Promoter Sequences

[0177] 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: 8-14 and 129-133. In some embodiments, the promoter comprises a polynucleotide sequence of any one of SEQ ID NOs: 8-14 and 129-133 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: 8-14 and 129-133. In some embodiments, the promoter consists of a polynucleotide sequence of any one of SEQ ID NOs: 8-14 and 129-133.

[0178] In some embodiments, the RNA polymerase III 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: 9-14. In some embodiments, the RNA polymerase in promoter comprises a polynucleotide sequence of any one of SEQ ID NOs: 9-14 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 any one of SEQ ID NOs: 9-14. In some embodiments, the RNA polymerase III promoter consists of a polynucleotide sequence of any one of SEQ ID NOs: 9-14.

[0179] 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 the 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 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a recombinant nucleic acid molecule comprising encoding a circular RNA without the promoter.

[0180] In some embodiments, the promoter is a tissue- or cell-specific promoter that induces transcription of a recombinant nucleic acid molecule encoding a circular RNA in a specific tissueor 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.

[0181] In some embodiments, a vector comprising the recombinant nucleic acid molecule comprises an external RNA polymerase III promoter (e.g., a U6+27 promoter), wherein the vector provides at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 100% higher circularization efficiency compared to a vector comprising an internal tRNA promoter sequence when transcribed in a cell. In some embodiments, a vector comprising the recombinant nucleic acid molecule comprises an external RNA polymerase in promoter (e.g., a U6+27 promoter), wherein the vector provides at least about 10% higher circularization efficiency compared to a vector comprising an internal tRNA promoter sequence when transcribed in a cell. In some embodiments, the internal tRNA promoter refers to the promoter of the unmodified trie scaffold. In some embodiments, the vector is an AAV vector.

[0182] In some embodiments, a vector comprising the recombinant nucleic acid molecule comprises an RNA polymerase III promoter (e.g., a U6+27 promoter), wherein the vector provides at least about 5%, at least about 10%. at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 100% higher circularization efficiency compared to a vector comprising an RNA polymerase II promoter when transcribed in a cell. In some embodiments, a vector comprising the recombinant nucleic acid molecule comprises an RNA polymerase III promoter (e.g., a U6+27 promoter), wherein the vector provides at least about 10% higher circularization efficiency compared to a vector comprising an RNA polymerase II promoter when transcribed in a cell. In some embodiments, the vector is an AAV vector.

[0183] In some embodiments, the recombinant nucleic acid molecule comprises a tissue-specific promoter. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0184] In some embodiments, the promoter is a liver-specific promoter, such as a TBG promoter or an Al AT promoter. In some embodiments, the promoter is a heart-specific promoter, such as a troponin T (TnT) promoter. In some embodiments, the promoter is a lung-specific promoter, such as a CC10 promoter, an SP-C promoter, or a FoxJl promoter. In some embodiments, the promoter is a central nervous system or brain-specific promoter, such as a synapsin promoter, a tyrosine hydroxylase promoter, or a CaMKII (Ca2+ / calmodulin-dependent protein kinase) promoter. In some embodiments, the promoter is a pancreas-specific promoter, such as an insulin promoter or an elastase-I promoter. In some embodiments, the promoter is a adipocyte specific promoter, such as an Ap2 promoter or an adiponectin promoter. In some embodiments, the promoter is a musclespecific promoter, such as a desmin promoter or an MHC promoter. In some embodiments, the promoter is an endothelial-cell specific promoter, such as an endothelin-I (ET -I) promoter or an Flt-I promoter. In some embodiments, the promoter is a retina-specific promoter, such as a VMD promoter.

[0185] In some embodiments, the promoter is a kidney-specific or cell-specific promoter. In some embodiments, the kidney-specific or cell-specific promoter is selected from any one of: a mouse polycystin 2 (PKD2) promoter, a human PKD2 promoter, a human uromodulin (UMOD) promoter, a mouse UMOD promoter, a fibrocystin promoter (PKHD1), a podocin (NPHS2) promoter, a nephrin (NPHS 1 ) promoter, a synaptopodin promoter, a WT1 promoter, a podocalyxin prompter, a PAX8 promoter, a gamma-glutamyltransferase (GGT) promoter, a sodium-glucose cotransporter-2 (SGLT2) promoter, a kidney androgen-regulated protein (KAP) promoter, a megalin promoter, a carbonic anhydrase II (CAII) promoter, an organic anion transporter 1 (OAT1 , SLC22A6) promoter, a sodium phosphate cotransporter 2a (NPT2a) promoter, an E-cadherin (ECAD) promoter, a glucose-6-phosphatase (G6Pase) promoter, a kidney-specific cadherin (KSP- C) promoter, an elongation factor- 1 alpha (EF-1α) promoter, a Na-K-Cl cotransporter 2 (NKCC2, SLC12A1) promoter, a WNK1 promoter, a WNK4 promoter, a v-type proton ATPase subunit Bl (ATP6V1B1) promoter, a thiazide-sensitive Na-Cl cotransporter (NCC) promoter, a calcium- sensing receptor (CaSR) promoter, an epithelial sodium channel (ENaC) promoter, a uromodulin promoter, an aquaporin 1 (AQP1) promoter, an aquaporin 2 (AQP2) promoter, a vasopressin receptor 2 (V2R) promoter, a transient receptor potential vanilloid 4 (TRPV4) promoter, a renin promoter, a parathyroid hormone receptor promoter, a smooth muscle alpha-actin (ACTA2) promoter, a COL4A1 promoter, a platelet-derived growth factor receptor beta (PDGFRfJ)promoter, a smooth muscle protein 22-a (SM22a) promoter, a smooth muscle myosin heavy chain (SM- MHC) promoter, a neuron-glial antigen 2 (NG2, chondroitin sulphate proteoglycan 4 (CSPG4)) promoter, a desmin promoter, a regulator of G-protein signaling- 5 (RGS5) promoter, a fibroblast-specific protein 1 (FSP1) promoter, a transcription factor 21 (TCF21) promoter, a COL1 A2 promoter, a COL3 A1 promoter, a vimentin promoter, a TIE2 promoter, a von Willebrand factor (vWF) promoter, a CD31 promoter, and a VE-cadherin promoter.

[0186] In some embodiments, the recombinant nucleic acid molecule comprises at least one RN A polymerase terminator. 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 (poly A) signal. In some embodiments, the RNA polymerase terminator comprises a polyT sequence. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0187] In some embodiments, the recombinant nucleic acid molecule comprises at least one RNA polymerase terminator, wherein the RNA polymerase terminator is a polyadenylation (poly A) signal sequence. The term “poly A signal” or “poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent 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 polyA, a human growth hormone polyA signal (hGHpA), a bovine growth hormone polyA signal (bGHpA), a rabbit β-globin polyA signal (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA signal known in the art In some embodiments, the polyA signal is a bovine growth hormone polyA signal (bGHpA). In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0188] In some embodiments, the recombinant nucleic acid molecule 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 sequence comprises at least 4, 5, 6, 7, or 8 thymine (T) nucleotides. In some embodiments, the polyT sequence comprises at least 4 T nucleotides. In some embodiments, the polyT sequence comprises4 T nucleotides (polyT4), 5 T nucleotides (polyT5), 6 T nucleotides (polyT6), 7 T nucleotides (polyT7), 8 T nucleotides (polyT8), 9 T nucleotides (polyT9), 10 T nucleotides (polyT10), 11 T nucleotides (polyT11), or 12 T nucleotides (polyT12). In some embodiments, the polyT sequence comprises 8 T nucleotides (polyT8). In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0189] Exemplary terminator sequences are listed in Table 13 below.Table 13. Exemplary RNA Polymerase Terminator Sequences

[0190] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase terminator, wherein the RNA polymerase terminator is a polyA signal. In some embodiments, the polyA signal is located 3’ of the tRNA 3’ trailer. In some embodiments, the polyA 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: 15. In some embodiments, the polyA signal comprises a polynucleotide sequence of SEQ ID NO: 15 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, the polyA signal comprises a polynucleotide sequence of SEQ ID NO: 15. In some embodiments, the polyA signal consists of a polynucleotide sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0191] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase terminator, wherein the RNA polymerase terminator is a polyT sequence. In some embodiments, the polyT sequence is located 3’ of the tRNA 3’ trailer. In some embodiments, thepolyT sequence 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 sequence comprises a polynucleotide sequence of TTTTTTTT. In some embodiments, the polyT sequence consists of a polynucleotide sequence of TTTTTTTT. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0192] In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase II promoter and a poly A signal. In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a poly A signal. In some embodiments, the recombinant nucleic acid molecule comprises a U6+27 promoter and a poly A signal. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0193] In some embodiments, the recombinant nucleic acid molecule comprises an H1.M11 promoter and a polyT sequence. In some embodiments, the recombinant nucleic acid molecule comprises an H1 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 U6+27 promoter and a poly A signal. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0194] In some embodiments, the recombinant nucleic acid molecule described herein comprises an untranslated region (UTR). UTRs are well-recognized in the art as playing crucial roles in post- transcriptional regulation of gene expression, including stability, cellular localization, and translation efficiency. In some embodiments, the recombinant nucleic acid molecule described herein comprises a 5’ UTR Exemplary 5’ UTRs for use in this invention include, but are not limited to, human beta globin, Xenopus laevis alpha globin, Xenopus laevis beta globin, human alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1 A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, and theadenovirus tripartite leader. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0195] In some embodiments, the recombinant nucleic acid molecule described herein comprises a 3’ UTR Exemplary 3’ UTRs for use in this invention include, but are not limited to, human beta globin, Xenopus laevis alpha globin, Xenopus laevis beta globin, human alpha globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNF alpha, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic ringspot virus, woodchuck hepatitis virus post-post-translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus, and Venezuelan equine encephalitis virus. In some embodiments, the recombinant nucleic acid molecule is in a vector, and the vector is an AAV vector.

[0196] In some embodiments, a vector comprising the recombinant nucleic acid molecule comprises a reporter gene to monitor the effectiveness of vector-mediated gene expression. Examples of reporter genes include, but are not limited to, fluorescent proteins of various colors (including green fluorescent protein (GFP) and red fluorescent protein (RFP)), E. colt β- galactosidase (LacZ), and various forms of luciferase (Luc)). In some embodiments, a vector comprising the recombinant nucleic acid molecule comprises a GFP reporter gene. In some embodiments, the vector is an AAV vector.

[0197] In some embodiments, the present disclosure provides an engineered tRNA sequence comprising: (i) a tRNA 5’ leader and a tRNA 3’ trailer; (ii) a tRNA 5’ exonic element and a tRNA 3’ exonic element; (iii) a tRNA 5’ intronic element and / or a tRNA 3’ intronic element; (iv) a 5’ RE site and a 3’ RE site; (v) a 5’ ligation motif and a 3’ ligation motif and (vi) a cargo. In some embodiments, the engineered tRNA sequence further comprises at least one spacer. In some embodiments, the engineered tRNA sequence further comprises a 5’ spacer and a 3’ spacer. In some embodiments, the cargo is encoded in an intronic region of the engineered tRNA sequence. In some embodiments, the tRNA is a precursor tRNA (i.e., pre-tRNA), which is a substrate for tRNA splicing. In some embodiments, the cargo is incorporated into a circular RNA following tRNA splicing.

[0198] In some embodiments, the present disclosure provides an engineered tRNA sequence comprising: (i) a tRNA 5’ leader and a tRNA 3’ trailer; (ii) a tRNA 5’ exonic element and a tRNA 3’ exonic element; (iii) a tRNA 3’ intronic element; (iv) a 5’ RE site and a 3’ RE site; (v) a 5’ ligation motif and a 3’ ligation motif and (vi) a cargo. In some embodiments, the engineered tRNA sequence further comprises at least one spacer. In some embodiments, the engineered tRNA sequence further comprises a 5’ spacer and a 3’ spacer. In some embodiments, the cargo is encoded in an intronic region of the tRNA. In some embodiments, the tRNA is a precursor tRNA (i.e., pre- tRNA), which is a substrate for tRNA splicing. In some embodiments, the cargo is incorporated into a circular RNA following tRNA splicing.

[0199] In some embodiments, the engineered tRNA sequence comprises, from 5’ to 3 ’, (i) a tRNA 5’ leader; (ii) a tRNA 5’ exonic element; (iii) a tRNA 5’ intronic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a cargo; (vii) a 3’ ligation motif; (viii) a 3’ RE site; (ix) a tRNA 3’ intronic element; (x) a tRNA 3’ exonic element; and (xi) a tRNA 3’ trailer. In some embodiments, the engineered tRNA sequence comprises a promoter 5’ to the tRNA 5’ leader. In some embodiments, the engineered tRNA sequence comprises a terminator 3’ to the tRNA 3’ trailer.

[0200] In some embodiments, the engineered tRNA sequence comprises, from 5 ’ to 3 ’ , (i) a tRNA 5’ leader; (ii) a tRNA 5’ exonic element; (iii) a tRNA 5’ intronic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (viii) a 3’ ligation motif; (xi) a 3’ RE site; (x) a tRNA 3’ intronic element; (xi) a tRNA 3’ exonic element; and (xii) a tRNA 3’ trailer. In some embodiments, the engineered tRNA sequence comprises a promoter 5’ to the tRNA 5’ leader. In some embodiments, the engineered tRNA sequence comprises a terminator 3’ to the tRNA 3’ trailer.

[0201] In some embodiments, the engineered tRNA sequence comprises, from 5’ to 3 ’, (i) a tRNA 5’ leader; (ii) a tRNA 5’ exonic element; (iii) a 5’ RE site; (iv) a 5’ ligation motif; (v) a cargo; (vi) a 3’ ligation motif; (vii) a 3’ RE site; (viii) a tRNA 3’ intronic element; (ix) a tRNA 3’ exonic element; and (x) a tRNA 3’ trailer. In some embodiments, the engineered tRNA sequence comprises a promoter 5’ to the tRNA 5’ leader. In some embodiments, the engineered tRNA sequence comprises a terminator 3’ to the tRNA 3’ trailer.

[0202] In some embodiments, the engineered tRN A sequence comprises, from 5 ’ to 3 ’ , (i) a tRNA 5’ leader; (ii) a tRNA 5’ exonic element; (iii) a 5’ RE site; (iv) a 5’ ligation motif; (v) a 5’ spacer;(vi) a cargo; (vii) a 3’ spacer; (viii) a 3’ ligation motif; (ix) a 3’ RE site; (x) a tRNA 3’ intronic element; (xi) a tRNA 3’ exonic element; and (xii) a tRNA 3’ trailer. In some embodiments, the engineered tRNA sequence comprises a promoter 5’ to the tRNA 5’ leader. In some embodiments, the engineered tRNA sequence comprises a terminator 3’ to the tRNA 3’ trailer.

[0203] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) a promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) a RNA polymerase terminator.

[0204] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) an RNA polymerase III promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) an RNA polymerase III terminator.

[0205] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) an RNA polymerase in promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) an RNA polymerase II terminator.

[0206] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) an RNA polymerase II promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) an RNA polymerase II terminator.

[0207] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) a U6+27 promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) a polyT sequence.

[0208] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) a U6+27 promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) a bGHpA signal.

[0209] In some embodiments, the recombinant nucleic acid molecule comprises, from 5’ to 3’, (i) a CMV promoter; (ii) a tRNA 5’ leader; (iii) a tRNA 5’ exonic element; (iv) a 5’ RE site; (v) a 5’ ligation motif; (vi) a 5’ spacer; (vii) a cargo; (viii) a 3’ spacer; (ix) a 3’ ligation motif; (x) a 3’ RE site; (xi) a tRNA 3’ intronic element; (xii) a tRNA 3’ exonic element; (xiii) a tRNA 3’ trailer; and (xiv) a bGHpA signal.

