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

Abstract

The present disclosure relates generally to methods and systems for producing RNA payloads in living cells by encapsulating the RNA payloads in viral coat proteins to form virus-like particles (VLPs); more specifically, engineering viral origin of assembly sequences (OAS) to increase stability, yield, and / or translation efficiency of RNA extracted from these VLPs.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 551,012, filed February 7, 2024, and U.S. Provisional Application No. 63 / 682,989, filed August 14, 2024, pursuant to 35 USC §119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] By referencing and incorporating into the sequence list

[0004] The material in the attached sequence list is hereby incorporated by reference in this application. The attached sequence list XML file, named SBIO1110-2WO,Sequence Listing ST26.xml, was created on February 4, 2025 and is 47,509 bytes in size. Technical Field

[0005] This disclosure generally relates to methods and systems for generating RNA payloads in living cells, which is achieved by encapsulating the RNA payload in viral capsid proteins to form virus-like particles (VLPs); more specifically, to engineering viral assembly origin sequences (OAS) to increase the stability, yield, and / or translation efficiency of RNA extracted from these VLPs. Background Technology

[0006] A growing understanding of RNA function and its crucial role in health and disease has spurred the development of various commercial and research applications for RNA. Currently, the only method for producing therapeutic-grade mRNA is through in vitro transcription (IVT). This process requires a series of reactions, starting with the enzymatic linearization of a plasmid DNA template, followed by purification. The template is then transcribed by recombinant T7 polymerase to produce RNA molecules. Modified nucleotides can be added to the reaction at this stage. RNA 5' capping is introduced either through co-translation or by using a second reaction with a capping enzyme. During DNA template transcription, immunogenic double-stranded RNA byproducts are produced due to transcriptional failure of the T7 enzyme. These dsRNA contaminants reduce the efficacy of therapeutic RNA and induce pro-inflammatory responses. Therefore, IVT production of mRNA molecules for therapeutic use requires multiple separate reactions and numerous purification steps, resulting in costly and complex GMP manufacturing processes.

[0007] In addition, purified RNA obtained using in vitro transcription methods is often unstable and easily degraded.

[0008] For years, researchers have been studying how viruses package and protect their genomic RNA. Their research has identified viral RNA segments that act as “packaging signals” and bind the viral genomic RNA to viral capsid proteins; this encapsulation protects the viral genomic RNA from degradation in host cells.

[0009] In filamentous viruses such as tobacco mosaic virus (TMV), the packaging sequence, known as the assembly origin sequence (OAS), is located approximately 1 kb from the 3' end of its genomic RNA. The OAS sequence forms a hairpin structure that inserts into the disc-shaped loop of the TMV capsid protein (TMV CP), thereby initiating the assembly of the TMV virus-like particle (VLP) into a helical filamentous capsid structure. PJG Butler, *Philosophical Transactions of the Royal Society of London*, 354: 537-550 (1999); see also PJG Butler, *Journal of General Virology*, 65 (Part 2): 253-279, (1984).

[0010] In vitro TMV capsid assembly studies using recombinant capsid monomers have shown that adding nucleotides to the OAS on either side of the minimum required 75-nucleotide stem-loop may affect VLP assembly and stability, and thus the VLP's stability and ability to protect the encapsulated RNA from nucleases. See PCT Publication WO 2015118183. Native TMV RNA contains a single copy of the OAS. Researchers have artificially inserted two or more copies of the TMV OAS into the RNA for packaging, where these additions alter the capsid morphology. These altered morphologies deviate from the native filamentous structure of TMV and result in the mRNA being exposed to nucleases and / or RNases. Eber et al., Nanoscale, Vol. 7: 344-355 (2015); Gallie et al., Virology 158, 473-476 (1987).

[0011] Therefore, there is still a need for systems that produce therapeutic-quality RNA that bypass the aforementioned limitations of IVT and can take advantage of the inherent production benefits derived from the characteristics of virus-like particles. Summary of the Invention

[0012] In a first aspect, this disclosure provides a segregated DNA sequence capable of being transcribed into an RNA sequence comprising a 5'-UTR, a heterologous RNA payload, and a 3'-UTR in a 5' to 3' sequence, wherein the 3'-UTR contains two or more tobacco mosaic virus (TMV) packaging signals.

[0013] In some embodiments of the first aspect, the isolated DNA sequence is double-stranded.

[0014] In some embodiments of the first aspect, the 3'-UTR of the transcribed RNA sequence contains only two TMV packaging signals.

[0015] In some embodiments of the first aspect, each pair of adjacent TMV packaging signals in the 3'UTR is 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 adjacent TMV packaging signals may exist in tandem (i.e., no nucleotides separate them). In some more specific embodiments of the first aspect, any two adjacent 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 adjacent 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 adjacent 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 adjacent TMV packaging signals in the 3'-UTR are separated by less than 15 nucleotides.

[0016] Each of the two or more TMV packaging signals available herein comprises an RNA sequence consisting of a 75-nucleotide sequence as shown in SEQ ID NO: 1, which encodes the minimal TMV assembly origin sequence identified by: DR Turner et al., *Journal of Molecular Biology* 209(3), 407-422 (1989). In some embodiments of the first aspect, each of the two or more TMV packaging signals is independently selected from any of SEQ ID NO: 1-5, and an RNA sequence comprising SEQ ID NO: 1 and having at least 90% sequence identity with any of SEQ ID NO: 1-5. In some more specific embodiments of the first aspect, each TMV packaging signal is independently 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, and even more specific, embodiments of the first aspect, each TMV packaging signal is the RNA sequence of SEQ ID NO: 4. In some, and even more specific, embodiments of the first aspect, the 3'-UTR contains two TMV packaging signals, and each TMV packaging signal is the RNA sequence of SEQ ID NO: 2. In some, and even more specific, embodiments of the first aspect, the 3'-UTR contains two TMV packaging signals, and each TMV packaging signal is the RNA sequence of SEQ ID NO: 3. In some, and even more specific, embodiments of the first aspect, the 3'-UTR contains two TMV packaging signals, and each TMV packaging signal is the RNA sequence of SEQ ID NO: 4.

[0017] In some specific embodiments of the first aspect, the 3'-UTR is derived from a naturally occurring RNA sequence corresponding to the 3'-UTR of human α-globin (SEQ ID NO: 6). In some embodiments, the 3'-UTR is an RNA sequence comprising SEQ ID NO: 6 and an RNA sequence corresponding to two or more TMV packaging sequences located at and / or scattered therein at the 5' and 3' ends of SEQ ID NO: 6. In some specific embodiments of the first aspect, the 3'-UTR is derived from a modification of a naturally occurring DNA sequence encoding the 3'-UTR of human α-globin, corresponding to the RNA sequence shown in SEQ ID NO: 7. In some embodiments, the 3'-UTR is an RNA sequence comprising SEQ ID NO: 7 and an RNA sequence corresponding to two or more TMV packaging sequences located at and / or scattered therein at the 5' and 3' ends of SEQ ID NO: 7. In some embodiments, the 3'-UTR is an RNA sequence comprising SEQ ID NO: 7 and two or more TMV packaging sequences corresponding to and / or scattered therein at the 5' and 3' ends of SEQ ID NO: 7, and further corresponding to one or more poly(A) segments. In some, or even more specific, embodiments of the first aspect, the 3'-UTR is an RNA sequence comprising any of SEQ ID NO: 8-16, or an RNA sequence having at least 90% sequence identity with any of SEQ ID NO: 8-16 and comprising an RNA sequence corresponding to at least two copies of the minimum TMV packaging sequence of SEQ ID NO: 1. In some, or even more specific, embodiments of the first aspect, the 3'-UTR is an RNA sequence comprising any of SEQ ID NO: 8-16.

[0018] In some embodiments of the first aspect, the heteroRNA payload encodes a polypeptide or protein. In some embodiments of the first aspect, the heteroRNA payload encodes a mammalian polypeptide or protein. In some embodiments of the first aspect, the heteroRNA payload encodes a therapeutically useful mammalian polypeptide or protein.

[0019] In some embodiments of the first aspect, the heterologous RNA payload is non-coding (e.g., it does not encode a polypeptide sequence).

[0020] In some embodiments of the first aspect, the 5'-UTR is an RNA sequence containing the nucleotide sequence of SEQ ID NO: 17.

[0021] In a second aspect, this disclosure provides an expression vector comprising an isolated DNA sequence as described in any embodiment of the first aspect; and a promoter operatively linked to said DNA sequence.

[0022] 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 the nucleic acid sequence of SEQ ID NO: 18, a TEF1 promoter having the nucleic acid sequence of SEQ ID NO: 19, or a promoter having a nucleic acid sequence having at least 90% sequence identity with 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 the nucleic acid sequence of SEQ ID NO: 18 or a TEF1 promoter having the nucleic acid sequence of SEQ ID NO: 19.

[0023] 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, the nucleic acid sequence being operatively linked to a GAP promoter having the nucleic acid sequence of SEQ ID NO: 18 or a promoter having a nucleic acid sequence having at least 90% sequence identity with 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, the nucleic acid sequence being operatively linked to a GAP promoter having the nucleic acid sequence of SEQ ID NO: 18.

[0024] In a third aspect, this disclosure provides an isolated host cell containing the expression vector described in any embodiment of the second aspect.

[0025] In a fourth aspect, this disclosure provides an isolated host cell comprising: (i) a first expression vector comprising an isolated DNA sequence according to any embodiment of the first aspect; and a promoter operatively linked thereto to the DNA sequence; and (ii) a second expression vector comprising a nucleic acid sequence encoding a TMV viral capsid protein operatively linked to the promoter.

[0026] 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 fourth aspect, the promoter in the second expression vector is a GAP promoter having the nucleic acid sequence of SEQ ID NO: 18 or a promoter having a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 18. In some more specific embodiments of the fourth aspect, the promoter in the second promoter is a GAP promoter having the nucleic acid sequence of SEQ ID NO: 18.

[0027] In some embodiments of the third and fourth aspects, the host cell is a yeast cell, insect cell, plant cell, or mammalian cell. In some more specific embodiments 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 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 the third and fourth aspects, the host cell is a plant cell, namely *Nicotiana benthamiana*. In some more specific embodiments of each of the third and fourth aspects, the host cell is a mammalian cell selected from Chinese hamster ovary cells and HEK293T cells. In some embodiments of the third and fourth aspects, the host cell is not a *Yarrowia lipolytica* cell. In some embodiments of the third and fourth aspects, the host cell is not a Yeast cell.

[0028] In a fifth aspect, this disclosure provides a method for generating a heterologous RNA payload in a cell, the method comprising culturing a host cell according to any one of the third aspects under conditions for generating virus-like particles, thereby generating a heterologous RNA payload, the virus-like particles comprising the heterologous RNA payload bound to and encapsulated thereon with the TMV viral capsid protein.

