WO2024254546A2 - Virus-like particles comprising circular mrna expression systems and methods of use thereof - Google Patents

Abstract

The present invention relates to a virus-like particle (VLP). The virus-like particle comprising a circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3' to the first ligation sequence; a second ligation sequence positioned 3' to the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s); and a plurality of one or more proteins that can self-assemble into a nanoparticle. Also disclosed are compositions and methods of making and using such virus-like particles.

Description

VIRUS-LIKE PARTICLES COMPRISING CIRCULAR MRNA EXPRESSION SYSTEMS AND METHODS OF USE THEREOF

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/506,997, filed June 8, 2023, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under 1F31NS125945-04 awarded by National Institute of Neurological Disorders and Stroke. The government has certain rights in the invention.

FIELD

[0003] The present invention relates to virus-like particles comprising circular mRNA expression systems and methods of use thereof.

BACKGROUND

[0004] All mRNA therapeutics have a limited duration of expression due to the relatively short half-life of mRNA in the cytoplasm. This problem can be reduced for in vitro synthesized mRNAs by synthesizing them as circular mRNAs (Wesselhoeft et al., “Engineering Circular RNA for Potent and Stable Translation in Eukaryotic Cells,” Nat. Commun. 9:2629 (2018)). The circular mRNAs are synthesized by ligating the 3’ and 5’ ends using enzymatic methods or permuted self-splicing introns (Qu et al., “Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants,” Cell 185: 1728-1744. el6 (2022); Puttaraju et al., “Group I Permuted Intron- Exon (PIE) Sequences Self-Splice to Produce Circular Exons,” Nucleic Acids Res. 20:5357-5364 (1992); and Obi et al., “The Design and Synthesis of Circular RNAs,” Methods 196:85-103 (2021)). Circular mRNAs utilize an internal ribosome entry site (IRES) for recruiting translational machinery since they lack a 5’ cap (Chen & Sarnow, “Initiation of Protein Synthesis by the Eukaryotic Translational Apparatus on Circular RNAs,” Science 268:415-417 (1995)). Circular RNAs are known to be highly stable (Cocquerelle et al., “Mis-Splicing Yields Circular RNA Molecules,” Faseb 77: 155-160 (1993) and Jeck et al., “Circular RNAs are Abundant, Conserved, and Associated with ALU Repeats,” RNA 19: 141-157 (2013)) since they cannot be degraded by exonucleases (Ibrahim et al., “RNA Recognition by 3 '-to-5' Exonucleases: The Substrate Perspective,” Biochim Biophys Acta 1779:256-265 (2008)). Thus, therapeutic circular mRNA can be used in place of linear mRNA to achieve prolonged expression of the encoded protein (Wesselhoeft et al., “Engineering Circular RNA for Potent and Stable Translation in Eukaryotic Cells,” Nat. Commun. 9:2629 (2018); Qu et al., “Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants,” Cell 185: 1728-1744. el6 (2022); and Chen et al., “Engineering Circular RNA for Enhanced Protein Production,” Nat. BiotechnoL 41 :293 (2023)). [0005] Another major challenge of mRNA therapeutics is achieving mRNA delivery to specific cell types. When administered systemically, mRNAs are taken up primarily by the liver (Pardi et al., “Expression Kinetics of Nucleoside-Modified mRNA Delivered in Lipid Nanoparticles to Mice by Various Routes,” J. Control Release 217:345-351 (2015)). Since many applications require mRNA delivery to other tissues, an important objective is to devise strategies for the cell-type specific delivery of therapeutic mRNA beyond the liver.

[0006] One emerging approach for delivering mRNAs to specific cell types is virus-like particles (VLPs). VLPs comprise the major structural proteins of a virus needed to assemble a viral capsid, but do not package viral genomic material. VLPs can be designed to package and deliver specific mRNAs (Lu et al., “Delivering SaCas9 mRNA by Lentivirus-Like Bionanoparticles for Transient Expression and Efficient Genome Editing,” Nucleic Acids Research 47:e44-e44 (2019) and Prel et al., “Highly Efficient In Vitro and In Vivo Delivery of Functional RNAs Using New Versatile MS2-Chimeric Retrovirus-Like Particles,” Molecular Therapy - Methods & Clinical Development 2: 15039 (2015)). Instead of mRNAs synthesis in vitro, mRNAs are expressed in mammalian cells and directed to enter VLPs during assembly. These VLPs are produced with a nucleocapsid protein fused to the MS2 coat protein (MCP). The nucleocapsid protein then recruits MS2 hairpin-containing mRNAs into the VLP (Lu et al., “Delivering SaCas9 mRNA by Lentivirus-Like Bionanoparticles for Transient Expression and Efficient Genome Editing,” Nucleic Acids Research 47:e44-e44 (2019); Prel et al., “Highly Efficient In Vitro and In Vivo Delivery of Functional RNAs Using New Versatile MS2-Chimeric Retrovirus-Like Particles,” Molecular Therapy - Methods & Clinical Development 2: 15039 (2015); and Segal et al., “Mammalian Retrovirus-Like Protein PEG10 Packages its own mRNA and can be Pseudotyped for mRNA Delivery,” Science 373:882-889 (2021), which is hereby incorporated by reference in its entirety).

[0007] A major advantage of VLPs is that they can be “pseudotyped,” which is a process where the surface proteins are replaced to modify the VLP tropism (Cronin et al., “Altering the Tropism ofLentiviral Vectors Through Pseudotyping,” Curr. Gene Ther. 5:387-398 (2005); Naldini et al., “In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector,” Science 272:263-267 (1996); and Hamilton et al., “Targeted Delivery of CRISPR-Cas9 and Transgenes Enables Complex Immune Cell Engineering,” Cell Rep 35: 109207 (2021)). An additional advantage is that VLPs deliver mRNA into the cytosol (Stein et al., “pH-Independent HIV Entry into CD4-Positive T Cells Via Virus Envelope Fusion to the Plasma Membrane,” Cell 49:659-668 (1987)), rather than endosomes. When mRNA is delivered using a lipid nanoparticle, only a small amount of mRNA “escapes” from the endosome into the cytoplasm (Maugeri et al., “Linkage Between Endosomal Escape of LNP-mRNA and Loading into EVs for Transport to Other Cells,” Nature Communications 10:4333 (2019)). Because VLPs deliver mRNA into the cytosol, relatively small amounts of mRNA can be used to achieve mRNA expression in target cells.

[0008] VLPs would become a more useful technology if the duration of expression of the therapeutic protein can be extended by delivering circular mRNA rather than linear mRNA. In order to achieve this, circular mRNAs need to be generated in cells. Previous work has found that the standard method for creating large circular RNAs, termed the backsplicing system (Liang et al., “Short Intronic Repeat Sequences Facilitate Circular RNA Production,” Genes Dev. 28:2233-2247 (2014), which is hereby incorporated by reference in its entirety), does not efficiently generate circular mRNA species and would therefore be an inefficient method for producing circular mRNA-containing VLPs (Jiang et al., “Overexpression-Based Detection of Translatable Circular RNAs is Vulnerable to Coexistent Linear RNA Byproducts,” Biochem. Biophys. Res. Commun. 558: 189-195 (2021) and Ho-Xuan et al., “Comprehensive Analysis of Translation from Overexpressed Circular RNAs Reveals Pervasive Translation from Linear Transcripts,” Nucleic Acids Res. 48: 10368-10382 (2020), which are hereby incorporated by reference in their entirety).

[0009] The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY

[0010] A first aspect of the present disclosure relates to a virus-like particle (VLP). The virus-like particle comprises a circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence; a second ligation sequence positioned 3’ to the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s); and a plurality of one or more proteins that can self-assemble into a nanoparticle.

[0011] Another aspect of the present disclosure relates to a vector encoding a translation system. The vector comprises a promoter and a nucleic acid sequence encoding an RNA molecule comprising: a first ribozyme; a first ligation sequence positioned 3’ to the first ribozyme; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence; a second ligation sequence positioned 3’ to the internal ribosomal entry site; and a second ribozyme positioned 3’ to the second ligation sequence.

[0012] Another aspect of the present disclosure relates to a system for producing viruslike particles (VLPs) comprising a circular RNA translation system. The system comprises a packaging vector encoding a plurality of one or more proteins that can self-assemble into a nanoparticle; an envelope vector; and a vector encoding a translation system according to the present disclosure.

[0013] Another aspect of the present disclosure relates to a method for producing a VLP comprising a circular RNA translation system. This method involves providing a host cell; transfecting the host cell with a system according to the present disclosure; and culturing the host cell under conditions suitable to express the packaging vector, the envelope vector, and the circular RNA expression vector in the host cell, where said culturing produces virus-like particles comprising a circular RNA translation system.

[0014] Another aspect of the present disclosure relates to a method of inducing an immune response against a pathogen. This method involves administering to a subject an effective dose of the virus-like particle (VLP) according to the present disclosure, a VLP produced using the system according to the present disclosure, or a VLP produced using a method according to the present disclosure.

[0015] Another aspect of the present disclosure relates to a method of treating a subject. This method involves administering the virus-like particle (VLP) according to the present disclosure, a VLP produced using the system according to the present disclosure, or a VLP produced using a method according to the present disclosure to a subject in need thereof, where upon said administering, the one or more peptide(s) is/are expressed in a cell of the subject, thereby treating the subject.

[0016] Another aspect of the present disclosure relates to a method of performing gene editing on a subject. This method involves administering a virus-like particle (VLP) according to the present disclosure, a VLP produced using a system according to the present disclosure, or a VLP produced using a method according to the present disclosure to a subject in need thereof, where the one or more peptide(s) comprise one or more gene editing protein(s) and where, upon said administering, the gene editing protein(s) is/are expressed in a cell of the subject, thereby editing the genome of the subject. [0017] Another aspect of the present disclosure relates to an RNA molecule comprising: a first ribozyme; a first ligation sequence positioned 3’ to the first ribozyme; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), and where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence, wherein the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof; a second ligation sequence positioned 3’ to the internal ribosomal entry site; and a second ribozyme positioned 3’ to the second ligation sequence.

[0018] Another aspect of the present disclosure relates to a circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide sequence(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the peptide sequence(s) is positioned 3’ to the first ligation sequence, where the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof; a second ligation sequence positioned 3’ to the internal ribosomal entry site coupled to the RNA molecule encoding the peptide sequence(s).

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1B are schematic illustrations showing embodiments of the design of a reporter for circular mRNA-specific translation. FIG. 1 A illustrates the design of the split nanoluciferase (nLuc system). The Large BiT (LgBiT) and Small BiT (SmBiT) can only produce luminescence when they are brought together by a protein tether. FIG. IB illustrates the construct design of the Tornado translation system using a split nLuc ORF. LgBiT tethered to the SmBiT produces luminescence. CJ=circularization junction.

[0020] FIGS. 2A-2F demonstrate that the Tornado translation system produces a circular mRNA. FIG. 2A is a schematic illustration showing embodiments of the construct design of Tornado translation and linear mRNA expression systems. All three mRNAs contain the same ORF. FIG. 2B is a northern blot showing RNA from HEK293T cells transfected with plasmids expressing the Tornado split nLuc mRNA (Tornado CMV-CVB3), linear cap-dependent split nLuc mRNA (Linear (Cap)), or linear cap-independent split nLuc mRNA (Linear (CVB3) treated with vehicle or RNase R to test whether the RNA is circular. Full blot image is shown in (FIG. 3B). FIG. 2C is a bar graph (left panel) and schematic (right panel) showing luminescence from HEK293T cells transfected with a plasmid expressing the Tornado translation system with a split nLuc mRNA (Tornado (CMV-CVB3)) and a similar transcript in which the 3’ Tornado ribozyme is mutated (termed “mutTornado (CMV-CVB3)”). FIG. 2D is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing the Tornado translation system (Tornado (CMV-CVB3)), the linear cap-dependent mRNA expression system (Linear (Cap)), and the linear CVB3 -dependent mRNA expression system (Linear (CVB3)). FIG. 2E is a bar graph showing luminescence from FIG. 2D normalized to RNA expression from FIG. 2B. FIG. 2F is a pair of graphs showing RNA and protein expression from stable cell lines expressing the linear cap-dependent mRNA expression system (Linear (Cap)) (triangle markers, A), the linear CVB3-dependent mRNA expression system (Linear (CVB3)) (square markers, ■), and Tornado translation system (Circular (CMV-CVB3)) (circle markers, •) under a tetracycline-responsive promoter. The cell lines were pulsed with tetracycline then measured for luminescence and RNA abundance periodically after replacing the media with tetracycline-free media. RNA was quantified by performing qRT-PCR with primers that amplify a 120 nt region ofthe LgBiT. RLU=Relative Luminescence units. CJ=Circularization junction. Data are presented as mean values +/- one SD (n=3 biological replicates). Significance was calculated using unpaired two-tailed student’s /-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05.

[0021] FIGS. 3 A-3C demonstrate that successful RNase R treatment can be used to quantify RNA expression levels. FIG. 3 A is an ethidium bromide stain of the northern blot shown in FIG. 2B, FIG. 7E. Disappearance of rRNA bands in RNase R treated samples shows successful RNase R treatment. Ethidium bromide stain shows similar loading of RNA samples. FIG. 3B shows the full blot image and quantification from the northern blot in FIG. 2B, FIG. 7E. The northern blot was used to answer two questions. First, it can show whether the Tornado translation system RNA is in circular form in FIG. 2B. Second, it can show the levels of RNA expression in FIG. 3C and in FIG. 7E. FIG. 3C is a bar graph showing RNA quantification from the northern blot. Quantification of RNA was done by multiplying the mean intensity by the area of the band from FIG. 3B.

[0022] FIGS. 4A-4L demonstrate that the Tornado translation system is a robust method for expression of a circular mRNA. FIG. 4A is a schematic of primer design for the convergent and divergent primers. The convergent primers amplify a sequence that is present when the mRNA is in both a linear and circular form. The divergent primers amplify a region that is only present when the mRNA is in circular form. FIG. 4B demonstrates that RT-PCR analysis confirms that the Tornado translation system expresses a circular mRNA. A gel of the PCR reaction using convergent and divergent primers on cDNA from HEK293T cells transfected with the Linear (CVB3) and Tornado (CMV-CVB3) plasmids is shown. Divergent primers create an amplicon from the cDNA from cells transfected with Tornado (CMV-CVB3) but not the Linear (CVB3) plasmid. The divergent amplicon is the expected size. It should be noted that the TapeStation ladder consistently is ~10 bp too low (see FIG. 4K, FIG. 12A). FIG. 4C shows that sanger sequencing confirms that Tornado translation system expresses a circular mRNA. The amplicon from FIG. 4B was sequenced. The sequence shown (TGGACTGTAGAACCATGCCG AGT (SEQ ID NO: 80)) aligns to the circularization junction. FIG. 4D is a graph showing that the Tornado translation system does not increase expression of innate immune genes compared to linear mRNA expression systems. RNA expression of innate immune markers RIG-I, IL6, and IFNP was quantified by doing RT-PCR on HeLa cells transfected with plasmids encoding the Tornado translation system (Tornado (CMV-CVB3)), the linear capdependent mRNA expression system (Linear (Cap)), and the linear CVB3 -dependent mRNA expression system (Linear (CVB3)). Poly I:C was used as a positive control. RNA expression was normalized to GAPDH. The lower panel shows the same data as the upper panel without Poly I:C. Data are presented as mean values +/- one SD (n = 3 biological replicates). FIG. 4E is a schematic of the Tornado circularization junction variants. The short, medium and long stems are 18, 26, and 49 base pairs respectively. Each stem is designed to have a bulge every ~10 nucleotides to avoid cleavage by Dicer. The stems are designed to flank the IRES (CVB3) and nLuc sequence to facilitate circularization. More efficient circularization is reflected by increased luminescence. FIG. 4F is a bar graph demonstrating that lengthening the circularization junction stem does not increase protein output from the Tornado translation system. Luminescence from HEK293T cells transfected with plasmids carrying the Tornado translation system with the short, medium, and long stems was quantified. All three constructs were expressed using the CMV promoter and used the CVB3 IRES to drive translation of the nLuc mRNA. The three stems produced similar levels of luminescence. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’ s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05. FIG. 4G is a graph showing that lengthening the circularization junction of the Tornado translation system does not increase expression of innate immune genes. RNA expression of innate immune markers RIG-I, IL6, and IFNP was quantified by doing RT-PCR on HeLa cells transfected with plasmids encoding the short, medium, and long Tornado translation system circularization junctions. Poly EC was used as a positive control. RNA expression was normalized to GAPDH. The lower panel shows the same data as the upper panel without Poly I:C. Data are presented as mean values +/- one SD (n = 3 biological replicates). FIG. 4H are bar graphs demonstrating that the Tornado translation system can be used in multiple cell types. Luminescence from HepG2 and ZR-75-1 cells transfected with plasmids expressing the Tornado translation system (Tornado (CMV-CVB3)), the linear cap-dependent mRNA expression system (Linear (Cap)), and the linear CVB3 -dependent mRNA expression system (Linear (CVB3)) was quantified. Tornado translation system produces luminescence in HepG2 and ZR-75-1 cells. Notably, the Tornado translation system produced similar levels of luminescence in ZR-75-1 cells as the linear cap-dependent mRNA expression system. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05. FIG. 41 is a bar graph showing that the Tornado translation system can circularize the SARS-CoV-2 spike protein mRNA (4719 nt). HEK293T cells were transfected with plasmids expressing the Tornado translation system and the linear mRNA expression system containing a spike protein insert. RNA was treated with vehicle or RNase R then quantified by doing qRT-PCR with primers that amplified a 124 nt region of the spike protein. The Tornado translation system produces an RNA that is circular as evidenced by its resistance to RNase R compared to the linear control. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05. FIG. 4J shows that northern blot confirms qRT-PCR results. HEK293T cells were transfected with plasmids expressing the Tornado translation system and the linear mRNA expression system containing a spike protein insert. RNA was treated with vehicle or RNase R then quantified by doing a northern blot using probes against the spike RNA. Tornado spike and Linear spike RNA was run on the same gel but cut in half for hybridization and downstream steps of the northern blot to ensure visualization of both Tornado spike and Linear spike RNA. The Tornado translation system produces an RNA that is circular as evidenced by its resistance to RNase R compared to the linear control. It should be noted that circular RNA can be degraded by RNase R, albeit less efficiently than linear RNA. This explains why the Tornado spike RNA is partially degraded by RNase R. Ethidium bromide stain of the membrane shows successful RNase R treatment. FIG. 4K shows that RT- PCR confirms that Tornado translation system expresses a circular spike mRNA. A gel of the PCR reaction using divergent primers on cDNA from HEK293T cells transfected with the Tornado spike plasmid was run. The divergent amplicon is the expected size. It should be noted that the TapeStation ladder consistently is ~10 bp too low (see FIG. 4B, FIG. 12A). FIG. 4L shows that sanger sequencing confirms that Tornado translation system expresses a circular spike mRNA. The amplicon from FIG. 4K was sequenced. The sequence shown (TGGACTGTAGAACCATGCCGAG (SEQ ID NO: 81)) aligns to the circularization junction. [0023] FIGS. 5A-5C demonstrate that the Tornado translation system expresses more circular mRNA than the backsplicing system. FIG. 5A is a schematic of the backsplicing reaction. Intron homology drives a backsplicing reaction that results in the formation of a circular RNA. FIG. 5B is a bar graph showing the luminescence from HEK293T cells transfected with plasmids expressing the Tornado translation (Tornado (CMV-CVB3)) and backsplicing (Backsplicing (CMV-CVB3)) systems expressing split nLuc mRNA. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n=3 biological replicates). Significance was calculated using unpaired two-tailed student’s Ltest.

****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05. FIG. 5C is a northern blot run with RNA from HEK293T cells transfected with plasmids expressing the Tornado translation (Tornado (CMV-CVB3)) and backsplicing (Backsplicing (CMV-CVB3)) systems treated with vehicle or RNase R to test whether the RNA is circular. The unspliced linear precursor for the backsplicing system is 1.8 kb. The uncleaved linear precursor for the Tornado translation system is 1.9 kb. Both the cleaved linear precursor for the Tornado translation system and the forward spliced linear RNA for the backsplicing system should run equivalent to their circular RNA counterparts as described previously (Abe et al., “Circular RNA Migration in Agarose Gel Electrophoresis,” Mol. Cell 82: 1768-1777 (2022), which is hereby incorporated by reference in its entirety). The lower molecular weight products from the Tornado (CMV-CVB3) likely represent alternative conformations/non-denatured products. The lower molecular weight products from the Linear (Cap) likely represent partially degraded products. Ethidium bromide- stained blot is shown in (FIG. 6B).