[0210] In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence that is 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 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: 200-232, 236, 238-240, and 264-276. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NOs: 200- 232, 236, 238-240, and 264-276 with one or more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, 72, 84, 96, or more mutations. In some embodiments, the recombinant nucleic acid molecule comprises a polynucleotide sequence of any one of SEQ ID NOs: 200-232, 236, 238-240, and 264-276. In some embodiments, the recombinant nucleic acid molecule consists of a polynucleotide sequence of any one of SEQ ID NOs: 200-232, 236, 238-240, and 264-276.Table 14. Exemplary Trie CassettesTable 15. Additional Exemplary CassettesNonviral and Viral Vectors

[0211] In some embodiments, a vector comprises the engineered tRNA sequence encoding a circular RNA described herein. In some embodiments, the vector is double-stranded. In some embodiments, the vector is single-stranded. In some embodiments, the vector is self- complementary. In some embodiments, the vector is a non- viral vector. In some embodiments, the vector is a viral vector.

[0212] In some embodiments, the vector comprises an engineered tRNA sequence encoding the circular RNAs described herein, wherein the vector is a vesicle-based or membrane-based vector. In some embodiments, the vector is a liposome or other similar vesicle (e.g., a lipid nanoemulsion or lipid nanoparticle). Liposomes are spherical vesicular structures consisting of a monolayer or multilamellar lipid bilayer surrounding an inner aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, the liposomes are anionic, neutral, or cationic. Methods of preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. patent No. 6,693,086, the teachings of which are incorporated herein by reference for the preparation of multilamellar vesicle lipids).

[0213] Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for the nucleic acid molecules described herein. In some embodiments, the lipid nanoparticles comprising the nucleic acid molecules described herein are nanostructured lipid carriers (NLCs), solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs), or lipid polymer nanoparticles (PLNs).

[0214] In some embodiments, the present disclosure provides a method of making a circular RNA, comprising: adding ribonucleotide triphosphates, inorganic pyrophosphatase, RNAase inhibitor, and an RNA polymerase to a vector in an appropriate reaction buffer, transcribing RNA from said vector, and allowing self-circularization of said transcribed RNA to produce a circular RNA of the invention.

[0215] In some embodiments, the present disclosure provides a method of producing circular RNA inside a cell, wherein the circular RNA is transcribed in the cytoplasm by a bacteriophage RNA polymerase or in the nucleus by host RNA polymerase II.

[0216] In some embodiments, a vector comprises the engineered tRNA sequence encoding a circular RNA described herein, and the vector is a viral vector. A number of viral based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors, γ-retroviral vectors, arenavirus vectors, alphavirus vectors, baculovirus vectors, vaccinia virus vectors, parvovirus vectors, poxvirus vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in other virology and molecular biology manuals. In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector.

[0217] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) vector encoding an engineered tRNA sequence, wherein the engineered tRNA sequence comprises: i) a tRNA 5’ leader and a tRNA 3’ trailer; ii) a tRNA 5’ exonic element and a tRNA 3’ exonic element; iii) a 5’ RE site and a 3’ RE site; iv) a tRNA 5’ intronic element and / or tRNA 3’ intronic element; v) a 5’ ligation motif and a 3’ ligation motif; and v) a cargo, wherein the cargo is incorporated into a circular RNA following tRNA splicing. In some embodiments, a spacer sequence is located 5’ and / or 3’ of the cargo. In some embodiments, an ITR sequence is located 5’ of the tRNA leader or promoter. In some embodiments, an ITR sequence is located 3’ of the tRNA trailer or RNA polymerase terminator.

[0218] In some embodiments, the AAV vector is a chimeric AAV vector, a single-stranded AAV vector, or a self-complementary AAV vector. In some embodiments, the viral vector is a specific AAV serotype. In some embodiments, the viral vector is a human AAV serotype.

[0219] The rAAV vector can be derived from any AAV serotype or variants thereof. In some embodiments, the rAAV vector is derived from AAV1 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV2 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV2i8 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV3 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV3-B or a variant thereof. In some embodiments, the rAAV vector is derived from AAV4 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV5 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV6 or a variant thereof. In someembodiments, the rAAV vector is derived from AAV7 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV8 or a variant thereof. In some embodiments, the rAAV vector is derived from AAVrh8 or a variant thereof. In some embodiments, the rAAV vector is derived from AAVrh8R or a variant thereof. In some embodiments, the rAAV vector is derived from AAV9 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV10 or a variant thereof. In some embodiments, the rAAV vector is derived from AAVrhlO or a variant thereof. In some embodiments, the rAAV vector is derived from AAV11 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV12 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV13 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV-DJ or a variant thereof. In some embodiments, the rAAV vector is derived from AAV LK03 or a variant thereof. In some embodiments, the rAAV vector is derived from AAVrh74 or a variant thereof. In some embodiments, the rAAV vector is derived from AAV44-9 or a variant thereof. In some embodiments, the rAAV vector is derived from or a variant thereof. In some embodiments, the rAAV vector is derived from avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any combination, derivative, or variant thereof. See also, Fields et al., Virology, Vol. 2, 4thedition; and Gao et al., (2004) J Virology 78:6381-6388; and Moris et al., Virology 33:375-383, each of which are herein incorporated by reference in their entirety.

[0220] The genomic sequences of various AAV serotypes, as well as the sequences of the native terminal repeats, Rep proteins, and capsid subunits are known in the art Such sequences may be found in the literature or in public databases such as the GenBank Database. See, e.g., GenBank Accession Numbers NC 044927, NC_002077, NC 001401, NC_001729, NC .001863, NC 001829, NC 001862, NC_000883, NC 001701, NC .001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, JOI 901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC 001540, AF513851, AF513852, AY530579. See also, e.g., Srivistava et al. (1983) J Virology 45:555; Chiorini et al. (1998) J. Virology 71 :6823; Chiorini et al (1999) J Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 1 1854; Moris et al. (2004) Virology 33:375-383; International Patent Publications WO2000 / 028061, WO 1999 / 061601, WO 1998 / 011244; WO 2019 / 094486; and U.S. Pat. No.6,156,303. These disclosures are incorporated by reference in their entirety.Table 16. Exemplary AAV Vectors

[0221] In some embodiments, the AAV vector is a self-complementary AAV (scAAV) vector.Self-complementary AAV vectors contain complementary sequences that spontaneously anneal to form a double-stranded DNA genome when entering a target cell.

[0222] In some embodiments, the viral vector is a pseudotyped AAV vector. A pseudotyped AAV vector, as used herein, refers to a genetically engineered vector derived from AAV that has been modified by incorporating the capsid from a different AAV serotype or virus. In some embodiments, a pseudotyped AAV vector comprises an AAV backbone derived from one serotype and an AAV capsid derived from another serotype. Specific examples of such pseudotyped AAV vectors, include, but are not limited to, vectors comprising an AAV2-derived genome with anAAV1 -derived capsid; an AAV2-derived genome with an AAV4-derived capsid; an AAV2- derived genome with an AAV5-derived capsid; an AAV2-derived genome with an AAV8-derived capsid; or an AAV2-derived genome with an AAV9-derived capsid. In some embodiments, the capsid is derived from another type of virus, such as a parvovirus.

[0223] In some embodiments, the viral vector is a chimeric viral vector (e.g., a chimeric AAV vector). As used herein, the term “chimeric viral vector” refers to a genetically engineered vector derived from a virus that has been modified by incorporating genetic material from other viruses or organisms. For example, in some embodiments, an AAV vector incorporates elements from different AAV serotypes or other viruses to overcome the limitations of the original AAV vector, such as pre-existing immunity in the host population. In some embodiments, the chimeric viralvector comprises viral portions from two or more viruses. In some embodiments, the chimeric viral vector is genetically engineered to increase transduction efficiency, selectivity, or a combination thereof. In some embodiments, the chimeric viral vector comprises capsid proteins from two or more serotypes. Chimeric viral vectors are further described in International Patent Appl. No. WO 2000 / 028004.

[0224] In some embodiments, the AAV vectors provided herein comprise a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector.

[0225] In some embodiments, the AAV vector comprises one or more AAV backbone elements. As used herein, an “AAV backbone element” refers to AAV genomic elements required for the bioactivity of AAV vectors. AAV backbone elements include, but are not limited to, packaging elements for the AAV vector to be assembled into an AAV particle, viral replication elements, reporter elements, and elements for expression of the circular RNA-encoding sequences described herein.

[0226] In some embodiments, the rAAV vectors described herein comprise one or more inverted terminal repeats (ITRs). An ITR is a specific DNA sequence found in adeno-associated viruses that is crucial for viral replication, packaging, and stability of the AAV genome. In some embodiments, the rAAV vector comprises a first ITR and a second ITR In some embodiments, the first ITR flanks the 5 ’ end of the recombinant nucleic acid molecule, and the second ITR flanks the 3’ end of the recombinant nucleic acid molecule.

[0227] In some embodiments, the 5’ and / or 3’ ITRs used in the vectors described herein have a wild-type nucleotide sequence. In some embodiments, the 5’ and / or 3’ ITRs used in the vectors described herein are not wild-type, but instead, comprise, e.g. , an insertion, deletion, or substitution of one or more nucleotides. AAV ITRs provided herein may be derived from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

[0228] In some embodiments, the 5’ and 3’ ITRs, which flank the recombinant nucleic acid molecule, are identical and derived from the same AAV serotype. In some embodiments, the 5’and 3’ ITRs, which flank the recombinant nucleic acid molecule, are different and / or derived from different AAV serotypes.

[0229] In some embodiments, the AAV vectors described herein comprise a Flip and / or Flop ITR The term “Flip ITR” or “Flop ITR,” as used herein, refers to the orientation of the ITR determined by the arrangement of its palindromic sequences. A single ITR comprises palindromic arms (A- A’, B-B’, and C-C’) that give the ITR its characteristic T-shape. The arrangement of the B-B’ and C-C’ palindromic sequences determines the orientation of the ITR, which can be either in the “Flip” or “Flop” orientation. In some embodiments, the AAV vector comprises a 5’ Flip ITR and a 3’ Flop ITR In some embodiments, the AAV vector comprises a 5’ Flop ITR and a 3’ Flip ITR In some embodiments, the AAV vector comprises a 5’ Flip ITR and a 3’ Flip ITR In some embodiments, the AAV vector comprises a 5’ Flop ITR and a 3’ Flop ITR In some embodiments, the 5’ and 3’ Flip and / or Flop ITRs are derived from the same AAV serotype. In some embodiments, the 5’ and 3’ Flip and / or Flop ITRs are derived from a different AAV serotype.

[0230] Exemplary ITRs are provided in Table 17 below.Table 17. Exemplary ITRs

[0231] In some embodiments, the 5’ ITR 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 leastabout 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 134. In some embodiments, the 5’ ITR comprises a polynucleotide sequence of SEQ ID NO: 134 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’ ITR comprises a polynucleotide sequence of SEQ ID NO: 134. In some embodiments, the 5’ ITR consists of a polynucleotide sequence of SEQ ID NO: 134.

[0232] In some embodiments, the 3’ ITR 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: 135. In some embodiments, the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 135 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’ ITR comprises a polynucleotide sequence of SEQ ID NO: 135. In some embodiments, the 3’ ITR consists of a polynucleotide sequence of SEQ ID NO: 135.

[0233] In some embodiments, the 3’ ITR 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: 199. In some embodiments, the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 199 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’ ITR comprises a polynucleotide sequence of SEQ ID NO: 199. In some embodiments, the 3’ ITR consists of a polynucleotide sequence of SEQ ID NO: 199.

[0234] In some embodiments, the 5’ ITR 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: 134; and the 3’ ITR 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: 135. In some embodiments, the 5’ ITR comprises a polynucleotide sequence of SEQ ID NO: 134 with oneor more mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations; and the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 135 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’ ITR comprises a polynucleotide sequence of SEQ ID NO: 134; and the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 135. In some embodiments, the 5’ ITR consists of a polynucleotide sequence of SEQ ID NO: 134; and the 3’ ITR consists of a polynucleotide sequence of SEQ ID NO: 135.

[0235] In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 80% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 85% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 91% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 92% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 93% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 94% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 95% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 96% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 98% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence that is at least about 99% identical to SEQ ID NO: 135. In some embodiments, the second ITR comprises a nucleic acid sequence of SEQ ID NO: 135.

[0236] In some embodiments, the ITR comprises any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired function such as replication, packaging, integration, and the like). In some embodiments, the ITR is an AAV ITR or a non-AAV ITR. For example, non-AAV ITR sequences such as those of other parvoviruses (e.g., canine parvovirus, mouse parvovirus, or human parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion,and / or addition. Further the ITR can be partially or completely synthetic, such as a “double-D sequence” as described in U.S. Pat. No. 5,468,745.

[0237] In some embodiments, the rAAV vector comprising the engineered tRNA sequence flanked by AAV ITRs can be constructed by directly inserting the engineered tRNA sequence into an AAV genome, e.g., into an excised AAV open reading frames, and certain portions of the AAV genome can optionally be deleted, as described in, e.g., WO 1993 / 003769; Kotin (1994) Human Gene Therapy 5: 793-801; Shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al. (1994) J. Exp. Med. 179: 1867-1875.

[0238] In some embodiments, AAV ITRs are excised from an AAV genome or from an AAV vector containing such ITRs and then inserted into the vector containing the engineered tRNA sequence using standard ligation techniques.

[0239] In some embodiments, the method described herein expresses the engineered tRNA sequence in one or more cell types in a kidney. In some embodiments, the method described herein expresses the engineered tRNA sequence in one or more regions or parts of a kidney. In some embodiments, the kidney cell is a mesangial cell. In some embodiments, the kidney cell is a podocyte. In some embodiments, the kidney cell is a glomerular endothelial cell. In some embodiments, the kidney cell is a parietal cell. In some embodiments, the kidney cell is a proximal tubule cell. In some embodiments, the kidney cell is a descending limb cell. In some embodiments, the kidney cell is a thin ascending limb cell and / or a thick ascending limb cell. In some embodiments, the kidney cell is a distal convoluted tubule cell. In some embodiments, the kidney cell is a connecting tubule cell. In some embodiments, the kidney cell is a collecting duct cell. In some embodiments, the kidney cell is a principal cell. In some embodiments, the kidney cell is a intercalated cell. In some embodiments, the kidney cell is a vascular smooth muscle cell. In some embodiments, the kidney cell is an endothelial cell. In some embodiments, the kidney cell is a fibroblast. In some embodiments, the kidney cell is an immune cell. In some embodiments, the immune cell is a natural killer cell, a T cell, a B cell, a macrophage, and / or a monocyte. In some embodiments, the AAV capsid has tropism for one or more regions or parts of a kidney. In some embodiments, the region or part of the kidney is the cortex. In some embodiments, the region or part of the kidney is the medulla. In some embodiments, the region or part of kidney is the renal column. In some embodiments, the region or part of the kidney is the renal pyramid. In someembodiments, the region or the part of the kidney is the distal tubule (e.g., a distal convoluted tubule). In some embodiments, the region or the part of the kidney is the proximal tubule (e.g., a proximal convoluted tubule). In some embodiments, the region or the part of the kidney is the collecting tubule. In some embodiments, the region or the part of the kidney is the Loop of Henle. In some embodiments, the region or the part of the kidney is the thick ascending limb. In some embodiments, the region or the part of the kidney is the glomerulus. In some embodiments, the region or part of the kidney is the renal pelvis. In some embodiments, the region or part of the kidney is the major calyx. In some embodiments, the region or part of kidney is the minor calyx. In some embodiments, the region or part of the kidney is the papillae. In some embodiments, the region or part of the kidney is the ureter. In some embodiments, the region or part of kidney is the proximal tubule.Recombinant Viral Particles

[0240] In some embodiments, the viral vector genomes described herein are packaged into viral particles, which are used to deliver the engineered tRNA sequence encoding the circular RNA to target cells. Viral particles of the present invention can be produced using any method known in the art, e.g., expression from a baculovirus (Brown et al. (1994) Virology 198:477-488). In some embodiments, the viral vector genome is an AAV vector genome packaged into an AAV particle.

