WO2011002522A2 - Methods of making and using synthetic viruses - Google Patents

Methods of making and using synthetic viruses Download PDF

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Publication number
WO2011002522A2
WO2011002522A2 PCT/US2010/001895 US2010001895W WO2011002522A2 WO 2011002522 A2 WO2011002522 A2 WO 2011002522A2 US 2010001895 W US2010001895 W US 2010001895W WO 2011002522 A2 WO2011002522 A2 WO 2011002522A2
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protein
virus
synthetic
lipid bilayer
rna
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WO2011002522A3 (en
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Philippe-Alexandre Gilbert
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Medimmune, Llc
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/00051Methods of production or purification of viral material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/00061Methods of inactivation or attenuation

Definitions

  • the present invention relates to synthetic viruses, and methods and devices for making and using such synthetic viruses.
  • Synthetic viruses can be fabricated by introducing internal, external and transmembrane proteins to a lipid bilayer before budding of the bilayer to capture a core nucleic acid assembly forming a viable synthetic virus particle.
  • Live attenuated viruses have long been used successfully to eradicate infectious disease and protect humans against viral infection.
  • Many current vaccines are produced by growing the virus in vivo, e.g., egg-based live attenuated Influenza vaccine (LAIV) or on mammalian host cells.
  • LAIV egg-based live attenuated Influenza vaccine
  • this presents problems in removal of unwanted residual host cell DNA and protein.
  • egg-grown viruses exhibit differences in antigenicity from viruses from isolated clinical specimens or grown in mammalian cell lines.
  • VLP virus-like particles
  • PSV pseudoviruses
  • the present inventions include synthetic viruses, methods of producing synthetic viruses, and devices for fabricating synthetic viruses.
  • the synthetic viruses can be fabricated by establishing a bilayer lipid membrane across an aperture, introducing a transmembrane protein to anchor other membrane components, introducing an affinity protein to a first side of the membrane to target the virion to the desired host cell, introducing an M protein to the second side to facilitate budding of the membrane, and introducing a nucleic acid core encoding a viable attenuated virus to the second side of the membrane. Budding of the membrane from the aperture can thus capture the nucleic acid core to provide a functional synthetic virus.
  • the synthetic virus can comprise a nucleic acid core encoding a virus and surrounded with a lipid bilayer membrane incorporating an outer protein directing the virus to a host cell.
  • Devices for production of the synthetic virus can include an aperture supporting a membrane between two chambers, and means to deliver proteins and a nucleic acid core to either side of the membrane.
  • the methods for preparing a synthetic virus can include, e.g., forming a synthetic lipid bilayer across an aperture separating a first chamber from a second chamber; delivering a first protein to the lipid bilayer from the first chamber, wherein the first protein associates to bind with the lipid bilayer or with a second protein embedded in the lipid bilayer; delivering a third protein to the lipid bilayer from the second chamber, wherein the third protein associates with the lipid bilayer or with the second protein; delivering an RNA core structure to the lower chamber; and, budding the lipid bilayer from the second chamber into the first chamber to form a liposome enclosing the RNA core structure and comprising the first protein on an outer surface of the liposome.
  • Budding of the liposome from the aperture can produce the synthetic virus wherein the first protein, on the outside of the of the synthetic virus, specifically binds a host cell surface receptor to direct delivery of the RNA core into the cell where the RNA is transcribed and translated to produce live virus from the host cell.
  • the synthesis of the virus is typically performed entirely in vitro, thus dramatically reducing purification issues.
  • the synthetic virus can be placed in storage until used, or the synthetic virus can be administered immediately after budding is completed.
  • the second protein is different from the first protein and also different from the third protein.
  • the second protein can be delivered to the lipid bilayer from the first chamber or from the second chamber.
  • the RNA core structure can optionally bind with the third protein and/or with the lipid bilayer.
  • the proteins and core interact through specific affinity interactions.
  • the lipid bilayer comprises lipid rafts, e.g., enriched in cholesterol, sphingolipids, and/or membrane proteins.
  • the quantity or variety of synthetic viruses produced can be increased by budding the liposomes from an array of apertures.
  • the array apertures are arranged in series along a channel and/or arranged in two or more parallel channels.
  • the second protein can be a transmembrane protein, e.g., providing an affinity target on one or both sides of the membrane.
  • the transmembrane protein can display an affinity for the first protein (e.g., outer affinity targeting protein), for the third protein (e.g., M protein) and/or for the RNA core.
  • the outside exposed surface protein can be an affinity protein, such as, e.g., a viral receptor, a cell surface antigen, a host cell surface protein, a cell surface ligand, and/or an antibody variable domain.
  • the third protein, on the inside surface of the membrane can be, e.g., a member of the M protein family or homologous sequence, e.g., having a segment with an affinity for a target determinant on the RNA core structure.
  • the membrane when the internal proteins and nucleic acid core are bound to the membrane, the membrane can be budded from the aperture to enclose the core. Budding can be induced by applying a force across the membrane, e.g., an osmotic pressure differential, a hydrostatic pressure differential, a voltage differential and/or the like.
  • budding can be induced when an adequate amount of an M protein has been applied to the lipid bilayer.
  • budding is facilitated by application of M protein to one side of the membrane, and initiated or completed by application of the pressure differential across the membrane.
  • the nucleic acid core structure (e.g., RNA core) can include, e.g., RNA encoding a virus, a scaffold, and RNA binding proteins.
  • the scaffold proteins can provide structure and ligand binding targets complimentary to a protein bound to the inner side of the membrane.
  • the RNA binding proteins a positive charge to stabilize and condense the nucleic acid in the core, and can also can have scaffolding protein functions.
  • the synthetic virus product of the method can contact an appropriate host cell to initiate replication of multiple copies of a progeny virus encoded by the nucleic acid of the synthetic virus.
  • the progeny viruses can be non-viable (e.g., but have desirable antigenic properties), can be attenuated live viruses, or can appear as a typical
  • the progeny virus is a live attenuated virus, such as, e.g., a paramyxovirus, a pnemovirus, an
  • orthomyxovirus a retrovirus, a morbillivirus and/or the like.
  • the methods of synthesizing virus particles can optionally include steps to control the average particle size of the particles.
  • the particle size can be adjusted by selective filtration of the bedded particles.
  • the synthetic liposomes flow from the first chamber through a "Y" channel having two asymmetric flow rates to create a pinched flow wherein smaller liposomes are fractionated from larger liposomes.
  • the present invention includes synthetic viruses produced by the methods of synthesizing viruses, described herein.
  • the synthetic virus is other than a virus produced in a living host cell.
  • sterile compositions of the synthetic virus e.g., sterile filtered taking advantage of the small size (e.g., 200 nm or less diameter) of the synthetic virus particles.
  • the synthetic virus can include, e.g., a liposome, a first protein associated with an outer surface of the liposome, a second protein inserted into a membrane of the liposome, a third protein associated with an inner surface of the liposome and a viral RNA core inside the liposome.
  • the synthetic virus can functionally direct production of a live virus when the liposome is delivered to a host cell.
  • the synthetic virus has a lipid bilayer comprising phosphatidylcholine (PC ) and/or
  • the synthetic virus is attenuated, cold adapted and/or temperature sensitive. In another embodiment, the synthetic virus is characterized by an average diameter of 0.2 ⁇ m or less, or 0.1 ⁇ m or less, or 0.05 ⁇ m or less.
  • the device can include, e.g., a first chamber, a second chamber, an aperture between the first chamber and the second chamber; a lipid bilayer traversing the aperture between the chambers.
  • the bilayer can have an affinity protein associated with the lipid bilayer on a first bilayer side facing the first chamber and a RNA core structure associated with the lipid bilayer on a second bilayer side facing the second chamber.
  • the device can include an array of apertures, and the apertures can have a diameter ranging from, e.g., 10 nm to 250 nm, or from 10 nm to 100 nm, or from 10 nm to 75 nm, or from 10 nm to 50 nm, or from 50 nm to 75 nm, or from 75 nm to 100 nm, or from 100 nm to 150 nm, or from 150 nm to 250 nm.
  • synthetic virus refers to a virus that is fabricated in vitro, as taught herein.
  • a virus assembled in a host cell, or by budding from the surface of a living host cell, is not considered a synthetic virus.
  • the mere presence of synthetic (man made) constituents in a virus does not in itself make the virus a synthetic virus of the present inventions.
  • Synthetic viruses of the invention include a synthetic lipid bilayer is, e.g., a lipid bilayer fabricated in vitro, as taught herein.
  • binding refers to attachment of one component with another.
  • the attachment can be by an affinity interaction between the components, binding based on non-specific interactions or can include covalent bonding.
  • binding between protein constituents is due to specific affinity interactions, while binding between proteins and lipid bilayers typically comprises hydrophobic interactions between certain protein segments and lipid components of a membrane.
  • RNA core structure includes at least an RNA strand encoding components of a virus and at least one protein binding the RNA, typically providing a more compact structure than the RNA alone.
  • the RNA core structure can include encoding RNA and a positively charged RNA binding protein.
  • the core (or liposome interior) can include an enzyme, such as a polymerase.
  • a protein of the core can include a segment with an affinity for a membrane protein, e.g., so that the core will bind to the surface of the membrane.
  • affinity e.g., with regard to interactions between synthetic virion components, refers to attractive interactions that bind one component to another.
  • Affinity can be specific, e.g., as in the classic lock and key models described for affinity interactions, e.g., between antigen and antibody, or receptor and ligand.
  • Affinity can be non-specific, e.g., with components associating non-specifically through attractive hydrophobic interactions or ionic interactions.
  • a lipid bilayer can have a nonspecific affinity for segments of a protein rich in hydrophobic amino acids (e.g., the inserted segments of a transmembrane protein).
  • the term "attenuated”, with regard to a virus refers to a viable virus with reduced virulence.
  • an attenuated virus in a vaccine is still viable (not “killed"), stimulating an immune response and creating immunity but not causing significant pathology.
  • a “segment” of a protein is a linear portion of the protein amino acid chain.
  • a hydrophobic segment typically includes peptide sequences enriched in hydrophobic amino acids, such as, e.g., Ala, VaI, De, Leu, Met, Phe, Tyr, and Tip.
  • Hydrophilic segments typically include peptide sequences rich in polar or ionized amino acids, such as, e.g., Lys, Arg, His, Asp, GIu, Ser, Thr, Arg and GIt. Segments can have amino acid sequences providing specific affinity, e.g., by expressing ligand or receptor functions.
  • the invention can include proteins that are "derived from” a parental molecule.
  • the term "derived from” refers to a component that is isolated from or made using a specified molecule or organism, or information from the specified molecule or organism.
  • a polypeptide that is derived from a second polypeptide can include an amino acid sequence that is identical or substantially similar to the amino acid sequence of the second polypeptide.
  • the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis.
  • the mutagenesis used to derive polypeptides can be intentionally directed or intentionally random, or a mixture of each.
  • mutagenesis of a polypeptide to create a different polypeptide derived from the first can be a random event, e.g., caused by polymerase infidelity, and the identification of the derived polypeptide can be made by appropriate screening methods.
  • Mutagenesis of a polypeptide typically entails manipulation of the polynucleotide that encodes the polypeptide.
  • a "matrix protein”, as used herein, refers to a protein matrix protein (Ml) of influenza virus, homologous proteins, other proteins of similar function from other viruses, such as the matrix proteins of respiratory syncytial virus (RSV) or proteins at least 90% identity, or 95% identity, or at least 98% identity to the influenza Ml protein.
  • M proteins are typically bifunctional proteins that mediate the encapsidation of RNA-nucleoprotein cores into the membrane envelope.
  • M proteins typically bind both membrane and RNA simultaneously e.g., acting as both a nucleic acid binding protein and as a modifier of membrane topology).
  • the X-ray crystal structure of the N-terminal portion of type A influenza virus Ml— amino acid residues 2-158— has been determined at 2.08 A resolution.
  • the protein forms a dimer.
  • a highly positively charged region on the dimer surface is suitably positioned to bind RNA while the hydrophobic surface opposite the RNA binding region is involved in interactions with the membrane.
  • the membrane-binding hydrophobic surface could be buried or exposed after a conformational change.
  • Homology of proteins and/or protein sequences can be the result of derivation from a common ancestral protein or protein sequence. Homology can be inferred, e.g., from structural and functional characteristics and from the percent identity of a putative homologous protein of homologous region of a protein. That is, homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity (percent identity) between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology.
  • sequence similarity e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, can also be used to establish homology.
  • Methods for determining sequence similarity percentages e.g., BLASTP and BLASTN using default parameters are described herein and are generally available.
  • Figures IA to IE describe a step by step assembly process of producing a synthetic virus from a lipid bilayer at an exemplary aperture.
  • Figure IA shows a schematic diagram of a synthetic lipid bilayer formed in an aperture. The upper and lower compartments are also exemplified.
  • Figure IB represents the delivery of various components to the synthetic lipid bilayer. The first (TM) and second (X) proteins are delivered via the upper chamber while the third protein (M) is delivered to the bottom chamber.
  • Figure 1C represents the putative associated of the various proteins in a synthetic lipid bilayer.
  • Figure ID represents the delivery of the RNA core to the bottom chamber. Once the RNA core is delivered, the synthetic virus is ready to be formed.
  • Figure IE represents the budding process to produce a functional synthetic virus, which can deliver a functional RNA cargo capable of directing virus production and replication.
  • Figure 2 is a schematic diagram of an exemplary microfluidic device configured to generate a synthetic virus.
  • a pair of microfluidic channels are separated by an aperture of controlled size.
  • the microfluidic channels permit the transport of various fluid phase units and their content to either side of the aperture.
  • the fluid phase units can sequentially be introduced in the upper and lower chambers. Control of pressure at the micro scale can permit precise handling. Evaporation and gravity are considered negligible in this environment. Air phase separating of the liquid is possible and permits washing and segregation of different components.
  • Figure 3A to 3D are schematic diagrams showing a fluidic device and process of synthesizing a synthetic virus.
  • Figure 4 shows a schematic diagram of constituents of the RNA core and its assembly.
  • the RNA is complexed with a scaffold, such as actin.
  • the RNA: scaffold mix can be further complexed with certain functional proteins (e.g., having affinity for a membrane protein) to form an exemplary RNA core.
  • Figure 5 is a chart of Reynold number versus Q volumetric flow showing the effect of Reynold number of flow through microchannels of various diameters.
  • Figure 6 is a chart of pressure versus aperture diameter showing theoretical failure pressure point based on lipid bilayer thickness.
  • the present inventions include synthetic viruses, methods of fabricating synthetic viruses, and devices configured to practice the methods of synthesizing synthetic virions.
  • the methods generally include preparation of a lipid bilayer across an aperture; introduction of protein structures on and into the bilayer; application of a viral genome- encoding nucleic acid to a surface of the bilayer; and, budding the bilayer into a liposome, thus, capturing the nucleic acid in a synthetic virus.
  • the synthetic viruses typically comprise a spherical or rod-like lipid bilayer membrane traversed with a transmembrane protein; an affinity protein (specific for a host cell receptor) bound to the outer surface of the membrane; a third protein bound to the inside surface of the membrane and typically influencing the membrane curvature and having affinity for a core structure; and, a nucleic acid core structure inside the membrane and comprising at least one nucleic acid binding protein that tends to compact the nucleic acid.
  • the devices include at least an aperture between a first and second chamber. The device is configured to provide an interface to • establish a lipid bilayer and populate the bilayer with the protein and nucleic acid components of a synthetic virus.
  • a difficulty in the creation of synthetic viruses is the establishment at the micro scale level of a stable lipid bilayer that will encompass an RNA core and produce an infectious virus unit.
  • Common liposome technologies typically rely on bulk micelle formation by agitation or shear to provide a bilayer membrane capsule (liposome) of phospholipids entrapping a soluble component within. Such methods can result in capture of small size plasmid vectors or RNAi segments, proteins, chemical drugs within the liposomes.
  • insertion of a full length viral RNA into liposomes capable of a productive infection does not result.
  • entrapment of large size nucleic acids (> 15 kb) into liposomes has not been shown.
  • a more controlled assembly of particles e.g., employing affinity interactions between the core and lipid shell components, is required to provide viable particles of the proper size.
  • Such a template has the advantage of allowing sequential micro-injection of assembly materials in predetermined steps, providing the necessary control in preparation of synthetic viruses.
  • Membrane lipid rafts are defined as cholesterol and sphingolipid-rich microdomains in the exoplasmic leaflet of the cellular plasma membrane (Laliberte et al, J. Virol. 81: 10636-10648; 2007).
  • the tight packing of cholesterol molecules among the saturated fatty acid tails of sphingolipids creates a local membrane organization, referred to as "liquid-ordered", which possesses properties different from those of the surrounding bulk plasma membrane.
  • Membrane lipid rafts usually function in membrane trafficking events and cell-cell attachment and serve as signal transduction platforms in many cell types. Membrane lipid rafts are known to be sites of virus assembly and release based on the fact that lipid raft-associated molecules are found in purified virions and that lipid rafts are cholesterol and sphingolipid-enriched microdomain found in cell membranes. Viral proteins will assemble at the base of the lipid-raft micro- domain to be specifically captured in the virus budding mechanism.
  • M proteins matrix proteins, which can independently provide for lipid-based virus like particle (VLP) formation (G ⁇ mez-Puertas et al., J. Virol: 74, 11538-11547; 2000) from a lipid bilayer.
  • VLP virus like particle
  • proteins encoded by certain M segments appear to be involved in modulating filamentous versus spherical virion morphology (Hughey et al., Virology 212: 411-421; 1995).
  • the M protein when expressed alone, assembles bilayers into VLPs, which are released into the culture medium.
  • the M protein operates to impose a curvature on the cell membrane.
  • M protein budding formation is virus dependent and will occur either in specific high cholesterol rich regions or else in non-lipid raft membranes. Initiation of bud formation requires outward curvature of the plasma membrane, which can be stimulated by the accumulation of the M protein at the inner leaflet of the lipid bilayer. The virus bud is then extruded until the inner core is enveloped. The budding process is completed when the membranes fuse at the base of the bud and the enveloped virus particle is released following fission from the cell membrane.
  • the M protein is the driving force behind this process because budding from the host cell cannot occur in the absence of this protein, yet it alone can induce the formation of virus-like particles under these conditions.
  • the M protein also contains a late domain that has been identified in several viral matrix proteins and is responsible for recruiting host factors required for the late stages of viral budding.
  • the basic residues in this domain have also been implicated in RNA binding.
  • the extent to which the membrane is extruded before pinching off occurs affects the size and shape of the virus particle.
  • influenza virus particles are either spherical or filamentous, and this characteristic morphology is genetically linked to the M segment.
  • the M protein induced membrane curvature is not dependent on the presence of cholesterol in the cell membrane.
  • the steps to assemble a synthetic virus should be composed of sequential events related to: the creation of a lipid bilayer, the insertion of viral surface protein, the association of surface protein with matrix (M) protein, and the entrapment of viral core RNA.
  • M surface protein with matrix
  • Methods to assemble a synthetic virus are described herein to generate synthetic viruses containing a genetic payload capable of directing the production of virus in a host cell.
  • methods of the invention comprise:
  • RNA core structure comprising viral RNA, RNA binding proteins, and a lipophilic component (and/or specific affinity component) to the lower chamber
  • a liposome inducing budding from the aperture from the lower chamber; whereby a liposome is released into the upper chamber comprising the lipid bilayer with the second protein on the outer surface, and the third protein and RNA core on the inside of the liposome.
  • a liposome can be a synthetic virus capable of specifically delivering the RNA core, by an affinity interaction between the second protein and a host cell receptor, to a host cell where the RNA can instruct production of a live non-synthetic virus product.
  • methods of the invention comprise forming a synthetic lipid bilayer in the aperture of the device, such that separate upper and lower chambers are formed.
  • the formed chambers are positioned to facilitate interaction of different fluid phases with the synthetic lipid bilayer surfaces.
  • an aqueous phase, an organic phase, or an air phase can interact with the synthetic lipid bilayer in sequence, as desired.
  • the synthetic lipid bilayer can comprise a mixture of phospholipids, proteins, glycoproteins, cholesterol, sphingosine, and/or other components.
  • Synthetic lipid bilayers have been constructed using various techniques such as those found in Suzuki et al, Lab Chip ⁇ 2004, 4:501-505; Malmstadt et al., Nano Letters 2006, 1961-1965; Ide and Ichikawa, Biosensors and Isoelectrics, 2005, 21:672-677.; Suzuki et al. Langmuir 2006, 22:1937-1942, each is incorporated by reference in its entirety.
  • a functional synthetic virion can be prepared as follows. Various virion components can be added to one side or the other of a lipid bilayer, an RNA core
  • a phospholipid preparation 10 in an upper chamber 11 is applied to a water phase 12 in a lower chamber 13.
  • Trans-membrane (TM) protein units 14 (Figure IB) pre-included in the lipid preparation (or, optionally, present in the water phase) can establish a cross-linking unit between the viral surface and matrix proteins. Assuming that the TM unit is lipophilic, its orientation could be adjusted by changing the pH of the buffers.