[0029] In some embodiments of the fifth aspect, the method further comprises isolating the virus-like particles from the host cells. 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.

[0030] In a sixth aspect, this disclosure provides an isolated RNA molecule encoded by an isolated DNA sequence as described in the first aspect.

[0031] In a seventh aspect, this disclosure provides an isolated virus-like particle generated by the method according to the fifth aspect.

[0032] In an eighth aspect, this disclosure provides an isolated heterologous RNA molecule produced by the method according to the fifth aspect. In some embodiments of the eighth aspect, the isolated heterologous RNA molecule is purified from the isolated VLP of the seventh aspect.

[0033] In a ninth aspect, this 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 can be used as a 3'-UTR in an RNA sequence encoding the 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 NO: 7 can also be used as a 3'-UTR without the insertion of two or more TMV OAS. For example, the RNA sequence of SEQ ID NO: 7 can be used as a 3'-UTR in a standard in vitro transcription (IVT) system. In some embodiments of the ninth aspect, the DNA sequence capable of being transcribed into the RNA sequence 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 the protein of interest (e.g., luciferase, GFP, COVID-19 spike protein, another therapeutically useful protein); and a DNA sequence capable of being transcribed into SEQ ID NO: 7. Such a template may also contain a promoter (e.g., the T7 promoter) and / or a sequence capable of being transcribed at the 5' end into an appropriate mRNA capping signal (e.g., Cap-1 cap) and / or a sequence capable of being transcribed at the 3' end into a polyA tail. Attached Figure Description

[0034] The various objects, features and advantages of the disclosed subject matter can be more fully appreciated when considered in conjunction with the following detailed description of the subject matter, in which the same reference numerals identify the same elements.

[0035] Figure 1 The plasmid map of TMV capsid (an expression vector used to express TMV capsid proteins in Saccharomyces cerevisiae) was depicted.

[0036] Figure 2 A plasmid map of GFP UTR-4 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the EGFP coding sequence, and the 3'-UTR of SEQ ID NO: 8) was depicted.

[0037] Figure 3 A plasmid map of GFP UTR-3 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the EGFP coding sequence, and the 3'-UTR of SEQ ID NO: 23) was depicted.

[0038] Figure 4 A plasmid map of GFP UTR-1 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the firefly luciferase coding sequence, and the 3'-UTR of SEQ ID NO: 22) was depicted.

[0039] Figure 5 A plasmid map of Luc UTR-1 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the firefly luciferase coding sequence, and the 3'-UTR of SEQ ID NO: 22) was depicted.

[0040] Figure 6 A plasmid map of Luc UTR-3 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the firefly luciferase coding sequence, and the 3'-UTR of SEQ ID NO: 23) was depicted.

[0041] Figure 7 A plasmid map of Luc UTR-4 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the firefly luciferase coding sequence, and the 3'-UTR of SEQ ID NO: 8) was depicted.

[0042] Figure 8A denatured SDS-PAGE gel of crude lysate from Tni cells transfected with recombinant baculovirus encoding both a TMV capsid protein and an RNA sequence is described. The RNA sequence comprises, in 5' to 3' order, the 5'-UTR of SEQ ID NO: 17, an 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). An arrow on the right side of the depicted gel indicates the expected target size (17 kDa) of the TMV capsid protein. Molecular weight markers are shown in the leftmost lane, and the arrows and numbers indicate the approximate size of the markers.

[0043] Figure 9A-9I The inset images depict transmission electron microscopy (TEM) images of TMV VLPs from transformed or transfected cells. Inset A depicts a TMV VLP containing wild-type, native TMV RNA from an expression extract of *Saccharomyces cerevisiae*. Insets BD depict TMV VLPs containing UTR-4 (which contains two TMV packaging signals) as the 3'-UTR in Sf9 insect cells (Inset B), Tni insect cells (Inset C), and *Saccharomyces cerevisiae* cells (Inset D). Insets E and F depict TMV VLPs containing UTR-1 as the 3'-UTR in either Sf9 insect cells (Inset E) or Tni insect cells (Inset F). Inset GI depicts a TMV VLP containing UTR-3 as the 3'-UTR in Sf9 insect cells (Inset G), Tni insect cells (Inset H), and *Saccharomyces cerevisiae* cells (Inset I). All images were collected using a Joel JEM-1400Flash TEM.

[0044] Figure 10 Denatured SDS-PAGE gels of clarified yeast lysate supernatants from yeast cells transfected with TMV capsid and GFP UTR-4 plasmid were depicted before (“s”) and after PEG precipitation (“PEGs” indicates supernatant after PEG precipitation; “PEGp” indicates precipitate after PEG precipitation). The 17 kDa TMV capsid protein was evident in both the cell lysate supernatant before PEG treatment and the PEG precipitate, but not in the PEG supernatant. Lanes labeled “M” contain molecular weight markers, the size of which is indicated on the left side of the gel.

[0045] Figure 11 The results of ultracentrifugation based on CsCl density depict the resuspension of the clear lysate from yeast cells transfected with TMV capsids and GFP UTR-4 plasmid. Arrows indicate blurred bands representing VLPs.

[0046] Figure 12 Denatured SDS-PAGE gels from various fractions obtained by ultracentrifugation based on CsCl density were depicted. Arrows indicate bands representing approximately 17.5 kDa of the TMV capsid protein. Lanes labeled "M" contain molecular weight markers, the size of which is indicated on the left side of the gel.

[0047] Figures 13A-13B The inset figures depict agarose gel analysis of TMVVLPs produced in yeast transfected with TMV capsids and either GFP UTR-3 (inset A) or GFP UTR-4 (inset B) plasmids before (without benzonase) treatment and after treatment. Molecular weight markers are shown on the left side of each gel. Arrows indicate bands corresponding to RNA of the expected size encoded by GFP UTR-3 or GFP UTR-4.

[0048] Figures 14A-14B This is a bar graph depicting the results of THP-1 twin-cell transfection, derived from RNA isolated from TMV VLPs produced in yeast transfected with plasmid TMV capsids and GFP UTR-1, GFP UTR-3, or GFP UTR-4. Prior to RNA transfection, THP-1 twin cells were differentiated into macrophage-like cells using standard phorbol 12-myristate 13-acetate treatment. Inset A depicts the eGFP expression level, expressed in relative fluorescent units (RFU), in THP-1 twin cells transfected with RNA isolated from each of the GFP UTR-1, GFP UTR-3, or GFP UTR-4 transfected yeasts. Figure B depicts the immunogenicity of RNA isolated from VLPs purified from two different batches of GFP UTR-4 transfected yeast to THP-1 twin cells, as measured by interferon pathway-stimulated Lucia enzyme expression (RLU Lucia), compared to a poly(dA:dT) control. Lipofectamine, a chemical transfection reagent TM It was used for all samples and was also included as a control containing only lipofectamine, since background immunostimulation is known to be caused by the transfection agent.

[0049] Figures 15A-15C It is a bar graph depicting the amount of luciferase produced from RNA transcribed in vitro from a DNA template that is transfected into THP-1 (inset A), HepG2 (inset B), or HEK293 (inset C) cells containing natural α-globin 3'-UTR (SEQ ID NO: 6) or a variant thereof (SEQ ID NO: 7).

[0050] Figure 16This is a bar graph depicting the amount of luciferase produced in THP-1 double cells transfected with RNA isolated from TMV VLPs produced from Yersinia lipolytica cells, which were transfected with a plasmid YL-TMV capsid and a plasmid encoding RNA having a single TMV OAS (Luc OAS) directly located at the 3' of the luciferase coding sequence or two TMV OAS sequences (Luc UTR-4) located in the middle of the 3'-UTR and separated by 13 nucleotides.

[0051] Figure 17 A map of plasmid YL-Luc OAS (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the firefly luciferase coding sequence, and the 3'-UTR of SEQ ID NO: 29) was depicted.

[0052] Figure 18 A map of plasmid YL-Luc UTR-4 (an expression vector for expressing RNA molecules containing the 5'-UTR of SEQ ID NO: 17, the firefly luciferase coding sequence, and the 3'-UTR of SEQ ID NO: 8) in Yersinia lipophila cells was depicted.

[0053] Figure 19 A map of plasmid YL-TMV-capsid (an expression vector for expressing TMV capsid proteins in Yersinia lipophila) was drawn. Detailed Implementation

[0054] Details of one or more variations of the subject matter described herein are set forth in the accompanying drawings and description below. Other features and advantages of the subject matter described herein will become apparent from the description, drawings, and claims. However, the disclosed subject matter is not limited to any particular embodiment disclosed.

[0055] definition

[0056] As used herein, the term "DNA sequence capable of being transcribed into an RNA sequence" refers to a polydeoxyribonucleotide that serves as a template for transcription into an RNA sequence when contacted with an RNA polymerase. Such RNA polymerases can be added to the DNA sequence in vitro (e.g., in in vitro translation) or can be present in host cells containing such DNA sequences. RNA polymerases that perform this type of transcription and are present in host cells can be naturally encoded by the host cell or can be recombinantly generated in host cells containing such DNA sequences.

[0057] As used herein, the term "5'-UTR" or "5'-untranslated region" refers to a polynucleotide sequence that is not translated into an amino acid sequence and is located at the 5' end or upstream of the starting point of the RNA sequence serving as a heteroRNA payload. In embodiments, when the heteroRNA payload encodes a polypeptide, the functional RNA sequence begins at the start codon, and the 5'-UTR terminates at a nucleotide preceding the start codon. The 5'UTR typically contains secondary and tertiary structures, as well as other sequence elements that regulate the stability of the transcribed RNA and, in some embodiments, modulate the translatability of the heteroRNA payload.

[0058] As used herein, the term "3'-UTR" or "3'-untranslated region" refers to a polynucleotide sequence that is not translated into an amino acid sequence and is located at the 3' end or downstream of the RNA sequence serving as a heteroRNA payload. In some embodiments, when the heteroRNA payload encodes a polypeptide, the functional RNA sequence ends with a stop codon, and the 3'-UTR begins with the nucleotide immediately following the stop codon. The 3'-UTR typically contains secondary and tertiary structures as well as other sequence elements that can affect the stability of the transcribed RNA molecule and, in some embodiments, affect the translatability of the heteroRNA payload. In some, but not all, embodiments, the 3'-UTR contains a polyadenylated tail.

[0059] As used herein, the term "heterologous RNA payload" means an RNA molecule that is not naturally present in tobacco mosaic virus and is active at the RNA level (e.g., not translated into protein) or by encoding a polypeptide (e.g., as mRNA) during translation. The heterologous mRNA payloads disclosed herein are intended for therapeutic, preventative, diagnostic, or research use in cells, cell lysates, in vitro translation systems, tissues, or organisms.

[0060] As used herein, the term "TMV packaging signal" refers to an RNA sequence that can bind to the capsid protein of tobacco mosaic virus.

