US20130167267A1 - Processes using vlps with capsids resistant to hydrolases - Google Patents

Processes using vlps with capsids resistant to hydrolases Download PDF

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US20130167267A1
US20130167267A1 US13/725,184 US201213725184A US2013167267A1 US 20130167267 A1 US20130167267 A1 US 20130167267A1 US 201213725184 A US201213725184 A US 201213725184A US 2013167267 A1 US2013167267 A1 US 2013167267A1
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capsid
sequence
ribozyme
vlp
seq
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Juan Pedro Humberto Arhancet
Juan P. Arhancet
Kimberly Delaney
Kathleen B. Hall
Neena Summers
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Apse Inc
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Apse Inc
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Priority to US13/725,184 priority Critical patent/US20130167267A1/en
Assigned to APSE, LLC reassignment APSE, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DELANEY, Kimberly, HALL, Kathleen B., SUMMERS, NEENA, ARHANCET, JUAN P., ARHANCET, Juan Pedro Humberto
Publication of US20130167267A1 publication Critical patent/US20130167267A1/en
Priority to US14/279,793 priority patent/US9181531B2/en
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Definitions

  • the invention relates to virus-like particles, and in particular to methods and compositions using viral capsids as nanocontainers for producing, isolating and purifying heterologous nucleic acids and proteins.
  • VLPs Virus-like particles
  • viruses are particles derived in part from viruses through the expression of certain viral structural proteins which make up the viral envelope and/or capsid, but VLPs do not contain the viral genome and are non-infectious.
  • VLPs have been derived for example from the Hepatitis B virus and certain other viruses, and have been used to study viral assembly and in vaccine development.
  • Viral capsids are composed of at least one protein, several copies of which assemble to form the capsid.
  • the viral capsid is covered by the viral envelope.
  • Such viral envelopes are comprised of viral glycoproteins and portions of the infected host's cell membranes, and shield the viral capsids from large molecules that would otherwise interact with them.
  • the capsid is typically said to encapsidate the nucleic acids which encode the viral genome and sometimes also proteins necessary for the virus' persistence in the natural environment.
  • the capsid must be disassembled. Such disassembly happens under conditions normally used by the host to degrade its own as well as foreign components, and most often involves proteolysis.
  • Viruses take advantage of normal host processes such as proteolytic degradation to enable that critical part of their cycle, i.e. capsid disassembly and genome release.
  • Nucleic acids including siRNA and miRNA, have for the most part been manufactured using chemical synthesis methods. These methods are generally complex and high cost because of the large number of steps needed and the complexity of the reactions which predispose to technical difficulties, and the cost of the manufacturing systems. In addition, the synthetic reagents involved are costly and so economy of scale is not easily obtained by simply increasing batch size.
  • the present disclosure provides a virus-like particle (VLP) comprising a capsid enclosing at least one heterologous cargo molecule and a packing sequence.
  • a VLP may further comprise at least one ribozyme enclosed by the capsid.
  • the heterologous cargo molecule may comprise an oligonucleotide, or an oligoribonucleotide.
  • a VLP may comprise a one or more ribozymes, and a ribozyme may be flanked by the packing sequence and the oligoribonucleotide to form a nucleic acid construct.
  • a VLP may comprise a plurality of the nucleic acid constructs.
  • oligoribonucleotide may be a short RNA selected from siRNA, shRNA, sshRNA, lshRNA and miRNA.
  • a VLP may comprise at least two ribozymes, wherein each ribozyme is selected to cut one end of the short RNA.
  • a VLP may further comprise a linker consisting of at least 1 to 100 nucleotides, the linker comprising at least 40% A's or at least 40% U's, wherein the linker links the oligoribonucleotide and the packing sequence, or the oligoribonucleotide and the ribozyme.
  • Ribozymes may be selected for example from a Hammerhead ribozyme and a Hepatitis Delta V ribozyme.
  • a Hammerhead ribozyme may be a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the oligoribonucleotide.