[0024] FIGS. 6A-6B demonstrate that the Tornado translation system produces more circular RNA than the backsplicing system. FIG. 6A shows that the Tornado translation system produces more circular RNA than the backsplicing system. HEK293T cells were transfected with plasmids expressing the Tornado translation system and backsplicing system containing a ZKSCAN1 exon 2/3 insert. RNA was treated with vehicle or RNase R to test whether the RNA is circular. The Tornado translation system produces an RNA that is primarily in circular form. The backsplicing system produces an RNA that is primarily in a linear form. Ethidium bromide- stained blot shows successful RNase R treatment. FIG. 6B shows an ethidium bromide stain of the northern blot shown in FIG. 5C. Disappearance of rRNA bands in RNase R treated samples shows successful RNase R treatment.

[0025] FIGS. 7A-7F show that the Tornado translation system produces the most protein using a CMV-CVB3 promoter and IRES combination. FIG. 7A is a graph showing luminescence from HEK293T cells transfected with plasmids expressing the CVB3 or EMCV IRES. Both constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the split nLuc ORF. FIG. 7B is a schematic showing Pol III termination signal 1 (UCUUU sequence in Termination signal 1 inset) and Pol III termination signal 2 (UUUU sequence in Termination signal 2 inset). Mutations that render the IRES compatible with a Pol III promoter are indicated in each inset (Termination signal 1 inset indicates a U— >A termination signal mutation in mutEMCV; Termination signal 2 inset indicates a U— >C termination signal mutation in mutEMCV), and the compensatory mutations to conserve the function of the IRES is shown in the Termination signal 2 inset (A— G compensatory mutation in mutEMCV). The mutant EMCV (termed “mutEMCV”) contains the mutations identified in both insets for termination signal 1 and 2. SEQ ID NO: 82 (AGGGGUCUUUCCCCU) in inset 1 and SEQ ID NO: 83 (GAACCACGGGGACGUGGUUUU) in inset 2 are identified. FIG. 7C is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing EMCV IRES mutants. Both constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the split nLuc ORF. FIG. 7D is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing the mutant EMCV (mutEMCV) and wild-type EMCV (EMCV) IRESs. Both constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the split nLuc ORF. FIG. 7E shows the quantification of RNA from HEK293T cells transfected with plasmids expressing Pol Il-driven (CMV CVB3) and Pol Ill-driven (U6 mutEMCV) Tornado translation systems by northern blot and calculation of pixel intensity with ImageLab software (FIG. 3A-3C). Full blot image is shown in FIG. 3B. FIG. 7F is a graph showing luminescence from HEK293T cells transfected with plasmids expressing Pol Il-driven (CMV- CVB3) and Pol Ill-driven (U6-mutEMCV) Tornado translation systems with a split nLuc ORF. RLU=Relative Luminescence units. Data are presented as mean values +/- one SD (n=3 biological replicates). Significance was calculated using unpaired two-tailed student’s /-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05.

[0026] FIGS. 8A-8H demonstrate that the Pol Ill-driven Tornado translation system produces less protein than the Pol Il-driven Tornado translation system. FIG. 8A is a bar graph demonstrating that the CVB3 IRES produces a similar amount of protein as the HRV-B3 IRES. Luminescence from HEK293T cells transfected with plasmids expressing the CVB3 or HRV-B3 IRES was quantified. Both constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with an nLuc ORF. The CVB3 and HRV-B3 IRES produce similar levels of luminescence. Using a P value cutoff of < 1, the CVB3 IRES produced more luminescence than the HRV-B3 IRES. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. FIG. 8B shows mF old structural predictions showing that the falcon picornavirus maintains the structure of the stem loop that contains the EMCV termination signal 2 mutation but does not have a Pol III termination signal. Termination signal (TTTT), Termination signal mutation (G), and Compensatory mutation (C) are indicated with arrows. SEQ ID NO: 84 (CCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAA) and SEQ ID NO: 85 (CCCACCAGCCCACGGGAGTGGGCTTTCCTTAAA) are identified in the left and right panels, respectively. FIG. 8C is a graph showing that mutEMCV produces more protein than the wildtype CVB3 in a Pol Ill-driven Tornado translation system. Luminescence was quantified from HEK293T cells transfected with plasmids expressing the CVB3 (U6-CVB3) and mutEMCV (U6-mutEMCV) IRES. Both constructs were expressed using a Pol Ill-driven (U6) Tornado translation system with the split nLuc ORF. The mutEMCV IRES produced ~15-fold more luminescence than the CVB3 IRES. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’ s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05. FIG. 8D is a schematic showing that HCV IRES and CSFV IRES are similar in structure, but not sequence. CSFV has a similar structure as the HCV IRES yet lacks the Pol III termination element that is present in HCV. Pol III termination signal in HCV IRES is shown in inset in left panel. SEQ ID NO: 86 is identified in FIG. 8D. FIG. 8E is a graph showing that mutEMCV produces more protein than the CSFV in a Pol Ill-driven Tornado translation system. Luminescence was quantified from HEK293T cells transfected with plasmids expressing the CSFV (U6-CSFV) and mutEMCV (U6-mutEMCV) IRES. Both constructs were expressed using a Pol Ill-driven (U6) Tornado translation system with the split nLuc ORF. The CSFV IRES produced 3 -fold less luminescence than the mutEMCV IRES. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05. FIG. 8F are images showing that the Pol Ill-driven Tornado translation system shows similar nuclear and cytoplasmic distribution as the Pol II- driven Tornado translation system and the linear cap-dependent mRNA expression system. Since Pol III transcripts are generally retained in the nucleus, whether the reason for low protein output from the Pol Ill-driven Tornado translation system was due to nuclear retention was evaluated. Fluorescence in situ hybridization was performed using a probe against the LgBiT region (green) of the mRNA and a control probe against the nuclear non-coding RNA NEAT1 (red). DAPI stain is shown in blue. Zoomed in images show the nuclear (white) and cytoplasmic (yellow) borders. As expected, NEAT1 was almost exclusively in the nucleus. However, the Pol Ill-driven Tornado translation system (U6-mutEMCV) is partially in the cytoplasm and partially in the nucleus which is similar to the distribution of the Pol Il-driven Tornado translation system (CMV-CVB3) and the linear cap-dependent mRNA expression system (Linear (Cap)). Scale bars = 24 pm. FIG. 8G is a graph showing that the Pol Ill-driven Tornado translation system shows a similar ratio of nuclear to cytoplasmic puncta as the Pol II- driven Tornado translation system and the linear cap-dependent mRNA expression system. The ratio of cytoplasmic to nuclear puncta from FIG. 8F was quantified. The Pol Ill-driven Tornado translation system (U6-mutEMCV) is -50% in the cytoplasm and 50% in the nucleus which is similar to the distribution of the Pol Il-driven Tornado translation system (CMV-CVB3) and the linear cap-dependent mRNA expression system (Linear (Cap)). FIG. 8H is a graph showing that the constitutive transport element (CTE) RNA sequence does not increase protein expression from the Pol Ill-driven Tornado translation system. Luminescence was quantified from HEK293T cells transfected with plasmids expressing the Pol Ill-driven Tornado translation system (U6-mutEMCV) with and without the CTE RNA sequence. The CTE sequence did not increase protein expression from the Pol Ill-driven Tornado translation system. The decreased protein expression from the Pol Ill-driven Tornado translation system compared to the Pol II- driven Tornado translation system is therefore not due to nuclear retention. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05.

[0027] FIGS. 9A-9G demonstrate that continuous translation does not improve the protein output from the Tornado translation system. FIG. 9A is a schematic showing the design of a non-continuous and continuous translation system. FIG. 9B is a Table showing viral IRESs contain several stop codons in all frames. FIG. 9C is a schematic showing mutations required to make the HCV IRES compatible with continuous translation. Stop codons (UAG in left inset, UGA in right inset, UAG in right inset) are shown. Shown are mutations that render the IRES compatible with continuous translation (G— C in stop codon UAG in left inset; U— >G in stop codon UGA in right inset; G— >U in stop codon UAG in right inset), and the compensatory mutation (C— G in left inset) to preserve the predicted structure of the IRES. The mutant HCV (termed “mutHCV”) contains the identified STOP codon mutations in mutHCV and compensatory mutations in mutHCV. SEQ ID NO: 87 (GCCUGAUAGGGU) is identified in FIG. 9C. FIG. 9D is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing the mutHCV non-continuous translation system (mutHCV STOP) and the wild-type HCV (termed “wtHCV”) non-continuous translation system (wtHCV STOP). Both constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the split nLuc ORF. FIG. 9E is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing the mutHCV non-continuous translation system (mutHCV STOP), the mutHCV continuous translation system (mutHCV NO STOP), and the CVB3 non-continuous translation system (CVB3 STOP). All constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with a split nLuc ORF. FIG. 9F is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing the LIMA1 non- continuous translation system (LIMAl STOP), the /./AM / continuous translation system (LIMAl NO STOP), and the CVB3 non-continuous translation system (CVB3 STOP). All constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the split nLuc ORF. FIG. 9G is a bar graph showing luminescence from HEK293T cells transfected with plasmids expressing the /./AM / non-continuous translation system (LIMAl STOP), the /./AM / continuous translation system (LIMA l NO STOP), the /./AM / continuous translation system with a mutant AUG (LIMAl mutAUG) and the continuous translation system with no IRES (NO IRES NO STOP). All constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the split nLuc ORF. RLU=Relative Luminescence units. Data are presented as mean values +/- one SD (n=3 biological replicates). Significance was calculated using unpaired two-tailed student’s /-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05.

[0028] FIGS. 10A-10B demonstrate that endogenous IRES elements have minimal translational activity. FIG. 10A is a nucleotide sequence alignment showing that the LIMAl IRES only contains one start codon in frame with the split nLuc ORF. The start codon is indicated. SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90 are shown in FIG. 10A. FIG. 10B is a bar graph showing that alternative endogenous IRESs elements do not produce more protein than the LIMAl IRES. Luminescence from HEK293T cells transfected with plasmids expressing the putative IRESs from a previous screen were quantified. All constructs were expressed using a Pol Il-driven (CMV) Tornado translation system with the continuous split nLuc ORF. None of the IRES elements tested produced more protein than the LIMAl IRES. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 2 technical replicates).

[0029] FIGS. 11 A-l IE demonstrate that VLPs produced using the Tornado translation system exhibit increased level and duration of protein expression compared to conventional VLPs. FIG. 11 A is a schematic of the circular mRNA VLP system. FIG. 1 IB is a bar graph showing that RNA from VLPs that were produced using the Tornado translation system (Tornado nLuc-MS2), or the linear mRNA expression system (Linear nLuc-MS2) as the transfer plasmid was treated with vehicle or RNase R to test whether the RNA is circular. RNA quantification was done by qRT-PCR using primers that amplified a 126nt region of the nLuc gene. FIG. 11C is a graph showing luminescence from HEK293T cells transduced with VLPs that were produced using either the Tornado translation system (Tornado nLuc-MS2) (circle markers, •) or the linear mRNA expression system (Linear nLuc-MS2) (square markers, ■) at 5, 24, 48, and 72 hours after transduction. HEK293T cells were transduced at equal levels of VLP mRNA (FIG. 12C). FIG 1 ID is a schematic for showing cell-type specific delivery of circular mRNA using spike pseudotyped VLPs. FIG. 1 IE is a bar graph showing luminescence from HEK293T cells and ACE2-expressing HEK293T cells transduced with VSV-G pseudotyped or spike pseudotyped VLPs containing circular nLuc mRNA. RLU=Relative Luminescence units. Data are presented as mean values +/- one SD (n=3 biological replicates). Significance was calculated using unpaired two-tailed student’s /-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>0.05.

[0030] FIGS. 12A-12D demonstrate that the Tornado translation system can be used to package circular mRNA into VLPs. FIG. 12A shows a gel showing the results of RT-PCR analysis confirming that VLPs packaged using the Tornado translation system contain a circular mRNA. A gel of the PCR reaction using divergent primers on cDNA from Tornado nLuc-MS2 viral RNA was run. The divergent amplicon is the expected size. It should be noted that the TapeStation ladder consistently is ~10 bp too low (see FIG. 4B, FIG. 4K). FIG. 12B shows Sanger sequencing confirmation that VLPs packaged using the Tornado translation system package circular mRNA. The amplicon from FIG. 12A was sequenced. The sequence aligns to the circularization junction. SEQ ID NO: 91 (CGGTCGGCGTGGACTGTAGAACCATGCCGAGTG CG) is indicated. FIG. 12C is a bar graph showing VLP RNA titers. The VLPs that were produced using the Tornado translation system and the linear mRNA expression system were tittered using RNA quantification. Viral RNA was extracted from equal volumes of viral supernatant that was produced using the Tornado translation system (Tornado nLuc-MS2) and the linear mRNA expression system (Linear nLuc-MS2). Then, qRT-PCR was performed using primers that amplified a 126nt region of the nLuc gene. The linear mRNA expression system produced VLPs that were 15-fold more concentrated than the VLPs produced by the Tornado translation system. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. ****p<0.0001, ***p<0 001, **p<0.01, *p<0.05, n.s. p>.05. FIG. 12D is a bar graph demonstrating that VLPs produced using the Tornado translation system can be used to transduce SH-SY5Y cells. Luminescence from SH-SY5Y cells transduced with VLPs that were produced using either the Tornado translation system (Tornado nLuc-MS2) or the linear mRNA expression system (Linear nLuc-MS2) at hour 24 after transduction were quantified. Cells were transduced at equal levels of VLP mRNA. VLPs that were produced using the Tornado translation system can be used to transduce SH-SY5Y cells and produce ~5-fold more luminescence than the VLPs that were produced using the linear mRNA expression system. RLU = Relative Luminescence units. Data are presented as mean values +/- one SD (n = 3 biological replicates). Significance was calculated using unpaired two-tailed student’s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. p>.05.

DETAILED DESCRIPTION

Definitions

[0031] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0032] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods and/or steps of the type described herein and/or which will become apparent to a person of ordinary skill in the art upon reading this disclosure. In another example, reference to “a compound” includes both a single compound and a plurality of different compounds.

[0033] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 50%, or within 20%, or within 10%, or within 5% (or any amount or range within 5-50%) of a given value or range. The allowable variation encompassed by the term “about” or “approximately” may depend on the context.

[0034] The term “and/or” as used herein means that the listed features are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed features is used or present.

[0035] As will be understood by a person of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, as well as any value within a range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so on. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so on. As will also be understood by a person of ordinary skill in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges or specific values therein as discussed above. Finally, as will be understood by a person of ordinary skill in the art, and as discussed above, a range includes each individual value.

[0036] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” The methods, kits, systems, and/or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0037] In some embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0038] The term “complementary” when used in connection with nucleic acid, refers to the pairing of bases, A with T or U, and G with C. The term “complementary” refers to nucleic acid molecules that are completely complementary, that is, form A to T or U pairs and G to C pairs across the entire reference sequence, as well as molecules that are partially (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) complementary. [0039] The terms “nucleic acid” and “nucleotide” encompass both DNA and RNA unless specified otherwise.

[0040] The term “polypeptide,” “peptide”, or “protein” are used interchangeably and to refer to a polymer of amino acid residues. The terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).

[0041] The terms “express” and “expression” mean allowing or causing the information in a DNA sequence to become produced, for example producing an RNA by activating the cellular functions involved in transcription of a DNA sequence.

[0042] As used herein, the term “virus like particle” or “VLP” refers to a stable macromolecular assembly which comprises the major structural proteins of a virus needed to assemble a viral capsid, but do not package viral genomic material. VLPs can be designed to package and deliver specific mRNAs.

[0043] As used herein, the term “pseudotyped” refers to the replacement of any component of a VLP with that of a heterologous virus to modify the VLP tropism. A “pseudotyped VLP” denotes a recombinant VLP comprising one or more heterologous envelope proteins. For example, a pseudotyped lentiviral VLP comprises one or more envelope and/or spike proteins of non-lentiviral origin or one or more envelope and/or spike proteins which are of a different species or subspecies of lentivirus.

[0044] As used herein, the term “circular RNA” refers to a single stranded, covalently closed loop RNA molecule having no 5' or 3' ends.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, some embodiments of the methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0046] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Virus-Like Particles

[0047] A major problem with mRNA therapeutics is that mRNA is usually degraded within a few hours after entering the cytosol. New approaches for in vitro synthesis of circular mRNA have allowed increased levels and duration of protein synthesis from mRNA therapeutics due to the long half-life of circular mRNA. However, it remains difficult to genetically encode circular mRNAs in mammalian cells.

[0048] The present disclosure provides an improvement of virus-like particle technology whereby circular mRNAs rather than linear mRNAs are packaged into VLPs. The disclosed “Tornado translation system” utilizes the Tornado (Twister-optimized RNA for durable overexpression) circular RNA expression system to generate high levels of small circular RNA (Litke et al., “Highly Efficient Expression of Circular RNA Aptamers in Cells using Autocatalytic Transcripts,” Nat. BiotechnoL 37:667-675 (2019), which is hereby incorporated by reference in its entirety). The Examples of the present disclosure show that the “Tornado translation system” can be used to produce virus-like particles (VLPs) that contain circular mRNA, resulting in VLPs that exhibit markedly prolonged protein synthesis compared to VLPs that contain linear mRNAs. Overall, these experiments provide a new approach for delivery of circular mRNAs into target cells using VLPs.

[0049] Accordingly, a first aspect of the present disclosure relates to a virus-like particle (VLP). The virus-like particle comprises a circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence; a second ligation sequence positioned 3’ to the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s); and a plurality of one or more proteins that can selfassemble into a nanoparticle.

[0050] As used herein, an “internal ribosome entry site” or “IRES” refers to an internal site of an mRNA sequence which recruits the ribosome or other translation initiation machinery to enable translation initiation.

[0051] The IRES may be a wild-type internal ribosomal entry site or a modified internal ribosomal entry site. A “modified IRES sequence” or “mutant IRES sequence” refers to an IRES sequence comprising one or more modifications, which can be addition, deletion, substitution, and/or alteration of at least one (or more) nucleotide(s). Such modifications may result in the addition or deletion of structural elements (e.g., a stop codon or a transcription termination signal), lengthening or shortening of an existing stem, loop, c or pseudoknot, changes in the composition or structure of a loop(s), stem(s), pseudoknot(s), or any combination of these.

[0052] Exemplary IRES elements include, without limitation, CVB3 IRES (SEQ ID NO: 1), EMCV IRES (SEQ ID NO: 2), mutEMCV IRES (SEQ ID NO: 3), mutHCV IRES (SEQ ID NO: 4), CSFV IRES (SEQ ID NO: 5), HRV-B3 IRES (SEQ ID NO: 6), mutCVB3 IRES (SEQ ID NO: 7), and LIMA1 IRES (SEQ ID NO: 8). [0053] In some embodiments, the IRES sequence is selected from the group consisting of

SEQ ID NOs: 1-8, or a derivative thereof (Table 1).

Table 1: IRES Sequences

[0054] The Tornado system enables expressed circular RNAs to be converted into highly stable circular RNAs, resulting in circular RNA expression within cells at micromolar concentrations (see, e.g., U.S. Patent No. 11,756,183 to Jaffrey et al., which is hereby incorporated by reference in its entirety).

[0055] As described in more detail infra, Pol III promoters express higher levels of RNA than Pol II promoters. Thus, the Tornado translation system according to the present disclosure could benefit by using a Pol III promoter. FIG. 7B demonstrates that many commonly used IRES sequences such as CVB3 (SEQ ID NO: 1), EMCV (SEQ ID NO: 2), and HRV-B3 (SEQ ID NO: 6) comprise Pol III termination signals (e.g., UUUU, UCUUU, or UUUAU). Thus, in some embodiments, the IRES lacks a Pol III termination element. Such IRES sequences may be wildtype or modified IRES sequences. For example, the IRES sequence may be a modified IRES sequence which has been modified to remove one or more Pol III termination signal(s). In accordance with such embodiments, the modified IRES is mutEMCV IRES (SEQ ID NO: 3). [0056] Continuous translation of a circular RNA molecules requires an IRES that is in frame with an open reading frame (ORF) (e.g., an RNA molecule encoding one or more peptide(s)) lacking a stop codon. The term “stop codon” refers to a nucleotide triplet within mRNA that signals a termination of translation. Exemplary stop codons include, for example, UAG (in RNA)/TAG (in DNA) (also known as an “amber” stop codon), UAA/TAA (also known as an “ochre” stop codon), and UGA/TGA (also known as an “opal” or “umber” stop codon). Thus, in some embodiments, the IRES lacks a stop codon.

