Maturase protein fusion for phage display

Abstract

Expression vectors encoding a modified MS2 capsid, maturase protein (MP) fusion proteins, such as produced using the expression vectors of the present disclosure, virus-like particles (VLPs) comprising the MP fusion protein of the present disclosure. In an example, the expression vector can include a nucleic acid sequence encoding a maturation protein A (MP); and a nucleic acid sequence encoding a coat protein (CP), wherein one or more of the nucleic acid sequence encoding the MP and the nucleic acid sequence encoding the CP comprises a cloning site. The cloning site can be a ligation independent cloning site.

Description

MATURASE PROTEIN FUSION FOR PHAGE DISPLAYCROSS-REFERENCE(S) TO RELATED APPLICATION(S)

[0001] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 727,510, filed on December 3, 2024, the entire disclosure of which is disclosed herein in its entirety.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3915-P1374WO.UW_Seq Listing 20251118_v2.xml. The XML file is 85,608 bytes; was created on November 18, 2025; and is being submitted electronically via Patent Center with the filing of the specification.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with government support under Grant Nos. 5 R01 Al 145486-05 and 5R33AI140460-05, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0004] Phage-Like Particles (PLPs) or, more generally, Virus-Like Particles (VLPs), are engineered nanoparticles owing their structural elements to viruses but without the capacity for self-replication due to lacking a viable genome. Conventionally, PLPs comprise the capsid forming viral proteins, or their derivatives, and a consigned artificial nucleic acid. These particles are attractive platforms for biotechnology because they are self-assembling and amenable to diverse modifications by making alterations to their encoding plasmids or by chemical conjugation. Applications of PLPs include such high- impact topics as vaccines, drug delivery, gene editing, biopanning, and nucleic acid amplification test (NAAT) controls. The particles’ similarity to living viruses; capability of displaying antigens, proteins, or peptides on their surface; and programmable nucleic acid payload in their capsid makes them uniquely well suited for these fields.

[0005] Viral mimicry with a customizable nucleic acid sequence as cargo is an important advancement for NAAT-based diagnostics, where control substrates are used to establish the viability of a test’s chemical and mechanical operation. RNA templates areparticularly vulnerable to degradation by RNases, which are abundant in many sample matrices. A pathogen look-alike PLP capable of protecting a synthetic RNA template from degradation when spiked into a sample makes an important process control system.

[0006] There are minimalistic requirements of the bacteriophage Emesvirus zinderi (MS2) to form a capsid around a specified RNA strand and enabled the manufacture of PLPs for RNA NAATs. In order to assemble a complete capsid, MS2 requires only two proteins; the coat protein (CP) forms the primary shell of the particle, nominally selfassembling from 178 CPs sub-assembled as dimers into a T = 3 Icosahedral shell, and a single copy of maturation protein A (MP) which aids in packing of genomic material and is responsible for conferring infectivity of active MS2 virus.

[0007] Use of conventional PLPs suffers from complicated experimental protocols with low yields and limited surface display options. Accordingly, there is a need for easily modifiable, modular PLP systems providing easy purification and high yields and more advanced surface display capability.SUMMARY

[0008] The present disclosure provides, in various aspects, expression vectors, MP fusion proteins, and virus-like particles to address these and related challenges.

[0009] An advantage of the present disclosure relates to the reduction of time and effort to produce PLPs by enhancing the efficiency of the entire workflow, from the plasmid construction steps to the realization of purified particles, in the hope of making the technology more accessible to researchers in different fields of study. Design changes also aim to create a system that is easy to modify and has high-throughput potential for production, an attractive prospect for project development and manufacturing needs. In this regard, in an aspect, the present disclosure provides a peptide display fusion for MS2 PLPs, such as to provide enhanced functionalization.

[0010] In order to achieve these improvements, the present disclosure provides, in embodiments, expression plasmids configured to simplify cloning steps and dramatically increase the yield of CP during protein expression. This, in turn, allows reduced culture volumes and microliter-scale spin column-based affinity chromatography (AC) purification without sacrificing the quality of the final product. This provides PLP purification leveraging the chimeric fusion of an affinity tag in a surface-displayed region of the maturation protein or coat protein, such as a coat protein dimer.

[0011] Accordingly, in an aspect, the present disclosure provides an expression vector encoding a modified MS2 capsid. In an embodiment, the expression vector comprises a nucleic acid sequence encoding a maturation protein A (MP); and a nucleic acid sequence encoding a coat protein (CP), wherein one or more of the nucleic acid sequence encoding the MP and the nucleic acid sequence encoding the CP comprises a cloning site.

[0012] In an embodiment, the cloning site is an overlap homology cloning site. In an embodiment, the overlap homology cloning site is selected from sequence and ligation independent cloning (SLIC), ligation independent cloning (LIC), in-fusion, Gibson assembly, homology assembly cloning (HAC) sites, and combinations thereof.

[0013] In an embodiment, the cloning site is a LIC site. In an embodiment, the LIC site comprises one of the following sequences: CGTGTTGGTTGATAATGGC (SEQ ID NO. 1) -(insert) -GGTGATGTAACCGTAGCTCCATCTAACTTCG (SEQ ID NO. 50); GCATCAACTCTCCCGGT (SEQ ID NO. 2)-(insert)- GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 51); and GCATCAACTCCTCCGGT (SEQ ID NO. 3)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 52), wherein (insert) indicates a location of an insert.

[0014] In an embodiment, the cloning site is disposed in the nucleic acid sequence encoding the MP, and wherein the cloning site is disposed in a position corresponding to loop end regions of the MP.

[0015] In an embodiment, the nucleic acid sequence encoding the MP comprises a nucleic acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO. 4.

[0016] In an embodiment, the cloning site is disposed in at least one of the following residues of the sequence of SEQ ID NO. 4: 46-102, 235-255, 730-747, and 1001- 1033.

[0017] In an embodiment, the MP or the CP further comprises a nucleic acid sequence in the cloning site encoding an amino acid sequence comprising between 1 amino acid residue and about 1,000 amino acid residues.

[0018] In an embodiment, the amino acid sequence is an amino acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOS. 5 and 6.

[0019] In an embodiment, the cloning site is disposed in the nucleic acid encoding the MP and is a restriction site.

[0020] In an embodiment, the nucleic acid sequence encoding the CP is positioned 5’ of the nucleic acid sequence encoding MP.

[0021] In an embodiment, the expression vector comprises one or more ribosomal binding sites. In an embodiment, a ribosomal binding site of the one or more ribosomal binding sites is positioned 3’ of the nucleic acid sequence encoding the CP. In an embodiment, the one or more ribosomal binding sites comprise a sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOS. 7-12.

[0022] In an embodiment, the expression vector comprises a stop codon positioned 5’ of the nucleic acid sequence encoding CP.

[0023] In an embodiment, the nucleic acid sequence encoding the CP encodes for a CP dimer.

[0024] In another aspect, the present disclosure provides an MP fusion protein produced using the expression vector according to any embodiment of the present disclosure.

[0025] In another aspect, the present disclosure provides a virus-like particle (VLP) comprising the MP fusion protein according to any embodiment of the present disclosure.

[0026] In an embodiment, a ratio of CP:MP is in a range of about 5: 1 to about 90: 1.

[0027] In another aspect, the present disclosure provides an MP fusion protein comprising a heterologous amino acid sequence inserted at one or more of residues 16-34, 79-85, 244-249, and 335-344 of SEQ ID NO. 4. In embodiments, the heterologous amino acid sequence is inserted between and / or replaces one or more of residues 16-34, 79-85, 244-249, and 335-344 of SEQ ID NO. 4.

[0028] In an embodiment, the MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 14 and 17.

[0029] In an embodiment, the MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 15 and 18.

[0030] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summaryis not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.DESCRIPTION OF THE DRAWINGS

[0031] The foregoing aspects and many of the attendant advantages of the present disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0032] FIGURE 1 schematically illustrates expression cassette designs for plasmids and their PLP products, according to embodiments of the present disclosure. Plasmid designs: plH, p2H, p2S, p3H, and p4HS. Pt7: T7 promoter, RBS: Ribosomal binding site (various), mat: MS2 mat coding DNA sequence (CDS), mRNA: Transcription product, WT: Wild-Type regulatory region from MS2, CPD: Coat Protein Dimer CDS or Protein, His6: 6x polyhistidine tag, TRM: Terminator (various), Ctrl: Packaged sequence / RNA to be packaged (RT-qPCR target), pac: pac site sequence, MP: Maturation Protein A (mat CDS product), 2x pac: tandem pac sites, ST: Strep-tag®II, 2x ST: Twin- Strep-tag®. Moving from top to bottom, only changed features are annotated.

[0033] FIGURE 2A illustrates an MP comprising a wild-type MP mutated to include a ligation independent cloning (LIC) site, according to embodiments of the present disclosure;

[0034] FIGURE 2B illustrates an MP-Twin-Strep-tag® fusion protein comprising linkers, a LIC site, and Strep-tag®II sequences, according to embodiments of the present disclosure;

[0035] FIGURES 3A-3F provide images of SDS-PAGE of PLP purifications, according to embodiments of the present disclosure. FIGURES 3A, 3C, 3E show comparative purifications of particles by IMAC and / or ST, as noted. Std: the Color Prestained protein standard, Broad Range (NEB, P7719), U: uninduced, P: pellet / insoluble (induced), S: Sample / supernatant (induced), F: Flow-through / unbound sample, E: Elution, D: Dialyzed elution fraction, MP: Maturation protein (3A) CPD: single-chain-Coat- protein-Dimer (HIS6 or ST tag), MPS: MP-Twin-Strep-tag® fusion, unk: unknown contaminant. Figures 3B, 3D, 3F show comparative yields of replicate purifications of particles, as noted. D1-D4: dialyzed elution fraction replicates 1-4 for respective PLPs.Expected protein mass: CPD (His6): 28.148 kDa, CPD (ST): 28.365 kDa, MP: 43.982 kDa,MPS: 47.720 kDa.