[0241] In some embodiments, methods of producing AAV particles herein comprise packaging an engineered tRNA sequence encoding a circular RNA into an AAV vector. In some embodiments, methods of producing the AAV particle comprises: (a) contacting a cell with at least one nucleic acid molecule encoding: (i) a circular RNA; (ii) a replication gene; and (iii) a capsid gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging the nucleic acid encoding the circular RNA into the AAV particle.

[0242] In some embodiments, the present disclosure provides a method of expressing a circular RNA in a cell by introducing the AAV vector or AAV capsid or particle encoding the engineered tRNA sequence into the cell under conditions wherein the tRNA is transcribed and undergoes tRNA splicing to produce a byproduct circular RNA of interest. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a myoblast. In some embodiments, the cell is a kidney cell. In some embodiments, the cell is akidney cell derived from or localized in the ascending thin limb, connecting tubule, collecting duct, descending thin limb, distal tubule, proximal tubule, Loop of Henle, glomerulus, or thick ascending limb. In some embodiments, the kidney cell is an endothelial cell, a mesangial cell, a glomerular endothelial cell, a parietal cell, a proximal tubule cell, a descending limb cell, a thin and / or thick ascending limb cell, a convoluted tubule cell, a connecting tubule cell, a collecting duct cell, a fibroblast, an immune cell (e.g., a natural killer cell, a T cell, a B cell, a macrophage, or a monocyte), an intercalated cell, a parietal epithelial cell, a podocyte, a principal cell, a Schwann cell, a vascular smooth muscle cell, or a pericyte.

[0243] In some embodiments, the present disclosure provides a method of expressing a recombinant circular RNA in a tissue by introducing the AAV vector or AAV capsid or particle comprising the engineered tRNA sequence into the tissue under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing to produce a circular RNA of interest In some embodiments, the tissue is kidney tissue.

[0244] In some embodiments, the present disclosure provides an rAAV particle comprising the engineered tRNA sequence described herein, or the vector comprising the engineered tRNA sequence, and an AAV capsid. In general, an AAV capsid comprises three proteins, VP1, VP2 and VP3, each of which is encoded by splice variants of the AAV cap gene. VP2 and VP3 are truncated versions of VP1 and thus have sequences that also comprise VP1. Generally, the amino acid sequence of VP1 defines the serotype of the capsid. Thus, for example, if the VP1 capsid protein encodes for an AAV2 VP1 protein, AAV will be of the AAV2 serotype, whereas if the VP1 capsid protein encodes an AAV8 VP1 protein, the AAV will be of the AAV8 serotype.

[0245] In some embodiments, the AAV capsid comprises a VP1 capsid protein. In some embodiments, the AAV capsid comprises a VP2 capsid protein. In some embodiments, the AAV capsid comprises a VP3 capsid protein. In some embodiments, the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein, and / or a VP3 capsid protein. In some embodiments, the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein, and / or a VP3 capsid protein, wherein the capsid proteins of the rAAV particle are of the same serotype. In some embodiments, the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein, and a VP3 capsid protein, wherein the capsid proteins of the rAAV particle are of a different serotype.

[0246] In some embodiments, an AAV capsid protein (e.g., VP1, VP2 and / or VP3) is derived from a naturally occurring capsid protein. In some embodiments, an AAV capsid protein (e.g., VP1, VP2 and / or VP3) in the present rAAV particle is not a naturally occurring capsid protein.

[0247] In some embodiments, the variant capsid protein (e.g. , VP1 , VP2, and / or VP3) comprises one or more mutations, e.g., encoding one or more amino acid substitutions, amino acid deletions, or heterologous peptide insertions, compared to a corresponding wild-type, reference, or parent capsid protein such as a naturally occurring capsid protein from which it was derived. In some embodiments, the amino acid sequence of the AAV capsid protein (e.g., VP1, VP2, and / or VP3) is identical to the amino acid sequence of the wild-type, reference, or parent AAV capsid protein except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, e.g., except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the capsid variants increase tropism for a target tissue or cell compared to the tropism of the wild-type or parental capsid. In some embodiments, the capsid variant decreases tropism for an off-target tissue or cell compared to the tropism of the wild-type or parental capsid.

[0248] In some embodiments, the capsid protein (e.g., VP1, VP2, and / or VP3) is derived from the same serotype as the ITRs or a variant thereof. In some embodiments, the capsid protein (e.g., VP1, VP2, and / or VP3) is a different serotype than the ITRs.

[0249] In some embodiments, the capsid protein is a capsid protein of AAV1 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV2 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV2i8 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV3 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV3-B or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV4 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV5 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV6 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV7 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV8 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAVrh8 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAVrh8R or a variant thereof. In someembodiments, the capsid protein is a capsid protein of AAV9 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV10 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAVrh10 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV11 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV 12 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV13 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV-DJ or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV LK03 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAVrh74 or a variant thereof. In some embodiments, the capsid protein is a capsid protein of AAV44-9 or a variant thereof. In some embodiments, the capsid is an AAV9 capsid variant, such as AAV.kl3 or AAV.k20, as described in PCT Publication No. WO / 2024 / 206226, which is incorporated herein by reference in its entirety.

[0250] In some embodiments, the AAV vectors described herein comprise a modified capsid to alter the tropism of the vector. In some embodiments, the modified capsid comprises proteins or peptides of non-viral origin to alter the tropism of the vector. For example, in some embodiments, the capsid comprises a ligand of a particular receptor, or a receptor of a particular ligand, to target the vector to the cell type(s) expressing said receptor or ligand, respectively. In some embodiments, the modified capsid is altered to target the vector to a particular tissue.

[0251] In some embodiments, the AAV capsid is a variant of a parental wild-type AAV capsid, wherein the AAV capsid improves transfer and / or expression of the recombinant nucleic acid molecule described herein in one or more region(s) or part(s) of the kidney compared to the parental wild-type AAV capsid.

[0252] Exemplary AAV capsids with improved transfer and / or expression in kidney cells and kidney-related cells, as well as methods of using AAV particles comprising such AAV capsids to efficiently deliver a cargo to one or more region(s) or part(s) of kidney, is disclosed in PCT Publication No. WO / 2024 / 206226.

[0253] In some embodiments, the AAV capsid comprises one or more mutations, e.g., one or more amino acid substitutions, amino acid deletions, or heterologous peptide insertions, compared to a parental wild-type AAV capsid, such as a naturally-occurring capsid protein from which it was derived. In some embodiments, the amino acid sequence of the AAV capsid (e.g., VP1, VP2,and / or VP3) is identical to the amino acid sequence of the parental wild-type AAV capsid except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues, e.g., except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the AAV capsid is an AAV.kl3 capsid. In some embodiments, the AAV capsid is an AAV.k20 capsid. Exemplary AAV capsids with improved transfer and / or expression in kidney cells and kidney-related cells, as well as methods of using AAV particles comprising such AAV capsids to efficiently deliver a cargo to one or more regions or parts of kidney is disclosed in PCT Publication No. WO / 2024 / 206226.

[0254] In some embodiments, the rAAV comprises a vector comprising a transgene and an AAV capsid. In some embodiments, the AAV capsid is an AAV.kl3 capsid. In some embodiments, the AAV capsid comprises an amino acid sequence of SEQ ID NO: 137 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV capsid comprises the amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV capsid protein consists of the amino acid sequence of SEQ ID NO: 137 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0255] In some embodiments, the rAAV comprises a vector comprising a transgene and an AAV capsid. In some embodiments, the AAV capsid is an AAV.k20 capsid. In some embodiments, the AAV capsid comprises an amino acid sequence of SEQ ID NO: 138 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV capsid comprises the amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136. In some embodiments, the AAV capsid protein consists of the amino acid sequence of SEQ ID NO: 138 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0256] Exemplary amino acid sequences for AAV9 capsid variants are provided below. For wildtype AAV9, bolded residues indicate position 452-458 in the AAV9 VP1 subunit.Table 18. Exemplary AAV Capsids

[0257] In some embodiments, the AAV capsid transduces kidney cells. In some embodiments, the AAV capsid transduces one or more region(s) or part(s) of a kidney. In some embodiments, the AAV capsid has tropism for kidney cells. In some embodiments, the AAV capsid has tropism for one or more region(s) or part(s) of a kidney. In some embodiments, the region or part of the kidney is adrenal glands. In some embodiments, the region or part of the kidney is the cortex. In some embodiments, the region or part of the kidney is the medulla. In some embodiments, the region or part of kidney is the renal column. In some embodiments, the region or part of the kidney is the renal pyramid. In some embodiments, the region or part of the kidney is the renal pelvis. In some embodiments, the region or part of the kidney is the major calyx. In some embodiments, the region or part of kidney is the minor calyx. In some embodiments, the region or part of the kidneyis the papillae. In some embodiments, the region or part of the kidney is the ureter. In some embodiments, the region or part of kidney is the proximal tubule (e.g., proximal convoluted tubule) in the cortex. In some embodiments, the region or part of kidney is the distal tubule (e.g., a distal convoluted tubule). In some embodiments, the region or part of kidney is the collecting tubule. In some embodiments, the region or part of kidney is the Loop of Henle. In some embodiments, the region or part of kidney is the glomerulus. In some embodiments, the AAV capsid is an AAV.kl3 capsid. In some embodiments, the AAV capsid is an AAV.k20 capsid.

[0258] In some embodiments, the AAV capsid variant transduces one or more kidney cells, kidney-derived cell types, and / or kidney-related cell types more efficiently than that of the wildtype AAV capsid. In some embodiments, the AAV capsid variant increases or improves transduction efficiency in the kidney 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 the wild-type AAV capsid.

[0259] In some embodiments, the AAV.kl3 capsid transduces one or more kidney cells, kidney- derived cell types, and / or kidney-related cell types more efficiently than that of the wild-type AAV9 capsid. In some embodiments, the AAV.kl3 capsid increases or improves transduction efficiency in the kidney 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 the wild-type AAV9 capsid.

[0260] In some embodiments, the AAV.k20 capsid transduces one or more kidney cells, kidney- derived cell types, and / or kidney-related cell types more efficiently than that of the wild-type AAV9 capsid. In some embodiments, the AAV.k20 capsid increases or improves transduction efficiency in the kidney 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 about70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to the wild-type AAV9 capsid.

[0261] The rAAV particles described herein may be produced using any suitable method known in the art For example, a host cell (e.g., a mammalian cell) may be engineered to stably express the necessary components for AAV particle production. This can be achieved by integrating a plasmid (or multiple plasmids) comprising AAV rep and cap genes and a selectable marker, such as an antibiotic (e.g., neomycin or ampicillin) resistance gene, into the genome of the cell. The cell can be, e.g., an insect or mammalian cell, which can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the rAAV vector comprising the 5’ and 3’ AAV ITRs. The use of a selectable marker allows for large-scale production of the rAAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells. As yet another non-limiting example, both the viral vector containing the 5’ and 3’ AAV ITRs and the rep and cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the rAAV.

[0262] In some embodiments, host cells containing the rAAV vectors described herein are rendered capable of providing AAV helper functions to replicate and encapsulate the recombinant nucleic acid molecule provided herein flanked by the AAV ITRs to produce rAAV particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the rAAV vectors. In some embodiments, AAV helper functions include one or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.

[0263] AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the rAAV vector. For example, AAV helper constructs can be used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for productive AAV infection. Typically, AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. The AAV helper constructs can be in the form of, e.g., a plasmid, phage, transposon, cosmid, virus, or virion.

[0264] A helper virus for AAV refers to a virus that allows AAV to be replicated and packaged by a host cell. A helper virus provides helper functions that allow for the replication of AAV. A number of such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV). Examples of adenovirus helper functions for the replication of AAV include E1A functions, E1B functions, E2A functions, VA functions, and E4orf6 functions.

[0265] A preparation of AAV is said to be substantially free of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 102:1; at least about 104:1, at least about 106:1; or at least about 108:1. Preparations can also be free of equivalent amounts of helper virus proteins (i.e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form). Viral and / or cellular protein contamination can generally be observed as the presence of Coomassie staining bands on SDS gels (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2 and VP3).

[0266] In some embodiments, the AAV replication and capsid genes are provided by any method known in the art. In some embodiments, the AAV replication and capsid genes are on a single vector. In some embodiments, the AAV replication and capsid genes are on separate vectors. In some embodiments, the vector(s) comprising the AAV replication and / or capsid genes are viral (e.g., AAV, herpesvirus, or EBV) or non- viral vectors. In some embodiments, the vectors) comprising the AAV replication and / or capsid genes are AAV vectors. In some embodiments, the vector(s) comprising the AAV replication and / or capsid genes are chimeric AAV vectors (e.g., inserted into the E1A or E3 regions of a deleted adenovirus vector).

[0267] In some embodiments, the host cell is also capable of providing or is provided with non AAV-derived functions or “accessory functions” to produce rAAV particles. Accessory functions are non-AAV-derived viral and / or cellular functions upon which AAV is dependent for itsreplication, such as non- AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. In some embodiments, viral-based accessory functions can be derived from a known helper virus.

[0268] A variety of host cells can be used to produce rAAV particles described herein. Suitable host cells for producing AAV particles from the nucleic acid molecules and AAV vectors provided herein include microorganisms, yeast cells, insect cells, and mammalian cells. Typically, such cells can be, or have been, used as recipients of a heterologous nucleic acid molecule and can grow in, e.g., suspension culture and a bioreactor.

[0269] In some embodiments, the cell is a mammalian host cell, for example, a HEK293, HEK293T, A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.

[0270] In some embodiments, the cell is an insect cell, e.g., an Sf9, SF21, SF900+-, or Drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyxmori cell lines, Trichoplusia ni cell lines, such as High Five cells, or Lepidoptera cell lines, such as Ascalapha odorata cell lines. In some embodiments, insect cells are cells from the insect species, which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38. For example, large scale production of recombinant AAV in cells, including Sf9 insect cells, has been described by Kotin RM. Hum Mol Genet 20 (Rl): R2-R6 (2011) doi: 10.1093 / hmg / ddrl41. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith. A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures. Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex. (1986); King, L.A. and RD. Possee, The baculovirus expression system. Chapman and Hall, United Kingdom (1992); O’Reilly, D.R, L.K. Miller, V.A. Luckow, Baculovirus Expression Vectors: A Laboratory Manual. New York (1992); W.H Freeman and Richardson, C.D., Baculovirus Expression Protocols. Methods in Molecular Biology, volume 39 (1995).

[0271] In some embodiments, as a result of the infection of the host cell with a helper virus and / or an accessory function vector, a recombinant AAV particle is produced, and this rAAV particle is infectious, replication-defective, and includes an AAV protein shell that encapsulates a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs.

[0272] rAAV particles can be purified from the host cell using a purification method known in the art, such as chromatography, CsCl gradients, and other methods as described, for example, in U.S. Pat Nos. 6,989,264 and 8,137,948 and WO 2010 / 148143. In some embodiments, residual helper virus can be inactivated using known methods, e.g., by heating.Pharmaceutical Compositions

[0273] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the engineered tRNA sequence, the recombinant nucleic acid molecule, the vector, or the rAAV particle described herein. In some embodiments, the pharmaceutical composition is used in the prevention and / or treatment of a disease, disorder, or condition in a subject

[0274] In some embodiments, the engineered tRNA sequence, the vector, or the rAAV particle described herein are formulated as one or more pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises an engineered tRNA sequence encoding a circular RNA and a pharmaceutically acceptable carrier or excipient.