[0057] In some embodiments of the compositions, systems, and/or methods of the present disclosure, a portion of a first ligation sequence is complementary to a portion of a second ligation sequence. In accordance with such embodiments, the 3’ portion of the first ligation sequence is complementary to the 5’ portion of the second ligation sequence. The portion of the first ligation sequence complementary to the portion of the second ligation sequence may be at least 18, at least 26, or at least 49 nucleotides in length.

[0058] In the Tornado circular RNA expression system, an RNA ligase such as RtcB may catalyze the ligation of a first ligation sequence comprising a 5 ’-OH end and a second ligation sequence comprising a 2’, 3 ’-cyclic phosphate end to form a circular RNA molecule (see, e.g., Litke and Jaffrey, “Highly Efficient Expression of Circular RNA Aptamers in Cells using Autocatalytic Transcripts,” Nat. Biotechnol. 37(6): 667-675 (2019), which is hereby incorporated by reference in its entirety). Thus, following ligation by the RNA ligase, the first ligation sequence is joined to the second ligation sequence.

[0059] In some embodiments, the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

[0060] As used herein, the term “neoepitope” refers to a potentially immunogenic epitope present in a protein associated with a disease cause by a mutation in DNA. A “cancer neoepitope” refers to a tumor-specific antigen associated with a somatic mutation(s) (see, e.g., Wickstrbm et al., “Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor- Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display,” Front. Immunol. 10:2766 (2019), which is hereby incorporated by reference in its entirety). In some embodiments, the one or more peptides comprises an antigen, e.g., a cancer neoepitope. [0061] As described herein, Cas family proteins form a ribonucleoprotein complex with a guide RNA, which guides the Cas protein to a target DNA sequence. Suitable Cas proteins include Cas nuclease (Cas) proteins (z.e., Cas proteins capable of introducing a double strand break at a target nucleic acid sequence), Cas nickase (nCas) proteins (z.e., Cas protein derivatives capable of introducing a single strand break at a target nucleic acid sequence), and nuclease dead Cas (dCas) proteins (z.e., Cas protein derivatives that do not have any nuclease activity).

[0062] The Cas family protein may be selected from the group consisting of Cas9, nCas9, dCas9, Cas 12a, nCasl2a, dCasl2a, Cas 12b, nCasl2b, and dCasl2b.

[0063] In some embodiments, the Cas family protein is a Cas9 protein. As used herein, the term “Cas9 protein” or “Cas9” includes any of the recombinant or naturally-occurring forms of the CRISPR-associated protein 9 (Cas9) or variants or homologs thereof. In some embodiments, the Cas9 protein is substantially identical to the protein identified by the UniProt reference number Q99ZW2, G3ECR1, J7RUA5, A0Q5Y3, or J3F2B0 (which are hereby incorporated by reference in their entirety) or a variant or homolog having substantial identity thereto. For example, the Cas family protein may be an nCas9 protein or a dCas9 protein.

[0064] In some embodiments, the Cas family protein is a Casl2a protein. As used herein, the term “Cas 12a protein” or “Cas 12a” includes any of the recombinant or naturally- occurring forms of the CRISPR-associated protein 12 (Cas 12a) or variants or homologs thereof. In some embodiments, the Casl2a protein is substantially identical to the protein identified by the UniProt reference number A0Q7Q2, U2UMQ6, A0A7C6JPC1, A0A7C9H0Z9, or A0A7J0AY55 (which are hereby incorporated by reference in their entirety) or a variant or homolog having substantial identity thereto. For example, the Cas family protein may be an nCasl2a protein or a dCasl2a protein.

[0065] In some embodiments, the Cas family protein is a Casl2b protein. As used herein, the term “Cas 12b protein” or “Cas 12b” includes any of the recombinant or naturally- occurring forms of the CRISPR-associated protein 12 (Cas 12b) or variants or homologs thereof. In some embodiments, the Casl2b protein is substantially identical to the protein identified by the UniProt reference number T0D7A2, A0A6I3SPI6, A0A6I7FUC4, A0A6N9TP17, A0A6M1UF64, A0A7Y8V748, A0A7X7KIS4, A0A7X8X2U5, or A0A7X8UMW7 (which are hereby incorporated by reference in their entirety) or a variant or homolog having substantial identity thereto. For example, the Cas family protein may be an nCasl2b protein or a dCasl2b protein. [0066] As used herein, the term “chimeric antigen T-cell receptor” refers to a genetically engineered T cell receptor. In some embodiments, the one or more peptides comprises a chimeric antigen T-cell receptor.

[0067] FIG. 11 A is a schematic of one embodiment of an exemplary virus-like particle comprising a circular mRNA according to the present disclosure. This virus-like particle is produced in a system for producing virus-like particles according to the present disclosure. Such systems rely on the specific interaction between an protein-binding RNA aptamer (e.g., RNA aptamer MS2) and its cognate RNA-binding protein (e.g., MS2 coat protein (MCP)) to package a circular mRNA of interest; and comprises an envelope vector (e.g., a plasmid encoding a viral envelope and/or spike protein), a vector encoding a translation system according to the present disclosure (e.g., a transfer plasmid that encodes circular mRNA molecule with a protein-binding RNA aptamer (e.g., the MS2 stem loop in its 3’UTR), and a packaging vector encoding a plurality of one or more proteins that can assemble into a nanoparticle (e.g., an integrasedeficient packaging plasmid that expresses the cognate RNA-binding protein (e.g., MCP) fused to the N-terminus of the nucleocapsid protein). VLPs produced with this system comprise a nucleocapsid protein fused to the MS2 coat protein (MCP), which recruits MS2 hairpincontaining circular mRNAs into the VLP.

[0068] In some embodiments of the virus-like particle according to the present disclosure, the circular RNA molecule further comprises a stem loop that binds to a cognate RNA-binding protein, where the stem loop is positioned 3’ to the RNA molecule encoding the one or more peptide(s). Suitable protein-binding RNA aptamers are well known in the art and provided in Table 2.

Table 2: Suitable RNA Aptamer Stem Loop Sequences

[0069] In accordance with such embodiments, the stem loop is selected from the group consisting of MS2, PP7, BoxB, and Com. In some embodiments, the stem loop is a MS2 stem loop sequence.

[0070] The RNA aptamer PP7 is bound by the Pseudomonas aeruginosa PP7 bacteriophage coat protein see, e.g., Lu et al., “Delivering SaCas9 mRNA by Lentivirus-Like Bionanoparticles for Transient Expression and Efficient Genome Editing,” Nucleic Acids Research 47:e44-e44 (2019) and Lim et al., “Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomas Phage PP7,” J. Biol. Chem. 276(25):22507-22513 (2001), which are hereby incorporated by reference in their entirety). In some embodiments, the stem loop is a PP7 stem loop sequence.

[0071] The RNA aptamer boxB is bound by the X bacteriophage N protein (see, e.g., Braselmann et al., “Illuminating RNA Biology: Tools for Imaging RNA in Live Mammalian Cells,” Cell Chem. Biol. 27(8): 891-903 (2020), which is hereby incorporated by reference in its entirety). In some embodiments, the stem loop is a boxB stem loop sequence.

[0072] The RNA aptamer com is bound by aptamer binding protein Com (see, e.g., Lyu and Lu et al., “New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery,” Int. J. Mol. Sci. 23(15):8750 (2022), which is hereby incorporated by reference in its entirety). In some embodiments, the stem loop is a Com stem loop.

[0073] The nanoparticle may be a viral capsid or a viral capsid-like structure. The term “viral capsid” or “capsid” refers to the proteinaceous shell or coat of a virion or a virus-like particle. The term “viral nucleocapsid” or “nucleocapsid” refers to the capsid and its associated nucleic acid molecule (e.g., a circular RNA molecule according to the present disclosure). In the context of the present disclosure, viral capsids or nucleocapsids may encapsidate, protect, transport, and/or release into a host cell a circular RNA molecule (e.g., a circular mRNA molecule).

[0074] In some embodiments, the plurality of one or more proteins that can self-assemble into a nanoparticle comprises a polyprotein. Viral polyproteins may be cleaved into individual enzymes by viral or cellular enzymes. In some embodiments, the polyprotein comprises one or more proteins selected from the group of proteins consisting of: a nucleocapsid protein, a capsid protein, a matrix protein, a reverse transcriptase, a protease, and a defective integrase.

[0075] Suitable polyproteins include, without limitation, a retroviral group specific antigen (Gag) polyprotein, a mammalian group specific antigen (Gag)-like polyprotein, and derivatives thereof.

[0076] In some embodiments, the retroviral group specific antigen (Gag) polyprotein is a retroviral polyprotein. Retroviral Gag polyproteins are organized from the amino terminus to the carboxyl terminus, with domains that are cleaved into, e.g., matrix, capsid, and nucleocapsid proteins (see, e.g., Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Virion Proteins, which is hereby incorporated by reference in its entirety). [0077] In some embodiments, the retroviral group specific antigen is a Human Immunodeficiency Virus Type 1 (HIV-1) group specific antigen (Gag). The HIV-1 group specific antigen (Gag) comprises four main structural domains (matrix, capsid, nucleocapsid, p6) and two smaller spacer peptides (SP1 and SP2) see, e.g., Marie and Gordon, “The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity,” Int. J. Mol. Sci. 23(14): 7569 (2022), which is hereby incorporated by reference in its entirety). [0078] In some embodiments, the mammalian group specific antigen (Gag)-like polyprotein is PEG10. PEG10 is a homolog of Gag which preferentially binds and facilitates vesicular secretion of its own mRNA (Segel et al., “Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery,” Science 373(6557): 822- 889 (2021), which is hereby incorporated by reference in its entirety). The mRNA cargo of PEG10 can be reprogrammed by flanking an RNA molecule encoding one or more peptides with PeglO’s untranslated regions. PEG10 untranslated regions are shown in Table 3 below.

Table 3: PEG10 Untranslated Region (UTR) Sequences

[0079] The plurality of one or more proteins that can self-assemble into a nanoparticle may comprise(s) one or more structural protein(s). For example, the one or more structural protein(s) may be selected from the group consisting of capsid protein(s), nucleocapsid protein(s), matrix protein(s), and combinations thereof.

[0080] In some embodiments, the capsid protein(s) is/are non-retroviral capsid protein(s). Exemplary non-retroviral capsid protein(s) is/are selected from the group consisting of Herpes Simplex Virus (HSV) VP23, Herpes Simplex Virus (HSV) VP19C, Hepatitis B Virus (HBV) core antigen, Human Papillomavirus (HPV) LI, Human Papillomavirus (HPV) L2, and combinations thereof.

[0081] In some embodiments, at least one of the plurality of the one or more proteins that can self-assemble into a nanoparticle comprise(s) or is fused to an RNA-binding protein protein/domain (e.g., an RNA aptamer-binding protein/domain). Suitable exemplary RNA- binding proteins and their amino acid sequences are identified in Table 4.

Table 4: Suitable RNA-Binding Proteins

[0082] In some embodiments, the RNA-binding protein domain is selected from the group consisting of MS2 coat protein (MCP), Com, PCP, and N22.

[0083] The RNA-binding domain may be located at an N-terminus or at a C-terminus of the at least one of the plurality of the one or more proteins that can self-assemble into a nanoparticle comprising or fused to the RNA-binding domain.

[0084] In some embodiments, the virus-like particle (VLP) further comprises one or more envelope and/or spike protein(s).

[0085] Suitable viral envelope protein(s) include, without limitation, a vesicular stomatitis virus envelope protein, a rabies virus envelope protein, a measles virus envelope protein, a nipah virus envelope protein, a chickungunya virus envelope protein, and a sindbis virus envelope protein. [0086] In some embodiments, the one or more envelope and/or spike protein(s) are selected from the group consisting of a vesicular stomatitis virus G (VSV G) protein, RabV-G, Chickungunya virus E1ZE2, Sindbis virus E1ZE2, Measle virus H/F, and derivatives thereof. [0087] In some embodiments, the one or more envelope and/or spike protein(s) comprises a vesicular stomatitis virus G (VSV G) protein or a derivative thereof. In accordance with such embodiments, the one or more envelope and/or spike protein(s) may comprise mutant VSV-G (K47Q, R354A). In some embodiments the one or more envelope and/or spike protein(s) comprises mutant VSV-G (K47Q, R354A) in combination with a targeting molecule such as an antibody or scFv, TCR, or MHC peptide pair.

[0088] In some embodiments, the one or more envelope and/or spike protein(s) comprise a coronavirus spike protein (e.g., a SARS-CoV-2 spike protein).

[0089] The one or more envelope and/or spike proteins may comprise a fusion protein.

[0090] In some embodiments of the virus-like particles according to the present disclosure, the VLP further comprises a protein that is trafficked to the viral particle and/or cell surface membrane. In accordance with such embodiments, the protein that is trafficked to the viral particle and/or cell surface membrane is a ligand or target binding protein.

[0091] The protein that is trafficked to the viral particle and/or cell surface membrane may be selected from the group consisting of a single chain of MHC fused with beta-2 - microglobulin (B2M) and a covalently linked peptide; a single-chain antibody variable fragment fused to a transmembrane domain; and an antigen fused to a transmembrane domain.

Vectors Encoding a Translation System

[0092] Another aspect of the present disclosure relates to a vector encoding a translation system. The vector comprises a promoter and a nucleic acid sequence encoding an RNA molecule comprising: a first ribozyme; a first ligation sequence positioned 3’ to the first ribozyme; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence; a second ligation sequence positioned 3’ to the internal ribosomal entry site; and a second ribozyme positioned 3’ to the second ligation sequence.

[0093] The term “vector” is used interchangeably with “expression vector.” The term “vector” may refer to viral or non-viral, prokaryotic or eukaryotic, DNA or RNA sequences that are capable of being transfected into a cell, referred to as “host cell,” so that all or a part of the sequences are transcribed. Vectors are frequently assembled as composites of elements derived from different viral, bacterial, or mammalian genes. Vectors contain various coding and noncoding sequences, such as sequences coding for selectable markers, sequences that facilitate their propagation in bacteria, or one or more transcription units that are expressed only in certain cell types. For example, mammalian expression vectors often contain both prokaryotic sequences that facilitate the propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed only in eukaryotic cells. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.

[0094] Suitable IRES sequences are described in detail supra. In some embodiments, the IRES sequence is be selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof. For example, the IRES may be selected from the group consisting of CVB3 IRES (SEQ ID NO: 1), EMCV IRES (SEQ ID NO: 2), mutEMCV IRES (SEQ ID NO: 3), mutHCV IRES (SEQ ID NO: 4), CSFV IRES (SEQ ID NO: 5), HRV-B3 IRES (SEQ ID NO: 6), mutCVB3 IRES (SEQ ID NO: 7), and LIMA1 IRES (SEQ ID NO: 8), or derivatives thereof.

[0095] As described in more detail supra, the IRES may be a wild-type internal ribosomal entry site or a modified internal ribosomal entry site.

[0096] The term “promoter” is used interchangeably with “promoter element” and “promoter sequence.” Likewise, the term “enhancer” is used interchangeably with “enhancer element” and “enhancer sequence.” The term “promoter” refers to a minimal sequence of a transgene that is sufficient to initiate transcription of a coding sequence of the transgene. Promoters may be constitutive or inducible. A constitutive promoter is considered to be a strong promoter if it drives expression of a transgene at a level comparable to that of the cytomegalovirus promoter (CMV) (Boshart et al., “A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus,” Cell 41 :521 (1985), which is hereby incorporated by reference in its entirety). Promoters may be synthetic, modified, or hybrid promoters. Promoters may be coupled with other regulatory sequences/elements which, when bound to appropriate intracellular regulatory factors, enhance (“enhancers”) or repress (“repressors”) promoter-dependent transcription. A promoter, enhancer, or repressor, is said to be “operably linked” to a transgene when such element(s) control(s) or affect(s) transgene transcription rate or efficiency. For example, a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene. As used herein, the term “regulatory elements” is used interchangeably with “regulatory sequences” and refers to promoters, enhancers, and other expression control elements, or any combination of such elements. [0097] Promoters are positioned 5' (upstream) to the genes that they control. Many eukaryotic promoters contain two types of recognition sequences: TATA box and the upstream promoter elements. The TATA box, located 25-30 bp upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis at the correct site. In contrast, the upstream promoter elements determine the rate at which transcription is initiated. These elements can act regardless of their orientation, but they must be located within 100 to 200 bp upstream of the TATA box.

[0098] Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. Enhancer elements often remain active even if their orientation is reversed (Li et al., “High Level Desmin Expression Depends on a Muscle-Specific Enhancer,” J. Bio. Chem. 266( 10):6562-6570 (1991), which is hereby incorporated by reference in its entirety). Furthermore, unlike promoter elements, enhancers can be active when placed downstream from the transcription initiation site, e.g., within an intron, or even at a considerable distance from the promoter (Yutzey et al., “An Internal Regulatory Element Controls Troponin I Gene Expression,” Mol. Cell. Bio. 9(4): 1397-1405 (1989), which is hereby incorporated by reference in its entirety).

[0099] RNA polymerase II (Pol II) is a complex, 12-subunit enzyme which transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes (Schier and Taatjes, “Structure and mechanism of the RNA polymerase II transcription machinery,” Genes Dev. 34(7-8): 465-488 (2020), which is hereby incorporated by reference in its entirety. Suitable Pol II promoters include, without limitation, CMV, SV40, PGK, and HSV-TK. Suitable Poll II promoter sequences are shown in Table 5 below.

Table 5: Suitable Pol II Promoter Sequences

[0100] In some embodiments, the IRES is selected from the group consisting of CVB3 IRES (SEQ ID NO: 1), HRV-B3 IRES (SEQ ID NO: 6), EMCV IRES (SEQ ID NO: 2), mutHCV IRES (SEQ ID NO: 3), and LIMA1 IRES (SEQ ID NO: 8), or derivatives thereof [0101] FIG. 7A and FIG. 8A demonstrate that, when IRES constructs were expressed from a Pol II CMV promoter in the Tornado translation system, the CVB3 IRES (SEQ ID NO: 1) produced more protein than the EMCV IRES (SEQ ID NO: 2) and slightly more protein than the HRV-B3 IRES (SEQ ID NO: 6). Thus, in some embodiments, the Pol II promoter is a CMV promoter. [0102] In some embodiments, the promoter is a Pol III promoter. Suitable Pol III promoter sequences are provided in Table 6 below.

Table 6: Suitable Pol III Promoter Sequences

[0103] Suitable Pol III promoters include, without limitation, U6, 7SK, Hl, and derivatives thereof.

[0104] In some embodiments, where the vector comprises a Pol III promoter, the IRES may be selected from the group consisting of mutEMCV or classic swine fever virus (CSFV) IRES (SEQ ID NO:5), mutEMCV IRES (SEQ ID NO:3), mutCVB3 IRES (SEQ ID NO:7), and derivatives thereof.

[0105] In some embodiments, the IRES lacks a Pol III termination element and/or signal. [0106] The term “ribozyme” refers to an RNA sequence that hybridizes to a complementary sequence in a substrate RNA and cleaves the substrate RNA in a sequence specific manner at a substrate cleavage site. Typically, a ribozyme contains a catalytic region flanked by two binding regions. The ribozyme binding regions hybridize to the substrate RNA, while the catalytic region cleaves the substrate RNA at a substrate cleavage site to yield a cleaved RNA product. The nucleotide sequence of the ribozyme binding regions may be completely complementary or partially complementary to the substrate RNA sequence with which the ribozyme hybridizes. [0107] In some embodiments of the vectors according to the present disclosure, a portion of the first ligation sequence is complementary to a portion of the first ribozyme and a portion of the second ligation sequence is complementary to a portion of the second ribozyme (see, e.g., U.S. Patent No. 11,756,183 to Jaffrey et al., which is hereby incorporated by reference in its entirety).

