Methods and systems for RNA encapsulation to increase RNA stability and translation efficiency

Abstract

The present disclosure generally relates to methods and systems for producing RNA payloads in living cells by encapsulating them in viral capsid proteins to form viral like particles (VLPs), and more specifically to engineering viral origin of assembly sequences (OAS) to increase the stability, yield and / or translational efficiency of the RNA that is extracted from those VLPs.

Description

PATENT ATTONEY DOCKET NO. SBIO1110-2 WO METHODS AND SYSTEMS FOR RNA ENCAPSULATION TO INCREASE RNA STABILITY AND TRANSLATION EFFICIENCYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U. S. C. § 119(e) to U. S. Provisional Application No: 63 / 551,012 filed on February 7, 2024; and U. S. Provisional Application No: 63 / 682,989 filed on August 14, 2024; the contents of which are herein incorporated by reference in their entirety.INCORPORATION BY REFERENCE SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing xml file, name SBIO1110-2WO, Sequence Listing ST26.xml, was created on February 4, 2025, and is 47,509 bytes.FIELD OF THE DISCLOSURE

[0003] The present disclosure generally relates to methods and systems for producing RNA payloads in living cells by encapsulating them in viral capsid proteins to form viral like particles (VLPs), and more specifically to engineering viral origin of assembly sequences (OAS) to increase the stability, yield and / or translational efficiency of the RNA that is extracted from those VLPs.BACKGROUND

[0004] The growing understanding of RNA functions and their critical role in health and disease has promoted the development of various commercial and research applications of RNA.Currently, the only method used to produce therapeutic grade mRNA is through in vitro transcription (IVT). This process requires a sequence of reactions starting with the enzymatic linearization of a plasmid DNA template, followed by purification. The template is then transcribed by recombinant T7 polymerases to generate the RNA molecule. At this stage, modified nucleotides may be added to the reaction. RNA 5 ’ capping is either introduced co-translationally or through a second reaction using a capping enzyme. During DNA template transcription, immunogenic double stranded RNA side products are produced due to abortive transcriptions by the T7 enzyme. These dsRNA contaminants reduce the efficacy of a therapeutic RNA and evoke a proinflammatory response. The IVT generation of mRNA11616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO molecules for therapeutic use therefore requires multiple, separate reactions and extensive purification steps resulting in a costly and complex GMP manufacturing process.

[0005] In addition, purified RNA obtained using in vitro transcription methods tends to be unstable and vulnerable to degradation.

[0006] Researchers have for many years studied the ways in which viruses have packaged, and thus protected, their genomic RNA. Their studies have identified stretches of viral RNA that act as “packaging signals” and cause viral genomic RNA to associate with the viral capsid protein; this encapsulation protects the viral genomic RNA from degradation in the host cell.

[0007] In filamentous viruses, such as the Tobacco Mosaic Virus (TMV), the packaging sequence is termed an Origin of Assembly Sequence (OAS), located approximately 1 kb from the 3 ’ end of its genomic RNA. The OAS sequence forms a hairpin structure capable of inserting into the disk-shaped rings of the TMV capsid protein (TMV CP) that initiates the assembly of the TMV virus like particles (VLPs) into a helical filament-capsid structure. P. J. G. Butler, Philosophical Transactions of the Royal Society of London, 354: 537-550 (1999); see also P. J. G. Butler, J. Gen. Virol., 65 (Pt 2): 253-279, (1984).

[0008] In vitro TMV capsid assembly studies, using recombinantly produced capsid monomers, have shown that the addition of nucleotides to the OAS, on either side of the minimally required 75 nucleotide stem loop, can influence VLP assembly and stability, and affect the ability of the VLP to stabilize and shield encapsulated RNA from nucleases. See PCT publication W0 2015118183. Native TMV RNA contains a single copy of the OAS. Researchers have artificially inserted two or more copies of the TMV OAS into RNA for packaging where these additions altered the morphology of the capsid. These altered morphologies deviate from the natural filamentous structure of the TMV and result in exposure of the mRNA therein to nucleases and / or RNases. Eber et al., Nanoscale, Vol. 7: 344-355 (2015); Gallic et al., Virology 158,473-476 (1987).

[0009] Accordingly, there remains a need for systems that produce therapeutic quality RNA, which would bypass the aforementioned limitations of the IVT and which can take advantage of the inherent production advantages arising from properties of virus-like particles.SUMMARY OF THE INVENTION

[0010] In a first aspect, the disclosure provides an isolated DNA sequence capable of being transcribed to an RNA sequence comprising, in a 5’ to 3’ order, a 5’-UTR, a heterologous RNA21616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO payload, and a 3’-UTR, wherein the 3’-UTR comprises two or more tobacco mosaic virus (TMV) packaging signals.

[0011] In some embodiments of the first aspect, the isolated DNA sequence is double stranded.

[0012] In some embodiments of the first aspect, the 3’-UTR of the transcribed RNA sequence comprises only two TMV packaging signals.

[0013] In some embodiments of the first aspect, every two proximal TMV packaging signals in the 3’UTR are separated by less than 500, less than 450, less than 400, less than 350, less than 300, less than 250, less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 15, less than 10, or less than 5 nucleotides. In some embodiments, two proximal TMV packaging signals may exist in tandem (i.e., no nucleotides separating the two). In some more specific embodiments of the first aspect, any two proximal TMV packaging signals in the 3’UTR are separated by less than 100 nucleotides. In some more specific embodiments of the first aspect, any two proximal TMV packaging signals in the 3’-UTR are separated by less than 50 nucleotides. In some more specific embodiments of the first aspect, any two proximal TMV packaging signals in the 3’-UTR are separated by less than 20 nucleotides. In some more specific embodiments of the first aspect, any two proximal TMV packaging signals in the 3’-UTR are separated by less than 15 nucleotides.

[0014] Each of the two or more TMV packaging signal useful herein comprise an RNA sequence consisting of the 75 nucleotide sequence set forth in SEQ ID NO: 1, which encodes the minimal TMV origin of assembly sequence identified by D. R. Turner et al., J Mol Bio 209(3), 407-422 (1989). In some embodiments of the first aspect, each of the two or more TMV packaging signal is independently selected from any one of SEQ ID NOs:l-5, and an RNA sequence comprising SEQ ID NO:1 and having at least 90% sequence identity to any one of SEQ ID NOs: 1-5. In some more specific embodiments of the first aspect, each TMV packaging signal independently is an RNA sequence of SEQ ID NO:2 or SEQ ID NO:3. In some even more specific embodiments of the first aspect, each TMV packaging signal is an RNA sequence of SEQ ID NO:2. In some even more specific embodiments of the first aspect, each TMV packaging signal is an RNA sequence of SEQ ID NO:3. In some even more specific embodiments of the first aspect, each TMV packaging signal is an RNA sequence of SEQ ID NO:4. In some even more specific embodiments of the first aspect, the 3’-UTR comprises two TMV packaging signals and each TMV packaging signal is an RNA sequence of SEQ ID NO:2.31616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO In some even more specific embodiments of the first aspect, the 3’-UTR comprises two TMV packaging signals and each TMV packaging signal is an RNA sequence of SEQ ID NO:3. In some even more specific embodiments of the first aspect, the 3’-UTR comprises two TMV packaging signals and each TMV packaging signal is an RNA sequence of SEQ ID NO:4.

[0015] In some specific embodiments of the first aspect, the 3’-UTR is derived from the naturally occurring RNA sequence corresponding to the 3’-UTR of human alpha globin (SEQ ID NO:6). In some embodiments, the 3’-UTR is an RNA sequence that comprises SEQ ID NO:6 and RNA sequences corresponding to two or more TMV packaging sequences at the 5’ and 3’ ends of and / or interspersed within SEQ ID NO:6. In some specific embodiments of the first aspect, the 3’-UTR is derived from a modification of the naturally occurring DNA sequence encoding the 3’-UTR of human alpha globin, corresponding to the RNA sequence set forth in SEQ ID NO:7. In some embodiments, the 3’-UTR is an RNA sequence that comprises SEQ ID NO:7 and RNA sequences corresponding to two or more TMV packaging sequences at the 5’ and 3’ ends of and / or interspersed within SEQ ID NO:7. In some embodiments, the 3’-UTR is an RNA sequence that comprises SEQ ID NO:7 with RNA sequences corresponding to two or more TMV packaging sequences at the 5 ’ and 3 ’ ends of and / or interspersed within SEQ ID NO:7 and further corresponding to one or more poly(A) tracts. In some even more specific embodiments, of the first aspect, the 3’-UTR is an RNA sequence that comprises any one of SEQ ID NOs:8-16 or an RNA sequence having at least 90% sequence identity to any one of SEQ ID NOs:8-16 and comprising RNA sequences corresponding to at least two copies of the minimal TMV packaging sequence of SEQ ID NO: 1. In some even more specific embodiments, of the first aspect, the 3’-UTR is an RNA sequence that comprises any one of SEQ ID NOs:8-16.

[0016] In some embodiments of the first aspect, the heterologous RNA payload encodes a polypeptide or protein. In some embodiments of the first aspect, the heterologous RNA payload encodes a mammalian polypeptide or protein. In some embodiments of the first aspect, the heterologous RNA payload encodes a therapeutically useful mammalian polypeptide or protein.

[0017] In some embodiments of the first aspect, the heterologous RNA payload is non-coding (e.g., it does not encode a polypeptide sequence).

[0018] In some embodiments of the first aspect, the 5’-UTR is an RNA sequence comprising the nucleotide sequence of SEQ ID NO: 17.41616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0019] In a second aspect, the disclosure provides an expression vector comprising the isolated DNA sequence of any embodiment of the first aspect; and a promoter operatively linked to the DNA sequence.

[0020] In some embodiments of the second aspect, the promoter in the expression vector is a constitutive promoter. In some embodiments of the second aspect, the promoter in the expression vector is a GAP promoter or a TEF1 promoter. In some more specific embodiments of the second aspect, the promoter in the expression vector is a GAP promoter having a nucleic acid sequence of SEQ ID NO:18, a TEF1 promoter having a nucleic acid sequence of SEQ ID NO: 19, or a promoter having a nucleic acid sequence of at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 19. In some more specific embodiments of the second aspect, the promoter is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18, or a TEF1 promoter having a nucleic acid sequence of SEQ ID NO: 19.

[0021] In some embodiments of the second aspect, the expression further comprises a nucleic acid sequence encoding a TMV viral capsid protein operatively linked to a promoter. In some more specific embodiments of the second aspect, the expression further comprises a nucleic acid sequence encoding a TMV viral capsid protein operatively linked to a constitutive promoter. In some more specific embodiments of the second aspect, the expression further comprises a nucleic acid sequence encoding a TMV viral capsid protein operatively linked to a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18 or a promoter having a nucleic acid sequence of at least 90% sequence identity to SEQ ID NO: 18. In some more specific embodiments of the second aspect, the expression further comprises a nucleic acid sequence encoding a TMV viral capsid protein operatively linked to a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18.

[0022] In a third aspect, the disclosure provides an isolated host cell containing the expression vector of any embodiment of the second aspect.

[0023] In a fourth aspect, the disclosure provides an isolated host cell containing: (i) a first expression vector comprising the isolated DNA sequence of any embodiment of the first aspect; and a promoter operatively linked to the DNA sequence therein; and (ii) a second expression vector comprising a nucleic acid sequence encoding a TMV viral capsid protein operatively linked to a promoter.

[0024] In some more specific embodiments of the fourth aspect, the promoter in the second expression vector is a constitutive promoter. In some more specific embodiments of the fourth51616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO aspect, the promoter in the second expression vector is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18 or a promoter having a nucleic acid sequence of at least 90% sequence identity to SEQ ID NO: 18. In some more specific embodiments of the fourth aspect, the promoter in the second promoter is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18.

[0025] In some embodiments of each of the third and fourth aspects, the host cell is a yeast cell, an insect cell, a plant cell or a mammalian cell. In some more specific embodiments of each of the third and fourth aspects, the host cell is a yeast cell selected from Yarrowia lipolytica, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia pastoris. In some more specific embodiments of each of the third and fourth aspects, the host cell is an insect cell selected from Spodoptera frugiperda and Trichoplusia ni. In some more specific embodiments of each of the third and fourth aspects, the host cell is a plant cell that is Nicotiana benthamiana. In some more specific embodiments of each of the third and fourth aspects, the host cell is a mammalian cell selected from a Chinese hamster ovary cell and a HEK293T cell. In some embodiments of each of the third and fourth aspects, the host cell is other than a Yarrowia lipolytica cell. In some embodiments of each of the third and fourth aspects, the host cell is other than a Yarrowia sp. cell.

[0026] In a fifth aspect, the disclosure provides a method of producing a heterologous RNA payload in a cell comprising growing the host cell of any of the third or fourth aspect under conditions in which virus-like particles comprising the heterologous RNA payload bound to and encapsulated by the TMV viral capsid protein are produced, thereby producing a heterologous RNA payload.

[0027] In some embodiments of the fifth aspect, the method further comprises the isolating the virus-like particles from the host cell. In some embodiments of the fifth aspect, the method comprises treating the isolated virus-like particles with one or more RNases or nucleases that digest double-stranded RNA. In some embodiments of the fifth aspect, the method comprises purifying the heterologous RNA from the isolated virus-like particles.

[0028] In a sixth aspect, the disclosure provides an isolated RNA molecule encoded by the isolated DNA sequence of the first aspect.

[0029] In a seventh aspect, the disclosure provides an isolated virus-like particle produced by the method of the fifth aspect.61616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0030] In an eighth aspect, the disclosure provides an isolated heterologous RNA molecule produced by the method of the fifth aspect. In some embodiments of the eighth aspect, the isolated heterologous RNA molecule is purified from the isolated VLPs of the seventh aspect.

[0031] In a ninth aspect, the disclosure provides an isolated RNA sequence of SEQ ID NO:7 or a DNA sequence capable of being transcribed into the RNA sequence of SEQ ID NO.:7. The RNA sequence of SEQ ID NO:7 is useful as a 3 ’-UTR in an RNA sequence encoding a protein of interest. Certain uses of the RNA sequence of SEQ ID NO:7 as a 3 ’-UTR are described in the first through eighth aspects above. The RNA sequence of SEQ ID NOU is also useful as a 3’-UTR without the insertion of two or more TMV OAS. For example, the RNA sequence of SEQ ID NOU is useful as a 3 ’-UTR in a standard in vitro transcription (IVT) system. In some embodiments of the ninth aspect, a DNA sequence capable of being transcribed into the RNA sequence of SEQ ID NO.:7 is part of a DNA template comprising, in 5’-to-3’ order, a DNA sequence capable of being transcribed into a 5 ’-UTR (e.g., SEQ ID NO: 17), a DNA sequence encoding a protein of interest (e.g., luciferase, GFP, a COVID-19 spike protein, another therapeutically useful protein); and a DNA sequence capable of being transcribed into SEQ ID NOU. Such a template might also comprise a promoter (e.g., a T7 promoter) and / or a sequence capable of being transcribed into an appropriate mRNA capping signal (e.g., a Cap-1 cap) at the 5 ’-end and / or a sequence capable of being transcribed into a polyA tail at the 3 ’-end.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Various objectives, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.

[0033] FIG. 1 depicts a plasmid map of TMV Capsid, an expression vector used to express TMV capsid protein in S. cerevisiae.

[0034] FIG. 2 depicts a plasmid map of GFP UTR-4, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO:17, an EGFP coding sequence, and a 3’-UTR of SEQ ID NO:8.

[0035] FIG. 3 depicts a plasmid map of GFP UTR-3, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO:17, an EGFP coding sequence, and a 3’-UTR of SEQ ID NO:23.71616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0036] FIG. 4 depicts a plasmid map of GFP UTR-1, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO: 17, a firefly luciferase coding sequence, and a 3’-UTR of SEQ ID NO:22.

[0037] FIG. 5 depicts a plasmid map of Luc UTR-1, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO: 17, a firefly luciferase coding sequence, and a 3’-UTR of SEQ ID NO:22.

[0038] FIG. 6 depicts a plasmid map of Luc UTR-3, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO: 17, a firefly luciferase coding sequence, and a 3’-UTR of SEQ ID NO:23.

[0039] FIG. 7 depicts a plasmid map of Luc UTR-4, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO: 17, a firefly luciferase coding sequence, and a 3’-UTR of SEQ ID NO:8.

[0040] FIG. 8 depicts a denaturing SDS-PAGE gel from the crude lysate of Tni cells transfected with a recombinant baculovirus encoding both the TMV capsid protein and an RNA sequence comprising, in 5’-to-3’ order, the 5’-UTR of SEQ ID NO:17, a mRNA encoding EGFP, and one of UTR-1 (SEQ ID NO:22), UTR-3 (SEQ ID NO:23), or UTR-4 (SEQ ID NO:8). The arrow on the right side of the depicted gel indicated the expected target size (17 kDa) for the TMV capsid protein. Molecular weight markers are shown in the leftmost lane and arrows and numbers show the approximate size of the marker.