[0061] As used herein, the term "proximity TMV packaging signals" refers to the pair of TMV packaging signals that are closest to each other in an RNA molecule. For clarity, an RNA molecule contains three TMV packaging signals, named "1", "2", and "3" in the structure: 1-2-3, where each pair of adjacent TMV packaging signals is separated by less than XX nucleotides (where XX is an integer), meaning that "1" and "2" are separated by less than XX nucleotides, and "2" and "3" are separated by less than XX nucleotides. This does not imply or suggest that "1" and "3" are separated by less than XX nucleotides.

[0062] As used herein, the term "operably linked" refers to the linking of a first molecule to a second molecule, wherein the molecules are arranged such that the first molecule influences the function of the second molecule. The two molecules may or may not be part of a single, continuous molecule and may or may not be adjacent. For example, if a promoter regulates the transcription of a transcribed polynucleotide molecule payload in a cell, then the promoter is operably linked to the transcribed polynucleotide molecule. Additionally, if two parts of a transcriptional regulatory element are linked such that the transcriptional activation function of one part is not adversely affected by the presence of the other part, then the two parts are operably linked to each other. Two transcriptional regulatory elements can be operably linked to each other via an adapter nucleic acid (e.g., an intermediate non-coding nucleic acid), or they can be operably linked to each other in the absence of an intermediate nucleotide.

[0063] As used herein, the term "promoter" refers to a recognition site on DNA that is bound by RNA polymerase. Polymerase drives transcription of DNA operatively linked to such promoters. The term "constitutive promoter" refers to a promoter whose ability to bind to RNA polymerase is not significantly affected by environmental or other factors.

[0064] The "sequence identity percentage (%)" relative 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 those in the reference polynucleotide or polypeptide sequence after alignment and the introduction of vacancies (if necessary) to achieve the maximum sequence identity percentage. Alignment for determining the nucleic acid or amino acid sequence identity percentage can be performed in various ways within the capabilities of those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. For example, the sequence identity percentage value can be generated using the sequence comparison computer program BLAST. As an illustration, the sequence identity percentage of a given nucleic acid or amino acid sequence A with, and, or relative to a given nucleic acid or amino acid sequence B (which can alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percentage of sequence identity with, and, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows:

[0065] 100 multiplied by (fraction X / Y)

[0066] Where X is the number of nucleotides or amino acids that are scored as identical matches by a sequence alignment program (e.g., BLAST) in the alignment of A and B, and Y is the total number of nucleic acids in B. It should be understood that when 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 percentage of sequence identity between A and B will not be equal to the percentage of sequence identity between B and A.

[0067] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids, viscera, or artificial chromosomes), RNA vectors, viruses, or any other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use herein contain polydeoxyribonucleotide sequences that can be transcribed into RNA, along with additional sequence elements, such as those used for such transcription. As described herein, some vectors suitable for RNA transcription include vectors containing regulatory sequences, such as promoter and enhancer regions that direct transcription. Expression vectors suitable for use herein may also contain polynucleotides encoding markers for the selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0068] Overview

[0069] This disclosure provides isolated DNA sequences, expression vectors, host cells, and methods for generating RNA payloads in living cells, as well as the resulting RNA payloads. According to the methods described herein, the RNA payloads are prepared into virus-like particles (VLPs) containing the RNA payload. VLPs are composed of viral capsid proteins that self-assemble to mimic the structure of the natural viruses from which they are derived. VLPs are non-infectious because they lack the genetic material required for integration, reverse transcription, replication, and / or infection. Isolated VLPs containing RNA payloads are also provided herein.

[0070] DNA and RNA sequences

[0071] On one hand, this disclosure provides: (i) an isolated DNA sequence capable of being transcribed into an RNA molecule comprising a 5'-UTR, a heterologous RNA payload, and a 3'-UTR in a 5' to 3' sequence, wherein the 3'-UTR contains two or more tobacco mosaic virus (TMV) packaging signals; and (ii) a transcribed RNA molecule. This isolated DNA sequence is designed to be transcribed into a single RNA transcript (i.e., in host cells transfected or transformed with or containing such a DNA sequence). The RNA transcript can be packaged into TMV virus-like particles upon contact with the TMV capsid protein due to the interaction of the TMV packaging signals with the capsid protein. Furthermore, the applicant believes that, in some embodiments, the presence of two or more TMV packaging signals results in an unexpected improvement over a similar transcribed RNA sequence containing only one TMV packaging signal. Such improvements include one or more of the following: an increased number of TMV VLPs, an increased amount of RNA recovered from the VLPs, and improved quality of the RNA recovered from the VLPs in terms of stability and ability to be translated into proteins.

[0072] 5'-UTR

[0073] As described above, the RNA transcripts disclosed herein contain a 5'-UTR. The 5'-UTR sequence disclosed herein is a region of transcribed RNA that is not translated into an amino acid sequence and is not necessarily directly involved in the activity of the heterologous RNA payload, but includes regulatory elements that regulate 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 just before the start of the functional RNA sequence (e.g., the start codon when the heterologous RNA payload encodes a polypeptide).

[0074] Naturally occurring RNA transcripts typically contain a 5'-UTR. In some embodiments, the 5'UTR contains a nucleic acid sequence naturally present in transcripts encoding a heterologous RNA payload. In some embodiments, the 5'UTR contains a nucleic acid sequence not naturally associated with the encoded heterologous mRNA payload. In some embodiments, the 5'UTR contains a nucleic acid sequence with high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to a nucleic acid sequence naturally present in transcripts encoding a heterologous RNA payload, wherein those dissimilar nucleotides confer quantitative or qualitative improvements to the transcript (e.g., in terms of stability, function, and / or translatability). Modifications to the naturally occurring 5'-UTR that produce such improvements are known in the art.

[0075] 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 cells used to generate the VLP. For example, the 5'-UTR and RNA payload can be human, while the cells used to generate the VLP containing such RNA payloads are non-human cells, such as mouse cells, rat cells, hamster cells, insect cells, yeast cells, bacterial cells, or plant cells.

[0076] The 5'-UTR may have a sequence or structure that alters (e.g., increases, decreases, or prevents) the level of RNA payload translation into a polypeptide. In some embodiments, the 5'-UTR has a sequence or structure that promotes cell-type-specific translation of the RNA payload.

[0077] The 5'UTR may 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 a ribosome or autocleavage ribozyme and, once bound to the sequence, initiates the translation of an RNA payload into a polypeptide. In some embodiments, the ribosome-binding sequence may be a Kozak sequence. In some embodiments, the 5'UTR includes intron substitution to mediate RNA self-circularization.

[0078] The 5'-UTR sequence disclosed herein can differ from the 5'-UTR obtained in transcribed RNA through 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 in an expression vector for final transcription in a host cell, the expression vector itself can serve as a template to synthesize additional ribonucleotides added to the 5' end of the encoded 5'-UTR. In some embodiments, the 5'-UTR can be capped. Capping refers to the modification of the first (5'-last) transcribed nucleotide of the RNA transcript by an enzyme present in the cell that produces such a transcript. Capping typically introduces a methylated guanosine onto the first transcribed nucleotide. Capping blocks 5'-3' exonuclease-mediated degradation and recruits specific RNA processing, export, and translation factors. The presence of the cap and the specific modification of the cap depend on the cell in which the isolated DNA sequence disclosed herein is transcribed. In some embodiments, the cap may 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 methylation of the N6 position of adenosine (also referred to herein as 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 a cell population (e.g., cells that generate VLPs) that transcribes the isolated DNA sequences disclosed herein can be achieved by simultaneous recombinant expression of a native enzyme in such cell population and / or by a capping enzyme or a polynucleotide encoding a capping enzyme (e.g., a 2'O methyltransferase). Modification of the 5'-UTR cap can also be achieved after RNA isolation (i.e., from VLPs) by treatment with an isolated methyltransferase.

[0079] In some embodiments, the 5'-UTR is an RNA sequence containing the following nucleotide sequence:

[0080] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCCGCCACC (SEQ ID NO: 17).

[0081] 3'-UTR

[0082] As described above, the RNA transcripts disclosed herein contain a 3'-UTR. The 3'-UTR is a region of the resulting transcribed RNA that is not translated into an amino acid sequence and is not necessarily directly involved in the activity of the heterologous RNA payload, but includes regulatory elements that regulate 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., a stop codon when the heterologous RNA payload encodes a polypeptide) and continues to the end of the transcribed RNA.

[0083] Naturally occurring RNA transcripts typically contain a 3'-UTR. In some embodiments, the 3' UTR disclosed herein comprises a nucleic acid sequence naturally present in transcripts encoding a heterologous RNA payload. In some embodiments, the 3' UTR comprises a nucleic acid sequence not naturally associated with an encoded heterologous mRNA payload. In some embodiments, the 3' UTR comprises a nucleic acid sequence having high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) with a nucleic acid sequence naturally present in transcripts encoding a heterologous RNA payload, wherein those dissimilar nucleotides confer quantitative or qualitative improvements to the transcript (e.g., in terms of stability, function, and / or translatability). Modifications to the naturally occurring 3'-UTR that produce such improvements are known in the art.

[0084] In some embodiments, the 3'-UTR includes a sequence element that mediates binding to a protein involved in mRNA transport and / or translation. In some embodiments, the 3'-UTR may include a sequence that represses mRNA deadenylation. In some embodiments, the 3'-UTR may include a polyadenylation signal sequence. In some embodiments, the 3'-UTR may include a polyadenylation tail. In some embodiments, the 3'-UTR may include a nucleotide sequence that represses mRNA deadenylation. As a non-limiting example, the 3'-UTR may include one or more regions of human α-globin or the entire 3'-UTR, which corresponds to a DNA sequence:

[0085] GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 6)

[0086] Or its variants:

[0087] GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGAUGGGGGCACAGAAACUUAUUUCAGACUCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 7)

[0088] Two or more TMV packaging signals are located at one or both ends and / or scattered within such 3'-UTRs, and optionally contain one or more poly(A) segments of varying lengths. It should be understood that when generating DNA encoding a 3'-UTR containing a TMV packaging signal, particularly when the TMV packaging signal is inserted into a parental 3'-UTR (e.g., the 3'-UTR of SEQ ID NO: 6 or SEQ ID NO: 7), a clonal artificial product may be introduced if a nucleotide in the parental 3'-UTR is deleted or its position is altered, or if an additional nucleotide is inserted into the parental 3'-UTR. For example, in SEQ ID NO: 8 below, the G nucleotide at the 5' of the first TMV packaging sequence is shifted compared to the native α-globin 3'-UTR from which it originated in SEQ ID NO: 6. The position of G in SEQ ID NO: 6 is indicated in bold italics below. In SEQ ID NO: 8, G is shifted to the empty position indicated by an asterisk below, as indicated by the arrow:

[0089]

[0090] The applicant believes that this one nucleotide difference has the least impact on 3'-UTR function.