  • lipid bilayer ( Figure 1C).
  • the approximate height of the lipid bilayer can be a small as 5 nm or less, but can be modulated by changing the length of the phospholipid(s).
  • Micro-injection of a surface protein in the upper phase is performed to populate the exposed surface of the lipid bilayer with the protein.
  • the surface protein will be ultimately located in the outer shell surface of the synthetic virus when budding occurs in the final step.
  • various surface proteins can be added, e.g., by engineering them to have affinity for the exposed TM protein segment.
  • RNA cores 16 can optionally be prepared previously in a separate reaction, e.g., mixing the RNA with polymerase protein and core proteins to create functional core elements of the desired dimension. Diffusion and attachment of the RNA core from the bilayer interface can be inhibited by affinity interactions with natural or engineered segments of the M protein. Introduction of a final harvest upper phase layer and manipulation of the pressure ( ⁇ P ⁇ 0) can be performed to fully allow membrane curvature and final budding of a synthetic virion 17.
  • a lipid bilayer is typically formed by aligning ambiphilic molecules at an air/water interface to form a monolayer, then introducing an aqueous buffer at the air side of the monolayer, forcing the ambiphilic molecules to rearrange into a bilayer with
  • hydrophobic portions embedded between outer hydrophilic surfaces For example, an air/water interface can be generated by surface tension across an aperture.
  • An immiscible hydrophobic solvent containing, e.g., phospholipids can be layered over the interface. A lipid monolayer of the phospholipids will spontaneously form at the interface when the solvent contacts the buffer. The solvent can be removed, but the monolayer will remain in place.
  • an aqueous buffer is placed over the monolayer where the hydrophobic solvent had been, the monolayer will spontaneously reassemble into a lipid bilayer, e.g., with the polar phospholipid heads oriented toward the aqueous buffers on each side and with the lipid tails of the phospholipid hydrophobically associating inside the membrane.
  • the lipid bilayer can be formed by stepwise addition and removal of fluids within chambers on each side of the aperture, as described above.
  • the lipid bilayer can be formed in bulk processes, e.g., wherein suspensions or emulsions of the lipid and aqueous buffer are exposed to shear forces that result in production of liposomes.
  • such liposomes can be formed by blending lipid and water in a blender or homogenizer.
  • the liposome product typically is not of uniform size. Further, such processes are often too uncontrolled to allow consistent incorporation of desired proteins in desired orientations, or to consistently capture a nucleic acid the size of a viral genome.
  • the liquid phases can be introduced from channels flowing to and from the aperture, e.g., as shown in Figure 2.
  • An upper channel can bring liquid packets to or from the lipid bilayer and optionally bring a protein component to interact with other components at the aperture.
  • Liquid packets can be separated by air intervals in the channels to avoid product cross contamination between liquid packets.
  • a lipid monolayer can be formed at aperture 20 by flowing an aqueous solution 21 through lower channel 22 to lower chamber 23.
  • a hydrophobic solvent 24 carrying, e.g., a phospholipid can flow from upper channel 25 to contact the aqueous interface spontaneously generating a lipid monolayer with hydrophobic tails oriented up and the polar head oriented down.
  • the hydrophobic phase can be replaced by a flow of aqueous phase in the upper channel (optionally segmented with a gaseous phase) to cover the aperture.
  • Introduction of aqueous phase to the upper chamber can cause the lipid monolayer to spontaneously rearrange to form a classic lipid bilayer membrane with inner hydrophobic lipid tails sandwiched between outer polar heads.
  • one or more proteins are present in the hydrophobic solvent, e.g., with the phospholipid, even before the monolayer or bilayer is formed.
  • a transmembrane protein typically including at least one hydrophobic segment between two hydrophilic peptide segments
  • the protein can be in solution in the hydrophobic solvent and/or in the form of insoluble aggregates.
  • the protein can be other than a transmembrane protein, such as a protein having a hydrophobic segment that will ultimately be incorporated into the membrane with the hydrophobic segment inserted into the hydrophobic interior of the membrane. Delivering Proteins to the Membrane
  • the proteins can be delivered to the membrane by simple manual (e.g., pipetting) techniques, by automated filling of the upper and/or lower chambers through ports, or, preferably, by flowing the proteins in solution through a channel to contact the membrane at the aperture.
  • the membrane can be selectively populated with desired proteins at either or both surfaces. Access to the separate membrane surfaces can allow introduction of different proteins to opposite sides of the membrane. For example, it is often desirable to incorporate a protein with affinity for a host cell receptor on one side of the membrane, and to incorporate an M protein with a segment having affinity for a nucleic acid core on the other side. Alternately, both types of proteins can be incorporated, e.g., on both sides of the membrane.
  • methods of the invention comprise delivery of a first protein to the synthetic lipid bilayer.
  • the first protein can be a lipophilic or transmembrane protein (see, for example, Figure 1).
  • the first protein can display affinity for a second protein and/or a third protein.
  • the first protein can display an affinity for the RNA core used in the method.
  • the first protein is added to the lipid bilayer prior to the application of the second protein, third protein, and/or RNA core (or added to the lipid before membrane formation).
  • the first protein is added to the lipid bilayer after the second protein, third protein, and/or the RNA core.
  • the first protein is a transmembrane protein. Additionally, the first protein can display an affinity (e.g., a specific affinity) for the second protein, third protein, and/or RNA core. In some embodiments, the first protein can associate covalently or non- covalently with the second protein, third protein or the RNA core. In yet further
  • the first protein can associate covalently or non-covalently with constituents of the RNA core.
  • methods of the invention comprise delivery of a second protein to the upper chamber formed by the lipid bilayer (see Figure 3B).
  • the second protein can be a lipophilic or transmembrane protein.
  • the second protein can display affinity for the first protein and/or the third protein.
  • the second protein can display an affinity for the RNA core structure used in the method.
  • the second protein is added to the lipid bilayer prior to the application of the first protein, third protein, and/or RNA core.
  • the second protein is added to the lipid bilayer after the second protein, third protein, and/or the RNA core.
  • the second protein is a transmembrane protein.
  • the second protein can display an affinity for the first protein, third protein, and/or RNA core.
  • the second protein can associate covalently or non-covalently with the first protein, third protein or the RNA core.
  • the second protein can associate covalently or non-covalently with constituents of the RNA core.
  • the protein When a protein in an aqueous buffer is delivered to contact with the membrane, the protein can spontaneously integrate into the membrane, e.g., through nonspecific hydrophobic interactions.
  • the protein can naturally have hydrophobic segments, or be engineered to include hydrophobic segments, with a non-specific affinity for the lipid tails of the membrane molecules.
  • the thermodynamic free energy of the hydrophobic effect can favor the integration of the hydrophobic segments with the hydrophobic regions of the membrane.
  • the free energy driving the integration can be influenced by adjustments in the surrounding aqueous buffers, e.g., by addition of solvents and surfactants, and/or by adjustment of pH and ionic strength of the solutions.
  • the protein delivered to the membrane can bond to the membrane through specific affinity interactions, e.g., with another protein already present on the membrane.
  • the membrane can include a protein with specific binding partner, such as, e.g., an antigenic determinant, receptor, lectin, antigen, and/or the like.
  • the delivered protein can be an antibody, or preferably include one or more CDR regions comprising a binding domain specific to the determinant.
  • one protein can include a ligand and the other protein can be a structurally complimentary receptor.
  • one or both proteins can include a chelator (e.g., poly- histidine) so the proteins can associate through a chelation bond.
  • the protein can covalently bond to the membrane surface.
  • a first protein bound to the membrane can include a reactive group and the delivered protein can include a moiety reactive with the reactive group, so that the two proteins are covalently bonded on contact.
  • one or both proteins can include a linker group, such as a N-Hydroxysuccinimide (NHS) group.
  • NHS N-Hydroxysuccinimide
  • the two proteins can be bonded together in the presence of a bidentate bridging linker.
  • one protein delivered to one side of the membrane is an M protein that can urge the planar membrane into a concave shape to ultimately facilitate budding of the membrane from the aperture.
  • M protein that can urge the planar membrane into a concave shape to ultimately facilitate budding of the membrane from the aperture.
  • a hydrostatic pressure differential ( ⁇ P > 0) can be used to prevent the premature curvature or budding of the membrane.
  • the second (outer affinity) protein is a viral receptor, a cell surface receptor, a cell surface ligand, or an antibody fragment that recognizes a cell surface antigen on a host cell.
  • the protein bound to the outside of the synthetic virion has a specific affinity for a desired target host cell.
  • the outer surface protein can include the hemagglutinin (HA) glycoprotein (or a structural analog or fragment of HA), which can specifically interact with the sialic acid containing glycans on a host cell surface.
  • HA hemagglutinin
  • Other specific host receptor/virus surface protein affinity interactions are known in the art.
  • other interacting pairs can include gpl20 interactions with CD4 and chemokine receptors on the T cell, HBV envelope proteins and liver cell receptors, herpes proteins and nerve cell receptors, and the like.
  • methods of the invention comprise delivery of a third protein to the lower chamber side of the at the lipid bilayer (see Figure 3C).
  • the third protein can be a lipophilic or transmembrane protein.
  • the third protein can display affinity for the first protein and/or the second protein.
  • the third protein can display an affinity for the RNA core structure used in the method.
  • the third protein can be added to the lipid bilayer prior to the application of the first protein, second protein, and/or RNA core.
  • the third protein is added to the lipid bilayer after the second protein, first protein, and/or the RNA core.
  • the third protein is a transmembrane protein.
  • the third protein may display an affinity for the first protein, second protein, and/or RNA core.
  • the third protein may associate covalently or non-covalently with the first protein, second protein or the RNA core.
  • the third protein may associate covalently or non-covalently with constituents of the RNA core.
  • the third protein in the methods of the invention is the Matrix (M) protein, the VP40 VLP protein, the ebola VP30 protein, a clathrin protein, a homologous protein or a derivative thereof from any virus.
  • M protein or derivative thereof is from any RNA virus.
  • the M protein is from the influenza virus or an M protein derivative thereof, e.g., derived using standard protein engineering techniques.
  • additional proteins can be bound to the membrane, complexed with the nucleic acid core, or captured within the liposome during the budding process.
  • the nucleic acid is RNA
  • the RNA core structure can be delivered to the lipid bilayer membrane in much the same way described above with regard to proteins. In most cases, the RNA core structure is delivered only to one side of the membrane, e.g., the side that will ultimately line the inside of the budded synthetic virus.
  • the RNA core can be delivered in a solution (or suspension) to the membrane surface, e.g., by pipetting onto the surface, filling an associated chamber through a port, or by flowing onto the membrane from a channel.
  • the nucleic acid can be delivered to the membrane surface as an essentially free naked nucleic acid strand.
  • the lipid bilayer can include ambiphilic molecules having hydrophilic heads with a positive charge.
  • the interaction between the nucleic acid and membrane can be moderated, e.g., by adjustment of the ambient pH.
  • Nucleic acid core structure proteins can be introduced to interact with the nucleic acid bound to the membrane, e.g., to further bind, stabilize and compact the nucleic acid. That is, the core scaffolding and nucleic acid binding proteins can be complexed with the nucleic acid in situ on the membrane.
  • the nucleic acid core includes one or more core proteins that provide structures providing, e.g., scaffolding, nucleic acid binding, charge neutralization and/or capsid functions.
  • the core proteins can include positive charges to allow compact coiling of the nucleic acid.
  • the proteins can be configured as scaffolding to direct the shape of the nucleic acid core.
  • the core proteins can include sequences providing affinity interactions between the core and the membrane or with membrane proteins.
  • Exemplary core proteins can be, without limitation, e.g., actin, herpes capsid proteins and scaffolding protein, influenza protein NSl, VP3 scaffolding protein, RSV F protein, SARS CoV accessory protein 7a, the SH3 domain, RNA binding protein HuR, HTV accessory protein Vpr, Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), HTV p24, homologs and engineered derivatives thereof.
  • the proteins of the nucleic acid core can be recombinant proteins, purified proteins, or proteins from crude lysates.
  • the M protein can function both in RNA binding and in membrane budding.
  • the M protein can have hydrophobic segments that interact with the lipid bilayer and a positively charged segment that associates with nucleic acids.
  • the methods can include binding the M protein to the nucleic acid (with M protein acting as a nucleic acid binding protein) to provide a core structure, then introducing the core structure to one side of the lipid bilayer membrane surface.
  • the M protein can be partially inserted into the membrane at the hydrophobic segment, while the positively charged segment remains exposed in the aqueous environment of the lower chamber.
  • the nucleic acid can be introduced to the lower chamber where it interacts with the positively charged M protein segments to bind and form a core in situ at the membrane.
  • methods of the invention comprise the delivery of an
  • RNA core which is comprised of a functional RNA molecule capable of directing virus generation and propagation in a host cell, a scaffold to hold the RNA molecule and accessory RNA binding proteins to facilitate the transcription or translation of the RNA molecule.
  • methods and compositions of the invention comprise an RNA core, which can comprise RNA molecules, RNA binding proteins and optionally, a scaffold protein (for example, but not limited to actin).
  • the RNA core may include only which elements are required to maintain a functional RNA molecule that will drive the production of a live virus when delivered to a host cell.
  • the RNA core may be preassembled prior to delivery to the synthetic lipid bilayer, e.g., as shown in Figure 4.
  • the RNA core can be assembled in the lower chamber formed by the synthetic lipid bilayer.
  • the RNA core can display an affinity for any one of the first, second or third proteins in the synthetic virus.
  • any entity of the RNA core can also have an affinity to the synthetic lipid bilayer.
  • the RNA core is added to the lower chamber prior to any of the proteins in the methods. In other embodiments, the RNA core is added after the first, second or third proteins in the method. In other embodiments, the RNA core associates covalently or non-covalently with the first, second, or third proteins, or alternatively directly with the lipid bilayer.
  • a synthetic virus is generated when the lipid bilayer membrane buds free from the aperture, enclosing the nucleic acid within.
  • methods of the invention result in the formation of a liposome comprising a second protein on the outer edge of the bilayer, a third protein on the inner edge of the bilayer and an RNA core present inside the center of the liposome.
  • the budded synthetic virus can be harvested from the fluid in the upper chamber by aspiration or can flow in a channel to a product chamber of a fluidic device.
  • Viral budding mechanisms for certain classes of viruses use the lipid raft
  • LR lipid raft
  • M protein is the only viral component essential for virus-like particle (VLP) formation (G ⁇ mez-Puertas et al., J. Virol. 74: 11538- 11547; 2000).
  • M protein when expressed alone, can induce membrane assembly into VLPs, which can be released into a culture medium.
  • the M protein operates to impose curvature on the cell membrane.
  • the M protein budding formation is virus dependent and occurs either in specific high cholesterol rich regions or the non-lipid raft membranes (Laliberte et al., J. Virol. 81: 10636-10648; 2007).
  • Budding can be initiated by providing a pressure differential across the lipid bilayer membrane and/or by application of an M protein to one side of the membrane. It is possible to provide budding by simply providing an adequate amount of an M protein, e.g., without application of mechanical pressures across the aperture. In many embodiments, the budding is at least facilitated by the delivery of the M protein to the lower chamber. In many other embodiments, budding is influenced by changes in the hydrostatic pressure or osmotic pressure between the upper and lower chambers across the lipid bilayer.
  • an M protein is introduced to the lower side of the membrane, causing the membrane to deflect upwards into the upper chamber; the nucleic acid core is introduced into the inside cavity of the membrane, and a higher pressure is applied to the lower chamber to expel the synthetic virus into the upper chamber. As the virus is being expelled, the membrane spontaneously closes on the under side to provide a sealed envelope around the captured nucleic acid.
  • the budded synthetic virus can be further processed.
  • an outer protein can be introduced to the virus for incorporation to the outer surface by hydrophobic interactions or specific affinity interactions.
  • the synthetic virus products can be selected for size, e.g., by filtration through a membrane of controlled pore size, or flowing the product past a side channel that receives only particles less than a certain size.
  • the newly synthesized viruses particles can be stored in dry (e.g., lyophilized or spray dried) form, or as a liquid suspension.
  • the particles can be immediately (e.g., before any drying step, freezing step, refrigeration step, holding in container separate from those of the synthesizer hardware; or in less than 12 hours, 6 hours, 1 hour, 15 minutes 5 minutes, or less than 1 minute) introduced to a host cell for production of progeny virions.
  • the synthetic virions are immediately administered (e.g., by injection or inhalation, e.g., as a vaccine) to an animal, e.g., straight from the device in which the synthetic virus was synthesized.
  • the synthetic virus can bind to a host cell and release a nucleic acid encoding production of progeny virions.
  • the product virions can be, e.g., non-viable virus particles, attenuated live viruses, or essentially typical live active forms of a naturally occurring virus.
  • the synthetic virus can infect cells in vitro to produce a desired virus, e.g., an attenuated virus to be administered as a vaccine.
  • the synthetic virus can be ⁇ administered directly to an animal, e.g., producing progeny attenuated or non-viable viruses in the animal that elicit a desired immune response to a microbial pathogen.
  • the synthetic virus has outer proteins with a specific affinity for a particular host cell of interest.
  • the protein outer viral membrane surface specifically interacts with complementary receptors on the host cell membrane.
  • the cell membrane is punctured as the virus membrane fuses with the host cell membrane and the viral core is injected into the host cell's cytoplasm.
  • the core nucleic acid encodes all the elements necessary to reproduce itself, package progeny nucleic acids in a capsid or membrane.
  • the core nucleic acid encodes proteins with activities that promote release of progeny viruses from the host cell by budding or lysis of the host cell.
  • the synthetic virus interacts non-specifically with a host cell, e.g., to enter by endocytosis.
  • a host cell e.g., to enter by endocytosis.
  • Many cells naturally take in resources from the environment or attack non-self particles by engulfing them into the cell. For example, many cells (particularly cells of the reticuloendothelial system and macrophages) will engulf particles they encounter.
  • the synthetic viruses of the present invention can include surface proteins (e.g., opsonization sequences) that trigger endocytosis into a host cell. Once inside the cell the synthetic virus nucleic acids can be translated proteins and/or release enzymes that result in a take over of the host cell metabolism and manufacture of the desired progeny virus particles.
  • Synthetic viruses of the invention can be synthetic viruses prepared according to methods described above.
  • a synthetic virus composition can comprise, e.g., a lipid bilayer capsule, a first protein (typically a transmembrane protein) inserted into the bilayer, a second protein (typically presenting an affinity for a host target surface protein) associated with the outer surface of the bilayer capsule, a third protein (typically a functional M protein) associated with the inner surface of the bilayer capsule, and a viral nucleic acid core encoding production of a live virus or virus like particle.
  • a first protein typically a transmembrane protein
  • second protein typically presenting an affinity for a host target surface protein
  • a third protein typically a functional M protein
  • the invention also encompasses the synthetic viruses generated by the methods presented herein.
  • the invention further encompasses compositions of progeny viruses resulting from infection of host cells with the synthetic viruses.
  • synthetic viruses can be produced to produce
  • a live virus such as a Rotavirus, a Coronavirus, SARS, Norwalk virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Barley Yellow Dwarf virus, Poliovirus, Hepatitis A virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Hepatitis E virus, Tobacco Mosaic virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mubs virus, Nipah virus, Hendra virus, Rabies virus, Lassa virus, Hantavirus, Parainfluenza virus, and Influenza virus.
  • viral pathogens include but are not limited to: adenovirdiae (e.g.,
  • herpesviridae e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6
  • leviviridae e.g., levi virus, enterobacteria phase MS2, allolevirus
  • poxviridae e.g., chordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxvirinae
  • papovaviridae e.g., polyomavirus and papillomavirus
  • paramyxoviridae e.g., paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measles virus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.
  • human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus (e.g., Sindbis virus) and rubi virus (e.g., rubella virus)), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa virus), coronaviridae (e.g., coronavirus and torovirus), and/or the like.
  • flaviviridae e.g., hepatitis C virus
  • the lipid bilayers are synthetic bilayers initially assembled across an aperture of a man made device.
  • the bilayers can include lipid constituents typically found in natural membranes, as well as constituents that are not normally found in natural membranes.
  • the lipid bilayers can be populated with outer, inner and/or transmembrane proteins.
  • the lipid bilayer capsule surrounds a nucleic acid that encodes, e.g., a wild type viral genome, an attenuated virus genome, and/or instructions to generate a non-viable virus or virus like particle.
  • the lipid bilayer typically consists primarily of a thin layer of amphipathic lipids which spontaneously arrange so that the hydrophobic "tail” regions are shielded from the surrounding polar fluid, causing the more hydrophilic "head” regions to associate with the cytosolic and extracellular faces of the resulting bilayer.
  • This forms a continuous, spherical or rod shaped lipid bilayer.
  • the lipids in the membrane can include, e.g., phospholipids, glycolipids, and/or cholesterols.