[0041] FIGS. 9A-9I panels depict transmission electron microscopy images of TMV VLPs from transformed or transfected cells. Panel A depicts TMV VLPs containing wild-type, native TMV RNA from a S. cerevisiae expression extract. Panels B-D depict TMV VLPs containing UTR-4, which comprises two TMV packaging signals, as the 3’-UTR in Sf9 insect cells (Panel B), Tni insect cells (Panel C) and S. cerevisiae yeast cells (Panel D). Panels E and F depict TMV VLPs containing UTR-1 as the 3’-UTR in Sf9 insect cells (Panel E) or Tni insect cells (Panel F).Panels G-I depict TMV VLPs containing UTR-3 as the 3’-UTR in Sf9 insect cells (Panel G), Tni insect cells (Panel H), and S. cerevisiae yeast cells (Panel I). All images were collected with a Jeol JEM-1400Flash transmission electron microscopy.

[0042] FIG. 10 depicts a denaturing SDS-PAGE gel of the supernatant of clarified yeast lysate from yeast cells transfected with the TMV Capsid and the GFP UTR-4 plasmids both before (“s”) and after PEG precipitation (“PEGs” represents the supernatant following PEG precipitation; “PEGp” represents the pellet following PEG precipitation). TMV capsid protein is81616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO apparent at 17 kDa in both the pre-PEG treated cell lysate supernatant and the PEG pellet, but not in the PEG supernatant. The lane designated as “M” contains molecular weight markers, the size of which is indicated to the left of the gel.

[0043] FIG. 11 depicts the result of CsCl density-based ultracentrifugation of resuspended pellets from PEG precipitated, clarified lysate from yeast cells transfected with the TMV Capsid and the GFP UTR-4 plasmids. The arrow shows the cloudy band representing the VLPs.

[0044] FIG. 12 depicts a denaturing SDS-PAGE gel of various fractions from the CsCl densitybased ultracentrifugation. The arrow shows the band at about 17.5 kDa representing the TMV capsid protein. The lane designated as “M” contains molecular weight markers, the size of which is indicated to the left of the gel.

[0045] FIGS. 13A-13B panels depict agarose gel analysis of TMV VLPs produced from yeast transfected with the TMV Capsid and either the GFP UTR-3 (panel A) or the GFP UTR-4 (panel B) plasmids before (No Benz) and after treatment with benzonase. Molecular weight markers are shown in the left land of each gel. Arrows denote the band corresponding to the RNA of expected size encoded by GFP UTR-3 or GFP UTR-4.

[0046] FIGS. 14A-14B are bar graphs depicting THP-1 Dual cell results from transfections with RNA isolated from TMV VLPs produced in yeast transfected with the plasmids TMV Capsid and either GFP UTR-1, GFP UTR-3 or GFP UTR-4. The THP-1 Dual cells were differentiated into macrophage-like cells, using standard phorbol 12-myristate 13-acetate treatment, before transfection with the RNAs. Panel A depicts the level of eGFP expression expressed in relative fluorescence units (RFU) in THP-1 Dual cells transfected with RNA isolated each of GFP UTR-1, GFP UTR-3 or GFP UTR-4 transfected yeast. Panel B depicts the immunogenic effect of RNA isolated from VLPs purified from two different batches of GFP UTR-4 transfected yeast on THP-1 Dual cells as measured by interferon pathway-stimulated Lucia enzyme expression (RLU Lucia) as compared to a poly(dA:dT) control. The chemical transfection reagent Lipofectamine™ was used for all samples and is also included as lipofectamine-only control due to the background immune stimulation known to result from transfection agents.

[0047] FIGS. 15A-15C panels, are bar graphs depicting the amount of luciferase produced from in vitro transcribed RNA from a DNA template comprising either a native alpha-globin 3’-UTR (SEQ ID NO:6) or a variant thereof (SEQ ID NOU) transfected into THP-1 (panel A), HepG2 (panel B), or HEK293 (panel C) cells.91616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO

[0048] FIG. 16 is a bar graph depicting the amount of luciferase produced in THP-1 Dual cells transfected with RNA isolated from TMV VLPs produced in Yarrowia lipolytica cells transfected with the plasmid YL-TMV Capsid and a plasmid encoding either an RNA having a single TMV OAS located immediately 3 ’ to the luciferase coding sequence (Luc OAS) or two TMV OAS sequences located in the middle of the 3’-UTR and spaced apart 13 nucleotides (Luc UTR-4).

[0049] FIG. 17 depicts a map of plasmid YL-Luc OAS, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO: 17, a firefly luciferase coding sequence, and a 3’-UTR of SEQ ID NO:29 in Yarrowia lipolytica cells.

[0050] FIG. 18 depicts a map of plasmid YL-Luc UTR-4, an expression vector used to express an RNA molecule comprising a 5’-UTR of SEQ ID NO:17, a firefly luciferase coding sequence, and a 3’-UTR of SEQ ID NO:8 in Yarrowia lipolytica cells.

[0051] FIG. 19 depicts a map of plasmid YL-TMV-Capsid, an expression vector used to express TMV capsid protein in Y. lipolytica.DETAILED DESCRIPTION

[0052] The details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features and advantages of the subject matter described herein will be apparent from the description and drawings, and from the claims. The disclosed subject matter is not, however, limited to any particular embodiment disclosed.

[0053] Definitions

[0054] The term “DNA sequence capable of being transcribed to a RNA sequence” as used herein means a polydeoxyribonucleotide that serves as a template for transcription when contacted with an RNA polymerase into an RNA sequence. Such RNA polymerase may be added to the DNA sequence in vitro such as in in vitro translation or may be present in a host cell that harbors such DNA sequence. The RNA polymerase that performs such transcription and is present in a host cell may be naturally encoded by the host cell or may be recombinantly produced in a host cell that harbors such DNA sequence.

[0055] The term “5’-UTR” or “5 ’-untranslated region” as used herein means a polyribonucleotide sequence that is not translated into an amino acid sequence and is located 5’ or upstream from the beginning of an RNA sequence that is the heterologous RNA payload. In101616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO embodiments when the heterologous RNA payload encodes a polypeptide, the beginning of a functional RNA sequence is a start codon and the 5’-UTR ends at the nucleotide preceding that start codon. The 5’UTR typically comprises secondary and tertiary structures and other sequence elements that regulate the stability of the transcribed RNA and, in some embodiments, the translatability of the heterologous RNA payload.

[0056] The term “3’-UTR” or “3 ’-untranslated region” as used herein means a polyribonucleotide sequence that is not translated into an amino acid sequence and is located 3’ or downstream from the end of an RNA sequence that is the heterologous RNA payload. In some embodiments when the heterologous RNA payload encodes a polypeptide, the end of a functional RNA sequence is a termination codon and the 3’-UTR begins at the nucleotide immediately following that termination codon. The 3’-UTR typically comprises secondary and tertiary structures and other sequence elements that can impact the stability the transcribed RNA molecule and, in some embodiments, the translatability of the heterologous RNA payload. In some, but not all, embodiments, the 3’-UTR comprises a polyadenylation tail.

[0057] The term “heterologous RNA payload” as used herein means an RNA molecule that is not found naturally in Tobacco Mosaic Virus and that has activity at the RNA level itself (e.g., without being translated into a protein), or by encoding upon translation a polypeptide (e.g., as a mRNA). The heterologous mRNA payloads disclosed herein are intended for therapeutic, prophylactic, diagnostic or research use in a cell, cell lysate, in vitro translation system, a tissue, or an organism.

[0058] The term “TMV packaging signal” as used herein means an RNA sequence that can bind to a Tobacco Mosaic Virus capsid protein.

[0059] The term “proximal TMV packaging signals” as used herein refers to a pair of TMV packaging signals that are closest to one another in an RNA molecule. For clarity, an RNA molecule comprising three TMV packaging signals, designated as “1”, “2” and “3” in the structure: 1-2-3, wherein every two proximal TMV packaging signals are separated by less than XX nucleotides (wherein XX is an integer), means that “1” and “2” are separated from one another by less than XX nucleotides and that “2” and “3” are separated from one another by less than XX nucleotides. It does not mean or imply that “1” and “3” are separated from one another by less than XX nucleotides.

[0060] As used herein, the term “operably linked” refers to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of111616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polydeoxyribonucleotide molecule if the promoter modulates transcription of the transcribable polydeoxyribonucleotide molecule payload in a cell. Additionally, two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.

[0061] As used herein, the term "promoter" refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the DNA to which such promoter is operably linked. The term “constitutive promoter” refers to a promoter whose ability to bind an RNA polymerase is not significantly affected by environmental or other factors.

[0062] “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0063] 100 multiplied by (the fraction X / Y)

[0064] where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where121616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0065] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, cosmid, or artificial chromosome, an RNA vector, a virus, or any other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are described in, e.g., Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use herein contain a poly deoxyribonucleotide sequence that can be transcribed into RNA, as well as, e.g., additional sequence elements used for such transcription. Certain vectors that can be used for the transcription of RNA as described herein include vectors that contain regulatory sequences, such as promoter and enhancer regions, which direct transcription. The expression vectors suitable for use herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0066] Overview

[0067] The present disclosure provides isolated DNA sequences, expression vectors, host cells and methods useful for generating RNA payloads in living cells and the resulting RNA payloads. According to the methods described herein, the RNA payloads are prepared as viral-like particles (VLPs) containing the RNA payloads. VLPs are composed of viral capsid proteins that self-assemble, mimicking the structure of the native viruses from which they are derived. VLPs are non-infectious as they lack genetic material required for integration, reverse transcription, replication and / or infection. Isolated VLPs comprising RNA payloads are also provided herein.

[0068] DNA and RNA Sequences

[0069] In one aspect, the disclosure provides: (i) an isolated DNA sequence capable of being transcribed to an RNA molecule comprising, in a 5’ to 3’ order, a 5’-UTR, a heterologous RNA payload, and a 3’-UTR, wherein the 3’-UTR comprises two or more tobacco mosaic virus (TMV) packaging signals; and (ii) the transcribed RNA molecule. This isolated DNA sequence131616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO is designed to be transcribed into a single RNA transcript (i.e., in a host cell transfected or transformed with or harboring such DNA sequence). That RNA transcript is capable of being packaged into a TMV virus-like particle when contacted with TMV capsid protein due to the interaction of the TMV packaging signals with the capsid protein. Moreover, applicants believe that in some embodiments, the presence of two or more TMV packaging signals results in unexpected improvements over similar transcribed RNA sequences comprising only one TMV packaging signal. Such improvements include one or more of: improved quantity of TMV VLPs, improved quantity of RNA recovered from VLPs, and improved quality of RNA recovered from VLPs in terms of stability and translatability into protein.

[0070] 5 ’-UTR

[0071] As set forth above, the RNA transcripts disclosed herein comprise a 5’-UTR. The 5’-UTR sequences disclosed herein are a region of the transcribed RNA that is not translated into an amino acid sequence, nor necessarily directly involved in the activity of the heterologous RNA payload, but include regulatory elements that modulate the stability of the transcribed RNA and its translation (e.g., if the RNA payload encodes a polypeptide). The 5’ UTR typically begins at the transcription start site and continues to just before the beginning of the functional RNA sequence (e.g., the start codon when the heterologous RNA payload encodes a polypeptide).

[0072] Naturally occurring RNA transcripts typically comprise a 5 ’-UTR. In some embodiments, the 5’ UTR comprises a nucleic acid sequence that is naturally present in the transcript of the encoded heterologous RNA payload. In some embodiments, the 5’ UTR comprises a nucleic acid sequence that is not naturally associated with the encoded heterologous mRNA payload. In some embodiments, the 5 ’ UTR comprises a nucleic acid sequence that has high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to the nucleic acid sequence that is naturally present in the transcript of the encoded heterologous RNA payload, wherein those non-identical nucleotides impart a quantitative or qualitative improvement to the transcript (e.g., in terms of stability, function and / or translatability).Modifications to naturally occurring 5’-UTRs that result in such improvements are known in the art.

[0073] As a non-limiting example, the 5 ’-UTR can be a human 5 ’-UTR and the heterologous RNA payload can be a human RNA payload. In some embodiments, the 5 ’ UTR can be heterologous to the cell used to produce the VLPs. For example, the 5 ’-UTR and the RNA141616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO payload can be human whereas the cell used to produce the VLP comprising such RNA payload is a non-human cell e.g., a mouse cell, a rat cell, a hamster cell, an insect cell, a yeast cell, a bacterial cell, or a plant cell.

[0074] The 5’ UTR can have a sequence or structure that alters the level of translation (e.g., increases, decreases, or prevents) of the RNA payload into a polypeptide. In some embodiments, the 5 ’-UTR has a sequence or structure that promotes a cell type-specific translation of the RNA payload.

[0075] The 5’ UTR can include one or more ribosome binding sequences. As used herein, the term “ribosome binding sequence” refers to a nucleic acid sequence that is recognized by the ribosome or a self-cleaving ribozyme, and upon binding to the sequence, can initiate translation of the RNA payload into a polypeptide. In some embodiments, a ribosome binding sequence can be a Kozak sequence. In some embodiments, the 5’ UTR includes permuted introns to mediate RNA self-circularization.

[0076] The 5 ’-UTR sequences disclosed herein may differ from the resulting 5 ’-UTR in the transcribed RNA by the 5’ extension and / or modification of the encoded 5 ’-UTR. In some embodiments, when the DNA sequence encoding the 5 ’-UTR disclosed herein is placed into an expression vector for ultimate transcription in a host cell, the expression vector itself may template additional ribonucleotides that are added to the 5’-end of the encoded 5’-UTR. In some embodiments, the 5 ’-UTR may be capped. Capping refers to a modification of the first (5 ’-most) transcribed nucleotide of an RNA transcript by enzymes present in the cell producing such transcript. Capping typically introduces a methylated guanosine onto that first transcribed nucleotide. Capping blocks 5 '-3' exonuclease-mediated degradation and recruits specific RNA processing, export and translation factors. The presence of a cap and the specific modification of the cap is dependent upon the cell in which the isolated DNA sequences disclosed herein are transcribed. In some embodiments, the cap can be a 7-methylguanosine (m7G) cap. In some embodiments the 2’ ribose position of the first cap-proximal nucleotide of the 5 ’-UTR is methylated to form a Cap 1 structure. In some embodiments, the 5 ’-UTR includes a methylation in the N6-position of adenosine (also herein N6-methyladenosine or m6A). In some embodiments, the 2’ ribose position of the second cap-proximal nucleotide of the 5 ’-UTR is methylated to form a Cap 2 structure. Capping of the 5 ’-UTR in the population of cells transcribing the isolated DNA sequences disclosed herein (e.g., the cells producing the VLPs) can be achieved by native enzymes in such population of cells and / or by concurrent recombinant151616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO expression of capping enzymes or polynucleotides encoding capping enzymes e.g., a 2’0 methyl-transferase enzyme. Modification of the 5’-UTR cap may also be achieved after the RNA is isolated (i.e., from VLPs) through treatment with isolated methyl transferases.

[0077] In some embodiments, the 5’-UTR is an RNA sequence comprising the following nucleotide sequence:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC (SEQ ID NO: 17).

[0078] 3'-UTR

[0079] As set forth above, the RNA transcripts disclosed herein comprise a 3’-UTR. The 3’-UTR is a region of the resulting transcribed RNA that is not translated into an amino acid sequence, nor necessarily directly involved in the activity of the heterologous RNA payload but includes regulatory elements that modulate the stability of the transcribed RNA and its translation (e.g., if the RNA payload encodes a polypeptide). The 3’ UTR typically begins immediately after the sequence encoding the heterologous RNA payload (e.g., the termination codon when the heterologous RNA payload encodes a polypeptide) and continues to the end of the transcribed RNA.

[0080] Naturally occurring RNA transcripts typically comprise a 3 ’-UTR. In some embodiments, the 3’ UTR disclosed herein comprises a nucleic acid sequence that is naturally present in the transcript of the encoded heterologous RNA payload. In some embodiments, the 3 ’ UTR comprises a nucleic acid sequence that is not naturally associated with the encoded heterologous mRNA payload. In some embodiments, the 3 ’ UTR comprises a nucleic acid sequence that has high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to the nucleic acid sequence that is naturally present in the transcript of the encoded heterologous RNA payload, wherein those non-identical nucleotides impart a quantitative or qualitative improvement to the transcript (e.g., in terms of stability, function and / or translatability). Modifications to naturally occurring 3’-UTRs that result in such improvements are known in the art.

