EP4658801A1 - In vitro reconstituted enveloped virus-like particles (evlps) for rna gene delivery - Google Patents

Abstract

The invention provides compositions of matter comprising a lipid enveloped virus capsid protein and a ribonucleic acid, as well as methods for using such compositions. In illustrative compositions, the lipid enveloped virus capsid protein envelops the ribonucleic acid so as to for a capsid that can inhibit the degradation of the ribonucleic acid (e.g. by RNAses). A method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell is also provided. Typically, the method comprises the steps of combining the mammalian cell with a composition of matter described herein under conditions selected to allow the lipid enveloped virus capsid to contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell.

Description

IN VITRO RECONSTITUTED ENVELOPED VIRUS-LIKE PARTICLES (EVLPS) FOR RNA GENE DELIVERY

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63/482,704, filed February 1, 2023, entitled “IN VITRO RECONSTITUTED ENVELOPED VIRUS-LIKE PARTICLES (EVLPS) FOR RNA GENE DELIVERY”, which applications are incorporated by reference herein. This application is related to U.S. Patent No.: 9,605,031, the contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under 1716925, 0714411, and 1051517 awarded by the National Science Foundation. The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to gene delivery and more particularly, methods and compositions related to the delivery of ribonucleic acid to mammalian cells.

BACKGROUND OF THE INVENTION

The use of animal virus-like particles (VLPs) as vectors for the delivery of genes to mammalian cells has been explored for many years and by a large variety of very different approaches. For example, U.S. Patent No. 9,605,031 describes the use of in vitro reconstituted virus-like particles (VLPs) for the packaging and subsequent delivery of self-replicating RNA genes to mammalian cells. That invention exploited the exceptional ability of the capsid protein of a particular plant virus — cowpea chlorotic mottle virus (CCMV) — to spontaneously self-assemble around RNA molecules with a wide range of length and sequence. The resulting nucleocapsids, each consisting of a single RNA molecule surrounded by a rigid single-protein-thick shell ("capsid"), are perfectly monodisperse (26 nm in diameter), stable against aggregation, protective against RNases, and release their RNA content in mammalian cells for translation by ribosomes. When a gene of interest in mRNA form is packaged in tandem ("in cis") with a gene encoding an RNA-dependent RNA polymerase (RdRp) — i.e., when the packaged RNA is a "replicon" — the gene of interest is amplified (replicated) up to one-million-fold by the RdRp before being translated, thereby giving rise to strong protein expression.

There is a need in the art for new and/or improved virus-like particles that are useful as vectors for the delivery of genes to mammalian cells.

SUMMARY OF THE INVENTION

Embodiments of the invention disclosed herein provide new and/or improved virus-like particles that are useful as vectors for the delivery of genes to mammalian cells by wrapping animal virus-like particles (VLPs) within a lipid bilayer envelope. Embodiments of the invention provide further protection of the VLP RNA contents as well as a further enhancement of the functionalization, targeting, and uptake of these VLPs. In the invention disclosed herein, a primary function of the capsid is to protect the RNA, while that of the lipid envelope is to hide it from the immune system and to provide for ready functionalization with ligands, for purposes of targeting and uptake by specific cells. The resulting lipid enveloped virus-like particles (EVLPs) are totally non-infectious, yet are powerful mimics of many mammalian viruses (including some of the most virulent human pathogens such as SARS, Dengue, and Zika). Consequently, embodiments of the invention provide optimized in vitro reconstituted vector/platforms for gene delivery.

Embodiments of the invention can be compared with a wide range of VLPs, liposomes, and virosomes known in the art. RNA-containing liposomes have been described in the art, but these particles are much less-well-characterized (e.g., much more polydisperse) and much less stable (against aggregation, and against nucleases, etc.) than the EVLPs disclosed herein. Similarly, previously described nucleocapsid- packaged-RNA-containing viral envelopes - virosomes - suffer from the same disadvantages, and additionally involve potentially dangerous viral-envelope components. In contrast, the in vitro assembly of EVLPs from purified components as disclosed herein avoids the use of cell cultures - thereby assuring that no unwanted RNA or proteins are involved - and provides monodisperse, well-defined, and robust particles that are functionalized for targeting of and uptake by mammalian cells. In this context, one unique aspect — and distinct advantage — of the invention is its ability to deliver genes to mammalian cells within a lipid-bilayer-wrapped nucleocapsid self-assembled from lipid, viral capsid protein and RNA molecules, all carried out in vitro (e.g., from purified components).

The present invention provides methods and materials that involve using lipid enveloped capsids of plant viruses as vectors for gene delivery. More particularly, the present invention involves the novel approach of using lipid enveloped in vitro reconstituted plant-virus-derived vectors for the packaging and delivery of ribonucleic acid (RNA) genes to mammalian cells. In one aspect of the present invention, a lipid enveloped cowpea chlorotic mottle virus (CCMV) capsid protein (CP) is exploited for its unique ability to spontaneously self-assemble around heterologous RNA molecules of widely varying length and sequence. The resulting lipid enveloped nucleocapsids - one molecule of RNA surrounded by a rigid single-protein-thick shell - are perfectly monodisperse, stable against aggregation, protect their RNA content against RNases, and release their RNA content in the cytoplasm of targeted mammalian cells.

Embodiments of the invention include compositions of matter comprising lipid enveloped viral capsid proteins and a ribonucleic acid. The lipid enveloped capsid proteins form a capsid that envelops the ribonucleic acid, thereby inhibiting degradation of the ribonucleic acid. Typically in these compositions, the ribonucleic acid encodes a polypeptide, for example a therapeutic polypeptide or a polypeptide that facilitates cellular imaging (e.g. a fluorescent protein). Optionally, the ribonucleic acid encodes a plurality of polypeptides, for example a plurality of therapeutic polypeptides and/or a plurality of polypeptides that facilitate cellular imaging. In some embodiments of the invention, the lipid enveloped virus capsid protein is coupled to a polypeptide that binds a molecule expressed on the surface of a mammalian cell (e.g. so as to facilitate targeting of a specific cellular lineage).

Another embodiment of the invention is a method of making a ribonucleic acid packaged within a lipid enveloped viral capsid, the method comprising combining viral capsid protein and a ribonucleic acid in a solution in vitro and wrapping this capsid in a lipid envelope so as to form a composition as disclosed herein. In these methods, relative amounts of lipid compositions, virus capsid protein and ribonucleic acid that are combined are controlled so as to control packaging of the ribonucleic acid in the virus capsid. For example, in some embodiments of the invention, relative amounts of a cowpea chlorotic mottle virus capsid protein and ribonucleic acid are controlled so that there are at least 10 cowpea chlorotic mottle virus capsid proteins for every 100 nucleotides of ribonucleic acid molecule length (e.g. 150 capsid proteins for a 1,500 nt ribonucleotide). In some embodiments of the invention, relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid are controlled so that the mass ratio of cowpea chlorotic mottle virus capsid proteins to ribonucleic acids is at least 6: 1.

In illustrative working embodiments of the invention, EVLPs were generated via a neutral-plus-cationic lipid wrapping of wildtype CCMV nucleocapsids, and of in vitro self-assembled virus-like particles involving purified CCMV capsid protein and heterologous RNA. In other embodiments, we have also wrapped - using neutral- plus-anionic lipid mixtures - in vitro reconstituted VLPs made with capsid protein from the closely-related brome mosaic virus (BMV), for which the magnitude and sign of the charge on the outside of the VLP can be very different from that of CCMV. It is significant that the charge on the VLP exterior is independent of the charge or nature of its contents; it is negative for pHs in excess of the capsid isoelectric point (pH=3.7 for CCMV, and 5.1 for BMV), thereby enabling wrapping of the VLP by positively or negatively charged lipid bilayers depending on the pH and on the capsid protein.