[0275] The pharmaceutical compositions described herein are formulated according to known methods to prepare pharmaceutically useful compositions, whereby the circular RNA or viral particle comprising the circular RNA is combined in a mixture with a pharmaceutically acceptable carrier or excipient Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994. The precise nature of the carrier or excipient will depend on the route of administration, which may be oral, or by injection, e.g., intravenous, subcutaneous, or intramuscular.

[0276] In some embodiments, viral particles comprising the circular RNAs described herein are formulated as one or more pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises a viral particle comprising a viral vector or genomeencoding a circular RNA described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises an AAV particle comprising an AAV vector or genome encoding a circular RNA described herein and a pharmaceutically acceptable carrier or excipient.

[0277] In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier. Illustrative pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers that may be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, such as vegetable oils and injectable organic esters.

[0278] In some embodiments, the pharmaceutical composition comprises at least one additional pharmaceutically acceptable excipient Non-limiting examples of a pharmaceutically acceptable excipient include arginine, arginine sulfate, citric acid, glycerol, hydrochloric acid, mannitol, methionine, polysorbate, sodium chloride, sodium citrate, sodium hydroxide, sorbitol, sucrose, trehalose, and / or water.

[0279] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include both acid and base addition salts. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2- oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. In some embodiments, organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. Suitable salts are further described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

[0280] In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable buffer. Non-limiting examples of suitable buffers include acetate, citrate, histidine, phosphate, histidine, Tris, tartrate, glycine, glutamate, and succinate buffers. In some embodiments, the pharmaceutical composition comprises an aqueous carrier comprising a pharmaceutically acceptable buffer. In some embodiments, the pharmaceutical composition comprises a salt and / or powder, such as, a freeze-dried, lyophilized, dehydrated, and / or cryodesiccated composition comprising a pharmaceutically acceptable buffer (e.g., sodium citrate).

[0281] In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable surfactants, emulsifying agents and lubricants, preservative agents, cryoprotective agents, antioxidants, clarifying agents, suspending agents, thickening agents, chelating agents, wetting agents, dispersing agents, stabilizers, isotonic agents, tonicity-adjusting agents, coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants.

[0282] In some embodiments, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0283] In some embodiments, the pharmaceutical compositions described herein are formulated in one of the following dosage forms: an intravenous dosage form, an intramuscular dosage form, an intraperitoneal dosage form, a subcutaneous dosage form, an oral dosage form, an intranasal dosage form, a suppository dosage form, an intradermal dosage form, a parenteral dosage form, a retro-ureteral dosage form, an intrathecal dosage form, a direct dosage form, or a topical dosage form. In some embodiments, the pharmaceutical compositions described herein are formulated in an intravenous dosage form. In some embodiments, the pharmaceutical compositions described herein are formulated in a subcutaneous dosage form. In some embodiments, the pharmaceutical compositions described herein are formulated in an intramuscular dosage form.

[0284] The pharmaceutical compositions provided herein can be provided as a controlled release or sustained release system. In some embodiments, a pump may be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987); Buchwald et al., Surgery 88: 507-16 (1980); and Saudek et al, N. Engl. J. Med. 321: 569-74 (1989)). In some embodiments, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent or a composition provided herein (see, e.g. , Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drue Bioavailability. Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23: 61-126 (1983); Levy et al., Science 228: 190-92 (1985); During et al., Ann. Neurol. 25: 351-56 (1989); Howard et al., J. Neurosurg. 71: 105-12 (1989); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), polyglycolides (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly (lactide-co-glycolides) (PLGA), and polyorthoesters. In some embodiments, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the kidney, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, Science 249: 1527-33 (1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy &Oncology 39: 179-89 (1996); Song et al., PDA J. of Pharma. Sci. &Tech. 50: 372-97 (1995); Cleek et al., Pro. Int’l. Symp. Control. Rel. Bioact Mater. 24: 853-54 (1997); and Lam etal., Proc. Int’l. Symp. Control Rel. Bioact. Mater. 24: 759- 60 (1997)).

[0285] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 18th edition.

[0286] In order for the pharmaceutical compositions to be used for in vivo administration, they are preferably sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions provided herein generally can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.Methods and Uses

[0287] In some embodiments, the engineered tRNA sequence, the recombinant nucleic acid molecule, the vector or rAAV particle described herein, or pharmaceutical composition thereof, are used in the prevention and / or treatment of a disease, disorder, or condition in a subject.

[0288] In some embodiments, the present disclosure provides a method of expressing a circular RNA in a subject, comprising administering an effective amount of the engineered tRNAsequences of the present disclosure, the vectors or rAAV particles of the present disclosure, or pharmaceutical compositions of the present disclosure, wherein the effective amount is an amount that reduces at least one symptom of disease or condition in the subject.

[0289] In some embodiments, the present disclosure provides a method of expressing a circular RNA in a target cell or tissue. In some embodiments, the method comprises introducing into the cell or the tissue the engineered tRNA sequences of the present disclosure, the vectors or rAAV particles of the present disclosure, or pharmaceutical compositions of the present disclosure, under conditions wherein the circular RNA molecule is produced.

[0290] In some embodiments, 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 shortterm (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. The effective amount of the pharmaceutical compositions administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex, and diet of the patient; the timing of administration and route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts. Dosage amount and interval can be adjusted individually to provide plasma levels of the compound(s) that are sufficient to maintain therapeutic or prophylactic effect In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of active compound(s) cannot be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

[0291] In some embodiments, the engineered tRNA sequence, the vector or rAAV particle described herein, or pharmaceutical composition thereof, is used in the prevention and / or treatment of a kidney disease. In some embodiments, the kidney disease is chronic kidney disease. In some embodiments, the kidney disease is end-stage renal disease. In some embodiments, the kidney disease is a genetic kidney disease. In some embodiments, the kidney disease is autosomal dominant polycystic kidney disease (ADPKD). In some embodiments, the kidney disease is autosomal recessive PKD (ARPKD). In some embodiments, the kidney disease is ADPKD1. In some embodiments, the kidney disease is ADPKD2. In some embodiments, the kidney disease is autosomal dominant tubulointerstitial kidney disease (ADTKD).

[0292] In some embodiments, the vector or viral particle provided herein is administered at the concentration in vector genomes per milliliter (vg / mL) in the range of about 102vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of 102vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of 102vg / mL to about 1010vg / mL. In some embodiments, the concentration is in the range of 102vg / mL to about 105vg / mL. In some embodiments, the concentration is in the range of 105vg / mL to about 1010vg / mL. In some embodiments, the concentration is in the range of 105vg / mL to about 108vg / mL. In some embodiments, the concentration is in the range of 107vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of 107vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of 107vg / mL to about 1010vg / mL. In some embodiments, the concentration is in the range of 109vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of 109vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of 1011vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of 1011vg / mL to about 1013vg / mL. In some embodiments, the concentration is about 105vg / mL. In some embodiments, the concentration is about 106vg / mL. In some embodiments, the concentration is about 107vg / mL. In some embodiments, the concentration is about 108vg / mL. In some embodiments, the concentration is about 109vg / mL. In some embodiments, the concentration is about 1010vg / mL. In some embodiments, the concentration is about 1011vg / mL. In some embodiments, the concentration is about 1012vg / mL. In some embodiments, the concentration is about 1013vg / mL. In some embodiments, the concentration is about 1014vg / mL. In some embodiments, the concentration is about 1015vg / mL.

[0293] In some embodiments, the vector or viral particle provided herein is administered in a volume between about 0.1 mL and about 80 mL, for example between about 1 mL and about 80 mL, between about 10 mL and about 80 mL, between about 20 mL and about 80 mL, between about 40 mL and about 80 mL, between about 60 mL and about 80 mL, between about 80 mL and about 100 mL, between about 100 mL and about 200 mL, between about 200 mL and about 250 mL, between about 200 mL and about 300 mL, between about 300 mL and about 400 mL, between about 400 mL and about 500 mL, between about 0.3 mL and about 30 mL, between about 0.5 mL and about 30 mL, between about 1 mL and about 30 mL, between about 5 mL and about 30 mL, between about 0.1 mL and about 20 mL, between about 0.1 mL and about 10 mL, between about 0.1 mL and about 5.0 mL, between about 0.1 mL and about 2.0 mL, between about 0.1 mL and about 1.0 mL, between about 0.1 mL and about 0.8 mL, between about 0.1 mL and about 0.6 mL, between about 0.1 mL and about 0.4 mL, between about 0.1 mL and about 0.2 mL, between about 0.2 mL and about 1.0 mL, between about 0.2 mL and about 0.8 mL, between about 0.2 mL and about 0.6 mL, between about 0.2 mL and about 0.4 mL, between about 0.4 mL and about 1.0 mL, between about 0.4 mL and about 0.8 mL, between about 0.4 mL and about 0.6 mL, between about 0.6 mL and about 1.0 mL, between about 0.6 mL and about 0.8 mL, between about 0.8 mL and about 1.0 mL, or about 0.1 mL, about 0.2 mL, about 0.4 mL, about 0.6 mL, about 0.8 mL, about 1.0 mL, about 5.0 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, about 125 mL, about 150 mL, about 175 mL, about 200 mL, about 225 mL, about 250 mL, about 275 mL, about 300 mL, about 325 mL, about 350 mL, about 375 mL, about 400 mL, about 425 mL, about 450 mL, about 475 mL, about 500 mL, or more.

[0294] In some embodiments, the vector or viral particle provided herein is administered to a subject at a dose of 1x102to 1x1020vector genomes (vg). In some embodiments, the dose is about 1x102to 1x1018vg. In some embodiments, the dose is about 1x102to 1x1016vg. In some embodiments, the dose is about lxl C^to 1x1014vg. In some embodiments, the dose is about 1x102to 1x1016vg. In some embodiments, the dose is about 1x102to 1x1010vg. In some embodiments, the dose is about 1x102to 1x1016vg. In some embodiments, the dose is about 1x102to 1x105vg. In some embodiments, the dose is about 1x105to 1x1012vg. In some embodiments, the dose is about 1x105to 1x1010vg. In some embodiments, the dose is about 1x105to 1x108vg. In some embodiments, the dose is about 1x107to 1x1018vg. In some embodiments, the dose is about 1x107to 1x1016vg. In some embodiments, the dose is about 1x107to 1x1014vg. In some embodiments, the dose is about 1x107to 1x1012vg. In some embodiments, the dose is about 1x107to 1x1010vg. In some embodiments, the dose is about 1x107to 1x108vg. In some embodiments, the dose is about 1x109to 1x1018vg. In some embodiments, the dose is about 1x109to 1x1016vg. In some embodiments, the dose is about 1x109to 1x1014vg. In some embodiments, the dose is about 1x109to 1x1012vg. In some embodiments, the dose is about 1x109to 1x1010vg. In some embodiments, the dose is about 1x1011to 1x1018vg. In some embodiments, the dose is about 1x1011to 1x1016vg. In some embodiments, the dose is about 1x1011to 1x1014vg. In some embodiments, the dose is about 1x1011to 1x1012vg. In some embodiments, the dose is about 1x1013to 1x1018vg. In some embodiments, the dose is about 1x1013to 1x1016vg. In some embodiments, the dose is about 1x1013to 1x1014vg. In some embodiments, the dose is about 1x1015to 1x1018vg. In some embodiments, the dose is about 1x1015to 1x1016vg. In some embodiments, the dose is about 1x105vg. In some embodiments, the dose is about 1x106vg. In some embodiments, the dose is about 1x107vg. In some embodiments, the dose is about 1x108vg. In some embodiments, the dose is about 1x109vg. In some embodiments, the dose is about 1x1010vg. In some embodiments, the dose is about 1x1011vg. In some embodiments, the dose is about 1x1012vg. In some embodiments, the dose is about 1x1013vg. In some embodiments, the dose is about 1x1014vg. In some embodiments, the dose is about 1x1015vg. In some embodiments, the dose is about 1x1016vg. In some embodiments, the dose is about 1x1017vg. In some embodiments, the dose is about 1x1018vg. In some embodiments, the dose is about 1x1019vg. In some embodiments, the dose is about 1x1020vg. In some embodiments, the dose is the total dose. In some embodiments, the dose is for each administration.

[0295] In some embodiments, the dose of the pharmaceutical composition comprising the circular RNAs described herein is between about 102to about 1020particles to a subject in need thereof. In some embodiments, the dose of the pharmaceutical composition comprising the circular RNAs described herein is between about 105to about 1015particles to a subject in need thereof. In some embodiments, the dose of the pharmaceutical composition comprising the circular RNAs described herein is between about 108to about 1013particles to a subject in need thereof. In some embodiments, the dose of the pharmaceutical composition comprising the circular RNAs described herein is at least about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013, about 1014, about 1015, about 1016, about 1017,about 1018, about 1019, about 1020particles, or any range therebetween to a subject in need thereof. In some embodiments, the particles are lipid nanoparticles.

[0296] In some embodiments, the nucleic acid molecule encoding the circular RNA described herein, or pharmaceutical composition thereof, is delivered to a subject in need thereof by methods known in the art. Such methods include, but are not limited to, transfection (e.g., lipid-mediated cationic polymers, calcium phosphate, dendrimers), electroporation or other methods of disrupting a membrane (e.g., nuclear transfection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, particle bombardment (“gene gun”), cell extrusion, light transfection, protoplast fusion, puncture infection, magnetic transfection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, or any combination thereof.

[0297] In some embodiments, the pharmaceutical compositions described herein are administered to subjects by one or more administration routes. Possible administration routes include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, oral, intranasal, intrasynovial, parenteral, intrapulmonary, transdermal, intrathecal, topical, and intralesional routes. In some embodiments, the administration route is selected from the group consisting of: intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, retroureteral administration, and oral administration. In some embodiments, the pharmaceutical composition is administered intravenously to a subject (e.g., by intravenous infusion). In some embodiments, the pharmaceutical composition is administered by retrograde ureteral infusion. In some embodiments, the pharmaceutical composition is administered through the renal vein. In some embodiments, the pharmaceutical composition is administered through the renal artery. In some embodiments, the pharmaceutical composition is administered using a catheter. In some embodiments, the pharmaceutical composition is administered subcutaneously to a subject. In some embodiments, the pharmaceutical composition is administered retro-ureterally to a subject In some embodiments, the pharmaceutical composition is administered intrathecally to a subject. In some embodiments, the pharmaceutical composition is administered intramuscularly to a subject In some embodiments, administration route is direct, local, or systemic.

[0298] For prevention and treatment purposes, the pharmaceutical compositions described herein can be administered to a subject in a single bolus delivery, via continuous delivery (e.g., continuoustransdermal delivery) over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily, weekly, monthly, or yearly basis).Dosages may be administered in single or multiple administrations, including, e.g., multiple weekly, bi-weekly, monthly, or yearly administrations. In some embodiments, a single dose of the pharmaceutical composition comprising the circular RNA is administered to a subject in need thereof. In some embodiments, the subject receives two or more doses of the pharmaceutical composition comprising the circular RNA. In some embodiments, the subject receives two or more doses of the pharmaceutical composition comprising the circular RNA, wherein consecutive doses are separated by at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least a week. In some embodiments, the subject receives two or more doses of the pharmaceutical composition comprising the circular RNA, wherein consecutive doses are separated by a period of at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least one month, at least two months, or at least three months. In some embodiments, two or more doses of the pharmaceutical composition comprising the circular RNA are administered to a subject in need thereof separated by a period of about one week to about two weeks, about two weeks to about four weeks, about one month to about two months, about two months to about four months, or about one month to about six months. In some embodiments, the duration of the administration of the pharmaceutical composition comprising the circular RNA is at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least one year, at least two years, at least three years, at least four years, or at least five years. In some embodiments, administration of the pharmaceutical composition comprising the circular RNA is on an irregular basis as indicated by monitoring clinical symptoms of the disease in a subject in need thereof. In some embodiments, administration of the pharmaceutical composition comprising the circular RNA is up to the lifetime of a subject in need thereof.