[0091] In some embodiments, the 3'-UTR is an RNA sequence comprising any one of the following nucleotide sequences in Table 1. In Table 1, the nucleotides shown in bold are portions of the RNA sequence corresponding to the TMV packaging signal.

[0092] Table 1. 3'-UTR sequences

[0093]

[0094]

[0095]

[0096] HeteroRNA payload

[0097] As mentioned above, the transcribed RNA sequences disclosed in this paper contain heterologous RNA payloads.

[0098] RNA payloads can have any length, for example, at least 9 nucleotides. For example, the length of an RNA payload 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, or 400 nucleotides. One, 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 some embodiments, the length of the RNA payload may be less than 50,000, 45,000, 40,000, 35,000, 30,000, 25,000, 20,000, 15,000, 10,000, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides.In some embodiments, the length of 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.

[0099] RNA payloads can be derived from naturally occurring RNA, or they can be synthetic or chimeric RNA.

[0100] 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 non-limiting example, the mammalian RNA payload can be a human RNA payload. In some embodiments, the RNA payload can be homologous to the cell population used to generate a VLP containing the RNA payload. In some embodiments, the RNA payload can be heterologous to the cell population used to generate a VLP containing the RNA payload. As used herein, "homologous" means two entities derived from the same species, and "heterologous" means two entities derived from different species. For example, the RNA payload can be a human RNA payload, and the cell population can be a human cell population (e.g., a human cell line). As another example, the RNA payload can be a human RNA payload, and the cell population can be a hamster cell population (e.g., a hamster-derived cell line, such as CHO cells).

[0101] In some embodiments, the RNA payload may be a self-amplifying RNA payload, which may include nucleotide sequences encoding non-structural proteins. These non-structural proteins may be, for example, nsP1, nsP2, nsP3, and / or nsP4 of the alphavirus genus.

[0102] RNA-encoding payload

[0103] RNA payloads can be RNA-coding payloads. As used herein, “RNA-coding payload” means an RNA payload capable of being translated into a protein-coding vector (e.g., in vitro, in vivo, in situ, or ex vivo). RNA-coding payloads will begin with a start codon and end with a stop codon sequence. In some embodiments, RNA-coding payloads include introns. Introns can facilitate nuclear output of ORF payloads. In one embodiment, an ORF payload includes substitutional introns for generating circular RNA.

[0104] The RNA-coding payload can have any length. In some embodiments, the length of the RNA-coding payload can be at least 30 nucleotides. In some embodiments, the length of the RNA-coding payload can be at least 300 nucleotides. In some embodiments, the length of the RNA-coding payload can be at least 4500 nucleotides. For example, the length of the RNA-encoding payload 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 some embodiments, the length of the encoding RNA payload may be less than 50,000, 45,000, 40,000, 35,000, 30,000, 25,000, 20,000, 15,000, 10,000, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, or 30 nucleotides.In some embodiments, the length of the encoding 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.

[0105] In some embodiments, in the cell population used to prepare a VLP containing an RNA-encoding payload, the RNA-encoding payload is not translated or is translated to a minimum into a polypeptide or protein sequence.

[0106] The encoding RNA payload may encode, for example, bacterial proteins or peptides, genome editing enzymes (e.g., Cas9, transposases, megnucleases, base editors, or retrotransposons), leader editors, DNA writers, viral proteins or peptides, eukaryotic transcription factors (e.g., mammalian transcription factors), growth factors, antibodies, peptides containing one or more viral or bacterial epitopes or antigens, enzymes, or therapeutically useful mammalian peptides (e.g., therapeutically useful human peptides). In some embodiments, the encoding RNA payload encodes a neoantigen. In some embodiments, the neoantigen is a mammalian cancer neoantigen. In some embodiments, the encoding RNA payload encodes a therapeutically useful human peptide. In some embodiments, the encoding RNA payload encodes a therapeutically useful human enzyme. In some embodiments, the encoding RNA payload encodes a therapeutically useful human enzyme that can be used to treat diseases or conditions characterized by reduced activity of the natural form of such enzymes.

[0107] In some embodiments, the encoding RNA payload may encode a peptide that can be used for immunotherapy. In some embodiments, the peptide may be a chimeric antigen receptor (CAR), such as an anti-c-Met CAR or an anti-CD19 CAR. In some embodiments, the encoding RNA payload may encode an immunomodulatory protein, such as a cytokine or a co-stimulatory molecule peptide. In some embodiments, the encoding RNA payload may encode a peptide that can be used as an immunotherapeutic vaccine. For example, the peptide may be the full length or a portion of a protein such as p53, survivin, gp100, tyrosinase, mTRP-2, MAGE-A3, MAGE-C2, PSCA, PSMA, PSA, STEAP1, NY-ESO-1, 5T4, MAGE-C1, telomerase, WT1, hTERT, and / or pp65.

[0108] In some embodiments, the RNA-encoding payload may encode a protein or peptide for protein substitution in hereditary diseases, lung diseases, hematologic disorders, orthopedic diseases, neurological diseases, metabolic diseases, and / or cardiovascular diseases. The RNA-encoding payload may encode methylmalonyl-CoA mutase, propionyl-CoA carboxylase, ornithine carbamoyltransferase, α1-antitrypsin, CFTR, factor VIII, factor IX, VEGFA, and / or regions or portions thereof.

[0109] In some embodiments, the RNA-encoding payload may encode gene-editing nucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, base editors, leader editors, DNA writers, and / or clustered regular-spaced short palindromic repeat (CRISPR)-associated protein nuclease systems.

[0110] In some embodiments, the encoding RNA payload may encode a peptide associated with the treatment of an infectious disease. The encoding RNA payload may encode a vaccine for an infectious disease. For example, the encoding RNA payload may encode a peptide that can be used as a COVID-19 vaccine. Such peptides may contain the complete protein or a portion thereof, such as, but not limited to, S-2P (and its variants), a receptor-binding domain, a trimerized receptor-binding domain, and / or a transmembrane spike (S) protein. In some embodiments, the encoding RNA payload may encode a rabies vaccine comprising a rabies virus glycoprotein. In some embodiments, the encoding RNA payload may encode an influenza vaccine comprising a hemagglutinin glycoprotein or a portion thereof. In some embodiments, the encoding RNA payload may encode an HIV vaccine comprising one or more of HIV Gag, Nef, Vpr, Rev proteins or portions thereof. In some embodiments, the encoding RNA payload may encode an anti-chikungunya virus antibody. As a non-limiting example, the encoding RNA payload may be the SARS-CoV-2 spike protein or a region or portion thereof.

[0111] In some embodiments, the RNA payload may encode bacterial antigens.

[0112] In some embodiments, the RNA payload may encode an immunoglobulin (IgG) for use in passive immunotherapy.

[0113] In some embodiments, the sequence encoding the RNA payload may be optimized to facilitate the translation of the RNA payload in cells, cell populations, or organisms intended for administration once the RNA is isolated from the cells used for VLP generation. In some embodiments, the sequence encoding the RNA payload may be optimized to increase the half-life of the RNA payload. In one embodiment, codon optimization or uridine deletion of the RNA payload may be performed to reduce immunogenicity in cells, cell populations, or organisms intended for administration of the RNA.

[0114] non-coding RNA payload

[0115] The RNA payloads used in this paper can be non-coding RNA payloads. RNA molecules that are not translated into encoded peptides, polypeptides, or proteins are referred to as non-coding RNAs in this paper. Non-coding RNA payloads can be short or long. Short non-coding RNA payloads can be 200, 150, 100, 75, 50, 25 nucleotides or less in length. Non-restrictive examples of short non-coding RNAs include microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small Cajal body-specific RNAs (scaRNAs), trans-activating CRISPR (tracr) RNAs, small nuclear RNAs (snRNAs), U-rich snRNAs (snRNAs), small nucleolar RNAs (snoRNAs), and / or Y RNAs.

[0116] Long non-coding RNAs can be longer than 200 nucleotides (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 RNAs include long intergenic ncRNAs (lincRNAs), naturally antisense transcript (NAT) RNAs, and / or circular RNAs (circRNAs). In one embodiment, the RNA payload is RNA with substitution introns to circularize the RNA to form circRNA.

[0117] Non-coding RNAs include, but are not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), signal recognition particle RNA (SRPRNA), antisense RNA (aRNA), microRNA (miRNA), small interfering RNA (siRNA), Y RNA, telomerase RNA and / or piwi-interacting RNA (piRNA), and RNA encoding RNA-dependent RNA polymerase complexes for self-amplification.

[0118] RNA payloads can be single-stranded or double-stranded. On the one hand, RNA payloads are single-stranded. Encoding RNA payloads are typically single-stranded.

[0119] TMV Packaging Signal

[0120] As described above, the transcribed RNA sequences disclosed herein contain two or more tobacco mosaic virus (TMV) packaging signals present in the 3'-UTR. In TMV, packaging signals are commonly referred to in the prior art as assembly origin sequences or OAS. The role of the TMV packaging signals in the transcribed RNA molecules described herein is to bind to TMV capsid proteins expressed in the same cells as the transcribed RNA, which allows the RNA to be encapsulated by TMVVLPs generated by the TMV capsid proteins in such cells. It is believed that the packaging signals may also play a role in VLP assembly and formation.

[0121] The size range of each of the two or more TMV packaging signals available in this document can independently be 75 to approximately 500 nucleotides in length, 75 to 300 nucleotides in length, 75 to 250 nucleotides in length, 100 to 250 nucleotides in length, or 100 to 200 nucleotides in length, and each will contain nucleotides corresponding to the following RNA sequences:

[0122] UGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCA (SEQ ID NO: 1).

[0123] SEQ ID NO: 1 is a 75-ribonucleotide sequence considered the minimum sequence necessary for binding to the TMV shell protein and allowing VLP assembly. In some embodiments, the sequence of the TMV packaging signal has 100% sequence identity with the sequence in naturally occurring TMV. In some embodiments, each of two or more TMV packaging signals independently corresponds to one of SEQ ID NO: 1-5 shown in Table 2 below, or a nucleotide sequence containing SEQ ID NO: 1 and having at least 90% sequence identity with any of SEQ ID NO: 1-5.

[0124] Table 2.

[0125]

[0126] In some embodiments, each of the two or more TMV packaging signals is independently one of SEQ ID NO: 1-5. In some embodiments, each of the two or more TMV packaging signals is identical and is one of SEQ ID NO: 1-5. In some embodiments, each of the two or more TMV packaging signals is identical 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.

[0127] As described above, in some embodiments, the distance between any two adjacent TMV packaging signals in the 3'UTR is 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 is evident that the maximum distance between two adjacent TMV packaging signals cannot exceed the length of the 3'-UTR in which they reside.