Embodiments of the invention can carry out wrapping of the VLPs by a special double-hydration procedure that we have developed for this purpose. Experimental results of this are shown in Figure 15), from methods that utilized neutral lipid (DMPC), cationic lipid (DOTAP,) and cholesterol in an approximate mole ratio of 6:3 : 1. An aliquot of a chloroform solution of the lipid mixture is evaporated to dryness by a flow of dry nitrogen gas. Next, the lipid is hydrated by the addition of buffer solution containing CCMV VLPs, and the solution is sonicated to form liposomes in the presence of these VLPs. Electron micrographs show that, at this point, relatively few of the particles are enveloped. To increase the yield of enveloped capsids the solution is again evaporated to dryness with nitrogen, ensuring that the VLPs become imbedded/dispersed in the lipid membrane. The film is then rehydrated with buffer solution and, as shown in Figure 15, the large majority of capsids have now become enclosed by a lipid bilayer; a lone “naked”, unenveloped, capsid is seen at the very bottom (see asterisk). Small aggregates of capsids are also wrapped, but this is not a problem because these double- and triple-VLPs encapsulated in an EVLP are likely to be taken up by cells just as easily as are the canonical (i.e., single-VLP) EVLPs and their RNA content made available just as efficiently to the ribosomal/translational machinery.

Yet another embodiment of the invention is a method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell, the method comprising combining the mammalian cell with a composition comprising a ribonucleic acid within a lipid enveloped virus capsid, and then allowing the lipid enveloped virus capsid to contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell (e.g. a mammalian cancer cell, a mammalian cell of a selected lineage or the like). Typically in these methods, the ribonucleic acid is at least 100 nucleotides in length and is not made by a virus. Typically, the ribonucleic acid encodes at least one polypeptide that is expressed in the mammalian cell following delivery of the ribonucleic acid into the cytoplasm of the mammalian cell. In one illustrative embodiment of the invention, the ribonucleic acid encodes a mammalian (e.g. human) polypeptide, for example a polypeptide useful in a therapeutic regimen. In another illustrative embodiment of the invention, the ribonucleic acid encodes a polypeptide selected for its ability to facilitate imaging of the mammalian cell.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates schematics of the relevant RNA reagents, (i) Wt SINV genome. The blocks labeled NS and S are the open reading frames coding for the replicase proteins and the structural proteins ([CP] and [GPs]). denotes the packaging signal, a sub-sequence in NS responsible for the preferential packaging of the RNA by its capsid protein. The hook-arrow denotes the promoter sequence controlling transcription of the downstream (structural, S) genes, (ii) GP-replicon RNA. Same as i, except for deletion of the CP gene, (iii) DIfEYFP] RNA. Derived from I upon deletion of most of the NS ORF and replacement of the structural genes (S ORF) with EYFP ORF. The signal is not present, but the cis-acting elements — the 5' and 3' untranslated regions (UTRs) needed for replication by the NS complex — are retained.

Figure 2 illustrates (A) Quantification of VLPs by densitometry. The densitometry plot quantifies the amount of RNA packaged in VLPs by measuring the EtBr fluorescence intensity from CCMV bands of known concentration. (B) Agarose gel electrophoresis of synthesized VLPs, naked DIfEYFP] 1800nt-RNA, and CCMV particle standards with known concentrations. Electrophoresis was carried out in a 1% agarose gel and in virus buffer (VB: see Methods section); the gel was stained with EtBr. (+) and (-) designations refer to the presence or absence of RNase A. The CCMV standards prepared by adding 10 pL of CCMV at concentrations of 93 (1), 69.8 (2), and 46.5 (3) ng/pL, respectively, correspond to concentrations of RNA (within the virions) of 20, 15, and 10 ng/pL.

Figure 3 illustrates the characterization of the VLP size distribution. Left: Distributions of diameters of VLPs (cross-hatched) and of wt CCMV virions (grey). Right: A negative-stain TEM micrograph of the VLPs assembled from CCMV CP+DI[EYFP] RNA. Scale bar = 100 nm.

Figure 4 illustrates that VLPs can release their RNA content into the cytoplasm of transfected cells. (A) Determination of Transduction Efficiency by Flow Cytometry. The left bars show the fractions of positive cells for duplicate sets of transfections of VLPs (red) and naked RNA (black) that were not treated with RNase A. The middle bars show the effect of adding RNase A after mixing with Lipofectamine-2000 (RN2, see Example section). The right bars show the result of incubation with RNase A before addition of Lipofectamine (RN1). From the precision of the cytometry measurements (0.002), an application of the Student t-test shows that the differences between the RNA and VLP measurements are significant with a probability of at least 95%. (B) Fluorescence micrographs of representative fluorescent cell densities for each scenario. Panel (iv) in (B) shows that transfected VLPs can deliver their RNA content into the cytoplasm of mammalian cells, where the RNA is involved in downstream processes, as is the case for the naked RNA control (B(i)). As expected, the naked VLPs overlaid on cells without any Lipofectamine could not transduce cells with EYFP.

Figure 5 illustrates the packaging of viruses with single-stranded RNA genomes. The packaging of the genome occurs spontaneously, via self-assembly - no pressure, no work. In one illustrative example, Cowpea Chlorotic Mottle Virus (CCMV) is shown. Each identical 28nm-capsid consists of 180 copies of one protein, and contains a different molecule of the viral RNA genome - RNA1, RNA2, or RNA3 (+RNA4) - each about 3000nt long.

Figure 6 shows graphs illustrating that super-stoichiometric excess of protein binds in physiological (pH 7, 1=0. IM) buffer (the graphs depict experimental results of (a) CP alone, (b) RNA alone, (c) CP and RNA, I=1M, (d) CP and RNA, 1=0. IM).

Figure 7 shows negative stain and cryo-electron microscopy images illustrating that neutral pH assemblies yield protein-decorated RNAs, not VLPs; pH needs to be lowered.

Figure 8 shows negative stain and cryo-electron microscopy images illustrating that pH lowering “turns on” lateral interactions between bound capsid proteins.

Figure 9 provides a diagram illustrating that electrostatic interactions are dominant in driving virus and VLP assembly. All lengths and sequences of RNA, from lOOnt to 10,000nt can be completely packaged by CCMV capsid protein CP, if the CP:RNA mass ratio is high enough. This threshold mass ratio (6: 1) is the same for all RNA lengths, corresponding to approximately 10 CP per lOOnt (+10 charges/CP N-terminus; -1 charge/nt). /.c., this (“magic”) ratio involves matching the RNA charge with the N-terminal capsid protein (CP) charge.

Figure 10 is a collection of images illustrating that CCMV protein can selfassemble with RNAs of many lengths. “Undersized” RNAs (less than 2000 nts) lead to many RNAs per capsid. “Oversized” RNAs (more than 4000nts) lead to many capsids per RNA (about 3000 bases per capsid).

Figure 11 is a graph illustrating the competition (for CCMV capsid protein) between 3234nt BMV viral NRA1 and several heterologous NRAs. Packing efficiency = (mass of competitor RNA packaged)/(mass of BMV RNA1 packaged). 500nt RNA is co-packaged. lOOOnt and 1500nt RNA cannot compete at all. Note that CCMV RNA1 is less efficiently packaged than BMV RNA1 by CCMV capsid protein. Figure 12 is a diagram illustrating a process in which plant (CCMV) capsids release mRNA for expression in mammalian (BHK) cells. This takes advantage of the robustness of CCMV nucleocapsids and of the promiscuity of their capsid protein. Plant (CCMV) capsids can release mRNA for expression in mammalian (BHK) cells. In a first step, lipofectamine-2000 is complexed with CCMV VLP containing DIfEYFP] RNA. In a second step, intact VLP is transfected into the cell. In a third step, VLP disassembles and delivers RNA to the cytoplasm. In a fourth step, replication machinery provided by GP-Rep vector amplifies DIfEYFP] RNA. In a fifth step, translation of EYFP occurs. In a sixth step, cell fluorescence results.