[0299] In some embodiments, dosages are administered to multiple organs to a subject in need thereof. For example, in some embodiments, a dose of the pharmaceutical composition comprising the circular RNA described herein is administered to a kidney using retroureteral administrationfollowed by a subsequent dose of the pharmaceutical composition administered to the other kidney using retroureteral administration.

[0300] In some embodiments, the AAV vector encoding a circular RNA, or pharmaceutical composition thereof, is introduced into cells ex vivo for the purpose of administering the modified cell to a subject in need thereof. In some embodiments, the cells are removed from a subject in need thereof and the nucleic acid molecule encoding the circular RNA, or pharmaceutical composition thereof, is introduced to the cells, and then the modified cells are administered back to the subject in need thereof. In some embodiments, the cells are removed from a “donor subject,” or other suitable source, and the nucleic acid molecule encoding the circular RNA, or pharmaceutical composition thereof, is introduced to the cells, and then the modified cells are administered to a subject in need thereof (i.e., a “recipient subject”). Methods of manipulating a subject’s cells ex vivo are known in the art (see, e.g., U.S. patent No. 5,399,346). Suitable cells for ex vivo nucleic acid delivery are described herein. Dosages of the cells to administer to a subject will vary upon the age, condition, and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 102to about 108cells or at least about 103to about 106cells will be administered per dose in a pharmaceutically acceptable carrier.

[0301] In some embodiments, the engineered tRNA sequences of the present disclosure, the vectors or rAAV particles of the present disclosure, or the pharmaceutical compositions of the present disclosure are administered in combination with additional therapeutic composition(s). In some embodiments, the pharmaceutical compositions disclosed herein and the additional therapeutic composition(s) are administered simultaneously. In some embodiments, the pharmaceutical compositions disclosed herein are administered before the additional therapeutic compositions). In some embodiments, the pharmaceutical compositions disclosed herein are administered after the additional therapeutic composition(s).

[0302] In some embodiments, the circular RNA described herein is expressed in a cell or tissue of the subject following administration of the pharmaceutical composition. In some embodiments, the expression of the circular RNA is increased in a cell or tissue of the subject compared to baseline (i.e. before treatment with the pharmaceutical composition comprising the engineered tRNA sequence described herein). In some embodiments, the expression of the circular RNA isincreased by at least about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 500-fold, about 1000-fold in a cell or tissue of the subject compared to baseline.

[0303] In some embodiments, the circular RNA reduces expression of at least one pathogenic protein. In some embodiments, the circular RNA directly reduces expression of at least one pathogenic protein. In some embodiments, the circular RNA indirectly reduces expression of at least one pathogenic protein. In some embodiments, the circular RNA reduces expression of at least one pathogenic protein by at least about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90- fold, about 100-fold, about 500-fold, about 1000-fold, or more in a transduced target cell or tissue compared to an untransduced target cell or tissue.

[0304] In some embodiments, the circular RNA increases expression of a non-pathogenic pre- mRNA or mRNA isoform. In some embodiments, the circular RNA directly increases expression of a non-pathogenic pre-mRNA or mRNA isoform. In some embodiments, the circular RNA indirectly increases expression of a non-pathogenic pre-mRNA or mRNA isoform. In some embodiments, the circular RNA increases expression of a non-pathogenic pre-mRNA or mRNA isoform by at least about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 500-fold, about 1000-fold, or more in a transduced target cell or tissue compared to an untransduced target cell or tissue.

[0305] In some embodiments, the circular RNA increases expression of a non-pathogenic protein isoform. In some embodiments, the circular RNA directly increases expression of a non- pathogenic protein isoform. In some embodiments, the circular RNA indirectly increases expression of a non-pathogenic protein isoform. In some embodiments, the circular RNA increases expression of a non-pathogenic protein isoform by at least about 2-fold, about 5-fold, about 10- fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 500-fold, about 1000-fold, or more in a transduced target cell or tissue compared to an untransduced target cell or tissue.Kits and Articles of Manufacture

[0306] Also provided herein are kits or articles of manufacture for carrying out methods described herein. At least one component of the kit or article of manufacture is the engineered tRNA sequences of the present disclosure, the recombinant nucleic acid molecules of the present disclosure, the vectors or rAAV particles of the present disclosure, or the pharmaceutical compositions of the present disclosure.

[0307] In some embodiments, the kit or article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing, and / or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle).

[0308] In some embodiments, the label or package insert indicates that at least one component of the kit or article of manufacture is used for treating the disorder of choice. In some embodiments, the label or package insert comprises instructions for practicing the methods provided herein. The instructions are generally recorded on a suitable recording medium. In some embodiments, the instructions may be printed on a substrate, such as paper or plastic, etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit or article of manufacture that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded.

[0309] In some embodiments, the kit or article of manufacturer further comprises other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.EMBODIMENTSEmbodiment Section 1

[0310] Embodiment 1. The present disclosure provides an engineered tRNA sequence, comprising: i) a tRNA 5’ leader and a tRNA 3’ trailer; ii) a tRNA 5’ exonic element and a tRNA3’ exonic element; iii) a tRNA 5’ intronic element and / or a tRNA 3’ intronic element; iv) a 5’ restriction endonuclease (RE) site and 3’ RE site; v) a 5’ ligation motif and a 3’ ligation motif; and vi) a cargo.

[0311] Embodiment 2. The engineered tRNA sequence of embodiment 1, wherein the cargo is incorporated into a circular RNA following tRNA splicing.

[0312] Embodiment 3. The engineered tRNA sequence of embodiment 1 or 2, wherein the engineered tRNA sequence is derived from a gene encoding a tyrosine tRNA, an isoleucine tRNA, a leucine tRNA, or an arginine tRNA.

[0313] Embodiment 4. The engineered tRNA sequence of embodiment 3, wherein the gene is a human gene.

[0314] Embodiment 5. The engineered tRNA sequence of embodiment 3, wherein the gene is a mouse gene.

[0315] Embodiment 6. The engineered tRNA sequence of any one of embodiments 1 to 5, wherein the tRNA 5’ leader comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 1, 29-54, and 139.

[0316] Embodiment 7. The engineered tRNA sequence of any one of embodiments 1 to 5, wherein the tRNA 3’ trailer comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 2, 55-80, and 140.

[0317] Embodiment 8. The engineered tRNA sequence of any one of embodiments 1 to 7, wherein the 5’ RE site comprises a polynucleotide sequence with at least about 60%, at least about 75%, at least about 85%, or 100% identity to GCGGCCGC or SEQ ID NO: 142.

[0318] Embodiment 9. The engineered tRNA sequence of any one of embodiments 1 to 8, wherein the 3’ RE site comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 4.

[0319] Embodiment 10. The engineered tRNA sequence of any one of embodiments 1 to 9, wherein the engineered tRNA sequence comprises the tRNA 3’ intronic element but does not comprise the tRNA 5’ intronic element.

[0320] Embodiment 11. The engineered tRNA sequence of embodiment 10, wherein the tRNA 3 ’ intronic element comprises a polynucleotide sequence with at least about 75%, or 100% identityto any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC.

[0321] Embodiment 12. The engineered tRNA sequence of any one of embodiments 1 to 9, wherein the engineered tRNA sequence comprises the tRNA 5’ intronic element and the tRNA 3’ intronic element.

[0322] Embodiment 13. The engineered tRN A sequence of embodiment 12, wherein the tRN A 5 ’ intronic element comprises a polynucleotide sequence of TG, CT, GA, or GC.

[0323] Embodiment 14. The engineered tRNA sequence of embodiment 12 or 13, wherein the tRNA 3’ intronic element comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs:101-104 and 141.

[0324] Embodiment 15. The engineered tRNA sequence of any one of embodiments 1 to 14, wherein the engineered tRNA sequence is cleaved by tRNA splicing endonuclease (TSEN) complex during tRNA splicing.

[0325] Embodiment 16. The engineered tRNA sequence of any one of embodiments 1 to 15, wherein the engineered tRNA sequence comprises a polynucleotide sequence that interacts with an RNA ligase.

[0326] Embodiment 17. The engineered tRNA sequence of any one of embodiments 1 to 16, wherein the 5’ ligation motif and the 3’ ligation motif comprise a binding site for an RNA ligase.

[0327] Embodiment 18. The engineered tRNA sequence of embodiment 16 or 17, wherein the RNA ligase is RtcB.

[0328] Embodiment 19. The engineered tRNA sequence of any one of embodiments 1 to 18, wherein the 5’ ligation motif and the 3’ ligation motif stabilize the secondary structure formed by the linear engineered tRNA sequence prior to and during tRNA splicing.

[0329] Embodiment 20. The engineered tRNA sequence of any one of embodiments 1 to 19, wherein the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 6, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO:7.

[0330] Embodiment 21. The engineered tRNA sequence of any one of embodiments 1 to 19, wherein the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 7, and whereinthe 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 6.

[0331] Embodiment 22. The engineered tRNA sequence of any one of embodiments 1 to 21, wherein the engineered tRNA sequence comprises an acceptor stem sequence.

[0332] Embodiment 23. The engineered tRNA sequence of embodiment 22, wherein the acceptor stem sequence comprises a polynucleotide sequence with at least about 70%, at least about 85%, or 100% identity to any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU.

[0333] Embodiment 24. The engineered tRNA sequence of any one of embodiments 1 to 23, wherein the tRNA 5’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 25 and 81-86.

[0334] Embodiment 25. The engineered tRNA sequence of any one of embodiments 1 to 24, wherein the tRNA 3 ’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 26 and 87-100.

[0335] Embodiment 26. The engineered tRNA sequence of any one of embodiments 1 to 25, comprising a polynucleotide sequence with 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% identity to SEQ ID NO: 27, wherein the polynucleotide sequence is upstream of the cargo.

[0336] Embodiment 27. The engineered tRNA sequence of any one of embodiments 1 to 25, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 109-118, and 145-164, wherein the polynucleotide sequence is upstream of the cargo.

[0337] Embodiment 28. The engineered tRNA sequence of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to SEQ ID NO:28, wherein the polynucleotide sequence is downstream of the cargo.

[0338] Embodiment 29. The engineered fRNA sequence of any one of embodiments 1 to 27, comprising a polynucleotide sequence with at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 190-192, wherein the polynucleotide sequence is upstream of the cargo sequence.

[0339] Embodiment 30. The engineered tRNA sequence of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 119-128, and 170-189, wherein the polynucleotide sequence is downstream of the cargo.

[0340] Embodiment 31. The engineered tRNA sequence of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 193-196, wherein the polynucleotide sequence is downstream of the cargo.

[0341] Embodiment 32. The engineered tRNA sequence of any one of embodiments 1 to 27, wherein i) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:29, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of A GCA, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 87, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 55; ii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:31, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGTG, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 88, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 57; iii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:35, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGA, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 90, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 61;iv) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:37, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGGC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 92, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:63; v) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:38, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO: 82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGCT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO:93, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 64; vi) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:41, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:84, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGTC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 96, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 67; vii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:43, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 97, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 69; viii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:45, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO:26, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 71; ix) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:47, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:85, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 73; orx) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:49, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:85, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:75.

[0342] Embodiment 33. The engineered tRNA sequence of any one of embodiments 1 to 32, wherein the cargo encodes a therapeutic protein.

[0343] Embodiment 34. The engineered tRNA sequence of any one of embodiments 1 to 32, wherein the cargo encodes a therapeutic RNA.

[0344] Embodiment 35. The engineered tRNA sequence of embodiment 34, wherein the therapeutic RNA is an antisense oligonucleotide, a ribozyme, a siRNA, a shRNA, a miRNA, a tough decoy, a miRNA sponge, a self-amplifying RNA, a guide RNA, an activating RNA, or a repressive RNA.

[0345] Embodiment 36. The engineered tRNA sequence of embodiment 34 or 35, wherein the therapeutic RNA is a miRNA sponge.

[0346] Embodiment 37. The engineered tRNA sequence of embodiment 34 or 35, wherein the therapeutic RNA is a tough decoy.

[0347] Embodiment 38. The engineered tRNA sequence of any one of embodiments 1 to 37, wherein the engineered tRNA sequence comprises a 5’ spacer and / or a 3’ spacer.

[0348] Embodiment 39. The engineered tRNA sequence of embodiment 38, wherein the 5 ’ spacer and / or the 3’ spacer comprise a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs: 105- 108.

[0349] Embodiment 40. A recombinant nucleic acid molecule comprising the engineered tRNA sequence of any one of embodiments 1 -39.

[0350] Embodiment 41. The recombinant nucleic acid molecule of embodiment 40, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter or an RNA polymerase II promoter.

[0351] Embodiment 42. The recombinant nucleic acid molecule of embodiment 41, wherein the RNA polymerase III promoter is an external RNA polymerase III promoter selected from the group consisting of H1, H1.M11, 7SK, U6, U6+27, and U6+1.

[0352] Embodiment 43. The recombinant nucleic acid molecule of embodiment 41, wherein the RNA polymerase III promoter is an internal tRNA promoter.

[0353] Embodiment 44. The recombinant nucleic acid molecule of any one of embodiments 40 to 43, wherein the recombinant nucleic acid molecule comprises an RNA polymerase terminator.

[0354] Embodiment 45. The recombinant nucleic acid molecule of embodiment 44, wherein the RNA polymerase terminator comprises a polyT sequence or a polyadenylation signal.

[0355] Embodiment 46. The recombinant nucleic acid molecule of any one of embodiments 41 to 45, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyT sequence.

[0356] Embodiment 47. The recombinant nucleic acid molecule of embodiment 46, wherein the recombinant nucleic acid molecule comprises an H1 promoter and the polyT sequence.

[0357] Embodiment 48. The recombinant nucleic acid molecule of embodiment 46, wherein the recombinant nucleic acid molecule comprises an H1.M11 promoter and the polyT sequence.

[0358] Embodiment 49. The recombinant nucleic acid molecule of embodiment 46, wherein the recombinant nucleic acid molecule comprises a U6 promoter and the polyT sequence.

[0359] Embodiment 50. The recombinant nucleic acid molecule of embodiment 46, wherein the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyT sequence.

[0360] Embodiment 51. The recombinant nucleic acid molecule of any one of embodiments 44 to 50, wherein the RNA polymerase terminator comprises at least 4 T nucleotides.

[0361] Embodiment 52. The recombinant nucleic acid molecule of any one of embodiments 44 to 51, wherein the RNA polymerase terminator comprises 8 T nucleotides.

[0362] Embodiment 53. The recombinant nucleic acid molecule of any one of embodiments 41 to 45, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyadenylation signal.

[0363] Embodiment 54. The recombinant nucleic acid molecule of embodiment 53, wherein the recombinant nucleic acid molecule comprises an H1 promoter and the polyadenylation signal.

[0364] Embodiment 55. The recombinant nucleic acid molecule of embodiment 53, wherein the recombinant nucleic acid molecule comprises an H1. M11 promoter and the polyadenylation signal.

[0365] Embodiment 56. The recombinant nucleic acid molecule of embodiment 53, wherein the recombinant nucleic acid molecule comprises a U6 promoter and the polyadenylation signal.

[0366] Embodiment 57. The recombinant nucleic acid molecule of embodiment 53, wherein the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyadenylation signal.

[0367] Embodiment 58. The recombinant nucleic acid molecule of any one of embodiments 44, 45, and 53 to 57, wherein the RNA polymerase terminator comprises a polynucleotide sequence with 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% identity to SEQ ID NO: 15.