  • the bilayer can include a phosphatidic acid (PA), phosphatidylethanolamine (e.g., cephalin - PE),
  • phosphatidylcholine e.g., lecithin - PC
  • PS phosphatidylserine
  • phosphoinosi tides such as, e.g: phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP)
  • ceramide phosphorylcholine e.g., sphingomyelin - SPH
  • ceramide phosphorylethanolamine Cer-PE
  • ceramide phosphorylglycerol e.g., ceramide phosphorylglycerol
  • the lipid bilayer can be synthesized to incorporate, e.g., glyceroglycolipids, galactolipids, sulfolipids (SQDG), glycosphingolipids, cerebrosides, alactocerebrosides, glucocerebrosides, glucobicaranateoets, gangliosides, globosides, sulfatides, glycophosphosphingolipids, and/or glycosylphosphatidylinositols.
  • the lipid bilayer will include cholesterol and/or cholesterol derivatives, such as, e.g., cholesterol esters and sterols.
  • the lipids of the bilayer can be from purified sources and formulated in controlled proportions for introduction into the bilayer from a hydrophobic solvent at the aperture, as described above.
  • the lipids can be provided as relatively crude extracts from natural membranes.
  • the synthetic virus preparations of the invention can include virus particles range in size (average diameter) from less than about 50 nm to more than about 1000 nm; from 100 nm to 500 nm; or about 200 nm or less. In many other embodiments, the average virus particle is about 200 nm in diameter, large enough to contain a genomic virus nucleic acid and small enough to pass through standard sterilization filters.
  • At least two proteins are typically integrated into the lipid bilayer membrane of the synthetic virus.
  • the synthetic virus requires a host cell targeting protein on the outer surface and a nucleic acid binding protein on the inside.
  • a transmembrane protein is utilized, e.g., as an anchor in the membrane for attachment of other proteins inside and/or outside the membrane, and as part of a lipid raft system.
  • proteins of the synthetic viruses are routinely discussed herein as separate proteins, a single protein sequence can actually provide multiple functions, such as (nucleic acid binding, anchoring, core scaffolding, modification of membrane topology, raft assembly, and/or target host cell affinity). Alternately, these functions can be provided by two peptides, three peptides, four peptides, or more.
  • a protein typically bound by the methods to the outside of the synthetic virus membrane will have a specific affinity for a host cell surface protein (act as a ligand to a host cell receptor).
  • the outer affinity protein can be independently incorporated into the lipid bilayer through a hydrophobic segment of the protein.
  • the outer affinity protein can be covalently anchored to a transmembrane protein or bound to a transmembrane protein through a mutual affinity interaction.
  • the outer affinity protein can be covalently anchored to a transmembrane protein or bound to a transmembrane protein through a mutual affinity interaction.
  • transmembrane protein can include an exposed (e.g., hydrophilic segment) engineered to have a specific affinity for the host cell.
  • an exposed (e.g., hydrophilic segment) engineered to have a specific affinity for the host cell No matter how the outer protein is bound to the lipid bilayer, it typically includes, e.g., a sequence functioning to provide a specific affinity for the desired host cell or host cell surface protein.
  • a protein exposed on the outside of the synthetic virus can include functional affinity sequences, such as, e.g., ligands, receptors, antigens, antibodies, lectins, antibody domains, nucleic acid binding domains, enzyme binding domains, enzyme substrates, metal chelators and/or the like.
  • the synthetic virus does not have a specific affinity for a specific host cell or specific host cell surface feature. However, the synthetic virus can still contact and fuse with a competent host cell, e.g., through hydrophobic interactions with host cell membranes or by endocytosis.
  • the synthetic lipid bilayer includes at least one transmembrane protein.
  • the transmembrane protein can function to anchor other virus components with the bilayer.
  • the transmembrane protein can associate with other membrane proteins and/or membrane lipids in a lipid raft complex.
  • the transmembrane protein can include at least one hydrophobic segment, and typically at least two hydrophilic segments.
  • the transmembrane function can be engineered based on the transmembrane domains of many well-characterized transmembrane proteins, or based on knowledge in the mature art of protein engineering. For example, transmembrane proteins can be the same as, or engineered from known viral transmembrane proteins.
  • Transmembrane proteins can be, e.g., influenza virus neuraminidase (NA), transmembrane protein R-peptide, MLV transmembrane protein pl5E, foamy virus transmembrane proteins, FTTV-I transmembrane protein gp41, transmembrane protein gp30, and the like, or a derivative thereof having more than 90%, 95%, 98% or 99% identity with such natural transmembrane proteins.
  • NA influenza virus neuraminidase
  • MLV transmembrane protein pl5E MLV transmembrane protein pl5E
  • foamy virus transmembrane proteins FTTV-I transmembrane protein gp41
  • transmembrane protein gp30 and the like, or a derivative thereof having more than 90%, 95%, 98% or 99% identity with such natural transmembrane proteins.
  • transmembrane structures can be found in membrane signaling complexes, neuron signaling proteins and ion transport proteins.
  • functional transmembrane peptide sequences can be found or derived from, e.g., ATP synthase alpha/beta subunits; ATP synthase gamma subunit, ATP synthase subunit C, ATP8B1, Asialoglycoprotein, bacterial antenna complex, rhodopsins, CDl Ic, cadherin, calcium ATPase, chloride channels, cytochrome b, cytochrome b5, cytochrome c oxidase subunit ⁇ , cytochrome c oxidase subunit HI, disulfide bond formation protein B, FXYD6, fumarate reductase, glycophorin, hemolysin, integrin, ion channel families, JAML, Ll protein, lactose permease, leukot
  • photosynthetic reaction center protein family photosystem II light-harvesting protein, the potassium channel tetramerisation domain, proton ATPase, SERCA, SNARE protein, SecY protein, SeI- 12, sodium-hydrogen antiporter, sodium/proton antiporter 1,
  • sodium:neurotransmitter symporter the solute carrier family, sulfatase, syntaxin, the TIM/TOM complex, translocase of the inner membrane, the transmembrane domain of ABC transporters, V-ATPase and/or the like.
  • Protein functions on the inner lipid bilayer surface can include, e.g., binding to the bilayer, binding of the nucleic acid core, and facilitating budding of the bilayer form the aperture.
  • a single protein can include segments providing all these functions.
  • members of the M protein family can have sequences functioning to bind, package and stabilize a nucleic acid, sequences that insert and anchor into the bilayer, and sequences acting to induce a curvature in the bilayer.
  • Peptide segments that bind the bilayer typically have hydrophobic sequences.
  • Peptide segments that bind the nucleic acid typically are rich in positively charged amino acids, such as, e.g., lysine and arginine.
  • Nucleic acid cores typically include a nucleic acid encoding a virus or virus like particle, and one or more proteins functioning to bind and package the nucleic acid.
  • the nucleic acid of the core can be DNA or RNA; single stranded or double stranded.
  • the nucleic acid can be a native natural genomic nucleic acid from a live or attenuated virus.
  • the nucleic acid can be synthesized in vitro.
  • the nucleic acid encodes all proteins necessary to direct production of a desired virus product in a living host cell.
  • the nucleic acid core is simply the functional nucleic acid bound to the charged surface of the lipid membrane.
  • the lipid bilayer can include abundant positively charged, such as, e.g., membranes comprising
  • lipid bilayers can capture a nucleic acid on the inside surface.
  • the nucleic acid core can optionally be formed by an interaction between the nucleic acid and a positively charged protein bound to the inner surface of the lipid bilayer.
  • Viral RNA cores of the invention can include purified viral RNA that is attenuated.
  • the RNA can be complexed to a scaffold protein, such as actin.
  • RNA: scaffold mix can be further complexed with other functional proteins to form an exemplary RNA core.
  • the nucleic acid core is assembled before the nucleic acid is delivered to the inside surface (surface that will be inside the synthetic virus on budding) of the lipid bilayer.
  • the nucleic acid can be previously bound with a nucleocapsid protein (NP) to provide a core before delivery to the lipid bilayer.
  • NP nucleocapsid protein
  • Other core proteins useful in preparation of core structures can include lentiviral core proteins, adeno-associated virus (AAV) core proteins, and retrovirus core proteins.
  • additional proteins are captured or bound within the synthetic virus.
  • the synthetic virus can include enzymes with activities that function to allow the virus to gain entry to the host cell and/or replicate inside the host cell.
  • additional proteins can include, e.g., transcriptase complex, proteases, and reverse transcriptase.
  • the progeny virus products of the methods are encoded by the nucleic acid of the synthetic virus.
  • the progeny virus can effectively present as a normal natural virus.
  • the progeny virus expresses at least one protein that is a recombinant sequence engineered into the synthetic virus nucleic acid.
  • the progeny virus is essentially the same as a pathogenic virus of interest, except it is encoded to express as an attenuated virus.
  • Such progeny virus products can be useful, e.g., in the production of vaccines.
  • Methods of attenuation are well- known in the art - see e.g., US Patents 5,578,473; 5,840,520; 5,820,871; 6,033,886, each of which are incorporated by reference in their entireties.
  • the progeny virus is engineered to be heat sensitive.
  • the virus can fail to replicate at temperatures above 37°C, so that it will not replicate if it causes a fever in a human.
  • the virus can be sensitive to
  • a progeny virus sensitive to temperatures above 30°C, 32°C, or 35 0 C could live in the nose or upper respiratory tract, but not in deeper tissues of the body.
  • the progeny virus is engineered to be cold adapted.
  • the virus can fail to replicate well at temperatures above room temperature, so that it will replicate well in a human upper respiratory tract.
  • the virus can be adapted for optimal growth at 20°C, 25°C, 27 0 C or 3O 0 C.
  • the methods of the invention comprise the production of a live viruses wherein the live virus, e.g., a Rotavirus, a Coronavirus, SARS, Norwalk virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Barley Yellow Dwarf virus, Poliovirus, Hepatitis A virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Hepatitis E virus, Tobacco Mosaic virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mubs virus, Nipah virus, Hendra virus, Rabies virus, Lassa virus, Hantavirus, Parainfluenza virus, Influenza virus and/or the like.
  • the live virus e.g., a Rotavirus, a Coronavirus, SARS, Norwalk virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Barley Yellow Dwarf virus, Poliovirus, Hepatitis A virus,
  • Devices of the invention for preparing synthetic viruses generally include, e.g., an aperture between two compartments and a means to deliver solutions or suspensions to the compartments to interact at an interface at the aperture.
  • the devices can include channels to flow the solutions or suspensions to the interface, and to remove the solutions or suspensions from the interface.
  • the solutions can include, e.g., aqueous buffers to establish the interface at the aperture, solvents to deliver ambiphilic lipids to the interface to form a monolayer, and additional aqueous buffers containing proteins and/or nucleic acids for delivery and binding at the lipid bilayer across the aperture.
  • Microfluidic devices can be used to practice the methods of the invention.
  • an aqueous suspension of transmembrane protein 33 flows from lower microfluidic channel 34 to contact the lower side of the bilayer (optionally, from the upper channel).
  • Hydrophobic segments of the transmembrane protein can be incorporated within the bilayer, e.g., through hydrophobic interactions.
  • a bolus of a ligand protein 35 in aqueous solution 36 can flow through upper microfluidic channel 37 to contact the upper side of the bilayer in the aperture, as shown in Figure 3B.
  • the ligand proteins can interact with the membrane and/or the transmembrane protein (e.g., by hydrophobic or specific affinity interactions) to bind to the upper surface of the bilayer.
  • the upper aqueous layer can be removed by a bolus of air in the upper chamber.
  • a third protein 38 in an aqueous solution can flow from the lower channel to contact the lower surface of the bilayer, as shown in Figure 3C.
  • an aqueous bolus of M protein in solution can flow to contact and bind to the lower side of the lipid bilayer, e.g., through hydrophobic interactions and/or affinity interactions with the transmembrane protein.
  • Flows in the bottom channel can remove the previous solution and introduce a solution containing a RNA core structure 39.
  • the RNA core structure can remain in place to be physically captured in the budding step.
  • the RNA core structure can interact (e.g., through hydrophobic interactions and/or specific affinity interactions) to bind on the under side of the lipid bilayer, where it can be surrounded within the lipid bilayer during the budding process.
  • the present inventions include devices to practice the methods of the invention.
  • a device can have an aperture, or a series of apertures (otherwise known as an array of apertures) suitable for the assembly of synthetic lipid bilayers.
  • Apertures of the invention are typically circular or ovoid across the plane and have a diameter ranging from about 10 nm to 1000 nm, from 50 nm to 500 nm, from 100 nm to 200 nm, or about 150 nm.
  • the device includes two or more apertures, e.g., 2 to more than 10 6 apertures, from 10 to 10 5 apertures, from 100 to 10 4 apertures, or about 1000 apertures.
  • the apertures can be arranged in arrays, e.g., parallel or serial arrays arranged along shared channels or chambers, or having separate dedicated channels providing desired fluid flows to each aperture.
  • the channels are microfluidic channels.
  • the channels preferably include at least one cross-sectional dimension ranging from about 1 nm to 1000 nm, from about 5 nm to 500 nm, from 10 nm to 200 nm, or about 100 nm.
  • the device includes two or more channels configured to deliver fluids to one or both sides of two or more apertures.
  • the devices can include, e.g., 2 to more than 10 6 channels, from 10 to 10 5 channels, from 100 to 10 4 channels, or about 500 channels.
  • the channels can sequentially deliver and remove fluids (solutions, suspensions and/or gasses) to apertures of the invention.
  • the channels can be configured to provide recirculating of the fluids, so they may be reused in sequence.
  • the devices can include chambers, e.g., on each side of each aperture.
  • fluidic channels can widen out adjacent to an aperture to form a chamber next to the aperture.
  • a chamber can be an adjacent channel section that is not widened.
  • chambers can be provided on each side of the aperture, even in embodiments where no flow through channels are provided.
  • the devices include a lipid bilayer traversing an aperture.
  • the lipid bilayers can be as described above in the Methods and Virus sections.
  • the bilayers can be two essentially parallel and planar layers of ambiphilic molecules aligned with more polar (e.g., hydroxyl or phosphate groups) structures at the surfaces, and with lipid "tail" hydrophobic chain structures forming an inner hydrophobic membrane layer.
  • the lipid bilayer can be populated with surface and/or transmembrane proteins.
  • the devices include one or more affinity proteins associated with the lipid bilayer.
  • the affinity proteins can be as described above.
  • the affinity proteins can include surface or transmembrane proteins with affinities for, e.g., a host cell surface receptor.
  • Other affinity proteins associated with the bilayer can be proteins having affinity for a transmembrane protein (allowing the protein to be anchored at the bilayer), proteins having an affinity for a nucleic acid or nucleic acid core (binding the nucleic acid core to the bilayer).
  • the devices can include nucleic acid core structures, e.g., as previously described herein.
  • the nucleic acid core can be an RNA core structure.
  • the core structure can include at least one protein with a binding interaction (typically a ionic interaction) with the nucleic acid.
  • the core structure can include a protein (which can be the same protein as the nucleic acid binding protein) that acts as a scaffolding to compactly package and stabilize the nucleic acid, e.g., in a tight capsid form.
  • the core structure can include one or more proteins that has a functional affinity interaction with another protein bound on the inside of the lipid bilayer, thus capturing and binding the core on the lipid bilayer (this protein can be the same protein providing the binding and/or scaffolding functions).
  • Biological materials are placed over or under an aperture by controlled injection.
  • the first step (STEP 1) requires the micro-injection of a phospholipid membrane preparation through the upper channel over an already existing water phase.
  • Transmembrane (TM) protein units are already present in the water phase and/or pre-included in the lipid preparation.
  • the lipophilic nature of the TM units permits a direct spontaneous incorporation.
  • the orientation of the TM elements can be modulated by adjusting the environment to cytoplasmic pH values or alternatively, by modifying the TM primary amino acid sequence.
  • the second step (STEP 2) consists of the micro-injection of the ligand (L) proteins in the upper phase.
  • the upper phase is used to populate the exposed surface of the synthetic virus with various populations of L proteins.
  • the introduction of the liquid upper phase will hypothetically reconstitute the lipid bilayer.
  • the approximate height of the lipid bilayer is 5 nm.
  • the lipid bilayer is estimated to be resistant to microchannel pressure up to 100 Pa.
  • the third step of assembly simulates the surface budding mechanism of a virus. Removal of the upper phase prevents membrane curvature. Use of end-to-end pressure may also achieve the same effect ( ⁇ P pressure (air or water) > 0) can prevent premature curvature of membrane.
  • ⁇ P pressure air or water
  • M matrix
  • the fourth step (STEP 4) consists of the injection of a RNA pre-assembled core.
  • the RNA core is prepared previously in a separate reaction. Diffusion and attachment of RNA core to M proteins obeys the same kinetic rules as for small proteins. The introduction of the upper phase layer and a pressure of ⁇ P ⁇ 0 fully allows membrane curvature at this final step.
  • a microfluidic process simulation permitted us to gather the physical parameters of a microfluidic virion assembly system early in the system design.
  • LAIV Physical parameters and system design.
  • the design of a LAIV should take into consideration industrial production standards, such as requirements that therapeutics and vaccines be compatible with sterile filtration, e.g., through 0.2 ⁇ m pore filter. While full aseptic procedures would allow the production of higher molecule size (> 200 nm diameter), we believe it would be economically more suitable to have a product that can be sterile filtered.
  • the amount of lipid required to prepare a virus can be estimated from a simple surface equation. Assuming a diameter of 0.2 ⁇ m, the virus surface would be 0.126 ⁇ m . This value can be used to define the size of the aperture at the interface of the two layers. An additional 20% surface area can be included in the aperture in order to adjust for any product loss occurring at the budding and pinching reaction. Optimal aperture size would therefore be in the order of about of 0.15 urn 2 . Control of virus dimensions can be enforced by using specific aperture values as long as the virus diameter is lower than 0.2 um. Suzuki et al. (2004) described a microchannel design wherein apertures are fabricated and aligned along the tubular section of a channel.
  • D is the diffusion coefficient
  • k is the Boltzmann's constant (1.38E23 JK "1 )
  • T is the absolute temperature
  • is the viscosity of the fluid and r the particle radius
  • t is the time of diffusion
  • L the path length of the molecule.
  • the volume of the reaction chamber can also be modulated, but could be in theory of infinite size (assuming a constant molecule concentration) as long as the particle concentration at the lipid bi-layer interface is unchanged.
  • the budding mechanism can be substantially influenced by the aggregation of a limited but unknown amount of M protein and the virus volume is also influenced by to the amount of M protein and the amount and type of lipid molecules.
  • Re is the Reynold number
  • Q the volumetric flow rate
  • D the hydraulic diameter of a pipe
  • v the kinematic viscosity
  • A the pipe cross-functional area.
  • microchannel flux could be increased up to 1.5 mL/min while still maintaining the Re value in the laminar flow range definition (Re ⁇ 4000).
  • the liquid phase replacement will therefore not disrupt the lipid bilayer interface.
  • V z ,max ( ⁇ P/4 ⁇ l)R 0 2 (Equation 4)
  • RO is the diameter of the microchannel
  • 1 is the path length
  • ⁇ P is the pressure difference
  • is the dynamic viscosity
  • Figure 6 shows the theoretical failure pressure point based on lipid bilayer thickness. Based on the maximum stress value on a bilayer membrane (Kusube et al., Colloids Surf B Biointerfaces 42: 79-88, 2005) and the poisson's ratio value of 0.0001 Mpa and 0.3, respectively, the failure pressure point can be assessed in Pascal. At aperture diameters lower than 80 ⁇ m, the pressure that can be used in the microchannel system and at the site of reaction will increase with the bilayer thickness. An increase from 5 to 10 nm thickness in the lipid bilayer will result in more than 5-fold increase in pressure resistance. This estimation shows that high throughput assembly is possible and can be modulated at different level. Yamada et al.
  • lipid, protein, RNA A major challenge of the synthetic assembly platform is the availability of component material in the appropriate format and quality (lipid, protein, RNA).
  • Functional lipid bi-layers have also been fabricated diphytanyl phosphatidylcholine (DPhPC).
  • DPhPC diphytanyl phosphatidylcholine
  • DPhPC is a molecule which has ether linkage instead of ordinary ester one between the hydrophobic chains and the glycerol backbone. While various lipid solutions are often available and well characterized as to hydrophobic characteristics, viral proteins and RNA are typically less available and characterized. Nucleic acids specific to a virus of interest often need to be produced independently.
  • Various biological protein production methods can be used to generate the M protein, TM, ligand and core viral proteins in single or separate reactions.
  • Synthetic or biologically produced proteins and nucleic acids usually need to be purified in order to reach the desired quality and minimize the introduction of undesired proteins into synthetic virions, particularly for use in vaccines or therapeutics for administration to humans.
  • cell-free protein synthesis will be feasible for commercial protein production and could even be integrated directly into the microchannel system.
  • Simple scale-up technologies for synthetic protein production have already being developed (Swartz, J. Ind. Microbiol. Biotechnol. 33: 476-485, 2006) but biological methods are still more economical.