Figure 13 is a diagram illustrating another process in which plant (CCMV) capsids release mRNA for expression in mammalian (BHK) cells. In a first step, lipofectamine-2000 is complexed with CCMV VLP containing EYFP -replicon RNA. They should also release mammalian (Sindbis) replicon RNA, if the size and charge of the 9000nt-replicon are reduced, with no need for replicon in trans. In a second step, intact VLP is transfected into the cell. In a third step, VLP disassembles and delivers RNA to the cytoplasm. Translation of EYFP occurs and then cell fluorescence results.

Figure 14 is a transmission electron microscopy (TEM) image of CCMV capsids (containing the DNA oligo agonist for Herpes vaccine) wrapped by a neutral/cationic lipid membrane. This image shows that in vztro-reconstituted CCMV VLPs can be wrapped in vitro by bilayer membrane, to make enveloped virus-like particles (EVLPs) for gene and vaccine delivery.

Figure 15. TEM image of EVLPs prepared from CCMV virions as described herein following two successive lipid film rehydrations, with a mix of neutral and cationic lipid and cholesterol. The majority of EVLPs involve the unilamellar-bilayer- wrapping of single nucleocapsids, with two instances of a pair of capsids being wrapped and one instance of a triple; the asterisk denotes a “naked” capsid. DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

Many of the pertinent techniques and methodologies described or referenced herein with regards to the present invention are readily available in the literature. These include: genetic engineering protocols for point-site mutation of capsid protein (e.g., to insert cysteines on the capsid exterior for ligand binding) and for fusions of it with polypeptide domains desirable for targeting and uptake by specific cells; molecular biology techniques (e.g., agrobacterium transfer of mutated capsid protein genes to host plants) for scaling up the synthesis of capsid protein for self-assembly reactions with RNA genes and gene of interest (GOI)-containing RNA replicons; and mouse models for in vivo testing of the in vitro reconstituted vectors described herein (e.g., of the delivery of ferritin genes and their expression in targeted cell tissue for MRI molecular imaging).

The present invention involves the use of plant viral capsids as vectors for gene delivery. More particularly, the present invention involves the use of in vitro reconstituted plant-virus-derived vectors for the packaging and delivery of ribonucleic acid (RNA) genes into mammalian cells. Generally, such plant virus capsids - synthesized from purified components - contain replicon RNA with a gene of interest that is expressed at a high copy number when the capsid is delivered to the cytoplasm of mammalian cells.

As disclosed herein, embodiments of the invention include VLPs enveloped by a lipid composition (e.g., a membrane comprising a lipid bilayer). In embodiments of the invention, one can start by reconstituting VLPs of this kind from purified CCMV capsid protein and the RNA replicon of interest, using our standard CCMV in vitro reconstitution protocols. One can then wrap or envelope them in a lipid composition, making the corresponding EVLP using the wrapping technique described herein. Artisans can do so by:

(1) Using charged lipids to drive the wrapping of VLs, as explained herein; or (2) Using chemical conjugation chemistries, to avoid the use of charged lipids. In illustrative embodiments, artisans can include in the lipid formulation some maleimide cholesterol or maleimide neutral lipid for conjugating unique cysteines in the capsid protein (we have already made recombinant capsid protein with a unique cysteine inserted into an “outside” loop of capsid protein, and have demonstrated the in vitro self-assembly competence of these proteins.) Artisans can also include lipids with head groups linked to an NHS-ester moiety, so that they can be conjugated to naturally-occurring lysines (of which there are 5 per CCMV protein subunit) exposed on the exterior of the VLP, to further enhance the wrapping of VLPs by lipid bilayer.

In addition to using conjugation chemistries to wrap the VLPs through the inner leaflet of the lipid bilayer, maleimide and NHS-ester lipids in the outer leaflet can also be used to conjugate the EVLPs themselves to targeting ligands, for purposes of functionalizing the lipid envelope with antibody and/or other polypeptide moieties, e.g., xcll for targeting antigen-presenting dendritic cells, or epithelial growth factor (EGF) for generic binding to cancer cells overexpressing EGF receptors (EGFRs), or anti-CA19-9 for specific binding of the marker for pancreatic cancer cells, etc. Further embodiments involve the conjugation of these VLPs with polypeptide ligands for targeting and uptake of the VLPs by one or more subsets of cells (e.g. a particular lineage). Important examples include the targeted delivery of cancer and viral vaccines in RNA form, of MRI contrast agents (e.g., iron-sequestering ferritin protein) in RNA form, and microRNA (e.g., miR34a, for down-regulating oncogene expression). DNA and RNA viruses have qualitatively different life cycles due to the fact that their genomes (double-stranded DNA and single-stranded RNA) are fundamentally different physical objects. More explicitly, the former (DNA) is a stiff, linear, polymer, while the latter (RNA) is a flexible, branched polymer. As a consequence, for example, DNA viruses are strongly pressurized, requiring for their formation considerable expenditure of energy, while RNA viruses can self-assemble spontaneously, resulting in their infectivities being associated with very different mechanisms of genome packaging and delivery.

In one aspect of the present invention, cowpea chlorotic mottle virus (CCMV) capsid protein (CP) is exploited for its unique ability to spontaneously self-assemble around heterologous RNA molecules of widely varying length and sequence. CCMV is a spherical virus that can be reconstituted ‘from scratch’, i.e., synthesized “zw vitro" from its purified components, namely the viral RNA genome and capsid protein. Furthermore, the capsid protein will package, via spontaneous self-assembly, not only its viral genome, but also all kinds of other RNA molecules. The resulting virus-like particles are stable against aggregation and their protein shells protect their RNA cargo against nuclease (RNase) enzymes. Processes for manipulating CCMV capsid proteins are known in the art and described, for example, in U.S. Patent Nos. 6,180,389, 7,928,290, and United States Patent Application Publication Nos. 20090093019 and 20120174263, the contents of which are incorporated by reference.

Unlike mammalian viruses - the majority of whose nucleocapsids are enveloped by an extra layer of protection in the form of a viral envelope - plant viruses are almost without exception “just” genetic material (DNA or RNA) surrounded by a shell composed of the capsid protein (CP). Because the viral genome is protected only by this protein shell, the resulting nucleocapsid (nucleic acid packaged inside a capsid) is significantly more robust than its counterpart in enveloped animal viruses. The nucleocapsids disclosed herein are perfectly monodisperse (in one example, 26 nm in diameter), stable against aggregation, protect their RNA content against RNases, and release their RNA content in the cytoplasm of target mammalian cells.

Further, an RNA gene of interest (GOI) packaged in accordance with the present invention can be expressed in mammalian cells following replication by RNA-dependent RNA polymerases (RDRPs) provided in trans. If the packaged molecule is a replicon containing a GOI, the RNA may be replicated up to one- million-fold (by the RDRP enzymes for which it encodes) before the GOI is translated and its protein product synthesized in high copy number. In one or more embodiments, the replicon and target gene may be separated into two molecules and each packaged separately into single capsids.

A variety of replicons that can be adapted for use with embodiments of the invention are known in the art (see, e.g. U.S. Patent Nos. 6,462,255 and 7,807,868 and U.S. Patent Publication Nos.20030119182, 20080282426 and 20060253939, the contents of which are incorporated by reference herein). In one or more embodiments, a replicon from a Nodamura virus is used. The replicon is short enough to be packaged in a single capsid, even when a reporter gene is added to it. The capsid protein provided can package into a single protective shell any RNA with lengths between 2000 and 4000 nucleotides, without it needing to be genetically engineered with a “packaging signal”. Moreover, there are a wide variety of methods for functionalization to various icosahedral capsids, allowing them to be targeted to specific cells.