  • the ribozyme may be a mutant Hepatitis Delta V ribozyme capable of cleaving its connection with the oligoribonucleotide at a rate at most about 50% the rate of a wild type Hepatitis Delta V ribozyme.
  • Such a mutant HDV ribozyme may have for example a nucleic acid sequence selected from SEQ ID Nos: 10-18.
  • VLP's may comprise a capsid which comprises a wild type viral capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4, or a capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid may comprise a wild type Enterobacteria phage MS2 capsid protein having the amino acid sequence of SEQ ID NO: 3.
  • VLP's may comprise a heterologous cargo molecule comprising a peptide or polypeptide.
  • a VLP may further comprise an oligonucleotide linker coupling the heterologous cargo peptide or polypeptide molecule and the viral capsid.
  • the oligonucleotide linker may be an oligoribonucleotide comprising a ribozyme sequence.
  • the heterologous cargo molecule may comprise a bi-molecular cargo molecule comprising a bifunctional polynucleotide comprising a first aptamer sequence which specifically binds a bioactive small molecule having a molecular weight of about 1,500 Da or less and a second aptamer sequence for binding a packing sequence of the capsid.
  • the VLP may further comprise the bioactive small molecule bound to the first aptameric sequence.
  • the bioactive small molecule may comprise and herbicide or a pesticide, which may selected for example from atrazine, acetamipridphorate, profenofos, isocarbophos and omethoateas.
  • the present disclosure provides a nucleic acid construct comprising a nucleotide sequence that encodes a short RNA, a ribozyme and a packing sequence.
  • the short RNA may be for example an siRNA or an shRNA.
  • the nucleic acid construct may further comprise a linking nucleotide sequence of 4 to 100 nucleotides of which at least 40% are A's or at least 40% are T's, wherein the linking nucleotide sequence is flanked by the packing-coding sequence and by the short RNA-coding sequence.
  • the nucleic acid construct may further comprise a linking nucleotide sequence of 4 to 100 nucleotides of which at least 40% are A's or at least 40% are U's, wherein the linking nucleotide sequence is flanked by the ribozyme and the short RNA-encoding sequence.
  • the ribozyme sequence may be flanked by the short RNA and the packing sequence.
  • the present disclosure also encompasses a vector comprising any such nucleic acid constructs, and host cells comprising such a vector, as well as host cell stably transformed with such a vector.
  • Host cells may be a bacterial cell, such as but not limited to an Escherichia coli cell, a plant cell, a mammalian cell, an insect cell, a fungal cell or a yeast cell.
  • a host cell may further be stably transfected with a second vector comprising a second nucleic acid sequence encoding a viral capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the second nucleic acid sequence may encode for example a viral protein encoding a viral capsid having at least 40% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a nucleic acid construct as described herein may also encode a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3).
  • the ribozyme in such a nucleic acid construct may be for example a Hammerhead ribozyme, .a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the short RNA, a Hepatitis Delta V ribozyme, or a mutant Hepatitis Delta V ribozyme capable of cleaving its connection with the short RNA at a rate at most 50% the rate of a wildtype Hepatitis Delta V ribozyme.
  • An non-limiting but exemplary mutant HDV ribozyme has a nucleic acid sequence selected from SEQ ID NOs: 10-18.
  • the present disclosure also encompasses a plant or plant tissue transformed to contain a nucleic acid construct described herein, and seed or progeny of such a plant or plant tissue, wherein the seed or progeny comprises the nucleic acid construct.
  • the present disclosure provides a composition
  • a composition comprising: a) a plurality of virus-like particles each comprising a viral capsid enclosing at least one heterologous cargo molecule; and b) one or more cell lysis products present in an amount of less than 4 grams for every 100 grams of capsid present in the composition, wherein the cell lysis products are selected from proteins, polypeptides, peptides and any combination thereof.
  • the capsid is for example resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid may comprise a capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid may comprises a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3).