[0036] FIGURES 4A-4F illustrate verification of PLP structure and function, according to embodiments of the present disclosure. (4A) RT-qPCR quantitation of RNA and DNA templates in copy number per pL of the dialyzed samples. Error bars represent standard error. (4B) RNase protection assay of particle designs containing ST fusions. Relative degradation of RNA template from intact or heat-lysed particles with and without RNase treatment. (4C) Dynamic light scattering measurements of median particle diameters as an average across replicates. P1H produced only a single sample passing quality control for DLS measurements. (4D) TEM of p2H-IMAC PLPs. (4E) TEM of p2S- ST PLPs. (4F) TEM of p4HS-IMAC PLPs.DETAILED DESCRIPTION

[0037] In various aspects, the present disclosure provides expression vectors encoding a modified MS2 capsid, maturase protein (MP) fusion proteins, such as produced using the expression vectors of the present disclosure, virus-like particles (VLPs) comprising the MP fusion protein of the present disclosure.

[0038] As provided further herein, the present disclosure provides plasmid vectors for MS2 derived phage-like particles (PLPs) that streamline vector manipulations for rapid prototyping of new particles; an alternative affinity tag validation for purification, and a high-throughput low-volume spin column purification methodology. Internal fusion sites in MS2 maturation protein A were previously passive elements of the MS2 capsid in prior PLP designs. The chimeric maturation proteins described herein are suitable for use for surface display of an affinity tag and purification, increasing the number of available peptide display sites for the MS2 PLP platform.

[0039] As described further herein, use of a cloning site, such as a LIC -based cloning system, allows single-step, high-specificity incorporation of RNA packaged sequences facilitating the rapid development of new control particles and avoiding the challenges of multi-step restriction cloning. As provided herein, LIC sequences were also engineered into the areas flanking fusion tag sites, so they are similarly revised with ease. Reconfiguration of expression cassette and regulatory elements to generate a favorable ratio of CPD and MP meaningfully increases the concentrations of PLPs in culture lysates. Enabled by increased yields of PLPs, the methodology for adapting a common low-volume affinity chromatography spin column format to PLPs provided herein dramaticallysimplifies and expedites the purification workflow. The microliter-volume spin columns, in turn, increase throughput; accordingly, smaller culture volumes are needed, and dozens of different PLPs can be prepared at once using a standard benchtop micro-centrifuge. PLP purification of the type and scale provided herein can be completed comfortably in under an hour. Further expanding the toolkit, the use of the Streptag®!! AC technology with capsid protein fusions increases available chemistries for purification in the same format. Even with low-volume spin column AC purifications, yields were as high as ~4xlOnencapsidated template copies from a single preparation of 3.75 mL of processed culture volume - enough for -400 million NAAT control reactions at 1,000 copies per reaction. At scale, these yields are also sufficiently high for biopanning, where lxlOlo-lxlO12particles are commonly used for a selection cycle. Furthermore, the entire process from PCR of fragments to QC of purified particles can be completed in under four days. This can be time saving for some applications, such as rapidly producing mock targets for the development of diagnostic NAATs for emerging or hazardous RNA viruses or producing process controls for those same assays. In this regard, the present disclosure provides a methodological approach to PLP production that is fast and flexible for a wide variety of research and manufacturing applications.

[0040] The MP protein fusion sites of the present disclosure are demonstrated with a Twin- Strep-tag® AC purification and can support chimeric polypeptide display of sequences at least 88 amino acids in length, notable because expression of fragments larger than 30 amino acids in the CPD can be destabilizing to capsid formation. Larger CPD fusions are also not universally tolerated in the CPD due to the particle’s critical structural dependence on the protein for capsid formation, a complication that may be mitigated with MP-fusions. Compatibility with SpyTag and SpyCatcher further increase the flexibility of display without requiring concessions to their design. MP chimeras are suitable to expand the role of MS2-based PLPs by enabling both rapid purification of particles and polypeptide display or, alternatively, display of multiple peptides. This has considerable practicality for biopanning and vaccine development in particular, where a display of binders or antigens is helpful, and an accessible and mutable platform can rapidly accelerate the development and production of high-quality materials. Indeed, the multiple expression cassettes (e.g., p4HS) were specifically designed to facilitate these future applications by decoupling the CPD and MP so their mRNAs can be programmed with pac sites individually, and thus the particles can readily carry the coding sequence for modifications to the CPD, MP or tertiaryRNA with minimal difficulty. Combined features for vector manipulation, efficient production, synthesis of multiple libraries, rapid iteration, and surface display, make otherwise complex PLP or phage-based applications more approachable in less specialized settings.

[0041] In various aspects, the present disclosure comprises expression vectors, MP fusion proteins, and virus-like particles (VLPs).

[0042] In an embodiment, the expression vectors of the present disclosure comprise recombinant expression vectors. “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the present disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the present disclosure includes other expression vectors that serve equivalent functions, such as viral vectors.

[0043] In an embodiment, the expression vector encodes a modified MS2 capsid, wherein the expression vector comprises a nucleic acid sequence encoding a maturation protein A (MP); and a nucleic acid sequence encoding a coat protein (CP), wherein one or more of the nucleic acid sequence encoding the MP and the nucleic acid sequence encoding the CP comprises a cloning site.

[0044] In an embodiment, the expression vector comprises a tertiary promoter with non-coding packaged sequence. In an embodiment, the expression vector comprises a tertiary coding sequence, or alternatively, no tertiary sequence at all (e.g. if the MP is packed).

[0045] In an embodiment, the cloning site is an overlap homology cloning site.

[0046] In an embodiment, the overlap homology cloning site is selected from sequence and ligation independent cloning (SLIC), ligation independent cloning (LIC), infusion, Gibson assembly, homology assembly cloning (HAC) sites, and combinations there.

[0047] In an embodiment, the cloning site is a LIC site.

[0048] In an embodiment, the LIC site comprises one of the following sequences:

[0049] CGTGTTGGTTGATAATGGC (SEQ ID NO. l)-(insert)-GGTGATGTAACCGTAGCTCCATCTAACTTCG (SEQ ID NO. 50);

[0050] GCATCAACTCTCCCGGT (SEQ ID NO. 2)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 51);

[0051] GCATCAACTCCTCCGGT (SEQ ID NO. 3)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 52);

[0052] wherein (insert) indicates a location of an insert. As described further herein, the inserts can include nucleic acid encoding amino acid sequences of varying length (e.g., 1-1000 amino acid residues in length) for, for example, display on VLPs.

[0053] In an embodiment, the LIC site comprises a nucleic acid sequence that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NOS: 1-3 and 50-52.

[0054] In an embodiment, the cloning site is disposed in the nucleic acid sequence encoding the MP, and wherein the cloning site is disposed in a position corresponding to loop end regions of the MP. In an embodiment, the cloning site is disposed in a beta-turn region.

[0055] In an embodiment, the nucleic acid sequence encoding the MP comprises a nucleic acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to the following sequence:

[0056] ATGCGAGCTTTTAGTACCCTTGATAGGGAGAACGAGACCTTC GTCCCCTCCGTTCGCGTTTACGCGGACGGTGAGACTGAAGATAACTCATTCTC TTTAAAATATCGTTCGAACTGGACTCCCGGTCGTTTTAACTCGACTGGGGCCA AAACGAAACAGTGGCACTACCCCTCTCCGTATTCACGGGGGGCGTTAAGTGT CACATCGATAGATCAAGGTGCCTACAAGCGAAGTGGGTCATCGTGGGGTCGC CCGTACGAGGAGAAAGCCGGTTTCGGCTTCTCCCTCGACGCACGCTCCTGCTA CAGCCTCTTCCCTGTAAGCCAGAACTTGACTTACATCGAAGTGCCGCAGAAC GTTGCGAACCGGGCGTCGACCGAAGTCCTGCAAAAGGTCACCCAGGGTAATT TTAACCTTGGTGTTGCTTTAGCAGAGGCCAGGTCGACAGCCTCACAACTCGCG ACGCAAACCATTGCGCTCGTGAAGGCGTACACTGCCGCTCGTCGCGGTAATT GGCGCCAGGCGCTCCGCTACCTTGCCCTAAACGAAGATCGAAAGTTTCGATC AAAACACGTGGCCGGCAGGTGGTTGGAGTTGCAGTTCGGTTGGTTACCACTA ATGAGTGATATCCAGGGTGCCTATGAGATGCTTACGAAGGTTCACCTTCAAG AGTTTCTTCCTATGAGAGCCGTACGTCAGGTCGGTACTAACATCAAGTTAAAT GGCCGTCTGTCGTATCCAGCTGCAAACTTCCAGACAACGTGCAACATATCGC GACGTATCGTGATATGGTTTTACATAAACGATGCACGTTTGGCATGGTTGTCG TCTCTAGGTATCTTGAACCCACTAGGTATAGTGTGGGAAAAGGTGCCTTTCTC ATTCGTTGTCGACTGGCTCCTACCTGTAGGTAACATGCTCGAGGGCCTTACGG CCCCCGTGGGATGCTCCTACATGTCAGGAACAGTTACTGACGTAATAACGGG TGAGTCCATCATAAGCGTTGACGCTCCCTACGGGTGGACTGTGGAGAGACAG GGCACTGCTAAGGCCCAAATCTCAGCCATGCATCGAGGGGTACAATCCGTAT GGCCAACAACTGGCGCGTACGTAAAGTCTCCTTTCTCGATGGTCCATACCTTA GATGCGTTAGCATTAATCAGGCAACGGCTCTCTAGATAA (SEQ ID NO. 4). In an embodiment, the cloning site is disposed at least one of the following residues of SEQ ID NO. 4: 46-102, 235-255, 730-747, and 1001-1033. As described further herein, such residues are positioned at the ends of beta sheets. Without wishing to be bound by any particular theory, it is understood that such end portions are not involved in or less involved in MP protein structure and / or formation and are, therefore, in part more amenable to substitution and / or addition of heterologous amino acid sequences while still being capable of forming VLPs / PLPs.

[0057] As used herein, “at least 55% identical” means that the polypeptide differs in its full length amino acid sequence by 45% or less (including any substitutions, deletions, additions, or insertions) from the sequences described herein.