In embodiments of the invention, a composition of matter is provided comprising a lipid enveloped cowpea chlorotic mottle virus capsid protein (CCMV CP) and a ribonucleic acid (RNA). The ribonucleic acid is not derived from a cowpea chlorotic mottle virus and the cowpea chlorotic mottle virus capsid protein envelops the ribonucleic acid so as to inhibit degradation of the ribonucleic acid. In one or more embodiments, the ribonucleic acid does not need to be engineered with a packaging signal and/or an origin of assembly. A packaging signal or origin of assembly is commonly used for the preferential packaging of ribonucleic acid by a capsid protein. See, e.g. Smith et al., 2007 and Mahraj et al., 2014, which describe Tobacco Mosaic virus (TMV) coat protein (CP) reassembly onto RNA that requires a TMV origin-of-assembly (OA) (see, e.g. SEQ ID NO: 2). The ability to package any RNA at all, without needing it to be genetically engineered with a packaging signal or origin of assembly, is a major advantage that eliminates the complexities, costs, and increased size associated with incorporating a packaging signal or origin of assembly into a replicon or RNA.

Embodiments of the invention include compositions of matter comprising lipid enveloped cowpea chlorotic mottle virus capsid proteins and a ribonucleic acid. In these compositions, the ribonucleic acid is at least 100 nucleotides in length and is not derived from a cowpea chlorotic mottle virus. The cowpea chlorotic mottle virus capsid proteins form a capsid that envelops the ribonucleic acid, thereby inhibiting degradation of the ribonucleic acid. In certain embodiments of the invention the composition further comprises a mammalian cell. Typically in these compositions, the ribonucleic acid encodes a polypeptide, for example a therapeutic polypeptide or a polypeptide that facilitates cellular imaging (e.g. a iron-sequestering ferritin protein, a green fluorescent protein or the like). Optionally, the ribonucleic acid encodes a plurality of polypeptides, for example a plurality of therapeutic polypeptides and/or a plurality of polypeptides that facilitate cellular imaging. In an illustrative embodiment, the ribonucleic acid encodes a plurality of polypeptides including a RNA-dependent RNA polymerase. In some embodiments of the invention, the cowpea chlorotic mottle virus capsid protein is coupled to a polypeptide binds a molecule expressed on the surface of a mammalian cell (e.g. so as to facilitate targeting of a specific cellular lineage, a cancer cell or the like).

In typical embodiments of the invention, the ribonucleic acid in the composition is between 100 and 10,000 nucleotides in length; and/or comprises at least 1,000 nucleotides, at least 2,000 nucleotides, at least 3,000 nucleotides, at least 4,000 nucleotides, or at least 5,000 nucleotides; and/or does not include a signal sequence that modulates packaging of the ribonucleic acid by the capsid proteins (e.g. a viral origin-of-assembly element). Typically, the capsid envelopes a single ribonucleic acid molecule. In certain embodiments of the invention, the relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid that are combined together are selectively controlled to form a composition wherein there are at least 10 cowpea chlorotic mottle virus capsid proteins for every 100 nucleotides of ribonucleic acid length. In some embodiments of the invention, the relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid that are combined together are controlled to form a composition wherein the mass ratio of cowpea chlorotic mottle virus capsid proteins to ribonucleic acids is at least 6: 1. As shown by the data presented in Figures 9-11, by controlling the relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid in this manner, the packaging of selected ribonucleic acids can be controlled.

Optionally, the lipid enveloped cowpea chlorotic mottle virus capsid protein is coupled to a polypeptide that is not derived from a cowpea chlorotic mottle virus, for example a polypeptide that binds a molecule expressed on the surface of a mammalian cell (e.g. a cowpea chlorotic mottle virus capsid protein coupled to a polypeptide comprising an antibody epitope that can bind a cellular protein on the surface of the mammalian cell). In one or more embodiments, the ribonucleic acid does not include a “packaging signal” used for the preferential packaging of ribonucleic acid by a capsid protein. In one illustrative embodiments, the packaging signal comprises AAGAAGUCG of SEQ ID NO: 2.

Another embodiment of the invention is a method of making a ribonucleic acid packaged within a lipid enveloped cowpea chlorotic mottle virus capsid, the method comprising combining a lipid composition, cowpea chlorotic mottle virus capsid protein and a ribonucleic acid in a first buffer solution in vitro so as to form a composition as disclosed herein. In these methods, relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid that are combined are controlled so as to control packaging of the ribonucleic acid in the cowpea chlorotic mottle virus capsid. For example, in some embodiments of the invention, relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid are controlled so that there are at least 10 cowpea chlorotic mottle virus capsid proteins for every 100 nucleotides of ribonucleic acid molecule length. In some embodiments of the invention, relative amounts of cowpea chlorotic mottle virus capsid protein and ribonucleic acid are controlled so that the mass ratio of cowpea chlorotic mottle virus capsid proteins to ribonucleic acids is at least 6: 1. Optionally, the method further comprises coupling the cowpea chlorotic mottle virus capsid with a membrane comprising a lipid, for example a bilayered membrane that envelops the cowpea chlorotic mottle virus capsid.

The methods can further comprise dialyzing the first buffer solution against a second buffer solution; and/or treating the capsid with a ribonuclease. For example, the methods can comprise the steps of incubating the cowpea chlorotic mottle virus capsid protein and the ribonucleic acid together in a solution having a neutral pH and then dialyzing the incubated mixture of step (a) within a buffer solution. In typical embodiments the first buffer solution has a neutral pH (e.g. a pH between 7.0 and 7.4), and the second buffer solution has a pH below 5.0. In one illustrative embodiment of the invention, one can mix capsid protein and RNA (ranging in length from lOOnt to 10,000nt) in pH 7.2 (1=0. IM) buffer, incubate the mixture overnight, and then dialyze against pH 4.8 (1=0. IM) buffer. In these methods, one can treat assemblies with RNase to remove unpackaged RNA. One can then analyze the particles by gel electrophoresis, sucrose gradients, and electron microscopies (and/or by packaging fluorescently labeled RNAs etc.). In addition, as disclosed herein, the relative amounts or ratios of combined RNAs and capsid proteins can be controlled to allow “Head-to-Head” packaging competition experiments. By doing this, one can mix equal masses of two RNAs of different lengths with sufficient protein to completely package only one of them (see, e.g. the data presented in FIG. 11).

Yet another embodiment of the invention is a method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell, the method comprising combining the mammalian cell with a composition comprising a ribonucleic acid enveloped by a cowpea chlorotic mottle virus capsid which is itself enveloped by a lipid composition, and then allowing the cowpea chlorotic mottle virus capsid to contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell (e.g. a mammalian cancer cell, a mammalian cell of a selected lineage or the like). Typically in these methods, the ribonucleic acid is at least 100 nucleotides in length and is not derived from a cowpea chlorotic mottle virus or other virus. Typically, the ribonucleic acid encodes at least one polypeptide that is expressed in the mammalian cell following delivery of the ribonucleic acid into the cytoplasm of the mammalian cell. In one illustrative embodiment of the invention, the ribonucleic acid encodes a mammalian (e.g. human) polypeptide, for example a polypeptide useful in a therapeutic regimen. In another illustrative embodiment of the invention, the ribonucleic acid encodes a polypeptide selected for its ability to facilitate imaging of the mammalian cell. In certain embodiments of the invention, the ribonucleic acid encodes a RNA-dependent RNA polymerase.