[0081] In some embodiments, a 3 ’-UTR includes sequence elements mediating binding to proteins involved in mRNA trafficking, and / or translation. In some embodiments, a 3 ’-UTR can include sequences repressing the deadenylation of mRNA. In some embodiments, a 3 ’-UTR can include a polyadenylation signal sequence. In some embodiments, a 3 ’-UTR can include a polyadenylation tail. In some embodiments, a 3 ’-UTR can include a nucleotide sequence repressing the deadenylation of mRNA. As a non-limiting example, a 3 ’-UTR can include one 161616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO or more regions of, or the entire 3'-UTR of human a-globin, which corresponds to the DNA sequence:GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCC UCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(SEQ ID NO:6)or a variant thereof:GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCC UCCCCUUCCUGCACCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCGUACC CCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:7)with the two or more TMV packaging signals located at one or both ends and / or interspersed within such 3’-UTR and optionally containing one or more poly(A) tracts of varying lengths. It will be understood that in creating DNA encoding TMV packaging signal-containing 3’-UTRs, especially where the TMV packaging signal is inserted within the parent 3’-UTR (e.g., the 3’-UTR of SEQ ID NO:6 or SEQ ID NO:7), cloning artifacts may be introduced where one nucleotide of the parent 3’-UTR is deleted or shifted in location, or where one additional nucleotide is inserted the parent 3’-UTR. For example, in SEQ ID NO:8, below, the G nucleotide located 5 ’ to the first TMV packaging sequence is shifted compared to the native a-globin 3’-UTR of SEQ ID NO:6, from which it is derived. The location of that G in SEQ ID NO:6 is indicated by italics and bolding below. In SEQ ID NO:8 that G is shifted as shown by the arrow to the gap indicated with an asterisk below:I I GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCC*CCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ. ID NO:6)Applicant believes that this one nucleotide difference has minimal effect on 3’-UTR function.

[0082] In some embodiments, the 3’-UTR is an RNA sequence comprising any of the following nucleotide sequences in Table 1. In Table 1, bolded nucleotides are the portions of the RNA sequence that correspond to the TMV packaging signals.

[0083] Table 1. 3 ’-UTR Sequences171616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO SEQ. ID NO: RNA Sequence8 GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCGU UGUUUAUAGAAAUAAUAUAAAAUUAGGUUUGAGAGAGAAGAUUACAAACGUGAGAG ACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCC CUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAGAGUGAUGUCC GCAAAGGGAAAAAUACCUUCCUGCACCCUUGUUUAUAGAAAUAAUAUAAAAUUAGGU UUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAA GUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGA UCUCGAACCGGAAAAAAGAGUGAUGUCCGCAAAGGGAAAAAUAUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAA9 GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCGG UUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGA AGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCG AUCUCGAACCGGCCUUCCUGCACCCGUUUGAGAGAGAAGAUUACAAACGUGAGAGACG GAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUA UGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGUACCCCCGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGCAAAAAAAAA10 GUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAG AAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUC GAUCUCGAACCGGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCG UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG UUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGA AGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCG AUCUCGAACCGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA11 GUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAG AAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUC GAUCUCGAACCGGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCG UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGUUUGAGAGAGAAGAUUACA AACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUG GAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGGCUGGAGC CUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCA CCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCGUACCCCCGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA181616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO SEQ. ID NO: RNA Sequence12 GUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAG AAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUC GAUCUCGAACCGGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCG UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGUUUGAGAGAGAAGAUUACA AACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUG GAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGG13 GUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAG AAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUC GAUCUCGAACCGGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCG UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCGUUUGAGAGAGAAGAUUACA AACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUG GAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAA14 UUGUUUAUAGAAAUAAUAUAAAAUUAGGUUUGAGAGAGAAGAUUACAAACGUGAGA GACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUC CCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAGAGUGAUGUCC GCAAAGGGAAAAAUACCUUCCUGCACCCUUGUUUAUAGAAAUAAUAUAAAAUUAGGU UUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAA GUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGA UCUCGAACCGGAAAAAAGAGUGAUGUCCGCAAAGGGAAAAAUAGCUGGAGCCUCGGUG GCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGAUGG GGGCACAGAAACUUAUUUCAGACUCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC15 GUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAG AAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUC GAUCUCGAACCGGCCUUCCUGCACCCGUUUGAGAGAGAAGAUUACAAACGUGAGAGAC GGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCU AUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGGCUGGAGCCUCGGUGGCCUA GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGAUGGGGGC ACAGAAACUUAUUUCAGACUCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGC16 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG U U UG AG AG AG AAG AU U AC AAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAU GGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGCCUUCCU GCACCCGUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUAC AGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAA GUUUCGAUCUCGAACCGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA191616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO

[0084] Heterologous RNA Payload

[0085] As set forth above, the transcribed RNA sequences disclosed herein comprise a heterologous RNA payload.

[0086] RNA payload can be of any length, for example at least 9 nucleotides in length. For example, the RNA payload length can be at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, or 50000 nucleotides in length. In some embodiments, the RNA payload can be fewer than 50000, 45000, 40000, 35000, 30000, 25000, 20000, 15000, 10000, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 nucleotides in length. In some embodiments, the RNA payload can be 10-20, 15-25, 20-30, 25-35, 30 - 35, 35 - 40, 40 - 45, 45 - 50, 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75, 75 - 80, 80 - 85, 85 - 90, 90 - 95, 95 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 350, 350 - 400, 400 - 450, 450 - 500, 500 - 550, 550 - 600, 600 - 650, 650 - 700, 700 - 750, 750 - 800, 800 - 850, 850 - 900, 900 - 950, 950 - 1000, 1000 - 1500, 1500 - 2000, 2000 - 2500, 2500 - 3000, 3000 - 3500, 3500 - 4000, 4000 - 4500, 4500 - 5000, 5000 - 10000, 10000 - 15000, 15000 - 20000, 20000 - 25000, 25000 - 30000, 30000 - 35000, 35000 - 40000, 40000 - 45000, or 45000 - 50000 nucleotides in length.

[0087] The RNA payload can be derived from a naturally occurring RNA or can be a synthetic or chimeric RNA.

[0088] The RNA payload can be a eukaryotic RNA payload or a prokaryotic RNA payload. In some embodiments, the eukaryotic RNA payload can be a mammalian RNA payload. As a nonlimiting example, the mammalian RNA payload can be a human RNA payload. In some embodiments, the RNA payloads can be homologous to the population of cells used to produce the VLPs containing the RNA payload. In some embodiments, the RNA payloads can be heterologous to the population of cells used to produce the VLPs containing the RNA payloads. As used herein, “homologous” refers to two entities that are derived from the same species, whereas “heterologous,” refers to two entities that are derived from different species. For example, the RNA payload can be a human RNA payload and the population of cells can be a human population of cells (e.g., a human-derived cell line). As another example, the RNA payload can be a human RNA payload and the population of cells can be a hamster population of cells (e.g., a hamster-derived cell line, e.g., CHO cells).201616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO

[0089] In some embodiments, the RNA payload can be self-amplifying RNA payload, which can include a sequence of nucleotides coding for nonstructural proteins. The nonstructural proteins can be, for example, nsPl, nsP2, nsP3 and / or nsP4 of alphaviruses.

[0090] Coding RNA payload

[0091] The RNA payload can be a coding RNA payload. As used herein a “coding RNA payload” refers to an RNA payload that is capable of being translated to an encoded protein, e.g., in vitro, in vivo, in situ, or ex vivo. A coding RNA payload will begin with a start codon and end with a stop codon sequence. In some embodiments, the coding RNA payload includes introns. Introns can aid in nuclear export of the ORF payload. In one embodiment, the ORF payload includes permuted introns for production of circular RNA.

[0092] The coding RNA payload can be of any length. In some embodiments, the coding RNA payload can be at least 30 nucleotides in length. In some embodiments, the coding RNA payload can be at least 300 nucleotides in length. In some embodiments, the coding RNA payload can be at least 4500 nucleotides in length. For example, the coding RNA payload length can be at least 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, or 50000 nucleotides in length. In some embodiments, the coding RNA payload can be fewer than 50000, 45000, 40000, 35000, 30000, 25000, 20000, 15000, 10000, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, or 30 nucleotides in length. In some embodiments, the coding RNA payload can be 30 - 35, 35 - 40, 40 - 45, 45 - 50, 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75, 75 - 80, 80 - 85, 85 - 90, 90 -95, 95 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 350, 350 - 400, 400 - 450, 450 -500, 500 - 550, 550 - 600, 600 - 650, 650 - 700, 700 - 750, 750 - 800, 800 - 850, 850 - 900, 900 -950, 950 - 1000, 1000 - 1500, 1500 - 2000, 2000 - 2500, 2500 - 3000, 3000 - 3500, 3500 - 4000, 4000 - 4500, 4500 - 5000, 5000 - 10000, 10000 - 15000, 15000 - 20000, 20000 - 25000, 25000 -30000, 30000 - 35000, 35000 - 40000, 40000 - 45000, or 45000 - 50000 nucleotides in length.

[0093] In some embodiments, the coding RNA payload is not translated or minimally translated to a polypeptide or a protein sequence in a population of cells that are used to prepare VLPs containing the coding RNA payload.

[0094] A coding RNA payload can encode, for example, a bacterial protein or polypeptide, a genome editing enzyme (e.g., Cas9, transposase, meganuclease, Base Editor or retrotransposon), a prime editor, a DNA writer, a virus protein or polypeptide, a eukaryotic transcription factor211616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO (e.g., a mammalian transcription factor), a growth factor, an antibody, a polypeptide containing one or more viral or a bacterial epitopes or antigens, an enzyme, or a therapeutically useful mammalian polypeptide (e.g., a therapeutically useful human polypeptide). In some embodiments, the coding RNA payload encodes a neo-antigen. In some embodiments, the neoantigen is a mammalian cancer neo-antigen. In some embodiments, the coding RNA payload encodes a therapeutically useful human polypeptide. In some embodiments, the coding RNA payload encodes a therapeutically useful human enzyme. In some embodiments, the coding RNA payload encodes a therapeutically useful human enzyme that can be used to treat a disease or condition characterized by reduced activity of the native form of such enzyme.

[0095] In some embodiments, a coding RNA payload can encode a polypeptide useful in immunotherapy. In some embodiments, the polypeptide can be a chimeric antigen receptor (CAR), such as an anti c-Met CAR or an anti CD 19 CAR. In some embodiments, the coding RNA payload can encode an immunomodulatory protein, such as a cytokine, or a costimulatory molecules polypeptide. In some embodiments, the coding RNA payload can encode a polypeptide that serves as a vaccine for immunotherapy. For example, the polypeptide can be a full-length or a portion of a protein such as, p53, survivin, gplOO, tyrosinase, mTRP-2, MAGE-A3, MAGE-C2, PSCA, PSMA, PSA, STEAP1, NY-ESO-1, 5T4, MAGE-CI, telomerase, WT1, hTERT, and / or pp65.

[0096] In some embodiments, a coding RNA payload can encode a protein, or a polypeptide for protein replacement in a genetic disorder, a lung disease, a hematological disease, an orthopedic disease, a neurogenic disease, a metabolic disorder, and / or a cardiovascular disease. A coding RNA payload can code for a methyl malonyl-CoA mutase, propionyl-CoA carboxylase, ornithine transcarbamylase, alpha 1 -antitrypsin, CFTR, Factor VIII, Factor IX, VEGFA, and / or a region or a portion thereof.

[0097] In some embodiments, the coding RNA payload can encode a gene editing nuclease, e.g., zinc finger nuclease, transcription activator-like effector nuclease, a Base Editor, a prime editor a DNA writer, and / or the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein nuclease system.

[0098] In some embodiments, the coding RNA payload can encode a polypeptide related to the treatment of an infectious disease. A coding RNA payload can encode a vaccine for infectious diseases. For example, a coding RNA payload can encode a polypeptide useful as a COVID-19 vaccine. Such polypeptide can contain the entire protein or a portion thereof such as, but not221616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO limited to, S-2P (and variants thereof), receptor binding domain, trimerized receptor binding domain, and / or transmembrane spike (S) protein. In some embodiments, a coding RNA payload can encode a rabies vaccine comprising Rabies virus glycoprotein. In some embodiments, a coding RNA payload can encode an influenza vaccine comprising the Hemagglutinin glycoprotein or a portion thereof. In some embodiments, a coding RNA payload can encode a HIV vaccine comprising one or more of HIV Gag, Nef, Vpr, Rev proteins or a portion thereof. In some embodiments, a coding RNA payload can encode an anti-Chikungunya antibody. As a non-limiting example, a coding RNA payload can be the SARS-CoV-2 spike protein or a region or a portion thereof.

[0099] In some embodiments, RNA payload can encode a bacterial antigen.

[0100] In some embodiments, the RNA payload can encode an immunoglobulin (IgG) for use in passive immunotherapy.

[0101] In some embodiments, the sequence of coding RNA payloads can be optimized to promote the translation of the coding RNA payload in a cell, population of cells, or organism which is intended to be administered the RNA once that RNA is isolated from the cells used for VLP production. In some embodiments, the sequence of coding RNA payloads can be optimized to increase the half-life of the RNA payload. In one embodiment, the coding RNA payload is codon optimized or uridine depleted to reduce immunogenicity in a cell, population of cells, or organism which is intended to be administered the RNA.

[0102] Non-coding RNA payload

[0103] RNA payloads useful herein can be non-coding RNA payloads. RNA molecules that are not translated to an encoded peptide, polypeptide, or protein are referred to herein as non-coding RNAs. The non-coding RNA payloads can be short non-coding RNA payload or long noncoding RNA payload. The short non-coding RNA payload can be 200, 150, 100, 75, 50, 25 nucleotides or less in length. Non-limiting examples of short non-coding RNA include, micro-RNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small Cajal body-specific RNAs (scaRNAs), trans-activating CRISPR (tracr) RNA, small nuclear RNA (snRNA), U-rich snRNA (snRNA), small nucleolar RNA (snoRNA), and / or Y RNAs.

[0104] The long non-coding RNAs can be greater than 200 nucleotides in length (e.g., about 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or more). Non-limiting examples of long non-coding RNA include, long intergenic ncRNA (lincRNA), natural antisense transcript (NAT) RNA, and / or circular RNA (circRNA). In one231616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO embodiment, the RNA payload is RNA with permuted introns to circularize the RNA to form a circRNA.

[0105] The non-coding RNAs include, but are not limited to, transfer RNAs (tRNA), ribosomal RNAs (rRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), signal recognition particle RNA (SRP RNA), antisense RNA (aRNA), micro-RNA (miRNA), small interfering RNA (siRNA), Y RNA, telomerase RNA, and / or piwi-interacting RNA (piRNA), RNAs encoding RNA-dependent RNA polymerase complexes for self-amplification.

[0106] The RNA payload can be a single stranded RNA payload or a double stranded RNA payload. In one aspect, the RNA payload is single stranded. Coding RNA payloads are typically single stranded.

[0107] TMV Packaging Signal

[0108] As set forth above, the transcribed RNA sequences disclosed herein comprise two or more tobacco mosaic virus (TMV) packaging signals present in the 3’-UTR. In TMV, the packaging signal is often referred to in the prior art as the origin of assembly sequence, or OAS. The role of the TMV packaging signals in the transcribed RNA molecules herein is to bind to TMV capsid protein expressed in the same cell as the transcribed RNA, which allows that RNA to be encapsidated by TMV VLPs produced in such cells from the TMV capsid protein. It is believed that the packaging signals may also play a role in VLP assembly and formation.

[0109] Each of the two or more TMV packaging signals useful herein may independently range in size from 75 to about 500 nucleotides in length, 75 to 300 nucleotides 75-250 nucleotides, 100-250 nucleotides, or 100-200 nucleotides in length and each will comprise the nucleotides corresponding to the following RNA sequence:UGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGG AAGAUGUCCCUAUGUCGAUCA (SEQ ID NO:1).

[0110] SEQ ID NO:1 is a 75 ribonucleotide sequence recognized as the minimal sequence necessary to bind to TMV coat protein and allow assembly of VLPs. In some embodiments, the sequence of the TMV packaging has 100% sequence identity to a sequence present in naturally occurring TMV. In some embodiments, each of the two or more TMV packaging signals independently corresponds to one of SEQ ID NOs:l-5 set forth in Table 2, below, or a nucleotide sequence comprising SEQ ID NO:1 and having at least 90% sequence identity to any one of SEQ ID NOs:l-5.241616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0111] Table 2.SEQ. ID NO. RNA Sequence1 UGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAG AUGUCCCUAUGUCGAUCA2 GUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAG AAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUC GAUCUCGAACCGG3 UUGUUUAUAGAAAUAAUAUAAAAUUAGGUUUGAGAGAGAAGAUUACAAACGUGAGA GACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUC CCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAGAGUGAUGUC CGCAAAGGGAAAAAUA4 AUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAG UUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUU5 GCAAGUUUUAGUUAAUAUUAGAAAUGUGAAGAUGUCAGCGGGUUUCUGUCCGCUUU CUCUGGAGUUUGUGUCGGUGUGUAUUGUUUAUAGAAAUAAUAUAAAAUUAGGUUUG AGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUC GUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCU CGAACCGG

[0112] In some embodiments, each of the two or more TMV packaging signals independently is one of SEQ ID NOs:l-5. In some embodiments, each of the two or more TMV packaging signals is the same and is one of SEQ ID NOs: 1-5. In some embodiments, each of the two or more TMV packaging signals is the same and is SEQ ID NO:1. In some embodiments, each of the two or more TMV packaging signals is SEQ ID NO:2. In some embodiments, each of the two or more TMV packaging signals is SEQ ID NO:3. In some embodiments, each of the two or more TMV packaging signals is SEQ ID NO:4. In some embodiments, each of the two or more TMV packaging signals is SEQ ID NO:5. In some embodiments the RNA contains only two TMV packaging signals.