[0108] In some embodiments, a portion of the first ligation sequence is complementary to a portion of the second ligation sequence. The portion of the first ligation sequence complementary to the portion of the second ligation sequence may be at least 18, at least 26, or at least 49 nucleotides in length.

[0109] In some embodiments, each of the first ribozyme and the second ribozyme comprises a sequence that may be cleaved to produce a 5'-OH end and a 2', 3 '-cyclic phosphate end. In accordance with such embodiments, each of the first ribozyme and the second ribozyme is a self-cleaving ribozyme. Self-cleaving ribozymes are known in the art and are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of self-cleaving ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site (see, e.g., Weinberg et al., “New Classes of SelfCleaving Ribozymes Revealed by Comparative Genomics Analysis,” Nat. Chem. Biol. 11(8): 606-610 (2015) and Lee et al., “Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes,” Molecules 22(4):E678 (2017), which are hereby incorporated by reference in their entirety).

[0110] Each of the first ribozyme and the second ribozyme may be independently selected from the group consisting of Hammerhead, Hairpin, Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Vgl, glucosamine-6-phosphate synthase (glmS), Twister, Twister Sister, Hatchet, Pistol ribozymes, engineered synthetic ribozymes, or derivatives thereof (see, e.g., Harris et al., “Biochemical Analysis of Pistol Self-Cleaving Ribozymes,” RNA 21(11): 1852-8 (2015), which is hereby incorporated by reference in its entirety)).

[oni] In some embodiments, the first ribozyme is a P3 Twister ribozyme and the second ribozyme is a Pl Twister ribozyme. Twister ribozymes comprise three essential stems (Pl, P2, and P4), with up to three additional ones (P0, P3, and P5) of optional occurrence. Three different types of Twister ribozymes have been identified depending on whether the termini are located within stem Pl (type Pl), stem P3 (type P3), or stem P5 (type P5) (see, e.g., Roth et al., “A Widespread Self-Cleaving Ribozyme Class is Revealed by Bioinformatics,” Nature Chem. Biol. 10(l):56-60 (2014), which is hereby incorporated by reference in its entirety). The fold of the Twister ribozyme is predicted to comprise two pseudoknots (T1 and T2, respectively), formed by two long-range tertiary interactions (see Gebetsberger et al., “Unwinding the Twister Ribozyme: from Structure to Mechanism,” WIREs RNA 8(3):el402 (2017), which is hereby incorporated by reference in its entirety).

[0112] In some embodiments, the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

[0113] In some embodiments, the RNA molecule further encodes a stem loop that binds to a cognate RNA-binding protein, wherein the stem loop is positioned 3’ to the ligation sequence. Suitable stem loops are described supra and include, without limitation, MS2, PP7, BoxB, and Com.

[0114] The IRES may lack or comprise a stop codon.

Pharmaceutical Compositions Comprising Virus-Like Particles

[0115] The virus-like particles according to the present disclosure may be formulated as pharmaceutical compositions for administration to a subject.

[0116] The pharmaceutical compositions may include a “pharmaceutically acceptable inert carrier,” and this expression is intended to include one or more inert excipients, which include, for example and without limitation, starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, disintegrating agents, and the like. If desired, tablet dosages of the disclosed compositions may be coated by standard aqueous or nonaqueous techniques. “Pharmaceutically acceptable carrier” also encompasses controlled release means.

[0117] Pharmaceutical compositions may also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, colorants, flavors, desiccants, plasticizers, dyes, and the like. Any such optional ingredient must be compatible with a circular RNA molecule as disclosed herein or a DNA construct as disclosed herein to insure the stability of the formulation. The composition may contain other additives as needed including, for example, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, starch, xylitol, mannitol, myoinositol, and the like, and hydrates thereof, and amino acids, for example, alanine, glycine, and betaine, and peptides and proteins, for example, albumen.

[0118] Examples of excipients for use as the pharmaceutically acceptable carriers and the pharmaceutically acceptable inert carriers and the aforementioned additional ingredients include, but are not limited to, binders, fillers, disintegrants, lubricants, anti-microbial agents, and coating agents.

[0119] Pharmaceutical compositions provided by the present disclosure include compositions wherein the virus-like particles according to the present disclosure is contained in a therapeutically effective amount, z.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the disease, condition, or disorder being treated. When administered in methods to treat a disease, condition, or disorder, such compositions will contain an amount of the virus-like particle as disclosed herein to achieve the desired result, e.g., induction of an immune response or treatment of a subject for a disease or condition (e.g., by reducing, eliminating, or slowing the progression of a symptom of a disease or condition). Determination of a therapeutically effective amount of a virus-like particle as disclosed herein is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.

[0120] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the virus which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of undesirable microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride.

[0121] Sterile injectable solutions are prepared by incorporating the virus-like particle in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. [0122] Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present viruses can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Systems for Producing VLPs Comprising Circular RNA Translation Systems

[0123] Another aspect of the present disclosure relates to a system for producing viruslike particles (VLPs) comprising a circular RNA translation system. The system comprises a packaging vector encoding a plurality of one or more proteins that can self-assemble into a nanoparticle; an envelope vector; and a vector encoding a translation system according to the present disclosure.

[0124] The system for producing virus-like particles according to the present disclosure relies on the self-assembly of viral structural proteins (e.g., capsid, nucleocapsid, matrix, envelope, and/or spike proteins) in virus-like particles, as well as the specific interaction between an RNA aptamer (e.g., a protein-binding RNA aptamer) and its cognate RNA-binding protein (e.g., an RNA aptamer-binding protein).

[0125] Viral capsid proteins are structural components of virus or virus-like particles which may bind to and package nucleic acid molecules. In some embodiments of the system for producing virus-like particles according to the present disclosure, the VLP comprises viral capsid proteins which have self-assembled into a viral capsid or viral capsid-like structures. In accordance with such embodiments, the viral capsid proteins are fused to an RNA-binding protein that recognizes a cognate RNA aptamer.

[0126] The plurality of the one or more proteins that can self-assemble into a nanoparticle may comprise a polyprotein. Suitable polyproteins are described supra. In some embodiments, the polyprotein is selected from the group consisting of a retroviral group specific antigen (Gag) polyprotein (e.g., Human Immunodeficiency Virus Type 1 (HIV-1) group specific antigen (Gag)), a mammalian group specific antigen (Gag)-like polyprotein (c.g, PEG10), and derivatives thereof.

[0127] As described supra, the polyprotein may comprise one or more proteins selected from the group of proteins consisting of a nucleocapsid protein, a capsid protein, a matrix protein, a reverse transcriptase, a protease, and a defective integrase. [0128] In some embodiments, the plurality of one or more proteins that can self-assemble into a nanoparticle comprise(s) one or more structural protein(s). For example, the one or more structural protein(s) may be selected from the group consisting of capsid protein(s), nucleocapsid protein(s), matrix protein(s), and combinations thereof. In accordance with such embodiments, the capsid protein(s) is/are non-retroviral capsid protein(s). Suitable exemplary non-retroviral capsid protein(s) may be selected from the group consisting of Herpes Simplex Virus (HSV) VP23, Herpes Simplex Virus (HSV) VP19C, Hepatitis B Virus (HBV) core antigen, Human Papillomavirus (HPV) LI, Human Papillomavirus (HPV) L2, and combinations thereof.

[0129] Suitable RNA aptamers and their cognate RNA-binding proteins are well known in the art (see, e.g., Jiang et al., “Multiplexed Gene Engineering Based on dCas9 and gRNA- tRNA Array Encoded on Single Transcript,” Int. J. Mol. Sci. 24(10): 8535 (2023), which is hereby incorporated by reference in its entirety) and are provided in Table 7.

Table 7: Protein-Binding RNA Aptamers and their Cognate RNA-Binding Proteins

[0130] In some embodiments, the RNA-binding protein is selected from the group consisting of MS2 coat protein (MCP), Com, PCP, and N22.

[0131] In the system for producing virus-like particles according to the present disclosure, at least one of the plurality of the one or more proteins that can self-assemble into a nanoparticle comprise(s) or is fused to an RNA-binding protein or RNA-binding protein domain. In some embodiments, the RNA-binding protein domain is located at an N-terminus or a C- terminus of at least one of the plurality of the one or more proteins that can self-assemble into a nanoparticle comprising or fused to the RNA-binding domain.

[0132] An exemplary system according to the present disclosure comprising a packaging vector encoding a plurality of one or more proteins that can self-assemble into a nanoparticle (e.g., capsid, nucleocapsid, and matrix proteins), an envelope vector encoding a viral envelope and/or spike protein, and a vector encoding a translation system according to the present disclosure is shown in FIG. 11 A. In this exemplary system, the packaging vector encodes a nucleocapsid protein fused to a MS2 coat protein (MCP) and the vector encoding the translation system encodes a circular mRNA molecule comprising the MS2 aptamer sequence. Since it contains the MS2 aptamer sequence, upon expression, this RNA will be packaged into VLPs by binding to the MCP domain in the nucleocapsid protein.

[0133] In accordance with the system for producing virus-like particles according to the present disclosure, the envelope vector may encode one or more envelope and/or spike protein(s). Suitable viral envelope and/or spike protein(s) are provided in Table 8.

Table 8: Suitable Viral Envelope and/or Spike Proteins

[0134] Suitable viral envelope protein(s) include, without limitation, a vesicular stomatitis virus envelope protein, a rabies virus envelope protein, a measles virus envelope protein, a nipah virus envelope protein, a chickungunya virus envelope protein, and a sindbis virus envelope protein.

[0135] In some embodiments, the one or more envelope and/or spike protein(s) are selected from the group consisting of a vesicular stomatitis virus G (VSV G) protein, RabV-G, Chickungunya virus E1ZE2, Sindbis virus E1ZE2, Measle virus H/F, and derivatives thereof. [0136] In some embodiments, the one or more envelope and/or spike protein(s) comprises a vesicular stomatitis virus G (VSV G) protein or a derivative thereof. For example, the one or more envelope and/or spike protein(s) may comprise mutant VSV-G (K47Q, R354A). [0137] In some embodiments, the one or more envelope and or spike proteins comprise a coronavirus spike protein.

[0138] In some embodiments, the one or more envelope and/or spike proteins comprises a fusion protein.

[0139] In some embodiments, the system further comprises a vector encoding a protein that is trafficked to the viral particle and/or cell surface membrane. In accordance with such embodiments, the protein that is trafficked to the viral particle and/or cell surface membrane is a ligand or target binding protein.

[0140] The protein that is trafficked to the viral particle and/or cell surface membrane may be selected from the group consisting of a single chain of MHC fused with beta-2 - microglobulin (B2M) and a covalently linked peptide; a single-chain antibody variable fragment fused to a transmembrane domain; and an antigen fused to a transmembrane domain.

Methods for Producing a VLP Comprising a Circular RNA Translation System [0141] Another aspect of the present disclosure relates to a method for producing a VLP comprising a circular RNA translation system. This method involves providing a host cell; transfecting the host cell with a system according to the present disclosure; and culturing the host cell under conditions suitable to express the packaging vector, the envelope vector, and the circular RNA expression vector in the host cell, where said culturing produces virus-like particles comprising a circular RNA translation system. [0142] The host cell may comprises an endogenous RNA ligase. In some embodiments, the endogenous RNA ligase has the ability to catalyze the circularization of a ribonucleic acid molecule having a 5'-OH and a 2',3'-cyclic phosphate. In accordance with some embodiments, the endogenous RNA ligase is RtcB. It will be recognized that there are some enzymes that are related in function to RtcB, but not in sequence to RtcB. In some embodiments, the RNA ligase is any RNA ligase that detects 5'-OH and 2'-3 '-cyclic phosphate ends.

[0143] The cell may be a eukaryotic cell. Exemplary eukaryotic cells include a yeast cell, an insect cell, a fungal cell, a plant cell, and an animal cell (e.g., a mammalian cell). Suitable mammalian cells include, for example without limitation, human, non-human primate, cat, dog, sheep, goat, cow, horse, pig, rabbit, and rodent cells. The host cell is preferably present either in a cell culture (ex vivo) or in a whole living organism (in vivo).

[0144] In some embodiments, the host cell is a mammalian cell line. Suitable mammalian cell lines are well known in the art and include, without limitation, HEK293T cells, HEK293FT cells, and derivatives thereof.

[0145] Suitable methods of introducing RNA molecules into cells are well known in the art and include, but are not limited to, the use of transfection reagents (e.g., FuGENE® transfection reagent), electroporation, microinjection, calcium phosphate transfection, DEAE- Dextran, and liposome-mediated transfection.

[0146] Culturing of host cells can be performed under known culture conditions. For example, when host cell are mammalian cells or derived from mammalian cells, culturing at a temperature of 30 to 37° C, humidity 95%, and CO2 concentration 5 to 10% is exemplified, but the methods of producing virus-like particles according to the present disclosure are not limited to such conditions. Culturing may be carried out at a temperature, humidity, or CO2 concentration outside the above range as long as desired host growth or desired virus-like particle production by the host cell can be achieved. The culture period is also not particularly limited, and may be any culture period in which desired host cell growth or desired virus-like particle production can be achieved. In some embodiments, culturing is carried out for at least 6 hours to at least 12 hours to at least 168 hours, or any amount therebetween. Thus, in some embodiments, culturing is carried out for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, at least 168 hours. In some embodiments, the culturing is carried out for 72 hours.

[0147] In some embodiments, culturing the transfected host cells involves culturing the host cells in a cell culture medium and changing the cell culture medium following transfection. In accordance with such embodiments, the cell culture medium may be changed at least once at about 6 hours following transfection, about 12 hours following transfection, about 18 hours following transfection, or about 24 hours following transfection. In some embodiments, the cell culture medium is changed at least once at about 24 hours following transfection of the host cell. [0148] In some embodiments, culturing the transfected host cells results in secretion of virus-like particles into a cell culture medium. Thus, in some embodiments, the method further involves collecting the cell culture medium and purifying the produced virus-like particles comprising the circular RNA translation system from the cell culture medium. The cell culture medium may be collected at least once, at least twice, or at least three times following host cell transfection. In accordance with such embodiments, the cell culture medium may be collected any time following transfection of the host cells. In some embodiments, the cell culture medium is collected at about 24 hours following transfection, at about 36 hours following transfection, at about 48 hours following transfection, at about 60 hours following transfection, at about 72 hours following transfection, at about 84 hours following transfection, or at about 96 hours following transfection. For example, the cell culture medium may be collected at about 72 hours following transfection.

[0149] Methods of purifying virus-like particles following collection of cell culture medium from transfected host cells are well known in the art and include, without limitation, centrifugation, ultracentrifugation, filtration, ion-exchange chromatography, and dialysis (see, e.g., Gonzalez-Dominguez et al., “A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles,” Vaccines 9(10): 1154 (2021) and Arevalo et al., “Expression and Purification of Virus-Like Particles for Vaccination,” J. Vis. Exp. 112: 54041 (2016), which are hereby incorporated by reference in their entirety).

[0150] In some embodiments, purifying the virus-like particles following collection of cell culture medium involves removing cell debris. This can be carried out by, e.g., centrifugation at about 500 g for about 10 minutes followed by, e.g., ultracentrifugation, concentration, diafiltration, filtration, ion-exchange chromatography, dialysis, and/or combinations thereof.

Methods of Inducing an Immune Response Against A Pathogen

[0151] Another aspect of the present disclosure relates to a method of inducing an immune response against a pathogen. This method involves administering to a subject an effective dose of a virus-like particle (VLP) according to the present disclosure, a VLP produced using the system according to the present disclosure, or a VLP produced using a method according to the present disclosure.

[0152] In some embodiments, the pathogen is a viral pathogen, a prokaryotic pathogen, or a eukaryotic pathogen.

[0153] Suitable viral pathogens include, without limitation, Adenovirus, Andes virus, Chikungunya virus, Coconut Creek virus, Coxsackievirus, Crimean-Congo Hemorrhagic Fever virus, Cytomegalovirus, Dengue virus, Eastern Equine Encephalitis virus, Ebola virus, Epstein- Barr virus, Hantavirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Human Immunodeficiency Virus 1 (HIV-1), Human Immunodeficiency Virus 2 (HIV-2), Human Papillomavirus (HPV), Influenza A virus, Influenza B virus, Japanese Encephalitis virus, Junin virus, La Crosse virus, Lassa fever virus, Marburg virus, Measles virus, MERS-CoV, Mumps virus, Nipah virus, Norovirus, Parvovirus Bl 9, Poliovirus, Rabies virus, Respiratory Syncytial Virus (RSV), Rhinovirus, Ross River virus, Rotavirus, Rubella virus, SARS-CoV, SARS-CoV-2, Severe Fever with Thrombocytopenia Syndrome virus, Varicella-Zoster Virus (VZV), Venezuelan Equine Encephalitis virus, West Nile virus, Yellow Fever virus, and Zika virus.

[0154] Suitable prokaryotic pathogens include, without limitations, gram-positive and gram-negative bacteria. In some embodiments, the pathogen is a gram-positive bacteria selected from the group consisting of Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Nocardia asteroids, Propionibacterium acnes, Rhodococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, and Streptococcus pyogenes.

[0155] In some embodiments, the pathogen is a gram-negative bacteria selected from the group consisting of Acinetobacter baumannii, Bordetella pertussis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Coxiella burnetii, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia prowazekii, Salmonella enterica, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholerae, Yersinia enter ocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0156] In some embodiments, the pathogen is a eukaryotic pathogen. Suitable eukaryotic pathogens include, without limitation, protozoan parasites such as Cryptosporidium spp., Cyclospora cayelanenensis. Entamoeba histolytica, Giardia inleslinalis, Plasmodium falciparum, Plasmodium malariae, Toxoplasma gondii, and Trypanosoma cruzi (see, e.g., Hague, R., “Human Intestinal Parasites,” J. Health PopuL Nutr. 25(4): 384-391 (2007), which is hereby incorporated by reference in its entirety).

[0157] Additional suitable eukaryotic pathogens include, without limitation, helminth parasites such as Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, Trichuris trichiura (see, e.g., Geiger et al., “Necator americanus and Helminth Co-Infections: Further Down-Modulation of Hookworm-Specific Type 1 Immune Responses,” PLoSNegL Trop. Dis. 5(9): el280 (2011), which is hereby incorporated by reference in its entirety).

[0001] In some embodiments of the methods of inducing an immune response against a pathogen according to the present disclosure, the term “subject” refers to any subject for whom the induction of an immune response against a pathogen is desired, particularly humans. The subject may be a mammalian subject, for example, a human subject. Suitable human subjects include, without limitation, children, adults, and elderly subjects. The mammalian subject may also be non-human, such as a bovine, ovine, porcine, feline, equine, murine, canine, lapine, etc. In some embodiments, the subject is a non-human primate.

[0158] In some embodiments, the subject is a non-mammalian subject, for example, an avian subject or an insect.

[0159] As used herein, the term “immune response” refers to the development in a subject of a humoral and/or a cellular immune response. A “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells.

[0160] An immune response may include one or more of the following effects: the production of antibodies by B-cells and/or the activation of suppressor, cytotoxic, or helper T- cells and/or T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity and/or mediate antibodycomplement, or antibody-dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays well known in the art. [0161] Administrating an effective dose of a virus-like particle (VLP) according to the present disclosure, a VLP produced using the system according to the present disclosure, or a VLP produced using a method according to the present disclosure may be effective to induce a humoral and/or cellular immune response against a pathogen in the subject.

[0162] In some embodiments of the methods of inducing an immune response described herein, the immune response is a humoral immune response. The presence of a humoral immune response can be determined and monitored by testing a biological sample (e.g., blood, plasma, serum, urine, saliva feces, CSF or lymph fluid) from a subject for the presence of antibodies directed to a component of the virus-like particle administered to the subject or the presence of antibodies directed to, e.g., the pathogen of interest.

[0163] In some embodiments of the methods of inducing an immune response described herein, the immune response is a cellular immune response. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte) assays which are known in the art.

Methods of Treating a Subject

[0164] Another aspect of the present disclosure relates to a method of treating a subject. This method involves administering the virus-like particle (VLP) according to the present disclosure, a VLP produced using the system according to the present disclosure, or a VLP produced using a method according to the present disclosure to a subject in need thereof, where upon said administering, the one or more peptide(s) is/are expressed in a cell of the subject, thereby treating the subject.

[0002] The terms “treat”, “treating”, “treatment”, and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process, or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process, or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject, e.g., patient, population. Accordingly, a given subject or subject, e.g., patient, population may fail to respond or respond inadequately to treatment.