  • the heterologous cargo molecule may comprise an oligonucleotide which may be an oligoribonucleotide.
  • each virus-like particle may further comprise at least one ribozyme, wherein the ribozyme is flanked by the packing sequence and the oligoribonucleotide to form a nucleic acid construct, and each virus-like particle may comprise a plurality of the nucleic acid constructs.
  • the ribozyme may be for example a Hammerhead ribozyme, .a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the short RNA, a Hepatitis Delta V ribozyme, or a mutant Hepatitis Delta V ribozyme capable of cleaving its connection with the short RNA at a rate at most 50% the rate of a wildtype Hepatitis Delta V ribozyme.
  • An non-limiting but exemplary mutant HDV ribozyme has a nucleic acid sequence selected from SEQ ID NOs: 10-18.
  • the VLP's in such a composition may further comprise a linking nucleotide sequence of 4 to 100 nucleotides of which at least 40% are A's or at least 40% are T's, wherein the linking nucleotide sequence is flanked by the packing-coding sequence and by the short RNA-coding sequence, or a linking nucleotide sequence of 4 to 100 nucleotides of which at least 40% are A's or at least 40% are U's, wherein the linking nucleotide sequence is flanked by the ribozyme and the short RNA-encoding sequence.
  • the ribozyme sequence may be flanked by the short RNA and the packing sequence.
  • the VLP's in such a composition may f comprise a heterologous cargo molecule comprising a peptide or polypeptide.
  • Such VLP's in a composition may further comprise an oligonucleotide linker coupling the heterologous cargo molecule and the viral capsid.
  • the oligonucleotide linker may be an oligoribonucleotide comprising a ribozyme sequence.
  • the cell lysis products may be present in an amount of less than 0.5 grams, less than 0.2 grams or less than 0.1 grams.
  • the present disclosure provides method for isolating and purifying a target cargo molecule, the method comprising: (a) obtaining a whole cell lysate comprising a plurality of virus-like particles (VLPs) each comprising a capsid enclosing at least one target cargo molecule, wherein the capsids are resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4; (b) subjecting the VLP's to hydrolysis using a peptide bond hydrolase category EC 3.4, for a time and under conditions sufficient for at least 60, at least 70, at least 80, or at least 90 of every 100 individual polypeptides present in the whole cell lysate but not enclosed by the capsids to be cleaved, while at least 60, at least 70, at least 80, or at least 90 of every 100 capsids present in the whole cell lysate before such hydrolysis remain intact following the hydrolysis.
  • VLPs virus-like particles
  • the capsids may each comprises a viral capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsids may each comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3).
  • the heterologous cargo molecule may comprise an oligonucleotide which may be an oligoribonucleotide, or a peptide or a polypeptide.
  • An oligoribonucleotide may be selected for example from siRNA, shRNA, sshRNA, lshRNA and miRNA.
  • each virus-like particle may further comprise a ribozyme, wherein the ribozyme is flanked by the packing sequence and the oligoribonucleotide to form a nucleic acid construct.
  • the method may further comprise purification of the capsids following hydrolysis. Purification may include at least one of a liquid-liquid extraction step, a crystallization step, a fractional precipitation step, and an ultra filtration step.
  • the present disclosure also encompasses a composition produced by such a method.
  • the present disclosure provides a method for protecting a target molecule from hydrolysis in a whole cell lyste following intracellular production of the target molecule in a host cell, the method comprising: (a) selecting a viral capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4; (b) stably transfecting the host cell with a first vector comprising a nucleic acid sequence encoding a viral protein forming the viral capsid, and a second vector comprising a nucleic acid sequence comprising a ribozyme flanked by a packing sequence and an siRNA sequence; and (c) maintaining the cells for a time and under conditions sufficient for the transformed cells to express and assemble capsids encapsidating the ribozyme flanked by the packing sequence and the siRNA sequence.