[0113] As set forth above, in some embodiments, every two proximal TMV packaging signals in the 3’-UTR are separated by less than 500, less than 450, less than 400, less than 350, less than 300, less than 250, less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 15, less than 10, or less than 5 nucleotides. It will be readily apparent that the maximum distance between two proximal TMV packaging signals can be no greater than the length of the 3’-UTR within which they reside.251616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO

[0114] Expression Vectors and Promoters

[0115] In some embodiments, the DNA sequences disclosed herein are double-stranded and are incorporated into an expression vector. In order to allow cells harboring such vectors to transcribe RNA therefrom, the expression vector must include a promoter that is operatively linked to the DNA sequence. The promoter can be a strong, weak, constitutive, inducible, cell type specific promoter, or a tissue specific promoter. The promoter can be a constitutive promoter or an inducible promoter. The selection of the promoter can be optimized to the cell type and / or the nature of the heterologous RNA payload. In some embodiments, the promoter can be optimized for a eukaryotic cell. For example, the promoter can be optimized for a mammalian cell such as a human cell. As another example, the promoter can be optimized for a yeast cell.

[0116] Non-limiting examples of a constitutive promoter includes, TDH3 promoter, CCW12 promoter, PGK1 promoter, HHF2 promoter, TEF1 promoter, TEF2 promoter, YEF3 promoter, RPL3 promoter, RPL15 A promoter, RPL4 promoter, RPL8B promoter, PDA1 promoter, HHF1 promoter, HTB2 promoter, RPL18B promoter, ALD6 promoter, PAB1 promoter, RET2 promoter, RNR1 promoter, SAC6 promoter, RNR2 promoter, POP6 promoter, RAD27 promoter, PSP2 promoter, TPI1 promoter, ENO2 promoter, ADH1 promoter, CYC1 promoter, GAP promoter, GPD promoter, REV1 promoter, SV40 promoter, hCMV-IE promoter, mCMV-IE promoter, RSV-LTR promoter, MMTV-LTR promoter, MoMLV-LTR promoter, Ad2MLP-TPL promoter, hUBC promoter, hEF-la promoter, mPGK promoter, β-Lactin promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, GDS promoter, ADH1 promoter, Ubi promoter, or CAG promoter.

[0117] Non-limiting examples of an inducible promoter includes, GALI promoter, GAL 10 promoter, TetO7 promoter, PhlO6 promoter, LuxO5 promoter, Met promoter, TPS1 promoter, HXT7 promoter, ADH2 promoter, SSA1 promoter, Z3V promoter, CUP1 promoter, mMT-I promoter, hMT-II promoter, hMT-IIA promoter, AlcA promoter, LexA promoter, or Ptet promoter.

[0118] The choice of promoter can determine the level of RNA transcribed from the DNA sequence and under what conditions, if any, such transcription can occur. This allows for fine tuning the level of transcribed RNA so that it is optimized for the formation of VLPs encapsulating such RNA (e.g., in a cell that co-expresses TMV coat protein).261616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0119] In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a GAP promoter. In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a GAP promoter comprising the DNA sequence:TCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTC CTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCC AAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAG TTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGA AAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATA GGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACG GGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCA ATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCT CTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCC CTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCT ATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACA CCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA (SEQ ID NO: 18).

[0120] In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a TEF1 promoter. In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a TEF1 promoter comprising the DNA sequence:CCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCG GACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTC CCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAA AAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATC ACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCA TTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTT GTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAAT CTAAG (SEQ ID NO: 19).

[0121] In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a TEF promoter further comprising upstream activation sequences (UAS), such as described in M Larroude et al., Microbial Biotechnology (2019) 12(6), 1249-1259.

[0122] In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a derivative of the P2_8UASxpr-TEF (M Larroude et al., Microbial Biotechnology (2019) 12(6), 1249-1259) promoter and comprises the DNA sequence:GCTTCGATCGCGTATCGATGATACGCGTCCATGGTGAGGTGTCTCACAAGTGCCGTG CAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGG TTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTC271616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO CCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTC CCGCCCACCTCGATCCGACATGTTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCC CCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCC ACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTT GCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTC GATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTC TCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCGATCC GGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTT GTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGGCAT GCACTGATCACGGGCAAAAGTGCTTCGATAGAGAGGGGGTTGGCGGCGCATTTGTG TCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGAC CCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACA CTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTG TACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTAC TGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGT CCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAG(“UAS8-2”; SEQ ID NO:28).

[0123] In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a Pl_4UASxpr-TEF (M Larroude et al., supra) promoter comprising the DNA sequence:ACGGCGATACGCGTATCGATACGCGTGCATGCTGAGGTGTCTCACAAGTGCCGTGC AGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGT TGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCC CGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCC CGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCC CACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCA CCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTG CTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCG ATCCGGCATGCACTGATCACGGGCAAAAGTGCGTTCGATAGAGAGGGGGTTGGCGG CGCATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCA GTAGCGGACCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCC GGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGT281616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO TCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAA AATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAA AGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCAC ACCCGAAGGATCCAATG (“UAS4-1”; SEQ ID NO:30).

[0124] In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a pTEF promoter comprising the DNA sequence:ACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGAC CCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACAT ATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTG GAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCC ATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAG CCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTC CTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTC AA (SEQ ID NO:34)

[0125] The expression vectors disclosed herein can include other polynucleotides in addition to those encoding promoter and those that are transcribed to the RNA molecule. For example, the expression vectors may include one or more of an origin of replication, an antibiotic resistance gene, an auxotrophic marker, a gene encoding one or one or more proteins that enhance transcription and / or stability of the encoded RNA molecule, 5’-UTR sequences that are added onto the 5 ’-end of the RNA, and / or 3’-UTR sequences that are added onto the 3 ’-end of the RNA. Antibiotic resistance genes include, but are not limited to, an ampicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene. Auxotrophic markers include, but are not limited to, HIS3, LEU2, TRP1, and URA3. Proteins that enhance transcription or RNA stability include, but are not limited to, RNA polymerases; DNA polymerases; RNA cap transferase enzymes, such as a RNA guanine -N7-methyltrasferase, a cap 0 methyltransferase (RNMT), and cap methyltransferases 1 and 2 (CMTrl / 2); endoribonuclease inhibitors; enzymes that increase the content or abundance of modified nucleotides such as methyl-transferase enzymes and pseudouridine synthase; an RNAse inhibitor; and / or a ribozyme. Ribonucleotides that may be added to the 5’ end of the 5’-UTR by the expression vector include, but are not limited to, nucleotides that bind transcription factors, nucleotides that aid in translation of a heterologous coding RNA payload, nucleotides that aid in the stability of a heterologous RNA payload, and nucleotides that are the target of RNA capping enzymes. Ribonucleotides that may291616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO be added to the 3’ end of the 3’-UTR sequences by the expression vector include, but are not limited to, nucleotides that encode a polyadenylation signal, nucleotides that encode a poly(A) tail, nucleotides that aid in translation of a heterologous coding RNA payload, nucleotides that aid in the stability of a heterologous RNA payload, and nucleotides that aid in the transport of resulting RNA molecule and nucleotides that are terminators. Terminators are RNA sequences that usually occur at the end of a transcript and cause transcription to stop. Terminators can also impact the half-life of the RNA transcript. The choice of expression vector can depend, in part, on the cell type that ultimately harbors it, as well as the cell type in which it is propagated (if different from the harboring cell type). Non-limiting examples of expression vectors useful herein include ESC-HIS, ESC-LEU, ESC-TRP, ESC-URA, JMP62-URA, JMP62-HIS, CIB8644, CIB8788, RK-flag-USP19, and / or TetO7-CSII-CR5.

[0126] The choice of terminator can depend in part on the promoter used to cause transcription of the heterologous RNA molecule, as well as other factors known in the art. In particular, useful terminators at the 3 ’-end of the heterologous RNA molecule include the CYC1 terminator:AUCCGCUCUAACCGAAAAGGAAGGAGUUAGACAACCUGAAGUCUAGGUCCCUAU UUAUUUUUUUAUAGUUAUGUUAGUAUUAAGAACGUUAUUUAUAUUUCAAAUUU UUCUUUUUUUUCUGUACAGACGCGUGUACGCAUGUAACAUUAUACUGAAAACCU UGCUUGAGAAGGUUUUGGGACGCUCGAAG (SEQ ID NO:25)the TI Tef (M Larroude et al., supra) terminator:UCUAGCUGCUUGUACCUAUGCAACCCCAGUUUGUUAAAAAUUAGUAGUCAAAAA CUUCUGAGUUAAAAAAAAAAAAAAAAGCUU (“TTEF”; SEQ ID NO:26),the T2-3_Lip2 (M Larroude et al., supra terminator:GGAUGUGUCUGUGGUAUCUAAGCUAUUUAUCACUCUUUACAACUUCUACCUCAA CUAUCUACUUUAAUAAAUGAAUAUCGUUUAUUCUCUAUGAUUACUGUAUAUGCG UUCCUCUAAGACAGAGU (“TLip2-2”; SEQ ID NO:27), andthe Tl-3_Lip2 (M Larroude et al., supra terminator:GUGUCUGUGGUAUCUAAGCUAUUUAUCACUCUUUACAACUUCUACCUCAACUAU CUACUUUAAUAAAUGAAUAUCGUUUAUUCUCUAUGAUUACUGUAUAUGCGUUCC UCUAAGACA (SEQ ID NO:35).301616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO

[0127] TMV Capsid Protein

[0128] In order to generate TMV VLPs, the RNA sequences described herein (e.g., the RNA that comprises in a 5’ to 3’ order, a 5’-UTR, a heterologous RNA payload, and a 3’-UTR, wherein the 3’-UTR comprises two or more tobacco mosaic virus (TMV) packaging signals) must be co-expressed in a cell with TMV capsid protein. The TMV capsid protein may be a naturally occurring TMV capsid protein or a variant thereof that is capable of forming a VLP and encapsidating the RNA sequence. Variants of TMV capsid proteins are known in the art. In some embodiments, the TMV capsid protein co-expressed in a cell has the amino acid sequence: MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQVT VRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRVDDAT VAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO:21).

[0129] In some embodiments, the TMV capsid protein is encoded by the DNA sequence:ATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCATCAGCGTGGGCCG ACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGAAATCAGTTTCAAACAC AACAAGCTCGAACTGTCGTTCAAAGACAATTCAGTGAGGTGTGGAAACCTTCACCA CAAGTAACTGTTAGGTTCCCTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGT ATTAGACCCGCTAGTCACAGCACTGTTAGGTGCATTCGACACTAGAAATAGAATAA TAGAAGTTGAAAATCAGGCGAACCCCACGACTGCCGAAACGTTAGATGCTACTCGT AGAGTAGACGACGCAACGGTGGCCATAAGGAGCGCGATAAATAATTTAATAGTAG AATTGATCAGAGGAACCGGATCTTATAATCGGAGCTCTTTCGAGAGCTCTTCTGGTT TGGTTTGGACCTCTGGTCCTGCAACTTGA (SEQ ID NO:20),or a DNA sequence having at least 50% sequence identity to and encoding the same amino acid sequence as SEQ ID NO:20.

[0130] In some embodiments the TMV capsid protein amino acid sequence may comprise up to 10 amino acid insertions, deletions or substitutions as long as the resulting polypeptide is capable of forming intact TMV VLPs with the RNA molecules disclosed herein that encapsidate such RNA molecules. In some embodiments, the amino acid substitutions are conservative amino acid substitutions, which are well known in the art.

[0131] The TMV capsid protein may be expressed in a cell through the use of an expression vector encoding that protein. In some embodiments, the expression vector encoding the TMV capsid protein is separate from the expression vector encoding the RNA sequences described herein. In some embodiments, the expression vector encoding the TMV capsid protein is the same as the expression vector encoding the RNA sequences described herein (e.g., both the RNA sequence and TMV capsid protein are encoded on a single expression vector).311616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0132] Expression of the TMV capsid protein from an expression vector requires that the DNA sequence encoding that protein be operatively linked to a promoter. Such promoter can be the same promoter that drives transcription of the RNA sequence described herein (e.g., a second copy of that promoter or a single copy of a promoter that is capable to transcribing both the TMV capsid protein encoding DNA and the RNA sequence, e.g., a bidirectional promoter), or a different promoter. The promoter driving TMV capsid protein expression can be a strong, weak, constitutive, inducible, cell type specific promoter, or a tissue specific promoter. The promoter can be a constitutive promoter or an inducible promoter. The selection of the promoter can be optimized to the cell type in which the capsid protein is expressed. In some embodiments, the promoter can be optimized for a eukaryotic cell. For example, the promoter can be optimized for a mammalian cell such as a human cell. As another example, the promoter can be optimized for a yeast cell. Specific choices of promoters may be selected from the list of constitutive and inducible promoters set forth above. In some embodiments, the promoter used to drive expression of TMV capsid protein is a GAP promoter. In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a GAP promoter comprising the DNA sequence of SEQ ID NO: 18. In some embodiments, the promoter used in an expression vector to drive transcription of the encoded RNA is a UAS4-1 promoter comprising the DNA sequence of SEQ ID NO:30.

[0133] In some embodiments, the RNA transcript encoding the TMV capsid protein comprises a terminator at its 3 ’-end. In some embodiments, the terminator is a TTEF terminator or SEQ ID NO:32.

[0134] Host Cells

[0135] As set forth above, the disclosure provides a host cell harboring an expression vector encoding the RNA sequence disclosed herein and an expression vector encoding the TMV capsid protein. As detailed above, in some embodiments, the host cell will harbor a single expression vector that encodes both the RNA sequence and the TMV capsid protein. In other embodiments, the host cell will harbor two separate expression vectors - one that encodes the RNA sequence disclosed herein and another that encodes the TMV capsid protein.

[0136] In some embodiments, the host cell can express, either naturally or recombinantly, enzymes and proteins capable of promoting transcription of, modifying and / or stabilizing the RNA sequence. Such enzymes and proteins include, but not limited to, RNA polymerases,321616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO DNA polymerases, RNA cap transferase enzymes, RNA ligase, RNase inhibitors, and / or enzymes that increase the content of modified nucleotides. Some non-limiting examples of specific enzymes or proteins that may be expressed include 2’-0 methyl-transferase enzyme or enzyme complex, which facilitates the addition of a methyl group at the 2'-0 position of the first nucleotide adjacent to the cap structure at the 5' end of the RNA; pseudouridine synthase that facilitates the site-specific isomerization of uridines into pseudouridine or N1 -methylpseudouridines; a RNA guanine-N7-methyltrasferase; a cap 0 methyltransferase (RNMT); a cap methyltransferase 1 or 2 (CMTrl or 2); a ribozyme; Tobacco mosaic virus RNA-dependent polymerase; a Brome mosaic virus RNA polymerase; a Turnip crinkle virus RNA polymerase; a Potato virus X RNA polymerase; a cowpea mild mottle virus RNA polymerase; an MS2 bacteriophage RNA polymerase; a T7 RNA polymerase; a T3 RNA polymerase; and an SP6 RNA polymerase. The host cell may also express, either naturally or recombinantly, an auxotrophic marker. Non-limiting examples of auxotrophic markers include HIS3, LEU2, TRP, MET 15, and / or URA3. As set forth above, any of these enzymes or proteins may also be encoded on an expression vector that also encodes the RNA sequence, an expression vector that also encodes the TMV capsid protein, or on an expression vector that also encodes both the RNA sequence and the TMV capsid protein.

[0137] The host cell of the disclosure can be a plant cell, an insect cell, a yeast cell, or a mammalian cell, population of yeast cells, plant cells, insect cells, mammalian cells or any combination thereof.

[0138] In some embodiments, the host cell is a plant cell. A plant cell useful as a host cell of the disclosure can be a Nicotiana spp. plant cell. For example, the plant cell can be a Nicotiana tabacum plant cell or a Nicotiana benthamiana plant cell. Other non-limiting examples of plant cells useful herein are potato plant cells, tomato plant cells, lettuce plant cells, carrot plant cells, com plant cells, soybean plant cells, or spinach plant cells.