[0058] In an embodiment, the nucleic acid encoding the MP or the CP further comprises a nucleic acid sequence in the cloning site encoding an amino acid sequence comprising between 1 amino acid residue and about 1,000 amino acid residues.

[0059] In an embodiment, the amino acid sequence is an amino acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to the following sequences:

[0060] MRAF STLDRENETF VP S VRVY ADGETEDNSF SLKYRSNWTPGRF NSTGAKTKQWHYPSPYSRGALSVTSIDQGAYKRSGSSWGRPYEEKAGFGFSLDA RSCYSLFPVSQNLTYIEVPQNVANRASTEVLQKVTQGNFNLGVALAEARSTASQL ATQTIALVKAYTAARRGNWRQALRYLALNEDRKFRSKHVAGRWLELQFGWLPL MSDIQGAYEMLTKVHLQEFLPMRAVRQVGTNIKLNGRLSYPAANFQTTCNISRRI VIWFYINDARLAWLSSLGILNPLGIVWEKVPFSFVVDWLLPVGNMLEGLTAPVG CSYMSGTVTDVITGESIISVDAPYGWTVERQGTAKAQISAMHRGVQSVWPTTGA YVKSPFSMVHTLDALALIRQRLSR (SEQ ID NO. 5); and

[0061] MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAY KVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMEL TIPIFAT NSDCELIVKAMQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGHHHHHHGDVT VAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVG GVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO. 6).

[0062] In an embodiment, the cloning site is disposed in the nucleic acid encoding the MP and is a restriction site.

[0063] In an embodiment, the nucleic acid sequence encoding the CP is positioned 5’ of the nucleic acid sequence encoding MP.

[0064] In an embodiment, the expression vector further comprises one or more ribosomal binding sites.

[0065] In an embodiment, a ribosomal binding site of the one or more ribosomal binding sites is positioned 3’ of the nucleic acid sequence encoding the CP.

[0066] In an embodiment, the one or more ribosomal binding sites comprise a sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to the following sequences:

[0067] TTTGTTTAACTTTAAGAAGGAGA (SEQ ID NO. 7);

[0068] ATTAAAGAGGAGAAA (SEQ ID NO. 8);

[0069] TC AC AC AGGAAAG (SEQ ID NO . 9);

[0070] AAAGAGGAGAAA (SEQ ID NO. 10);

[0071] CTTTAGGAGGT (SEQ ID NO. 11); and

[0072] AGGAGAT (SEQ ID NO. 12).

[0073] In an embodiment, the expression vector further comprises a stop codon positioned 5’ of the nucleic acid sequence encoding CP.

[0074] In an embodiment, the nucleic acid sequence encoding the CP encodes for a CP dimer.

[0075] In an another aspect, the present disclosure provides an MP fusion protein produced using the expression vector according to any embodiment of the present disclosure.

[0076] In an aspect, the present disclosure provides a virus-like particle (VLP) comprising the MP fusion protein according to any embodiment of the present disclosure.

[0077] As used herein, the term “virus-like particle” refers to a VLP resembling the structure of a bacteriophage, being non replicative and noninfectious, and usually lacking one or more viral genes needed for propagation of the bacteriophage as an infectious virus. The VLPs of RNA bacteriophages typically also lack the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. In examples, the VLP is a phage-like particle (PLP).

[0078] This definition also encompasses VLPs of bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and noninfectious virus-like particles of a bacteriophage.

[0079] A VLP can comprise the capsid structure formed from the self-assembly of one or more subunits of RNA bacteriophage CP and MP and, in embodiments, comprising mRNA for the CP and MP, and optionally containing host RNA.

[0080] In an embodiment, a ratio of CP:MP in the VLP is in a range of about 5: 1 to about 90: 1.

[0081] In an embodiment, the VLPs of the present disclosure comprise a heterologous peptide inserted into the MP. In an embodiment, the heterologous peptide is inserted into a loop end region of the MP. As described further herein, such inserted heterologous peptides can be useful for a variety of functions, such as purification, display, and the like. Additionally, because of the placement of the inserts, the size and length of the inserted heterologous peptides can vary widely.

[0082] A “heterologous” peptide is a peptide which is an identifiable segment of a polypeptide that is not found in association with the larger polypeptide in nature.

[0083] In an embodiment, the VLPs of the present disclosure comprise an MP fusion protein comprising a heterologous amino acid sequence inserted at one or more of residues 16-34, 79-85, 244-249, and 335-344 of SEQ ID NO. 4.

[0084] In an embodiment, the MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 14, 17, 54, and 56.

[0085] In an embodiment, the MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 15 and 18.

[0086] In an embodiment, the VLP comprises a payload, such RNA molecules disposed in an interior of the VLP. In an embodiment, the payload comprises a fragment of an infectious disease genome (e.g., a portion of an RNA virus, such as HIV), or several fragments concatenated (e.g., an influenza fragment, an RSV fragment, a Norovirus fragment, and / or multiple fragments from the same genome), to simulate multiple pathogens for assay QC assessment. In an embodiment, the packaged payload comprises an engineered reference sequence, either in whole or in part, in order to be differentiable from known pathogens. For example, this may be a pathogen sequence corresponding to a nucleic acid amplification diagnostic assay target with the detectable element replaced with a synthetic sequence, such as a unique and differentiable signal from the actual target. In an embodiment, the target is the target of a PCR assay, with the position of the detecting probe replaced with an artificial target, so that the fragment is amplified using the same PCR materials and system, but detected by a dedicated and differentiable probe. In an embodiment, the packaged payload sequence may also be randomized to create a library or pool of sequences, which can then be associated with the PLP plasmid sequence, so that the packaged sequence can act as a reporter or barcode for changes within the VLP plasmid and that can be recovered from the VLP itself.

[0087] In an embodiment, the payload comprises sequences encoding the MP and / or CP mRNA (depending on vector configuration), rather than a specified non-coding packaged sequence. To achieve this, the dedicated packaged sequence transcription cassette can be removed by eliminating the promoter element (nominally T7), the control cloning site sequences (e.g. homology tags), the packing signals, and the terminator associated with the control cloning site. The packing signals (pac sites) are inserted into the 3’ untranslated region (i.e., between the stop codon and transcription terminator) of the transcript containing the PLP capsid protein to be packaged (the coat protein dimer, maturation protein, or both; transcript may be polycistronic). Thus, the coding transcript for this / these protein(s) will be directed to be packaged within the PLP. This configuration has applications in phage / PLP display, wherein the sequence coding for an MP fusion and / or a CP / CPD fusion can be packaged within the particle and allow the displayed peptide or protein coding sequence(s) to be recovered from the RNA within the particle.

[0088] An alternative to this approach is to convert the control transcription cassette into an expression cassette by incorporating a ribosomal binding site (RBS) between the promoter and the control insert. In this instance, the packaged sequence is a coding sequence for a peptide or protein fused with a protein affinity element such as Spytag, SpyCatcher, or other protein-protein interaction domain. The cognate affinity element (e.g., SpyTag for SpyCatcher) is then fused to the MP or CPD, so that the now- expressed protein associates with the surface of the PLP capsid for phage display (a secondary display strategy, e.g. “spyDisplay”). In summary, the packaged sequence is expressed and displayed on the particle via its binding or reaction with the fusion, and the encoding RNA would be packaged within the PLP. This may also be a polycistronic approach where the packaged RNA may be the same as the RNA encoding for one or more of capsid proteins (CP / CPD / MP), or different, or be associated with multiple expressed proteins. In an embodiment, a displayed peptide is a cell-targeting peptide (intended to promote binding or uptake of the particle into a cell), an immunogenic peptide (intended to create an immune response), a therapeutic peptide, or a combination of those (e.g., one on CPD and one on MP).

[0089] In an embodiment, the packaged RNA is a coding sequence for an immunogenic peptide or a therapeutic peptide that can be expressed in vivo.

[0090] In various embodiments, the VLPs / PLPs of the present disclosure can be in the form of vaccines. In one embodiment, the PLP / VLP is suitable for use as a vaccine.

[0091] For a nucleic acid vaccine, the RNA payload (AKA the “control sequence”) of the PLP / VLP can comprise a gene or genes that are expressible by the targeted host organism (e.g., humans, farm animals). This gene or genes can encode a known antigen or antigens, or fragments of antigens, so that when expressed the presence of the antigen initiates a neutralizing immune response. To enhance stability and functionality of the payload RNA, functional elements such as (but not limited to) ribosomal binding sites (e.g. an internal ribosome entry site (IRES)), and RNA stabilizing structures (polyadenylation, hairpin structures, etc), might be incorporated into the payload.

[0092] In the case of antigen subunit type vaccines, the PLP may be modified to display known pathogen antigens, in whole or in part, in the MP and / or CP fusion sites for surface display. These antigens would then be recognizable by the host to initiate an immune response.

[0093] In some embodiments, the PLP vaccine comprises both an RNA vaccine payload and surface displayed antigens.

[0094] In some embodiments, the PLP displays cell-penetrating and / or targeted affinity peptides or proteins in the CP or MP fusion sites on its surface. These modifications could facilitate the vaccine particles entry into host cells to initiate an immune response.

[0095] In various embodiments, the VLPs / PLPs of the present disclosure are suitable for RNA-based therapeutics. As above, the PLP may carry an RNA containing expressible genes and may include CP or MP surface display modifications to facilitate host cell targeting or uptake. In the present embodiment, rather than encoding an antigen, the VLP carries the coding sequence for therapeutic protein(s) intended to treat disease or augment capability.

[0096] In various embodiments, the VLP / PLPs of the present disclosure are suitable for drug delivery. A PLP carrying CP or MP surface modifications may be used as a drug delivery platform. The particles of the present disclosure can be disassembled in- vitro (using, e.g., changes in pH) and reassemble the PLP / VLPs in the presence of a drug or other therapeutic agent (DNA, protein, chemical, etc.) to compartmentalize the drug. As before, the surface modifications may be engineered to facilitate cell targeting or penetration / entry for targeted drug delivery to specific tissues (e.g. a tumor).