  • RNA segmentation of the influenza virus adds a level of difficulty as RNA packaging with proteins will be more complex.
  • RNA viruses by replacing the genetic template by non-segmented RNA or plasmid DNA.
  • RNA cores should preferably condense the RNA and protein to an aggregate smaller than 100 nm in diameter.
  • a plasmid DNA template could be used as a simpler alternative to package the necessary genetic information, as an alternate over RNA encoding, without the help of any starting protein material.
  • LAFV assembly chips can contain all the raw material necessary for the construction of the synthetic virus. An appropriate amount of raw material could be inserted in distinct compartments on the chip to produce the exact dose required for the patient.
  • a microfluidic apparatus can enable LAFV assembly on a microchip stored at 4 0 C. It is also possible that these chips be reduced in size as they will be able to handle volumes in the range of the femtolitre, e.g. the size of a virus.
  • the first testing consisted of diluting a 10Ox concentration of CDLC down to 50x, 10x, 5x, and Ix and processing these samples through the Microfluidizer. However, due to time constraints, only the 5Ox experiment was completed. This experiment was done on the M-11OP Microfluidizer processor at 30,000 psi for two passes with the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration.
  • the second set of tests was performed by varying the ratio of two lipids, PC and PE, in DI water or WFI and processing with the M-11OP Microfluidizer processor. Samples were mixed with stir bars until the lipid powder was no longer visible in the mixture.
  • the third set of tests consisted of replacing the DI water or WFT with one of three different protein medias: BRlO-VLPOl Medi559 (17MarlO - #3), RSVsF CHO 8D4.1.6 Clone #15 (14DecO9), or RSVsF CHO 8D4.1.7 Clone #16 (14DecO9). Samples were mixed with stir bars until the powder was no longer visible in the mixture.
  • Table 1 Listed below are the processing conditions and the resulting particle size measurements of the first set of experiments with 50x concentration of CDLC. All samples were processed on the M-11OP Microfluidizer processor with the Fl 2 Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration at 30,000 psi.
  • Table 2 Listed below are the processing conditions and the resulting particle size measurements of the second set of experiments with PC and PE in DI water or WFI. The weight of the water in all the formulations was 100 g. All samples were processed on the M-11OP Microfluidizer processor with the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC. The experiments highlighted in green were the most successful and the basis for the third set of experiments.
  • Table 3 Listed below are the processing conditions and the resulting particle size measurements of the third set of experiments with PC and PE in protein media. The weight of all the protein media samples was 200 g. All samples were processed on the M- 11OP Microfluidizer processor with the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC. Post processing analysis by Medlmmune will determine the success of these experiments. [0142] Comments:
  • particle size is a strong function of the components in the formulation. Since these formulations had not been finalized, further improvements towards the particle size goals can be achieved with further research and testing.
  • a gas removal step may be required for formulations containing MPLA-PHAD prior to processing with the Microfluidizer processor.
  • APM Auxiliary processing module/ interaction chamber used as either a pre-processing chamber for a solid dispersion application, or as a backpressure module to create a backpressure for Y-chamber applications.
  • CDLC Chemically Defined Lipid Concentrate, a lipid from Gibco.
  • DLS Dynamic light scattering.
  • IXC Interaction chamber; a cylindrical module with a specific orifice and channel design thru which fluid is conducted at high pressures to control shear rates.
  • PC L-a-Phosphatidylcholine, a lipid from Avanti Polar Lipids, Inc.
  • PE L-a-Phosphatidylethanolamine, Transphosphatidylated, a lipid from
  • SLS Static laser scattering.
  • Figure A1 Shown above is the particle size distribution of the 5Ox concentration CDLC, before (dotted) and after (solid) processing. The processed sample experienced two passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration.
  • Figure B1 Shown above is the particle size distribution from Experiment B. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. Size Distribution by Intensity
  • Microfluidizer processor at 20,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. Size Distribution by Intensity
  • Figure B3 Shown above is the particle size distribution from Experiment D. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 20,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. The last sample (black) experienced the first three passes at 20,000 psi and the final pass at 30,000 psi. Size Distribution by Intensity
  • Figure B4 Shown above is the particle size distribution from Experiment E. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 20,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. The last sample (blue) experienced the first three passes at 20,000 psi and the final pass at 10,000 psi. Size Distribution by Intensity
  • Figure B5 Shown above is the particle size distribution from Experiment F This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 20,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. Appendix C
  • Figure C1 Shown above is the particle size distribution from Experiment G. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration.
  • the first sample (red) was the
  • Figure C2 Shown above is the particle size distribution from Experiment H. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. Size Distribution by Intensity
  • Figure C3 Shown above is the particle size distribution from Experiment I. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. The last sample (black) experienced the first three passes at 20,000 psi and the final pass at 10,000 psi. Size Distribution by Intensity
  • Figure C4 Shown above is the particle size distribution from Experiment J. This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration. Size Distribution by Intensity
  • Figure C5 Shown above is the particle size distribution from Experiment K This sample was processed for various passes on the M-110P
  • Microfluidizer processor at 30,000 psi through the F12Y (75 ⁇ m) - H30Z (200 ⁇ m) IXC configuration.

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Abstract

This invention provides synthetic viruses, and methods and devices to prepare synthetic viruses. In the methods, a lipid bilayer membrane is established at an aperture between tow compartments. Fluids contact the membrane to deliver proteins and a nucleic acid core to the membrane before budding. The devices include a lipid bilayer across an aperture with an affinity protein on one side and a nucleic acid core bound to the other side.

Description

METHODS OF MAKING AND USING SYNTHETIC VIRUSES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of a prior U.S. Provisional
Application number 61/222,570, METHODS OF MAKING AND USING SYNTHETIC VIRUSES, by Philippe-Alexandre Gilbert, filed July 2, 2009. The full disclosure of the prior application is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to synthetic viruses, and methods and devices for making and using such synthetic viruses. Synthetic viruses can be fabricated by introducing internal, external and transmembrane proteins to a lipid bilayer before budding of the bilayer to capture a core nucleic acid assembly forming a viable synthetic virus particle.
BACKGROUND OF THE INVENTION
[0003] Live attenuated viruses have long been used successfully to eradicate infectious disease and protect humans against viral infection. Many current vaccines are produced by growing the virus in vivo, e.g., egg-based live attenuated Influenza vaccine (LAIV) or on mammalian host cells. However, this presents problems in removal of unwanted residual host cell DNA and protein.
[0004] Live attenuated Influenza vaccines are currently produced, entirely from viruses inoculated and grown in the allantoic cavity of 9- to 11-day-old embryonated chicken eggs. Egg production presents enormous challenges related to its scalability, safety, product consistency and cost-effectiveness. For example, manufacture involving eggs is not sufficiently flexible to allow vaccine supplies to be rapidly expanded, especially in the face of an impending pandemic such as the one experienced in 2009 with the Swine flu. Other problems arise from the infections of progenitor flocks that adversely affect egg supplies, and from the manufacturing process itself, where breakdowns in sterility can contaminate large batches of viral allantoic fluid. In addition, egg-grown viruses exhibit differences in antigenicity from viruses from isolated clinical specimens or grown in mammalian cell lines. These concerns and the probable need for increased manufacturing capabilities have been brought into focus in recent years (Mayrhofer et al., J. Virol. 83: 5192-5203; 2009).
[0005] Because of these methodological and operational hurdles, the virus production industry has attempted to shift from egg environment to cultured cell
environment. Alternative approaches involving the use of anchorage-dependent and anchorage-independent preparations of the African Green monkey kidney (Vero), Madin- Darby canine kidney (MDCK) and other cell lines have been pursued by multiple companies in recent years. Yields comparable to those obtained in embryonated eggs have been achieved. These improvements have occurred in parallel with newer technologies that allow the growth of cells in synthetic media that do not contain animal serum, in order to allay the concerns of regulators about the potential for spread of various contaminants such as transmissible spongiform encephalopathy (BSE). While cell production may be useful in production of protein-based vaccines, virus yield has continued to be a problem, e.g., in the field of attenuated live virus vaccines. Although the cell-based processes offer significant technical advantages over the traditional egg-based processes, it remains challenging to significantly reduce residual host cell DNA and proteins, control the product heterogeneity and minimize the process variability. Production of live virus still requires an enormous effort of characterization and safety as virus amplification relies either on eggs or cell based products. Despite the broad spectrum of applications, all the described platforms depend solely on cellular replication to produce either protein or virus antigens.
[0006] A major problem in conventional antigen production is the pressing need for higher yields. Scale up of egg harvesting and improvements in viral titers can only go so far. The industry is compelled to consider fundamentally different virus production processes. Some advances have been made in production of synthetic antigen carriers, but the technologies remain impractical for commercial vaccine production. Multiple variations on the making of virus-like particles (VLP) and pseudoviruses (PSV) have been generated in the past decades. The current market demand for these technologies is primarily vaccination and gene therapy. Immune clearance is an issue tentatively addressed by creating new chimeras with different antigen presentation properties. Despite of the advances in VLP & PSV technology, there is currently no synthetic platform available for the making of infectious viruses. Moreover, assembly of lipid viruses from virus parts with the intention to regain infectivity has not yet been demonstrated. Reports of spontaneous in vitro virus protein-RNA complexes have been reported. However, such processes are inefficient and still present purification issues.
[0007] Accordingly, there is a need to develop methods to produce synthetic viruses capable of delivering a payload of genetic material to direct the production of live attenuated viruses in a host cell. It would be desirable to have a means to produce such synthetic viruses in large amounts. A defined composition synthetic virus could provide substantial benefits in the realm of live attenuated vaccines. The present invention provides these and other features that will be apparent upon review of the following.
SUMMARY OF THE INVENTION
[0008] The present inventions include synthetic viruses, methods of producing synthetic viruses, and devices for fabricating synthetic viruses. The synthetic viruses can be fabricated by establishing a bilayer lipid membrane across an aperture, introducing a transmembrane protein to anchor other membrane components, introducing an affinity protein to a first side of the membrane to target the virion to the desired host cell, introducing an M protein to the second side to facilitate budding of the membrane, and introducing a nucleic acid core encoding a viable attenuated virus to the second side of the membrane. Budding of the membrane from the aperture can thus capture the nucleic acid core to provide a functional synthetic virus. The synthetic virus can comprise a nucleic acid core encoding a virus and surrounded with a lipid bilayer membrane incorporating an outer protein directing the virus to a host cell. Devices for production of the synthetic virus can include an aperture supporting a membrane between two chambers, and means to deliver proteins and a nucleic acid core to either side of the membrane.
[0009] The methods for preparing a synthetic virus can include, e.g., forming a synthetic lipid bilayer across an aperture separating a first chamber from a second chamber; delivering a first protein to the lipid bilayer from the first chamber, wherein the first protein associates to bind with the lipid bilayer or with a second protein embedded in the lipid bilayer; delivering a third protein to the lipid bilayer from the second chamber, wherein the third protein associates with the lipid bilayer or with the second protein; delivering an RNA core structure to the lower chamber; and, budding the lipid bilayer from the second chamber into the first chamber to form a liposome enclosing the RNA core structure and comprising the first protein on an outer surface of the liposome. Budding of the liposome from the aperture can produce the synthetic virus wherein the first protein, on the outside of the of the synthetic virus, specifically binds a host cell surface receptor to direct delivery of the RNA core into the cell where the RNA is transcribed and translated to produce live virus from the host cell. The synthesis of the virus is typically performed entirely in vitro, thus dramatically reducing purification issues. The synthetic virus can be placed in storage until used, or the synthetic virus can be administered immediately after budding is completed.
[0010] In other embodiments, the second protein is different from the first protein and also different from the third protein. The second protein can be delivered to the lipid bilayer from the first chamber or from the second chamber. The RNA core structure can optionally bind with the third protein and/or with the lipid bilayer. In some cases, the proteins and core interact through specific affinity interactions. In certain embodiments, the lipid bilayer comprises lipid rafts, e.g., enriched in cholesterol, sphingolipids, and/or membrane proteins.
[0011] In some embodiments, the quantity or variety of synthetic viruses produced can be increased by budding the liposomes from an array of apertures. For example, the array apertures are arranged in series along a channel and/or arranged in two or more parallel channels.
[0012] The methods can introduce a number of different functional proteins to the membrane. For example, the second protein can be a transmembrane protein, e.g., providing an affinity target on one or both sides of the membrane. The transmembrane protein can display an affinity for the first protein (e.g., outer affinity targeting protein), for the third protein (e.g., M protein) and/or for the RNA core. The outside exposed surface protein can be an affinity protein, such as, e.g., a viral receptor, a cell surface antigen, a host cell surface protein, a cell surface ligand, and/or an antibody variable domain. The third protein, on the inside surface of the membrane can be, e.g., a member of the M protein family or homologous sequence, e.g., having a segment with an affinity for a target determinant on the RNA core structure.
[0013] The when the internal proteins and nucleic acid core are bound to the membrane, the membrane can be budded from the aperture to enclose the core. Budding can be induced by applying a force across the membrane, e.g., an osmotic pressure differential, a hydrostatic pressure differential, a voltage differential and/or the like.
Optionally, budding can be induced when an adequate amount of an M protein has been applied to the lipid bilayer. In many cases, budding is facilitated by application of M protein to one side of the membrane, and initiated or completed by application of the pressure differential across the membrane.
[0014] The nucleic acid core structure (e.g., RNA core) can include, e.g., RNA encoding a virus, a scaffold, and RNA binding proteins. The scaffold proteins can provide structure and ligand binding targets complimentary to a protein bound to the inner side of the membrane. The RNA binding proteins a positive charge to stabilize and condense the nucleic acid in the core, and can also can have scaffolding protein functions.
[0015] The synthetic virus product of the method can contact an appropriate host cell to initiate replication of multiple copies of a progeny virus encoded by the nucleic acid of the synthetic virus. The progeny viruses can be non-viable (e.g., but have desirable antigenic properties), can be attenuated live viruses, or can appear as a typical
representation of a normal naturally generated virus. In other embodiments, the progeny virus is a live attenuated virus, such as, e.g., a paramyxovirus, a pnemovirus, an
orthomyxovirus, a retrovirus, a morbillivirus and/or the like.
[0016] The methods of synthesizing virus particles can optionally include steps to control the average particle size of the particles. For example, the particle size can be adjusted by selective filtration of the bedded particles. In one embodiment, the synthetic liposomes flow from the first chamber through a "Y" channel having two asymmetric flow rates to create a pinched flow wherein smaller liposomes are fractionated from larger liposomes. [0017] The present invention includes synthetic viruses produced by the methods of synthesizing viruses, described herein. The synthetic virus is other than a virus produced in a living host cell. Other embodiments include sterile compositions of the synthetic virus, e.g., sterile filtered taking advantage of the small size (e.g., 200 nm or less diameter) of the synthetic virus particles. The synthetic virus can include, e.g., a liposome, a first protein associated with an outer surface of the liposome, a second protein inserted into a membrane of the liposome, a third protein associated with an inner surface of the liposome and a viral RNA core inside the liposome. The synthetic virus can functionally direct production of a live virus when the liposome is delivered to a host cell. In many embodiments, the synthetic virus has a lipid bilayer comprising phosphatidylcholine (PC ) and/or
phosphatidylethanolamine (PE). In some embodiments, the synthetic virus is attenuated, cold adapted and/or temperature sensitive. In another embodiment, the synthetic virus is characterized by an average diameter of 0.2 μm or less, or 0.1 μm or less, or 0.05 μm or less.
[0018] Devices to prepare the synthetic viruses are included in the present inventions. The device can include, e.g., a first chamber, a second chamber, an aperture between the first chamber and the second chamber; a lipid bilayer traversing the aperture between the chambers. The bilayer can have an affinity protein associated with the lipid bilayer on a first bilayer side facing the first chamber and a RNA core structure associated with the lipid bilayer on a second bilayer side facing the second chamber. The device can include an array of apertures, and the apertures can have a diameter ranging from, e.g., 10 nm to 250 nm, or from 10 nm to 100 nm, or from 10 nm to 75 nm, or from 10 nm to 50 nm, or from 50 nm to 75 nm, or from 75 nm to 100 nm, or from 100 nm to 150 nm, or from 150 nm to 250 nm.
DEFINITIONS
[0019] Unless otherwise defined herein or below in the remainder of the
specification, all technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. [0020] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular devices or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a component" can include a combination of two or more components; reference to "lipid" can include mixtures of lipids, and the like.
[0021] Although many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, the exemplified materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
[0022] As used herein, the term "synthetic virus" with regard to a virus refers to a virus that is fabricated in vitro, as taught herein. A virus assembled in a host cell, or by budding from the surface of a living host cell, is not considered a synthetic virus. The mere presence of synthetic (man made) constituents in a virus does not in itself make the virus a synthetic virus of the present inventions. Synthetic viruses of the invention include a synthetic lipid bilayer is, e.g., a lipid bilayer fabricated in vitro, as taught herein.
[0023] As used herein, the terms "binds" and "binding" refer to attachment of one component with another. The attachment can be by an affinity interaction between the components, binding based on non-specific interactions or can include covalent bonding. Typically, in the present synthetic virions and methods of their fabrication, binding between protein constituents is due to specific affinity interactions, while binding between proteins and lipid bilayers typically comprises hydrophobic interactions between certain protein segments and lipid components of a membrane.
[0024] As used herein, the term "associates" refers to a physical interaction between two or more components, tending to hold them in contact. For example, components of a synthetic virus can associate through covalent bonding, ionic interactions, hydrophobic interactions, specific affinity interactions and/or the like. [0025] As used herein, the term "RNA core structure" includes at least an RNA strand encoding components of a virus and at least one protein binding the RNA, typically providing a more compact structure than the RNA alone. For example the RNA core structure can include encoding RNA and a positively charged RNA binding protein.
Further, the core (or liposome interior) can include an enzyme, such as a polymerase. A protein of the core can include a segment with an affinity for a membrane protein, e.g., so that the core will bind to the surface of the membrane.
[0026] As used herein, the term "affinity", e.g., with regard to interactions between synthetic virion components, refers to attractive interactions that bind one component to another. Affinity can be specific, e.g., as in the classic lock and key models described for affinity interactions, e.g., between antigen and antibody, or receptor and ligand. Affinity can be non-specific, e.g., with components associating non-specifically through attractive hydrophobic interactions or ionic interactions. For example, a lipid bilayer can have a nonspecific affinity for segments of a protein rich in hydrophobic amino acids (e.g., the inserted segments of a transmembrane protein).
[0027] As used herein, the term "attenuated", with regard to a virus, refers to a viable virus with reduced virulence. For example, an attenuated virus in a vaccine is still viable (not "killed"), stimulating an immune response and creating immunity but not causing significant pathology.
[0028] A "segment" of a protein, as used herein, is a linear portion of the protein amino acid chain. A hydrophobic segment typically includes peptide sequences enriched in hydrophobic amino acids, such as, e.g., Ala, VaI, De, Leu, Met, Phe, Tyr, and Tip.
Hydrophilic segments typically include peptide sequences rich in polar or ionized amino acids, such as, e.g., Lys, Arg, His, Asp, GIu, Ser, Thr, Arg and GIt. Segments can have amino acid sequences providing specific affinity, e.g., by expressing ligand or receptor functions.
[0029] In one aspect, the invention can include proteins that are "derived from" a parental molecule. As used herein, the term "derived from" refers to a component that is isolated from or made using a specified molecule or organism, or information from the specified molecule or organism. For example, a polypeptide that is derived from a second polypeptide can include an amino acid sequence that is identical or substantially similar to the amino acid sequence of the second polypeptide. In the case of polypeptides, the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis. The mutagenesis used to derive polypeptides can be intentionally directed or intentionally random, or a mixture of each. The
mutagenesis of a polypeptide to create a different polypeptide derived from the first can be a random event, e.g., caused by polymerase infidelity, and the identification of the derived polypeptide can be made by appropriate screening methods. Mutagenesis of a polypeptide typically entails manipulation of the polynucleotide that encodes the polypeptide.
[0030] A "matrix protein", as used herein, refers to a protein matrix protein (Ml) of influenza virus, homologous proteins, other proteins of similar function from other viruses, such as the matrix proteins of respiratory syncytial virus (RSV) or proteins at least 90% identity, or 95% identity, or at least 98% identity to the influenza Ml protein. M proteins are typically bifunctional proteins that mediate the encapsidation of RNA-nucleoprotein cores into the membrane envelope. M proteins typically bind both membrane and RNA simultaneously e.g., acting as both a nucleic acid binding protein and as a modifier of membrane topology). The X-ray crystal structure of the N-terminal portion of type A influenza virus Ml— amino acid residues 2-158— has been determined at 2.08 A resolution. The protein forms a dimer. A highly positively charged region on the dimer surface is suitably positioned to bind RNA while the hydrophobic surface opposite the RNA binding region is involved in interactions with the membrane. The membrane-binding hydrophobic surface could be buried or exposed after a conformational change.