[0139] In some embodiments, the host cell is an insect cell. An insect cell useful as a host cell of the disclosure can be a Spodoptera spp. insect cell, a Trichoplusia spp. insect cell, or a Drosophila spp. insect cell. As a non-limiting example, the insect cell can be a Spodoptera frugiperda cell. In one embodiment, the insect cell can be a Trichoplusia ni cell. In another embodiment, the insect cell can be a Drosophila melanogaster cell. Non-limiting examples of other insect cells useful herein can be a Sf9, Sf21, BTI-TN-5B1-4, or S2 cell.331616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0140] In some embodiments, the host cell is a mammalian cell. Non-limiting examples of a mammalian cell useful as a host cell of the disclosure include a Chinese Hamster Ovary cell (CHO), a mouse myeloma cells (NS0), a baby hamster kidney cell (BHK), and / or a HEK293 cell.

[0141] In some embodiments, the host cell is a yeast cell. Non-limiting examples of a yeast cell useful as a host cell of the disclosure include Yarrowia lipolytica, Kluyveromyces marxianus, Saccharomyces cerevisiae and / or Pichia pastoris. In some embodiments, the yeast cells can be Saccharomyces cerevisiae and / or Pichia pastoris. In some embodiments the yeast cell is naturally or has been genetically modified to be devoid of retrotransposons e.g., Tyl, Ty2, Ty3, Ty4, and / or a Ty5 retro transposon.

[0142] Methods of Producing VLPs Containing the Heterologous RNA Payload

[0143] In some aspects, the disclosure provides a method of producing a heterologous RNA payload in a cell comprising growing a host cell described above and expressing both an RNA described herein (e.g., comprising a 5’-UTR, a heterologous RNA payload, and a 3’-UTR) and a TMV capsid protein. The expressed RNA molecule will bind to the TMV capsid protein through the two or more TMV packaging signals present in the RNA molecule, resulting in the self-assembly and production of virus-like particles encapsidating the RNA comprising. The VLPs can then be isolated from the host cell by cell lysis followed by centrifugation and treatment of the soluble material with PEG to precipitate the VLPs. The precipitated VLPs may be further purified by density gradient centrifugation. Even further purification of the VLPs may be achieved through the use of ultrafiltration, membrane filter centrifugation and / or size exclusion chromatography.

[0144] In some embodiments, the isolated VLPs are treated with one or more enzymes that digest nucleic acids or polynucleotides. Nucleic acids or polynucleotides produced by the host cell, whether naturally occurring or the result of transcription from one or more expression vectors harbored by the host cell, is not desirable and therefore it is advantageous to remove it from the isolated VLPs. Such nucleic acids or polynucleotides may stick to or are associated with the exterior of the VLP. Thus, treatment of the VLP with an enzyme that digests these species provides a method of removing such contaminants while the VLP protects the encapsidated RNA comprising the heterologous RNA payload from attack by such enzymes.341616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0145] Isolated VLPs Containing RNA Comprising a Heterologous RNA Payload and Isolated RNA therefrom.

[0146] In some aspects, the disclosure provides VLPs comprising a RNA molecule disclosed herein isolated from the host cell.

[0147] In some aspects, the disclosure provides an isolated RNA molecule disclosed herein.

[0148] In some aspects, the disclosure provides an RNA molecule disclosed herein isolated from the VLPs encapsidating such RNA.

[0149] Once the VLPs have been isolated and optionally treated with an enzyme that digests nucleic acids or polynucleotides external to the TMV VLP, the encapsidated RNA therein can be extracted. Such extraction may be achieved using commercially available RNA-extraction columns or other RNA extraction methodologies known in the art. The extracted RNA may then be used directly or further formulated to administer to a subject in need thereof.

[0150] In some aspects, the encapsidated RNA is not extracted from the VLPs and the VLPs themselves are used directly or are further formulated as an agent to deliver the RNA therein to a subject in need thereof.

[0151] All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof.

[0152] It should be understood that although the present methods and compositions have been specifically disclosed by embodiments and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0153] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.351616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0154] Whenever a range is given in the specification, for example, a temperature range, a time range, a composition, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods.

[0155] In addition, where features or aspects of the compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. Similarly, any two or more embodiments of a particular aspect set forth herein may be combined even if such specific combination is not specifically disclosed.EXAMPLES

[0156] Example 1. Production of VLPs in Yeast

[0157] A plasmid comprising a DNA sequence encoding TMV capsid protein (CP) (DNA sequence - SEQ ID NO:20; CP amino acid sequence - SEQ ID NO:21) operatively linked to a GAP promoter (SEQ ID NO: 18) was prepared using standard cloning methods. A map of this plasmid (TMV Capsid) is shown in FIG. 1.

[0158] A second set of plasmids were prepared comprising DNA that is capable of being transcribed into an RNA molecule comprising a firefly luciferase or enhanced green fluorescence protein (EGFP) mRNA as the heterologous RNA payload flanked by the 5’-UTR of SEQ ID NO: 17 and various 3’ UTRs containing one or two copies of a TMV packaging sequence as indicated in Table 3 below.

[0159] The 3 ’-UTRs containing only one copy of the TMV packaging sequence had the following RNA sequences. Bolded nucleotides represent the TMV packaging sequence.

[0160] UTR-1:GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCC UCCCCUUCCUGCACCCGGUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAG GGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCC UAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGUACCCCCGUGGUCU UUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAA (SEQ ID NO:22).361616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO

[0161] UTR-3:GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCC UCCCCUUCCUGCACCCGUUGUUUAUAGAAAUAAUAUAAAAUUAGGUUUGAGAGA GAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGU UGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUC UCGAACCGGAAAAAAGAGUGAUGUCCGCAAAGGGAAAAAUAUACCCCCGUGGUC UUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAA (SEQ ID NO:23).

[0162] Table 3. Content of various plasmids used to transcribe RNA encapsidated by TMV VLPsPlasmid Name Promoter Transcribed 5'- Protein encoded Transcribed 3'- UTR by transcribed UTRRNA payloadGFP UTR-1 TEF1 (SEQ. ID SEQ ID NO:17 eGFP UTR-1 (SEQ ID NO:19) NO:22) GFP UTR-3 TEF1 (SEQ ID SEQ ID NO:17 eGFP UTR-3 (SEQ ID NO:19) NO:23) GFP UTR-4 TEF1 (SEQ ID SEQ ID NO:17 eGFP UTR-4 (SEQ ID NO:19) NO:8)Luc UTR-1 TEF1 (SEQ ID SEQ ID NO:17 Firefly luciferase UTR-1 (SEQ ID NO:19) NO:22)Luc UTR-3 TEF1 (SEQ ID SEQ ID NO:17 Firefly luciferase UTR-3 (SEQ ID NO:19) NO:23)Luc UTR-4 TEF1 (SEQ ID SEQ ID NO:17 Firefly luciferase UTR-4 (SEQ IDNO:19) NO:8)

[0163] Maps of the plasmids set forth above are shown in FIGs. 2-7.

[0164] S.cerevisiae yeast production cells (BY 4741) were cultured in YPD (1% [w / v] yeast extract, 2% [w / v] peptone, 2% [w / v] dextrose) media before dual transformation with both a TMV-CP and an mRNA-UTR plasmid using the Yeast Transformation Kit (Sigma- Aldrich Cat. YEAST 1-1KT) with salmon sperm ssDNA (Abeam Cat. ab229278) and following the manufacturer's instructions. Reactions were incubated at 30°C for 30 mins followed by heatshock treatment before cells were plated onto solid auxotrophic drop-out media (0.17% [w / v] yeast nitrogen base without supplemented amino acids and nitrogen bases, 0.5% [w / v] ammonium sulfate, 0.14% [w / v] of Yeast Synthetic Drop-out Medium Supplements without histidine, leucine, tryptophan and uracil, 2% [w / v] dextrose, 50 mg / L tryptophan, 50 mg / L uracil). After 4 days at 30°C, positive selection of plasmid-containing cells was performed. Glycerol stocks of the transformed yeasts were cultured by inoculating cells into yeast nitrogen371616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO base with the same yeast auxotrophic drop-out medium. Yeasts were cultured for a total of 5 days at 30°C with constant shaking. Cells were harvested by centrifugation and supernatants removed, cells were then resuspended, and the centrifugation repeated. Cells were next resuspended in TEN buffer (50 mM Tris pH 7,4; 500 mM NaCl; 2mM EDTA; 1 protease inhibitor (Roche Cat.04693124001). Cells were lysed using mechanical lysis by glass bead beating (TissueLyserll, 425-600 µm, Sigma- Aldrich, Cat. G8772). Cell lysates containing the VLPs were then collected and centrifuged to remove beads.

[0165] Example 2. Production of VLPs in Insect Cells

[0166] The Tobacco mosaic virus (TMV) capsid protein coding sequence (SEQ ID NO:20) was synthesized and directly cloned into baculovirus transfer vectors under promoter control. DNA Cassettes encoding firefly luciferase, enhanced green fluorescence protein (EGFP) or Sars-Cov-2 spike protein as the heterologous RNA payload flanked by the transcribed 5’-UTR of SEQ ID NO: 17 and various transcribed 3’ UTRs containing one or two copies of a TMV packaging sequence (UTR-1 (c SEQ ID NO:22), UTR-3 (SEQ ID NO:23), or UTR-4 (SEQ ID NO:8)) were subcloned into the baculovirus transfer vectors already containing the SEQ ID NO:20, using standard molecular cloning techniques.

[0167] Recombinant baculoviruses were produced by co-transfection with each of the generated transfer vectors using either Sf9 or Tni insect cells and ESF 921 medium (Expression Technologies). Reaction mixtures, each containing flasKQ AC ULTRA™ viral DNA (Oxford Expression Technologies), the transfer vectors and the transfection reagent baculoFECTIN II™(Oxford Expression Technologies), were used to infect the insect cells that were then seeded onto dishes and incubated at 28°C for 5 days in medium. The medium, now containing the corresponding P0 virus stock, was harvested into sterile tubes. To prepare the Pl virus stock, Sf9 or Tni cells were inoculated with the appropriate amount of corresponding P0 virus stock. The virus-infected shake cultures were incubated for 5 days at 28°C, followed by harvesting the Pl virus stock containing medium into sterile tubes.

[0168] The infectious titres of TMV-mRNA-UTR baculoviruses were determined by baculoQUANT™(Oxford Expression Technologies) qPCR. Sf9 cultures and Tni cultures seeded into shake flasks were set up and each culture was inoculated with one of the Pl virus stocks described. TMV-VLPs were extracted from Sf9 or Tni cell cultures by first lysing the cells by381616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO sonication before clarification by centrifugation. FIG. 8 shows a representative SDS-PAGE gel from lysate of transfected Tni cells producing TMV encapsulated mRNAs.

[0169] TMV VLP structures produced from transformed yeast described in Example 1, or from transfected Sf9 or Tni cells described in this example were observed by TEM (FIG. 9). All of the VLPs from each of the cells and containing one (UTR-1 and UTR-3) or two (UTR-4) copies of the TMV packaging sequence showed intact native TMV-like rods. This is in contrast to the prior art that showed that multiple copies of the TMV packaging sequence resulted in altered capsid morphology. Eber et al., Nanoscale, Vol. 7: 344-355 (2015); Gallic et al., Virology 158,473-476 (1987).

[0170] Example 3. Purification of VLPs

[0171] Clarified soluble material containing VLPs from either yeast cell or insect cell lysates was transferred to centrifuge tubes and PEG master solution (20% (w / v) PEG 6000, IM NaCl) was added to each sample resulting in a final concentration of 4% (w / v) PEG 6000, 200mM NaCl. The samples were gently mixed before incubation overnight at 4°C using gentle rotation. Samples were then spun at 13,000 x g for 45 minutes at 20°C to pellet the visible precipitated material. Samples were examined by denaturing SDS-PAGE analysis to confirm the presence of TMV capsid protein both in the lysate and the PEG pellet. See FIG. 10.

[0172] Density-based VLP purification was next performed by resuspending PEG-precipitated pellets into PBS before the addition of CsCl solution (35% w / v) and centrifugation at 180,000 x g. VLPs were visible in the CsCl ultracentrifugation tube as a blue band. FIG. 11 shows this for an exemplary yeast cell lysate. The solution was fractionated by serial removal of 500 pL aliquots and the presence of TMV capsid was analyzed by denaturing SDS-PAGE of each aliquot. FIG. 12 shows this for certain fractions of an exemplary yeast cell lysate prep. Capsidcontaining fractions were pooled and transferred into PBS using membrane filter centrifugation. Total capsid protein concentrations were measured by BCA assay (Pierce BCA kit, Thermo Scientific Cat. 23225). TMV VLPs containing a heterologous RNA payload have also been successfully purified from crude cell lysate by size-exclusion chromatography: CIMultus™ OH (2 pm) 1.5 M to 50 mM potassium phosphate buffer pH 7 gradient.391616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0173] Example 4. Extraction of RNA from VLPs

[0174] VLPs in PBS were treated with a nuclease enzyme or a mixture of enzymes (Benzonase or specific cocktails of RNases) designed to digest double-stranded RNA in the presence of MgCh for 1 hour at 37°C. Digestion reactions were terminated by the addition of EDTA. VLPs were then exchanged into PBS by membrane centrifugal filtration before the encapsidated RNA was extracted using commercial RNA-extraction columns based on chaotropic salt silica membrane separation principles (RNeasy Mini kit, Qiagen, Cat. 74104, Monarch RNA cleanup, NEB, Cat. T2040L). FIG. 13, panels A and B show agarose gel images of RNA samples extracted from TMV VLPs produced in yeast transfected with the TMV Capsid and either the GFP UTR-3 or the GFP UTR-4 plasmids, before and after benzonase treatment. The RNA band of expected size encoded by either the GFP UTR-3 or the GFP UTR-4 plasmids are highlighted by arrows. These results confirm the TEM images showing that the VLPs formed by and encapsidating RNA containing one or two OAS packaging sequences were intact structures that protected the RNA from digestion. RNA species that were eliminated by benzonase treatment were not encapsulated and are assumed to be adhered to the outside of the VLP or are contaminating RNA species in the VLP preps.

[0175] Table 4. VLP protein and encapsidated RNA from yeast transfected with plasmids encoding one or two TMV packaging signals.RNA-encoding plasmid Total purified VLP Total RNA massRNA: VLP mass ratio (%) used for transfection protein mass (pg) isolated from VLPs (ng)GFP UTR-1 467.2 134.7 0.03GFP UTR-4 203.4 610.3 0.30

[0176] Example 5. Functionality and Immunogenicity of VLP-extracted RNA in THP-1 Monocytes.

[0177] THP-1 Dual™ cells NF-KB-SEAP IRF-LUC Reporter Monocytes (InvivoGen cat.thpd-nfis) were modified to express lucia luciferase enzyme when the interferon regulatory factor pathway is stimulated as a result of TLR-3, MDA5, RIG-1 or cCAS receptor activation. THP-1 Dual™ cells were cultured using standard mammalian cell culture techniques in RPMI 1640 + 10% FBS (heat inactivated) with 1% Pen / Strep and the selective antibiotic Normocin. At cell passage P4, the THP-1 monocytes were differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) (Thermo Fisher Cat. J63916). THP-1 monocytes were seeded into flat bottomed well plates before treatment with PMA at a final concentration of 50 ng / mL in401616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO growth medium for 3 hours at 37°C. This treatment medium was then removed and replaced with growth medium before the cells were incubated for 3 days. The resulting adherent cells were gently washed into fresh media before use in transfection assays. RNA extracted from yeast TMV VLPs produced from each of GFP UTR-1, GFP UTR-3 and GFP UTR-4 was prepared for transfection with Lipofectamine (Invitrogen Cat. LMRNA008) reagent according to the manufacturer's instructions and immediately added to cells at a final concentration of 125 ng of RNA per well. Poly (dA:dT) (InvivoGen cat.tlrl-patn) was used as an immunogenicity positive control transfected with lipofectamine at 125 ng per well. EGFP and lucia enzyme expression was measured after 24 hours. Expressed eGFP measurements were taken directly from cells in the culture plate using a plate reader at ex. 480 nm and em. 520 nm. (PHERAstar FSX). Lucia luciferase, expressed upon the stimulation of the interferon regulatory pathway, was measured from cell supernatant samples according to the QUANTI-Luc 4 Reagent coelenterazine -based luminescence assay (InvivoGen Cat.rep-qlc4gl). All bioluminescence assays were measured by plate reader. The results of these experiments are shown in FIG. 14 panels A and B.

[0178] Example 6. A variant of a human alpha-globin 3’-UTR sequence containing a partial internal repeat enhances expression over the native human alpha-globin 3’ sequence in IVT.