[0368] Embodiment 59. The recombinant nucleic acid molecule of any one of embodiments 1 to 58, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 200-232, 236, 238-240, and 264-276.

[0369] Embodiment 60. The recombinant nucleic acid molecule of any one of embodiments 1 to 25, 27, 29 to 59, wherein the circularization efficiency of the recombinant nucleic acid molecule is at least about 10% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively.

[0370] Embodiment 61. The recombinant nucleic acid molecule of any one of embodiments 1 to 25, 27, 29 to 59, wherein the abundance of the resulting circular RNA is at least about 100% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively.

[0371] Embodiment 62. The recombinant nucleic acid molecule of embodiment 41 or 42, wherein the recombinant nucleic acid molecule comprises an external RNA polymerase III promoter, wherein a vector comprising the external RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an internal tRNA promoter when transcribed in a cell.

[0372] Embodiment 63. The recombinant nucleic acid molecule of any one of embodiments 41 to 43, wherein the recombinant nucleic acid molecule comprises an RNA polymerase JU promoter, wherein a vector comprising the RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an RNA polymerase II promoter when transcribed in a cell.

[0373] Embodiment 64. An adeno-associated virus (AAV) vector encoding the engineered tRNA sequence of any one of embodiments 1-39 or the recombinant nucleic acid molecule of any one of embodiments 40-63.

[0374] Embodiment 65. The AAV vector of embodiment 64, wherein the engineered tRNA sequence or recombinant nucleic acid molecule is flanked by AAV inverted terminal repeats (ITRs).

[0375] Embodiment An AAV particle comprising the AAV vector of embodiment 64 or 65 and a capsid protein.

[0376] Embodiment 67. The AAV particle of embodiment 66, wherein the capsid protein is a capsid protein of AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or a variant thereof.

[0377] Embodiment 68. The AAV particle of embodiment 66 or 67, wherein the capsid protein is an AAV9 capsid variant.

[0378] Embodiment 69. The AAV particle of any one of embodiments 66-68, wherein the capsid protein is an AAV.kl3 capsid protein.

[0379] Embodiment 70. The AAV particle of embodiment 69, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 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: 136.

[0380] Embodiment 71. The AAV particle of embodiment 69 or 70, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0381] Embodiment 72. The AAV particle of any one of embodiments 69 to 71, wherein the AAV.kl 3 capsid protein consists of an amino acid sequence of SEQ ID NO: 137 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0382] Embodiment 73. The AAV particle of any one of embodiments 69 to 72, wherein the AAV.kl3 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.

[0383] Embodiment 74. The AAV particle of any one of embodiments 66-68, wherein the capsid protein is an AAV.k20 capsid protein.

[0384] Embodiment 75. The AAV particle of embodiment 74, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 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: 136.

[0385] Embodiment 76. The AAV particle of embodiment 74 or 75, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0386] Embodiment 77. The AAV particle of any one of embodiments 74 to 76, wherein the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 138 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0387] Embodiment 78. The AAV particle of any one of embodiments 74 to 77, 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 AAV9 capsid.

[0388] Embodiment 79. The AAV particle of any one of embodiments 66 to 78, wherein the capsid protein is a variant of a parental wild-type capsid protein, and wherein the capsid protein improves gene transfer and / or expression in one or more region(s) or part(s) of kidney when compared to the parental wild-type capsid protein.

[0389] Embodiment 80. A composition comprising the engineered tRNA sequence of any one of embodiments 1 to 39, the recombinant nucleic acid molecule of any one of embodiments 40 to 63, the AAV vector of embodiment 64 or 65, or the AAV particle of any one of embodiments 66 to 79, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0390] Embodiment 81. A kit, comprising: the engineered tRNA sequence of any one of embodiments 1 to 39, the recombinant nucleic acid molecule of any one of embodiments 40 to 63, the AAV vector of embodiment 64 or 65, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 80.

[0391] Embodiment 82. The kit of embodiment 81, wherein the kit comprises an instruction for delivering the AAV vector, the AAV particle, or the composition to a target cell or tissue.

[0392] Embodiment 83. A method of expressing a recombinant circular RNA in a cell, comprising introducing the engineered tRNA sequence of any one of embodiments 1 to 39, the recombinant nucleic acid molecule of any one of embodiments 40 to 63, the AAV vector of embodiment 64 or 65, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 80 into the cell under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

[0393] Embodiment 84. The method of embodiment 83, wherein the cell is a mammalian cell.

[0394] Embodiment 85. The method of embodiment 83 or 84, wherein the cell is a human cell.

[0395] Embodiment 86. The method of any one of embodiments 83 to 85, wherein the cell is a myoblast.

[0396] Embodiment 87. The method of any one of embodiments 83 to 85, wherein the cell is a kidney cell.

[0397] Embodiment 88. A method of expressing a recombinant circular RNA in a tissue, comprising introducing the engineered tRNA sequence of any one of embodiments 1 to 39, the recombinant nucleic acid molecule of any one of embodiments 40 to 63, the AAV vector of embodiment 64 or 65, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 80 into the tissue under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

[0398] Embodiment 89. A method of expressing a recombinant circular RNA in a subject, comprising administering an effective amount of the engineered tRNA sequence of any one of embodiments 1 to 39, the recombinant nucleic acid molecule of any one of embodiments 40 to 63, the AAV vector of embodiment 64 or 65, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 80 to the subject, wherein the effective amount is an amount that reduces at least one symptom of a disease or condition in the subject

[0399] Embodiment 90. A method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the engineered tRNA sequence of any one of embodiments 1 to 39, the recombinant nucleic acid molecule of any one of embodiments 40 to 63, the AAV vector of embodiment 64 or 65, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 80 to the subject.

[0400] Embodiment 91. The method of embodiment 90, wherein the subject is a human subject.

[0401] Embodiment 92. The method of embodiment 90 or 91, wherein the subject has autosomal dominant polycystic kidney disease (ADPKD).

[0402] Embodiment 93. The method of embodiment 92, wherein the subject has ADPKD 1.

[0403] Embodiment 94. The method of embodiment 92, wherein the subject has ADPKD2.

[0404] Embodiment 95. The method of any one of embodiments 90 to 94, wherein the method inhibits or ameliorates renal cyst developmentEmbodiment Section 2

[0405] Embodiment 1. The present disclosure provides a recombinant nucleic acid molecule, comprising: i) a tRNA 5’ leader and a tRNA 3’ trailer; ii) a tRNA 5’ exonic element and a tRNA 3’ exonic element; iii) a tRNA 5’ intronic element and / or a tRNA 3’ intronic element; iv) a 5’ restriction endonuclease (RE) site and 3’ RE site; v) a 5’ ligation motif and a 3 ’ ligation motif; and vi) a cargo.

[0406] Embodiment 2. The recombinant nucleic acid molecule of embodiment 1, wherein the cargo is incorporated into a circular RNA following tRNA splicing.

[0407] Embodiment 3. The recombinant nucleic acid molecule of embodiment 1 or 2, wherein the tRNA 5’ leader, the tRNA 3’ trailer, the tRNA 5’ exonic element, the tRNA 3’ exonic element, the tRNA 5 ’ intronic element and / or the tRNA 3 ’ intronic element are derived from a gene encoding a tyrosine tRNA, an isoleucine tRNA, a leucine tRNA, or an arginine tRNA.

[0408] Embodiment 4. The recombinant nucleic acid molecule of embodiment 3, wherein the gene is a human gene.

[0409] Embodiment 5. The recombinant nucleic acid molecule of embodiment 3, wherein the gene is a mouse gene.

[0410] Embodiment 6. The recombinant nucleic acid molecule of any one of embodiments 1 to 5, wherein the tRNA 5’ leader comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 1, 29-54, and 139.

[0411] Embodiment 7. The recombinant nucleic acid molecule of any one of embodiments 1 to 5, wherein the tRNA 3’ trailer comprises a polynucleotide sequence with at least about 90%, atleast about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 2, 55-80, and 140.

[0412] Embodiment 8. The recombinant nucleic acid molecule of any one of embodiments 1 to7, wherein the 5’ RE site comprises a polynucleotide sequence with at least about 60%, at least about 75%, at least about 85%, or 100% identity to GCGGCCGC or SEQ ID NO: 142.

[0413] Embodiment 9. The recombinant nucleic acid molecule of any one of embodiments 1 to8, wherein the 3’ RE site comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 4.

[0414] Embodiment 10. The recombinant nucleic acid molecule of any one of embodiments 1 to9, wherein the recombinant nucleic acid molecule comprises the tRNA 3 ’ intronic element but does not comprise the tRNA 5’ intronic element.

[0415] Embodiment 11. The recombinant nucleic acid molecule of embodiment 10, wherein the tRNA 3’ intronic element comprises a polynucleotide sequence with at least about 75%, or 100% identity to any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC.

[0416] Embodiment 12. The recombinant nucleic acid molecule of any one of embodiments 1 to 9, wherein the recombinant nucleic acid molecule comprises the tRNA 5’ intronic element and the tRNA 3’ intronic element.

[0417] Embodiment 13. The recombinant nucleic acid molecule of embodiment 12, wherein the tRNA 5’ intronic element comprises a polynucleotide sequence of TG, CT, GA, or GC.

[0418] Embodiment 14. The recombinant nucleic acid molecule of embodiment 12 or 13, wherein the tRNA 3 ’ intronic element comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs:101-104 and 141.

[0419] Embodiment 15. The recombinant nucleic acid molecule of any one of embodiments 1 to14, wherein the recombinant nucleic acid molecule is cleaved by tRNA splicing endonuclease (TSEN) complex during tRNA splicing.

[0420] Embodiment 16. The recombinant nucleic acid molecule of any one of embodiments 1 to15, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence that interacts with an RN A ligase.

[0421] Embodiment 17. The recombinant nucleic acid molecule of any one of embodiments 1 to 16, wherein the 5’ ligation motif and the 3’ ligation motif comprise a binding site for an RNA ligase.

[0422] Embodiment 18. The recombinant nucleic acid molecule of embodiment 16 or 17, wherein the RNA ligase is RtcB.

[0423] Embodiment 19. The recombinant nucleic acid molecule of any one of embodiments 1 to18, wherein the 5’ ligation motif and the 3’ ligation motif stabilize the secondary structure formed by the linear recombinant nucleic acid molecule prior to and during tRNA splicing.

[0424] Embodiment 20. The recombinant nucleic acid molecule of any one of embodiments 1 to19, wherein the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 6, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 7.

[0425] Embodiment 21. The recombinant nucleic acid molecule of any one of embodiments 1 to 19, wherein the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 7, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO:6.

[0426] Embodiment 22. The recombinant nucleic acid molecule of any one of embodiments 1 to 21, wherein the recombinant nucleic acid molecule comprises an acceptor stem sequence.

[0427] Embodiment 23. The recombinant nucleic acid molecule of embodiment 22, wherein the acceptor stem sequence comprises a polynucleotide sequence with at least about 70%, at least about 85%, or 100% identity to any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU.

[0428] Embodiment 24. The recombinant nucleic acid molecule of any one of embodiments 1 to23, wherein the tRNA 5’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 25 and 81-86.

[0429] Embodiment 25. The recombinant nucleic acid molecule of any one of embodiments 1 to24, wherein the tRNA 3’ exonic element comprises a polynucleotide sequence with at least about85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 26 and 87-100.

[0430] Embodiment 26. The recombinant nucleic acid molecule of any one of embodiments 1 to 25, comprising a polynucleotide sequence with 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% identity to SEQ ID NO: 27, wherein the polynucleotide sequence is upstream of the cargo.

[0431] Embodiment 27. The recombinant nucleic acid molecule of any one of embodiments 1 to 25, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 109-118, and 145-164, wherein the polynucleotide sequence is upstream of the cargo.

[0432] Embodiment 28. The recombinant nucleic acid molecule of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to SEQ ID NO:28, wherein the polynucleotide sequence is downstream of the cargo.

[0433] Embodiment 29. The recombinant nucleic acid molecule of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 190-192, wherein the polynucleotide sequence is upstream of the cargo sequence.

[0434] Embodiment 30. The recombinant nucleic acid molecule of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 119-128, and 170-189, wherein the polynucleotide sequence is downstream of the cargo.

[0435] Embodiment 31. The recombinant nucleic acid molecule of any one of embodiments 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 193-196, wherein the polynucleotide sequence is downstream of the cargo.

[0436] Embodiment 32. The recombinant nucleic acid molecule of any one of embodiments 1 to 27, wherein i) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:29, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGCA, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 87, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:55; ii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:31, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGTG, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 88, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 57; iii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:35, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGA, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 90, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 61 ; iv) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:37, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGGC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 92, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:63; v) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:38, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGCT, the tRNA 3’exonic element comprises a polynucleotide sequence of SEQ ID NO:93, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 64; vi) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:41, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:84, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGTC, the tRNA 3’exonic elementcomprises a polynucleotide sequence of SEQ ID NO: 96, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 67; vii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:43, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 97, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 69; viii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:45, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO:26, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 71; ix) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:47, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:85, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 73; or x) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:49, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:85, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 75.

[0437] Embodiment 33. The recombinant nucleic acid molecule of any one of embodiments 1 to 32, wherein the cargo encodes a therapeutic protein.

[0438] Embodiment 34. The recombinant nucleic acid molecule of any one of embodiments 1 to 32, wherein the cargo encodes a therapeutic RNA.

[0439] Embodiment 35. The recombinant nucleic acid molecule of embodiment 34, wherein the therapeutic RNA is an antisense oligonucleotide, a ribozyme, a siRNA, a shRNA, a miRNA, a tough decoy, a miRNA sponge, a self-amplifying RNA, a guide RNA, an activating RNA, or a repressive RNA.

[0440] Embodiment 36. The recombinant nucleic acid molecule of embodiment 34 or 35, wherein the therapeutic RNA is a miRNA sponge.

[0441] Embodiment 37. The recombinant nucleic acid molecule of embodiment 34 or 35, wherein the therapeutic RNA is a tough decoy.

[0442] Embodiment 38. The recombinant nucleic acid molecule of any one of embodiments 1 to 37, wherein the recombinant nucleic acid molecule comprises a 5’ spacer and / or a 3’ spacer.

[0443] Embodiment 39. The recombinant nucleic acid molecule of embodiment 38, wherein the 5’ spacer and / or the 3’ spacer comprise a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs: 105-108.

[0444] Embodiment 40. The recombinant nucleic acid molecule of any one of embodiments 1- 39, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter or an RNA polymerase II promoter.

[0445] Embodiment 41. The recombinant nucleic acid molecule of embodiment 40, wherein the RNA polymerase III promoter is an external RNA polymerase III promoter selected from the group consisting of H1, H1.M11, 7SK, U6, U6+27, and U6+1.

[0446] Embodiment 42. The recombinant nucleic acid molecule of embodiment 40, wherein the RNA polymerase III promoter is an internal tRNA promoter.

[0447] Embodiment 43. The recombinant nucleic acid molecule of any one of embodiments 40 to 43, wherein the recombinant nucleic acid molecule comprises an RNA polymerase terminator.

[0448] Embodiment 44. The recombinant nucleic acid molecule of embodiment 43, wherein the RNA polymerase terminator comprises a polyT sequence or a polyadenylation signal.

[0449] Embodiment 45. The recombinant nucleic acid molecule of any one of embodiments 40 to 45, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyT sequence.

[0450] Embodiment 46. The recombinant nucleic acid molecule of embodiment 45, wherein the recombinant nucleic acid molecule comprises an H1 promoter and the polyT sequence.

[0451] Embodiment 47. The recombinant nucleic acid molecule of embodiment 45, wherein the recombinant nucleic acid molecule comprises an H1.M11 promoter and the polyT sequence.