[0097] In various embodiments, the VLPs / PLPs of the present disclosure are suitable for biopanning.

[0098] In embodiments, the CPD displays a purification tag and the MP displays a library of peptides, and the packaged RNA contains the coding sequence for the MP peptide. In an embodiment, the MP displays a purification tag and the CPD displays a library of peptides, and the packaged RNA contains the coding sequence for the CPD peptide

[0099] The PLPs may be used as a biopanning platform for discovery of protein affinity reagents or characterization of natural or artificial binding interactions. In this case, PLP displays a peptide, polypeptide or protein of interest (POI) in its CP or MP position and may be combined with other surface modifications (e.g., an affinity tag) in the CP or MP. This POI might be a naturally occurring sequence, or a synthetically engineered protein or protein scaffold. The particle would be configured such the mRNA encoding the POI sequence was directed to be packaged as a payload by the “pac” operator. As an example, if the POI were an MP fusion, the mRNA encoding the MP would carry the pac sequence(es). Alternatively, if the POI were expressed as a protein with e.g. Spytag / SpyCatcher to self-assemble with a Spytag / Spy catcher modification in the CP or MP, that protein encoding mRNA would be packaged in the PLP. In this way, the PLP would contain the encoding information for the POI surface displayed element. The POI gene might consist of a single sequence, a small collection of sequences, or a library with nearly unlimited sequences (e.g. 1E9+). Libraries of sequences could be cloned individually into the vector or could be cloned from pools of encoding DNA fragments (e.g. as degenerate oligonucleotide sequences, synthetic gene pools, experimentally derived DNA fragments, etc.), which would then be expressed to create libraries of PLPs harboring variant surface displayed proteins and carrying the mRNA that encodes those sequences. This material could be used for biopanning-like discovery, wherein the PLPs are selected for their affinity to a surface immobilized antigen or antigens. More specifically, PLP libraries in solution would be applied to a surface immobilized antigen (e.g., polystyrene beads or plates, or via antibody intermediates, etc.). The PLP solution would be removed, and the antigen substrate washed. PLPs displaying POIs with high affinity would be retained on the antigen during this process. The identity and sequence information from the retained / high-affinity POIs could then be recovered from the retained PLPs by standard molecular biology processes and manipulations (e.g. extraction, RT-PCR, sequencing). Recovered payload sequences could be used to create a secondarylibrary of vectors and PLPs to continue the iterative biopanning process, with the highest affinity POIs being enriched over cycles / time.

[0100] In another aspect, the present disclosure provides recombinant host cells comprising the nucleic acid expression vectors of the disclosure. The host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected or transduced. Such transfection and transduction of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.). A method of producing a polypeptide according to the present disclosure is an additional part of the disclosure. The method comprises the steps of (a) culturing a host according to this aspect of the present disclosure under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide. The expressed polypeptide can be recovered from the cell free extract, cell pellet, or recovered from the culture medium. Methods to purify recombinantly expressed polypeptides are well known to a person ordinarily skilled in the art.

[0101] TERMINOLOGY

[0102] Unless stated otherwise, experimental hypotheses or forward-looking models and statements are not intended to be binding on the applicant or exhaustive of the range of possible experimental hypotheses or forward-looking models and statements, but rather are intended to be illustrative, non-limiting examples for aiding those in the art in the understanding and practice of elements of the disclosure.

[0103] All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells:A Manual of Basic Technique, 2ndEd. (R. I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0104] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.

[0105] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0106] As used throughout the present application, the term “polypeptide” is used in its broadest sense to refer to a sequence of subunit amino acids, whether naturally occurring or of synthetic origin. The polypeptides of the present disclosure may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, or glycosylation. Such linkage can be covalent or non-covalent as is understood by those of skill in the art. The polypeptides may be linked to any other suitable linkers, including but not limited to any linkers that can be used for purification or detection (such as FLAG or His tags).

[0107] All embodiments of any aspect of the present disclosure can be used in combination, unless the context clearly dictates otherwise.

[0108] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0109] Unless the context clearly requires otherwise, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

[0110] Unless the context clearly requires otherwise, the phrase “consisting of’ excludes any element, step, or ingredient not specified.[OHl] If an element is described or claimed herein such that it “comprises” a feature, that description or claim also includes embodiments wherein the element “consists essentially of’ and embodiments wherein the element “consists of’ the feature, unless something else is specifically stated to the contrary.

[0112] A nucleic acid is a polymer of monomer units or “residues”. The monomer subunits, or residues, of the nucleic acids each contain a nitrogenous base (i.e., nucleobase), a five-carbon sugar, and a phosphate group. The identity of each residue is typically indicated herein with reference to the identity of the nucleobase (or nitrogenous base) structure of each residue. Canonical nucleobases include adenine (A), guanine (G), thymine (T), uracil (U) (in RNA instead of thymine (T) residues) and cytosine (C). However, the nucleic acids of the present disclosure can include any modified nucleobase, nucleobase analogs, and / or non-canonical nucleobase, as are well-known in the art. Modifications to the nucleic acid monomers, or residues, encompass any chemical change in the structure of the nucleic acid monomer, or residue, that results in a noncanonical subunit structure. Such chemical changes can result from, for example, epigenetic modifications (such as to genomic DNA or RNA), or damage resulting from radiation, chemical, or other means. Illustrative and nonlimiting examples of noncanonical subunits, which can result from a modification, include uracil (for DNA), 5-methylcytosine, 5-hydroxymethylcytosine, 5- formethylcytosine, 5-carboxycytosine b-glucosyl-5-hydroxymethylcytosine, 8- oxoguanine, 2-amino-adenosine, 2-amino-deoxyadenosine, 2-thiothymidine, pyrrolo- pyrimidine, 2-thiocytidine, or an abasic lesion. An abasic lesion is a location along the deoxyribose backbone but lacking a base. Known analogs of natural nucleotides hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphorothioate DNA. The five-carbon sugar to which the nucleobases are attached can vary depending on the type of nucleic acid. For example, the sugar is deoxyribose in DNA and is ribose in RNA. In some instances herein, the nucleic acid residues can also be referred with respect to the nucleoside structure, such as adenosine, guanosine, 5-methyluridine, uridine, and cytidine. Moreover, alternative nomenclature for the nucleoside also includes indicating a “ribo” or deoxyrobo” prefix before the nucleobase to infer the type of five-carbon sugar. For example, “ribocytosine” as occasionally used herein is equivalent to a cytidine residue because it indicates thepresence of a ribose sugar in the RNA molecule at that residue. A nucleic acid polymer can be or comprise a deoxyribonucleotide (DNA) polymer, or a ribonucleotide (RNA) polymer. The nucleic acids can also be or comprise a PNA polymer, or a combination of any of the polymer types described herein (e.g., contain residues with different sugars).

[0113] Unless otherwise stated or the context clearly requires otherwise, methods of the disclosure can be performed, in whole or in part, in any order of steps, including steps that are performed subsequently, in parallel, and in combination. In addition, methods can be performed, in whole or in part, by humans optionally assisted by one or more machines such as one or more computational devices or systems (e.g., computer(s)). In at least some instances, methods can be performed by one or more humans with little or no substantive assistance by one or more machines. In at least some other instances, methods can be performed by one or more humans with substantive assistance by one or more machines, and in at least some instances, one or more machines can perform methods autonomously or semi-autonomously.

[0114] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0115] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0116] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0117] All of the references cited herein are incorporated by reference. Aspects of the disclosure can be modified, if necessary, to employ the systems, functions, and concepts of the cited references and disclosure to provide yet further embodiments of thedisclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0118] It will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications can be made without deviating from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as stated by the claims.

[0119] TABLE 1 : Polynucleotide and polypeptide sequences of the present disclosure.EXAMPLESEXAMPLE 1 : MATERIALS AND METHODS

[0120] Plasmid construction

[0121] Expression vectors containing the elements for PLP production were constructed by cloning combinations and variations of the expression and control transcription cassettes into our mini-plasmid pEN6, an in-house native protein expression LIC vector. The pEN6 backbone was designed in Genious 8.1.9 (Dotmatics) using publicly available genetic elements, principally from the iGEM Registry of Standard BiologicalParts, as well as de novo sequence. The antecedent plasmid was ordered as a synthetic DNA fragment (gBlocks™, Integrated DNA Technologies (IDT)), enzymatically circularized and transformed into E. coli. Several iterations of changes have been introduced using routine muta-genesis methodology to generate the current embodiment of the pEN6 vector. The PLP cassette elements were similarly made with Geneious using relevant genes derived from MS2 (GenBank accession number: NC 001417) in combination with known genetic elements and de novo sequence. The Coat Protein Dimer sequence was designed with extensive silent mutations in repeat elements to aid in synthesis, manipulation, and plasmid stability. LIC sites within coding sequences were created by introducing silent mutations where possible, or with favored substitutions when not possible. gBlock (IDT) gene fragments corresponding to the CPD, MP, and packaged sequence cloning site with its associated pac sequence were ordered and assembled by overlap-extension PCR (OE- PCR) into expression cassettes. The assembled cassettes were cloned by ligation independent cloning (LIC) into pEN6 and then modified over multiple iterations using combinations of site-directed muta-genesis and OE-PCR to generate the study’s plasmid variants. It is expected these plasmids could be manufactured by gene synthesis to reproduce them as a whole.

[0122] PCR, site-directed mutagenesis, and vector manipulation

[0123] For routine vector manipulations such as preparation of linearized plasmid backbones for LIC, preparation of fragment inserts for LIC (affinity tag gene fragments, packaged sequence), site-directed mutagenesis, and OE-PCR, Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs® (NEB), M0493) was used, per manufacturer’s guidance. All oligos necessary for vector manipulation used in this study were sourced from IDT. Circularization of linear PCR products following mutagenesis or assembly was accomplished with KLD Enzyme Mix (NEB, M0554). Amplified fragments and cultured plasmids were purified as necessary by column-based cleanup and extraction Kits (NEB, T1030, T1020, and T1010).