[0003] In some embodiments of the methods of treating a subject according to the present disclosure, the term “subject” refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The subject may be a mammalian subject, an amphibian subject, an avian subject, a fish, or a reptilian subject.

[0004] In some embodiments, the subject is a mammalian subject. The terms “mammal” or “mammalian subject” for purposes of the methods described herein refers to any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. [0005] In some embodiments, the mammalian subject is a human subject. The human subject may be an infant, a child, an adolescent, an adult, or a geriatric subject.

[0006] In some embodiments, the methods of the present disclosure find use in experimental animals, in veterinary application, and in the development of animal models, including, but not limited to, rodents including mice, rats, hamsters, and primates.

[0165] In accordance with the methods of the present disclosure, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0166] The VLPs or pharmaceutical composition(s) comprising the VLPs of the disclosure can be administered alone or can be co-administered to a subject. Co-administration is meant to include simultaneous or sequential administration of the VLPs or pharmaceutical composition(s) comprising the VLPs of the disclosure individually or in combination (more than one compound or agent). Thus, the VLPs or pharmaceutical composition(s) comprising the VLPs of the disclosure can also be combined, when desired, with other active substances (e.g., to induce an immune response or to treat a subject). The VLPs or pharmaceutical composition(s) comprising the VLPs of the disclosure can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. The VLPs or pharmaceutical composition(s) comprising the VLPs of the disclosure of the present disclosure may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760, which are hereby incorporated by reference in their entirety. The VLPs or pharmaceutical composition(s) comprising the VLPs of the disclosure can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see, e.g., Rao, J. Biomater Sci. Polym. Ed. 7:623-645 (1995), which is hereby incorporated by reference in its entirety; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863 (1995), which is hereby incorporated by reference in its entirety); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669674, 1997, which is hereby incorporated by reference in its entirety).

[0167] The dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g. symptoms of cardiomyopathy or neurodegeneration such as Parkinson’s disease and severity of such symptoms), kind of concurrent treatment, complications from the disease being treated or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the present disclosure. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.

[0168] For any VLP or pharmaceutical composition(s) comprising a VLP of the disclosure described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.

[0169] Therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0170] Dosages may be varied depending upon the requirements of the subject and the VLP or pharmaceutical composition(s) comprising the VLPs of the disclosure being employed. The dose administered to a subject, in the context of the present disclosure should be sufficient to effect a beneficial response in the subject over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached.

[0171] Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

Methods of Performing Gene Editing on a Subject

[0172] Another aspect of the present disclosure relates to a method of performing gene editing on a subject. This method involves administering a virus-like particle (VLP) according to the present disclosure, a VLP produced using a system according to the present disclosure, or a VLP produced using a method according to the present disclosure to a subject in need thereof, where the one or more peptide(s) comprise one or more gene editing protein(s) and where, upon said administering, the gene editing protein(s) is/are expressed in a cell of the subject, thereby editing the genome of the subject.

[0173] In some embodiments, the one or more gene editing protein(s) comprises a Cas family protein. Suitable Cas family proteins are well known in the art and include, without limitation, dCas proteins and nCas proteins, as described supra. In some embodiments, the Cas family protein is a dCas family protein.

[0174] In some embodiments, the one or more gene editing protein(s) is/are fused to an additional protein. The additional protein may be selected from the group consisting of a reverse transcriptase, an adenosine deaminase, a cytidine deaminase, and a transposase/ recombinase.

[0175] The method may further involve administering a guide RNA. As used herein, the term “guide RNA” or “gRNA” refers to a ribonucleotide sequence capable of binding a nucleoprotein, thereby forming a ribonucleoprotein complex. The guide RNA may comprise (i) a DNA-targeting sequence that is complementary to a target nucleic acid sequence and (ii) a binding sequence for the Cas protein (e.g., Cas9 nuclease, Cas9 nickase, dCas9, Casl2a nuclease, Casl2a nickase, or dCasl2a).

[0176] In some embodiments, the VLP further includes a guide RNA packaged into the VLP using a lentiviral packaging signal (psi).

[0177] Suitable subjects include, without limitation, a mammalian subject (e.g., a human subject). In some embodiments, the subject is a non-mammalian subject. Suitable mammalian and non-mammalian subjects are described above.

RNA Molecules

[0178] Another aspect of the present disclosure relates to an RNA molecule comprising: a first ribozyme; a first ligation sequence positioned 3’ to the first ribozyme; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), and where the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence, wherein the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof; a second ligation sequence positioned 3’ to the internal ribosomal entry site; and a second ribozyme positioned 3’ to the second ligation sequence.

[0179] Suitable first ribozyme sequences, first ligation sequences, IRES sequences, RNA molecules encoding one or more peptides, second ligation sequences, and second ribozyme sequences are described supra.

[0180] The IRES may be a wild-type internal ribosomal entry site or a modified internal ribosomal entry site. In some embodiments, the IRES lacks a Pol III termination element.

[0181] In some embodiments, a portion of the first ligation sequence may be complementary to a portion of the first ribozyme and a portion of the second ligation sequence may be complementary to a portion of the second ribozyme.

[0182] In some embodiments, a portion of the first ligation sequence is complementary to a portion of the second ligation sequence. The portion of the first ligation sequence complementary to the portion of the second ligation sequence may be at least 18 nucleotides in length, at least 26 nucleotides in length, and at least 49 nucleotides in length.

[0183] In some embodiments, each of the first ribozyme and the second ribozyme comprises a sequence that may be cleaved to produce a 5'-OH end and a 2', 3 '-cyclic phosphate end. [0184] Each of the first ribozyme and the second ribozyme may be independently selected from the group consisting of Hammerhead, Hairpin, Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Vgl, glucosamine-6-phosphate synthase (glmS), Twister, Twister Sister, Hatchet, Pistol ribozymes, engineered synthetic ribozymes, or derivatives thereof. In some embodiments, the first ribozyme is a P3 Twister ribozyme and the second ribozyme is a Pl Twister ribozyme.

[0185] In some embodiments, the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

[0186] The RNA molecule may further comprise a stem loop that binds to a cognate RNA-binding protein, wherein the stem loop is positioned 3’ to the ligation sequence. For example, the stem loop is selected from the group consisting of MS2, PP7, BoxB, and Com. [0187] In some embodiments, the IRES lacks a stop codon.

Circular RNA Molecules

[0188] Another aspect of the present disclosure relates to a circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide sequence(s), where the internal ribosomal entry site coupled to the RNA molecule encoding the peptide sequence(s) is positioned 3’ to the first ligation sequence, where the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof; a second ligation sequence positioned 3’ to the internal ribosomal entry site coupled to the RNA molecule encoding the peptide sequence(s).

[0189] The RNA molecules according to the present disclosure may be synthesized (e.g., by chemical synthesis) or in vitro transcribed (e.g., from a Tornado vector) (see, e.g., Litke and Jaffrey, “Highly Efficient Expression of Circular RNA Aptamers in Cells using Autocatalytic Transcripts,” Nat. Biotechnol. 37(6):667-675 (2019) and U.S. Patent Application Publication No. 2021/0340542 to Jaffrey et al., which are hereby incorporated by reference in their entirety). Circular RNA may then be purified by standard methods.

[0190] Suitable first ligation sequences, IRES sequences, RNA molecules encoding one or more peptides, and second ligation sequences are described supra. [0191] The IRES may be a wild-type internal ribosomal entry site or a modified internal ribosomal entry site. In some embodiments, the IRES lacks a Pol III termination element.

[0192] In some embodiments, the IRES lacks a Pol III termination element.

[0193] In some embodiments, a portion of the first ligation sequence is complementary to a portion of the second ligation sequence. The portion of the first ligation sequence complementary to the portion of the second ligation sequence may be at least 18 nucleotides in length, at least 26 nucleotides in length, and at least 49 nucleotides in length.

[0194] In some embodiments, the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

[0195] The RNA molecule may further comprise a stem loop that binds to a cognate RNA-binding protein, where the stem loop is positioned 3’ to the ligation sequence. For example, the stem loop may be selected from the group consisting of MS2, PP7, BoxB, and Com.

[0196] In some embodiments, the IRES lacks a stop codon.

EXAMPLES

[0197] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.

Materials and Methods for Examples 1 - 10

Cell Lines and Culture

[0198] HepG2 (ATCC HB-8065, male, hepatocellular carcinoma), HEK293T (ATCC CRL-11268, gender unknown, embryo kidney tissue), Flip-In-293 cells (ThermoFisher #R75007, gender unknown, embryo kidney tissue), and HeLa cells (ATCC CCL-2, female, cervical carcinoma) were cultured with x 1 DMEM (ThermoFisher #11995-065) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin under standard tissue culture conditions. ZR-75-1 (ATCC CRL-1500, female, breast carcinoma) were cultured using RPMI 1640 Medium with no phenol red (ThermoFisher #11835030) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin under standard tissue culture conditions. SH-SY5Y (ATCC CRL-2266, female, metastatic bone tumor) were cultured with F-12/DMEM (ThermoFisher #11320033), 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin. Cells were cultured at 37 °C and 5% CO2 and passaged every 2-3 days. Cell lines have not been authenticated. Reagents and Resources

[0199] Unless otherwise stated, all reagents were from Sigma-Aldrich except for cell culture reagents, which were from Invitrogen. These reagents were used without further purification. Table 9 provides reagents and resources used in Examples 1-10. Table 9. Reagents and Resources

¹ Liang & Wilusz, “Short Intronic Repeat Sequences Facilitate Circular RNA Production,”

Genes Dev 28:2233-2247 (2014); ² Crawford et al., “Protocol and Reagents for Pseudotyping

Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays,” Viruses 12

(2020); ³ Didier Trono; ⁴ Lu et al., “Delivering SaCas9 mRNA by Lentivirus-Like Bionanoparticles for Transient Expression and Efficient Genome Editing,” Nucleic Acids

Research 47:e44-e44 (2019); ⁵ Litke, J.L. & Jaffrey, S.R., “Highly Efficient Expression of Circular RNA Aptamers in Cells Using Autocatalytic Transcripts,” Nature Biotechnology 37:667-675 (2019); ⁶ Chan et al., “Engineering Human ACE2 to Optimize Binding to the Spike Protein of SARS Coronavirus 2,” Science 369: 1261-1265 (2020). Table 10. Primers, plasmids, and gene blocks

Tornado Stem Design

[0200] The Tornado circularization junction stem was designed using mfold (http://www.unafold.org/mfold/applications/ma-folding-form.php). Stems were designed to have bulges every ~10 bp.

Split nLuc Design

[0201] Split nLuc ORF was codon optimized to avoid Pol III termination signals using the codon optimization tool from Integrated DNA Technologies. The frame of the Tornado circularization stem was chosen to avoid stop codons. To improve the sensitivity of the protein readout, the split nLuc contained a C-terminal 2x Glutamine degron.

Cloning Pol III Transcripts

[0202] Split nLuc, IRES, and partial Tornado sequences were chemically synthesized as gene blocks (Integrated DNA Technologies), then cloned into the Notl and SacII sites of the pAV-U6+27-Tornado-Broccoli plasmid (Addgene #124360). Split nLuc ORF was codon optimized to be compatible with a Pol III promoter. Changing the IRES was done by cloning a gene block into the EcoRI and BsiWI internal restriction sites. All plasmids were sequenced (Psomagen) to verify identity.

Cloning Pol II Transcripts

[0203] Split nLuc, nLuc, spike, IRES, and Tornado sequences were chemically synthesized as gene blocks (Integrated DNA Technologies). Gene blocks were cloned into the BamHI and Xhol sites of pcDNA3.1+ vector backbone. Changing the IRES was done by cloning a gene block into the EcoRI and BsiWI internal restriction sites. Minor alterations such as stop codon insertion or IRES point mutations were done using a QuikChange Site-directed mutagenesis kit II (Agilent #200523) according to manufacturer’s instructions. All plasmids were sequenced (Psomagen) to verify identity. Cloning of Backsplicing System Sequences

[0204] A gene block containing the same exact sequence as the CMV-CVB3 Tornado translation system was synthesized (Integrated DNA Technologies) and cloned into the EcoRV and SacII restriction site of the pcDNA3.1(+) CircRNA Mini Vector (Addgene #60648). The Tornado circularization stems were included at the 5’ end of the sequence to ensure the ORF from the Tornado Translation system would match the ORF from the backsplicing-based system. After cloning, this plasmid was sequenced (Psomagen) to verify identity.

Cell Culture and Transfection

[0205] HepG2 (ATCC HB-8065), HEK293T (ATCC CRL-11268), and HeLa cells (ATCC CCL-2) were cultured with x 1 DMEM (ThermoFisher #11995-065) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin under standard tissue culture conditions. ZR-75 (ATCC CRL-1500) were cultured using RPMI 1640 Medium with no phenol red (ThermoFisher #11835030) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin under standard tissue culture conditions. SH-SY5Y (ATCC CRL- 2266) were cultured with F-12/DMEM (ThermoFisher #11320033), 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin. Cells were cultured at 37 °C and 5% CO2 and passaged every 2-3 days. TrypLE Express (ThermoFisher #12604013) was used to lift cells for passaging. Cells were plated at a density of 2xl0⁴ cells/cm² 20 hours before transfection. Cells were transfected using a 3 : 1 ratio of FuGENE (Promega #E5911) to DNA in OptiMEM I Reduced Serum Media (ThermoFisher, #31985). Unless otherwise stated, all cells were harvested 72 hours after transfection for downstream applications.

Protein Expression Analysis

[0206] Cells were harvested by directly lifting cells with xi phosphate buffered saline (PBS) (ThermoFisher, no. 10010031). For luminescence assays, cells were harvested 72 hours after transfection unless otherwise stated. Media was aspirated off cells then cells were resuspended in PBS. 50pl of cell suspension was transferred to a flat-bottomed white-walled 96- well plate (Corning). Nano-Gio Luciferase Assay System (Promega #N1110) reagent was prepared according to manufacturer's instructions. 50ul of Nano-Gio reagent was added to each well of cell suspensions. The plate was gently shaken, then luminescence detection was done using SpectraMax iD3 (Molecular Devices) machine with SoftMax Pro (v.7.1) software using the luminescence acquisition settings (Endpoint luminescence, 96 Well Standard opaque plate, integration time 1000 ms, 1 mm read height). RNA Extraction

[0207] RNA was harvested from cultured cells by removing media and detaching enzymatically or directly lifting cells with * 1 phosphate buffered saline (PBS) (ThermoFisher, #10010031). Cell suspensions were mixed with TRIzol LS Reagent (Invitrogen, no. 10296010), then frozen and stored at -20 °C or purified immediately according to the manufacturer’s instructions.

RNase R Reactions

[0208] Following RNA extraction, RNA concentration was quantified using a NanoDrop 2000 (Thermo Scientific). Equal concentrations of RNA were added to two tubes and were treated with RNase R (Biosearch Technologies RNR07250) according to manufacturer's instructions. Following RNase R reaction, RNA was purified using the RNA Clean & Concentrator kit (Zymo Research, no. R1015).

Northern Blot

[0209] Following RNA extraction or RNase R reactions, RNA was blotted using the NorthemMax kit (ThermoFisher #AM1940) according to manufacturer’s instructions. Antisense DNA probes designed to bind to either the LgBiT, ZKSCAN1 exon2/3 or spike RNA were synthesized with a 5’Biotin (Integrated DNA Technologies). Band detection was done using the Chemiluminescent Nucleic Acid Detection Module (ThermoFisher #89880) according to manufacturer’s instructions. The blot was imaged using ChemiDoc MP imager (Bio-Rad) with the chemiluminescent band detection setting. RNA quantification from northern blots was done using Image Lab (v.5.2.1) software. qRT-PCR for Split nLuc

[0210] Following RNA extraction or RNase R reactions, RNA was treated with DNase (ThermoFisher #EN0521) according to the manufacturer's instructions. RNA was then directly used for cDNA synthesis with the Superscript III kit (ThermoFisher #12574026). cDNA was diluted 1 : 10 added to Eppendorf twin. tec 96 real-time PCR Plate (Eppendorf #0030132700) along with iQ Syber Green Supermix (Bio-Rad #1708880) and primers. Primers designed to amplify a 150nt region in the LgBiT region and reference primers for GAPDH were chemically synthesized (Integrated DNA Technologies). qPCR was done using the Eppendorf Realplex qPCR machine. RNA quantification was done by using the 2'^[ACt(target) ’ ^(reference)] m^od.

RT-PCR with Divergent Primers and Sanger Sequencing

[0211] RNA was reverse transcribed as described above. Convergent primers amplified a region within the ORF of the RNA. Divergent primers amplified a region that spanned the circularization junction (see Table 10 for exact primer sequences). PCR reactions were done using Phusion High-Fidelity DNA polymerase (NEB #M0530S) then run on a High Sensitivity D1000 ScreenTape (Agilent #5067-5584) using a 4150 TapeStation system (Agilent #G2992AA). For sequencing, PCR reactions were PCR purified using QIAquick PCR Purification Kit (Qiagen #28104) then submitted to Psomagen for Sanger sequencing.

NCBI BLAST Searches

[0212] EMCV IRES sequence was aligned against viral (taxid: 10239) reference genomes (refseq genomes) with the somewhat similar (blastn) program on NCBI BLAST.

RNA/Protein Half-Life Experiments

[0213] Flip-In-293 cells (ThermoFisher #R75007) were cultured with x 1 DMEM (ThermoFisher #11995-065) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin under standard tissue culture conditions. Cell lines that stably express the Tornado (CMV-CVB3), Linear (CVB3), and Linear (Cap) mRNAs were made by using the Flp-In T-Rex Core Kit (ThermoFisher #K6500-01). Cells were plated at a density of 2xl0⁴ cells/cm² 16 hours before tetracycline treatment. Cells were then treated with 1 pg/mL tetracycline (Santa Cruz Biotechnology) for 12 hours then the media was changed to tetracycline-free media. Cells were harvested at 0, 5, 10, 24, 48, and 72 hours after tetracycline withdrawal. Cells were harvested by directly lifting cells with x 1 phosphate buffered saline (PBS) (ThermoFisher #10010031) then split in half for both protein quantification and RNA quantification.

[0214] qRT-PCR RNA quantification normalizes the target RNA level to the reference RNA level. This means the level of tet-inducible RNA expression will appear to go down over time as the HEK293T cells divide and make more GAPDH while the tet-inducible gene is no longer being expressed. RNA quantification was therefore adjusted to the cell count at every time point. Cell counts were obtained using Countess 3 automated cell counter (ThermoFisher). In addition, the RNA expression level needs to be normalized to the level of RNA expressed when no tetracycline is added. The equation for this normalization is as follows:

where x is the hour that the RNA was harvested, and RNA expression was calculated using the qRT-PCR for split nLuc method.

VLP Production

[0215] MCP -modified packaging plasmid pspAX2-D64-NC-MCP (Addgene #122944),

VSVg envelope plasmid pMD2.G (Addgene #12259) or spike envelope plasmid (Addgene #158762), and transfer plasmid Tornado/Linear nLuc-MS2 were transfected into 80% confluent HEK293T cells at a ratio of 3: 1.5:4.5. 3 pg of total DNA was transfected into each well of a 6- well plate. Cells were transfected using a 3 : 1 ratio of FuGENE (Promega #E5911) to DNA in OptiMEM I Reduced Serum Media (ThermoFisher, #31985). Media was changed 24 hours after transfection. VLPs were harvested and filtered through a 45-micron filter three days after transfection.

VLP Transduction

[0216] Unconcentrated VLPs were diluted according to their viral RNA titers (see “RNA isolation from VLPs and RT-qPCR analysis”) in fresh media and added to 5 x 10⁴ cells in a 12-well plate. At the first collection time, cells were washed with 1ml of x 1 phosphate buffered saline (PBS) (ThermoFisher #10010031) before harvesting. Media for subsequent time points was replaced with fresh media after the first collection time point. At each collection time point, cells were harvested and subject to protein expression analysis (see “Protein expression analysis” section).

Generating ACE-2 HEK293T Cells

[0217] HEK293T cells in a 6-well plate were transfected with 2pg/well of a plasmid encoding ACE-2 (Addgene #145171) then replated into a 12-well plate (GenClone #25-106) using media with Geneticin (Thermo Fisher #10131035) 24 hours after transfection. The cells were treated with VLPs 24 hours later.