In other embodiments of the invention, a method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell is provided. The method comprises the steps of combining the mammalian cell with the composition of matter described herein under conditions selected to allow the lipid enveloped virus capsid protein to contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell. In various embodiments, the ribonucleic acid encodes at least one polypeptide that is expressed in the mammalian cell following delivery of the ribonucleic acid into the cytoplasm of the mammalian cell.

As illustrated in the Example section below, the in vitro reconstituted viruslike particles disclosed herein - whose protein shells are made of plant viral, Cowpea Chlorotic Mottle Virus (CCMV), capsid protein - are capable of releasing their RNA cargo in mammalian cells. In the illustrative example, hybrid virus-like particles, assembled in vitro from Cowpea Chlorotic Mottle Virus (CCMV) capsid protein (CP) and a heterologous RNA derived from a mammalian virus (Sindbis), were shown to be capable of releasing their RNA in the cytoplasm of mammalian cells. Moreover, the reporter genes included in the RNA were expressed, resulting in a high level of protein synthesis.

In another aspect of the present invention, the cytoplasmic entry of the RNA- containing nucleocapsid can be facilitated by conjugating the CCMV capsid protein with a ligand that binds to target cells and induces endocytosis. Important examples include the targeted delivery of vaccines in RNA form, MRI contrast agents (e.g., iron-sequestering ferritin protein) in RNA form, and a variety of therapeutic proteins (e.g., super oxide dismutase (SOD)) in RNA form.

Work by many others in the art has shown that the outside of the protein shells of plant viruses like CCMV can be chemically modified so that they target and are taken up by specific mammalian cells. Thus, chemical and biological techniques may be used for mutating the CCMV capsid protein so that the exterior surface of the nucleocapsid is functionalized for targeting and uptake by specific cells. For example, if the capsid protein ligand involved is an antibody to a specific cancer-cell-presenting antigen, and the GOI codes for ferritin, one is able to test directly in mouse models the efficacy of MRI to produce contrast enhancement in cells due to the vector.

In another embodiment of the invention, a method of making the composition of matter described above is provided. The method comprises combining the lipid composition, the virus capsid proteins and the ribonucleic acid in a solution in vitro. The method further comprises the steps of (a) incubating the virus capsid protein and the ribonucleic acid together in a solution having a neutral pH and (b) dialyzing the incubated mixture of step (a) within a buffer solution.

In other embodiments of the invention, a method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell is provided. The method comprises the steps of combining the mammalian cell with the composition of matter described herein under conditions selected to allow the cowpea chlorotic mottle virus capsid protein contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell. In various embodiments, the ribonucleic acid encodes at least one polypeptide that is expressed in the mammalian cell following delivery of the ribonucleic acid into the cytoplasm of the mammalian cell.

In certain instances, the method comprises intravenous injection of aqueous solutions of the RNA-containing functionalized nucleocapsids disclosed herein. Experimental animal models, such as mouse models, may be used to further test and optimize the efficacy of the methods for delivering, in separate experiments, the following proteins: influenza vaccines, ferritin for MRI contrast, and superoxide dismutase.

One unique aspect - and a distinct advantage - of the present invention is the delivery of genes to mammalian cells within the capsid of a self-assembling plant virus rather than packaging them in a mammalian virus or in liposomes. The in vitro assembly from purified components avoids the use of cell cultures and provides monodisperse, well-defined, and robust particles that are functionalized for targeting of and uptake by mammalian cells.

The present invention is also significant in that, for example, instead of trying to deliver desired proteins (e.g., vaccines or enzymes or toxins) to specific cells, it is more powerful to deliver the RNA genes for these proteins to the cells. By packaging a GOI-containing replicon, the present invention can be used to deliver - not a single protein, but rather - a self-amplifying RNA gene for that protein, thereby ensuring a high level of expression. Accordingly, the present invention allows for the usage of well-characterized, stable, long-shelf-life, in vitro synthesized, virus-like particles to deliver RNA genes to targeted cells.

The Example section below illustrates packaging an RNA gene of interest in virus-like particles, getting the particle into mammalian cells, and providing in trans an RNA replicon molecule that replicates the RNA gene a million-fold before the gene is translated into protein. However, in certain embodiments the RNA gene and the replicon may be packaged as a single RNA molecule, so that the resulting viruslike particle is capable - all by itself - of protecting, delivering, amplifying, and expressing the target gene in specific mammalian cells. In one illustrative example, a single RNA molecule with two open reading frames - one containing the RDRP genes of Nodamura virus and the other the EYFP GOI - is packaged by a CCMV capsid protein. Transfection of these reconstituted nucleocapsids result directly in EFYP expression. In other embodiments, the replicon and target gene are packaged separately into single capsids.

EXAMPLE

Reconstituted plant viral capsids can release genes to mammalian cells

This example illustrates in vitro packaging by CCMV capsid protein of an RNA gene coding for a fluorescent protein, EYFP (see also, Azizgolshani, et al. Virology 441 (2013) 12-17; http://www.elsevierblogs.com/virology/?p=65). The resulting nucleocapsids are delivered by transfection to mammalian (baby hamster kidney [BHK]) cells, with RDRPs provided in trans, and EYFP expression (fluorescence) observed directly.

The nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. This example demonstrates that hybrid virus-like particles, assembled in vitro from CCMV CP and a heterologous RNA derived from a mammalian virus (e.g. Nodamura, Sindbis), are capable of releasing their RNA in the cytoplasm of mammalian cells. This result demonstrates the use of plant viral capsids as vectors for gene delivery and expression in mammalian cells. Furthermore, the CCMV capsid protects the packaged RNA against nuclease degradation and serves as a robust external scaffold with many possibilities for further functionalization and cell targeting.

The use of animal virus-like particles (VLPs) as vectors for the delivery of genes to mammalian cells has been explored for many years and by a large variety of very different approaches (Schaffer et al., 2008; Lavillette et al., 2001; Soong et al., 2000). This example examines the possibility of using a well-characterized plant RNA virus for expression of heterologous genes in mammalian cells. Unlike mammalian viruses - the majority of whose nucleocapsids are enveloped by an extra layer of protection in the form of a viral envelope - plant viruses are almost without exception “just” genetic material (DNA or RNA) surrounded by a shell composed of the capsid protein (CP). Because the viral genome is protected only by this protein shell, the resulting nucleocapsid (nucleic acid packaged inside a capsid) is significantly more robust than its counterpart in enveloped animal viruses.

Consider, for example, the “superfamily” of single-stranded RNA viruses comprised of bromoviruses (infecting plants) and alphaviruses (infecting animals), and whose members include Cowpea Chlorotic Mottle Virus (CCMV) and Sindbis Virus (SINV), respectively (Strauss and Strauss, 1994). The nucleocapsids of both CCMV (Bancroft and Hiebert, 1967; Zhao et al., 1995; Fox et al., 1998) and SINV (Wengler et al., 1982; Tellinghuisen et al., 1999; Mukhopadhyay et al., 2002; Giocochea et al., 2007; Cheng and Mukhopadhyay, 2011) have been reconstituted in vitro from purified components. However, the nucleocapsids of CCMV are much more stable in solution than those of SINV. Furthermore, in contrast to SINV nucleocapsids that have evolved to depend on their viral envelope for protection against nucleases, CCMV capsids protect their RNA content against digestion.