[0031] Homology of proteins and/or protein sequences can be the result of derivation from a common ancestral protein or protein sequence. Homology can be inferred, e.g., from structural and functional characteristics and from the percent identity of a putative homologous protein of homologous region of a protein. That is, homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity (percent identity) between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.
[0032] Directions given with reference to devices of the invention are to be construed according to their common meaning. However, because gravity has no substantial influence on the flow characteristics and interactions of the devices, terms "upper" and "lower" can typically be exchanged while retaining the function being described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figures IA to IE describe a step by step assembly process of producing a synthetic virus from a lipid bilayer at an exemplary aperture. Figure IA shows a schematic diagram of a synthetic lipid bilayer formed in an aperture. The upper and lower compartments are also exemplified. Figure IB represents the delivery of various components to the synthetic lipid bilayer. The first (TM) and second (X) proteins are delivered via the upper chamber while the third protein (M) is delivered to the bottom chamber. Figure 1C represents the putative associated of the various proteins in a synthetic lipid bilayer. Figure ID represents the delivery of the RNA core to the bottom chamber. Once the RNA core is delivered, the synthetic virus is ready to be formed. Figure IE represents the budding process to produce a functional synthetic virus, which can deliver a functional RNA cargo capable of directing virus production and replication.
[0034] Figure 2 is a schematic diagram of an exemplary microfluidic device configured to generate a synthetic virus. A pair of microfluidic channels are separated by an aperture of controlled size. The microfluidic channels permit the transport of various fluid phase units and their content to either side of the aperture. The fluid phase units can sequentially be introduced in the upper and lower chambers. Control of pressure at the micro scale can permit precise handling. Evaporation and gravity are considered negligible in this environment. Air phase separating of the liquid is possible and permits washing and segregation of different components. [0035] Figure 3A to 3D are schematic diagrams showing a fluidic device and process of synthesizing a synthetic virus.
[0036] Figure 4 shows a schematic diagram of constituents of the RNA core and its assembly. Firstly, the RNA is complexed with a scaffold, such as actin. The RNA: scaffold mix can be further complexed with certain functional proteins (e.g., having affinity for a membrane protein) to form an exemplary RNA core.
[0037] Figure 5 is a chart of Reynold number versus Q volumetric flow showing the effect of Reynold number of flow through microchannels of various diameters.
[0038] Figure 6 is a chart of pressure versus aperture diameter showing theoretical failure pressure point based on lipid bilayer thickness.
DETAILED DESCRIPTION
[0039] The present inventions include synthetic viruses, methods of fabricating synthetic viruses, and devices configured to practice the methods of synthesizing synthetic virions. The methods generally include preparation of a lipid bilayer across an aperture; introduction of protein structures on and into the bilayer; application of a viral genome- encoding nucleic acid to a surface of the bilayer; and, budding the bilayer into a liposome, thus, capturing the nucleic acid in a synthetic virus. The synthetic viruses typically comprise a spherical or rod-like lipid bilayer membrane traversed with a transmembrane protein; an affinity protein (specific for a host cell receptor) bound to the outer surface of the membrane; a third protein bound to the inside surface of the membrane and typically influencing the membrane curvature and having affinity for a core structure; and, a nucleic acid core structure inside the membrane and comprising at least one nucleic acid binding protein that tends to compact the nucleic acid. The devices include at least an aperture between a first and second chamber. The device is configured to provide an interface to • establish a lipid bilayer and populate the bilayer with the protein and nucleic acid components of a synthetic virus.
[0040] A difficulty in the creation of synthetic viruses is the establishment at the micro scale level of a stable lipid bilayer that will encompass an RNA core and produce an infectious virus unit. Common liposome technologies typically rely on bulk micelle formation by agitation or shear to provide a bilayer membrane capsule (liposome) of phospholipids entrapping a soluble component within. Such methods can result in capture of small size plasmid vectors or RNAi segments, proteins, chemical drugs within the liposomes. However, insertion of a full length viral RNA into liposomes capable of a productive infection does not result. In a simple mixed solution system, entrapment of large size nucleic acids (> 15 kb) into liposomes has not been shown. A more controlled assembly of particles, e.g., employing affinity interactions between the core and lipid shell components, is required to provide viable particles of the proper size. Further, we propose use of a flat two-dimensional lipid bi-layer stabilized between two controlled buffer zones creates a better template for virus assembly. Such a template has the advantage of allowing sequential micro-injection of assembly materials in predetermined steps, providing the necessary control in preparation of synthetic viruses.
[0041] We have devised a synthetic virus assembly platform based on the lipid-raft virus budding mechanism. Mimicry of the virus budding mechanisms can lead to the establishment of a set of functional micro-reactions enabling the assembly of functional synthetic viruses. Membrane lipid rafts are defined as cholesterol and sphingolipid-rich microdomains in the exoplasmic leaflet of the cellular plasma membrane (Laliberte et al, J. Virol. 81: 10636-10648; 2007). The tight packing of cholesterol molecules among the saturated fatty acid tails of sphingolipids creates a local membrane organization, referred to as "liquid-ordered", which possesses properties different from those of the surrounding bulk plasma membrane. Specific proteins, such as raft-organizing, acylated, and
glycosylphosphatidylinositol anchored proteins, partition into these domains, resulting in their concentration into patches within the plasma membrane. Membrane lipid rafts usually function in membrane trafficking events and cell-cell attachment and serve as signal transduction platforms in many cell types. Membrane lipid rafts are known to be sites of virus assembly and release based on the fact that lipid raft-associated molecules are found in purified virions and that lipid rafts are cholesterol and sphingolipid-enriched microdomain found in cell membranes. Viral proteins will assemble at the base of the lipid-raft micro- domain to be specifically captured in the virus budding mechanism. [0042] Key components of virus budding mechanisms are matrix (M) proteins, which can independently provide for lipid-based virus like particle (VLP) formation (Gόmez-Puertas et al., J. Virol: 74, 11538-11547; 2000) from a lipid bilayer. In flu viruses, proteins encoded by certain M segments appear to be involved in modulating filamentous versus spherical virion morphology (Hughey et al., Virology 212: 411-421; 1995). The M protein, when expressed alone, assembles bilayers into VLPs, which are released into the culture medium. The M protein operates to impose a curvature on the cell membrane. M protein budding formation is virus dependent and will occur either in specific high cholesterol rich regions or else in non-lipid raft membranes. Initiation of bud formation requires outward curvature of the plasma membrane, which can be stimulated by the accumulation of the M protein at the inner leaflet of the lipid bilayer. The virus bud is then extruded until the inner core is enveloped. The budding process is completed when the membranes fuse at the base of the bud and the enveloped virus particle is released following fission from the cell membrane. The M protein is the driving force behind this process because budding from the host cell cannot occur in the absence of this protein, yet it alone can induce the formation of virus-like particles under these conditions. The M protein also contains a late domain that has been identified in several viral matrix proteins and is responsible for recruiting host factors required for the late stages of viral budding. The basic residues in this domain have also been implicated in RNA binding. The extent to which the membrane is extruded before pinching off occurs affects the size and shape of the virus particle. Generally, influenza virus particles are either spherical or filamentous, and this characteristic morphology is genetically linked to the M segment.
[0043] It appears that the M protein induced membrane curvature is not dependent on the presence of cholesterol in the cell membrane. Based on the main characteristic of the classical virus assembly mechanism, we propose that the steps to assemble a synthetic virus should be composed of sequential events related to: the creation of a lipid bilayer, the insertion of viral surface protein, the association of surface protein with matrix (M) protein, and the entrapment of viral core RNA. By decomposing and reducing the viral assembly steps to a minimal amount, a high throughput platform can be used to repetitively assemble substantial numbers of synthetic viruses. METHODS OF FABRICATING SYNTHETIC VIRUSES
[0044] Methods to assemble a synthetic virus are described herein to generate synthetic viruses containing a genetic payload capable of directing the production of virus in a host cell.
[0045] In some embodiments, methods of the invention comprise:
a) formation of a synthetic lipid bilayer in a device comprising an aperture wherein the bilayer across the aperture separates an upper chamber from a lower;
b) delivery of a first protein to the bilayer wherein the first protein comprises a lipophilic segment that inserts into or across the bilayer;
c) delivery of a second protein to the upper chamber wherein said second protein associates with the lipid bilayer and/or the first protein; d) delivery of a third protein to the lower chamber, wherein the third protein associates with the lipid bilayer and/or the first protein;
e) delivery of an RNA core structure comprising viral RNA, RNA binding proteins, and a lipophilic component (and/or specific affinity component) to the lower chamber; and,
f) inducing budding from the aperture from the lower chamber; whereby a liposome is released into the upper chamber comprising the lipid bilayer with the second protein on the outer surface, and the third protein and RNA core on the inside of the liposome. Such a liposome can be a synthetic virus capable of specifically delivering the RNA core, by an affinity interaction between the second protein and a host cell receptor, to a host cell where the RNA can instruct production of a live non-synthetic virus product.
[0046] In some embodiments, methods of the invention comprise forming a synthetic lipid bilayer in the aperture of the device, such that separate upper and lower chambers are formed. The formed chambers are positioned to facilitate interaction of different fluid phases with the synthetic lipid bilayer surfaces. For example, an aqueous phase, an organic phase, or an air phase can interact with the synthetic lipid bilayer in sequence, as desired. The synthetic lipid bilayer can comprise a mixture of phospholipids, proteins, glycoproteins, cholesterol, sphingosine, and/or other components. Synthetic lipid bilayers have been constructed using various techniques such as those found in Suzuki et al, Lab ChipΛ 2004, 4:501-505; Malmstadt et al., Nano Letters 2006, 1961-1965; Ide and Ichikawa, Biosensors and Isoelectrics, 2005, 21:672-677.; Suzuki et al. Langmuir 2006, 22:1937-1942, each is incorporated by reference in its entirety.
[0047] Evaluation and application of current micro-fluidic technology show that our virus assembly model can be established in a microchannel chip. Suzuki et al. (Biomed Microdevices 11: 17-22; 2009; Lab Chip 4: 502-505; 2004; and Langmuir 22: 1937-1942; 2006) reconstituted a lipid bilayer with a micro-fluidic system in a manner that is suitable for automated processing. While rudimentary, this system provides evidence that micro- channels can be installed around a reaction chamber and that flow can be applied to the micro-channels in an alternate way to introduce various buffers and solutions. In this microfluidic environment, the flow of material is usually laminar (not turbulent), independent of gravity and compression, and is dominated by viscous forces.
[0048] A functional synthetic virion can be prepared as follows. Various virion components can be added to one side or the other of a lipid bilayer, an RNA core
surrounded by the bilayer, and the synthetic virion budded from the aperture (see Figures IA to IE). The micro-injection of a phospholipid preparation to the upper channel over a water phase in the lower chamber can originally create a lipid monolayer across the aperture. In Figure IA, a phospholipid preparation 10 in an upper chamber 11 is applied to a water phase 12 in a lower chamber 13. Trans-membrane (TM) protein units 14 (Figure IB) pre-included in the lipid preparation (or, optionally, present in the water phase) can establish a cross-linking unit between the viral surface and matrix proteins. Assuming that the TM unit is lipophilic, its orientation could be adjusted by changing the pH of the buffers. Next, addition of a water phase in the upper channel will induce the instantaneous formation of a lipid bilayer (Figure 1C). The approximate height of the lipid bilayer can be a small as 5 nm or less, but can be modulated by changing the length of the phospholipid(s). Micro-injection of a surface protein in the upper phase is performed to populate the exposed surface of the lipid bilayer with the protein. The surface protein will be ultimately located in the outer shell surface of the synthetic virus when budding occurs in the final step. With such a versatile system, various surface proteins can be added, e.g., by engineering them to have affinity for the exposed TM protein segment. Alternately, diffusion and fusion of the protein hydrophobic regions into liposomes can populate them with viral proteins, without the need of a special affinity techniques. Addition of the matrix (M) protein 15 in the lower phase will result in the diffusion of the protein and its association to the TM unit 14.
Injection of pre-assembled RNA core elements 16 finally completes assembly of components (Figure ID). The RNA cores can optionally be prepared previously in a separate reaction, e.g., mixing the RNA with polymerase protein and core proteins to create functional core elements of the desired dimension. Diffusion and attachment of the RNA core from the bilayer interface can be inhibited by affinity interactions with natural or engineered segments of the M protein. Introduction of a final harvest upper phase layer and manipulation of the pressure (ΔP < 0) can be performed to fully allow membrane curvature and final budding of a synthetic virion 17.
Forming Lipid Bilavers Across an Aperture
[0049] A lipid bilayer is typically formed by aligning ambiphilic molecules at an air/water interface to form a monolayer, then introducing an aqueous buffer at the air side of the monolayer, forcing the ambiphilic molecules to rearrange into a bilayer with
hydrophobic portions embedded between outer hydrophilic surfaces. For example, an air/water interface can be generated by surface tension across an aperture. An immiscible hydrophobic solvent containing, e.g., phospholipids, can be layered over the interface. A lipid monolayer of the phospholipids will spontaneously form at the interface when the solvent contacts the buffer. The solvent can be removed, but the monolayer will remain in place. When an aqueous buffer is placed over the monolayer where the hydrophobic solvent had been, the monolayer will spontaneously reassemble into a lipid bilayer, e.g., with the polar phospholipid heads oriented toward the aqueous buffers on each side and with the lipid tails of the phospholipid hydrophobically associating inside the membrane.
[0050] The lipid bilayer can be formed by stepwise addition and removal of fluids within chambers on each side of the aperture, as described above. Optionally, the lipid bilayer can be formed in bulk processes, e.g., wherein suspensions or emulsions of the lipid and aqueous buffer are exposed to shear forces that result in production of liposomes. For example, such liposomes can be formed by blending lipid and water in a blender or homogenizer. However, the liposome product typically is not of uniform size. Further, such processes are often too uncontrolled to allow consistent incorporation of desired proteins in desired orientations, or to consistently capture a nucleic acid the size of a viral genome.
[0051] Instead of batch injection of liquid phases into chambers at the aperture, in one embodiment, the liquid phases (and optionally gas phases) can be introduced from channels flowing to and from the aperture, e.g., as shown in Figure 2. An upper channel can bring liquid packets to or from the lipid bilayer and optionally bring a protein component to interact with other components at the aperture. Liquid packets can be separated by air intervals in the channels to avoid product cross contamination between liquid packets. For example, a lipid monolayer can be formed at aperture 20 by flowing an aqueous solution 21 through lower channel 22 to lower chamber 23. A hydrophobic solvent 24 carrying, e.g., a phospholipid can flow from upper channel 25 to contact the aqueous interface spontaneously generating a lipid monolayer with hydrophobic tails oriented up and the polar head oriented down. The hydrophobic phase can be replaced by a flow of aqueous phase in the upper channel (optionally segmented with a gaseous phase) to cover the aperture. Introduction of aqueous phase to the upper chamber can cause the lipid monolayer to spontaneously rearrange to form a classic lipid bilayer membrane with inner hydrophobic lipid tails sandwiched between outer polar heads.
[0052] In some embodiments, one or more proteins are present in the hydrophobic solvent, e.g., with the phospholipid, even before the monolayer or bilayer is formed. For example, a transmembrane protein (typically including at least one hydrophobic segment between two hydrophilic peptide segments) can be included in the hydrophobic solvent so that the protein is incorporated into the membrane as it is formed. The protein can be in solution in the hydrophobic solvent and/or in the form of insoluble aggregates. Optionally, the protein can be other than a transmembrane protein, such as a protein having a hydrophobic segment that will ultimately be incorporated into the membrane with the hydrophobic segment inserted into the hydrophobic interior of the membrane. Delivering Proteins to the Membrane
[0053] The proteins can be delivered to the membrane by simple manual (e.g., pipetting) techniques, by automated filling of the upper and/or lower chambers through ports, or, preferably, by flowing the proteins in solution through a channel to contact the membrane at the aperture. After the membrane is assembled across the aperture, the membrane can be selectively populated with desired proteins at either or both surfaces. Access to the separate membrane surfaces can allow introduction of different proteins to opposite sides of the membrane. For example, it is often desirable to incorporate a protein with affinity for a host cell receptor on one side of the membrane, and to incorporate an M protein with a segment having affinity for a nucleic acid core on the other side. Alternately, both types of proteins can be incorporated, e.g., on both sides of the membrane.
[0054] The delivery of protein molecules to lipid bilayers has been accomplished through various processes, including for example but not limited to, a GPI anchor, incorporation of a lipophilic segment, or a transmembrane protein segment. In some embodiments, methods of the invention comprise delivery of a first protein to the synthetic lipid bilayer. The first protein can be a lipophilic or transmembrane protein (see, for example, Figure 1). The first protein can display affinity for a second protein and/or a third protein. Optionally, the first protein can display an affinity for the RNA core used in the method. In some embodiments, the first protein is added to the lipid bilayer prior to the application of the second protein, third protein, and/or RNA core (or added to the lipid before membrane formation). In other embodiments, the first protein is added to the lipid bilayer after the second protein, third protein, and/or the RNA core. In yet other
embodiments, the first protein is a transmembrane protein. Additionally, the first protein can display an affinity (e.g., a specific affinity) for the second protein, third protein, and/or RNA core. In some embodiments, the first protein can associate covalently or non- covalently with the second protein, third protein or the RNA core. In yet further
embodiments, the first protein can associate covalently or non-covalently with constituents of the RNA core.
[0055] In some embodiments, methods of the invention comprise delivery of a second protein to the upper chamber formed by the lipid bilayer (see Figure 3B). In some embodiments, the second protein can be a lipophilic or transmembrane protein. The second protein can display affinity for the first protein and/or the third protein. Optionally, the second protein can display an affinity for the RNA core structure used in the method. In some embodiments, the second protein is added to the lipid bilayer prior to the application of the first protein, third protein, and/or RNA core. In other embodiments, the second protein is added to the lipid bilayer after the second protein, third protein, and/or the RNA core. In yet other embodiments, the second protein is a transmembrane protein.
Additionally, the second protein can display an affinity for the first protein, third protein, and/or RNA core. In some embodiments, the second protein can associate covalently or non-covalently with the first protein, third protein or the RNA core. In yet further embodiments, the second protein can associate covalently or non-covalently with constituents of the RNA core.
[0056] When a protein in an aqueous buffer is delivered to contact with the membrane, the protein can spontaneously integrate into the membrane, e.g., through nonspecific hydrophobic interactions. The protein can naturally have hydrophobic segments, or be engineered to include hydrophobic segments, with a non-specific affinity for the lipid tails of the membrane molecules. In such a case, the thermodynamic free energy of the hydrophobic effect can favor the integration of the hydrophobic segments with the hydrophobic regions of the membrane. The free energy driving the integration can be influenced by adjustments in the surrounding aqueous buffers, e.g., by addition of solvents and surfactants, and/or by adjustment of pH and ionic strength of the solutions.
[0057] Optionally, the protein delivered to the membrane can bond to the membrane through specific affinity interactions, e.g., with another protein already present on the membrane. For example, the membrane can include a protein with specific binding partner, such as, e.g., an antigenic determinant, receptor, lectin, antigen, and/or the like. The delivered protein can be an antibody, or preferably include one or more CDR regions comprising a binding domain specific to the determinant. In another embodiment, one protein can include a ligand and the other protein can be a structurally complimentary receptor. In another embodiment, one or both proteins can include a chelator (e.g., poly- histidine) so the proteins can associate through a chelation bond. [0058] Optionally, the protein can covalently bond to the membrane surface. In some embodiments, a first protein bound to the membrane can include a reactive group and the delivered protein can include a moiety reactive with the reactive group, so that the two proteins are covalently bonded on contact. For example, one or both proteins can include a linker group, such as a N-Hydroxysuccinimide (NHS) group. Alternately the two proteins can be bonded together in the presence of a bidentate bridging linker.
[0059] In many embodiments, one protein delivered to one side of the membrane is an M protein that can urge the planar membrane into a concave shape to ultimately facilitate budding of the membrane from the aperture. In some cases, it can be advisable to take steps to prevent budding after the M protein had been delivered, until all virion components are in place. For example, removal of the upper phase can prevent membrane curvature.
Alternately, a hydrostatic pressure differential (ΔP > 0) can be used to prevent the premature curvature or budding of the membrane.
[0060] In some embodiments, the second (outer affinity) protein is a viral receptor, a cell surface receptor, a cell surface ligand, or an antibody fragment that recognizes a cell surface antigen on a host cell. In a typical embodiment, the protein bound to the outside of the synthetic virion has a specific affinity for a desired target host cell. For example, the outer surface protein can include the hemagglutinin (HA) glycoprotein (or a structural analog or fragment of HA), which can specifically interact with the sialic acid containing glycans on a host cell surface. Other specific host receptor/virus surface protein affinity interactions are known in the art. For example, other interacting pairs can include gpl20 interactions with CD4 and chemokine receptors on the T cell, HBV envelope proteins and liver cell receptors, herpes proteins and nerve cell receptors, and the like.