[0128] Expression vectors and promoters

[0129] In some embodiments, the DNA sequence disclosed herein is double-stranded and integrated into an expression vector. To allow cells containing such a vector to transcribe RNA from it, the expression vector must include a promoter operatively linked to the DNA sequence. The promoter can be a strong promoter, a weak promoter, a constitutive promoter, an inducible promoter, a cell-type-specific promoter, or a tissue-specific promoter. The promoter can be constitutive or inducible. The choice of promoter can be optimized based on the cell type and / or the nature of the heterologous RNA payload. In some embodiments, the promoter can be optimized for eukaryotic cells. For example, the promoter can be optimized for mammalian cells such as human cells. As another example, the promoter can be optimized for yeast cells.

[0130] Non-limiting examples of constitutive promoters include the TDH3 promoter, CCW12 promoter, PGK1 promoter, HHF2 promoter, TEF1 promoter, TEF2 promoter, YEF3 promoter, RPL3 promoter, RPL15A 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, and ENO2 promoter. The promoters include: 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-1α promoter, mPGK promoter, β-actin promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrosis protein promoter, CaMKIIa promoter, GDS promoter, ADH1 promoter, Ubi promoter, or CAG promoter.

[0131] Non-limiting examples of inducible promoters include the GAL1 promoter, GAL10 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.

[0132] The choice of promoter can determine the level of RNA transcribed from a DNA sequence, and under what conditions, if any, such transcription can occur. This allows for fine-tuning of the transcribed RNA level, enabling optimization of the formation of VLPs encapsulating such RNA (e.g., in cells co-expressing the TMV coat protein).

[0133] In some embodiments, the promoter used to drive transcription of the encoded RNA in the expression vector is a GAP promoter. In some embodiments, the promoter used to drive transcription of the encoded RNA in the expression vector is a GAP promoter, which comprises the following DNA sequence:

[0134] (SEQ IDNO: 18).

[0135] In some embodiments, the promoter used to drive transcription of the encoded RNA in the expression vector is the TEF1 promoter. In some embodiments, the promoter used to drive transcription of the encoded RNA in the expression vector is the TEF1 promoter, which comprises the following DNA sequence:

[0136] CCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAA AAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG (SEQ ID NO: 19).

[0137] In some embodiments, the promoter used in the expression vector to drive transcription of the encoded RNA is a TEF promoter that further includes an upstream activation sequence (UAS), as described in M ​​Larroude et al., Microbial Biotechnology (2019) 12(6), 1249-1259.

[0138] In some embodiments, the promoter used in the expression vector to drive transcription of the encoded RNA is a derivative of the P2_8UASxpr-TEF promoter (M Larroude et al., Microbial Biotechnology (2019) 12(6), 1249-1259) and contains the following DNA sequence:

[0139]

[0140] In some embodiments, the promoter used in the expression vector to drive transcription of the encoded RNA is the P1_4UASxpr-TEF (M Larroude et al., ibid.) promoter, which contains the following DNA sequence:

[0141] ACGGCGATACGCGTATCGATACGCGTGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGGCATGCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGAGGGTTGTTCCCGCCCACCTCGATCCGGCATGCACTGATCACGGGCAAAAGTGCGTTCGATAGAGAGGGGGTTGGCGGCGCATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGACCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAGGATCCAATG ("UAS4-1"; SEQ ID NO: 30).

[0142] In some embodiments, the promoter for driving transcription of the encoded RNA in the expression vector is the pTEF promoter, said promoter comprising the following DNA sequence:

[0143] ACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTCAA(SEQ ID NO: 34)

[0144] In addition to those encoding promoters and those transcribed into RNA molecules, the expression vectors disclosed herein may include other polynucleotides. For example, expression vectors may include one or more origins of replication, antibiotic resistance genes, auxotrophic markers, genes encoding one or more proteins that enhance transcription and / or stability of the encoded RNA molecule, 5'-UTR sequences added to the 5' end of RNA, and / or 3'-UTR sequences added to the 3' end of RNA. Antibiotic resistance genes include, but are not limited to, ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes. 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 transferases, such as RNA guanine-N7-methyltransferase, cap O-methyltransferase (RNMT), and cap methyltransferases 1 and 2 (CMTr1 / 2); endonuclease inhibitors; enzymes that increase the content or abundance of modified nucleotides, such as methyltransferases and pseudouridine synthases; RNase inhibitors; and / or ribozymes. Ribonucleotides that can be added to the 5' end of the 5'-UTR via expression vectors include, but are not limited to, nucleotides that bind to transcription factors, nucleotides that facilitate the translation of heterologous RNA-encoding payloads, nucleotides that contribute to the stability of heterologous RNA-encoding payloads, and nucleotides that serve as targets for RNA capping enzymes. Ribonucleotides that can be added to the 3' end of the 3'-UTR sequence via expression vectors include, but are not limited to, nucleotides encoding polyadenylation signals, nucleotides encoding poly(A) tails, nucleotides that facilitate the translation of heterologous RNA-encoding payloads, nucleotides that contribute to the stability of heterologous RNA-encoding payloads, nucleotides that contribute to the transport of the resulting RNA molecule, and nucleotides that serve as terminators. A terminator is an RNA sequence that typically appears at the end of a transcript and stops transcription. Terminators can also affect the half-life of the RNA transcript. The choice of expression vector can depend in part on the cell type in which it will ultimately be contained, and on the cell type in which it will proliferate (if different from the cell type containing it). Non-limiting examples of expression vectors available in this paper include ESC-HIS, ESC-LEU, ESC-TRP, ESC-URA, JMP62-URA, JMP62-HIS, CfB8644, CfB8788, RK-flag-USP19 and / or Tet07-CSII-CR5.

[0145] The choice of terminator can depend in part on the promoter used to transcribe the heterologous RNA molecule, as well as other factors known in the art. Specifically, available terminators at the 3' end of the heterologous RNA molecule include the CYC1 terminator:

[0146] AUCCGCUCUAACCGAAAAGGAAGGUUAGACAACCUGAAGUCUAGGUCCCUAUUUAUUUUUUAUAGUUAUGUUAGUAUUAAGAACGUUAUUUAUAUUUCAAAUUUUCUUUUUUUCUGUACAGACGCGUGUACGCAUGUAACAUUAUACUGAAAACCUUGCUUGAGAAGGUUUUGGGACGCUCGAAG (SEQ ID NO: 25)

[0147] T1_Tef (M Larroude et al., ibid.) Terminator:

[0148] UCUAGCUGCUUGUACCUAUGCAACCCCAGUUUGUUAAAAAUUAGUAGUCAAAAACUUCUGAGUUAAAAAAAAAAAAAAAAGCUU ("TTEF"; SEQ ID NO: 26),

[0149] T2-3_Lip2 (M Larroude et al., ibid.) Termination component:

[0150] and

[0151] T1-3_Lip2 (M Larroude et al., ibid.) Termination component:

[0152] GUGUCUGUGGUAUCUAAGCUAUUUAUCACUCUUUACAACUUCUACCUCAACUAUCUACUUUAAUAAAUGAAUAUCGUUUAUUCUCUAUGAUUACUGUAUAUGCGUUCCUAAGACA (SEQ ID NO: 35).

[0153] TMV capsid protein

[0154] To generate a TMV VLP, the RNA sequence described herein (e.g., RNA comprising a 5'-UTR, a heterologous RNA payload, and a 3'-UTR in a 5' to 3' sequence, wherein the 3'-UTR contains two or more tobacco mosaic virus (TMV) packaging signals) must be co-expressed in the cell with a TMV capsid protein. The TMV capsid protein can be a naturally occurring TMV capsid protein or a variant thereof capable of forming a VLP and encapsulating the RNA sequence. Variants of the TMV capsid protein are known in the art. In some embodiments, the TMV capsid protein co-expressed in the cell has the following amino acid sequence:

[0155] MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQVTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRVDDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO: 21).

[0156] In some embodiments, the TMV capsid protein is encoded by the following DNA sequence:

[0157] ATGTCTTACAGTATCACTCCATCTCAGTTCGTGTTCTTGTCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGAAATCAGTTTCAAACACAACAAGCTCGAACTGTCGTTCAAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCCCTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGT CACAGCACTGTTAGGTGCATTCGACACTAGAAATAGAATAATAGAAGTTGAAAATCAGGCGAACCCCACGACTGCCGAAACGTTAGATGCTACTCGTAGAGTAGACGACGCAACGGTGGCCATAAGGAGCGCGATAAATAATTTAATAGTAGAATTGATCAGAGGAACCGGATCTTATAATCGGAGCTCTTTCGAGAGCTCTTCTGGTTTGGTTTGGACCTCTGGTCCTGCAACTTGA (SEQ ID NO: 20),

[0158] Or a DNA sequence that has at least 50% sequence identity with SEQ ID NO: 20 and encodes the same amino acid sequence as SEQ ID NO: 20.

[0159] In some embodiments, the amino acid sequence of the TMV capsid protein may include up to 10 amino acid insertions, deletions, or substitutions, provided that the resulting polypeptide can form a complete TMV VLP with the RNA molecule disclosed herein, the complete TMV VLP encapsulating such RNA molecule. In some embodiments, the amino acid substitutions are conserved amino acid substitutions, as is well known in the art.

[0160] The TMV capsid protein can be expressed in cells using an expression vector encoding the protein. In some embodiments, the expression vector encoding the TMV capsid protein is separate from the expression vector encoding the RNA sequence described herein. In some embodiments, the expression vector encoding the TMV capsid protein is the same as the expression vector encoding the RNA sequence described herein (e.g., both the RNA sequence and the TMV capsid protein are encoded on a single expression vector).

[0161] Expression of the TMV capsid protein from an expression vector requires the DNA sequence encoding the protein to be operatively linked to a promoter. Such a promoter can be the same promoter that drives transcription of the RNA sequence described herein (e.g., a second copy of the promoter or a single copy of a promoter capable of transcribing both the DNA and RNA sequences encoding the TMV capsid protein (e.g., a bidirectional promoter)) or a different promoter. The promoter driving TMV capsid protein expression can be a strong promoter, a weak promoter, a constitutive promoter, an inducible promoter, a cell type-specific promoter, or a tissue-specific promoter. The promoter can be constitutive or inducible. The selection of the promoter can be optimized for the cell type expressing the capsid protein. In some embodiments, the promoter can be optimized for eukaryotic cells. For example, the promoter can be optimized for mammalian cells such as human cells. As another example, the promoter can be optimized for yeast cells. The specific selection of the promoter can be chosen from the list of constitutive and inducible promoters shown above. In some embodiments, the promoter used to drive TMV capsid protein expression is the GAP promoter. In some embodiments, the promoter for driving transcription of the encoded RNA in the expression vector is the GAP promoter, which comprises the DNA sequence of SEQ ID NO: 18. In some embodiments, the promoter for driving transcription of the encoded RNA in the expression vector is the UAS4-1 promoter, which comprises the DNA sequence of SEQ ID NO: 30.