Equally important, CCMV capsid protein has been shown to be capable of packaging, via spontaneous in vitro self-assembly, a wide range of non-viral cargo, including: heterologous RNAs (Hiebert et al., 1968; Bancroft et al., 1969; Rao, 2006), synthetic anionic polymers (Douglas and Young, 1998; Hu et al., 2008; Brasch and Cornelissen, 2012, Cadena-Navaetal., 2011), mineralized salts(Douglas andYoung, 1998), negatively charged colloidal particles such as gold nanoparticles (Chen et al., 2006) and oil-in-water nanoemulsion droplets (Chang et al., 2008), fluorescent proteins (Minten et al., 2009), and pH-controlled chromophores (Brasch et al., 2011). In addition, by functionalizing and chemically modifying their capsids, closely-related plant viruses like Cowpea Mosaic Virus (CPMV) have been used for targeting of and uptake by specific mammalian cells for imaging and drug delivery (Destito et al., 2007; Gonzales et al., 2009; Yildiz et al., 2011; Steinmetz, 2010; Wu et al., 2012). Similarly, MS2 VLPs containing a variety of drugs, siRNAs, and toxins have been conjugated with peptide ligands and shown to produce selective cytotoxicity in cancer cells (Ashley et al., 2011).

Plant-derived VLPs, however, have not been used for direct gene delivery and expression, although a recent study (Li et al., 2012) reports the transfection of tobacco mosaic virus (TMV) virions into HeLa cells with the resulting expression of TMV capsid protein; see also the 2007 Virology and 2014 Int. J. Mol. Sci. papers of McCormick and co-workers. Interestingly, high-level expression of mammalian genes in plant hosts, mediated by Agrobacterium plasmids containing the translationenhancement elements from messenger RNAs of the plant virus CPMV, has been demonstrated for various target proteins and vaccines (Sainsbury and Lomonossoff, 2008). The insect virus baculovector system has also been effectively used for expression of mammalian genes in a wide variety of insect and mammalian hosts (Chen et al., 2011), but unlike the system we describe here it cannot be reconstituted in vitro and must be prepared by recombinant plasmid engineering in cell culture.

There have been no attempts to use a spherical plant viral capsid to deliver heterologous genes for expression in mammalian cells, even though there are several independent demonstrations of the internalization of plant virus by such cells. Examples include the membrane-protein-mediated uptake of CPMV by mouse vascular endothelial cells (Koudelka et al., 2009) and the uptake of brome mosaic virus (BMV) by human bronchial epithelial cells (Jung et al., 2011).

The possibility of uptake and release of the contents of a plant capsid in a mammalian cell has been suggested in the case of red clover necrotic mosaic virus (RCNMV) (Lockney et al., 2011). In particular, capsids of purified virus loaded with doxorubicin were functionalized with HeLa cell -targeting peptides, and the survivability of HeLa cells overlaid by them shown to decrease with doxorubicin concentration. While disassembly of the capsids in the HeLa cells was not directly established, doxorubicin was argued to be released by the same divalent cation- controlled mechanism as known to occur with RCNMV RNA in plant cell hosts.

In light of there being no direct demonstration of spherical plant viruses disassembling and thereby releasing their contents in animal cells, the question considered is whether heterologous genes in spherical plant VLPs can be made available to a mammalian cell and their protein products synthesized?

In answering this question, and thereby providing a new platform for gene delivery to mammalian cells, many unique in vitro reconstitution properties of the spherical plant virus, CCMV is exploited. In particular — in contradistinction to the CPMV and RCNMV examples mentioned above — the purified capsid protein of CCMV is capable of efficiently packaging a large range of RNA lengths and sequences, including arbitrary transgenes of interest. If these in vitro synthesized VLPs can be shown to disassemble and release their RNA contents in mammalian cells, then the stage is set for functionalizing the capsids to target those cells in vivo.

The strategy is as follows: A well-characterized VLP consisting of a reporter ssRNA molecule packaged inside a CCMV capsid is reconstituted in vitro. To this end, a SINV-derived defective interfering RNA (DIfEYFP], 1800nt: Fig. liii) is designed, which upon transcription produces the mRNA for expression of high levels of enhanced yellow fluorescent protein (EYFP). The VLPs containing DIfEYFP] are transfected into a monolayer of baby hamster kidney (BHK) cells. The machinery for the transcription and replication of the DIfEYFP] RNA is supplied by a SINV-like particle (GP-Rep vector: Fig. lii) providing the replicase proteins that will efficiently replicate the DIfEYFP] RNA molecule if and only if the encapsidated DIfEYFP] RNA is released from the VLPs upon internalization. Therefore, the expression of EYFP in vector-infected, VLP -transfected, cells can report on the successful release of the encapsidated RNA for transcription, replication and translation.

Fig. 1 shows schematically the coding and regulatory sequences of the full SINV RNA genome (i), highlighting the two open reading frames (ORFs) coding for the nonstructural (NS) genes (RNA-dependent RNA polymerase) and structural (S) genes (capsid protein(CP) and membrane glycoproteins (GP)). Also shown is the SINV-derived glycoprotein “replicon” (GP-Rep) RNA (ii), obtained by deleting the CP gene in the full SINV genome (see Methods section). A defective-interfering (DI) RNA molecule (iii) is derived from it by deleting most of the nonstructural ORF and replacing the structural genes by the EYFP gene (see Methods section).

Results and discussion

VLP synthesis and characterization

As described in the Methods section below, DIfEYFP] RNA and an excess of CCMV CP are subjected to the standard protocol for in vitro reconstitutions of RNA and CCMV CP. Basically, they are incubated together at neutral pH and then dialyzed against pH 4.8 buffer solution.

The resulting mixture was run in a 1% agarose electrophoresis gel, and ethidium bromide (EtBr) staining revealed no bands associated with free (unpackaged) RNA. Rather, the only band observed was one corresponding to VLPs (Fig. 2B, VLP[-]) which are resistant to RNase digestion (Fig. 2B, VLP[+]). Note that this RNase treatment was sufficient for complete digestion of naked RNA (Fig. 2B, RNA). Comparing the band intensity with lanes containing a set of CCMV standards of known concentration (Fig. 2A) showed that the assembled VLPs contained a concentration of RNA equal to 18ng/ml. Furthermore, the VLPs were homogeneous (see, however, discussion below TEM data) and had electrophoretic mobilities corresponding to well-formed VLPs.

The assembly mix was imaged by negative-stain transmission electron microscopy (TEM). The images showed spherical VLPs (Fig. 3, micrograph) with a distribution of diameters corresponding to roughly equal numbers of “pseudo T=2” and T=3 capsids, overlapping the range of wt CCMV virions (Fig. 3, histogram) — in agreement with what was previously found for packaging of 2000-nt RNA into CCMV VLPs (Cadena-Nava et al., 2012). CCMV VLPs can release their RNA content in the cytoplasm of mammalian cells Having characterized the plant virus VLPs containing DIfEYFP] RNA, mammalian cells were then transfected with them, using Lipofectamine-2000 (see Methods section). The transfected cells were incubated for 30 min to allow for internalization of the VLPs and for release of their RNA in the cytoplasm. At this point, GP-Rep vector was added at a multiplicity of infection (MOI) of 100 in order to produce a high copy number of EYFP mRNA from DIfEYFP] RNAs that had been released from their CCMV capsids and successfully delivered to the cytoplasm. The intracellular fluorescence signal generated by the subsequent translation of this amplified mRNA into EYFP could then be used to report on the level of VLP delivery and the release of its RNA.

In parallel, equal amounts of the naked DIfEYFP] RNA were transfected by Lipofectamine-2000 into BHK cells under similar conditions. In addition, a sample consisting of “naked” VLPs (i.e., VLPs in final assembly buffer without Lipofectamine) was incubated with BHK cells.

The transfected cells were imaged by fluorescence microscopy to estimate the number of cells positive for EYFP. The number of EYFP-expressing cells was also quantified by flow cytometry; the transduction efficiencies are shown as a function of RNase treatment in Fig. 4A.