[0179] RNA production

[0180] Double-stranded DNA templates that produce a luciferase-encoding RNA sequence during IVT, were generated from plasmids containing, in a 5’-to-3’ order, a DNA sequence capable of being transcribed into the 5’-UTR of SEQ ID NO:17 minus the 5’-most G, a luciferase coding sequence (SEQ ID NO:24), and a DNA sequence capable of being transcribed into the 3’-UTR of either SEQ ID NO:6 or SEQ ID NO:7. Primers were designed to target the sequence only between the beginning of the 5’-UTR to the end of the 3’-UTR of the luciferase RNA encoding template within the plasmid backbone. The primers also added 5’ to the 5’-UTR a T7 promoter with an additional AG at its 3’ end (SEQ ID NO:25) to enable the use of the CleanCap® Reagent AG, which replaces the 5’ G deleted from the 5’-UTR. The CleanCap® Reagent AG was used during the IVT reaction to generate cap-1 capped RNA. At the 3’ end of the 3’-UTR, the primers added an 80 nucleotide-long polyadenosine tail. The positive-sense strand of the resulting DNA templates had the nucleotide sequences set forth in SEQ ID NO:26411616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO and SEQ ID NO:27. PCR reactions were then used to amplify the template using High-Fidelity Master Mix (New England Biolabs, (NEB Cat: M0541S)), following the manufacturer's protocol. These DNA templates were purified (GeneJET® PCR Purification Kit, Thermo Fisher Scientific) and transcribed into RNA using the HiScribe® T7 mRNA Kit (NEB, Cat E2080S)) in the presence of CleanCap® reagent AG at 4mM. All other reagents were used in accordance with the HiScribe kit instructions. The reaction was incubated at 37°C for 150 min before termination with DNase I and a further 30 min incubation. The RNA was then extracted from the reaction mixture using Oligo d(T)25 beads (NEB) following the manufacturer's instructions. The eluted RNA was then further purified using Monarch RNA Cleanup Kit (NEB) before analysis by capillary electrophoresis. The successful production and the correct length of the RNA was confirmed by capillary electrophoresis before transfection.

[0181] Cell Transfection

[0182] THP-1 Dual™ cells were cultured using standard mammalian cell culture techniques in RPMI 1640 + 10% FBS (heat inactivated) with 1% Pen / Strep and the selective antibiotic Normocin. At cell passage P4, the THP-1 monocytes were differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) (Thermo Fisher Cat. J63916). THP-1 monocytes were seeded into flat-bottomed well plates before treatment with PMA at a final concentration of 50 ng / mL in growth medium for 3 hours at 37°C. This treatment medium was then removed and replaced with growth medium before the cells were incubated for 3 days. The resulting adherent cells were gently washed into fresh media before use in transfection assays. IVT-generated RNA was prepared for transfection with Lipofectamine (Invitrogen Cat.LMRNA008) reagent according to the manufacturer's instructions and immediately added to cells at a final concentration of 125 ng of RNA per well.

[0183] Adherent HEK cells were cultured in DMEM + 10% FBS with 1% Pen / Strep and were passaged every 2-4 days, using trypsin-EDTA to detach the cells from the plate. Cells were then seeded in 96-well plates using OptiMEM serum-free media and were left to adhere for at least 2 hours before transfection with RNA.

[0184] HepG2 were treated in a similar manner using MEM instead of DMEM for the initial culturing prior to transfection. For transfection, RNA samples were prepared with Lipofectamine (Invitrogen, Cat. LMRNA008) reagent according to the manufacturer's instructions and immediately added to cells at a final concentration of 125 ng of RNA per well.421616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO

[0185] Analysis of Expression Products

[0186] After 24 hours of incubation at 37°C the cells were assessed for transfection efficiency by the expression of the firefly luciferase reporter gene. Luciferase expression was analyzed by the measurement of bioluminescence resulting from the addition of the substrate D-luciferin to transfected cells expressing luciferase. The protocol from the kit Promega Luciferase Assay System (Cat: E1501) was followed; in short, cells were treated with lysis buffer in the 96 well culture plates, and 50 µL of the lysate was transferred to a white, opaque plate. The substrate D-luciferin was added to the lysate and bioluminescence was measured immediately using a plate reader. The results are shown in FIG. 15, panels A-C. For each of the cell lines tested, the presence of the modified a-globin 3’-UTR (SEQ ID NO:7) resulted in a substantial increase in expression product over the native a-globin 3’-UTR (SEQ ID NO:6).

[0187] Example 7. Dual TMV OAS in the 3’-UTR Increases Quality of RNA Produced in Yarrowia lipolytica

[0188] We compared the effect of dual TMV OAS sequences in the 3’-UTR versus a single TMV OAS located at the 5 ’-end of the 3’-UTR (i.e., immediately following the luciferase coding region) on the ability of the encoded luciferase to be expressed in THP-1 cells.

[0189] RNA-Containing VLP Production in Y. lipolytica

[0190] Y. lipolytica yeast production cells (POlh or POld) were cultured in YPD (1% [w / v] yeast extract, 2% [w / v] peptone, 2% [w / v] D(+) glucose) media until efficient biomass was achieved. Cells were then prepared for transformation by transferral into 0.1M lithium acetate buffer pH 6. Plasmids were restriction digested using rCutSmart buffer (NEB, Cat. B6004S) withNotl-HF (NEB, Cat. R3189L). Reactions were incubated at 37 °C for 2 hours, followed by 20 min at 65°C, then stored on ice until transformation. Transformation was achieved by the addition of digested plasmid YL-Luc OAS (FIG. 17) or YL-Luc UTR-4 (FIG. 18), together with digested plasmid YL-TMV-Capsid (FIG. 19) and the transformation mix: salmon sperm ssDNA (0.3 mg / mL Abeam Cat. ab229278), 40% PEG3350, 0.05M DTT and 0.1M lithium acetate pH 6, before incubation at 39 °C for 60 min. YL-Luc OAS and YL-Luc UTR-4 each comprise, in a 5’-to-3’ order, a pTEF promoter (SEQ ID NO:34), a DNA sequence capable of being transcribed into the 5’-UTR of SEQ ID NO:17, and the luciferase coding sequence of SEQ ID NO:24. Luc UTR-4 further comprises a DNA sequence capable of being transcribed into the 3’-UTR of SEQ ID NO:8 (UTR-4) at the 3 ’-end of the luciferase coding sequence, while Luc OAS further431616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO comprises a DNA sequence capable of being transcribed into the 3’-UTR of SEQ ID NO:29 at the 3 ’-end of the luciferase coding sequence. Each of Luc OAS and Luc UTR-4 further comprises a Tl-3 Lipt2 terminator (SEQ ID NO:35). YL-TMV-Capsid encodes the TMV capsid protein coding sequence (SEQ ID NO:21) whose expression was driven by a pTEF promoter (SEQ ID NO:34) and include a Tl-3 Lipt2 terminator (SEQ ID NO:35). The transformed cells were then isolated and plated onto solid auxotrophic drop-out media (0.17% [w / v] yeast nitrogen base without supplemented amino acids and nitrogen bases, 0.5% [w / v] ammonium sulfate, 2% [w / v] D(+)-glucose, 0.19% [w / v] Yeast Synthetic Drop-out Medium Supplements without uracil) plates and colonies were selected after 4 days. Glycerol stocks of the transformed yeasts were cultured by inoculating cells into yeast nitrogen base with yeast synthetic drop-out medium supplemented with glucose. Yeasts were cultured for a total of 3 days at 30°C with constant shaking. Cells were harvested by centrifugation and supernatants were removed. The yeast cells were then resuspended, and the centrifugation was repeated. Cells were next resuspended in PBS buffer pH 7.2 with protease inhibitor (Roche Cat.04693124001). The cell suspension was then treated with lyticase (Sigma Cat: L2524) at 750 U / mL and was incubated at 37°C for 1 hour before lysis. Yeast cells were lysed using mechanical lysis by glass bead beating (TissueLyserll, 425-600 µm, Sigma- Aldrich, Cat.G8772) or shearing lysis using Microfluidizer® (LM20, Micro fluidics).

[0191] Purification ofVLPs by Density Centrifugation

[0192] Clarified soluble material containing VLPs from yeast cell lysates was transferred to centrifuge tubes and PEG master solution (20% (w / v) PEG 6000, IM NaCl) was added to each sample resulting in a final concentration of 4% (w / v) PEG 6000, 200mM NaCl. The samples were gently mixed before incubation overnight at 4°C using gentle rotation. Samples were then centrifuged at 13,000 x g for 45 minutes at 20°C to pellet the visible precipitated material.Samples were examined by denaturing SDS-PAGE to confirm the presence of TMV capsid protein both in the lysate and the PEG pellet. Density-based VLP purification was next performed by resuspending PEG-precipitated pellets into PBS before adding CsCl solution (35% w / v) and centrifugation at 180,000 x g. VLPs were visible in the CsCl ultracentrifugation tube as a blue band. The solution was fractionated by serial removal of 500 pL aliquots and the presence of TMV capsid was analyzed by denaturing SDS-PAGE of each aliquot. Capsid-containing fractions were pooled and transferred into PBS (150 mM) pH 7.2 using membrane filter441616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO centrifugation. Total capsid protein concentrations were measured by BCA assay (Pierce BCA kit, Thermo Scientific Cat. 23225).

[0193] Purification ofVLPs by Chromatography

[0194] TMV-RNA particles were also purified from cell lysate by size-exclusion chromatography using a CIMultus™ OH (2 pm) column with 1.5 M potassium phosphate buffer pH 7 as the equilibration buffer and 50 mM potassium phosphate buffer pH 7 as the elution buffer. Fractions containing TMV VLPs were collected and pooled before being confirmed for purity by SDS-PAGE analysis. Pooled fractions were transferred into PBS (150 mM) pH 7.2 using membrane filter centrifugation before concentration and total capsid protein concentrations were measured by BCA assay (Pierce BCA kit, Thermo Scientific Cat. 23225).

[0195] RNA extraction from VLPs, THP-1 Cell Transfection with Extracted RNA and Analysis of Expression Products

[0196] RNA was isolated from the purified VLPs and as described in Example 5. THP-1 Dual™ cells were prepared for transfection and transfected with the isolated RNA as described in Example 5. Luciferase expression in THP-1 cells was measured as described in Example 6. The results showed that the presence of dual TMV OAS sequences in the RNA resulted in a substantial increase in Luciferase expression as compared to a single TMV OAS located immediately 3’ to the luciferase coding sequence (FIG. 16).