[0162] In some embodiments, the RNA transcript encoding the TMV capsid protein includes a terminator at its 3' end. In some embodiments, the terminator is the TTEF terminator or SEQ ID NO: 32.

[0163] host cells

[0164] As described above, this disclosure provides a host cell containing an expression vector encoding an RNA sequence disclosed herein and an expression vector encoding a TMV capsid protein. As detailed above, in some embodiments, the host cell will contain a single expression vector encoding both the RNA sequence and the TMV capsid protein. In other embodiments, the host cell will contain two separate expression vectors—one encoding the RNA sequence disclosed herein and the other encoding the TMV capsid protein.

[0165] In some embodiments, the host cell may naturally or recombinantly express enzymes and proteins that can promote transcription, modification, and / or stabilize RNA sequences. Such enzymes and proteins include, but are not limited to, RNA polymerases, DNA polymerases, RNA cap transferases, RNA ligases, RNase inhibitors, and / or enzymes that increase the content of modified nucleotides. Some non-limiting examples of specific enzymes or proteins that can be expressed include 2'-O methyltransferases or enzyme complexes that promote the addition of a methyl group at the 2'-O position of the first nucleotide adjacent to the cap structure at the 5' end of RNA; pseudouridine synthases that promote site-specific isomerization of uridine to pseudouridine or N1-methyl-pseudouridine; RNA guanine-N7-methyltransferases; cap O-methyltransferases (RNMT); cap methyltransferase 1 or 2 (CMTr1 or 2); ribozymes; tobacco mosaic virus RNA-dependent polymerase; bromeliad mosaic virus RNA polymerase; turnip shrub virus RNA polymerase; potato virus X RNA polymerase; cowpea mild mottle virus RNA polymerase; MS2 phage RNA polymerase; T7 RNA polymerase; T3 RNA polymerase; and SP6 RNA polymerase. Host cells can also express auxotrophic markers naturally or recombinantly. Non-limiting examples of auxotrophic markers include HIS3, LEU2, TRP, MET15, and / or URA3. As described above, any of these enzymes or proteins may also be encoded on expression vectors that also encode RNA sequences, expression vectors that also encode TMV capsid proteins, or expression vectors that encode both RNA sequences and TMV capsid proteins.

[0166] The host cell disclosed herein may be a plant cell, an insect cell, a yeast cell, or a mammalian cell, a population of yeast cells, a population of plant cells, a population of insect cells, a population of mammalian cells, or any combination thereof.

[0167] In some embodiments, the host cell is a plant cell. Plant cells that can be used as host cells in this disclosure may be tobacco plant cells. For example, the plant cell may be a tobacco plant cell or a *Tobacco Benzoica* plant cell. Other non-limiting examples of plant cells available herein are potato plant cells, tomato plant cells, lettuce plant cells, carrot plant cells, corn plant cells, soybean plant cells, or spinach plant cells.

[0168] In some embodiments, the host cell is an insect cell. Insect cells that can be used as host cells in this disclosure may be cells of the gray-winged armyworm, the white armyworm, or fruit fly. As a non-limiting example, the insect cell may be a fall armyworm cell. In one embodiment, the insect cell may be a white armyworm cell. In another embodiment, the insect cell may be a black-bellied fruit fly cell. Other non-limiting examples of insect cells that may be used herein may be Sf9, Sf21, BTI-TN-5B1-4, or S2 cells.

[0169] In some embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used as host cells in this disclosure include Chinese hamster ovary cells (CHO), mouse myeloma cells (NSO), young hamster kidney cells (BHK), and / or HEK293 cells.

[0170] In some embodiments, the host cell is a yeast cell. Non-limiting examples of yeast cells that can be used as the host cell of this disclosure include *Yarrowia lipolytica*, *Kluyveromyces martensii*, *Saccharomyces cerevisiae*, and / or *Pichia pastoris*. In some embodiments, the yeast cell may be *Saccharomyces cerevisiae* and / or *Pichia pastoris*. In some embodiments, the yeast cell is natural or has been genetically modified to lack retrotransposons (e.g., Ty1, Ty2, Ty3, Ty4, and / or Ty5 retrotransposons).

[0171] Methods for generating VLPs containing heterologous RNA payloads

[0172] In some aspects, this disclosure provides a method for generating a heterologous RNA payload in cells, the method comprising culturing the host cells described above and expressing both the RNA described herein (e.g., containing a 5'-UTR, a heterologous RNA payload, and a 3'-UTR) and a TMV capsid protein. The expressed RNA molecule binds to the TMV capsid protein via two or more TMV packaging signals present in the RNA molecule, thereby inducing the self-assembly and generation of virus-like particles encapsulating the RNA. The VLP can then be isolated from the host cells by cell lysis, followed by centrifugation and treatment of the soluble material with PEG to precipitate the VLP. The precipitated VLP can be further purified by density gradient centrifugation. Further purification of the VLP can be achieved by using ultrafiltration, membrane filtration centrifugation, and / or size exclusion chromatography.

[0173] In some embodiments, 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 contained in the host cell, are undesirable and therefore their removal from the isolated VLPs is advantageous. Such nucleic acids or polynucleotides can adhere to or be associated with the VLP. Therefore, treating the VLPs with enzymes that digest these substances provides a method for removing such contaminants while protecting the capsidized RNA containing the heterologous RNA payload from attack by such enzymes.

[0174] The isolated VLP contains RNA with a heterologous RNA payload, and RNA isolated from it.

[0175] In some respects, this disclosure provides a VLP comprising the RNA molecules isolated from host cells as disclosed herein.

[0176] In some respects, this disclosure provides for the isolated RNA molecules disclosed herein.

[0177] In some respects, this disclosure provides RNA molecules disclosed herein isolated from VLPs encapsulating such RNA.

[0178] Once the VLP is isolated, and optionally treated with an enzyme that digests the nucleic acids or polynucleotides outside the TMV VLP, the capsidated RNA can be extracted. This extraction can be performed using commercially available RNA extraction columns or other RNA extraction methods known in the art. The extracted RNA can then be used directly or further formulated for administration to subjects in need.

[0179] In some cases, instead of extracting capsidized RNA from VLPs, the VLPs themselves are used directly, or further formulated into a drug to deliver the RNA therein to subjects in need.

[0180] All patents, patent applications, and other scientific or technical writings mentioned anywhere else herein are incorporated herein in their entirety by reference. The embodiments described illustratively herein may be practiced without the presence of any one or more elements, or one or more limitations, specifically or not specifically disclosed herein. Therefore, for example, in each case herein, the terms “comprising,” “substantially consisting of,” and “composed of” may be replaced by any of the other two terms while retaining their ordinary meaning. The terms and expressions used are used as descriptive rather than restrictive terms and are not intended to exclude any equivalents or portions thereof of the features shown and described.

[0181] It should be understood that although the methods and compositions of the present invention have been specifically disclosed through examples and optional features, those skilled in the art may make modifications and variations to the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the compositions and methods as defined in the specification and appended claims.

[0182] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein may be specifically excluded from the claims.

[0183] Whenever ranges are given in the specification, such as temperature ranges, time ranges, compositional or concentration ranges, all intermediate ranges and sub-ranges, as well as all individual values ​​included within a given range, are intended to be included in this disclosure. It should be understood that any sub-range or individual value included in the ranges or sub-ranges herein may be excluded from this aspect. It should be understood that any element or step included in the specification herein may be excluded from the claimed compositions and methods.

[0184] Furthermore, when features or aspects of compositions and methods are described in accordance with the Markush group or other alternative groups, those skilled in the art will recognize that the compositions and methods are therefore also described in accordance with any single member or subgroup 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 combinations are not specifically disclosed.

[0185] Example

[0186] Example 1. Production of VLPs in yeast

[0187] A plasmid was prepared using standard cloning methods. The plasmid contains a DNA sequence (DNA sequence - SEQ ID NO: 20; CP amino acid sequence - SEQ ID NO: 21) encoding the TMV capsid protein (CP), operably linked to the GAP promoter (SEQ ID NO: 18). A map of this plasmid (TMV capsid) is shown in [the image / image / description]. Figure 1 As shown in the image.

[0188] Prepare a second set of plasmids containing DNA capable of being transcribed into RNA molecules, wherein the RNA molecules contain firefly luciferase or enhanced green fluorescent protein (EGFP) mRNA as a heterologous RNA payload with a 5'-UTR side-attached to SEQ ID NO: 17 and one or two copies of a 3' UTR containing a TMV packaging sequence, as indicated in Table 3 below.

[0189] A single copy of the 3'-UTR containing the TMV packaging sequence has the following RNA sequence. Nucleotides shown in bold represent the TMV packaging sequence.

[0190] UTR-1: .

[0191] UTR-3: GCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUUGUUUAUAGAAAUAAUAUAAAAUUAGGUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACUUAC AGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGGCUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAGAGUGAUGUCCGCAAAGGGAAAAAUAUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAA (SEQ ID NO: 23).

[0192] Table 3. Content of various plasmids used for transcription of RNA encapsulated by TMV VLP

[0193]

[0194] The plasmid map mentioned above is in Figure 2-7 As shown in the image.

[0195] Before double transformation with TMV-CP and mRNA-UTR plasmids, *Saccharomyces cerevisiae* production cells (BY 4741) were cultured in YPD medium (1% [w / v] yeast extract, 2% [w / v] peptone, 2% [w / v] glucose) using a yeast transformation kit (Sigma-Aldrich, catalog number YEAST1-1KT) and salmon sperm ssDNA (Abogen, catalog number ab229278) following the manufacturer's instructions. The reaction solution was incubated at 30°C for 30 minutes, followed by heat shock treatment, and then the cells were plated on solid auxotrophic medium (0.17% [w / v] yeast nitrogen base, no supplemented amino acids and nitrogen base, 0.5% [w / v] ammonium sulfate, 0.14% [w / v] yeast synthesis auxotrophic medium supplement, no histidine, leucine, tryptophan and uracil, 2% [w / v] glucose, 50 mg / L tryptophan, 50 mg / L uracil). Positive selection of plasmid-containing cells was performed after 4 days at 30°C. Transformed yeast glycerol stock was cultured by seeding cells into a yeast nitrogen base containing the same yeast auxotrophic deficient medium. The yeast was cultured at 30°C with constant shaking for a total of 5 days. Cells were collected by centrifugation, and the supernatant was removed. The cells were then resuspended and centrifuged repeatedly. Next, the cells were resuspended in TEN buffer (50 mM Tris pH 7.4; 500 mM NaCl; 2 mM EDTA; 1 mM protease inhibitor (Roche catalog number 04693124001). Cells were lysed mechanically by beating with glass beads (TissueLyser II, 425–600 μm, Sigma-Aldrich, catalog number G8772). The cell lysate containing VLPs was then collected and centrifuged to remove the beads.