[0124] Ligation independent cloning

[0125] Overhang generation reactions for vector and insert fragments used in LIC included 500 ng of purified PCR Product with 2 pL NEBuffer™ r2.1 (NEB, B6002), 0.5 pL of 100 mM deoxynucleotide (dGTP or dCTP) (NEB, N0446), 0.5 pL of 0.2 M Dithiothreitol, and 0.5 pL (1.4 units (U)) of T4 DNA polymerase (NEB, M0203) in a final volume of 20 pL. Reactions were incubated at 22 °C for 30 min followed by incubation at80 °C for 20 min. Overhangs for short (<200 bp) inserts were realized by ordering partially complimentary synthetic oligonucleotide strands (IDT) and annealing them in NEBuffer™ r2.1 by heating them to 90 °C and cooling them to room temperature. Vector and insert fragments with overhangs were combined at approximately 1 :3 ratio with a total volume of 1.5-5 pL. Reactions were incubated at room temperature for 5 min, then supplemented with 1 pL of 25 mM Ethylenediaminetetraacetic acid, and further incubated for 5 min before transformation.

[0126] Cloning, expression, and lysis

[0127] Plasmids were transformed into T7 Express lysY / Iq Competent E. coli (High Efficiency) (NEB, C3013I) per the manufacturer’s protocol. Select clones were sequence-verified by Sanger sequencing or whole plasmid sequencing (GENEWIZ® Sanger or Plasmid-EZ). Starter cultures were grown from validated clones by inoculating 4 mL LB Broth (Fisher, BP9722) containing 100 pg / mL carbenicillin (“growth media”) and incubating at 30 °C overnight (approx. 16 h) while shaking at 200 RPM. 11 mL of growth media were inoculated with 100 pL of starter culture and incubated at 37 °C while shaking at 200 RPM for use as expression cultures. Expression experiments were conducted as specified in the manufacturer’s recommended protocol, except that immediately prior to induction with isopropyl [3-d-l -thiogalactopyranoside, 1 mL of culture was partitioned as a negative induction control. Following expression, cells were immediately harvested by centrifugation at 1,500 RCF for 15 min and growth media was siphoned off. The cell pellet was resuspended in 1 mL of Lysis and Binding Buffer (NEB, S1427S) for IMAC or, alternatively, in 1 mL of Buffer W (IBA Lifesciences GmbH, 2- 1003-100) for Strep-tag®II (ST) purifications, and lysed by ultrasonic disruption with a Fisherbrand Model 120 Sonic Dismembrator (Thermo Fisher Scientific, FB120A110), while on ice, for 2 total minutes at 40% amplitude with 2 s on and 2 s off, pulse. Lysed samples were centrifuged at 12,000 RCF for 15 min to pellet insoluble material and cell debris, and the clarified lysate supernatant was collected.

[0128] NEBExpress® purification

[0129] PLPs were purified from crude lysates using NEBExpress® Ni Spin Columns (NEB, S1427) generally following the manufacturer’s protocol, reproduced in part here with optional steps and variances. Prepared columns were loaded with 500 pL of clarified lysate and incubated while rotating at 4 °C for 15 min, then centrifuged at 800 RCF for 1 min 30 s. Columns were next loaded with a nuclease solution containing 5 U ofDNase I (NEB, M0303), 62.5 U of RNase If (NEB, M0243) in 250 pL of DNase I Reaction buffer (NEB, B0303) and incubated at 37 °C for 10 min prior to centrifugation. Columns were washed three times with Wash Buffer containing 10 mM imidazole, otherwise as described in the product documentation. Particles were eluted as recommended, followed by dialysis of 150 pL of the first elution fraction in Tube-O-DIALYZER™, Micro 50K MWCO dialysis tubes (G-Biosciences®, 786-614) into 50-100 mL STLE buffer (10 mM tris-HCL pH7.5, 10 mM NaCl, 0.1 mM EDTA). Dialysis was completed with three buffer exchanges, allowing 2-4 h between exchanges, at room temperature while shaking at 50 RPM or over-night at 4 °C. Dialyzed samples were transferred to Eppendorf® Protein LoBind Tubes (Eppendorf®, 022431102) and stored at 4 °C. Sample concentrations were estimated as needed by A280 UV-Vis spectrophotometry (1 abs = 1 mg / mL).

[0130] Strep-Tactin® XT purification

[0131] PLPs were purified using a modified in-house column extraction using Strep-Tactin®XT 4Flow® resin (IB A Lifesciences GmbH, 2-5010-002). Emptied and cleaned columns from the NEBExpress® Ni Spin Column kit were recycled for Strep-tag® purification. The cleaned columns were loaded with 200 pL of Strep-Tactin®XT 4Flow® 50% suspension and centrifuged at 500 RCF for one minute to remove the storage buffer. The beads were washed by addition of 250 pL of lx Buffer W. 500 pL of clarified sample lysate supernatant was applied to the column, sealed, and rotated at 4 °C for 30 min or overnight for particles with CPD-Strep-tag®II fusion or MP-Twin-Strep-tag® fusion, respectively, to bind the PLPs. Columns were centrifuged at 500 RCF for one minute and the flow through was collected. An on-column nuclease treatment was applied as described for IMAC, and then the resin was washed three times with 250 pL of Buffer W. PLPs were eluted by applying 200 pL lx elution reagent Buffer BXT (IBA Lifesciences GmbH, 2- 1042-025), vortexing briefly, incubating at room temperature for 5 min, vortexing briefly again, and centrifuging at 700 RCF for 1 min to collect the eluant. Buffer exchange by dialysis was done as described for IMAC, above.

[0132] SDS-PAGE

[0133] SDS-PAGE experiments were carried out on a Mini-PROTEAN Tetra apparatus (Bio-Rad Laboratories, Inc, 1658004) with 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad Laboratories Inc, 4561096 and 4561095) and Tris / Glycine / SDS buffer (Bio-Rad Laboratories, Inc, 1610732) and were run at 200 V for 35 min. 5 pL of Color Prestained Protein Standard, Broad Range (NEB, P7719) were runfor protein size approximation. Gels were stained for visualization with QC Colloidal Coomassie Stain (Bio-Rad Laboratories, Inc, 1610803) per the manufacturer’s instructions. Samples were processed for electrophoresis by the addition of 4x Laemmli Sample Buffer (lx final) (NEB, 1610747) and 0.2 M Dithiothreitol (0.05 M final) and heating to 90 °C for 10 min. All samples were run in volume equivalent quantities for direct comparison. Insoluble fractions were solubilized in 1 mL 8 M urea prior to sample preparations. ImageJ 1.53e software was used for Gel Densitometry analysis.

[0134] RT-qPCR

[0135] RT-qPCR was executed in a three-step process: lysis, reverse transcription, and qPCR. Particles were lysed in a solution containing: 2 pL of PLPs, 37 pL of TLE / IDTE pH 8.0 buffer (IDT, 11-05-01-09), and 1 pL (40 U) of RNasin® Plus Ribonuclease Inhibitor (Promega Corporation, N2611), with a final volume of 40 pL. Samples were heated to 65 °C for 5 min to lyse, then stored on ice until needed. The reverse transcription reaction mix was prepared containing: 13.75 pL Molecular Biology Grade Water (Coming®, 46-000-CM), 2 pL lOx Isothermal Amplification Buffer (NEB, B0537S), 0.5 pL 10 mM dNTP Solution Mix (NEB, N0447S), 1 pL of 10 pM RT primer, 0.5 pL (20 U) RNasin® Plus Ribonuclease Inhibitor, 0.25 pL (0.75 U) WarmStart® RTx Reverse Transcriptase (NEB, M0380S), and 2 pL of PLP lysate. Reverse transcription reactions were incubated at 51 °C for 10 min followed by 80 °C for 10 min and resultant cDNA was stored at -20 °C until needed. Reverse transcriptase negative reactions were prepared by substituting water for the reverse transcriptase and were otherwise handled identically. At each step, additional negative no-template control (NTC) reactions were generated by substituting the appropriate buffer for the PLP sam-ple. qPCR reactions were prepared as follows: 11.58 pL molecular grade water, 4 pL 5x Reaction Buffer (MGQuest, EP032), 0.4 pL dNTP Solution Mix, 0.8 pL 10 pM Forward Primer, 0.8 pL Reverse Primer, 0.3 pL EvaGreen® Dye 20x in Water (Biotium, 31000), 0.12 pL (0.6 U) GemTaq™ Hot Start DNA Polymerase (MGQuest, EP032), and 2 pL of cDNA sample. qPCR reactions were run on a CFX-96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, #1851196 with #1845096). loglO standard curves with copy number inputs ranging from 2E7-2E2 of a dsDNA fragment corresponding to the packaged sequence, as well as negative and NTC reactions, were evaluated in each run. Data analysis was performed with Bio-Rad CFX Maestro Software 2.3 (Bio-Rad Laboratories, 12013758).

[0136] RNase protection assay

[0137] Three replicate samples of each particle type were made for appraisal of RNase resistance by mixing 2 pL of purified PLPs into 38 pL of TLE / IDTE pH 8.0 buffer. Samples were then split into 20 pL aliquots to create two sets of paired replicate samples. One set was stored on Ice, while the second set was heated at 65 °C for 5 min. Each sample was challenged with RNase digest reactions containing: 20.85 pL Molecular Biology Grade Water, 3 pL lOx DNase I Reaction buffer, 0.15 pL (7.5 U) RNase If, and 6 pL of sample. Reactions were Incubated at 37 °C for 10 min and then placed on ice, followed by immediate RNA purification with a Monarch® Total RNA Miniprep Kit (NEB, T2010). 30 pL of DNA / RNA Protection Reagent was mixed with each RNase digest reaction prior to RNA extraction as recommended for reaction cleanup with on-column DNase I treatment, and samples were eluted in 100 pL of nuclease-free water. cDNA was made from RNA as previously described, except 6 pL of RNA was used in the 20 pL reaction. Quantitation of cDNA was evaluated by qPCR as above, with a standard curve ranging from 2E6 to 2E1 copies per reaction.

