Patent Grant
10221399
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 30, 2015, is named 30435.277-US-U1 SL.txt and is 2,508 bytes in size.
The present invention relates generally to gene delivery and more particularly, methods and compositions related to the delivery of ribonucleic acid to mammalian cells.
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). Interestingly, however, plant-derived VLPs have not been used for direct gene delivery and expression. In particular, there have been no attempts to use a plant viral capsid to deliver heterologous genes for expression in mammalian cells. High-level expression of mammalian genes in plant hosts, mediated by Agrobacterium plasmids containing the translation-enhancement 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 such a system cannot be reconstituted in vitro and must be prepared by recombinant plasmid engineering in cell culture.
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.
The present invention provides methods and materials that involve using the capsids of plant viruses as vectors for gene delivery. More particularly, the present invention involves the novel approach of using 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 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 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 target mammalian cells.
Embodiments of the invention include compositions of matter comprising cowpea chlorotic mottle virus capsid proteins and ribonucleic acid. The cowpea chlorotic mottle virus 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 cowpea chlorotic mottle 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 cowpea chlorotic mottle virus capsid, the method comprising combining cowpea chlorotic mottle virus capsid protein and a ribonucleic acid in a 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 (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.
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, 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.
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.
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.
For example, 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.
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 “in 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. Pat. 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. Pat. 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 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 cowpea chlorotic mottle virus capsid proteins and 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
Optionally, the 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 cowpea chlorotic mottle virus capsid, the method comprising combining 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 100 nt to 10,000 nt) in pH 7.2 (I=0.1M) buffer, incubate the mixture overnight, and then dialyze against pH 4.8 (I=0.1M) 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
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, 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 cowpea chlorotic mottle 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 virus-like 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 cowpea chlorotic mottle virus capsid protein and the ribonucleic acid in a solution in vitro. The method further comprises the steps of (a) incubating the cowpea chlorotic mottle 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 MM 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 virus-like 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.
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). 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 and Young, 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 translation-enhancement 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 (DI[EYFP], 1800 nt:
VLP Synthesis and Characterization
As described in the Methods section below, DI[EYFP] 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 (
The assembly mix was imaged by negative-stain transmission electron microscopy (TEM). The images showed spherical VLPs (
CCMV VLPs Can Release Their RNA Content in the Cytoplasm of Mammalian Cells
Having characterized the plant virus VLPs containing DI[EYFP] 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 DI[EYFP] 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 DI[EYFP] 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
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. 4Biv 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
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.
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 DI[EYFP] RNA in conjunction with GP-Rep vector infection, we are able to exploit the powerful messenger RNA amplification scheme unique to plus-strand RNA viruses (Strauss and Strauss, 1994; Frolov et al., 1996). More explicitly, upon release from its VLP each DI[EYFP] 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.
Constructs and Reagents
Di[EYFP]-RNA
The DI[EYFP] 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 260 nm.
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 24 h.
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
DI[EYFP] 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 KCl, 5 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl pH 7.2) for 4 h, followed by dialysis against virus buffer (VB: 0.1M sodium acetate, 1 mM EDTA, pH4.8) for 16 h, 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 VLPs
A 6 μl 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 μL 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 1×1 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 of BHK Cell Culture
Prior to transfection, BHK-21 cells were grown as monolayers on 6-well plates at 37° 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 of BHK 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 μg of DI[EYFP] RNA was diluted into Opti-MEM to 300 μl for each transfection. Similarly, 3 μl of Lipofectamine-2000 was diluted into 300 μl 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× 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×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.
RNaseA assays
All RNase A digestion steps were carried out using 0.2 ng/μl 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 (
Flow Cytometry
Twenty-four hour post-transfection, the cells were washed with 1×PBS, trypsinized, and resuspended in 1×PBS buffer with 2% FCS at a density of 3×106 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.
See, e.g. Allison et al., Virology 172 (1), 321-330 (1989)
Tobacco Mosaic Virus Origin of Assembly
See, e.g. Goelet et al., Proc. Natl. Acad. Sci. USA (79) 5818-5822 (1982)
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.
This application is a divisional application that claims the benefit under 35 U.S.C. § 121 of U.S. patent application Ser. No. 14/540,816, filed Nov. 13, 2014, which claims the benefit under 35 U.S.C. Section 119(e) of U.S. Provisional Patent Application Ser. No. 61/906,061, titled “IN VITRO RECONSTITUTED PLANT VIRUS CAPSIDS FOR DELIVERING RNA GENES TO MAMMALIAN CELLS” filed Nov. 19, 2013, the contents of each of which are incorporated herein by reference.
This invention was made with Government support under Grant Number CHE1051507, awarded by the National Science Foundation. The Government has certain rights in the invention.
| Number | Name | Date | Kind |
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| 20120015899 | Lomonossoff et al. | Jan 2012 | A1 |
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| Annamalai, P., et al., “Dispensability of 3′ tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs”, Virology, vol. 332 (2005) issue 2, pp. 650-658. |
| Cadena-Nova, Ruden D., et al., “Self-Assembly of Viral Capsid Protein and RNA Molecules of Different Sizes: Requirement for a Specific High Protein/RNA Mass Ratio”, Journal of Virology, Mar. 2012, vol. 86, No. 6, pp. 3318-3326. |
| Mahraj, Payal D. et al. “Nanoparticle encapsidation of Flock House virus by auto assembly of Tobacco Mosaic virus coat protein”, International Journal of Molecular Sciences, 15, 2014. |
| Smith, Mark L. “Assembly of trans-encapsidated recombinant viral vectors engineered from Tobacco mosaic virus and Semliki Forest virus and their evaluation as immunogens”, Virology 358, 321-333, 2007. |
| Number | Date | Country | |
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| 20170183635 A1 | Jun 2017 | US |
| Number | Date | Country | |
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| 61906061 | Nov 2013 | US |
| Number | Date | Country | |
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| Parent | 14540816 | Nov 2014 | US |
| Child | 15449827 | US |