[0196] Example 2. Production of VLPs in insect cells

[0197] The tobacco mosaic virus (TMV) capsid protein coding sequence (SEQ ID NO: 20) was synthesized and directly cloned into a promoter-controlled baculovirus transfer vector. Using standard molecular cloning techniques, a DNA cassette encoding firefly luciferase, enhanced green fluorescent protein (EGFP), or Sars-Cov-2 spike protein was subcloned into a baculovirus transfer vector already containing SEQ ID NO: 20. The DNA cassette served as a heterologous RNA payload and was flanked by a transcribed 5'-UTR of SEQ ID NO: 17 and various transcribed 3' UTRs (UTR-1 (SEQ ID NO: 22), UTR-3 (SEQ ID NO: 23), or UTR-4 (SEQ ID NO: 8)) containing one or two copies of the TMV packaging sequence.

[0198] Recombinant baculoviruses were generated using Sf9 or Tni insect cells and ESF 921 medium (Expression Technologies) by co-transfection with each of the resulting transfer vectors. Each vector contained flashBAC ULTRA. TM Viral DNA (Oxford Expression Technologies), transfer vector, and baculoFECTIN II transfection reagent TM The reaction mixture (from Oxford Expression Technologies) was used to infect insect cells, which were then inoculated onto culture dishes and incubated in medium at 28°C for 5 days. The medium now containing the corresponding P0 virus stock solution was collected into sterile tubes. To prepare the P1 virus stock solution, Sf9 or Tni cells were inoculated with an appropriate amount of the corresponding P0 virus stock solution. The virus-infected shaking culture was incubated at 28°C for 5 days, and then the medium containing the P1 virus stock solution was collected into sterile tubes.

[0199] via baculoQUANT TM (Oxford Expression Technologies) qPCR was used to determine the infectious titer of TMV-mRNA-UTR baculovirus. Sf9 and Tni cultures were established and inoculated into shake flasks, with each culture inoculated with one of the described P1 virus stock solutions. TMV-VLP was extracted from the Sf9 or Tni cell cultures by first lysing the cells by sonication and then clarifying by centrifugation. Figure 8 A representative SDS-PAGE gel of lysates from transfected Tni cells that produced TMV-encapsulated mRNA is shown.

[0200] TMV VLP structures produced by the transformed yeast described in Example 1 or by the transfected Sf9 or Tni cells described in this example were observed by TEM (Fig. 9). All VLPs from each cell 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 contrasts with prior art, which shows that multiple copies of the TMV packaging sequence lead to altered capsid morphology. Eber et al., Nanoscale, Vol. 7: 344-355 (2015); Gallie et al., Virology 158, 473-476 (1987).

[0201] Example 3. Purification of VLP

[0202] Clear, soluble material containing VLP from yeast or insect cell lysates was transferred to centrifuge tubes, and a PEG stock solution (20% (w / v) PEG 6000, 1 M NaCl) was added to each sample to bring the final concentration to 4% (w / v) PEG 6000, 200 mM NaCl. The samples were gently mixed and incubated overnight at 4°C by gentle rotation. The samples were then centrifuged at 13,000 × g for 45 minutes at 20°C to allow visible precipitate to form. The samples were examined by denaturing SDS-PAGE analysis to confirm the presence of TMV capsid protein in both the lysates and the PEG precipitate. See also Figure 10 .

[0203] Next, density-based purification of VLP was performed by resuspending the PEG-precipitated precipitate in PBS, adding CsCl solution (35% w / v), and centrifuging at 180,000 × g. VLP was visible as blue bands in the CsCl ultracentrifuge tube. Figure 11 An exemplary case of yeast cell lysate is shown. The solution was fractionated by sequentially taking 500 µL aliquots, and the presence of the TMV capsid was analyzed by denaturing SDS-PAGE of each aliquot. Figure 12 The following diagram illustrates the fractions of an exemplary yeast cell lysate preparation. Fractions containing the capsid were combined and transferred to PBS via membrane filtration and centrifugation. Total capsid protein concentration was measured by BCA assay (Pierce BCA kit, Thermo Scientific catalog 23225). TMV VLPs containing heterologous RNA payloads were also successfully purified from crude cell lysates by size exclusion chromatography: CIMultus TM OH (2 μm) 1.5 M to 50 mM potassium phosphate buffer pH 7 gradient.

[0204] Example 4. Extracting RNA from VLP

[0205] The VLPs in PBS were treated with a nuclease or mixture of enzymes (benzonuclease or a specific RNase mixture) designed to digest double-stranded RNA in the presence of MgCl2 at 37°C for 1 hour. The digestion reaction was terminated by adding EDTA. The VLPs were then exchanged for PBS by membrane centrifugation before extraction of the capsidated RNA using a commercial RNA extraction column based on the principle of liquid-crystallized silica membrane separation (RNeasy microkit, Qiagen, catalog 74104; Monarch RNA cleanup, NEB, catalog T2040L). Figure 13, insets A and B, show agarose gel images of RNA samples extracted from TMV VLPs generated from yeast transfected with TMV capsids and GFP UTR-3 or GFP UTR-4 plasmids before and after benzonuclease treatment. Arrows highlight RNA bands of the expected size encoded by the GFP UTR-3 or GFP UTR-4 plasmids. These results confirmed the TEM images, which showed that the VLP, formed by and encapsulating RNA containing one or two OAS packaging sequences, is an intact structure protecting the RNA from digestion. RNA species eliminated by benzonuclease treatment were not encapsulated and are believed to have adhered to the exterior of the VLP or contaminated the RNA species in the VLP preparation.

[0206] Table 4. VLP proteins and capsidated RNA from yeast transfected with plasmids encoding one or two TMV packaging signals.

[0207]

[0208] Example 5. Function and immunogenicity of VLP-extracted RNA in THP-1 mononuclear cells.

[0209] When the interferon regulatory pathway is stimulated by activation of TLR-3, MDA5, RIG-1, or cCAS receptors, THP-1 Dual TM NF-κB-SEAP IRF-Luc reporter monocytes (InvivoGen catalog number thpd-nfis) were modified to express lucia luciferase. THP-1 Dual cells were cultured using standard mammalian cell culture techniques in RPMI 1640 + 10% FBS (heat-inactivated) containing 1% Pen / Strep and the selective antibiotic Normocin. TMCells. In cell passage P4, THP-1 monocytes were differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) (Thermo Fisher Scientific catalog number J63916). THP-1 monocytes were seeded into flat-bottomed plates and treated with PMA at a final concentration of 50 ng / mL in growth medium at 37°C for 3 hours. This treatment medium was then removed and replaced with growth medium, and the cells were cultured for 3 days. The resulting adherent cells were gently washed into fresh medium before use for transfection assays. RNA extracted from yeast TMV VLPs produced by each of GFP UTR-1, GFP UTR-3, and GFP UTR-4 was prepared according to the manufacturer's instructions using Lipofectamine reagent (Ingenieur catalog number LMRNA008) for transfection and immediately added to the cells at a final concentration of 125 ng RNA / well. Poly(dA:dT) (InvivoGen catalog number tlrl-patn) was used as an immunogenicity positive control transfected with lipofectamine at 125 ng / well. EGFP and lucia luciferase expression were measured 24 hours later. eGFP expression was measured directly from cells in the culture plate using a microplate reader at excitation wavelength (ex.) 480 nm and emission wavelength (em.) 520 nm (PHERAstar FSX). Lucia luciferase expression under interferon-regulated pathway stimulation was measured from cell supernatant samples according to the coelenterate-based bioluminescence assay using the QUANTI-Luc 4 reagent (InvivoGen catalog number rep-qlc4g1). All bioluminescence assays were performed using a microplate reader. The results of these experiments are shown in Figure 14, insets A and B.

[0210] Example 6. In IVT, variants of the human α-globin 3'-UTR sequence containing partial internal repeat sequences enhance expression relative to the native human α-globin 3' sequence.

[0211] RNA production

[0212] The double-stranded DNA template for generating the RNA sequence encoding luciferase during IVT is produced by a plasmid containing, in 5' to 3' order, a DNA sequence capable of being transcribed into the 5'-UTR of SEQ ID NO: 17 minus the G at the very 5' end, the luciferase-coding sequence (SEQ ID NO: 24), and a DNA sequence capable of being transcribed into the 3'-UTR of SEQ ID NO: 6 or SEQ ID NO: 7. Primers are designed to target only the sequence from the beginning of the 5'-UTR to the end of the 3'-UTR of the luciferase RNA-coding template within the plasmid backbone. The primers also have a T7 promoter added to the 5' end of the 5'-UTR and an additional AG added to its 3' end (SEQ ID NO: 25) to allow the use of CleanCap® reagent AG, which replaces the 5' G missing from the 5'-UTR. CleanCap® reagent AG is used to generate cap-1 capped RNA during the IVT reaction. At the 3' end of the 3'-UTR, an 80-nucleotide polyadenylated tail was added to the primer. The positive strand of the resulting DNA template had the nucleotide sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27. The template was then amplified by PCR using High Fidelity Master Mix (New England Biolabs, (NEB catalog number: M0541S)) according to 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, catalog number E2080S) in the presence of 4 mM CleanCap® reagent AG. All other reagents were used according to the HiScribe kit instructions. The reaction mixture was incubated at 37°C for 150 min, then terminated with DNase I and incubated for another 30 min. RNA was then extracted from the reaction mixture using Oligo d(T)25 beads (NEB) according to the manufacturer's instructions. The eluted RNA was then further purified using the Monarch RNA Cleanup Kit (NEB) before analysis by capillary electrophoresis. Successful RNA production and correct length were confirmed by capillary electrophoresis prior to transfection.

[0213] Cell transfection

[0214] THP-1 Dual cells were cultured using standard mammalian cell culture techniques in RPMI 1640 + 10% FBS (heat-inactivated) containing 1% Pen / Strep and the selective antibiotic Normocin. TMCells. In cell passage P4, THP-1 monocytes were differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) (Thermo Fisher Scientific catalog number J63916). THP-1 monocytes were seeded into flat-bottomed plates and treated with PMA at a final concentration of 50 ng / mL in growth medium at 37°C for 3 hours. This treatment medium was then removed and replaced with growth medium, and the cells were cultured for 3 days. The resulting adherent cells were gently washed into fresh medium before use for transfection assays. RNA generated from IVT was prepared for transfection using Lipofectamine reagent (Ingenieur catalog number LMRNA008) according to the manufacturer's instructions and immediately added to the cells at a final concentration of 125 ng RNA / well.

[0215] Adherent HEK cells were cultured in DMEM containing 1% Pen / Strep and 10% FBS, and passaged every 2–4 days. Cells were then separated from the plate using trypsin-EDTA. Cells were then seeded in 96-well plates using serum-free OptiMEM medium and allowed to adhere for at least 2 hours before RNA transfection.