[0197] Table 5. Sequences.SEQ. Description SEQ. ID Type of SequenceID (sensible) SequenceNO1 75 nt minimal RNA UGAGAGACGGAGGGCCCAUGGAACUUACAGA TMV AGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCpackaging CCUAUGUCGAUCAsequence2 127 nt TMV SEQ.082 RNA G U U U G AG AG AG AAG AU U ACAAACG U GAG AG A packaging CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG sequence UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC GG3 186 nt TMV SEQ.083 RNA UUGUUUAUAGAAAUAAUAUAAAAUUAGGUUU packaging GAGAGAGAAGAUUACAAACGUGAGAGACGGA sequence GGGCCCAUGGAACUUACAGAAGAAGUCGUUG AUGAGUUCAUGGAAGAUGUCCCUAUGUCGAU CAGGCUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAGAGUGAUGUCCGCAAAGGGAAAAAUA451616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO 4 99 nt TMV SEQ.098 RNA AUUACAAACGUGAGAGACGGAGGGCCCAUGG packaging AACU U ACAG AAG AAG UCG U UG AUG AG U UCAU sequence GGAAGAUGUCCCUAUGUCGAUCAGGCUUGCA AAGUUU5 234 nt TMV RNA GCAAGUUUUAGUUAAUAUUAGAAAUGUGAAG packaging AUGUCAGCGGGUUUCUGUCCGCUUUCUCUGG sequence AGUUUGUGUCGGUGUGUAUUGUUUAUAGAA AUAAUAUAAAAUUAGGUUUGAGAGAGAAGAU UACAAACGUGAGAGACGGAGGGCCCAUGGAAC UUACAGAAGAAGUCGUUGAUGAGUUCAUGGA AGAUGUCCCUAUGUCGAUCAGGCUUGCAAAG UUUCGAUCUCGAACCGG6 human alpha SEQ.038 RNA GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCC globin 3'-UTR UUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU (native) GCACCCGUACCCCCGUGGUCUUUGAAUAAAGU CUGAGUGGGCGGC7 modified SEQ.032 RNA GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCC human alpha UUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU globin 3'-UTR GCACCCGAUGGGGGCACAGAAACUUAUUUCAG ACUCACCCG U ACCCCCG UGG UCU U UG AAU AAA GUCUGAGUGGGCGGC8 3' UTR SEQ.028 RNA GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCC sequence with UUGGGCCUCCCCCCAGCCCCUCCUCCGUUGUU two TMV 186 UAUAGAAAUAAUAUAAAAUUAGGUUUGAGAG packaging AGAAGAUUACAAACGUGAGAGACGGAGGGCCC sequences AUGGAACUUACAGAAGAAGUCGUUGAUGAGU located within UCAUGGAAGAUGUCCCUAUGUCGAUCAGGCU a native a- UGCAAAGUUUCGAUCUCGAACCGGAAAAAAGA globin 3'-UTR GUGAUGUCCGCAAAGGGAAAAAUACCUUCCUG and short CACCCU UG U U U AU AG AAAU AAU AU AAAAU U A polyA tail GG U U UG AG AG AG AAG AU U ACAAACG UG AG AG (UTR-4) ACGGAGGGCCCAUGGAACUUACAGAAGAAGUC GUUGAUGAGUUCAUGGAAGAUGUCCCUAUGU CGAUCAGGCUUGCAAAGUUUCGAUCUCGAACC GGAAAAAAGAGUGAUGUCCGCAAAGGGAAAA AUAUACCCCCGUGGUCUUUGAAUAAAGUCUG AGUGGGCGGCAAAAAAAAA9 3' UTR SEQ.043 RNA GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCC sequence with UUGGGCCUCCCCCCAGCCCCUCCUCCGGUUUG two TMV 127 AGAGAGAAGAUUACAAACGUGAGAGACGGAG packaging GGCCCAUGGAACUUACAGAAGAAGUCGUUGA sequences UGAGUUCAUGGAAGAUGUCCCUAUGUCGAUC located within AGGCUUGCAAAGUUUCGAUCUCGAACCGGCCU a native a- UCCUGCACCCGUUUGAGAGAGAAGAUUACAAA globin 3'-UTR CGUGAGAGACGGAGGGCCCAUGGAACUUACA having a short GAAGAAGUCGUUGAUGAGUUCAUGGAAGAUG polyA tail UCCCUAUGUCGAUCAGGCUUGCAAAGUUUCG (UTR-5) AUCUCGAACCGGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAA 461616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO 10 3' UTR SEQ.053 RNA G U U U G AG AG AG AAG AU U ACAAACG U GAG AG A sequence with CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG two TMV 127 UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC packaging GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC sequences GGGCUGGAGCCUCGGUGGCCUAGCUUCUUGC flanking a CCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU modified a- CCUGCACCCGAUGGGGGCACAGAAACUUAUUU globin 3'-UTR CAGACUCACCCGUACCCCCGUGGUCUUUGAAU with a polyA AAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAA tail and AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA ending with a AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA polyA tail AAG U U UG AG AG AG AAG AU U ACAAACG U G AG A GACGGAGGGCCCAUGGAACUUACAGAAGAAG UCGUUGAUGAGUUCAUGGAAGAUGUCCCUAU GUCGAUCAGGCUUGCAAAGUUUCGAUCUCGA ACCGGAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA11 3' UTR SEQ.052 RNA G U U U GAG AG AG AAG AU U ACAAACG U GAG AG A sequence with CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG two copies of UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC a TMV 127 GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC packaging GGGCUGGAGCCUCGGUGGCCUAGCUUCUUGC sequence- CCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU modified a- CCUGCACCCGAUGGGGGCACAGAAACUUAUUU globin 3'-UTR CAGACUCACCCGUACCCCCGUGGUCUUUGAAU sequence and AAAGUCUGAGUGGGCGGCGUUUGAGAGAGAA ending with a GAUUACAAACGUGAGAGACGGAGGGCCCAUG polyA tail GAACUUACAGAAGAAGUCGUUGAUGAGUUCA UGGAAGAUGUCCCUAUGUCGAUCAGGCUUGC AAAGUUUCGAUCUCGAACCGGGCUGGAGCCUC GGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC CCCAGCCCCUCCUCCCCUUCCUGCACCCGAUGG GGGCACAGAAACUUAUUUCAGACUCACCCGUA CCCCCGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA12 3' UTR SEQ.044 RNA G U U U GAG AG AG AAG AU U ACAAACG U GAG AG A sequence with CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG two TMV 127 UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC packaging GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC sequences at GGGCUGGAGCCUCGGUGGCCUAGCUUCUUGC the 5' and 3' CCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU end of a CCUGCACCCGAUGGGGGCACAGAAACUUAUUU modified a- CAGACUCACCCGUACCCCCGUGGUCUUUGAAU globin 3'-UTR AAAGUCUGAGUGGGCGGCGUUUGAGAGAGAA sequence GAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCA471616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO UGGAAGAUGUCCCUAUGUCGAUCAGGCUUGC AAAGUUUCGAUCUCGAACCGG13 3' UTR SEQ.045 RNA G U U U G AG AG AG AAG AU U ACAAACG U GAG AG A sequence with CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG two TMV 127 UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC packaging GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC sequences at GGGCUGGAGCCUCGGUGGCCUAGCUUCUUGC the 5' and 3' CCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU end of a CCUGCACCCGAUGGGGGCACAGAAACUUAUUU modified a- CAGACUCACCCGUACCCCCGUGGUCUUUGAAU globin 3'-UTR AAAGUCUGAGUGGGCGGCGUUUGAGAGAGAA sequence and GAUUACAAACGUGAGAGACGGAGGGCCCAUG ending with a GAACUUACAGAAGAAGUCGUUGAUGAGUUCA polyA tail UGGAAGAUGUCCCUAUGUCGAUCAGGCUUGC AAAGUUUCGAUCUCGAACCGGAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAA14 3'-UTR with SEQ.042 RNA UUGUUUAUAGAAAUAAUAUAAAAUUAGGUUU two TMV 186 GAGAGAGAAGAUUACAAACGUGAGAGACGGA packaging GGGCCCAUGGAACUUACAGAAGAAGUCGUUG sequences AUGAGUUCAUGGAAGAUGUCCCUAUGUCGAU connected by CAGGCUUGCAAAGUUUCGAUCUCGAACCGGAA a linker and 5' AAAAGAGUGAUGUCCGCAAAGGGAAAAAUACC to a modified UUCCUGCACCCUUGUUUAUAGAAAUAAUAUA a-globin 3'- AAAU U AGG U U UG AG AG AGAAG AU U ACAAACG UTR sequence UGAGAGACGGAGGGCCCAUGGAACUUACAGA AGAAGUCGUUGAUGAGUUCAUGGAAGAUGUC CCUAUGUCGAUCAGGCUUGCAAAGUUUCGAU CUCGAACCGGAAAAAAGAGUGAUGUCCGCAAA GGGAAAAAUAGCUGGAGCCUCGGUGGCCUAG CUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUC CUCCCCUUCCUGCACCCGAUGGGGGCACAGAA ACUUAUUUCAGACUCACCCGUACCCCCGUGGU CUUUGAAUAAAGUCUGAGUGGGCGGC15 3'-UTR with SEQ.041 RNA G U U U GAG AG AG AAG AU U ACAAACG U GAG AG A two TMV 127 CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG packaging UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC sequences GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC connected by GGCCUUCCUGCACCCGUUUGAGAGAGAAGAU a linker and 5' UACAAACGUGAGAGACGGAGGGCCCAUGGAAC to a modified UUACAGAAGAAGUCGUUGAUGAGUUCAUGGA a-globin 3'- AGAUGUCCCUAUGUCGAUCAGGCUUGCAAAG UTR sequence UUUCGAUCUCGAACCGGGCUGGAGCCUCGGU GGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGAUGGGG GCACAG AAACU U AU U UCAG ACUCACCCG U ACC CCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC481616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO 16 3'-UTR with SEQ.039 RNA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA with two TMV AAAAAAAAG U U UG AG AG AG AAG AU U ACAAAC 127 packaging GUGAGAGACGGAGGGCCCAUGGAACUUACAG sequences AAGAAGUCGUUGAUGAGUUCAUGGAAGAUGU connected by CCCUAUGUCGAUCAGGCUUGCAAAGUUUCGA a linker and UCUCGAACCGGCCUUCCUGCACCCGUUUGAGA flanked on GAGAAGAUUACAAACGUGAGAGACGGAGGGC both the 5' CCAUGGAACUUACAGAAGAAGUCGUUGAUGA and 3' -end by GUUCAUGGAAGAUGUCCCUAUGUCGAUCAGG polyA CUUGCAAAGUUUCGAUCUCGAACCGGAAAAAA stretches AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA17 native 5'-UTR N / A RNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG AAAUAUAAGACCCCGGCGCCGCCACC18 GAP promoter DNA TCATTATCAATACTGCCATTTCAAAGAATACGTA AATAATTAATAGTAGTGAI 1 1 1 CCTAACTTTATTT AGTCAAAAAATTAGCCI 1 1 1 AATTCTGCTGTAACC CGTACATGCCCAAAATAGGGGGCGGGTTACACA GAATATATAACATCGTAGGTGTCTGGGTGAACA GTTTATTCCTGGCATCCACTAAATATAATGGAGC CCGCI 1 1 1 IAAGCTGGCATCCAGAAAAAAAAAGA ATCCCAGCACCAAAATATTG 1 1 1 1 C 1 1 CACCAACC ATCAGTTCATAGGTCCATTCTCTTAGCGCAACTAC AGAGAACAGGGGCACAAACAGGCAAAAAACGG GCACAACCTCAATGGAGTGATGCAACCTGCCTG GAGTAAATGATGACACAAGGCAATTGACCCACG CATGTATCTATCTCA 1 1 1 1 C 1 1 ACACCTTCTATTAC CTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAA AAAAGGTTGAAACCAGTTCCCTGAAATTATTCCC CTACTTGACTAATAAGTATATAAAGACGGTAGGT ATTGATTGTAATTCTGTAAATCTATTTCTTAAACT TCTTAAATTCTAC 1 1 1 IAIAG I IAG I CI 1 1 1 1 1 1 IA G 1 1 1 1 AAAACACCAAGAACTTAGTTTCGAATAAA CACACATAAACAAACAAA19 TEF1 DNA CCACACACCATAGCTTCAAAATG 1 1 1 C 1 AC 1 CL 1 1 promoter 1 1 1 1 AC 1 C 1 1 CCAGA 1 1 1 1 C 1 CGGACTCCGCGCAT CGCCGTACCACTTCAAAACACCCAAGCACAGCAT ACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCG TTAATTACCCGTACTAAAGGTTTGGAAAAGAAAA AAGACACCGCCTCG 1 1 1 C 1 1 1 1 1 C 1 1 CGTCGAAAA AGGCAATAAAAAI 1 1 1 1 AI CACG I 1 I CI 1 1 1 I CI 1 GAAAAI 1 1 1 1 1 1 1 1 1 I GAI 1 1 1 1 1 I CI CI 1 I CGATG ACCTCCCATTGATATTTAAGTTAATAAACGGTCAT CAAI 1 I CI CAAG I 1 I CAG I I I CAI 1 1 1 I CI I GTTCT ATTACAAC I 1 I l l i 1 ACTTCTTGCTCATTAGAAAG AAAGCATAGCAATCTAATCTAAG20 TMV Capsid DNA ATGTCTTACAGTATCACTACTCCATCTCAGTTCGT protein G 1 1 C 1 1 GTCATCAGCGTGGGCCGACCCAATAGAGencoding DNA TTAATTAATTTATGTACTAATGCCTTAGGAAATCA 491616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO GTTTCAAACACAACAAGCTCGAACTGTCGTTCAA AGACAATTCAGTGAGGTGTGGAAACCTTCACCA CAAGTAACTGTTAGGTTCCCTGACAGTGACTTTA AGGTGTACAGGTACAATGCGGTATTAGACCCGC TAGTCACAGCACTGTTAGGTGCATTCGACACTAG AAATAGAATAATAGAAGTTGAAAATCAGGCGAA CCCCACGACTGCCGAAACGTTAGATGCTACTCGT AGAGTAGACGACGCAACGGTGGCCATAAGGAG CGCGATAAATAATTTAATAGTAGAATTGATCAGA GGAACCGGATCTTATAATCGGAGCTCTTTCGAGA GCTCTTCTGGTTTGGTTTGGACCTCTGGTCCTGC AACTTGA21 TMV Capsid Protein MSYSITTPSQ. FVFLSSAWADPIELINLCTNALGNQ. F protein QTQQARTVVQRQFSEVWKPSPQVTVRFPDSDFK sequence VYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTT AETLDATRRVDDATVAIRSAINNLIVELIRGTGSYNR SSFESSSGLVWTSGPAT*22 3'-UTR with SEQ.027 RNA GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCC one TMV 127 UUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU packaging GCACCCGG U U UG AG AG AGAAG AU U ACAAACG sequence UGAGAGACGGAGGGCCCAUGGAACUUACAGA located within AGAAGUCGUUGAUGAGUUCAUGGAAGAUGUC a native a- CCUAUGUCGAUCAGGCUUGCAAAGUUUCGAU globin 3'-UTR CUCGAACCGGUACCCCCGUGGUCUUUGAAUAA and short AGUCUGAGUGGGCGGCAAAAAAAAA polyA tail(UTR-1)23 3'-UTR with SEQ.099 RNA GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCC one TMV 186 UUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU packaging GCACCCGUUGUUUAUAGAAAUAAUAUAAAAU sequence U AGG U U UG AG AGAG AAG AU U ACAAACG UG AG located within AGACGGAGGGCCCAUGGAACUUACAGAAGAAG a native a- UCGUUGAUGAGUUCAUGGAAGAUGUCCCUAU globin 3'-UTR GUCGAUCAGGCUUGCAAAGUUUCGAUCUCGA and short ACCGGAAAAAAGAGUGAUGUCCGCAAAGGGAA polyA tail AAAUAUACCCCCGUGGUCUUUGAAUAAAGUC (UTR-3) UGAGUGGGCGGCAAAAAAAAA24 Luciferase DNA ATGGAGGACGCCAAGAACATCAAGAAGGGCCCC coding GCCCCCTTCTACCCCCTGGAGGACGGCACCGCCG sequence GCGAGCAGCTGCACAAGGCCATGAAGCGGTACG CCCTGGTGCCCGGCACCATCGCCTTCACCGACGC CCACATCGAGGTGGACATCACCTACGCCGAGTA CTTCGAGATGAGCGTGCGGCTGGCCGAGGCCAT GAAGCGGTACGGCCTGAACACCAACCACCGGAT CGTGGTGTGCAGCGAGAACAGCCTGCAG 1 1 Cl 1 CATGCCCGTGCTGGGCGCCCTGTTCATCGGCGTG GCCGTGGCCCCCGCCAACGACATCTACAACGAG CGGGAGCTGCTGAACAGCATGGGCATCAGCCAG CCCACCGTGGTGTTCGTGAGCAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCC 501616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO ATCATCCAGAAGATCATCATCATGGACAGCAAG ACCGACTACCAGGGCTTCCAGAGCATGTACACCT TCGTGACCAGCCACCTGCCCCCCGGCTTCAACGA GTACGACTTCGTGCCCGAGAGCTTCGACCGGGA CAAGACCATCGCCCTGATCATGAACAGCAGCGG CAGCACCGGCCTGCCCAAGGGCGTGGCCCTGCC CCACCGGACCGCCTGCGTGCGGTTCAGCCACGC CCGAGACCCCATCTTCGGCAACCAGATCATCCCC GACACCGCCATCCTGAGCGTGGTGCCCTTCCACC ACGGCTTCGGCATGTTCACCACCCTGGGCTACCT GATCTGCGGCTTCCGGGTGGTGCTGATGTACCG GTTCGAGGAGGAGCTGTTCCTGCGGAGCCTGCA GGACTACAAGATCCAGAGCGCCCTGCTGGTGCC CACCCTGTTCAGCTTCTTCGCCAAGAGCACCCTG ATCGACAAGTACGACCTGAGCAACCTGCACGAG ATCGCCAGCGGCGGCGCCCCCCTGAGCAAGGAG GTGGGCGAGGCCGTGGCCAAGCGGTTCCACCTG CCCGGCATCCGGCAGGGCTACGGCCTGACCGAG ACCACCAGCGCCATCCTGATCACCCCCGAGGGC GACGACAAGCCCGGCGCCGTGGGCAAGGTGGT GCCCTTCTTCGAGGCCAAGGTGGTGGACCTGGA CACCGGCAAGACCCTGGGCGTGAACCAGCGGG GCGAGCTGTGCGTGCGGGGCCCCATGATCATGA GCGGCTACGTGAACAACCCCGAGGCCACCAACG CCCTGATCGACAAGGACGGCTGGCTGCACAGCG GCGACATCGCCTACTGGGACGAGGACGAGCACT TCTTCATCGTGGACCGGCTGAAGAGCCTGATCAA GTACAAGGGCTACCAGGTGGCCCCCGCCGAGCT GGAGAGCATCCTGCTGCAGCACCCCAACATCTTC GACGCCGGCGTGGCCGGCCTGCCCGACGACGAC GCCGGCGAGCTGCCCGCCGCCGTGGTGGTGCTG GAGCACGGCAAGACCATGACCGAGAAGGAGAT CGTGGACTACGTGGCCAGCCAGGTGACCACCGC CAAGAAGCTGCGGGGCGGCGTGGTGTTCGTGG ACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGG ACGCCCGGAAGATCCGGGAGATCCTGATCAAGG CCAAGAAGGGCGGCAAGATCGCCGTGTGA25 T7 promoter DNA GACGTAATACGACTCACTATAGwith thedinucleotideAG added tothe 3' end forCleancap®26 IVT DNA DNA GACGTAATACGACTCACTATAGGGAAATAAGAG template AGAAAAGAAGAGTAAGAAGAAATATAAGACCCC comprising a GGCGCCGCCACCATGGAGGACGCCAAGAACATC T7 promoter, AAGAAGGGCCCCGCCCCCTTCTACCCCCTGGAG an AG GACGGCACCGCCGGCGAGCAGCTGCACAAGGCC dinucleotide, ATGAAGCGGTACGCCCTGGTGCCCGGCACCATCa DNA GCCTTCACCGACGCCCACATCGAGGTGGACATCA 511616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO sequence CCTACGCCGAGTACTTCGAGATGAGCGTGCGGC corresponding TGGCCGAGGCCATGAAGCGGTACGGCCTGAACA to the 5'-UTR CCAACCACCGGATCGTGGTGTGCAGCGAGAACA of SEQ ID GCCTGCAGTTCTTCATGCCCGTGCTGGGCGCCCT NO:17 minus GTTCATCGGCGTGGCCGTGGCCCCCGCCAACGA two 5' CATCTACAACGAGCGGGAGCTGCTGAACAGCAT terminal G GGGCATCAGCCAGCCCACCGTGGTGTTCGTGAG nucleotides, CAAGAAGGGCCTGCAGAAGATCCTGAACGTGCA the luciferase GAAGAAGCTGCCCATCATCCAGAAGATCATCATC coding ATGGACAGCAAGACCGACTACCAGGGCTTCCAG sequence of AGCATGTACACCTTCGTGACCAGCCACCTGCCCC SEQ. ID NO:24, CCGGCTTCAACGAGTACGACTTCGTGCCCGAGA a DNA GCTTCGACCGGGACAAGACCATCGCCCTGATCAT sequence GAACAGCAGCGGCAGCACCGGCCTGCCCAAGG corresponding GCGTGGCCCTGCCCCACCGGACCGCCTGCGTGC to the 3'-UTR GGTTCAGCCACGCCCGAGACCCCATCTTCGGCAA of SEQ. ID CCAGATCATCCCCGACACCGCCATCCTGAGCGTG NO:6, and a GTGCCCTTCCACCACGGCTTCGGCATGTTCACCA DNA sequence CCCTGGGCTACCTGATCTGCGGCTTCCGGGTGGT corresponding GCTGATGTACCGGTTCGAGGAGGAGCTGTTCCT to a polyA tail GCGGAGCCTGCAGGACTACAAGATCCAGAGCGC CCTGCTGGTGCCCACCCTGTTCAGCTTCTTCGCCA AGAGCACCCTGATCGACAAGTACGACCTGAGCA ACCTGCACGAGATCGCCAGCGGCGGCGCCCCCC TGAGCAAGGAGGTGGGCGAGGCCGTGGCCAAG CGGTTCCACCTGCCCGGCATCCGGCAGGGCTAC GGCCTGACCGAGACCACCAGCGCCATCCTGATC ACCCCCGAGGGCGACGACAAGCCCGGCGCCGTG GGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTG GTGGACCTGGACACCGGCAAGACCCTGGGCGTG AACCAGCGGGGCGAGCTGTGCGTGCGGGGCCC CATGATCATGAGCGGCTACGTGAACAACCCCGA GGCCACCAACGCCCTGATCGACAAGGACGGCTG GCTGCACAGCGGCGACATCGCCTACTGGGACGA GGACGAGCACTTCTTCATCGTGGACCGGCTGAA GAGCCTGATCAAGTACAAGGGCTACCAGGTGGC CCCCGCCGAGCTGGAGAGCATCCTGCTGCAGCA CCCCAACATCTTCGACGCCGGCGTGGCCGGCCTG CCCGACGACGACGCCGGCGAGCTGCCCGCCGCC GTGGTGGTGCTGGAGCACGGCAAGACCATGACC GAGAAGGAGATCGTGGACTACGTGGCCAGCCA GGTGACCACCGCCAAGAAGCTGCGGGGCGGCG TGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGA CCGGCAAGCTGGACGCCCGGAAGATCCGGGAG ATCCTGATCAAGGCCAAGAAGGGCGGCAAGATC GCCGTGTGAGCTGGAGCCTCGGTGGCCTAGCTT CTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCC CTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAA 521616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA27 IVT DNA DNA GACGTAATACGACTCACTATAGGGAAATAAGAG template AGAAAAGAAGAGTAAGAAGAAATATAAGACCCC comprising a GGCGCCGCCACCATGGAGGACGCCAAGAACATC T7 promoter, AAGAAGGGCCCCGCCCCCTTCTACCCCCTGGAG an AG GACGGCACCGCCGGCGAGCAGCTGCACAAGGCC dinucleotide, ATGAAGCGGTACGCCCTGGTGCCCGGCACCATC a DNA GCCTTCACCGACGCCCACATCGAGGTGGACATCA sequence CCTACGCCGAGTACTTCGAGATGAGCGTGCGGC corresponding TGGCCGAGGCCATGAAGCGGTACGGCCTGAACA to the 5'-UTR CCAACCACCGGATCGTGGTGTGCAGCGAGAACA of SEQ. ID GCCTGCAGTTCTTCATGCCCGTGCTGGGCGCCCT NO:17 minus GTTCATCGGCGTGGCCGTGGCCCCCGCCAACGA two 5' CATCTACAACGAGCGGGAGCTGCTGAACAGCAT terminal G GGGCATCAGCCAGCCCACCGTGGTGTTCGTGAG nucleotides, CAAGAAGGGCCTGCAGAAGATCCTGAACGTGCA the luciferase GAAGAAGCTGCCCATCATCCAGAAGATCATCATC coding ATGGACAGCAAGACCGACTACCAGGGCTTCCAG sequence of AGCATGTACACCTTCGTGACCAGCCACCTGCCCC SEQ. ID NO:24, CCGGCTTCAACGAGTACGACTTCGTGCCCGAGA a DNA GCTTCGACCGGGACAAGACCATCGCCCTGATCAT sequence GAACAGCAGCGGCAGCACCGGCCTGCCCAAGG corresponding GCGTGGCCCTGCCCCACCGGACCGCCTGCGTGC to the 3'-UTR GGTTCAGCCACGCCCGAGACCCCATCTTCGGCAA of SEQ ID CCAGATCATCCCCGACACCGCCATCCTGAGCGTG NO:7, and a GTGCCCTTCCACCACGGCTTCGGCATGTTCACCA DNA sequence CCCTGGGCTACCTGATCTGCGGCTTCCGGGTGGT corresponding GCTGATGTACCGGTTCGAGGAGGAGCTGTTCCT to a polyA tail GCGGAGCCTGCAGGACTACAAGATCCAGAGCGC CCTGCTGGTGCCCACCCTGTTCAGCTTCTTCGCCA AGAGCACCCTGATCGACAAGTACGACCTGAGCA ACCTGCACGAGATCGCCAGCGGCGGCGCCCCCC TGAGCAAGGAGGTGGGCGAGGCCGTGGCCAAG CGGTTCCACCTGCCCGGCATCCGGCAGGGCTAC GGCCTGACCGAGACCACCAGCGCCATCCTGATC ACCCCCGAGGGCGACGACAAGCCCGGCGCCGTG GGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTG GTGGACCTGGACACCGGCAAGACCCTGGGCGTG AACCAGCGGGGCGAGCTGTGCGTGCGGGGCCC CATGATCATGAGCGGCTACGTGAACAACCCCGA GGCCACCAACGCCCTGATCGACAAGGACGGCTG GCTGCACAGCGGCGACATCGCCTACTGGGACGA GGACGAGCACTTCTTCATCGTGGACCGGCTGAA GAGCCTGATCAAGTACAAGGGCTACCAGGTGGC CCCCGCCGAGCTGGAGAGCATCCTGCTGCAGCA CCCCAACATCTTCGACGCCGGCGTGGCCGGCCTG CCCGACGACGACGCCGGCGAGCTGCCCGCCGCC GTGGTGGTGCTGGAGCACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTACGTGGCCAGCCA 531616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO GGTGACCACCGCCAAGAAGCTGCGGGGCGGCG TGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGA CCGGCAAGCTGGACGCCCGGAAGATCCGGGAG ATCCTGATCAAGGCCAAGAAGGGCGGCAAGATC GCCGTGTGAGCTGGAGCCTCGGTGGCCTAGCTT CTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCC CTTCCTGCACCCGATGGGGGCACAGAAACTTATT TCAGACTCACCCGTACCCCCGTGGTCTTTGAATA AAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA28 Modified DNA GCTTCGATCGCGTATCGATGATACGCGTCCATGG UAS8-2 TGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCC promoter CCACTTGCTTCTCTTTGTGTGTAGTGTACGTACAT TATCGAGAGGGTTGTTCCCGCCCACCTCGATCCG GCATGCTGAGGTGTCTCACAAGTGCCGTGCAGT CCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTA CGTACATTATCGAGAGGGTTGTTCCCGCCCACCT CGATCCGACATGTTGAGGTGTCTCACAAGTGCCG TGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGT AGTGTACGTACATTATCGAGAGGGTTGTTCCCGC CCACCTCGATCCGGCATGCTGAGGTGTCTCACAA GTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTT GTGTGTAGTGTACGTACATTATCGAGAGGGTTG TTCCCGCCCACCTCGATCCGGCATGCTGAGGTGT CTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGC TTCTCTTTGTGTGTAGTGTACGTACATTATCGAG AGGGTTGTTCCCGCCCACCTCGATCCGGCATGCT GAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCC CACTTGCTTCTCTTTGTGTGTAGTGTACGTACATT ATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGG CATGCACTGATCACGGGCAAAAGTGCTTCGATA GAGAGGGGGTTGGCGGCGCATTTGTGTCCCAAA AAACAGCCCCAATTGCCCCAATTGACCCCAAATT GACCCAGTAGCGGACCCAACCCCGGCGAGAGCC CCCTTCACCCCACATATCAAACCTCCCCCGGTTCC CACACTTGCCGTTAAGGGCGTAGGGTACTGCAG TCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTGCTTT GTGGTTGGGACTTTAGCCAAGGGTATAAAAGAC CACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCT CTCTCCTTGTCAACTCACACCCGAAG29 3'-UTR with RNA G U U U G AG AG AG AAG AU U ACAAACG U GAG AG A one TMV 127 CGGAGGGCCCAUGGAACUUACAGAAGAAGUCG packaging UUGAUGAGUUCAUGGAAGAUGUCCCUAUGUC sequence GAUCAGGCUUGCAAAGUUUCGAUCUCGAACC followed by GGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU 541616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO native a- CCUGCACCCGUACCCCCGUGGUCUUUGAAUAA globin 3'-UTR AGUCUGAGUGGGCGGC30 UAS4-1 DNA ACGGCGATACGCGTATCGATACGCGTGCATGCT promoter GAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCC CACTTGCTTCTCTTTGTGTGTAGTGTACGTACATT ATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGG CATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCC CGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACG TACATTATCGAGAGGGTTGTTCCCGCCCACCTCG ATCCGGCATGCTGAGGTGTCTCACAAGTGCCGT GCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTA GTGTACGTACATTATCGAGAGGGTTGTTCCCGCC CACCTCGATCCGGCATGCTGAGGTGTCTCACAAG TGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTG TGTGTAGTGTACGTACATTATCGAGAGGGTTGTT CCCGCCCACCTCGATCCGGCATGCACTGATCACG GGCAAAAGTGCGTTCGATAGAGAGGGGGTTGG CGGCGCATTTGTGTCCCAAAAAACAGCCCCAATT GCCCCAATTGACCCCAAATTGACCCAGTAGCGGA CCCAACCCCGGCGAGAGCCCCCTTCACCCCACAT ATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAA GGGCGTAGGGTACTGCAGTCTGGAATCTACGCT TGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCA TCCGGGTAACCCATGCCGGACGCAAAATAGACT ACTGAAAATTTTTTGCTTTGTGGTTGGGACTTTA GCCAAGGGTATAAAAGACCACCGTCCCCGAATT ACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACT CACACCCGAAGGATCCAATG31 CYC1 RNA AUCCGCUCUAACCGAAAAGGAAGGAGUUAGAC terminator AACCUGAAGUCUAGGUCCCUAUUUAUUUUUU UAUAGUUAUGUUAGUAUUAAGAACGUUAUU UAUAUUUCAAAUUUUUCUUUUUUUUCUGUA CAGACGCGUGUACGCAUGUAACAUUAUACUG AAAACCUUGCUUGAGAAGGUUUUGGGACGCU CGAAG32 TTEF RNA UCUAGCUGCUUGUACCUAUGCAACCCCAGUUU terminator GUUAAAAAUUAGUAGUCAAAAACUUCUGAGU U AAAAAAAAAAAAAAAAGCU U33 TLip2-2 RNA GGAUGUGUCUGUGGUAUCUAAGCUAUUUAUC terminator ACUCUUUACAACUUCUACCUCAACUAUCUACU UUAAUAAAUGAAUAUCGUUUAUUCUCUAUGA UUACUGUAUAUGCGUUCCUCUAAGACAGAGU34 pTEF DNA ACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAA promoter CAGCCCCAATTGCCCCAATTGACCCCAAATTGAC CCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCC TTCACCCCACATATCAAACCTCCCCCGGTTCCCAC ACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCT GGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACG 551616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO CAAAATAGACTACTGAAAATTTTTTGCTTTGTG GTTGGGACTTTAGCCAAGGGTATAAAAGACCAC CGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTC TCCTTGTCAACTCACACCCGAAATCGTTAAGCATT TCCTTCTGAGTATAAGAATCATTCAA35 Tl-3 Lipt2 RNA GUGUCUGUGGUAUCUAAGCUAUUUAUCACUC terminator UUUACAACUUCUACCUCAACUAUCUACUUUAAUAAAUGAAUAUCGUUUAUUCUCUAUGAUUACUGUAUAUGCGUUCCUCUAAGACA