[0061] In some embodiments, methods of the invention comprise delivery of a third protein to the lower chamber side of the at the lipid bilayer (see Figure 3C). In some embodiments, the third protein can be a lipophilic or transmembrane protein. The third protein can display affinity for the first protein and/or the second protein. Optionally, the third protein can display an affinity for the RNA core structure used in the method. In some embodiments, the third protein can be added to the lipid bilayer prior to the application of the first protein, second protein, and/or RNA core. In other embodiments, the third protein is added to the lipid bilayer after the second protein, first protein, and/or the RNA core. In yet other embodiments, the third protein is a transmembrane protein. Additionally, the third protein may display an affinity for the first protein, second protein, and/or RNA core. In some embodiments, the third protein may associate covalently or non-covalently with the first protein, second protein or the RNA core. In yet further embodiments, the third protein may associate covalently or non-covalently with constituents of the RNA core.
[0062] In specific embodiments, the third protein in the methods of the invention is the Matrix (M) protein, the VP40 VLP protein, the ebola VP30 protein, a clathrin protein, a homologous protein or a derivative thereof from any virus. In other embodiments, the M protein or derivative thereof, is from any RNA virus. In other embodiments, the M protein is from the influenza virus or an M protein derivative thereof, e.g., derived using standard protein engineering techniques.
[0063] In many embodiments additional proteins can be bound to the membrane, complexed with the nucleic acid core, or captured within the liposome during the budding process. For example, in many cases where the nucleic acid is RNA, it can be desirable to include a reverse transcriptase as a component of the synthetic virus.
Fabricating and Delivering the RNA Core Structure
[0064] The RNA core structure can be delivered to the lipid bilayer membrane in much the same way described above with regard to proteins. In most cases, the RNA core structure is delivered only to one side of the membrane, e.g., the side that will ultimately line the inside of the budded synthetic virus. The RNA core can be delivered in a solution (or suspension) to the membrane surface, e.g., by pipetting onto the surface, filling an associated chamber through a port, or by flowing onto the membrane from a channel.
[0065] In some cases, the nucleic acid can be delivered to the membrane surface as an essentially free naked nucleic acid strand. For example, the lipid bilayer can include ambiphilic molecules having hydrophilic heads with a positive charge. The interaction between the nucleic acid and membrane can be moderated, e.g., by adjustment of the ambient pH. Nucleic acid core structure proteins can be introduced to interact with the nucleic acid bound to the membrane, e.g., to further bind, stabilize and compact the nucleic acid. That is, the core scaffolding and nucleic acid binding proteins can be complexed with the nucleic acid in situ on the membrane.
[0066] In most cases, the nucleic acid core includes one or more core proteins that provide structures providing, e.g., scaffolding, nucleic acid binding, charge neutralization and/or capsid functions. The core proteins can include positive charges to allow compact coiling of the nucleic acid. The proteins can be configured as scaffolding to direct the shape of the nucleic acid core. The core proteins can include sequences providing affinity interactions between the core and the membrane or with membrane proteins. Exemplary core proteins can be, without limitation, e.g., actin, herpes capsid proteins and scaffolding protein, influenza protein NSl, VP3 scaffolding protein, RSV F protein, SARS CoV accessory protein 7a, the SH3 domain, RNA binding protein HuR, HTV accessory protein Vpr, Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), HTV p24, homologs and engineered derivatives thereof. The proteins of the nucleic acid core can be recombinant proteins, purified proteins, or proteins from crude lysates.
[0067] In one embodiment, the M protein can function both in RNA binding and in membrane budding. The M protein can have hydrophobic segments that interact with the lipid bilayer and a positively charged segment that associates with nucleic acids. The methods can include binding the M protein to the nucleic acid (with M protein acting as a nucleic acid binding protein) to provide a core structure, then introducing the core structure to one side of the lipid bilayer membrane surface. Alternately, the M protein can be partially inserted into the membrane at the hydrophobic segment, while the positively charged segment remains exposed in the aqueous environment of the lower chamber. Next, the nucleic acid can be introduced to the lower chamber where it interacts with the positively charged M protein segments to bind and form a core in situ at the membrane.
[0068] In many embodiments, methods of the invention comprise the delivery of an
RNA core which is comprised of a functional RNA molecule capable of directing virus generation and propagation in a host cell, a scaffold to hold the RNA molecule and accessory RNA binding proteins to facilitate the transcription or translation of the RNA molecule. In some embodiments, methods and compositions of the invention comprise an RNA core, which can comprise RNA molecules, RNA binding proteins and optionally, a scaffold protein (for example, but not limited to actin). In other embodiments, the RNA core may include only which elements are required to maintain a functional RNA molecule that will drive the production of a live virus when delivered to a host cell. In other embodiments, the RNA core may be preassembled prior to delivery to the synthetic lipid bilayer, e.g., as shown in Figure 4. In other embodiments, the RNA core can be assembled in the lower chamber formed by the synthetic lipid bilayer. In yet other embodiments, the RNA core can display an affinity for any one of the first, second or third proteins in the synthetic virus. Alternatively, any entity of the RNA core can also have an affinity to the synthetic lipid bilayer.
[0069] In some embodiments, the RNA core is added to the lower chamber prior to any of the proteins in the methods. In other embodiments, the RNA core is added after the first, second or third proteins in the method. In other embodiments, the RNA core associates covalently or non-covalently with the first, second, or third proteins, or alternatively directly with the lipid bilayer.
Budding the Lipid Bilaver from the Aperture
[0070] A synthetic virus is generated when the lipid bilayer membrane buds free from the aperture, enclosing the nucleic acid within. In a specific embodiment, methods of the invention result in the formation of a liposome comprising a second protein on the outer edge of the bilayer, a third protein on the inner edge of the bilayer and an RNA core present inside the center of the liposome. The budded synthetic virus can be harvested from the fluid in the upper chamber by aspiration or can flow in a channel to a product chamber of a fluidic device.
[0071] Viral budding mechanisms for certain classes of viruses use the lipid raft
(LR) as a template for virus assembly. LRs are cholesterol and sphingolipid-enriched microdomains found in cell membranes. These specialized membrane microdomains can localize cellular processes by serving as organizing centers for the assembly of molecules, influencing membrane fluidity and membrane protein trafficking. Many viruses make use of lipid raft to complete their budding process. One of the main viral events in the making of viruses at the LR interface is the accumulation of viral matrix proteins that regulate the budding event. It has been shown that the matrix (M) protein is the only viral component essential for virus-like particle (VLP) formation (Gόmez-Puertas et al., J. Virol. 74: 11538- 11547; 2000). It was also shown that the M protein, when expressed alone, can induce membrane assembly into VLPs, which can be released into a culture medium. The M protein operates to impose curvature on the cell membrane. The M protein budding formation is virus dependent and occurs either in specific high cholesterol rich regions or the non-lipid raft membranes (Laliberte et al., J. Virol. 81: 10636-10648; 2007).
[0072] Budding can be initiated by providing a pressure differential across the lipid bilayer membrane and/or by application of an M protein to one side of the membrane. It is possible to provide budding by simply providing an adequate amount of an M protein, e.g., without application of mechanical pressures across the aperture. In many embodiments, the budding is at least facilitated by the delivery of the M protein to the lower chamber. In many other embodiments, budding is influenced by changes in the hydrostatic pressure or osmotic pressure between the upper and lower chambers across the lipid bilayer. In a particular embodiment, an M protein is introduced to the lower side of the membrane, causing the membrane to deflect upwards into the upper chamber; the nucleic acid core is introduced into the inside cavity of the membrane, and a higher pressure is applied to the lower chamber to expel the synthetic virus into the upper chamber. As the virus is being expelled, the membrane spontaneously closes on the under side to provide a sealed envelope around the captured nucleic acid.
[0073] In some embodiments, the budded synthetic virus can be further processed.
For example, an outer protein can be introduced to the virus for incorporation to the outer surface by hydrophobic interactions or specific affinity interactions. Optionally, the synthetic virus products can be selected for size, e.g., by filtration through a membrane of controlled pore size, or flowing the product past a side channel that receives only particles less than a certain size.
[0074] The newly synthesized viruses particles can be stored in dry (e.g., lyophilized or spray dried) form, or as a liquid suspension. Optionally, the particles can be immediately (e.g., before any drying step, freezing step, refrigeration step, holding in container separate from those of the synthesizer hardware; or in less than 12 hours, 6 hours, 1 hour, 15 minutes 5 minutes, or less than 1 minute) introduced to a host cell for production of progeny virions. In one embodiment, the synthetic virions are immediately administered (e.g., by injection or inhalation, e.g., as a vaccine) to an animal, e.g., straight from the device in which the synthetic virus was synthesized.
Virus Progeny from a Synthetic Virus Infection
[0075] The synthetic virus can bind to a host cell and release a nucleic acid encoding production of progeny virions. The product virions can be, e.g., non-viable virus particles, attenuated live viruses, or essentially typical live active forms of a naturally occurring virus. The synthetic virus can infect cells in vitro to produce a desired virus, e.g., an attenuated virus to be administered as a vaccine. Optionally, the synthetic virus can be administered directly to an animal, e.g., producing progeny attenuated or non-viable viruses in the animal that elicit a desired immune response to a microbial pathogen.
[0076] In certain embodiments, the synthetic virus has outer proteins with a specific affinity for a particular host cell of interest. When the synthetic virus contacts the host cell, the protein outer viral membrane surface specifically interacts with complementary receptors on the host cell membrane. The cell membrane is punctured as the virus membrane fuses with the host cell membrane and the viral core is injected into the host cell's cytoplasm. The core nucleic acid encodes all the elements necessary to reproduce itself, package progeny nucleic acids in a capsid or membrane. Typically the core nucleic acid encodes proteins with activities that promote release of progeny viruses from the host cell by budding or lysis of the host cell.
[0077] In some embodiments, the synthetic virus interacts non-specifically with a host cell, e.g., to enter by endocytosis. Many cells naturally take in resources from the environment or attack non-self particles by engulfing them into the cell. For example, many cells (particularly cells of the reticuloendothelial system and macrophages) will engulf particles they encounter. The synthetic viruses of the present invention can include surface proteins (e.g., opsonization sequences) that trigger endocytosis into a host cell. Once inside the cell the synthetic virus nucleic acids can be translated proteins and/or release enzymes that result in a take over of the host cell metabolism and manufacture of the desired progeny virus particles.
SYNTHETIC AND PROGENY VIRUSES
[0078] Synthetic viruses of the invention can be synthetic viruses prepared according to methods described above. A synthetic virus composition can comprise, e.g., a lipid bilayer capsule, a first protein (typically a transmembrane protein) inserted into the bilayer, a second protein (typically presenting an affinity for a host target surface protein) associated with the outer surface of the bilayer capsule, a third protein (typically a functional M protein) associated with the inner surface of the bilayer capsule, and a viral nucleic acid core encoding production of a live virus or virus like particle.
[0079] The invention also encompasses the synthetic viruses generated by the methods presented herein. The invention further encompasses compositions of progeny viruses resulting from infection of host cells with the synthetic viruses.
[0080] In methods of the invention, synthetic viruses can be produced to
functionally encode production of a live virus, such as a Rotavirus, a Coronavirus, SARS, Norwalk virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Barley Yellow Dwarf virus, Poliovirus, Hepatitis A virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Hepatitis E virus, Tobacco Mosaic virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mubs virus, Nipah virus, Hendra virus, Rabies virus, Lassa virus, Hantavirus, Parainfluenza virus, and Influenza virus. Further examples of viral pathogens include but are not limited to: adenovirdiae (e.g.,
mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levi virus, enterobacteria phase MS2, allolevirus), poxviridae (e.g., chordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxvirinae), papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae (e.g., paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measles virus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.g., pneumovirus, human respiratory synctial virus), and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus)), picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e.g., human hepatitis A virus), cardiovirus, and apthovirus), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, and oryzavirus), retroviridae (e.g., mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV-HTLV retroviruses, lentivirus (e.g. human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus (e.g., sindbis virus) and rubi virus (e.g., rubella virus)), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa virus), coronaviridae (e.g., coronavirus and torovirus), and/or the like.
Lipid Bilayer Capsules
[0081] The lipid bilayers are synthetic bilayers initially assembled across an aperture of a man made device. The bilayers can include lipid constituents typically found in natural membranes, as well as constituents that are not normally found in natural membranes. The lipid bilayers can be populated with outer, inner and/or transmembrane proteins. The lipid bilayer capsule surrounds a nucleic acid that encodes, e.g., a wild type viral genome, an attenuated virus genome, and/or instructions to generate a non-viable virus or virus like particle.
[0082] The lipid bilayer typically consists primarily of a thin layer of amphipathic lipids which spontaneously arrange so that the hydrophobic "tail" regions are shielded from the surrounding polar fluid, causing the more hydrophilic "head" regions to associate with the cytosolic and extracellular faces of the resulting bilayer. This forms a continuous, spherical or rod shaped lipid bilayer. The lipids in the membrane can include, e.g., phospholipids, glycolipids, and/or cholesterols. For example, the bilayer can include a phosphatidic acid (PA), phosphatidylethanolamine (e.g., cephalin - PE),
phosphatidylcholine (e.g., lecithin - PC), phosphatidylserine (PS), phosphoinosi tides (such as, e.g: phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP),
phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol triphosphate (PIP3)), phosphosphingolipids, ceramide phosphorylcholine (e.g., sphingomyelin - SPH), ceramide phosphorylethanolamine (Cer-PE), and/or ceramide phosphorylglycerol. Further, the lipid bilayer can be synthesized to incorporate, e.g., glyceroglycolipids, galactolipids, sulfolipids (SQDG), glycosphingolipids, cerebrosides, alactocerebrosides, glucocerebrosides, glucobicaranateoets, gangliosides, globosides, sulfatides, glycophosphosphingolipids, and/or glycosylphosphatidylinositols. In many cases, the lipid bilayer will include cholesterol and/or cholesterol derivatives, such as, e.g., cholesterol esters and sterols.
[0083] The lipids of the bilayer can be from purified sources and formulated in controlled proportions for introduction into the bilayer from a hydrophobic solvent at the aperture, as described above. Optionally, the lipids can be provided as relatively crude extracts from natural membranes.
[0084] The synthetic virus preparations of the invention can include virus particles range in size (average diameter) from less than about 50 nm to more than about 1000 nm; from 100 nm to 500 nm; or about 200 nm or less. In many other embodiments, the average virus particle is about 200 nm in diameter, large enough to contain a genomic virus nucleic acid and small enough to pass through standard sterilization filters.
Bilayer Bound Proteins
[0085] At least two proteins are typically integrated into the lipid bilayer membrane of the synthetic virus. In most embodiments, the synthetic virus requires a host cell targeting protein on the outer surface and a nucleic acid binding protein on the inside. Often a transmembrane protein is utilized, e.g., as an anchor in the membrane for attachment of other proteins inside and/or outside the membrane, and as part of a lipid raft system. We note however, that although proteins of the synthetic viruses are routinely discussed herein as separate proteins, a single protein sequence can actually provide multiple functions, such as (nucleic acid binding, anchoring, core scaffolding, modification of membrane topology, raft assembly, and/or target host cell affinity). Alternately, these functions can be provided by two peptides, three peptides, four peptides, or more.
[0086] For example, a protein typically bound by the methods to the outside of the synthetic virus membrane will have a specific affinity for a host cell surface protein (act as a ligand to a host cell receptor). The outer affinity protein can be independently incorporated into the lipid bilayer through a hydrophobic segment of the protein. Alternately, the outer affinity protein can be covalently anchored to a transmembrane protein or bound to a transmembrane protein through a mutual affinity interaction. Alternately, the
transmembrane protein can include an exposed (e.g., hydrophilic segment) engineered to have a specific affinity for the host cell. No matter how the outer protein is bound to the lipid bilayer, it typically includes, e.g., a sequence functioning to provide a specific affinity for the desired host cell or host cell surface protein. For example, a protein exposed on the outside of the synthetic virus can include functional affinity sequences, such as, e.g., ligands, receptors, antigens, antibodies, lectins, antibody domains, nucleic acid binding domains, enzyme binding domains, enzyme substrates, metal chelators and/or the like.
[0087] In some embodiments, the synthetic virus does not have a specific affinity for a specific host cell or specific host cell surface feature. However, the synthetic virus can still contact and fuse with a competent host cell, e.g., through hydrophobic interactions with host cell membranes or by endocytosis.
[0088] In many embodiments, the synthetic lipid bilayer includes at least one transmembrane protein. The transmembrane protein can function to anchor other virus components with the bilayer. The transmembrane protein can associate with other membrane proteins and/or membrane lipids in a lipid raft complex. The transmembrane protein can include at least one hydrophobic segment, and typically at least two hydrophilic segments. The transmembrane function can be engineered based on the transmembrane domains of many well-characterized transmembrane proteins, or based on knowledge in the mature art of protein engineering. For example, transmembrane proteins can be the same as, or engineered from known viral transmembrane proteins. Transmembrane proteins can be, e.g., influenza virus neuraminidase (NA), transmembrane protein R-peptide, MLV transmembrane protein pl5E, foamy virus transmembrane proteins, FTTV-I transmembrane protein gp41, transmembrane protein gp30, and the like, or a derivative thereof having more than 90%, 95%, 98% or 99% identity with such natural transmembrane proteins.
[0089] Other functional transmembrane structures can be found in membrane signaling complexes, neuron signaling proteins and ion transport proteins. For example, functional transmembrane peptide sequences can be found or derived from, e.g., ATP synthase alpha/beta subunits; ATP synthase gamma subunit, ATP synthase subunit C, ATP8B1, Asialoglycoprotein, bacterial antenna complex, rhodopsins, CDl Ic, cadherin, calcium ATPase, chloride channels, cytochrome b, cytochrome b5, cytochrome c oxidase subunit π, cytochrome c oxidase subunit HI, disulfide bond formation protein B, FXYD6, fumarate reductase, glycophorin, hemolysin, integrin, ion channel families, JAML, Ll protein, lactose permease, leukotriene C4 synthase, light-harvesting complexes, linker of activated T cells, list of human ATPase genes, the MAPEG family, MT-ATP6, the main subunit of cytochrome c oxidase, facilitators, intrinsic proteins, the mechanosensitive ion channel, the mitochondrial carrier, mitochondrial membrane transport protein, myelin proteolipid protein, the organic anion-transporting polypeptide, permease, the
photosynthetic reaction center protein family, photosystem II light-harvesting protein, the potassium channel tetramerisation domain, proton ATPase, SERCA, SNARE protein, SecY protein, SeI- 12, sodium-hydrogen antiporter, sodium/proton antiporter 1,
sodium:neurotransmitter symporter, the solute carrier family, sulfatase, syntaxin, the TIM/TOM complex, translocase of the inner membrane, the transmembrane domain of ABC transporters, V-ATPase and/or the like.
[0090] Protein functions on the inner lipid bilayer surface can include, e.g., binding to the bilayer, binding of the nucleic acid core, and facilitating budding of the bilayer form the aperture. As discussed above, a single protein can include segments providing all these functions. For example, members of the M protein family can have sequences functioning to bind, package and stabilize a nucleic acid, sequences that insert and anchor into the bilayer, and sequences acting to induce a curvature in the bilayer. Peptide segments that bind the bilayer typically have hydrophobic sequences. Peptide segments that bind the nucleic acid typically are rich in positively charged amino acids, such as, e.g., lysine and arginine.
Nucleic Acid Cores
[0091] Nucleic acid cores typically include a nucleic acid encoding a virus or virus like particle, and one or more proteins functioning to bind and package the nucleic acid. [0092] The nucleic acid of the core can be DNA or RNA; single stranded or double stranded. The nucleic acid can be a native natural genomic nucleic acid from a live or attenuated virus. Optionally, the nucleic acid can be synthesized in vitro. In other embodiments, the nucleic acid encodes all proteins necessary to direct production of a desired virus product in a living host cell.
[0093] In one embodiment, the nucleic acid core is simply the functional nucleic acid bound to the charged surface of the lipid membrane. For example, the lipid bilayer can include abundant positively charged, such as, e.g., membranes comprising
dihexadecyldimethyl ammonium bromide (DHDMAB). Such lipid bilayers can capture a nucleic acid on the inside surface.
[0094] As described above, the nucleic acid core can optionally be formed by an interaction between the nucleic acid and a positively charged protein bound to the inner surface of the lipid bilayer.
[0095] Viral RNA cores of the invention can include purified viral RNA that is attenuated. The RNA can be complexed to a scaffold protein, such as actin. The
RNA: scaffold mix can be further complexed with other functional proteins to form an exemplary RNA core.
[0096] In more other embodiments, the nucleic acid core is assembled before the nucleic acid is delivered to the inside surface (surface that will be inside the synthetic virus on budding) of the lipid bilayer. For example, the nucleic acid can be previously bound with a nucleocapsid protein (NP) to provide a core before delivery to the lipid bilayer. Other core proteins useful in preparation of core structures can include lentiviral core proteins, adeno-associated virus (AAV) core proteins, and retrovirus core proteins.