[0138] Dynamic light scattering

[0139] Dialyzed PLPs were diluted 1 : 10 into STLE buffer and particle sizes were measured with a Malvern Zetasizer Nano ZS. Specifically, 80 pL of diluted purified PLP solution was added into BRAND UV micro cuvettes (Sigma, BR759200) for measurement of protein using parameters appropriate for STLE buffer at 25 °C. Default number-based size distributions were reported for analysis.

[0140] Transmission electron microscopy (TEM)

[0141] PLPs were stained using the side-blot method with 0.5% phosphotungstic acid (pH = 7.2) for 10 s and images were captured using a Philips CM100 transmission electron microscope operating at 80 kV.EXAMPLE 2: RESULTS AND DISCUSSION

[0142] Plasmid engineering

[0143] A set of related expression vectors was created to explore possible refinements to the design of PLP-producing expression cassettes (Figure 1). Briefly, vector features are as follows: plH is conceptually similar (not identical) to the plasmid described by Mikel et al. (Mikel P, Vasickova P, Kralik P. One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles. Sci Rep. 2019;9(l):6377. doi: 10.1038 / s41598-019-39693-2, and Mikel P, Vasickova P, Kralik P. One-plasmid double-expression His-tag system for rapid production and easy purificationof MS2 phage-like particles. Sci Rep. 2017;7(l): 17501. doi: 10.1038 / s41598-017-17951- 5) but contains a LIC site for inserting the packaged sequence and an LIC site flanking the His6 affinity tag in the CPD to allow replacement of the His6 with a different insertion. P2H is derived from plH with a rearrangement inverting the order of CPD and MP polycistronic mRNA that further necessitated alterations to the regulatory elements of both proteins, as well as revisions to the packaged sequence cloning cassette including an additional pac sequence. P2S is a minor variation of p2H with the eight-residue Strep- tag®II (ST) replacing the CPD His6 tag by way of the LIC site. p3H converts the expression of PLP proteins from a polycistronic format to a multiple expression format by substituting a T22 terminator, T7 promoter, and an exogenous ribosomal binding site (RBS) between the CPD and MP CDSs. The multiple expression format influences relative expression yields and allows for alternative pac signal configurations in planned future work. Several design iterations were explored to tune the relative expression of the proteins, and MP is expected to be a limiting component in particle formation for p3H. p4HS is a derivation of p3H that employs a new LIC site at 1,001-1,033 bp of the MP by making several point mutations around an in-frame insertion site which subsequently hosted a Twin-Strep-tag® (tandem ST tags) flanked by short glycine-serine linker sequences. Example LIC cloning site and linker pairs are provided in SEQ ID NOS. 13 and 53, as well as SEQ ID NOS. 16 and 55.

[0144] A synthetic non-coding packaged sequence was cloned into the Packaged sequence LIC cloning site in all plasmid designs for RT-qPCR quantitation. For further information on plasmid sequences refer to the Data Availability statement.

[0145] Maturase internal fusion

[0146] A variety of MP fusion strategies were attempted for this study in order to incorporate a secondary AC tag, inclusive of N and C-terminal truncations that were surface accessible, and internal fusions of loops and beta turns within the P-sheet domain. Structural modeling was performed using AlphaFold2 (ColabFold vl.5.5: AlphaFold2 using MMseqs2) to identify candidate internal-fusion sites. These positions were modified in vector p3H by site-directed mutagenesis to constitute new LIC sites, and various polypeptide coding sequence inserts were cloned to verify their solubility and copurification with the His6-tagged CPD. All but one of these attempts resulted in insoluble or fragmented MP or otherwise failed to co-purify with the coat protein (data not shown). The successful arrangement involved the introduction of a mutagenized LIC site and fusionproteins between MP Ser334 and Val352, roughly corresponding to the position of an antiparallel beta-sheet loop (Figure 2A). MP insertions of SpyCatcher (an 88 amino acid fragment) and SpyTag were successful, while a fusion with EGFP in that position was unsuccessful (data not shown). To evaluate its potential functionality for affinity chromatography, a Hisl3-tag was cloned into the MP LIC site, and the CPD His6-tag was removed (“empty”) to ensure purification was via the MP fusion. This MP Hisl3-tag fusion appeared to be soluble, but purification by IMAC was unsuccessful in isolation of particles when evaluated by SDS-PAGE, as were several other related His-tag designs (comprising various numbers and lengths of His-tag, and linker configurations). This failure may be attributed to educed avidity between the PLP and IMAC matrix because of the net decreased surface display of His tags. As an alternative high-affinity tag purification strategy, a Twin- Strep-tag® with flanking linkers was cloned into the MP LIC site (Figure 2B). In conjunction with a CPD His6-tag, this approach produced p4HS PLPs which were able to be purified by ST AC as well as IMAC (Figure 3E). A variant of p4HS with empty CPD and MP LIC sites recovered only trace amounts of a particle when purified by ST spin column, despite high expression yields, which confirmed the specificity of the ST fusion AC purification.

[0147] Comparative purification results

[0148] For each of the expression vectors and AC method strategies compared in this study, four replicate cultures were grown in parallel and induced for overexpression, and PLPs were purified by a relevant AC method in a spin column format. Purification yields were compared by protein visualization in SDS-PAGE and quantification by RT- qPCR. Particle formation was also confirmed by dynamic light scattering (DLS) and select particle preparations were evaluated in an RNase protection assay and by transmission electron micros-copy (TEM). PLPs are referred to by their originating construct and AC purification method (“plasmid” - “AC method”).

[0149] SDS-PAGE

[0150] SDS-PAGE gels were run so that relevant comparisons of vector designs and purification strategies could be made in gel (Figure 3). Qualitative evaluation of IMAC purifications confirms the functionality of the spin column-based format and overall workflow for the generated particles, with the His6-tagged CPD consistently visible in the final dialysis fraction (Figure 3B, 3D, 3F). Purifications by ST AC (Figure 3C-F) clearly show recovery of the expected tagged protein (CDP-ST or MP-ST fusions, as appropriate),validating the Strep-tag®II AC strategy as well. Across all purifications, excluding plH, the co-purification of CPD and MP proteins in elution fractions is indicative of functional capsid proteins forming a complex, regardless of the location and type of the affinity tag used for purification. Concentrations of plH particles are presumably too low to accurately assess this behavior by SDS-PAGE. Gel densitometry estimations of the protein-mass- corrected ratios of MP to CPD and their fusions universally suggest a higher ratio of MP to CPD than the assumed value of 1 :89 (one per particle). Values ranged from approximately 1 :32 for p4HS-IMAC to 1 : 11 for p2H-IMAC, and while variability in this number is expected due to differences in the levels of relative expression and the purification approach, it implies that more than one maturase may be present per particle under all evaluated conditions. It may be possible to tune the density of MP on the surface of the particle.

[0151] Purities of PLPs following dialysis were acceptable, however, many smaller protein contaminants appear to be proportional to final PLP concentrations and are not removed by dialysis with a higher MW cutoff than their apparent size. It is possible these small contaminants may be cytosolic proteins encapsidated within the particle, or alternatively, irreversibility associated with the surface of the particle. A discrete contaminant band (Figure 3E and 3F, “unk”) of larger size than is expected for the MP fusion is observed with purification of p4HS-ST, but this band is not seen when purified by IMAC or with p2S-ST purifications. These observations suggest that an undesired MP expression product contaminant that is not capable of integrating into a PLP may be co- purified by ST purification of MP-Strep-tag® fusions.

[0152] It is qualitatively apparent that reconfiguration of the plH expression cassette, exemplified by reordering the mat and CPD genes, resulted in dramatically increased production of CPD in sample lysates (Figure 3, plH vs all others, “S” lanes), corresponding to an increase in eluted protein (Figure 3, plH vs all others, “E” lanes). This observation is supported by densitometry analysis of replicates; CPD levels of p3H-HIS are more than 60-fold higher than plH-HIS (Figure 3B). This is most likely due to CPD expression being driven by a high-efficiency RBS in p2H and its derivatives, as opposed to the native RBS in plH. Relative yield of MP is markedly reduced following the redesign of plH into p2H, p2S, p3H, and p4HS, as is best visualized by comparison of the insoluble fractions (Figure 3, lanes “P”), considering the solubility of MP not in complex with CPD is low (Figure 3A, plH-IMAC, lanes “P” and “S”). Comparison of p2H-IMAC and p2S-ST PLPs, or of p4HS-IMAC and p4HS-ST PLPs, reveal higher yield with IMAC relative to ST purifications. This yield discrepancy is likely due to the binding capacity of the specific resins used in this context. However, the maximum ST column binding capacity is unclear because the CPD-ST fusion observed in p2S preparation has reduced yield relative to p2H for unknown reasons (this result was repeated and confirmed). While the MP-ST appears to be in excess or the p4HS-ST purification, the presence of the unknown contaminant complicates the interpretation of binding capacity.

[0153] RT-qPCR

[0154] Thermal Lysis of PLPs followed by RT-qPCR proved to be an effective tool for quantitating control templates. Quantitative measurement of the control template largely corroborated the observation of proteins by SDS-PAGE but allowed assessment of packaged sequences and direct juxtaposition across all PLP preparations (Figure 4A). Comparison of plH-IMACto p2H-IMAC revealed a greater than 362-fold increase in the average copy number of packaged control with the revised polycistronic configuration of p2H (p = 0.0007). A multiple expression system for the capsid proteins, p3H-IMAC, showed a greater than 400-fold increase in the control template relative to plH-IMAC (p < 0.0001), but was not significantly different from p2H-IMAC (p = 0.5163). Use of Streptag®II labeled CPD and ST AC purification for p2S-ST also significantly increased control template concentration relative to the plH-IMAC purification, producing greater than a 115-fold increase (p < 0.0001), but was reduced approximately 0.32-fold relative to the His6-tagged p2H-IMAC under similar experimental conditions (format, proto-col, volumes of resin) (p = 0.0051). In like manner, the dual-tagged p4HS PLP yields, when purified by either IMAC or ST, were significantly increased relative to plH (~265-fold (p < 0.0001) and ~38-fold (p < 0.0001), respectively) and higher concentrations were recovered via IMAC vs. ST (~7.9-fold, p < 0.0001). The successful detection of RNA control templates by these methods is, in itself, evidence it is co-purified with the protein components, supporting the utility of the plasmid constructs in PLP production. Template concentration observations are strong support for a dramatic increase in the production of functional PLPs with the reconfiguration of the phage protein expression cassette. Template measurements also confirm the successful purification of functional PLPs with an ST AC protocol, with tags located either as an internal fusion in the conventional CPD site or in the MP internal fusion site described here. The RT-qPCR results are incongruous with gel densitometry in regard to the relative scale of the increase, e.g. for pHl-IMAC vsp3H-IMAC Gel densitometry suggests a ~60-fold increase, whereas RT-qPCR found a greater than 400-fold increase. It is possible this is a result of increased packaging efficiency due to the tandem pac sequences resulting in multiple templates being pack-aged per particle, or a limitation in the accuracy of gel densitometry as implemented here.