RNA Isolation from VLPs and RT-qPCR Analysis

[0218] RNA from VLPs was extracted using the QIAmp Viral RNA Mini Kit (Qiagen #52904) according to manufacturer’s instructions. RNA was then treated with RNase R (See RNase R reactions section) then DNAse (ThermoFisher #EN0521) according to the manufacturer's instructions. RNA was then directly used for cDNA synthesis with the Superscript III kit (ThermoFisher #12574026). cDNA was diluted 1 : 10 added to Eppendorf twin. tec 96 real-time PCR Plate (Eppendorf #0030132700) along with iQ Syber Green Supermix (Bio-Rad #1708880) and primers. Primers that amplified a 125nt amplicon within the nLuc gene were designed using Integrated DNA technologies. RNA abundance was calculated by using the 2-[Act(tar_get) - Act(reference)] _eq_uaq_{on w}j th

reference being a no RNA control. For RNase R reactions, RNA was quantified using the 2'^{(Ct +RNase R Cl} -^{RNase R}) equation.

RNA FISH

[0219] FISH was done according to the ViewRNA™ ISH Cell Assay Kit protocol (Thermo Fisher QVC0001). FISH probes for the LgBiT RNA were designed using the custom - I l l - branched DNA probe set tool. HEK293T cells were transfected with plasmids encoding the Tornado (U6-mutEMCV), Tornado (CMV-CVB3), and Linear (cap) expression systems then sub-cultured onto glass-bottomed 24-well plates (MatTek Corporation P24G-1.5-13-F) that were coated with poly-D-lysine (Cultrex 3429-100-01) then additionally coated with Cultrex Mouse Laminin I (Thermo Fisher 340001002) 24 hours after transfection. Fluorescence images were acquired with a CoolSnap HQ2 CCD camera through a 403 air objective (NA 0.75) mounted on a Nikon Eclipse TE2000-E microscope and analyzed with the NIS-Elements software. The LgBiT probe is a TYPE 4 probe, imaged using a 488 nm excitation (FITC). The NEAT1 probe is a TYPE 1 probe, imaged using a 550 nm excitation (TRITC). DAPI was imaged using a 358 nm excitation (DAPI). Cellular distribution was calculated by counting the number of cytoplasmic and nuclear puncta then dividing the number of nuclear puncta by the total number of puncta.

Immunogenicity Assays

[0220] HeLa cells (ATCC CCL-2) were plated at a density of 2xl0⁴ cells/cm² in a 6-well plate (Greiner #657160) 20 hours before transfection. 2 pg of plasmids encoding the Tornado (CMV-CVB3), Linear (Cap), and Linear (CVB3), Tornado (Short), Tornado (Medium), and Tornado (Long) expression systems or .02 ug of Poly(I:C) HMW (InvivoGen tlrl-pic) were transfected into the HeLa cells using a 3 : 1 ratio of FuGENE (Promega #E5911) to DNA in OptiMEM I Reduced Serum Media (ThermoFisher, #31985). RNA was extracted 20 hours after transfection. Expression of RIG- IFNfll, and IL6 was quantified using RT-qPCR and normalized to GAPDH expression.

Quantification and Statistical Analysis

[0221] All data are expressed as means ± SD with the number of independent experiments (n) listed for each experiment. Statistical analyses were performed using Excel (Microsoft) and Prism (GraphPad).

Example 1 - Design of a Reporter for Circular mRNA-Specific Translation

[0222] First, a construct where the circular mRNA, but not the precursor linear mRNA, can translate a reporter was developed. This construct uses split nano-luciferase (nLuc) composed of Large BiT (LgBiT) and Small BiT (SmBiT) (Dixon et al., “NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells,” ACS Chem. Biol. 11 :400-408 (2016), which is hereby incorporated by reference in its entirety). These components have low affinity for each other and only produce luminescence when they are artificially brought together, for example, by two proteins or by a protein linker (FIG. 1 A). This design was incorporated into the Tornado system, which involves synthesis of a linear RNA containing ribozymes at the 5’ and 3’ ends of the transcript. After the ribozymes undergo autocatalytic cleavage, the 5’ and 3’ ends are ligated by RtcB, an endogenous RNA ligase (Litke et al., “Highly Efficient Expression of Circular RNA Aptamers in Cells using Autocatalytic Transcripts,” Nat. BiotechnoL 37:667-675 (2019), which is hereby incorporated by reference in its entirety). In this design, luminescence will only occur when the mRNA is circularized and can be translated into a protein where the circularization junction tethers the SmBiT to the LgBiT (FIG. IB).

[0223] To confirm that this construct generates circular mRNA, a plasmid expressing the construct with a RNA polymerase II cytomegalovirus (CMV) promoter in the Tornado translation system was cloned. The Coxsackievirus B3 (CVB3) IRES, which has previously been used to drive translation of in iv'/ra-tran scribed circular mRNA was used (Wesselhoeft et al., “Engineering Circular RNA for Potent and Stable Translation in Eukaryotic Cells,” Nat. Commun. 9:2629 (2018), which is hereby incorporated by reference in its entirety). This construct is referred to as the CMV-CVB3 Tornado translation system. After transfection into HEK293T cells, a northern blot using probes against LgBiT revealed a single major band (FIG. 2B). To determine if this was the precursor linear RNA or the circular RNA, RNase R, which preferentially degrades linear RNA, was used (Abe et al., “Circular RNA Migration in Agarose Gel Electrophoresis,” Mol. Cell 82: 1768-1777 (2022), which is hereby incorporated by reference in its entirety). It was found that this band was resistant to RNase R, while a control linear RNA encoding the split nLuc (FIG. 2A) was largely degraded by RNase R treatment (FIG. 2B).

[0224] To ensure that the RNase R resistance of the RNA was not due to the structure of the CVB3 IRES, an additional linear control containing the CVB3 IRES was included (FIG. 2A). It was similarly found that the linear CVB3-driven RNA was degraded by RNase R. Finally, RT-PCR was performed over the circularization junction, which yielded a band of the expected size and sequence (FIGS. 4A-4C). These data are consistent with the idea that the Tornado translation system produces a predominantly circular mRNA.

[0225] As a final control to confirm that the luminescence derives from a circular RNA, the 3’ ribozyme sequence was deleted. This prevents formation of the 2’, 3 ’-cyclic phosphate at the 3’ end of the RNA, which is needed for RtcB-mediated circularization. This construct is referred to as “mutTomado”. Cells transfected with the mutTornado plasmid produced no luminescence (FIG. 2C).

[0226] While circular RNAs generated within the cell do not stimulate the innate immune system (Chen et al., “N6-Methyladenosine Modification Controls Circular RNA,” Immunity. Mol. Cell 76:96-109 (2019), which is hereby incorporated by reference in its entirety), whether the Tornado translation system stimulates the innate immune system was next investigated. It was found that the Tornado translation system did not stimulate the innate immune system more than the linear mRNA expression systems (FIG. 4D).

[0227] Since the Tornado system was originally designed to circularize small RNAs, whether lengthening the circularization junction would increase circular mRNA production was investigated. The length of the circularization junction was increased from 18 base pairs (bp), which is the length in the original Tornado construct, to 26 and 49 bp (FIG. 4E). A traditional nLuc was used in place of the split nLuc to ensure that the different peptides encoded by the circularization junction would not influence luminescence. It was found that the 26 or 49 bp circularization junctions did not provide a statistically significant increase in luminescence compared to the 18 bp circularization junctions (FIG. 4F). Importantly, lengthening the circularization junction did not increase the immunogenicity of the RNAs (FIG. 4G). Although not statistically significantly, the 26 bp stem produced the most luminescence and thus was used for subsequent experiments.

Example 2 - The Tornado Translation System Provides Lower, but more Persistent Levels of Protein Production than a Linear mRNA Translation System

[0228] Next, the overall protein expression level of the Tornado translation system was compared to a linear cap-dependent mRNA translation system (FIG. 2A). It was found that the Tornado translation system produced ~5 fold less luminescence than the linear mRNA translation system (FIG. 2D).

[0229] To determine if the lower amount of protein from the Tornado translation system was due to its reliance on the CVB3 IRES compared to the 5’ cap for the linear RNA, the circular RNA was compared to a linear construct that relies on CVB3 -dependent translation (FIG. 2 A). The CVB3 -dependent linear construct has the same split nLuc open reading frame (ORF) and CVB3 IRES as the Tornado translation system, but in a linear form. To ensure the CVB 3 -dependent linear translation system would not undergo cap-dependent translation, an upstream ORF that ended with a stop codon was included (FIG. 2A). The protein expression from the linear cap-dependent translation system was then compared to the linear CVB3- dependent translation system. It was found that the linear CVB 3 -dependent translation system produced ~10-fold less luminescence than the linear cap-dependent translation system (FIG. 2D). [0230] The expression level of each of the tested transcripts was next determined. To measure this, the RNA levels from the northern blot in FIG. 2B was quantified. The linear capdependent translation system had ~ 10-fold increased RNA expression compared to the linear CVB 3 -dependent transcript and ~3-fold increased RNA expression compared to the Tornado translation system (FIG. 3B). After normalizing the amount of luminescence to the RNA expression, it was found that both the linear and circular CVB 3 -dependent translation systems showed 3 -fold lower luminescence (FIG. 2E). Thus, the decreased protein expression from the Tornado translation system is due to the combination of decreased RNA expression of transcripts that contain the CVB3 IRES and decreased translational activity of the CVB3 IRES compared to cap-dependent translation.

[0231] Whether the Tornado translation system extends the duration of protein expression compared to a linear mRNA expression system was next investigated. Stable cell lines that expressed the Tornado translation system, linear cap-dependent translation system, and linear CVB3 -dependent translation system under a tetracycline-responsive promoter were generated. The stable cell lines were then pulsed with tetracycline for 12 hours to induce transcription, followed by wash out. Cells were harvested 0, 5, 10, 24, 48, and 72 hours later and assayed for RNA expression and luminescence. It was found that the Tornado translation system provided ~ 10-fold longer duration of luminescence and mRNA expression compared to both the linear cap-dependent and CVB3 -dependent translation systems (FIG. 2F). Thus, the Tornado translation system prolongs the duration of protein expression compared to a linear mRNA expression system encoding the same protein.

[0232] Next, whether the Tornado translation system can be used in other cell types was determined. HepG2 and ZR-75-1 cells were transfected with the Tornado translation system (CMV-CVB3) and detected luminescence in both these cell lines (FIG. 4H). The lower levels of luminescence is likely due to lower transfection efficiency.

Example 3 - The Tornado Translation System can Circularize Long mRNAs

[0233] To test whether the Tornado system could circularize long mRNAs, a Tornado construct designed to express a circular mRNA that encodes for the spike protein from SARS- CoV-2 was generated (Huang et al., “Structural and Functional Properties of SARS-CoV-2 Spike Protein: Potential Antivirus Drug Development for COVID-19,” Acta Pharmacol. Sin. 41 : 1141- 1149 (2020), which is hereby incorporated by reference in its entirety). This circular mRNA, including the CVB3 IRES, is 4719 nt long. HEK293T cells were transfected with either a plasmid expressing the Tornado spike mRNA or a plasmid expressing a linear spike RNA. It was found that the Tornado system circularized the spike mRNA, as evidenced by its resistance to RNase R compared to the linear spike mRNA (FIG. 41, FIG. 4J). It was further confirmed that the spike RNA is in circular form by performing RT-PCR and sanger sequencing over the circularization junction (FIG. 4K, FIG. 4L). Taken together, these results suggest that the Tornado system can therefore be used to circularize long mRNAs.

Example 4 - The Tornado Translation System Produces more Circular mRNA than the Backsplicing System

[0234] The amount of circular mRNA generated by the Tornado system was compared to the backsplicing system. The backsplicing system uses an exon comprising a gene of interest and intronic sequences from ZKSCAN1, a gene which normally produces a circular RNA through an endogenous backsplicing event (FIG. 5A) (Liang et al., “Short Intronic Repeat Sequences Facilitate Circular RNA Production,” Genes Dev. 28:2233-2247 (2014), which is hereby incorporated by reference in its entirety). The same split nLuc ORF and IRES (CVB3) was cloned from the Tornado translation system into the plasmid backbone for implementing the backsplicing system (Liang et al., “Short Intronic Repeat Sequences Facilitate Circular RNA Production,” Genes Dev. 28:2233-2247 (2014), which is hereby incorporated by reference in its entirety). Surprisingly, the Tornado translation system produced 220-fold more luminescence than the backsplicing system (FIG. 5B).

[0235] This result was validated by measuring the amount of circular mRNA generated by the two systems. Northern blotting was performed and the RNA was treated with RNase R to identify the circular products. The Tornado translation system expressed a predominantly circular product, while the backsplicing system created a predominantly linear band as evidenced by the disappearance of the band after RNase R treatment (FIG. 5C). Thus, the major product of the backsplicing system is a linear RNA. To confirm that the Tornado translation system produces more circular RNA than the backsplicing system, the northern blot experiment was repeated using an alternative insert — the ZKSCAN1 exon2/3 sequence. As expected, it was found that the Tornado translation system created a single predominant band, which was a circular RNA based on its resistance to RNase R. However, the backsplicing system created a predominantly linear band (FIG. 6A).

[0236] Notably, two studies recently reported that the backsplicing system expresses several linear transcripts (Jiang et al., “Overexpression-Based Detection of Translatable Circular RNAs is Vulnerable to Coexistent Linear RNA Byproducts,” Biochem. Biophys. Res. Commun. 558: 189-195 (2021) and Ho-Xuan et al., “Comprehensive Analysis of Translation from Overexpressed Circular RNAs Reveals Pervasive Translation from Linear Transcripts,” Nucleic Acids Res. 48: 10368-10382 (2020), which are hereby incorporated by reference in their entirety). Thus, the backsplicing system produces most of its RNA in a linear form while the Tornado system produces predominantly circular mRNA. These data suggest that the Tornado translation system is more effective for cell-based synthesis of circular mRNAs for VLPs or other applications.

Example 5 - Selection of the Pol Il-Compatible IRES

[0237] Possible IRESs were next compared to maximize protein expression from a Tornado-expressed circular mRNA. A recent study showed that the CVB3 IRES produces more protein than the EMCV IRES in in vitro-transcribed circular mRNA (Wesselhoeft et al., “Engineering Circular RNA for Potent and Stable Translation in Eukaryotic Cells,” Nat. Commun. 9:2629 (2018), which is hereby incorporated by reference in its entirety). The related human rhinovirus B3 (HRV-B3) IRES was reported to provide more protein production than the CVB3 IRES (Chen et al., “Engineering Circular RNA for Enhanced Protein Production,” Nature Biotechnology (2022), which is hereby incorporated by reference in its entirety. These IRESs were therefore compared in the Tornado translation system. Each construct was expressed from a Pol II (CMV) promoter. It was found that the CVB3 IRES produces more protein than the EMCV IRES and slightly more protein than the HRV-B3 IRES (FIG. 7A, FIG. 8A). Thus, the CVB3 IRES produces the highest level of protein expression from a Pol Il-driven Tornado translation system.

Example 6 - Selection of the Pol Il-Compatible IRES

[0238] The Tornado translation system could benefit by using a Pol III promoter because Pol III promoters express higher levels of RNA than Pol II promoters (Dieci, G. & Sentenac, A. “Facilitated Recycling Pathway for RNA Polymerase III,” Cell 84:245-252 (1996), which is hereby incorporated by reference in its entirety). A problem with the Pol III promoter is that Pol III termination signals, i.e., UUUU or closely related sequences such as UCUUU or UUUAU (Orioli et al., “Widespread Occurrence of Non-Canonical Transcription Termination by Human RNA Polymerase III,” Nucleic Acids Research 39:5499-5512 (2011), which is hereby incorporated by reference in its entirety), are found in commonly used IRESs such as CVB3 and EMCV. The EMCV, CVB3, and HRV-B3 IRESs have two, three, and four Pol III termination signals respectively (FIG. 7B, Table 2). The highly conserved U-stretch in CVB3 and HRV-B3 IRESs is responsible for binding directly to 18S rRNA (Bailey & Tapprich, “Structure of the 5' Nontranslated Region of the Coxsackievirus B3 Genome:Chemical Modification and Comparative Sequence Analysis,” Journal of Virology 81 :650-668 (2007) and Yang et al., “A Shine-Dalgamo-like Sequence Mediates in Vitro Ribosomal Internal Entry and Subsequent Scanning for Translation Initiation of Coxsackievirus B3 RNA,” Virology 305:31-43 (2003), which are hereby incorporated by reference in its entirety). Mutations in this sequence lead to abrogation of IRES activity (Yang et al., “A Shine-Dalgamo-like Sequence Mediates in Vitro Ribosomal Internal Entry and Subsequent Scanning for Translation Initiation of Coxsackievirus B3 RNA,” Virology 305:31-43 (2003), which is hereby incorporated by reference in its entirety). Thus, the focus was on the EMCV IRES.

[0239] The EMCV IRES contains two Pol III termination signals (FIG. 7B). The first comprises a sequence (UCUUU) that binds polypyrimidine-tract-binding protein (PTB), which is thought to be important for IRES function (FIG. 7B) (Kaminski & Jackson, “The Polypyrimidine Tract Binding Protein (PTB) Requirement for Internal Initiation of Translation of Cardiovirus RNAs is Conditional Rather than Absolute,” RNA 4:626-638 (1998), which is hereby incorporated by reference in its entirety). PTB can bind several U-rich sequences, with the most common sequence being UUCUCU,32 which is not a Pol III termination signal (Orioli et al., “Widespread Occurrence of Non-Canonical Transcription Termination by Human RNA Polymerase III,” Nucleic Acids Research 39:5499-5512 (2011), which is hereby incorporated by reference in its entirety). Therefore, the Pol III termination element was replaced with UUCUCU, as well as UCUCU, which has also been described as a PTB-binding motif, 32 and UCUAU, which is not a canonical PTB-binding motif (FIG. 7B) (Xue et al., “Genome-Wide Analysis of PTB-RNA Interactions Reveals a Strategy used by the General Splicing Repressor to Modulate Exon Inclusion or Skipping,” Mol. Cell 36:996-1006 (2009), which is hereby incorporated by reference in its entirety). To determine IRES activity, each EMCV variant was cloned into the Pol Il-driven Tornado translation system. Surprisingly, the UCUAU mutant produced the most luminescence despite not being a canonical PTB-binding sequence (FIG. 7C). [0240] To mutate the second Pol III termination signal a related IRES, the Falcon picornavirus, that has a related sequence but no Pol III termination element in this region was identified (FIG. 8B). The Falcon picornavirus sequence was then incorporated in place of the Pol III termination signal in the wild-type EMCV (wtEMCV) IRES (FIG. 7B).

[0241] The EMCV that incorporates both mutations (mutEMCV) was next compared to the parental EMCV IRES. The protein output was measured using the Pol II Tornado translation system, which transcribes regardless of Pol III termination elements. It was found that the wtEMCV IRES produced ~3-fold more luminescence than mutEMCV IRES (FIG. 7D). Thus, the mutations decreased the translational activity of the IRES, but the mutEMCV IRES still maintains translational activity. [0242] Next, the protein output of the mutEMCV IRES was compared to the WT CVB3 IRES in a Tornado translation system driven by U6, a Pol III promoter. It was found that the mutEMCV produced ~15-fold more luminescence than the CVB3 IRES, likely due to the Pol III termination signals in the CVB3 IRES preventing transcription of the full-length RNA (FIG.

8C). Taken together, these results show that the mutEMCV IRES can be used to drive translation from a Pol Ill-driven expression system.

[0243] Lastly, the translational activity of the mutEMCV IRES was compared to an IRES that naturally does not contain a Pol-III termination signal. The classical swine fever virus (CSFV) IRES lacks Pol III termination elements, and is therefore compatible with Pol III (FIG. 8D) (Sizova et al., “Specific Interaction of Eukaryotic Translation Initiation Factor 3 with the 50 Nontranslated Regions of Hepatitis C Virus and Classical Swine Fever Virus RNAs,” J. Virol. 72:4775-4782 (1998), which is hereby incorporated by reference in its entirety). It was found that the mutEMCV IRES provides >3-fold more luminescence than the CSFV IRES (FIG. 8E). Thus, mutEMCV is the best Pol Ill-compatible IRES tested.