The number of fluorescent cells is lower (by a factor of 5) in the case of VLP transfection than for RNA. Two explanations are plausible for this observation: either CCMV VLPs have a lower transfection efficiency than their corresponding RNA content, or not all of the VLPs disassemble to make their RNA content available for transcription, replication and translation. The second scenario — that only about a fifth of the VLPs are releasing their gene cargo, as opposed to 5 times fewer VLPs being transfected than RNAs — is consistent with experiments in which equal numbers of the same VLPs and RNA molecules were manually microinjected — rather than transfected — into the same BHK cells, and the VLPs were found to give rise to a factor of 5 fewer fluorescent cells. CCMV VLPs remain intact prior to cell entry and protect their RNA content against RNase digestion

Two variations on the transfection procedure involving RNase A were carried out in parallel to gain further information about the physical state of the VLPs during transfection. They are referred to as “RN1” and “RN2” and are described below:

RN1: Before the initial equilibration with Lipofectamine-2000, RNase A was added to both the packaged and naked RNA samples. This digestion step was designed to remove all naked full-length RNAs from solution and ensure that any transduction activity (reported by EYFP production) generated by the VLPs did not come from trace amounts of unpackaged RNA or from RNA that escaped from the VLPs during transfection. Essentially identical transduction efficiencies for VLPs with and without RNase digestion was observed (Fig. 4B iv and vi), indicating that unpackaged RNA was not involved. The complete absence of EYFP transduction in the digested naked RNA sample (see Fig. 4Biii) was also observed, confirming that the concentration of RNase A used was adequate for complete removal of all naked full-length RNA in solution.

RN2: RNase A was added immediately after the addition of Lipofectamine. This digestion step was designed to test whether Lipofectamine-2000 is capable of protecting unpackaged RNA against RNase A digestion. The results show (see Fig. 4) that adding RNase A at this step of the transfection procedure results in a large attenuation of the transduction activity of the naked RNA while having no effect on that of the VLPs, indicating again that VLPs are being transfected as intact capsids.

The intact state of the plant viral capsids, even after RNase treatment in the absence and presence of Lipofectamine, is only one measure of their robustness. Another is their “shelf-life” between synthesis and transfection: in the experiments described above, for example, the VLPs were left for 1 week at 4 °C before being subjected to RNase treatment and transfection. Conclusions

It has now been shown that plant VLPs formed in vitro from CCMV capsid protein and several-thousand-nt-long RNA: (1) are resistant to RNase; (2) can be transfected into mammalian cells, and (3) disassemble in these cells, resulting in the expression of the target transgene.

This demonstration establishes that plant virus capsids — here CCMV — are capable of protecting their RNA contents outside the cell, and yet making them available in the intracellular environment of mammalian cells. The CP as well as the RNA of a super-family of plus-strand RNA viruses like CCMV and SINV have been chosen — as opposed to minus-strand or retro RNA viruses — in order to avoid the complications of needing to package directly the delicate RNA-dependent RNA polymerase or reverse transcriptase proteins themselves. Further, with plus-strand viruses, all of the nucleic acid replication takes place in the cytoplasm rather than requiring trafficking of RNA into and out of the host cell nucleus. In addition, utilizing the DIfEYFP] RNA in conjunction with GP-Rep vector infection, we are able to exploit the powerful messenger RNA amplification scheme unique to plusstrand RNA viruses (Strauss and Strauss, 1994; Frolov et al., 1996). More explicitly, upon release from its VLP each DIfEYFP] RNA is not only transcribed into EYFP mRNA suitable for translation, but is also replicated by the RNA-dependent RNA polymerase (supplied by the GP-Rep vector) into many copies, each of which can in turn be transcribed again and used in the translation of the reporter EYFP. It is this enhancement that significantly increases the sensitivity for detecting RNA release from the VLPs in the cytoplasm.

These studies were undertaken to demonstrate that nucleocapsids of plant viral capsid protein — prepared as nuclease-resistant closed shells — are capable of making available heterologous RNA content to mammalian cells, and that RNA genes in this form can be expressed. Disassembly of these VLPs is most likely driven by their binding to ribosomes, as has been demonstrated in studies of bromoviruses (Roenhorst et al., 1989) and alphaviruses (Singh and Helenius, 1992). Here transfection of the VLPs is used to provide a “proof of principle” demonstration of RNA release and expression. The results of this work can now be directly combined with functionalization of the capsid protein for targeting and uptake by mammalian cells (Destito et al., 2007; Gonzales et al., 2009; Yildiz et al., 2011; Steinmetz, 2010; Wu et al., 2012; Koudelka et al., 2009; Jung et al., 2011; Lockney et al., 2011), thereby facilitating the use of reconstituted plant viral capsids for gene delivery.

Materials and methods

Constructs and reagents

Di[EYFP]-RNA

The DIfEYFP] construct was the result of (1) deletion of the region spanning BamHI and BspEI restriction sites on the NS ORF of SINV cDNA, and (2) replacement of the S ORF with nuclear EYFP ORF, using standard molecular biology protocols. The resulting plasmid — after linearization — was used as the template for in vitro transcription using Ambion Sp6 mMessage mMachine in vitro transcription kit. The RNA was purified with a Qiagen mini RNeasy kit, and quantified by UV absorption at 260nm.

GP-rep vector

First, two constructs, GP-Rep and DHBB plasmids, were prepared: GP-Rep was made by deleting the CP gene from the structural ORF in the SINV cDNA while DHBB was made by deleting the region between BamHI and BspEI sites on the NS ORF of SINV cDNA. These constructs were made into corresponding RNA transcripts, using in vitro transcription (see above). Next, the two RNAs were cotransfected into BHK cells using electroporation protocol. The vector particles were harvested from the medium on top of transfected cells after 24h. CCMV CP purification

CCMV was purified from infected California cowpea plant (Vigna ungiculata cv BlackEye) (Bancroft, 1970) and CP was isolated as described previously (Cadena- Nava et al., 2011; Annamalai and Rao, 2005). SDS-PAGE and MALDI-TOF were employed to ascertain that the purified protein was intact.

VLP assembly reactions

DIfEYFP] RNA was packaged by CCMV CP at a CP:RNA mass ratio of 6.5: 1 to ensure complete RNA packaging. Assembly was carried out by mixing CP and RNA in buffer B(1 M NaCl, 20 mM Tris-HCl pH 7.2, 1 mM EDTA, 1 mM DTT and 1 mM PMSF), followed by dialysis against RNA assembly buffer (RAB: 50 mM NaCl, 10 mM KC1, 5 mM MgCl₂, 1 mM DTT, 50 mM Tris-HCl pH 7.2) for 4h, followed by dialysis against virus buffer (VB: 0.1M sodium acetate, 1 mM EDTA, pH4.8) for 16h, all at 4 °C. Free (excess) CP was removed by washing the sample with VB in a 100 kDa Amicon centrifugal filtration unit at 3000 G at 4 °C.

Electron microscopy characterization of VLP s

A 6pl aliquot of the final assembly mixture was applied to a glow-discharged copper grid (400-mesh) that previously had been coated with Parlodion and carbon. The mix was spread onto the grid for 1 min, blotted with Whatman filter paper and then stained with 6 pL of 1% uranyl acetate for 1 min. Excess stain was removed by blotting with filter paper. The samples were stored overnight in a desiccator and analyzed with a JEM 1200-EX transmission electron microscope operated at 80 keV and equipped with a wide-angle (topmount) BioScan 600 W 1 x 1 K² pixel digital camera. The reported average diameter of VLPs is that of the geometric mean of two orthogonal measurements of the capsids obtained with ImageJ (U.S. National Institutes of Health) software from recorded images. Preparation ofBHK cell culture

Prior to transfection, BHK-21 cells were grown as monolayers on 6-well plates at37 °C in a CO2 incubator. EMEM (ATCC) supplied with 10% (v/v) FCS and Penicillin-streptomycin was used to grow and maintain cells.