[0216] HepG2 cells were treated in a similar manner, using MEM instead of DMEM, for initial culture prior to transfection. For transfection, RNA samples were prepared using Lipofectamine reagent (Ingenieur catalog number LMRNA008) according to the manufacturer's instructions and immediately added to cells at a final concentration of 125 ng RNA / well.

[0217] Analysis of expression products

[0218] After incubation at 37°C for 24 hours, cell transfection efficiency was assessed by the expression of the firefly luciferase reporter gene. Luciferase expression was analyzed by measuring the bioluminescence produced by adding the substrate D-luciferin to transfected cells expressing luciferase. The protocol of the Promega Luciferase Assay Kit (catalog number: E1501) was followed; in short, cells were treated with lysis buffer in 96-well plates, and 50 µL of lysate was transferred to a white opaque plate. The substrate D-luciferin was added to the lysate, and bioluminescence was immediately measured using a microplate reader. The results are shown in Figure 15, inset AC. For each cell line tested, the presence of modified α-globin 3'-UTR (SEQ ID NO: 7) significantly increased the expression product compared to the native α-globin 3'-UTR (SEQ ID NO: 6).

[0219] Example 7. The double TMV OAS in the 3'-UTR increased the quality of RNA produced in *Yarrowia lipolytica*.

[0220] The effects of double TMV OAS sequences in the 3'-UTR and single TMV OAS sequences located at the 5' end of the 3'-UTR (i.e., immediately following the luciferase coding region) on the expression capacity of the encoded luciferase in THP-1 cells were compared.

[0221] RNA-containing VLP production in Yersinia lipophila

[0222] Yeast extract (PO1h or PO1d) cells were cultured in YPD medium (1% [w / v] yeast extract, 2% [w / v] peptone, 2% [w / v] D(+) glucose) until sufficient biomass was obtained. Cells were then prepared for transformation by transferring to 0.1 M lithium acetate buffer at pH 6. Restriction digestion of the plasmid was performed using rCutSmart buffer (NEB, catalog number B6004S) containing NotI-HF (NEB, catalog number R3189L). The reaction solution was incubated at 37°C for 2 hours, followed by incubation at 65°C for 20 minutes, and then stored on ice until transformation was complete. Cells were then transformed by adding the digested plasmid YL-LucOAS (… Figure 17 ) or YL-Luc UTR-4 ( Figure 18 ), and digested plasmid YL-TMV-capsid ( Figure 19The transformation mixture consisted of salmon sperm ssDNA (0.3 mg / mL, Abogen, catalog number ab229278), 40% PEG3350, 0.05 M DTT, and 0.1 M lithium acetate at pH 6, followed by incubation at 39°C for 60 minutes to achieve transformation. YL-Luc OAS and YL-Luc UTR-4 each contained, in 5' to 3' order, a pTEF promoter (SEQ ID NO: 34), a DNA sequence transcribed into the 5'-UTR of SEQ ID NO: 17, and a luciferase-coding sequence of SEQ ID NO: 24. Luc UTR-4 further contained, at the 3' end of the luciferase-coding sequence, a DNA sequence transcribed into the 3'-UTR of SEQ ID NO: 8 (UTR-4), while Luc OAS further contained, at the 3' end of the luciferase-coding sequence, a DNA sequence transcribed into the 3'-UTR of SEQ ID NO: 29. Each of Luc OAS and Luc UTR-4 further includes the T1-3 Lipt2 terminator (SEQ ID NO: 35). YL-TMV-capsid encodes the TMV capsid protein coding sequence (SEQ ID NO: 21), whose expression is driven by the pTEF promoter (SEQ ID NO: 34) and includes the T1-3 Lipt2 terminator (SEQ ID NO: 35). Transformed cells were then isolated and plated onto solid auxotrophic medium (0.17% [w / v] yeast nitrogen base, without amino acid and nitrogen base supplementation, 0.5% [w / v] ammonium sulfate, 2% [w / v] D(+)-glucose, 0.19% [w / v] yeast synthesis auxotrophic medium supplement, uracil-free) plates, and colonies were selected after 4 days. Glyceryl stock of transformed yeast was cultured by inoculating cells into yeast nitrogen base with glucose-supplemented yeast synthesis auxotrophic medium. The yeast was cultured at 30°C with constant shaking for a total of 3 days. Cells were collected by centrifugation, and the supernatant was removed. The yeast cells were then resuspended and centrifuged repeatedly. Next, the cells were resuspended in PBS buffer (pH 7.2) containing a protease inhibitor (Roche catalog number 04693124001). The cell suspension was then treated with a 750 U / mL lysin (Sigma catalog number L2524) and incubated at 37°C for 1 hour prior to lysis. Yeast cells were lysed by mechanical lysis using glass bead beating or by shear lysis using a Microfluidizer® (LM20, Microfluidics) (TissueLyserII, 425-600 μm, Sigma-Aldrich, catalog number G8772).

[0223] VLP purified by density centrifugation

[0224] Clear, soluble material containing VLP from yeast cell lysates was transferred to centrifuge tubes, and PEG stock solution (20% (w / v) PEG 6000, 1 M NaCl) was added to each sample to make a final concentration of 4% (w / v) PEG 6000, 200 mM NaCl. The samples were gently mixed and incubated overnight at 4°C by gentle rotation. The samples were then centrifuged at 13,000 × g for 45 min at 20°C to allow visible precipitate to form. The samples were examined by denaturing SDS-PAGE to confirm the presence of TMV capsid protein in both the lysates and the PEG precipitate. Next, density-based VLP purification was performed by resuspending the PEG-precipitated precipitate in PBS and centrifuging at 180,000 × g before adding CsCl solution (35% w / v). VLP was visible as blue bands in the CsCl ultracentrifuge tubes. The solution was fractionated by sequentially taking 500 µL aliquots, and the presence of TMV capsid was analyzed by denaturing SDS-PAGE of each aliquot. Fractions containing capsid were combined and transferred to PBS (150 mM) pH 7.2 by membrane filtration centrifugation. Total capsid protein concentration was measured by BCA assay (Pierce BCA kit, Thermo Scientific catalog number 23225).

[0225] VLP purified by chromatography

[0226] Also using CIMultus TM TMV-RNA particles were purified from cell lysates using size exclusion chromatography on an OH (2 μm) 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 purity was confirmed by SDS-PAGE analysis. Prior to concentration, the pooled fractions were transferred to PBS (150 mM) pH 7.2 by membrane filtration centrifugation, and the total capsid protein concentration was measured by BCA assay (PierceBCA kit, Thermo Scientific catalog 23225).

[0227] RNA was extracted from VLPs, and the extracted RNA was used to transfect THP-1 cells, followed by analysis of the expression products.

[0228] RNA was isolated from the purified VLP as described in Example 5. THP-1 Dual RNA was prepared as described in Example 5. TMCells were transfected, and the cells were then transfected with isolated RNA. Luciferase expression in THP-1 cells was measured as described in Example 6. The results showed that the presence of a double TMV OAS sequence in the RNA significantly increased luciferase expression compared to a single TMV OAS immediately adjacent to the luciferase coding sequence 3'. Figure 16 ).

[0229] Table 5. Sequences.

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Therefore, the invention is limited only by the following claims.

Claims

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

2. The isolated DNA sequence according to claim 1, wherein the 3'-UTR contains two TMV packaging signals.

3. The isolated DNA sequence according to claim 1 or 2, wherein each pair of adjacent TMV packaging signals in the 3'-UTR is separated by less than 500 nucleotides.

4. The isolated DNA sequence of claim 3, wherein each pair of adjacent TMV packaging signals in the 3'-UTR is separated by less than 100 nucleotides.

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

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

3.

7. The isolated DNA sequence according to claim 6, wherein the 3'-UTR is an RNA sequence comprising any one of SEQ ID NO: 8-16.

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

8.

9. The isolated DNA sequence according to any one of claims 1 to 8, wherein the DNA sequence is double-stranded.

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 according to claim 10, wherein the promoter is a constitutive promoter.

12. The expression vector according to claim 11, wherein the promoter is a GAP promoter having the nucleic acid sequence of SEQ ID NO: 18, a TEF1 promoter having the nucleic acid sequence of SEQ ID NO: 19, a modified UAS8-2 promoter having the nucleic acid sequence of SEQ ID NO: 28, a UAS4-1 promoter having the nucleic acid sequence of SEQ ID NO: 30, or a promoter having a nucleic acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 18, 19, 28 or 30.

13. The expression vector according to claim 12, wherein the promoter is a GAP promoter having the nucleic acid sequence of SEQ ID NO: 18, a TEF1 promoter having the nucleic acid sequence of SEQ ID NO: 19, a modified UAS8-2 promoter having the nucleic acid sequence of SEQ ID NO: 28, or a UAS4-1 promoter having the 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 the DNA sequence of SEQ ID NO: 18, a UAS4-1 promoter having the nucleic acid sequence of SEQ ID NO: 30, or a promoter having a nucleic acid sequence having at least 90% sequence identity with 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 the DNA sequence of SEQ ID NO: 18 or a UAS4-1 promoter having the nucleic acid sequence of SEQ ID NO:

30.

18. An isolated host cell containing an expression vector according to any one of claims 10 to 17.

19. An isolated host cell containing an expression vector according to any one of claims 10 to 13; and further containing 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 the nucleic acid sequence of SEQ ID NO: 18 or a promoter having a nucleic acid sequence having at least 90% sequence identity with 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 the nucleic acid sequence of SEQ ID NO:

18.

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

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

25. The host cell according to claim 23, wherein the cell is an insect cell selected from the fall armyworm (Spodopterafrugiperda) and the white armyworm (Trichoplusia ni).

26. The host cell according to claim 23, wherein the cell is a plant cell, namely, Nicotiana benthamiana.

27. The host cell according to claim 23, wherein the cell is a mammalian cell selected from Chinese hamster ovary cells and HEK293T cells.

28. A method for producing an RNA molecule comprising a heterologous RNA payload in a cell, the method comprising culturing a host cell according to any one of claims 18 to 27 under conditions for producing virus-like particles, thereby producing the RNA molecule, the virus-like particles comprising the RNA molecule bound to and encapsulated thereon by a TMV viral capsid protein.

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

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 capsidated RNA from the isolated virus-like particles.

32. An isolated virus-like particle, which is produced by the method according to claim 29 or 30.

33. An isolated RNA molecule transcribed from an isolated DNA sequence according to any one of claims 1 to 9.

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

35. An isolated RNA molecule produced from isolated virus-like particles according to claim 32.

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

7.

37. A DNA template for in vitro transcription, said DNA template comprising the DNA sequence according to claim 36.

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 the 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 a DNA template according to any one of claims 37 to 39.

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