[0155] Assessment of template DNA contamination of purified particles was achieved by omission of reverse transcriptase enzyme during the reverse transcription reaction so that only contaminating DNA template would be measured. Template DNA contamination was found to range from a mean of 1,984 to 6,178 copies per pL (Figure 4A, dark grey) across experiments. Interestingly, the amount of DNA contamination was not significantly correlated to the PLP preparations themselves (ANOVA, p = 0.37), despite significant differences in the concentrations of recovered RNA templates and varying methods of purification. The implication is that the observed DNA contamination is a function of the column-based purification format, plausibly due to passive non-specific adsorption to the column materials. As a consequence, the proportion of DNA to RNA control template varied from as high as 0.11% for plH-IMAC, to as low as 0.000092% for p3H-IMAC. While plH-IMAC is most similar to that described by Mikel et al., the relative DNA contamination in this format is markedly worse than that account (-0.00010%). However, the revised plasmid configurations (P2, P3, and P4) produce approximately similar levels of DNA contamination to those reported by Mikel, notwithstanding the very different microliter-scale spin column AC methodology.

[0156] RNase protection assay

[0157] Protection from RNase-driven degradation is a feature of PLPs for most applications. While previous iterations have shown definitive RNase protection conferred by the CPD-His and MP protein constituents of the capsid, the CPD-ST and MP-ST fusions of this study have no precedent. To that end, p4HS-IMAC and p2S-ST were evaluated as representative PLPs for these modifications (Figure 4B). When intact PLPs were challenged with an RNase treatment most template was recovered while particles subjected to heat lysis prior to the RNase challenge lost a majority of RNA templates relative to no RNase control conditions (log transformed paired T-test, p4HS-IMAC p = 0.0021, p2S-ST p = 0.0014). Nearly perfect recovery of template from intact p4HS-IMAC particles treated with RNase (-102% recovered, p = 0.8534) confirms that the CPD-His6 protein of previous studies, as well as the MP-ST support intact RNase excluding PLPs. Conversely, it was found that p2S-ST had some observable template degradation in the unlysed condition withRNase vs. without RNase (-63% recovered, p = 0.0338). All PLPs were previously treated with RNase under nearly identical conditions during purification, so it is plausible that the loss may be the result of degradation during lysis for sample purification due to reduced stability of the capsid with an ST modification. A preliminary investigation of particle stability appears to support this possibility.

[0158] Dynamic light scattering

[0159] DLS measurements (Figure 4C) support a monodispersed particle approximately similar to the theoretical diameter of 27 nm. Statistical comparison was not made, due to differing methodology of measurement.

[0160] Transmission electron microscopy

[0161] TEM images show spherical particles with a diameter of approximately 20-30 nm for all three PLP preparations that were investigated (Figure 4D-F). These observations confirm uniform nanoparticle formation within the expected size range.

[0162] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the present disclosure.NON-LIMITING EMBODIMENTS

[0163] While general features of the disclosure are described and shown and particular features of the disclosure are set forth in the claims, the following non-limiting Embodiments relate to features, and combinations of features, that are explicitly envisioned as being part of the disclosure. The following non-limiting Embodiments contain elements that are modular and can be combined with each other in any number, order, or combination to form a new non-limiting Embodiment, which can itself be further combined with other non-limiting Embodiments.

[0164] 1. An expression vector encoding a modified MS2 capsid, the expression vector comprising:

[0165] a nucleic acid sequence encoding a maturation protein A (MP); and

[0166] a nucleic acid sequence encoding a coat protein (CP),

[0167] wherein one or more of the nucleic acid sequence encoding the MP and the nucleic acid sequence encoding the CP comprises a cloning site.

[0168] 2 The expression vector of Embodiment 1, wherein the cloning site is an overlap homology cloning site.

[0169] 3 The expression vector of Embodiment 2, wherein the overlap homology cloning site is selected from sequence and ligation independent cloning (SLIC), ligation independent cloning (LIC), in-fusion, Gibson assembly, homology assembly cloning (HAC) sites, and combinations there.

[0170] 4. The expression vector of Embodiment 1, wherein the cloning site is a LIC site.

[0171] 5. The expression vector of Embodiment 4, wherein the LIC site comprises one of the following sequences:

[0172] CGTGTTGGTTGATAATGGC (SEQ ID NO. l)-(insert) - GGTGATGTAACCGTAGCTCCATCTAACTTCG (SEQ ID NO. 50);

[0173] GCATCAACTCTCCCGGT (SEQ ID NO. 2)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 51); and

[0174] GCATCAACTCCTCCGGT (SEQ ID NO. 3)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 52),

[0175] wherein (insert) indicates a location of an insert.

[0176] 6. The expression vector of any one of Embodiments 1-5, wherein the cloning site is disposed in the nucleic acid sequence encoding the MP, and wherein the cloning site is disposed in a position corresponding to loop end regions of the MP.

[0177] 7. The expression vector of any of Embodiments 1-6, wherein the nucleic acid sequence encoding the MP comprises a nucleic acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to the following sequence:

[0178] ATGCGAGCTTTTAGTACCCTTGATAGGGAGAACGAGACCTTC GTCCCCTCCGTTCGCGTTTACGCGGACGGTGAGACTGAAGATAACTCATTCTC TTTAAAATATCGTTCGAACTGGACTCCCGGTCGTTTTAACTCGACTGGGGCCA AAACGAAACAGTGGCACTACCCCTCTCCGTATTCACGGGGGGCGTTAAGTGT CACATCGATAGATCAAGGTGCCTACAAGCGAAGTGGGTCATCGTGGGGTCGC CCGTACGAGGAGAAAGCCGGTTTCGGCTTCTCCCTCGACGCACGCTCCTGCTA CAGCCTCTTCCCTGTAAGCCAGAACTTGACTTACATCGAAGTGCCGCAGAAC GTTGCGAACCGGGCGTCGACCGAAGTCCTGCAAAAGGTCACCCAGGGTAATT TTAACCTTGGTGTTGCTTTAGCAGAGGCCAGGTCGACAGCCTCACAACTCGCG ACGCAAACCATTGCGCTCGTGAAGGCGTACACTGCCGCTCGTCGCGGTAATT GGCGCCAGGCGCTCCGCTACCTTGCCCTAAACGAAGATCGAAAGTTTCGATCAAAACACGTGGCCGGCAGGTGGTTGGAGTTGCAGTTCGGTTGGTTACCACTA ATGAGTGATATCCAGGGTGCCTATGAGATGCTTACGAAGGTTCACCTTCAAG AGTTTCTTCCTATGAGAGCCGTACGTCAGGTCGGTACTAACATCAAGTTAAAT GGCCGTCTGTCGTATCCAGCTGCAAACTTCCAGACAACGTGCAACATATCGC GACGTATCGTGATATGGTTTTACATAAACGATGCACGTTTGGCATGGTTGTCG TCTCTAGGTATCTTGAACCCACTAGGTATAGTGTGGGAAAAGGTGCCTTTCTC ATTCGTTGTCGACTGGCTCCTACCTGTAGGTAACATGCTCGAGGGCCTTACGG CCCCCGTGGGATGCTCCTACATGTCAGGAACAGTTACTGACGTAATAACGGG TGAGTCCATCATAAGCGTTGACGCTCCCTACGGGTGGACTGTGGAGAGACAG GGCACTGCTAAGGCCCAAATCTCAGCCATGCATCGAGGGGTACAATCCGTAT GGCCAACAACTGGCGCGTACGTAAAGTCTCCTTTCTCGATGGTCCATACCTTA GATGCGTTAGCATTAATCAGGCAACGGCTCTCTAGATAA (SEQ ID NO. 4).

[0179] 8. The expression vector of any of Embodiments 1-7, wherein the cloning site is disposed in at least one of the following residues of the sequence of SEQ ID NO. 4: 46-102, 235-255, 730-747, and 1001-1033.

[0180] 9. The expression vector of any of Embodiments 1-8, wherein the MP or the CP further comprises a nucleic acid sequence in the cloning site encoding an amino acid sequence comprising between 1 amino acid residue and about 1,000 amino acid residues.

[0181] 10. The expression vector of Embodiment 9, wherein the amino acid sequence is an amino acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to any of the following sequences:

[0182] MRAFSTLDRENETFVPSVRVYADGETEDNSFSLKYRSNWTPGRF NSTGAKTKQWHYPSPYSRGALSVTSIDQGAYKRSGSSWGRPYEEKAGFGFSLDA RSCYSLFPVSQNLTYIEVPQNVANRASTEVLQKVTQGNFNLGVALAEARSTASQL ATQTIALVKAYTAARRGNWRQALRYLALNEDRKFRSKHVAGRWLELQFGWLPL MSDIQGAYEMLTKVHLQEFLPMRAVRQVGTNIKLNGRLSYPAANFQTTCNISRRI VIWFYINDARLAWLSSLGILNPLGIVWEKVPFSFVVDWLLPVGNMLEGLTAPVG CSYMSGTVTDVITGESIISVDAPYGWTVERQGTAKAQISAMHRGVQSVWPTTGA YVKSPFSMVHTLDALALIRQRLSR (SEQ ID NO. 5); and

[0183] MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAY KVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFAT NSDCELIVKAMQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGHHHHHHGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVG GVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO. 6).