Lipofectamine transfections

Monolayers ofBHK cells were grown on six-well plates to 90% confluence. Transfection with Lipofectamine-2000 was carried out following a standard protocol normally used for the transfection of nucleic acids. Briefly, the protocol consisted of the following (5) steps:

(1) An aliquot of VLPs (or naked RNA) containing 1 pg of DIfEYFP] RNA was diluted into Opti-MEM to 300 pl for each transfection. Similarly, 3 pl of Lipofectamine-2000 was diluted into 300 pl Opti-MEM and the dilutions were incubated at room temperature for 5 min.

(2) The two diluted reagents were mixed and (RNA/VLP + Lipofectamine) complexes were allowed to form by incubating at room temperature for 30 min.

(3) The cells were washed with 1 x nuclease-free PBS before overlaying the transfectant onto the cells, followed by incubation at 37 °C in a CO2 incubator with occasional shaking to distribute the transfectant evenly on the cells.

(4) After 30 min of incubation, a 150-mL aliquot of GP-Rep vector at an MOI of > 100 was added to the cells in order to supply the replication machinery necessary for EYFP production. Longer incubation times did not enhance the transduction yield.

(5) After 4 h the transfectant/inoculums was removed and the cells were gently washed twice with pre-warmed 1 x PBS and 1 ml of 2% FCS medium was overlaid on the cells. The cells were left overnight at 37 °C in a CO2 incubator.

RNase A assays

All RNase A digestion steps were carried out using 0.2 ng/pl RNase A for 30 min at room temperature. For digestions that were performed during the transfection protocol (i.e. scenarios RN1 and RN2), this concentration of RNase A was maintained throughout each subsequent step of the protocol. These conditions were found to be sufficient to remove all full-length RNA band intensity from the agarose gel assay (Fig. 2B, RNA panel) as well as completely destroy the transfection efficiency of naked RNA by Lipofectamine-2000 (Fig. 4A and 4Biii, vi).

Flow cytometry

Twenty -four hour post-transfection, the cells were washed with 1 x PBS, trypsinized, and resuspended in 1 x PBS buffer with 2% FCS at a density of 3 x 10⁶ cells/ml. Analysis was performed using a Becton Dickinson SORP BD LSRII Analytic Flow Cytometer equipped with a 488-nm blue laser for excitation of EYFP. Yellow-green YFP fluorescence was collected after a 530/30 bandpass filter. FACSDIVa software was used to control the parameters during the run and to analyze collected data.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification.

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CAPSID PROTEIN COWPEA CHLOROTIC MOTTLE VIRUS

ACCESSION P03601

MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVE PIASGQGKAIKAWTGYSVSKWTASCAAAEAKVTSA ITI SLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSCVTETQTTAAAS FQVALAVADNSKDVVAAMY PEAFKGITLEQLTADLTIYLYSSAALTEGDVIVHLEVEHVRPTFDDS FTPVY ( SEQ ID NO : 1 ) See, e.g. Allison et al., Virology 172 (1), 321-330 (1989)

TOBACCO MOSAIC VIRUS ORIGIN OF ASSEMBLY guuiuigagaga gaagauuaca aacgugagag acggagggcc cauggaacuu acagaagaag ucguiuigauiga guucauiggaa gaugucccuia ugucgauicag gcuugcaaag uuiuicgauicuic gaaccgg ( SEQ ID NO : 2 )

See, e.g. Goelet et al., Proc. Natl. Acad. Sci. USA (79) 5818-5822 (1982)

CONCLUSION This concludes the description of the illustrative embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

Claims

1. A composition of matter comprising: virus capsid proteins disposed within a lipid envelope; and ribonucleic acid disposed within the virus capsid proteins; wherein: the ribonucleic acid is at least 100 nucleotides in length; the ribonucleic acid is not made by a virus; and the lipid enveloped virus capsid proteins form a capsid that envelops the ribonucleic acid, thereby inhibiting degradation of the ribonucleic acid.

2. The composition of claim 1, wherein the virus capsid proteins comprise cowpea chlorotic mottle virus capsid proteins.

3. The composition of claim 1, wherein the ribonucleic acid:

(a) encodes a RNA-dependent RNA polymerase;

(b) is between 100 and 10,000 nucleotides in length; and/or

(c) comprises at least 1,000 nucleotides, at least 2,000 nucleotides, at least 3,000 nucleotides, at least 4,000 nucleotides, or at least 5,000 nucleotides; and/or

(d) does not include a signal sequence that modulates packaging of the ribonucleic acid by the capsid proteins.

4. The composition of claim 1, wherein the relative amounts of virus capsid proteins and ribonucleic acid are such that there are at least 10 capsid proteins for every 100 nucleotides of ribonucleic acid length.

5. The composition of claim 1, wherein: the proteins are coupled to a polypeptide that is not derived from a virus; and the polypeptide binds a molecule expressed on the surface of a mammalian cell.

6. The composition of claim 1, wherein the lipid enveloped capsid envelopes a single ribonucleic acid molecule.

7. A method of making a ribonucleic acid packaged within a virus capsid, the method comprising combining a lipid composition, virus capsid protein and a ribonucleic acid, wherein: the capsid packages a single ribonucleic acid molecule; the capsid is enveloped by a composition comprising a lipid bilayer; the ribonucleic acid is at least 100 nucleotides in length; the ribonucleic acid encodes a gene or segment of polynucleotides not derived from a virus; and relative amounts of virus capsid proteins and ribonucleic acid combined in the method are selected so as to control packaging of the ribonucleic acid in the virus capsid.

8. The method of claim 7, wherein relative amounts of virus capsid protein and ribonucleic acid are selected so that there are at least 10 virus capsid proteins for every 100 nucleotides of ribonucleic acid molecule length.

9. The method of claim 7, wherein relative amounts of virus capsid protein and ribonucleic acid are selected so that the mass ratio of virus capsid proteins to ribonucleic acids is at least 6: 1.

10. The method of claim 7, wherein the method comprises: dialyzing the first buffer solution against a second buffer solution; and/or treating the capsid with a ribonuclease.

11. The method of claim 10, wherein: the first buffer solution has a pH between 7.0 and 7.4; and wherein the second buffer solution has a pH below 5.0.

12. The method of claim 7, wherein the method further comprises coupling the virus capsid with a membrane comprising a lipid.

13. The method of claim 12, wherein the membrane is a bilayered membrane that envelops the virus capsid.

14. A method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell, the method comprising:

(a) combining the mammalian cell with a composition comprising a ribonucleic acid enveloped by a virus capsid; wherein: the capsid is enveloped by a composition comprising a lipid bilayer; the ribonucleic acid is at least 100 nucleotides in length; the ribonucleic acid is not derived from a virus; and the virus capsid protein envelops the ribonucleic acid so as to inhibit RNAse nuclease degradation of the ribonucleic acid; and

(b) allowing the lipid enveloped virus capsid to contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell.

15. The method of claim 14, wherein the ribonucleic acid encodes at least one polypeptide that is expressed in the mammalian cell following delivery of the ribonucleic acid into the cytoplasm of the mammalian cell.

16. The method of claim 15, wherein the ribonucleic acid encodes a RNA- dependent RNA polymerase.

17. The method of claim 15, wherein the ribonucleic acid encodes a polypeptide selected for its ability to facilitate imaging of the mammalian cell.

18. The method of claim 15, wherein the ribonucleic acid encodes a polypeptide useful in a therapeutic regimen.

19. The method of claim 14, wherein the virus capsid protein is coupled to a polypeptide selected for its ability to bind a molecule expressed on the surface of the mammalian cell.

20. The method of claim 14, wherein the mammalian cell is a cancer cell.