[0452] Embodiment 48. The recombinant nucleic acid molecule of embodiment 45, wherein the recombinant nucleic acid molecule comprises a U6 promoter and the polyT sequence.

[0453] Embodiment 49. The recombinant nucleic acid molecule of embodiment 45, wherein the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyT sequence.

[0454] Embodiment 50. The recombinant nucleic acid molecule of any one of embodiments 43 to 49, wherein the RNA polymerase terminator comprises at least 4 T nucleotides.

[0455] Embodiment 51. The recombinant nucleic acid molecule of any one of embodiments 43 to 50, wherein the RNA polymerase terminator comprises 8 T nucleotides.

[0456] Embodiment 52. The recombinant nucleic acid molecule of any one of embodiments 40 to 44, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyadenylation signal.

[0457] Embodiment 53. The recombinant nucleic acid molecule of embodiment 52, wherein the recombinant nucleic acid molecule comprises an H1 promoter and the polyadenylation signal.

[0458] Embodiment 54. The recombinant nucleic acid molecule of embodiment 52, wherein the recombinant nucleic acid molecule comprises an H1. M11 promoter and the polyadenylation signal.

[0459] Embodiment 55. The recombinant nucleic acid molecule of embodiment 52, wherein the recombinant nucleic acid molecule comprises a U6 promoter and the polyadenylation signal.

[0460] Embodiment 56. The recombinant nucleic acid molecule of embodiment 52, wherein the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyadenylation signal.

[0461] Embodiment 57. The recombinant nucleic acid molecule of any one of embodiments 43, 44, and 52 to 56, wherein the RNA polymerase terminator comprises a polynucleotide sequence with 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% identity to SEQ ID NO: 15.

[0462] Embodiment 58. The recombinant nucleic acid molecule of any one of embodiments 1 to 57, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 200-232, 236, 238-240, and 264-276.

[0463] Embodiment 59. The recombinant nucleic acid molecule of any one of embodiments 1 to 25, 27, 29 to 58, wherein the circularization efficiency of the recombinant nucleic acid molecule is at least about 10% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively.

[0464] Embodiment 60. The recombinant nucleic acid molecule of any one of embodiments 1 to 25, 27, 29 to 58, wherein the abundance of the resulting circular RNA is at least about 100% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively.

[0465] Embodiment 61. The recombinant nucleic acid molecule of embodiment 40 or 41 , wherein the recombinant nucleic acid molecule comprises an external RNA polymerase III promoter, wherein a vector comprising the external RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an internal tRNA promoter when transcribed in a cell.

[0466] Embodiment 62. The recombinant nucleic acid molecule of any one of embodiments 40 to 42, wherein the recombinant nucleic acid molecule comprises an RNA polymerase in promoter, wherein a vector comprising the RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an RNA polymerase II promoter when transcribed in a cell.

[0467] Embodiment 63. An adeno-associated virus (AAV) vector encoding the recombinant nucleic acid molecule of any one of embodiments 1-39 or the recombinant nucleic acid molecule of any one of embodiments 40-63.

[0468] Embodiment 64. The AAV vector of embodiment 64, wherein the engineered tRNA sequence or recombinant nucleic acid molecule is flanked by AAV inverted terminal repeats (ITRs).

[0469] Embodiment 65. An AAV particle comprising the AAV vector of embodiment 63 or 64 and a capsid protein.

[0470] Embodiment 66. The AAV particle of embodiment 65, wherein the capsid protein is a capsid protein of AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or a variant thereof.

[0471] Embodiment 67. The AAV particle of embodiment 65 or 66, wherein the capsid protein is an AAV9 capsid variant.

[0472] Embodiment 68. The AAV particle of any one of embodiments 65-67, wherein the capsid protein is an AAV.kl3 capsid protein.

[0473] Embodiment 69. The AAV particle of embodiment 68, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 with 1, 2, 3, 4, or more amino acidsubstitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0474] Embodiment 70. The AAV particle of embodiment 68 or 69, wherein the AAV. kl 3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0475] Embodiment 71. The AAV particle of any one of embodiments 68 to 70, wherein the AAV.kl3 capsid protein consists of an amino acid sequence of SEQ ID NO: 137 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0476] Embodiment 72. The AAV particle of any one of embodiments 68 to 71, wherein the AAV.kl3 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.

[0477] Embodiment 73. The AAV particle of any one of embodiments 65-67, wherein the capsid protein is an AAV.k20 capsid protein.

[0478] Embodiment 74. The AAV particle of embodiment 73, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 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: 136.

[0479] Embodiment 75. The AAV particle of embodiment 73 or 74, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0480] Embodiment 76. The AAV particle of any one of embodiments 73 to 75, wherein the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 138 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

[0481] Embodiment 77. The AAV particle of any one of embodiments 73 to 76, 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 AAV9 capsid.

[0482] Embodiment 78. The AAV particle of any one of embodiments 65 to 77, wherein the capsid protein is a variant of a parental wild-type capsid protein, and wherein the capsid protein improves gene transfer and / or expression in one or more region(s) or part(s) of kidney when compared to the parental wild-type capsid protein.

[0483] Embodiment 79. A composition comprising the recombinant nucleic acid molecule of any one of embodiments 1 -62, the AAV vector of embodiment 63 or 64, or the AAV particle of any one of embodiments 65 to 78, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0484] Embodiment 80. A kit, comprising: the recombinant nucleic acid molecule of any one of embodiments 1-62, the AAV vector of embodiment 63 or 64, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 79.

[0485] Embodiment 81. The kit of embodiment 81, wherein the kit comprises an instruction for delivering the AAV vector, the AAV particle, or the composition to a target cell or tissue.

[0486] Embodiment 82. A method of expressing the recombinant nucleic acid molecule of any one of embodiments 1-62, the AAV vector of embodiment 63 or 64, the AAV particle of any one of embodiments 66 to 79, or the composition of embodiment 79 into the cell under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

[0487] Embodiment 83. The method of embodiment 82, wherein the cell is a mammalian cell.

[0488] Embodiment 84. The method of embodiment 82 or 83, wherein the cell is a human cell.

[0489] Embodiment 85. The method of any one of embodiments 82 to 84, wherein the cell is a myoblast.

[0490] Embodiment 86. The method of any one of embodiments 82 to 84, wherein the cell is a kidney cell.

[0491] Embodiment 87. A method of expressing a recombinant circular RNA in a tissue, comprising introducing the recombinant nucleic acid molecule of any one of embodiments 1-62, the AAV vector of embodiment 63 or 64, the AAV particle of any one of embodiments 65 to 78, or the composition of embodiment 79 into the tissue under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

[0492] Embodiment 88. A method of expressing a recombinant circular RNA in a subject, comprising administering an effective amount of the recombinant nucleic acid molecule of any one of embodiments 1-62, the AAV vector of embodiment 63 or 64, the AAV particle of any one of embodiments 65 to 78, or the composition of embodiment 79 to the subject, wherein the effective amount is an amount that reduces at least one symptom of a disease or condition in the subject.

[0493] Embodiment 89. A method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the recombinant nucleic acid molecule of any one of embodiments 1-62, the AAV vector of embodiment 63 or 64, the AAV particle of any one of embodiments 65 to 78, or the composition of embodiment 79 to the subject.

[0494] Embodiment 90. The method of embodiment 89, wherein the subject is a human subject.

[0495] Embodiment 91. The method of embodiment 89 or 90, wherein the subject has autosomal dominant polycystic kidney disease (ADPKD).

[0496] Embodiment 92. The method of embodiment 91, wherein the subject has ADPKD 1.

[0497] Embodiment 93. The method of embodiment 91 , wherein the subject has ADPKD2.

[0498] Embodiment 94. The method of any one of embodiments 89 to 93, wherein the method inhibits or ameliorates renal cyst developmentEXAMPLES

[0499] The present invention is further illustrated by reference to the following Examples. The Examples are illustrative and are not to be construed as restricting the scope of the invention in any way.Example 1: Design of Trie Scaffolds for Circular RNA Biogenesis

[0500] Trie scaffold expression cassettes were designed as shown in FIGs. 1A-1D and FIGs.2A- 2D. FIG. 1A and FIG. 2A depict schematic representations of engineered trie scaffold expression cassettes. The expression cassette encodes a linear engineered pre-tRNA comprising a cargo that is flanked by tRNA expression and splicing elements — including leader, trailer, exonic, and intronic elements — RE sites, and ligation motifs. The 3’ RE site depicted in FIGs. 1-2 contains a restriction enzyme site and additional stuffier nucleotides to accommodate interactions with the 5’ RE site below the BHB structure in the folded scaffold. The 5’ RE site depicted in FIG. 2 contains a restriction enzyme site and additional stuffier nucleotides to complete base-pairing with the 3’RE site. The 5’ and 3’ spacer sequences are engineered to be unstructured sequence elements surrounding the cargo and to create primer and probe binding sites for circRNA quantitation. The expression cassette also contains an RNA polymerase promoter and a terminator for optimal expression. The scaffold in FIG. 1 contains a four-nucleotide 3’ intronic sequence, whereas the scaffold in FIG. 2 contains a two-nucleotide 5’ intronic sequence and a 10-nucleotide 3’ intronic sequence adjacent to the exons. FIG. 1B and FIG. 2B show schematic representations of the components of the linear engineered pre-tRNA that is transcribed from the expression cassettes. FIG. 1C and FIG. 2C show the predicted secondary structures that are formed from the linear engineered pre-tRNA prior to splicing. The BHB-like motif is marked by a box. FIG. 1D and FIG. 2D show the components of circRNA that are formed following pre-tRNA splicing. The splice junction is between the 5’ RE site and the partial tRNA 3’ intronic element (FIG. 1D) or between the partial tRNA 5’ intronic element and the partial tRNA 3’ intronic element (FIG. 2D).

[0501] Exemplary sequences for the trie scaffold expression cassettes described herein are provided in Tables 1-15.Example 2: hTricY Scaffold with Modified Ligation Motifs

[0502] The objective of this study was to assess the necessity of engineered tRNA components within the hTricY (TRY-GTA3-1.1) scaffold for circRNA generation. All expression cassettes comprised the same circRNA sequence and used a CMV / polyA expression system. A circRNA expression cassette with a disrupted ligation motif was generated (PEX033) using standard methods commonly known in the art CircRNA expression levels were then measured in human embryonic kidney (HEK293T) cells following transfection with plasmids encoding the standard hTricY scaffold (PEX040) or the hTricY scaffold with the disrupted ligation motif (PEX033). A plasmid containing only cytomegalovirus promoter-driven green fluorescent protein (CMV-GFP) (PEX018) was used as a negative control lacking the hTricY scaffold and circRNA sequences. The number of precursor RNA copies per nanogram (FIG. 3A) and circRNA copies per nanogram (ng) (FIG. 3B) were measured by RT-dPCR and normalized to GFP. The circularization efficiency was then calculated by dividing the circRNA by total RNA abundance (circRNA + precursor RNA) and multiplying by 100 (FIG. 3C). Statistical significance was determined via two-tailed unpaired t-test. These results demonstrated that disrupting the ligation sequence in the engineered tRNA sequence significantly reduces circRNA abundance as well as circularization efficiency.Example 3: hTricY Scaffolds with Modified Leader and Trailer Regions

[0503] The leader and trailer sequences of pre-tRNA within the engineered hTricY (TRY-GTA3- 1.1) scaffold were evaluated for their importance in generating circular RNAs. Four CMV / polyA- driven expression cassettes all comprising the same circRNA sequence (Table 19) were generated: a standard hTricY scaffold (PEX040), a hTricY scaffold without a tRNA leader (PEX034), a hTricY scaffold without a tRNA trailer (PEX035), and a hTricY scaffold without a tRNA leader and trailer (PEX036). A CMV-GFP only cassette (PEX018) was used as a negative control lacking the hTricY scaffold and circRNA sequences.Table 19. Plasmid Constructs Encoding Trie Scaffolds

[0504] The constructs were transfected into HEK293T cells, and the number of precursor RNA copies per nanogram (FIG. 4A) and circRNA copies per nanogram (ng) of RNA (FIG. 4B) were measured by RT-dPCR and normalized to GFP. The circularization efficiency was then calculated by dividing the circRNA by total RNA abundance (circRNA + precursor RNA) and multiplying by 100 (FIG. 4C). Statistical significance was determined by one-way ANOVA, followed by Tukey’s multiple comparison test (all means compared to each other). Overall, the results showed that the leader and trailer sequences are essential for circRNA generation.Example 4: hTricY Scaffolds with Destabilizing Acceptor Stem Regions

[0505] The objective of this study was to assess whether modifying the acceptor stem region formed by the tRNA exons of the hTricY (TRY-GTA3-1.1) scaffold impacts circRNA production.

[0506] tRNAs and tRNA-like small RNAs, such as MEN0, that contain guanosines at the first two positions and an unstable acceptor stem generated by base-pairing mismatches or multiple Wobble base pairs are substrates for the post-transcriptional addition of CCACCA at their 3’ ends,targeting them for degradation. Therefore, weakening base-pairing interactions in the acceptor stem via mutations can lead to destabilization of the tRNA itself. Conversely, the mascRNA (MALAT1 -associated small cytoplasmic RNA), another tRNA-like small RNA, does not contain the necessary acceptor stem sequences that signal for its modification with CCACCA, making it a relatively stable molecule.

[0507] Expression cassettes encoding hTricY (TRY-GTA3-1.1) with modified acceptor stems formed by the tRNA exons were generated. The hTricY-CCTT>GGCC scaffold (PEX037) and hTricY-MENβ scaffold (PEX038) encode guanosines at the first and second positions of the acceptor stem and additional mismatched nucleotides in the acceptor stem, which are signals to promote post-transcriptional CCACCA addition to the 3’ end of tRNA. In the hTricY- CCTT>GGCC scaffold (PEX037), the first four nucleotides of the acceptor stem are replaced with ‘GGCC’. In the hTricY-MENβ scaffold (PEX038), the acceptor stem from the tRNA-like small RNA MENβ is swapped in place of the hTricY acceptor stem. The hTricY-Masc scaffold (PEX039) encodes a tRNA where the acceptor stem sequence is swapped for the acceptor stem of the mouse mascRNA. All expression cassettes comprised the same circRNA sequence and used a CMV / polyA expression system. A CMV-GFP only cassette (PEX018) was used as a negative control lacking the hTricY scaffold and circRNA sequences. The expression cassettes were transfected into HEK293T cells, and circRNA expression was assessed using RT-dPCR. Statistical significance was determined by one-way ANOVA, followed by Tukey’s multiple comparison test (all means compared to each other).

[0508] As shown in FIG. 5, expression cassettes with modified acceptor stem regions showed lower circular RNA abundance compared to the standard hTricY circularization scaffold (PEX040). These data demonstrated that integrity of the tRNA acceptor stem sequence is necessary for efficient production of circRNA.Example 5: hTricY Scaffolds with Pol III / PolyT Expression Systems

[0509] The objective of this study was to assess the effect of various RNA polymerase promoters and terminators on circularization efficiency with the hTricY scaffold (TRY-GTA3-1.1).

[0510] Various expression cassettes were desi...

Claims

CLAIMS1. An engineered tRNA sequence, comprising: i) a tRNA 5’ leader and a tRNA 3’ trailer; ii) a tRNA 5’ exonic element and a tRNA 3’ exonic element; iii) a tRNA 5’ intronic element and / or a tRNA 3’ intronic element; iv) a 5’ restriction endonuclease (RE) site and 3’ RE site; v) a 5’ ligation motif and a 3’ ligation motif; and vi) a cargo.

2. The engineered tRNA sequence of claim 1, wherein the cargo is incorporated into a circular RNA following tRNA splicing.