[0198] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.561616904083.2

Claims

1. PATENT ATTONEY DOCKET NO. SBIO1110-2 WOWhat is Claimed Is:

1. An isolated DNA sequence capable of being transcribed into an RNA sequence comprising, in a 5’ to 3’ order, a 5’-UTR, a heterologous RNA payload, and a 3’-UTR, wherein the 3’-UTR comprises two or more tobacco mosaic virus (TMV) packaging signals.

2. The isolated DNA sequence of claim 1, wherein the 3’-UTR comprises two TMV packaging signals.

3. The isolated DNA sequence of claim 1 or 2, wherein every two proximal TMV packaging signals in the 3’-UTR are separated by less than 500 nucleotides.

4. The isolated DNA sequence of claim 3, wherein every two proximal TMV packaging signals in the 3’-UTR are separated by less than 100 nucleotides.

5. The isolated DNA sequence of claim 1 or 2, wherein each TMV packaging signal is an RNA sequence independently comprising any one of SEQ ID NOs:l-5, and an RNA sequence comprising SEQ ID NO:1 and having at least 90% sequence identity to one of SEQ ID NOs:l-5.

6. The isolated DNA sequence of claim 5, wherein each TMV packaging signal independently is an RNA sequence comprising SEQ ID NO:2 or SEQ ID NO:3.

7. The isolated DNA sequence of claim 6, wherein the 3’-UTR is an RNA sequence comprising any one of SEQ ID NOs: 8-16.

8. The isolated DNA sequence of claim 7, wherein the 3’-UTR is an RNA sequence comprising SEQ ID NO:8.

9. The isolated DNA sequence of any one of claims 1-8, wherein the DNA sequence is double-stranded.571616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO 10. An expression vector comprising the double stranded DNA sequence of claim 9, and a promoter operatively linked to the double stranded DNA sequence.

11. The expression vector of claim 10, wherein the promoter is a constitutive promoter.

12. The expression vector of claim 11, wherein the promoter is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18, a TEF 1 promoter having a nucleic acid sequence of SEQ ID NO: 19, a modified UAS8-2 promoter having a nucleic acid sequence of SEQ ID NO:28, a UAS4-1 promoter having a nucleic acid sequence of SEQ ID NO:30 or a promoter having a nucleic acid sequence of at least 90% sequence identity to any one of SEQ ID NOs:18, 19, 28 or 30.

13. The expression vector of claim 12, wherein the promoter is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18, a TEF 1 promoter having a nucleic acid sequence of SEQ ID NO: 19, a modified UAS8-2 promoter having a nucleic acid sequence of SEQ ID NO:28, or a UAS4-1 promoter having a nucleic acid sequence of SEQ ID NO:30.

14. The expression vector of claim 13, further comprising a DNA sequence encoding a TMV viral capsid protein operatively linked to a promoter.

15. The expression vector of claim 14, wherein the promoter operatively linked to the nucleic acid sequence encoding the TMV viral capsid protein is a constitutive promoter.

16. The expression vector of claim 15, wherein the promoter operatively linked to the nucleic acid sequence encoding the TMV viral capsid protein is a GAP promoter having a DNA sequence of SEQ ID NO: 18, a a UAS4-1 promoter having a nucleic acid sequence of SEQ ID NO:30, or a promoter having a nucleic acid sequence of at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO:30.

17. The expression vector of claim 16, wherein the promoter operatively linked to the nucleic acid sequence encoding the TMV viral capsid protein is a GAP promoter having a DNA581616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO sequence of SEQ ID NO:18, or a UAS4-1 promoter having a nucleic acid sequence of SEQ ID NO:30.

18. An isolated host cell harboring the expression vector of any one of claims 10-17.

19. An isolated host cell harboring the expression vector of any one of claims 10-13; and additionally harboring an expression vector comprising a DNA sequence encoding a TMV viral capsid protein operatively linked to a promoter.

20. The host cell of claim 19, wherein the promoter operatively linked to the DNA sequence encoding the TMV viral capsid protein is a constitutive promoter.

21. The host cell of claim 20, wherein the promoter operatively linked to the nucleic acid sequence encoding the TMV viral capsid protein is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18 or a promoter having a nucleic acid sequence of at least 90% sequence identity to SEQ ID NO: 18.

22. The host cell of claim 21, wherein the promoter operatively linked to the nucleic acid sequence encoding the TMV viral capsid protein is a GAP promoter having a nucleic acid sequence of SEQ ID NO: 18.

23. The host cell of any one of claims 18-22, wherein the cell is a yeast cell, an insect cell, a plant cell or a mammalian cell.

24. The host cell of claim 23, wherein the cell is a yeast cell selected from Yarrowia lipolytica, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia pastoris.

25. The host cell of claim 23, wherein the cell is an insect cell selected from Spodoptera frugiperda and Trichoplusia ni.

26. The host cell of claim 23, wherein the cell is a plant cell that is Nicotiana benthamiana.591616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2 WO 27. The host cell of claim 23, wherein the cell is a mammalian cell selected from a Chinese hamster ovary cell and a HEK293T cell.

28. A method of producing an RNA molecule comprising a heterologous RNA payload in a cell comprising growing the host cell of any one of claims 18-27 under conditions in which virus-like particles comprising the RNA molecule bound to and encapsidated by the TMV viral capsid protein are produced, thereby producing the RNA molecule.

29. The method of claim 28, further comprising isolating the virus-like particles from the cell.

30. The method of claim 29, further comprising treating the isolated virus-like particles with one or more RNases or nucleases that digest double-stranded RNA.

31. The method of claim 29 or 30, further comprising purifying the encapsidated RNA from the isolated virus-like particles.

32. An isolated virus-like particle produced by the method of claim 29 or 30.

33. An isolated RNA molecule transcribed from the isolated DNA sequence of any one of claims 1-9.

34. An isolated RNA molecule produced by the method of any one of claims 28-31.

35. An isolated RNA molecule produced from the isolated virus-like particle of claim 32.

36. An isolated double-stranded DNA sequence capable of being transcribed into a 3’-UTR having the RNA sequence of SEQ ID NO:7.

37. A DNA template for in vitro transcription comprising the DNA sequence of claim 36.601616904083.2PATENT ATTONEY DOCKET NO. SBIO1110-2WO 38. The DNA template of claim 37, comprising, in 5’-to-3’ order, a promoter, a DNA sequence capable of being transcribed into a 5’-UTR, a DNA sequence encoding a protein of interest, and the DNA sequence of claim 36.

39. The DNA template of claim 38, further comprising a DNA sequence capable of being transcribed into a Cap-1 cap mRNA capping signal and located between the promoter and the DNA sequence capable of being transcribed into the 5’-UTR; and a DNA sequence capable of being transcribed into a polyA tail and located at the 3’ end of the DNA sequence of claim 36.

40. An isolated RNA molecule produced by in vitro transcription of the DNA template of any one of claims 37-39.611616904083.2

Images