Example 7 - The Tornado Translation System Produces the most Protein using a CMV-

CVB3 Promoter and IRES Combination

[0244] Whether protein expression is more efficient in HEK293T cells using Pol II and a wild-type IRES or Pol III and the mutated IRES was next investigated. Northern blotting using probes against the LgBiT showed a single prominent band with both constructs (FIG. 7E). The Pol Ill-driven (U6) Tornado translation system expressed over 10-fold more RNA than the Pol Il-driven (CMV) Tornado translation system (FIG. 7E). However, the luminescence from the Pol Il-driven system was ~6-fold higher than the Pol Ill-driven system (FIG. 7F). This is in part due to the decreased translational activity of the mutEMCV IRES (FIG. 7D). However, the 10- fold higher RNA levels from the Pol III system would be expected to cause higher protein expression despite the ~3-fold decreased translational activity of the mutEMCV IRES.

[0245] The possibility that the circular RNAs were retained in the nucleus when expressed using Pol III was considered. Therefore, fluorescence in-situ hybridization was used to quantify the nuclear and cytoplasmic localization of the Pol II and Pol Ill-driven Tornado translation systems. It was found that the distribution of the circular RNA was similar with both systems (FIG. 8F, FIG. 8G). Furthermore, the distribution was similar to a linear cap-dependent translation system (FIG. 8F, Fig. 8G). Addition of a constitutive export element (CTE) derived from the Mason-Pfizer monkey virus34 into the Pol Ill-driven (U6) Tornado translation system did not produce more luminescence (FIG. 8H). Thus, unknown factors besides nuclear retention account for the reduced protein output from the Pol Ill-driven Tornado translation system. Thus, the Pol III system generates more circular RNA, but this RNA is not efficiently translated due to the IRES and potentially other factors.

Example 8 - A Continuous Translation Construct does not Improve Protein Output

[0246] Whether the protein production could be increased using continuous translation was next investigated. Continuous translation can occur in a circular RNA when the ORF lacks a stop codon, and the ribosome continues translating past the ORF into the IRES, then back into the ORF (Abe et al., “Rolling Circle Amplification in a Prokaryotic Translation System using Small Circular RNA,” Angew. Chem. Int. Ed. Engl. 52:7004-7008 (2013), which is hereby incorporated by reference in its entirety). Continuous translation requires an IRES that is in frame with the ORF lacking a stop codon. In this way, the ribosome might circumambulate the circular RNA indefinitely, thus producing high yields of protein (FIG. 9 A) (Abe et al., “Rolling Circle Translation of Circular RNA in Living Human Cells,” Sci. Rep. 5: 16435 (2015) and Costello et al., “Continuous Translation of Circularized mRNA Improves Recombinant Protein Titer,” Metab. Eng. 52:284-292 (2019), which is hereby incorporated by reference in its entirety). Inclusion of a viral P2A sequence, which induces ribosome skipping and therefore cleaves the polypeptide chain (Liu et al., “Systematic Comparison of 2A Peptides for Cloning Multi-Genes in a Polycistronic Vector,” Sci. Rep. 7:2193 (2017), which is hereby incorporated by reference in its entirety), can ensure that the protein polymer created by continuous translation is separated into functional protein monomers.

[0247] Most IRES sequences cannot be used for continuous translation since they contain multiple stop codons in all three reading frames (FIG. 9B). The HCV IRES contains only three stop codons in one of its reading frames, which is the fewest stop codons of any of the examined IRESs in any frame (FIG. 9B). The first stop codon occurs in a stem whose structure, but not sequence, is conserved in related IRESs (Honda et al., “A Phylogenetically Conserved Stem- Loop Structure at the 5’ Border of the Internal Ribosome Entry Site of Hepatitis C Virus is Required for Cap -Independent Viral Translation,” J. Virol. 73: 1165-1174 (1999), which is hereby incorporated by reference in its entirety). Thus, a UAG to UAC mutation and a mutation on the complementary base to conserve the structure of the stem was made (FIG. 9C). The second two stop codons are adjacent (UGA UAG) and contain a conserved loop sequence (GAUA) (FIG. 9C) (Brown et al., “Secondary structure of the 5’ Nontranslated Regions of Hepatitis C Virus and Pestivirus Genomic RNAs,” Nucleic Acids Res. 20:5041-5045 (1992), which is hereby incorporated by reference in its entirety). The loop from UGAUAG was mutated to GGAUAU in order to maintain both the sequence of the conserved region and the G- U wobble base pair at the base of the loop, while removing both stop codons (FIG. 9C). These two mutations were used to create the mutant HCV (mutHCV) IRES that could be used for continuous translation.

[0248] To test whether the mutHCV IRES had decreased translational activity compared to the wild-type HCV (wtHCV) IRES, the mutHCV and the wtHCV IRESs were cloned into the Pol II non-continuous Tornado translation system, which contained a stop codon at the end of the split nLuc ORF (FIG. 9A). It was found that mutHCV IRES produced similar levels of luminescence as the wtHCV IRES (FIG. 9D). Thus, the mutHCV IRES retained its ability to drive translation and can be used for continuous translation.

[0249] Next, the efficiency of the mutHCV continuous translation system was tested. The mutHCV IRES was cloned into a continuous Tornado translation system where the IRES and split nLuc ORF contained no stop codons. A P2A sequence was included downstream of the IRES (FIG. 9A). Next, the protein expression from the mutHCV continuous translation system was compared to the mutHCV non-continuous translation system. Interestingly, only a 50% increase in luminescence was observed by using a continuous translation system which suggests that the ribosome was only able to circumambulate ~l-2 times (FIG. 9E). This likely reflects the highly structured HCV IRES, since structure can stop translation (Wen et al., “Following Translation by Single Ribosomes One Codon at a Time,” Nature 452:598-603 (2008); Chen et al., “Dynamics of Translation by Single Ribosomes through mRNA Secondary Structures,” Nat. Struct. Mol. Biol. 20:582-588 (2013); and Zheng et al., “Genome-Wide Double-Stranded RNA Sequencing Reveals the Functional Significance of Base-Paired RNAs in Arabidopsis,” PLoS Genet. 6:el001141 (2010), which are hereby incorporated by reference in their entirety). Furthermore, it was found that the CVB3 non-continuous translation system produced ~10-fold more luminescence than the continuous mutHCV translation system (FIG. 9E). Thus, the mutHCV continuous translation system does not improve the protein output from the Tornado translation system.

[0250] Recently, a high-throughput screen discovered thousands of endogenous IRES elements that drive translation of circular RNAs (Chen et. al., “Structured Elements Drive Extensive Circular RNA Translation,” Mol. Cell 81 :4300-4318 (2021), which is hereby incorporated by reference in its entirety). These endogenous IRESes drive translation using a small stem-loop structure. Moreover, many of the IRESes identified in the screen are free of stop codons in at least one frame due to their short length (<200 nt). The combined benefit of having a simple structure and not requiring mutations may allow these endogenous IRESes to drive continuous translation.

[0251] An IRES candidate was selected from the screen (Chen et. al., “Structured Elements Drive Extensive Circular RNA Translation,” Mol. Cell 81 :4300-4318 (2021), which is hereby incorporated by reference in its entirety) that exhibited high translational activity and lacked a stop codon in at least one frame — the LIMA1 IRES. The LIMA1 IRES was cloned into a continuous or non-continuous Pol Il-driven Tornado translation system using a split nLuc ORF and compared the translational output. Interestingly, a ~90-fold increase in luminescence from the LIMA1 continuous translation system was observed compared to the LIMA1 non-continuous translation system (FIG. 9F). However, the LIMA1 continuous translation system produced >15-fold less luminescence than the CVB3 non-continuous translation system (FIG. 9F). Thus, even though continuous translation markedly enhances the protein output of the LIMA1 IRES, its overall activity is still very low compared to the CVB3 non-continuous system.

[0252] Next, the identity of the AUG that is used in the LIMA1 continuous translation system was investigated. Since the LIMA1 continuous translation system does not have any stop codons, any start codon in the same frame as the split nLuc could serve as a start codon — even if it is not within the IRES. To identify the AUG, constructs where the AUG downstream of the LIMA1 IRES was mutated to CCC (LIMA1 mutAUG NO STOP), and where the entire LIMA1 IRES was deleted (NO IRES NO STOP) were generated. Importantly, the mutated AUG was the only AUG within the LIMA1 IRES that is in frame with the ORF (FIG. 10 A). Interestingly, it was found that the LIMA1 mutAUG continuous translation system showed 2.6-fold increased luminescence signal compared to the LIMA1 continuous translation system and the no IRES continuous translation system showed similar luminescence signal to the LIMA1 continuous translation system (FIG. 9G). This suggests that the protein output from the LIMA1 continuous translation system is likely occurring through a combination of a non-AUG start codon within the IRES and an AUG outside of the LIMA1 IRES. This further supports the hypothesis that the LIMA 1 -dependent translation is near the background levels of the Tornado Translation system. [0253] Whether alternative endogenous IRES elements from the previously mentioned screen (Chen et. al., “Structured Elements Drive Extensive Circular RNA Translation,” Mol. Cell 81 :4300-4318 (2021), which is hereby incorporated by reference in its entirety) would have higher translational output than the LIMA1 IRES was investigated. To test this, three additional endogenous IRESs (CEP50, TGFBRG, and CHD2) that exhibited high translational activity according to the screen (Chen et. al., “Structured Elements Drive Extensive Circular RNA Translation,” Mol. Cell 81 :4300-4318 (2021), which is hereby incorporated by reference in its entirety) and lacked a stop codon in at least one frame were selected. It was found that none of these endogenous IRESs produced more luminescence than the LIMA1 continuous translation system (FIG. 10B). Thus, endogenous IRES elements are unlikely to produce meaningful levels of protein expression despite being able to undergo continuous translation.

Example 9 - The Tornado Translation System can be used to Produce Circular mRNA- Containing VLPs

[0254] Next, VLPs carrying circular mRNAs were developed to achieve a longer duration of heterologous protein expression. To package circular mRNAs, the mRNA- containing lentiviral VLP system was used (Lu et al., “Delivering SaCas9 mRNA by Lentivirus- Like Bionanoparticles for Transient Expression and Efficient Genome Editing,” Nucleic Acids Res. 47:e44 (2019), which is hereby incorporated by reference in its entirety). This system comprises an envelope plasmid; a transfer plasmid that contains a gene of interest with an MS2 stem loop in its 3’UTR; and an integrase-deficient packaging plasmid that expresses MCP fused to the N-terminus of the nucleocapsid proteinl 1 (FIG. 11 A). This system was modified to package circular mRNAs in VLPs. To do so, a transfer plasmid was created by cloning a MS2 stem loop into the 3’UTR of the CMV-CVB3 Tornado translation system that expresses an nLuc gene (FIG. 11 A). Since it contains the MS2 sequence, this RNA will be packaged into VLPs by binding to the MCP domain in the nucleocapsid protein.

[0255] Although the Tornado translation system expresses a predominantly circular mRNA (see FIG. 1C), the possibility that linear precursors get packaged into the VLPs was investigated. To test this, RNA from VLPs produced using the Tornado translation system was subjected to RNase R. As a control, a linear mRNA VLP system with the same nLuc ORF and MS2 sequence as the circular mRNA was included. It was found that the viral RNA from VLPs packaged using the Tornado translation system was resistant to RNase R while the viral RNA from the control VLPs made with linear RNAs was degraded (FIG. 1 IB). To further confirm that the viral RNA from VLPs packaged using the Tornado translation system was circular RT- PCR was performed over the circularization junction and the amplicon was sequenced. The amplicon was the expected size and sequence (FIG. 12A, FIG. 12B). Thus, the VLPs produced with the Tornado translation system package circular mRNA. Example 10 - VLPs with Circular mRNA Exhibit Increased Level and Duration of Protein Expression

[0256] Whether VLPs produced using the Tornado translation system exhibit a longer duration of protein expression compared to VLPs produced using a linear mRNA expression system was next evaluated. To test this, HEK293T cells were transduced with equal amounts of infectious VLPs, as determined by the levels of nLuc mRNA in the VLP measured by qRT-PCR (FIG. 12C). At the initial 5-hour time point, the cells transduced with VLPs produced using the Tornado translation system produced a similar amount of luminescence as the cells transduced with VLPs produced using a linear mRNA expression system (FIG. 11C). However, at 24 hours after transduction and beyond, VLPs that were produced using the Tornado translation system produced >5-fold more luminescence than VLPs that were produced using the linear mRNA expression system (FIG. 11C). This result is consistent with the longer half-life of circular mRNA, which would allow prolonged synthesis and accumulation of protein over time.

[0257] It was also asked whether these VLPs could be used in other cell types. SH- SY5Y neuroblastoma cells, which similarly showed a ~5-fold increase in luminescence by using VLPs produced using the Tornado translation system compared to VLPs produced using linear mRNA expression system (FIG. 12D) were evaluated.

[0258] To show that VLPs produced using the Tornado translation system can be pseudotyped to achieve cell-type specificity, VLPs were pseudotyped using a spike protein from SARS-CoV-2 that enables infection of ACE-2-expressing cells (Crawford et al., “Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays,” Viruses 12:513 (2020), which is hereby incorporated by reference in its entirety). The luminescence from HEK293T cells and ACE-2-expressing HEK293T cells (Chan et al., “Engineering Human ACE2 to Optimize Binding to the Spike Protein of SARS Coronavirus 2,” Science 369: 1261-1265 (2020), which is hereby incorporated by reference in its entirety) transduced with spike-pseudotyped VLPs containing circular nLuc mRNA was next compared (FIG. 12D). The spike-pseudotyped cells showed selective delivery into ACE-2 expressing cells (FIG. 1 IE). Thus, VLPs produced using the Tornado translation system can be pseudotyped to achieve cell-type specificity.

Discussion of Examples 1 - 10

[0259] Examples 1-10 describe the development of VLPs with circular mRNAs, thus enabling VLPs to exert longer duration of protein expression compared to VLPs with linear mRNAs. This approach is enabled by the Tornado RNA circularization approach which was previously developed to circularize small RNA aptamers (Litke et al., “Highly Efficient Expression of Circular RNA Aptamers in Cells using Autocatalytic Transcripts,” Nat. BiotechnoL 37:667-675 (2019), which is hereby incorporated by reference in its entirety). Through several different types of optimizations, altered sequence requirements for the Tornado RNA, as well as specific promoters and IRESs that result in a translatable circular mRNAs that are efficiently packaged into VLPs were identified. Using this approach, VLPs produced using the Tornado translation system were shown to increase the duration and level of protein expression, thus demonstrating the potential use of this technology for mRNA delivery.

[0260] As part of this study, circular mRNA generated by the backsplicing system was evaluated (Liang et al., “Short Intronic Repeat Sequences Facilitate Circular RNA Production,” Genes Dev. 28:2233-2247 (2014), which is hereby incorporated by reference in its entirety). This method generates circular RNA by using the molecular mechanism that occurs with the endogenous genes, such as ZKSCAN1. The Examples disclosed herein demonstrate that the predominant product is a linear RNA, likely reflecting the more efficient forward splicing reaction. Other groups similarly found that the backsplicing system generated linear forward splicing products (Jiang et al., “Overexpression-Based Detection of Translatable Circular RNAs is Vulnerable to Coexistent Linear RNA Byproducts,” Biochem. Biophys. Res. Commun. 558: 189-195 (2021) and Ho-Xuan et al., “Comprehensive Analysis of Translation from Overexpressed Circular RNAs Reveals Pervasive Translation from Linear Transcripts,” Nucleic Acids Res. 48: 10368-10382 (2020), which are hereby incorporated by reference in their entirety). In contrast, the Tornado system according to the present disclosure readily generated large circular mRNAs (up to 4719 bp), with minimal detectable linear precursors. This reflects the highly efficient nature of circularization using the Tornado approach. Thus, for experiments requiring generation of small or large RNA circles in cells, the Tornado approach should be used.

[0261] The VLP system has the benefit that it can be pseudotyped to enable cell-type specific infection (Cronin et al., “Altering the Tropism of Lentiviral Vectors Through Pseudotyping,” Curr. Gene Ther. 5:387-398 (2005); Naldini et al., “In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector,” Science 272:263-267 (1996); and Hamilton et al., “Targeted Delivery of CRISPR-Cas9 and Transgenes Enables Complex Immune Cell Engineering,” Cell Rep. 35: 109207 (2021), which are hereby incorporated by reference in their entirety). Recent variants of the VLP system using endogenous retrotransposons (Segal et al., “Mammalian Retrovirus-Like Protein PEG10 Packages its own mRNA and can be Pseudotyped for mRNA Delivery,” Science 373:882-889 (2021), which is hereby incorporated by reference in its entirety), may be particularly useful for delivering mRNA without unwanted immune effects that would presumably occur using repeated dosing of current VLPs.

[0262] More circular mRNA is generated using a Pol III promoter compared to when a CMV promoter, which is a Pol II promoter, is used. The IRES was mutated to remove Pol III termination elements, which in turn reduced the activity of the IRES. Thus, the Pol III Tornado translation system should be used when a large amount of RNA is desired, such as the expression of a noncoding RNA. The Pol II Tornado translation system should be used when the highest level of protein expression is desired. In the future, it will be important to develop Pol Ill- compatible IRESs that are also highly efficient for translation initiation to take advantage of the high RNA expression seen with the Pol III system.

[0263] Cellular expression of circular mRNAs can be valuable for other applications. For example, plasmid therapeutics rely on the expression of the encoded mRNAs, but the plasmid DNA is often epigenetically silenced (Chen et al., “Silencing of Episomal Transgene Expression by Plasmid Bacterial DNA Elements in Vivo,” Gene Ther. 11 : 856-864 (2004), which is hereby incorporated by reference in its entirety), limiting its duration of action. Adenoviral vectors similarly deliver a DNA that is readily silenced (Brooks et al., “Transcriptional Silencing is Associated with Extensive Methylation of the CMV Promoter Following Adenoviral Gene Delivery to Muscle,” J. Gene Med. 6:395-404 (2004), which is hereby incorporated by reference in its entirety). Plasmid and adenoviral vector-based therapeutics could have a longer duration of action by using circular mRNAs expressed using the Tornado translation system.

[0264] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. A virus-like particle (VLP) comprising: a circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), wherein the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence; a second ligation sequence positioned 3’ to the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s); and a plurality of one or more proteins that can self-assemble into a nanoparticle.

2. The virus-like particle (VLP) of claim 1, wherein the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof.

3. The virus-like particle (VLP) of claim 1 or claim 2, wherein the IRES is a wild-type internal ribosomal entry site.

4. The virus-like particle (VLP) of claim 1 or claim 2, wherein the IRES is modified internal ribosomal entry site.

5. The virus-like particle (VLP) any one of claims 1 to 4, wherein the IRES lacks a Pol III termination element.

6. The virus-like particle (VLP) of any one of claims 1 to 5, wherein the IRES lacks a stop codon.

7. The virus-like particle (VLP) of any one of claims 1 to 6, wherein a portion of the first ligation sequence is complementary to a portion of the second ligation sequence.

8. The virus-like particle (VLP) of claim 7, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 18 nucleotides in length.

9. The virus-like particle (VLP) of claim 7, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 26 nucleotides in length.

10. The virus-like particle (VLP) of claim 7, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 49 nucleotides in length.

11. The virus-like particle (VLP) of any one of claims 1 to 10, wherein the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

12. The virus-like particle (VLP) of claim 11, wherein the one or more peptides comprises an antigen and wherein the antigen is a cancer neoepitope.

13. The virus-like particle (VLP) of claim 11, wherein the Cas family protein is selected from the group consisting of Cas9, nCas9, dCas9, Casl2a, nCasl2a, dCasl2a,

Cas 12b, nCasl2b, and dCasl2b.

14. The virus-like particle (VLP) of claim 11, wherein the one or more peptides comprises a chimeric antigen T-cell receptor.

15. The virus-like particle (VLP) of any one of claims 1 to 14, wherein the circular RNA molecule further comprises: a stem loop that binds to a cognate RNA-binding protein, wherein the stem loop is positioned 3’ to the RNA molecule encoding the one or more peptide(s).

16. The virus-like particle (VLP) of claim 15, wherein the stem loop is selected from the group consisting of MS2, PP7, BoxB, and Com.

17. The virus-like particle (VLP) of any one of claims 1 to 16, wherein the nanoparticle is a viral capsid.

18. The virus-like particle (VLP) of any one of claims 1 to 16, wherein the nanoparticle is a viral capsid-like structure.