[0097] In many cases, additional proteins are captured or bound within the synthetic virus. For example, the synthetic virus can include enzymes with activities that function to allow the virus to gain entry to the host cell and/or replicate inside the host cell. Such additional proteins can include, e.g., transcriptase complex, proteases, and reverse transcriptase. Virus Progeny
[0098] The progeny virus products of the methods are encoded by the nucleic acid of the synthetic virus. The progeny virus can effectively present as a normal natural virus. In other embodiments, the progeny virus expresses at least one protein that is a recombinant sequence engineered into the synthetic virus nucleic acid.
[0099] In many cases, the progeny virus is essentially the same as a pathogenic virus of interest, except it is encoded to express as an attenuated virus. Such progeny virus products can be useful, e.g., in the production of vaccines. Methods of attenuation are well- known in the art - see e.g., US Patents 5,578,473; 5,840,520; 5,820,871; 6,033,886, each of which are incorporated by reference in their entireties.
[0100] In certain embodiments, the progeny virus is engineered to be heat sensitive.
For example, the virus can fail to replicate at temperatures above 37°C, so that it will not replicate if it causes a fever in a human. Optionally, the virus can be sensitive to
temperatures below body temperature, so that the virus can only grow in cells at cooler body locations. For example, a progeny virus sensitive to temperatures above 30°C, 32°C, or 350C, could live in the nose or upper respiratory tract, but not in deeper tissues of the body.
[0101] In certain embodiments, the progeny virus is engineered to be cold adapted.
For example, the virus can fail to replicate well at temperatures above room temperature, so that it will replicate well in a human upper respiratory tract. Optionally, the virus can be adapted for optimal growth at 20°C, 25°C, 270C or 3O0C.
[0102] In some embodiments, the methods of the invention comprise the production of a live viruses wherein the live virus, e.g., a Rotavirus, a Coronavirus, SARS, Norwalk virus, Yellow Fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Barley Yellow Dwarf virus, Poliovirus, Hepatitis A virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Hepatitis E virus, Tobacco Mosaic virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mubs virus, Nipah virus, Hendra virus, Rabies virus, Lassa virus, Hantavirus, Parainfluenza virus, Influenza virus and/or the like. DEVICES FOR PREPARING SYNTHETIC VIRUSES
[0103] Devices of the invention for preparing synthetic viruses generally include, e.g., an aperture between two compartments and a means to deliver solutions or suspensions to the compartments to interact at an interface at the aperture. The devices can include channels to flow the solutions or suspensions to the interface, and to remove the solutions or suspensions from the interface. The solutions can include, e.g., aqueous buffers to establish the interface at the aperture, solvents to deliver ambiphilic lipids to the interface to form a monolayer, and additional aqueous buffers containing proteins and/or nucleic acids for delivery and binding at the lipid bilayer across the aperture.
[0104] Microfluidic devices can be used to practice the methods of the invention.
For example, as shown in Figure 3A, after assembly of a lipid bilayer 30 in the aperture 31 of a microfluidic device 32, an aqueous suspension of transmembrane protein 33 flows from lower microfluidic channel 34 to contact the lower side of the bilayer (optionally, from the upper channel). Hydrophobic segments of the transmembrane protein can be incorporated within the bilayer, e.g., through hydrophobic interactions. Next, a bolus of a ligand protein 35 in aqueous solution 36 can flow through upper microfluidic channel 37 to contact the upper side of the bilayer in the aperture, as shown in Figure 3B. The ligand proteins can interact with the membrane and/or the transmembrane protein (e.g., by hydrophobic or specific affinity interactions) to bind to the upper surface of the bilayer. The upper aqueous layer can be removed by a bolus of air in the upper chamber. A third protein 38 in an aqueous solution can flow from the lower channel to contact the lower surface of the bilayer, as shown in Figure 3C. For example, an aqueous bolus of M protein in solution can flow to contact and bind to the lower side of the lipid bilayer, e.g., through hydrophobic interactions and/or affinity interactions with the transmembrane protein. Flows in the bottom channel can remove the previous solution and introduce a solution containing a RNA core structure 39. The RNA core structure can remain in place to be physically captured in the budding step. Preferably, the RNA core structure can interact (e.g., through hydrophobic interactions and/or specific affinity interactions) to bind on the under side of the lipid bilayer, where it can be surrounded within the lipid bilayer during the budding process. [0105] The present inventions include devices to practice the methods of the invention. For example, a device can have an aperture, or a series of apertures (otherwise known as an array of apertures) suitable for the assembly of synthetic lipid bilayers.
Apertures of the invention are typically circular or ovoid across the plane and have a diameter ranging from about 10 nm to 1000 nm, from 50 nm to 500 nm, from 100 nm to 200 nm, or about 150 nm. In many embodiments, the device includes two or more apertures, e.g., 2 to more than 106 apertures, from 10 to 105 apertures, from 100 to 104 apertures, or about 1000 apertures. The apertures can be arranged in arrays, e.g., parallel or serial arrays arranged along shared channels or chambers, or having separate dedicated channels providing desired fluid flows to each aperture.
[0106] In embodiments where fluids are delivered in channels, it may be that the channels are microfluidic channels. For example, the channels preferably include at least one cross-sectional dimension ranging from about 1 nm to 1000 nm, from about 5 nm to 500 nm, from 10 nm to 200 nm, or about 100 nm. In many embodiments, the device includes two or more channels configured to deliver fluids to one or both sides of two or more apertures. For example, the devices can include, e.g., 2 to more than 106 channels, from 10 to 105 channels, from 100 to 104 channels, or about 500 channels. The channels can sequentially deliver and remove fluids (solutions, suspensions and/or gasses) to apertures of the invention. In some embodiments, the channels can be configured to provide recirculating of the fluids, so they may be reused in sequence.
[0107] The devices can include chambers, e.g., on each side of each aperture. For example, fluidic channels can widen out adjacent to an aperture to form a chamber next to the aperture. Alternately, a chamber can be an adjacent channel section that is not widened. Alternately, chambers can be provided on each side of the aperture, even in embodiments where no flow through channels are provided.
[0108] The devices include a lipid bilayer traversing an aperture. The lipid bilayers can be as described above in the Methods and Virus sections. For example, the bilayers can be two essentially parallel and planar layers of ambiphilic molecules aligned with more polar (e.g., hydroxyl or phosphate groups) structures at the surfaces, and with lipid "tail" hydrophobic chain structures forming an inner hydrophobic membrane layer. The lipid bilayer can be populated with surface and/or transmembrane proteins.
[0109] The devices include one or more affinity proteins associated with the lipid bilayer. The affinity proteins can be as described above. For example, the affinity proteins can include surface or transmembrane proteins with affinities for, e.g., a host cell surface receptor. Other affinity proteins associated with the bilayer can be proteins having affinity for a transmembrane protein (allowing the protein to be anchored at the bilayer), proteins having an affinity for a nucleic acid or nucleic acid core (binding the nucleic acid core to the bilayer).
[0110] The devices can include nucleic acid core structures, e.g., as previously described herein. For example, the nucleic acid core can be an RNA core structure. The core structure can include at least one protein with a binding interaction (typically a ionic interaction) with the nucleic acid. The core structure can include a protein (which can be the same protein as the nucleic acid binding protein) that acts as a scaffolding to compactly package and stabilize the nucleic acid, e.g., in a tight capsid form. The core structure can include one or more proteins that has a functional affinity interaction with another protein bound on the inside of the lipid bilayer, thus capturing and binding the core on the lipid bilayer (this protein can be the same protein providing the binding and/or scaffolding functions).
EXAMPLES
[0111] The following examples serve merely to illustrate the invention and are not intended to limit the invention in any way.
Example 1 - Generation of a Synthetic Virus
[0112] Biological materials are placed over or under an aperture by controlled injection. The first step (STEP 1) requires the micro-injection of a phospholipid membrane preparation through the upper channel over an already existing water phase. Transmembrane (TM) protein units are already present in the water phase and/or pre-included in the lipid preparation. The lipophilic nature of the TM units permits a direct spontaneous incorporation. The orientation of the TM elements can be modulated by adjusting the environment to cytoplasmic pH values or alternatively, by modifying the TM primary amino acid sequence.
[0113] The second step (STEP 2) consists of the micro-injection of the ligand (L) proteins in the upper phase. The upper phase is used to populate the exposed surface of the synthetic virus with various populations of L proteins. The introduction of the liquid upper phase will hypothetically reconstitute the lipid bilayer. The approximate height of the lipid bilayer is 5 nm. The lipid bilayer is estimated to be resistant to microchannel pressure up to 100 Pa.
[0114] The third step of assembly (STEP 3) simulates the surface budding mechanism of a virus. Removal of the upper phase prevents membrane curvature. Use of end-to-end pressure may also achieve the same effect (ΔP pressure (air or water) > 0) can prevent premature curvature of membrane. The addition of the matrix (M) protein to lower phase is also envisaged for this step. Diffusion and affinity attachment of the M protein to the TM protein occurs at the lipid membrane interface.
[0115] The fourth step (STEP 4) consists of the injection of a RNA pre-assembled core. The RNA core is prepared previously in a separate reaction. Diffusion and attachment of RNA core to M proteins obeys the same kinetic rules as for small proteins. The introduction of the upper phase layer and a pressure of ΔP < 0 fully allows membrane curvature at this final step.
Example 2 - Microfluidic Simulation
[0116] A microfluidic process simulation permitted us to gather the physical parameters of a microfluidic virion assembly system early in the system design. In this study, we initially gathered physical parameters pertaining to the system and assessed the system for potential liquid turbulences that could impede the assembly of the synthetic virus.
[0117] 1) Physical parameters and system design. The design of a LAIV should take into consideration industrial production standards, such as requirements that therapeutics and vaccines be compatible with sterile filtration, e.g., through 0.2 μm pore filter. While full aseptic procedures would allow the production of higher molecule size (> 200 nm diameter), we believe it would be economically more suitable to have a product that can be sterile filtered.
[0118] The amount of lipid required to prepare a virus can be estimated from a simple surface equation. Assuming a diameter of 0.2 μm, the virus surface would be 0.126 μm . This value can be used to define the size of the aperture at the interface of the two layers. An additional 20% surface area can be included in the aperture in order to adjust for any product loss occurring at the budding and pinching reaction. Optimal aperture size would therefore be in the order of about of 0.15 urn2. Control of virus dimensions can be enforced by using specific aperture values as long as the virus diameter is lower than 0.2 um. Suzuki et al. (2004) described a microchannel design wherein apertures are fabricated and aligned along the tubular section of a channel. The use of multiple apertures at the reaction site is also a possibility as long as there are no failure pressure points at the aperture level, e.g., originating from an increased total aperture surface that could disrupt the lipid bilayers. If this sort of system is truly independent of pressure, an interface zone can be envisioned between the upper and lower phases that would contain thousands of reaction sites at the point of contact. A lower specific value for the aperture size would help the system to withstand any pressure changes in the system. Pressure could also be better controlled by increasing the lipid chain length and the thickness of the lipid bilayer (see, Figure 6). The model we propose would benefit from a pressure controlled environment as the shape of the lipid bilayer could be adjusted to prevent premature budding of the M protein aggregates before the inclusion of the RNA core.
[0119] The assembly of all components are dependent of diffusion and ligand attachment. While ligand attachment can be easily modulated by increasing or decreasing protein affinity, diffusion is sensitive to a series of physical parameters, such as
temperature, viscosity and size of the molecule. Particle diffusion in such a small system is extremely rapid and can be rapidly estimated by calculating the coefficient of diffusivity from the Stokes-Einstein equation (Equation 1) and by estimating the diffusion time with simple linear diffusivity time estimation (Equation 2). For small molecules such as proteins (approximately 0.2 nm diameter), time of diffusion would be under 10"5 second with a constant of diffusivity of 1.10 x 10"6 m2/s and a path length of 25 um. Diffusion of larger molecule such an RNA core (approximately 100 nm diameter) would be approximately 0.1 second with a constant of diffusivity of 2.20 x 10"9 m2/s and a path length of 25 um.
Assuming that the ligand-ligand interaction is immediate at time of contact, assembly of multiple parts for the making of the LAIV could possibly be performed under one minute. Concentration of the viral parts in the liquid phase would have to evaluated and optimized in order to assess the impact on diffusivity and viscosity in the liquid phase.
The Stokes-Einstein equation for the calculation of the virus coefficient diffusivity is,
D = kT/6πηr (Equation 1)
And the diffusion time for a given molecule in a linear plane is
tD = L2/D (Equation 2)
Where D is the diffusion coefficient, where k is the Boltzmann's constant (1.38E23 JK"1), T is the absolute temperature, η is the viscosity of the fluid and r the particle radius, t is the time of diffusion, L the path length of the molecule.
[0120] The volume of the reaction chamber can also be modulated, but could be in theory of infinite size (assuming a constant molecule concentration) as long as the particle concentration at the lipid bi-layer interface is unchanged. The budding mechanism can be substantially influenced by the aggregation of a limited but unknown amount of M protein and the virus volume is also influenced by to the amount of M protein and the amount and type of lipid molecules.
[0121] 2) Volumetric flow and turbulences effects. One of the difficulties related to microfluidic and microchannel aperture is the possibility to generate volumetric flow turbulences by repetitively changing the buffers on both sides of the lipid layers.
Assessment of volumetric disturbances can be rapidly calculated with the Reynold number (Equation 3).
Re = QD/vA (Equation 3)
Where Re is the Reynold number, Q the volumetric flow rate, D the hydraulic diameter of a pipe, v is the kinematic viscosity, and A the pipe cross-functional area. [0122] At a temperature of 20°C and a microchannel flux of 0.1 ul/min, the Re value is 0.562, indicative of a laminar flow regimen. Assuming that the air interface between the reagents does not affect the laminar flow, such as system could therefore support a sequential viral assembly process. A microchannel diameter value of 50 μm will maintain a laminar flow in the system, but reduction of the diameter will increase the Re value to level that are still tolerable (Figure 5). In theory, the microchannel flux could be increased up to 1.5 mL/min while still maintaining the Re value in the laminar flow range definition (Re < 4000). The liquid phase replacement will therefore not disrupt the lipid bilayer interface. According to the maximum velocity equation, maximum velocity in a microchannel of 100 μm radius would be 0.25 m/s (Dp = 0.1 bar, 1 = 10 cm, h = 0.001 Pas) (equation 4).
Vz,max = (ΔP/4ηl)R0 2 (Equation 4)
Where RO is the diameter of the microchannel, 1 is the path length, ΔP is the pressure difference and η is the dynamic viscosity.
[0123] Figure 6 shows the theoretical failure pressure point based on lipid bilayer thickness. Based on the maximum stress value on a bilayer membrane (Kusube et al., Colloids Surf B Biointerfaces 42: 79-88, 2005) and the poisson's ratio value of 0.0001 Mpa and 0.3, respectively, the failure pressure point can be assessed in Pascal. At aperture diameters lower than 80 μm, the pressure that can be used in the microchannel system and at the site of reaction will increase with the bilayer thickness. An increase from 5 to 10 nm thickness in the lipid bilayer will result in more than 5-fold increase in pressure resistance. This estimation shows that high throughput assembly is possible and can be modulated at different level. Yamada et al. (Biotechnol Bioeng 88: 489-494; 2004 and Anal Chem 76: 5465-5471; 2004) demonstrated that a Y channel structure putting together two asymmetric flow rates would create a pinched-flow fractionation separating smaller from bigger particles. This phenomenon could be used to increase the homogeneity of the product by possibly separating the defective particles from the infectious units.
[0124] A major challenge of the synthetic assembly platform is the availability of component material in the appropriate format and quality (lipid, protein, RNA). The lipid solution used by Suzuki et al. (2004) was composed of asolectin dissolved in n-decane. Functional lipid bi-layers have also been fabricated diphytanyl phosphatidylcholine (DPhPC). DPhPC is a molecule which has ether linkage instead of ordinary ester one between the hydrophobic chains and the glycerol backbone. While various lipid solutions are often available and well characterized as to hydrophobic characteristics, viral proteins and RNA are typically less available and characterized. Nucleic acids specific to a virus of interest often need to be produced independently. Various biological protein production methods can be used to generate the M protein, TM, ligand and core viral proteins in single or separate reactions. Synthetic or biologically produced proteins and nucleic acids usually need to be purified in order to reach the desired quality and minimize the introduction of undesired proteins into synthetic virions, particularly for use in vaccines or therapeutics for administration to humans. We see that cell-free protein synthesis will be feasible for commercial protein production and could even be integrated directly into the microchannel system. Simple scale-up technologies for synthetic protein production have already being developed (Swartz, J. Ind. Microbiol. Biotechnol. 33: 476-485, 2006) but biological methods are still more economical.
[0125] One of the most challenging steps in creating synthetic viruses is to incorporate genetic information into the virion, e.g., a negative RNA strand providing a functional template for RNA replication and generation of messenger RNAs for protein synthesis. The RNA strand should be encapsulated with the necessary prime elements to start the virus replication in the cell cytoplasm. RNA segmentation of the influenza virus adds a level of difficulty as RNA packaging with proteins will be more complex.
Alternative genetic expression can be considered for complex RNA viruses, by replacing the genetic template by non-segmented RNA or plasmid DNA. Moreover the encapsulation of RNA cores should preferably condense the RNA and protein to an aggregate smaller than 100 nm in diameter.
[0126] A plasmid DNA template could be used as a simpler alternative to package the necessary genetic information, as an alternate over RNA encoding, without the help of any starting protein material.
[0127] Reverse genetics technology (plasmid rescue) is currently used to enhance safety, specificity, reliability and efficiency with which new vaccine strains can be produced. A synthetic virus platform can bypass this step and rely on pure PCR
amplification to generate the viral RNA segments that will be coated with nucleoprotein and bound with a polymerase complex.
[0128] To fully utilize VLP technology, a synergistic interaction between biomolecular design and bioprocess engineering would be required in order to increase yield, reduce process time and total cost. The model we propose has the advantage of addressing these important process factors for the design of LAFVs. A synthetic LAIV platform would also enhance flexibility in virus design by populating the virus surface, e.g., with selected receptors with known amount of proteins. The benefits in cell targeting would be immense. The high throughput capacity of the systems described herein would ensure control of virus population homogeneity, while maintaining robustness at the dosage level.
[0129] The use of defined raw material for protein, lipid and nucleic acid is a clear advantage for the release of defined product with a high degree of purity. The shift from biological products to synthetic product would be highly desirable in order to reduce the intermediary steps needed to purify the raw material for the LAIV assembly. Protein expression could be performed in non-mammalian system such as yeast to avoid
mammalian host cell protein contamination. Further, synthetic production of LAFVs offers the opportunity to increase stability by modulating their lipid composition or by establishing enhanced virus synthetic scaffolds.
[0130] The introduction of a microfluidic chip technology for the assembly of LAFV can also resolve long term storage issues by allowing the assembly of viruses on site of vaccination. LAFV assembly chips can contain all the raw material necessary for the construction of the synthetic virus. An appropriate amount of raw material could be inserted in distinct compartments on the chip to produce the exact dose required for the patient. A microfluidic apparatus can enable LAFV assembly on a microchip stored at 40C. It is also possible that these chips be reduced in size as they will be able to handle volumes in the range of the femtolitre, e.g. the size of a virus. Example 3 - Identification of Membrane Composition Candidates
[0131] Equipment:
-MicrofluidizerTM processors - M-IlOP
Interaction chambers - F12Y (75 μm) - H30Z (200 μm)
Microscope - Olympus BH-2 optical with attachments
Diluent - De-ionized water (17.1 MegaOhms)
Particle Size Analyzers - Horiba LA-910 (SLS) and Malvern Zetasizer Nano-S (DLS)
Refractive Index - 1.40 - O.Oli
Rotostator Mixer - IKA T25
[0132] Procedure:
There were three phases of particle size reduction testing at Microfluidics: a Chemically Defined Lipid Concentrate (CDLC) at various concentrations, different ratios of two lipids (PC and PE) in water, and the optimum ratios from the lipids in water experiments, tested in a protein media.
[0133] The first testing consisted of diluting a 10Ox concentration of CDLC down to 50x, 10x, 5x, and Ix and processing these samples through the Microfluidizer. However, due to time constraints, only the 5Ox experiment was completed. This experiment was done on the M-11OP Microfluidizer processor at 30,000 psi for two passes with the F12Y (75 μm) - H30Z (200 μm) IXC configuration.
[0134] The second set of tests was performed by varying the ratio of two lipids, PC and PE, in DI water or WFI and processing with the M-11OP Microfluidizer processor. Samples were mixed with stir bars until the lipid powder was no longer visible in the mixture.
Exp. B: 100 mg PC + 0 mg PE + 100 g DI water
Exp. C: 75 mg PC + 25 mg PE + 100 g DI water Exp. D: 50 mg PC + 50 mg PE + 100 g DI water
Exp. E: 25 mg PC + 75 mg PE + 100 g WH
Exp. F: 0 mg PC + 100 mg PE + 100 g WFI
These experiments were done at 20,000 or 30,000 psi with the Fl 2Y (75 μm) - H30Z (200 μm) IXC configuration for various numbers of passes and their particle size was measured. Select experiments were passed through a 0.2 μm PTFE or PES filter and measured again.