[0184] 11. The expression vector of any of Embodiments 1-10, wherein the cloning site is disposed in the nucleic acid encoding the MP and is a restriction site.

[0185] 12. The expression vector of any of Embodiments 1-12, wherein the nucleic acid sequence encoding the CP is positioned 5’ of the nucleic acid sequence encoding MP.

[0186] 13. The expression vector of any of Embodiments 1-12 further comprising one or more ribosomal binding sites.

[0187] 14. The expression vector of Embodiment 13, wherein a ribosomal binding site of the one or more ribosomal binding sites is positioned 3’ of the nucleic acid sequence encoding the CP.

[0188] 15. The expression vector of Embodiment 13, wherein the one or more ribosomal binding sites comprise a sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to any of the following sequences:TTTGTTTAACTTTAAGAAGGAGA (SEQ ID NO. 7)ATTAAAGAGGAGAAA (SEQ ID NO. 8)TCACACAGGAAAG (SEQ ID NO. 9)AAAGAGGAGAAA (SEQ ID NO. 10)CTTTAGGAGGT (SEQ ID NO. 10)AGGAGAT (SEQ ID NO. 11)

[0189] 16. The expression vector of any of Embodiments 1-15, further comprising a stop codon positioned 5’ of the nucleic acid sequence encoding CP.

[0190] 17. The expression vector of any of Embodiments 1-16, wherein the nucleic acid sequence encoding the CP encodes for a CP dimer.

[0191] 18. An MP fusion protein produced using the expression vector of any of Embodiments 1-17.

[0192] 19. A virus-like particle (VLP) comprising the MP fusion protein of Embodiment 18.

[0193] 20. The VLP of Embodiment 19, wherein a ratio of CP:MP is in a range of about 5 : 1 to about 90: 1.

[0194] 21. An MP fusion protein comprising:

[0195] a heterologous amino acid sequence inserted at one or more of residues 16-34, 79-85, 244-249, and 335-344 of SEQ ID NO. 4.

[0196] 22. The MP fusion protein of Embodiment 21, wherein MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 14, 17, 54, and 56.

[0197] 23. The MP fusion protein of any of Embodiments 21 and 22, wherein MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 15 and 18.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An expression vector encoding a modified MS2 capsid, the expression vector comprising: a nucleic acid sequence encoding a maturation protein A (MP); and a nucleic acid sequence encoding a coat protein (CP), wherein one or more of the nucleic acid sequence encoding the MP and the nucleic acid sequence encoding the CP comprises a cloning site.

2. The expression vector of Claim 1, wherein the cloning site is an overlap homology cloning site.

3. The expression vector of Claim 2, wherein the overlap homology cloning site is selected from sequence and ligation independent cloning (SLIC), ligation independent cloning (LIC), in-fusion, Gibson assembly, homology assembly cloning (HAC) sites, and combinations there.

4. The expression vector of Claim 1, wherein the cloning site is a LIC site.

5. The expression vector of Claim 4, wherein the LIC site comprises one of the following sequences:CGTGTTGGTTGATAATGGC (SEQ ID NO. 1)-(insert) GGTGATGTAACCGTAGCTCCATCTAACTTCG (SEQ ID NO. 50);GCATCAACTCTCCCGGT (SEQ ID NO. 2)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 51); andGCATCAACTCCTCCGGT (SEQ ID NO. 3)-(insert)-GGCGGGTGGAGTGTGGAGC (SEQ ID NO. 52), wherein (insert) indicates a location of an insert.

6. The expression vector of Claim 1, wherein the cloning site is disposed in the nucleic acid sequence encoding the MP, and wherein the cloning site is disposed in a position corresponding to loop end regions of the MP.

7. The expression vector of Claim 1, wherein the nucleic acid sequence encoding the MP comprises a nucleic acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to the following sequence: ATGCGAGCTTTTAGTACCCTTGATAGGGAGAACGAGACCTTCGTCCCCTCCGT TCGCGTTTACGCGGACGGTGAGACTGAAGATAACTCATTCTCTTTAAAATATC GTTCGAACTGGACTCCCGGTCGTTTTAACTCGACTGGGGCCAAAACGAAACA GTGGCACTACCCCTCTCCGTATTCACGGGGGGCGTTAAGTGTCACATCGATAG ATCAAGGTGCCTACAAGCGAAGTGGGTCATCGTGGGGTCGCCCGTACGAGGA GAAAGCCGGTTTCGGCTTCTCCCTCGACGCACGCTCCTGCTACAGCCTCTTCC CTGTAAGCCAGAACTTGACTTACATCGAAGTGCCGCAGAACGTTGCGAACCG GGCGTCGACCGAAGTCCTGCAAAAGGTCACCCAGGGTAATTTTAACCTTGGT GTTGCTTTAGCAGAGGCCAGGTCGACAGCCTCACAACTCGCGACGCAAACCA TTGCGCTCGTGAAGGCGTACACTGCCGCTCGTCGCGGTAATTGGCGCCAGGC GCTCCGCTACCTTGCCCTAAACGAAGATCGAAAGTTTCGATCAAAACACGTG GCCGGCAGGTGGTTGGAGTTGCAGTTCGGTTGGTTACCACTAATGAGTGATAT CCAGGGTGCCTATGAGATGCTTACGAAGGTTCACCTTCAAGAGTTTCTTCCTA TGAGAGCCGTACGTCAGGTCGGTACTAACATCAAGTTAAATGGCCGTCTGTC GTATCCAGCTGCAAACTTCCAGACAACGTGCAACATATCGCGACGTATCGTG ATATGGTTTTACATAAACGATGCACGTTTGGCATGGTTGTCGTCTCTAGGTAT CTTGAACCCACTAGGTATAGTGTGGGAAAAGGTGCCTTTCTCATTCGTTGTCG ACTGGCTCCTACCTGTAGGTAACATGCTCGAGGGCCTTACGGCCCCCGTGGG ATGCTCCTACATGTCAGGAACAGTTACTGACGTAATAACGGGTGAGTCCATC ATAAGCGTTGACGCTCCCTACGGGTGGACTGTGGAGAGACAGGGCACTGCTA AGGCCCAAATCTCAGCCATGCATCGAGGGGTACAATCCGTATGGCCAACAAC TGGCGCGTACGTAAAGTCTCCTTTCTCGATGGTCCATACCTTAGATGCGTTAG CATTAATCAGGCAACGGCTCTCTAGATAA (SEQ ID NO. 4).

8. The expression vector of Claim 1, wherein the cloning site is disposed in at least one of the following residues of the sequence of Claim 7:46-102, 235-255, 730-747, and 1001-1033.

9. The expression vector of Claim 1, wherein the MP or the CP further comprises a nucleic acid sequence in the cloning site encoding an amino acid sequence comprising between 1 amino acid residue and about 1,000 amino acid residues.

10. The expression vector of Claim 9, wherein the amino acid sequence is an amino acid sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to any of the following sequences:MRAF STLDRENETF VPS VRVYADGETEDNSF SLKYRSNWTPGRFNSTGAKTKQW HYPSPYSRGALSVTSIDQGAYKRSGSSWGRPYEEKAGFGFSLDARSCYSLFPVSQ NLTYIEVPQNVANRASTEVLQKVTQGNFNLGVALAEARSTASQLATQTIALVKA YTAARRGNWRQALRYLALNEDRKFRSKHVAGRWLELQFGWLPLMSDIQGAYE MLTKVHLQEFLPMRAVRQVGTNIKLNGRLSYPAANFQTTCNISRRIVIWFYINDA RLAWLSSLGILNPLGIVWEKVPFSFVVDWLLPVGNMLEGLTAPVGCSYMSGTVT DVITGESIISVDAPYGWTVERQGTAKAQISAMHRGVQSVWPTTGAYVKSPFSMV HTLDALALIRQRLSR (SEQ ID NO. 5); andMASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSA QNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKA MQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGHHHHHHGDVTVAPSNFANGV AEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWR SYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO. 6).

11. The expression vector of Claim 1, wherein the cloning site is disposed in the nucleic acid encoding the MP and is a restriction site.

12. The expression vector of Claim 1, wherein the nucleic acid sequence encoding the CP is positioned 5’ of the nucleic acid sequence encoding MP.

13. The expression vector of Claim 1 further comprising one or more ribosomal binding sites.

14. The expression vector of Claim 13, wherein a ribosomal binding site of the one or more ribosomal binding sites is positioned 3’ of the nucleic acid sequence encoding the CP.

15. The expression vector of Claim 13, wherein the one or more ribosomal binding sites comprise a sequence comprising at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to any of the following sequences:TTTGTTTAACTTTAAGAAGGAGA (SEQ ID NO. 7)ATTAAAGAGGAGAAA (SEQ ID NO. 8)TCACACAGGAAAG (SEQ ID NO. 9)AAAGAGGAGAAA (SEQ ID NO. 10)CTTTAGGAGGT (SEQ ID NO. 10)AGGAGAT (SEQ ID NO. 11)16. The expression vector of Claim 1, further comprising a stop codon positioned 5’ of the nucleic acid sequence encoding CP.

17. The expression vector of Claim 1, wherein the nucleic acid sequence encoding the CP encodes for a CP dimer.

18. An MP fusion protein produced using the expression vector of any of Claims 1-17.

19. A virus-like particle (VLP) comprising the MP fusion protein of Claim 18.

20. The VLP of Claim 19, wherein a ratio of CP:MP is in a range of about 5: 1 to about 90: 1.

21. An MP fusion protein comprising: a heterologous amino acid sequence inserted at one or more of residues 16-34, 79- 85, 244-249, and 335-344 of SEQ ID NO. 4.

22. The MP fusion protein of Claim 21, wherein MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 14 and 17.

23. The MP fusion protein of Claim 21, wherein MP fusion protein comprises an amino acid sequence according to one or more of SEQ ID NOS. 15 and 18.

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