19. The virus-like particle (VLP) of any one of claims 1 to 18, wherein the plurality of one or more proteins that can self-assemble into a nanoparticle comprises a polyprotein.

20. The virus-like particle (VLP) of claim 19, wherein the polyprotein is selected from the group consisting of a retroviral group specific antigen (Gag) polyprotein, a mammalian group specific antigen (Gag)-like polyprotein, and derivatives thereof.

21. The virus-like particle (VLP) of claim 20, wherein the polyprotein comprises one or more proteins selected from the group of proteins consisting of: a nucleocapsid protein, a capsid protein, a matrix protein, a reverse transcriptase, a protease, and a defective integrase.

22. The virus-like particle (VLP) of claim 20 or claim 21, wherein the retroviral group specific antigen (Gag) polyprotein is Human Immunodeficiency Virus Type 1 (HIV-1) group specific antigen (Gag).

23. The virus-like particle (VLP) of claim 20 or claim 21, wherein the mammalian group specific antigen (Gag)-like polyprotein is PEG10.

24. The virus-like particle (VLP) of any one of claims 1 to 18, wherein the plurality of one or more proteins that can self-assemble into a nanoparticle comprise(s) one or more structural protein(s).

25. The virus-like particle (VLP) of claims 24, wherein the one or more structural protein(s) is/are selected from the group consisting of capsid protein(s), nucleocapsid protein(s), matrix protein(s), and combinations thereof.

26. The virus-like particle (VLP) of claim 25, wherein the capsid protein(s) is/are non-retroviral capsid protein(s).

27. The virus-like particle (VLP) of claim 26, wherein the non-retroviral capsid protein(s) is/are selected from the group consisting of Herpes Simplex Virus (HSV) VP23, Herpes Simplex Virus (HSV) VP19C, Hepatitis B Virus (HBV) core antigen, Human Papillomavirus (HPV) LI, Human Papillomavirus (HPV) L2, and combinations thereof.

28. The virus-like particle (VLP) of any one of claims 1 to 27, wherein at least one of the plurality of one or more proteins that can self-assemble into a nanoparticle comprise(s) or is fused to an RNA-binding protein domain.

29. The virus-like particle (VLP) of claim 28, wherein the RNA-binding protein domain is selected from the group consisting of MS2 coat protein (MCP), Com, PCP, and N22.

30. The virus-like particle (VLP) of claim 28 or claim 29, wherein the RNA- binding domain is located at an N-terminus of the at least one of the plurality of one or more proteins that can self-assemble into a nanoparticle comprising or fused to the RNA-binding domain.

31. The virus-like particle (VLP) of claim 30, wherein the RNA-binding domain is located at a C-terminus of the at least one of the plurality of one or more proteins that can self-assemble into a nanoparticle comprising or fused to the RNA-binding domain.

32. The virus-like particle (VLP) of any one of claims 1 to 31 further comprising: one or more envelope and/or spike protein(s).

33. The virus-like particle (VLP) of claim 32, wherein the one or more envelope protein(s) is/are viral envelope and/or spike protein(s).

34. The virus-like particle (VLP) of claim 33, wherein the one or more viral envelope and/or spike protein(s) is/are selected from the group consisting of a vesicular stomatitis virus envelope protein, a rabies virus envelope protein, a measles virus envelope protein, a nipah virus envelope protein, a chickungunya virus envelope protein, and a sindbis virus envelope protein.

35. The virus-like particle (VLP) of claim 33 or claim 34, wherein the one or more envelope and/or spike protein(s) comprises a vesicular stomatitis virus G (VSV G) protein, a RabV-G, Chickungunya virus E1ZE2, Sindbis virus E1ZE2, Measle virus H/F, and derivatives thereof.

36. The virus-like particle (VLP) of any one of claims 32 to 35, wherein the one or more envelope and/or spike protein(s) comprises mutant VSV-G (K47Q, R354A).

37. The virus-like particle (VLP) of any one of claims 32 to 36, wherein the one or more envelope and/or spike proteins comprises a fusion protein.

38. The virus-like particle (VLP) of any one of claims 1 to 37, wherein the VLP further comprises: protein that is trafficked to the viral particle and/or cell surface membrane.

39. The virus-like particle (VLP) of claim 38, wherein the protein that is trafficked to the viral particle and/or cell surface membrane is a ligand or target binding protein.

40. The virus-like particle (VLP) of claim 38 or claim 39, wherein the protein that is trafficked to the viral particle and/or cell surface membrane is selected from the group consisting of a single chain of MHC fused with beta-2-microglobulin (B2M) and a covalently linked peptide; a single-chain antibody variable fragment fused to a transmembrane domain; and an antigen fused to a transmembrane domain.

41. A vector encoding a translation system comprising: a promoter and a nucleic acid sequence encoding an RNA molecule comprising: a first ribozyme; a first ligation sequence positioned 3’ to the first ribozyme; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), wherein the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence; a second ligation sequence positioned 3’ to the internal ribosomal entry site; and a second ribozyme positioned 3’ to the second ligation sequence.

42. The vector of claim 41, wherein the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof.

43. The vector of claim 41 or claim 42, wherein the IRES is a wild-type internal ribosomal entry site.

44. The vector of claim 41 or claim 42, wherein the IRES is modified internal ribosomal entry site.

45. The vector of any one of claims 41 to claim 44, wherein the promoter is a Pol II promoter.

46. The vector of claim 45, wherein the Pol II promoter selected from the group consisting of CMV, SV40, PGK, and HSV-TK.

47. The vector of claim 45 or claim 46 wherein the IRES is selected from the group consisting of CVB3 IRES (SEQ ID NO:1), HRV-B3 IRES (SEQ ID NO:6), EMCV IRES (SEQ ID NO:2), mutHCV IRES (SEQ ID NO:3), and LIMA1 IRES (SEQ ID NO:8), or derivatives thereof.

48. The vector of any one of claims 41 to 44, wherein the promoter is a Pol III promoter.

49. The vector of claim 48, wherein the Pol III promoter is selected from the group consisting of U6, 7SK, Hl, and derivatives thereof.

50. The vector of claim 48 or claim 49, wherein the IRES is selected from the group consisting of mutEMCV or classic swine fever virus (CSFV) IRES (SEQ ID NO:5), mutEMCV IRES (SEQ ID NO:3), mutCVB3 IRES (SEQ ID NO:7), and derivatives thereof.

51. The vector of any one of claims 48 to 50, wherein the IRES lacks a Pol III termination element and/or signal.

52. The vector of any one of claims 41 to 51, wherein a portion of the first ligation sequence is complementary to a portion of the first ribozyme and a portion of the second ligation sequence is complementary to a portion of the second ribozyme.

53. The vector of any one of claims 41 to 52, wherein a portion of the first ligation sequence is complementary to a portion of the second ligation sequence.

54. The vector of claim 53, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 18 nucleotides in length.

55. The vector of claim 53, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 26 nucleotides in length.

56. The vector of claim 53, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 49 nucleotides in length.

57. The vector of any one of claims 41 to 56, wherein each of the first ribozyme and the second ribozyme comprises a sequence that may be cleaved to produce a 5'- OH end and a 2', 3 '-cyclic phosphate end.

58. The vector of any one of claims 41 to 57, wherein each of the first ribozyme and the second ribozyme is independently selected from the group consisting of Hammerhead, Hairpin, Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Vgl, glucosamine- 6-phosphate synthase (glmS), Twister, Twister Sister, Hatchet, Pistol ribozymes, engineered synthetic ribozymes, or derivatives thereof.

59. The vector of any one of claims 41 to 58, wherein the first ribozyme is a P3 Twister ribozyme and the second ribozyme is a Pl Twister ribozyme.

60. The vector of any one of claims 41 to 59, wherein the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

61. The vector of any one of claims 41 to 60, wherein the RNA molecule further encodes: a stem loop that binds to a cognate RNA-binding protein, wherein the stem loop is positioned 3’ to the RNA molecule encoding the one or more peptide(s).

62. The vector of claim 61, wherein the stem loop is selected from the group consisting of MS2, PP7, BoxB, and com.

63. The vector of any one of claims 41 to 62, wherein the IRES lacks a stop codon.

64. A system for producing virus-like particles (VLPs) comprising a circular RNA translation system, said system comprising: a packaging vector encoding a plurality of one or more proteins that can selfassemble into a nanoparticle; an envelope vector; and a vector encoding a translation system according to any one of claims 41 to 63.

65. The system of claim 64, wherein the nanoparticle is a viral capsid.

66. The system of claim 64, wherein the nanoparticle is a viral capsid-like structure.

67. The system of any one of claims 64 to 66, wherein the plurality of one or more proteins that can self-assemble into a nanoparticle comprise(s) a polyprotein.

68. The system of claim 67, wherein the polyprotein is selected from the group consisting of a retroviral group specific antigen (Gag) polyprotein, a mammalian group specific antigen (Gag)-like polyprotein, and derivatives thereof.

69. The system of claim 68, wherein the polyprotein comprises one or more proteins selected from the group of proteins consisting of: a nucleocapsid protein, a capsid protein, a matrix protein, a reverse transcriptase, a protease, and a defective integrase.

70. The system of claim 68 or 69, wherein the retroviral group specific antigen (Gag) polyprotein is Human Immunodeficiency Virus Type 1 (HIV-1) group specific antigen (Gag).

71. The system of claim 68 or claim 69, wherein the mammalian group specific antigen (Gag)-like polyprotein is PEG10.

72. The system of any one of claims 64 to 66, wherein the plurality of one or more proteins that can self-assemble into a nanoparticle comprise(s) one or more structural protein(s).

73. The system of claim 72, wherein the one or more structural protein(s) is/are selected from the group consisting of capsid protein(s), nucleocapsid protein(s), matrix protein(s), and combinations thereof.

74. The system of claim 73, wherein the capsid protein(s) is/are non-retroviral capsid protein(s).

75. The system of claim 74, wherein the non-retroviral capsid protein(s) is/are selected from the group consisting of Herpes Simplex Virus (HSV) VP23, Herpes Simplex Virus (HSV) VP19C, Hepatitis B Virus (HBV) core antigen, Human Papillomavirus (HPV) LI, Human Papillomavirus (HPV) L2, and combinations thereof.

76. The system of any one of claims 64 to 75, wherein at least one of the plurality of one or more proteins that can self-assemble into a nanoparticle comprise(s) or is fused to an RNA-binding protein domain.

77. The system of claim 76, wherein the RNA-binding protein domain is selected from the group consisting of MS2 coat protein (MCP), Com, PCP, and N22.

78. The system of claim 76 or claim 77, wherein the RNA-binding domain is located at an N-terminus or C-terminus of at least one of the plurality of one or more proteins that can self-assemble into a nanoparticle comprising or fused to the RNA-binding domain.

79. The system of any one of claims 64 to 78, wherein the envelope vector encodes one or more envelope and/or spike protein(s).

80. The system of claim 79, wherein the one or more envelope and/or spike protein(s) is/are viral envelope protein(s).

81. The system of claim 80, wherein the one or more viral envelope and/or spike protein(s) is/are selected from the group consisting of a vesicular stomatitis virus envelope protein, a rabies virus envelope protein, a measles virus envelope protein, a nipah virus envelope protein, a chickungunya virus envelope protein, and a sindbis virus envelope protein.

82. The system of claim 80 or claim 81, wherein the one or more envelope and/or spike protein(s) comprises a vesicular stomatitis virus G (VSV G) protein, a RabV-G, Chickungunya virus E1ZE2, Sindbis virus E1ZE2, Measle virus H/F, and derivatives thereof.

83. The system of any one of claims 79 to 82, wherein the one or more envelope and/or spike protein(s) comprises mutant VSV-G (K47Q, R354A).

84. The system of any one of claims 79 to 83, wherein the one or more envelope and/or spike proteins comprises a fusion protein.

85. The system of any one of claims 64 to 84 further comprising: a vector encoding a protein that is trafficked to the viral particle and/or cell surface membrane.

86. The system of claim 85, wherein the protein that is trafficked to the viral particle and/or cell surface membrane is a ligand or target binding protein.

87. The system of claim 85 or 86, wherein the protein that is trafficked to the viral particle and/or cell surface membrane is selected from the group consisting of a single chain of MHC fused with beta-2-microglobulin (B2M) and a covalently linked peptide; a single-chain antibody variable fragment fused to a transmembrane domain; and an antigen fused to a transmembrane domain.

88. A method for producing a VLP comprising a circular RNA translation system, said method comprising: providing a host cell; transfecting the host cell with the system of any one of claims 64 to 87; and culturing the host cell under conditions suitable to express the packaging vector, the envelope vector, and the circular RNA expression vector in the host cell, wherein said culturing produces virus-like particles comprising a circular RNA translation system.

89. The method of claim 88, wherein the host cell is a eukaryotic host cell.

90. The method of claim 89, wherein the host cell is a mammalian host cell.

91. The method of claim 89, wherein the host cell is a mammalian cell line, optionally wherein the mammalian cell line is selected from the group consisting of HEK293T cells, HEK293FT cells, and derivatives thereof.

92. The method of any one of claims 88 to 91 further comprising: purifying the produced virus-like particles comprising the circular RNA translation system.

93. A method of inducing an immune response against a pathogen, said method comprising: administering to a subject an effective dose of the virus-like particle (VLP) of any one of claims 1 to 40, a VLP produced using the system of any one of claims 64 to 87, or a VLP produced using the method of any one of claims 88 to 92.

94. The method of claim 93, wherein the pathogen is a viral pathogen, a prokaryotic pathogen, or a eukaryotic pathogen.

95. The method of claim 93 or claim 94, wherein the subject is a mammalian subject.

96. The method of any one of claims 93 to 95, wherein the subject is a human subject.

97. The method of claim 93 or claim 94, wherein the subject is a nonmammalian subject.

98. The method of claim 97, wherein the subject is an avian subject or an insect.

99. A method of treating a subject, said method comprising: administering the virus-like particle (VLP) of any one of claims 1 to 40, a VLP produced using the system of any one of claims 64 to 87, or a VLP produced using the method of any one of claims 88 to 92 to a subject in need thereof, wherein upon said administering, the one or more peptide(s) is/are expressed in a cell of the subject, thereby treating the subject.

100. The method of claim 99, wherein the subject is a mammalian subject, an amphibian subject, an avian subject, a fish, or a reptilian subject.

101. The method of claim 100, wherein the subject is a human subject.

102. A method of performing gene editing on a subject, said method comprising: administering the virus-like particle (VLP) of any one of claims 1 to 40, a VLP produced using the system of any one of claims 64 to 87, or a VLP produced using the method of any one of claims 88 to 92 to a subject in need thereof, wherein the one or more peptide(s) comprise one or more gene editing protein(s) and wherein, upon said administering, the gene editing protein(s) is/are expressed in a cell of the subject, thereby editing the genome of the subject.

103. The method of claim 102, wherein the one or more gene editing protein(s) comprises a Cas family protein.

104. The method of claim 103, wherein the one or more gene editing protein(s) comprises a dead Cas family protein.

105. The method of any one of claims 102 to 104, wherein the one or more gene editing protein(s) is/are fused to an additional protein.

106. The method of claim 105, wherein the additional protein is selected from the group consisting of: a reverse transcriptase, an adenosine deaminase, a cytidine deaminase, and a transposase/recombinase.

107. The method of any one of claims 103 to 106 further comprising: administering a guide RNA.

108. The method of any one of claims 102 to 107, wherein the VLP further comprises: a guide RNA packaged into the VLP using a lentiviral packaging signal (psi).

109. The method of any one of claims 102 to 108 wherein the subject is a mammalian subject.

110. The method of claim 109, wherein the subject is a human subject.

111. The method of any one of claims 102 to 108, wherein the subject is a nonmammalian subject.

112. An RNA molecule comprising: a first ribozyme; a first ligation sequence positioned 3’ to the first ribozyme; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide(s), and wherein the internal ribosomal entry site coupled to the RNA molecule encoding the one or more peptide(s) is positioned 3’ to the first ligation sequence, wherein the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof; a second ligation sequence positioned 3’ to the internal ribosomal entry site; and a second ribozyme positioned 3’ to the second ligation sequence.

113. The RNA molecule of claim 112, wherein the IRES is a wild-type internal ribosomal entry site.

114. The RNA molecule of claim 112, wherein the IRES is modified internal ribosomal entry site.

115. The RNA molecule of any one of claims 112 to 114, wherein the IRES lacks a Pol III termination element.

116. The RNA molecule of any one of claims 112 to 115, wherein a portion of the first ligation sequence is complementary to a portion of the first ribozyme and a portion of the second ligation sequence is complementary to a portion of the second ribozyme.

117. The RNA molecule of any one of claims 112 to 116, wherein a portion of the first ligation sequence is complementary to a portion of the second ligation sequence.

118. The RNA molecule of claim 117, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 18 nucleotides in length.

119. The RNA molecule of claim 117, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 26 nucleotides in length.

120. The RNA molecule of claim 117, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 49 nucleotides in length.

121. The RNA molecule of any one of claims 112 to 120, wherein each of the first ribozyme and the second ribozyme comprises a sequence that may be cleaved to produce a 5'-OH end and a 2', 3 '-cyclic phosphate end.

122. The RNA molecule of any one of claims 112 to 121, wherein each of the first ribozyme and the second ribozyme is independently selected from the group consisting of Hammerhead, Hairpin, Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Vgl, glucosamine- 6-phosphate synthase (glmS), Twister, Twister Sister, Hatchet, Pistol ribozymes, engineered synthetic ribozymes, or derivatives thereof.

123. The RNA molecule of any one of claims 112 to 122, wherein the first ribozyme is a P3 Twister ribozyme and the second ribozyme is a Pl Twister ribozyme.

124. The RNA molecule of any one of claims 112 to 123, wherein the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

125. The RNA molecule of any one of claims 112 to 124, wherein the RNA molecule further comprises: a stem loop that binds to a cognate RNA-binding protein, wherein the stem loop is positioned 3’ to the RNA molecule encoding the one or more peptide(s).

126. The RNA molecule of claim 125, wherein the stem loop is selected from the group consisting of MS2, PP7, BoxB, and Com.

127. The RNA molecule of any one of claims 112 to 126, wherein the IRES lacks a stop codon.

128. A circular RNA molecule comprising: a first ligation sequence; an internal ribosomal entry site (IRES) coupled to an RNA molecule encoding one or more peptide sequence(s), wherein the internal ribosomal entry site coupled to the RNA molecule encoding the peptide sequence(s) is positioned 3’ to the first ligation sequence, wherein the IRES sequence is selected from the group consisting of SEQ ID NOs: 1-8, or a derivative thereof; a second ligation sequence positioned 3’ to the internal ribosomal entry site coupled to the RNA molecule encoding the peptide sequence(s).

129. The circular RNA molecule of claim 128, wherein the IRES is a wild-type internal ribosomal entry site.

130. The circular RNA molecule of claim 128, wherein the IRES is modified internal ribosomal entry site.

131. The circular RNA molecule of any one of claims 128 to 130, wherein the IRES lacks a Pol III termination element.

132. The circular RNA molecule of any one of claims 128 to 131, wherein a portion of the first ligation sequence is complementary to a portion of the second ligation sequence.

133. The circular RNA molecule of claim 132, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 18 nucleotides in length.

134. The circular RNA molecule of claim 132, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 26 nucleotides in length.

135. The circular RNA molecule of claim 132, wherein the portion of the first ligation sequence complementary to the portion of the second ligation sequence is at least 49 nucleotides in length.

136. The circular RNA molecule of any one of claims 128 to 135, wherein the one or more peptide(s) is/are selected from the group consisting of an antibody; an antigen such as a cancer neoepitope and a viral antigen; an enzyme or a gene editing protein such as a Cas family protein; a reverse transcriptase; a transposase/recombinase; a transcription factor; a chemokine; a receptor such as a chimeric antigen T-cell receptor; a channel; a structural protein; a motor protein; a transport protein; a signaling protein; a cytoskeletal protein; a chaperone protein; or any combination thereof.

137. The circular RNA molecule of any one of claims 128 to 136, wherein the RNA molecule further comprises: a stem loop that binds to a cognate RNA-binding protein, wherein the stem loop is positioned 3’ to the RNA molecule encoding the one or more peptide(s).

138. The circular RNA molecule of claim 137, wherein the stem loop is selected from the group consisting of MS2, PP7, BoxB, and Com.

139. The circular RNA molecule of any one of claims 128 to 138, wherein the IRES lacks a stop codon.