3. The engineered tRNA sequence of claim 1 or 2, wherein the engineered tRNA sequence is derived from a gene encoding a tyrosine tRNA, an isoleucine tRNA, a leucine tRNA, or an arginine tRNA.

4. The engineered tRNA sequence of claim 3, wherein the gene is a human gene.The engineered tRNA sequence of claim 3, wherein the gene is a mouse gene.

6. The engineered tRNA sequence of any one of claims 1 to 5, wherein the tRNA 5’ leader comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 1, 29-54, and 139.

7. The engineered tRNA sequence of any one of claims 1 to 5, wherein the tRNA 3’ trailer comprises a polynucleotide sequence with at least about 90%, at least about 92%, at least about 95%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 2, 55-80, and 140.

8. The engineered tRNA sequence of any one of claims 1 to 7, wherein the 5’ RE site comprises a polynucleotide sequence with at least about 60%, at least about 75%, at least about 85%, or 100% identity to GCGGCCGC or SEQ ID NO: 142.

9. The engineered tRNA sequence of any one of claims 1 to 8, wherein the 3 ’ RE site comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 4.

10. The engineered tRNA sequence of any one of claims 1 to 9, wherein the engineered tRNA sequence comprises the tRNA 3’ intronic element but does not comprise the tRNA 5’ intronic element.

11. The engineered tRNA sequence of claim 10, wherein the tRNA 3’ intronic element comprises a polynucleotide sequence with at least about 75%, or 100% identity to any one of AGGT, AGCA, AGTG, GGTG, GGGA, AGGA, GGTC, GGGC, GGCT, AGGC, AGAA, AGAC, GGCA, GGAC, and GGCC.

12. The engineered tRNA sequence of any one of claims 1 to 9, wherein the engineered tRNA sequence comprises the tRNA 5’ intronic element and the tRNA 3’ intronic element13. The engineered tRNA sequence of claim 12, wherein the tRNA 5’ intronic element comprises a polynucleotide sequence of TG, CT, GA, or GC.

14. The engineered tRNA sequence of claim 12 or 13, wherein the tRNA 3’ intronic element comprises a polynucleotide sequence with at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs: 101-104 and 141.

15. The engineered tRNA sequence of any one of claims 1 to 14, wherein the engineered tRNA sequence is cleaved by tRNA splicing endonuclease (TSEN) complex during tRNA splicing.

16. The engineered tRNA sequence of any one of claims 1 to 15, wherein the engineered tRNA sequence comprises a polynucleotide sequence that interacts with an RNA ligase.

17. The engineered tRNA sequence of any one of claims 1 to 16, wherein the 5’ ligation motif and the 3’ ligation motif comprise a binding site for an RNA ligase.

18. The engineered tRNA sequence of claim 16 or 17, wherein the RNA ligase is RtcB.

19. The engineered tRNA sequence of any one of claims 1 to 18, wherein the 5’ ligation motif and the 3’ ligation motif stabilize the secondary structure formed by the linear engineered tRNA sequence prior to and during tRNA splicing.

20. The engineered tRNA sequence of any one of claims 1 to 19, wherein the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 6, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO:7.

21. The engineered tRNA sequence of any one of claims 1 to 19, wherein the 5’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 7, and wherein the 3’ ligation motif comprises a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to SEQ ID NO: 6.

22. The engineered tRNA sequence of any one of claims 1 to 21 , wherein the engineered tRNA sequence comprises an acceptor stem sequence.

23. The engineered tRNA sequence of claim 22, wherein the acceptor stem sequence comprises a polynucleotide sequence with at least about 70%, at least about 85%, or 100% identity to any one of CCUUCGA, UCGGAGGA, GGCCCGA, UCGGACCG, GGCACUG, CAGUACCG, GACGCUG, and CGGUGUCU.

24. The engineered tRNA sequence of any one of claims 1 to 23, wherein the tRNA 5’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%,at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 25 and 81- 86.

25. The engineered tRNA sequence of any one of claims 1 to 24, wherein the tRNA 3’ exonic element comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 94%, at least about 97%, or 100% identity to any one of SEQ ID NOs: 26 and 87- 100.

26. The engineered tRNA sequence of any one of claims 1 to 25, comprising a polynucleotide sequence with 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% identity to SEQ ID NO: 27, wherein the polynucleotide sequence is upstream of the cargo.

27. The engineered tRNA sequence of any one of claims 1 to 25, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 109-118, and 145-164, wherein the polynucleotide sequence is upstream of the cargo.

28. The engineered tRNA sequence of any one of claims 1 to 27, comprising a polynucleotide sequence with 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% identity to SEQ ID NO:28, wherein the polynucleotide sequence is downstream of the cargo.

29. The engineered tRNA sequence of any one of claims 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 190-192, wherein the polynucleotide sequence is upstream of the cargo sequence.

30. The engineered tRNA sequence of any one of claims 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 119-128, and 170-189, wherein the polynucleotide sequence is downstream of the cargo.

31. The engineered tRNA sequence of any one of claims 1 to 27, comprising a polynucleotide sequence with 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% identity to any one of SEQ ID NOs: 193-196, wherein the polynucleotide sequence is downstream of the cargo.

32. The engineered tRNA sequence of any one of claims 1 to 27, wherein i) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:29, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGCA, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 87, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:55; ii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:31, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:81, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGTG, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 88, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 57; iii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:35, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGA, the tRNA 3’exonic element comprises a polynucleotide sequence of SEQ ID NO: 90, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 61; iv) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:37, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGGC, the tRNA 3’exonic elementcomprises a polynucleotide sequence of SEQ ID NO: 92, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO:63; v) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:38, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:82, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGCT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO:93, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 64; vi) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:41, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:84, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGTC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 96, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 67; vii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:43, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 97, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 69; viii) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:45, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:25, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGGT, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 26, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 71; ix) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:47, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:85, the tRNA 3’ intronic element comprises a polynucleotide sequence of AGAC, the tRNA 3 ’exonic element comprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 73; or x) the tRNA 5’ leader comprises a polynucleotide sequence of SEQ ID NO:49, the tRNA 5’ exonic element comprises a polynucleotide sequence of SEQ ID NO:85, the tRNA 3’ intronic element comprises a polynucleotide sequence of GGAC, the tRNA 3 ’exonic elementcomprises a polynucleotide sequence of SEQ ID NO: 98, and the tRNA 3’ trailer comprises a polynucleotide sequence of SEQ ID NO: 75.

33. The engineered tRNA sequence of any one of claims 1 to 32, wherein the cargo encodes a therapeutic protein.

34. The engineered tRNA sequence of any one of claims 1 to 32, wherein the cargo encodes a therapeutic RNA.

35. The engineered tRNA sequence of claim 34, wherein the therapeutic RNA is an antisense oligonucleotide, a ribozyme, a siRNA, a shRNA, a miRNA, a tough decoy, a miRNA sponge, a self-amplifying RNA, a guide RNA, an activating RNA, or a repressive RNA.

36. The engineered tRNA sequence of claim 34 or 35, wherein the therapeutic RNA is a miRNA sponge.

37. The engineered tRNA sequence of claim 34 or 35, wherein the therapeutic RNA is a tough decoy.

38. The engineered tRNA sequence of any one of claims 1 to 37, wherein the engineered tRNA sequence comprises a 5’ spacer and / or a 3’ spacer.

39. The engineered tRNA sequence of claim 38, wherein the 5’ spacer and / or the 3’ spacer comprise a polynucleotide sequence with at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% identity to any one of SEQ ID NOs: 105-108.

40. A recombinant nucleic acid molecule comprising the engineered tRNA sequence of any one of claims 1-39.

41. The recombinant nucleic acid molecule of claim 40, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter or an RNA polymerase II promoter.

42. The recombinant nucleic acid molecule of claim 41 , wherein the RNA polymerase III promoter is an external RNA polymerase III promoter selected from the group consisting of H1, H1.M11, 7SK, U6, U6+27, and U6+1.

43. The recombinant nucleic acid molecule of claim 41, wherein the RNA polymerase III promoter is an internal tRNA promoter.

44. The recombinant nucleic acid molecule of any one of claims 40 to 43, wherein the recombinant nucleic acid molecule comprises an RNA polymerase terminator.

45. The recombinant nucleic acid molecule of claim 44, wherein the RNA polymerase terminator comprises a polyT sequence or a polyadenylation signal.

46. The recombinant nucleic acid molecule of any one of claims 41 to 45, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyT sequence.

47. The recombinant nucleic acid molecule of claim 46, wherein the recombinant nucleic acid molecule comprises an H1 promoter and the polyT sequence.

48. The recombinant nucleic acid molecule of claim 46, wherein the recombinant nucleic acid molecule comprises an H1.M11 promoter and the polyT sequence.

49. The recombinant nucleic acid molecule of claim 46, wherein the recombinant nucleic acid molecule comprises a U6 promoter and the polyT sequence.

50. The recombinant nucleic acid molecule of claim 46, wherein the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyT sequence.

51. The recombinant nucleic acid molecule of any one of claims 44 to 50, wherein the RNA polymerase terminator comprises at least 4 T nucleotides.

52. The recombinant nucleic acid molecule of any one of claims 44 to 51, wherein the RNA polymerase terminator comprises 8 T nucleotides.

53. The recombinant nucleic acid molecule of any one of claims 41 to 45, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter and a polyadenylation signal.

54. The recombinant nucleic acid molecule of claim 53, wherein the recombinant nucleic acid molecule comprises an H1 promoter and the polyadenylation signal.

55. The recombinant nucleic acid molecule of claim 53, wherein the recombinant nucleic acid molecule comprises an H1.M11 promoter and the polyadenylation signal.

56. The recombinant nucleic acid molecule of claim 53, wherein the recombinant nucleic acid molecule comprises a U6 promoter and the polyadenylation signal.

57. The recombinant nucleic acid molecule of claim 53, wherein the recombinant nucleic acid molecule comprises a U6+27 promoter and the polyadenylation signal.

58. The recombinant nucleic acid molecule of any one of claims 44, 45, and 53 to 57, wherein the RNA polymerase terminator comprises a polynucleotide sequence with 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% identity to SEQ ID NO: 15.

59. The recombinant nucleic acid molecule of any one of claims 1 to 58, wherein the recombinant nucleic acid molecule comprises a polynucleotide sequence with at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 200-232, 236, 238-240, and 264-276.

60. The recombinant nucleic acid molecule of any one of claims 1 to 25, 27, 29 to 59, wherein the circularization efficiency of the recombinant nucleic acid molecule is at least about 10% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively.

61. The recombinant nucleic acid molecule of any one of claims 1 to 25, 27, 29 to 59, wherein the abundance of the resulting circular RNA is at least about 100% higher compared to an recombinant nucleic acid molecule comprising SEQ ID NO:27 and SEQ ID NO:28 upstream and downstream of the cargo, respectively.

62. The recombinant nucleic acid molecule of claim 41 or 42, wherein the recombinant nucleic acid molecule comprises an external RNA polymerase III promoter, wherein a vector comprising the external RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an internal tRNA promoter when transcribed in a cell.

63. The recombinant nucleic acid molecule of any one of claims 41 to 43, wherein the recombinant nucleic acid molecule comprises an RNA polymerase III promoter, wherein a vector comprising the RNA polymerase III promoter provides at least about 10% higher circularization efficiency compared to a vector comprising an RNA polymerase II promoter when transcribed in a cell.

64. An adeno-associated virus (AAV) vector encoding the engineered tRNA sequence of any one of claims 1 -39 or the recombinant nucleic acid molecule of any one of claims 40-63.

65. The AAV vector of claim 64, wherein the engineered tRNA sequence or recombinant nucleic acid molecule is flanked by AAV inverted terminal repeats (ITRs).

66. An AAV particle comprising the AAV vector of claim 64 or 65 and a capsid protein.

67. The AAV particle of claim 66, wherein the capsid protein is a capsid protein of AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or a variant thereof.

68. The AAV particle of claim 66 or 67, wherein the capsid protein is an AAV9 capsid variant.

69. The AAV particle of any one of claims 66-68, wherein the capsid protein is an AAV.kl3 capsid protein.

70. The AAV particle of claim 69, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 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: 136.

71. The AAV particle of claim 69 or 70, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

72. The AAV particle of any one of claims 69 to 71, wherein the AAV.kl3 capsid protein consists of an amino acid sequence of SEQ ID NO: 137 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

73. The AAV particle of any one of claims 69 to 72, wherein the AAV.kl3 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.

74. The AAV particle of any one of claims 66-68, wherein the capsid protein is an AAVk20 capsid protein.

75. The AAV particle of claim 74, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 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: 136.

76. The AAV particle of claim 74 or 75, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

77. The AAV particle of any one of claims 74 to 76, wherein the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 138 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 136.

78. The AAV particle of any one of claims 74 to 77, 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 AAV9 capsid.

79. The AAV particle of any one of claims 66 to 78, wherein the capsid protein is a variant of a parental wild-type capsid protein, and wherein the capsid protein improves gene transfer and / or expression in one or more region(s) or part(s) of kidney when compared to the parental wild-type capsid protein.

80. A composition comprising the engineered tRNA sequence of any one of claims 1 to 39, the recombinant nucleic acid molecule of any one of claims 40 to 63, the AAV vector of claim 64 or 65, or the AAV particle of any one of claims 66 to 79, and a pharmaceutically acceptable carrier, diluent, or excipient.

81. A kit, comprising: the engineered tRNA sequence of any one of claims 1 to 39, the recombinant nucleic acid molecule of any one of claims 40 to 63, the AAV vector of claim 64 or 65, the AAV particle of any one of claims 66 to 79, or the composition of claim 80.

82. The kit of claim 81, wherein the kit comprises an instruction for delivering the AAV vector, the AAV particle, or the composition to a target cell or tissue.

83. A method of expressing a recombinant circular RNA in a cell, comprising introducing the engineered tRNA sequence of any one of claims 1 to 39, the recombinant nucleic acid molecule of any one of claims 40 to 63, the AAV vector of claim 64 or 65, the AAV particle of any one of claims 66 to 79, or the composition of claim 80 into the cell under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

84. The method of claim 83, wherein the cell is a mammalian cell.

85. The method of claim 83 or 84, wherein the cell is a human cell.

86. The method of any one of claims 83 to 85, wherein the cell is a myoblast.

87. The method of any one of claims 83 to 85, wherein the cell is a kidney cell.

88. A method of expressing a recombinant circular RNA in a tissue, comprising introducing the engineered tRNA sequence of any one of claims 1 to 39, the recombinant nucleic acid molecule of any one of claims 40 to 63, the AAV vector of claim 64 or 65, the AAV particle of any one of claims 66 to 79, or the composition of claim 80 into the tissue under conditions wherein the engineered tRNA sequence is transcribed and undergoes tRNA splicing.

89. A method of expressing a recombinant circular RNA in a subject, comprising administering an effective amount of the engineered tRNA sequence of any one of claims 1 to 39, the recombinant nucleic acid molecule of any one of claims 40 to 63, the AAV vector of claim 64 or 65, the AAV particle of any one of claims 66 to 79, or the composition of claim 80 to the subject, wherein the effective amount is an amount that reduces at least one symptom of a disease or condition in the subject.

90. A method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the engineered tRNA sequence of any one of claims 1 to 39, the recombinant nucleic acid molecule of any one of claims 40 to 63, the AAV vector of claim 64 or 65, the AAV particle of any one of claims 66 to 79, or the composition of claim 80 to the subject91. The method of claim 90, wherein the subject is a human subject.

92. The method of claim 90 or 91, wherein the subject has autosomal dominant polycystic kidney disease (ADPKD).

93. The method of claim 92, wherein the subject has ADPKD1.

94. The method of claim 92, wherein the subject has ADPKD2.

95. The method of any one of claims 90 to 94, wherein the method inhibits or ameliorates renal cyst development.

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