[0135] The third set of tests consisted of replacing the DI water or WFT with one of three different protein medias: BRlO-VLPOl Medi559 (17MarlO - #3), RSVsF CHO 8D4.1.6 Clone #15 (14DecO9), or RSVsF CHO 8D4.1.7 Clone #16 (14DecO9). Samples were mixed with stir bars until the powder was no longer visible in the mixture.
Exp. G: 150 mg PC + 50 mg PE + 200 g BRIO
Exp. H: 150 mg PC + 50 mg PE + 200 g Clone #15
Exp. I: 200 mg PC + 0 mg PE + 200 g BRIO
Exp. J: 200 mg PC + 0 mg PE + 200 g Clone #15
Exp. K: 150 mg PC + 50 mg PE + 200 g Clone #16 + 50 mg
Monophosphoryl Lipid A (synthetic), PHAD (MPLA-PHAD)
These experiments were done at 20,000 or 30,000 psi with the F12Y (75 μm) - H30Z (200 μm) IXC configuration for various numbers of passes and their particle size was measured.
[0136] The additional ingredient (MPLA-PHAD) in the Exp. K formulation required a pre-mixing step of 1 minute at 8000 RPM on the IKA T25 rotostator mixer. Gas became entrained in the mixture during the premixing; however, it was removed by pulling a vacuum on the sample prior to processing with the Microfluidizer processor. [0137] Analysis:
Analysis involved the use of the Malvem Zetasizer NanoS (dynamic light scattering) and the Horiba LA910 (static laser scattering). Particle size analysis was done at the conclusion of each test. Microscope images were also taken of the samples after various stages of processing.
[0138] Results:
The results from the particle size analysis and the processing conditions are found in Tables 1 - 3. Particle size distributions are found in Appendices A - C (Attached).
[0139] Table 1: Listed below are the processing conditions and the resulting particle size measurements of the first set of experiments with 50x concentration of CDLC. All samples were processed on the M-11OP Microfluidizer processor with the Fl 2 Y (75 μm) - H30Z (200 μm) IXC configuration at 30,000 psi.
Figure imgf000045_0001
[0140] Table 2: Listed below are the processing conditions and the resulting particle size measurements of the second set of experiments with PC and PE in DI water or WFI. The weight of the water in all the formulations was 100 g. All samples were processed on the M-11OP Microfluidizer processor with the F12Y (75 μm) - H30Z (200 μm) IXC. The experiments highlighted in green were the most successful and the basis for the third set of experiments.
Figure imgf000045_0002
Figure imgf000046_0001
[0141] Table 3: Listed below are the processing conditions and the resulting particle size measurements of the third set of experiments with PC and PE in protein media. The weight of all the protein media samples was 200 g. All samples were processed on the M- 11OP Microfluidizer processor with the F12Y (75 μm) - H30Z (200 μm) IXC. Post processing analysis by Medlmmune will determine the success of these experiments.
Figure imgf000047_0001
[0142] Comments:
As discussed during the visit, particle size is a strong function of the components in the formulation. Since these formulations had not been finalized, further improvements towards the particle size goals can be achieved with further research and testing.
Additional testing will evaluate encapsulation and/or stability of the proteins in the processed samples.
A gas removal step may be required for formulations containing MPLA-PHAD prior to processing with the Microfluidizer processor.
[0143] Glossary:
APM: Auxiliary processing module/ interaction chamber used as either a pre-processing chamber for a solid dispersion application, or as a backpressure module to create a backpressure for Y-chamber applications.
CDLC: Chemically Defined Lipid Concentrate, a lipid from Gibco.
DLS: Dynamic light scattering.
IXC: Interaction chamber; a cylindrical module with a specific orifice and channel design thru which fluid is conducted at high pressures to control shear rates.
Pass: One (1) processing cycle.
PC: L-a-Phosphatidylcholine, a lipid from Avanti Polar Lipids, Inc.
PDI: Poly-dispersity Index.
PE: L-a-Phosphatidylethanolamine, Transphosphatidylated, a lipid from
Avanti Polar Lipids, Inc.
Psi: Pounds per square inch.
SLS: Static laser scattering.
Unprocessed: A sample that has not experienced any passes through the
Microfluidizer processor.
WFI: Water for injection. [0144] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
[0145] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, many of the techniques and apparatus described above can be used in various combinations and permutations, all of which cannot reasonably be recited individually in this document, but can be understood by one of skill in the art on review of this specification.
[0146] All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.
Appendix A
Particle size distributions from CDLC experiments
Figure imgf000050_0001
Diameter (μM)
Figure A1 : Shown above is the particle size distribution of the 5Ox concentration CDLC, before (dotted) and after (solid) processing. The processed sample experienced two passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration.
Appendix B
Particle size distributions from Experiments B - F: Dl water or WFI experiments (All figures show the distribution based on intensity (top) and volume (bottom))
Figure imgf000051_0003
Figure imgf000051_0001
Size (d.nm)
Record 5 20100406B 100 mg PC/100 ml WFI - 1 pass F12Y - H30Z 30,000 psi 1
Record 6 20100406B 100 mg PC/100 ml WFI - 2 passes F12Y - H30Z 30,000 psi 1 Record 7 20100406B 100 mg PC/100 ml WFI - 3 passes F12Y - H30Z 30,000 psi 1 Record 8 20100406B 100 mg PC/100 ml WFI - 3 passes F12Y - H30Z 30,000 psi - Filtered 1
Figure imgf000051_0002
Figure imgf000051_0004
0
Size (d.nm)
Record 5 20100406B 100 mg PC/100 ml WFI - 1 pass F12Y - H30Z 30,000 psi 1
Record 6 20100406B 100 mg PC/100 ml WFI - 2 passes F12Y - H30Z 30,000 psi 1
Record 7 20100406B 100 mg PC/100 ml WFI - 3 passes F12Y - H30Z 30,000 psi 1
Record 8 20100406B 100 mg PC/100 ml WFI - 3 passes F12Y - H30Z 30,000 psi - Filtered 1
Figure B1 : Shown above is the particle size distribution from Experiment B. This sample was processed for various passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. Size Distribution by Intensity
Figure imgf000052_0003
Figure imgf000052_0001
Size (d.nm)
Record 10 20100406C 75 mg PC+25 mg PE/100 ml WFI - 1 pass F12Y - H30Z 30,000 psi 1 Record 11 20100406C 75 mg PC+25 mg PE/100 ml WFI - 2 passes F12Y - H30Z 30,000 psi 1 Record 12 20100406C 75 mg PC+25 mg PE/100 ml WFI - 3 passes F12Y - H30Z 30,000 psi 1 Record 13 20100406C 75 mg PC+25 mg PE/100 ml WFI - 3 passes F12Y - H30Z 30,000 psi - filtered PES 1
Size Distribution by Volume
Figure imgf000052_0002
Size (d.nm)
Record 10 20100406C 75 mg PC+25 mg PE/100 ml WFI - 1 pass F12Y - H30Z 20,000 psi 1 Record 11 20100406C 75 mg PC+25 mg PE/100 ml WFI - 2 passes F12Y - H30Z 20,000 psi 1 Record 12 20100406C 75 mg PC+25 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1 Record 13 20100406C 75 mg PC+25 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi - filtered PES 1
Figure B2: Shown above is the particle size distribution from Experiment C This sample was processed for various passes on the M-110P
Microfluidizer processor at 20,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. Size Distribution by Intensity
Figure imgf000053_0002
10 100 1000 10000
Size (d.nm)
Record 14 20100406D 50 mg PC+50 mg PE/100 ml WFI - 1 pass F12Y- H30Z 20,000 psi 1 Record 15 20100406D 50 mg PC+50 mg PE/100 ml WFI - 2 passes F12Y - H30Z 20,000 psi 1 Record 16 20100406D 50 mg PC+50 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1 Record 17 20100406D 50 mg PC+50 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi +
1 p@30K 1
Size Distribution by Volume
Figure imgf000053_0001
0 1 10 100 1000 10000
Size (d.nm)
Record 14 20100406D 50 mg PC+50 mg PE/100 ml WFI - 1 pass F12Y - H30Z 20,000 psi 1 Record 15 20100406D 50 mg PC+50 mg PE/100 ml WFI - 2 passes F12Y - H30Z 20,000 psi 1 Record 16 20100406D 50 mg PC+50 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1 Record 17 20100406D 50 mg PC+50 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi +
1 p@30K 1
Figure B3: Shown above is the particle size distribution from Experiment D. This sample was processed for various passes on the M-110P
Microfluidizer processor at 20,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. The last sample (black) experienced the first three passes at 20,000 psi and the final pass at 30,000 psi. Size Distribution by Intensity
Figure imgf000054_0003
Figure imgf000054_0001
Size (d.nm)
Record 18: 20100406E 25 mg PC+75 mg PE/100 ml WFI - 1 pass F12Y - H30Z 20,000 psi 1 Record 19: 20100406E 25 mg PC+75 mg PE/100 ml WFI - 2 passes F12Y - H30Z 20,000 psi 1 Record 20: 20100406E 25 mg PC+75 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1 + 1P@10K 1
Size Distribution by Volume
Figure imgf000054_0004
Figure imgf000054_0002
Size (d.nm)
Record 18: 20100406E 25 mg PC+75 mg PE/100 ml WFI - 1 pass F12Y - H30Z 20,000 psi 1
- Record 19: 20100406E 25 mg PC+75 mg PE/100 ml WFI - 2 passes F12Y - H30Z 20,000 psi 1
- Record 20: 20100406E 25 mg PC+75 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1 + 1P@10K 1
Figure B4: Shown above is the particle size distribution from Experiment E. This sample was processed for various passes on the M-110P
Microfluidizer processor at 20,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. The last sample (blue) experienced the first three passes at 20,000 psi and the final pass at 10,000 psi. Size Distribution by Intensity
Figure imgf000055_0003
Figure imgf000055_0001
Size (d.nm)
Record 21 20100406E 0 mg PC+100 mg PE/100 ml WFI - 1 pass F12Y - H30Z 20,000 psi 1 Record 22 20100406E 0 mg PC+100 mg PE/100 ml WFI - 2 passes F12Y- H30Z 20,000 psi 1 Record 23 20100406E 0 mg PC+100 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1
Size Distribution by Volume
Figure imgf000055_0002
Figure imgf000055_0004
0 1 10 100 1000 10000
Size (d.nm)
Record 21 20100406F 0 mg PC+100 mg PE/100 ml WFI - 1 pass F12Y - H30Z 20,000 psi 1 Record 22 20100406F 0 mg PC+100 mg PE/100 ml WFI - 2 passes F12Y - H30Z 20,000 psi 1 Record 23 20100406F 0 mg PC+100 mg PE/100 ml WFI - 3 passes F12Y - H30Z 20,000 psi 1
Figure B5: Shown above is the particle size distribution from Experiment F This sample was processed for various passes on the M-110P
Microfluidizer processor at 20,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. Appendix C
Particle size distributions from Experiments G - K: protein media experiments (All figures show the distribution based on intensity (top) and volume (bottom))
Size Distribution by Intensity
ω
Figure imgf000056_0001
10 100 1000 10000
Size (d.nm)
Record 24. 20100406G - BR10-VLP01 no liposomes 1
Record 29: 20100406G - 150 mg PC+50 g BRIO-VLP01 - 1 pass F12Y - H30Z 30,000 psi 1
Record 30: 20100406G - 150 mg PC+50 g BRIO-VLP01 - 2 passes F12Y - H30Z 30,000 psi 1
Figure imgf000056_0002
0 1 10 100 1000 10000
Size (d.nm)
Record 24. 20100406G - BR10-VLP01 no liposomes 1
Record 29. 20100406G - 150 mg PC+50 g BRIO-VLP01 - 1 pass F12Y - H30Z 30,000 psi 1
Record 30: 20100406G - 150 mg PC+50 g BRIO-VLP01 - 2 passes F12Y - H30Z 30,000 psi 1
Figure C1 : Shown above is the particle size distribution from Experiment G. This sample was processed for various passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. The first sample (red) was the
unprocessed BR10 protein media and did pass throught the Microfluidizer processor. Size Distribution by Intensity
Figure imgf000057_0003
Figure imgf000057_0001
Size (d.nm)
Record 31: 20100406H - 150 mg PC+50 g RSVsF CHO-8D4.16 - 1 pass F12Y - H30Z 30,000 psi 1 Record 32 20100406H - 150 mg PC+50 g RSVsF CHO-8D4.16 - 2 passes F12Y- H30Z 30,000 psi 1
Size Distribution by Volume
Figure imgf000057_0002
Figure imgf000057_0004
0.1 10 100 1000 10000
Size (d.nm)
Record 31: 20100406H - 150 mg PC+50 g RSVsF CHO-8D4.16 - 1 pass F12Y - H30Z 30,000 psi 1 Record 32- 20100406H - 150 mg PC+50 g RSVsF CHO-8D4.16 - 2 passes F12Y- H30Z 30,000 psi 1
Figure C2: Shown above is the particle size distribution from Experiment H. This sample was processed for various passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. Size Distribution by Intensity
Figure imgf000058_0003
Figure imgf000058_0001
Size (d.nm)
Record 25: 201004061 - 200 mg PC+200 g BRIO-VLP01 - 1 pass F12Y- H30Z 20,000 psi 1 Record 26: 201004061 - 200 mg PC+200 g BRIO-VLP01 - 1 pass F12Y - H30Z 20,000 psi 1 Record 27: 201004061 - 200 mg PC+200 g BRIO-VLP01 - 3 passes F12Y - H30Z 20,000 psi 1 Record 28' 201004061 - 200 mg PC+200 g BRIO-VLP01 - 3 passes F12Y - H30Z 20,000 psi +
1 pass @ 30Kpsi 1
Size Distribution by Volume
Figure imgf000058_0002
Figure imgf000058_0004
0.1 10 100 1000 10000
Size (d.nm)
Record 25 201004061 - 200 mg PC+200 g BRIO-VLP01 - 1 pass F12Y - H30Z 20,000 psi 1
— Record 26 201004061 - 200 mg PC+200 g BRIO-VLP01 - 1 pass F12Y - H30Z 20,000 psi 1
— - - - Record 27 201004061 - 200 mg PC+200 g BRIO-VLP01 - 3 passes F12Y - H30Z 20,000 psi 1
— Record 28 201004061 - 200 mg PC+200 g BRIO-VLP01 - 3 passes F12Y - H30Z 20,000 psi +
1 pass @ 30Kpsi 1
Figure C3: Shown above is the particle size distribution from Experiment I. This sample was processed for various passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. The last sample (black) experienced the first three passes at 20,000 psi and the final pass at 10,000 psi. Size Distribution by Intensity
Figure imgf000059_0001
Size (d.nm)
Record 33 20100406J - 200 mg PC+200 ml RSVsF CHO-8D4 16 - 1 pass F12Y - H30Z 30,000 psi 1
Size Distribution by Volume
ω
§
Figure imgf000059_0002
0 1 10 100 1000 10000
Size (d nm)
Record 33 20100406J - 200 mg PC+200 ml RSVsF CHO-8D4 16 - 1 pass F12Y - H30Z 30,000 psi 1
Figure C4: Shown above is the particle size distribution from Experiment J. This sample was processed for various passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration. Size Distribution by Intensity
Figure imgf000060_0003
Figure imgf000060_0001
Size (d.nm)
Record 34 20100406K - 150 mg PC+50 mg PE + 50 mg MPLA-PHAD/200 ml RSVsF CHO-8D4 16 1 pass F12Y- H30Z 30,000 psi
Record 34 20100406K - 150 mg PC+50 mg PE + 50 mg MPLA-PHAD/200 ml RSVsF CHO-8D4 16 1 pass F12Y- H30Z 30,000 psi
Size Distribution by Volume
Figure imgf000060_0004
Figure imgf000060_0002
Size (d.nm)
Record 34 20100406K - 150 mg PC+50 mg PE + 50 mg MPLA-PHAD/200 ml RSVsF CHO-8D4 16 1 pass F12Y - H30Z 30,000 psi
Record 34 20100406K - 150 mg PC+50 mg PE + 50 mg MPLA-PHAD/200 ml RSVsF CHO-8D4 16 1 pass F12Y - H30Z 30,000 psi
Figure C5: Shown above is the particle size distribution from Experiment K This sample was processed for various passes on the M-110P
Microfluidizer processor at 30,000 psi through the F12Y (75 μm) - H30Z (200 μm) IXC configuration.

Claims

WHAT IS CLAIMED IS:
1. A method of making a viable synthetic virus, the method comprising:
a) forming a synthetic lipid bilayer across an aperture separating a first chamber from a second chamber;
b) delivering a first protein to the lipid bilayer from the first chamber, wherein the first protein associates to bind with the lipid bilayer or with a second protein embedded in the lipid bilayer;
c) delivering a third protein to the lipid bilayer from the second chamber, wherein the third protein associates with the lipid bilayer or with the second protein;
d) delivering an RNA core structure to the lower chamber; and,
e) budding the lipid bilayer from the second chamber into the first chamber to form a liposome enclosing the RNA core structure and comprising the first protein on an outer surface of the liposome, thereby forming the synthetic virus;
wherein the first protein of the synthetic virus specifically binds a host cell surface receptor to deliver the RNA core into the cell where RNA of the core encodes production of live virus from the host cell.
2. The method of claim 1, wherein the aperture has a diameter ranging from 10 nm to 250 nm.
3. The method of claim 1, wherein said budding comprises budding liposomes from an array of apertures.
4. The method of claim 3, wherein the array apertures are arranged in parallel channels.
5. The method of claim 1, wherein the second protein is a transmembrane protein.
6. The method of claim 5, wherein the transmembrane protein displays an affinity for said first protein, said third protein or said RNA core.
7. The method of claim 1, wherein the first protein is selected from the group consisting of: a viral receptor, a cell surface antigen, a host cell surface protein, a cell surface ligand, and an antibody variable domain.
8. The method of claim 1, wherein the third protein is an M protein.
9. The method of claim 1, wherein the third protein comprises a segment with an affinity for the RNA core structure.
10. The method of claim 1, wherein the RNA core comprises RNA, a scaffold, and RNA binding proteins.
11. The method of claim 1, wherein said budding is induced by a force selected from the group consisting of: an osmotic pressure differential, a hydrostatic pressure differential, and a voltage differential.
12. The method of claim 1, wherein said budding is induced by application of an M protein to the lipid bilayer.
13. The method of claim 1, wherein said method is performed sequentially, steps a through e.
14. The method of claim 1, wherein the live virus produced in a host cell is attenuated.
15. The method of claim 1, wherein the live virus is selected from the group consisting of: a paramyxovirus, a pnemovirus, an orthomyxovirus, a retrovirus, and a morbilli virus.
16. The method of claim 1, wherein said method is performed in vitro.
17. The method of claim 1, wherein said lipid bilayer comprises lipid rafts.
18. The method of claim 1, wherein the second protein is different from the first protein and different from the third protein.
19. The method of claim 1, wherein said method further comprises delivering the second protein to the lipid bilayer from the first chamber or from the second chamber.
20. The method of claim 1, wherein the RNA core structure binds with the third protein or with the lipid bilayer.
21. The method of claim 1, wherein said associating or binding is a specific affinity interaction.
22. The method of claim 1, further comprising flowing the liposomes in the first chamber through a "Y" channel having two asymmetric flow rates to create a pinched flow to fractionate smaller liposomes from larger liposomes.
23. A method comprising introducing the synthetic virus of claim 1 to the host cell.
24. The method of claim 23, wherein the synthetic virus is introduced to the cell immediately after said budding is induced.
25. The synthetic virus produced by the method of claim 1.
26. A sterile composition of the synthetic virus of claim 25.
27. A viable synthetic virus composition comprising, a liposome, a first protein associated with an outer surface of the liposome, a second protein inserted into a membrane of the liposome, a third protein associated with an inner surface of the liposome and a viral RNA core inside the liposome;
said core capable of functionally directing production of a live virus when the liposome is delivered to a host cell; and,
wherein the synthetic virus is other than a virus produced in a living host cell.
28. The synthetic virus of claim 27, wherein the lipid bilayer comprises
phosphatidylcholine (PC ) or phosphatidylethanolamine (PE).
29. The synthetic virus of claim 27, wherein the attenuated synthetic virus is cold adapted or temperature sensitive.
30. The synthetic virus of claim 27, wherein the virus is characterized by an average diameter of 0.2 μm or less.
31. A device comprising:
a first chamber;
a second chamber;
an aperture between the first chamber and the second chamber;
a lipid bilayer traversing the aperture between the chambers;
an affinity protein associated with the lipid bilayer on a first bilayer side facing the first chamber and a RNA core structure associated with the lipid bilayer on a second bilayer side facing the second chamber.
32. The device of claim 32, further comprising an array of said apertures.
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