  • the capsids may each comprises a viral capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • Step (c) may comprise (i) performing a first precipitation with ammonium sulfate followed by a first centrifugation to obtain a first precipitate and a first supernatant; and (ii) performing a second precipitation on the first supernatant with ammonium sulfate followed by a second centrifugation to obtain a second precipitate, wherein the second precipitate comprises at least about 70%, 80% or 90% by weight of the VLP's.
  • Step (c) may comprise (i) performing a first precipitation with ethanol followed by a first centrifugation to obtain a first precipitate and a first supernatant; and (ii) performing a second precipitation on the first supernatant with ammonium sulfate followed by a second centrifugation to obtain a second precipitate, wherein the second precipitate comprises at least about 70%, 80% or 90% by weight of the VLP's.
  • Step (c) may comprise ultracentrifuging the hydrolysate to obtain a precipitate comprising at least about 70%, 80% or 90% by weight of the VLP's.
  • the VLP's may each comprise a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • EC 3.4 which can comprise a capsid protein having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 45%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the heterologous cargo molecule enclosed by the VLP's may comprise an oligonucleotide which may be an oligoribonucleotide, or a peptide or a polypeptide.
  • An oligoribonucleotide may be selected for example from siRNA, shRNA, sshRNA, lshRNA and miRNA.
  • the VLP's may each further comprise a ribozyme as described herein, flanked by a packing sequence and the oligoribonucleotide to form a nucleic acid construct.
  • the present disclosure provides VLP's comprising a capsid enclosing at least one heterologous cargo molecule and a packing sequence wherein the capsid comprises a capsid protein which is a variant of wild type Enterobacteria phage MS2 capsid (SEQ ID NO: 3).
  • the capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO: 3) except that the A residue at position 1 is deleted, and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a 1-2 residue insertion in the 24-30 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid protein may be one which has the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single (1) residue insertion in the 10-18 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a linker segment may be a Gly-Ser linker selected from -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly-
  • the capsid may comprise the capsid protein concatenated with a third capsid monomer sequence which assembles into a capsid which resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid may comprise a first coat protein sequence in a concatenated dimer which is C-terminally truncated by 1 residue and the linker segments lengthened by the one residue or wherein the first and/or second coat protein sequence in a concatenated trimer is C-terminally truncated by 1 residues and which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the capsid may comprise a capsid protein having N- and C-terminal truncations and which is resistant to hydrolysis catalyzed by peptide bond hydrolase category EC 3.4.
  • FIG. 1 is a plot of Optical Density (OD; filled diamonds) and pH (open squares) over time, showing propagation of wild type MS2 bacteriophage (ATCC No. 15597-B1, from American Type Culture Collection, Rockville, Md.) in its E. Coli host (ATCC No. 15669).
  • FIG. 2 is a gel showing results of SDS-PAGE analysis of MS2 bacteriophage samples obtained following propagation in E. Coli and purified using Proteinase K and ultrafiltration, showing that Proteinase K purification yields phage purified to higher than 99% (band at 14 kDa corresponds to MS2 bacteriophage coat protein).
  • FIG. 3 is a gel showing results of SDS-PAGE analysis of partially purified MS2, showing complete degradation of the phage and results obtained after 1 ⁇ or 2 ⁇ ultrafiltration of the lysate (Lanes 4 and 6).
  • FIG. 4 is a gel showing results of SDS-PAGE analysis of MS2 samples purified using ultrafiltration and Proteinase K treatment.
  • FIG. 5 is a gel showing results of SDS-PAGE analysis of MS2 samples purified using Proteinase K treatment, precipitation at acidic conditions, precipitation using ethanol at basic and acidic conditions, and ultrafiltration.
  • FIG. 6 is a graph showing the UV spectrum of MS2 samples purified using Proteinase K treatment, precipitation at acidic conditions, precipitation using ethanol at basic and acidic conditions, and ultrafiltration.
  • FIG. 7 is a chromatograph of PCR products obtained from an MS2 sample following purification described for FIGS. 5 and 6 , chromatographed in 1.5% agarose gel stained with Ethidium Bromide (1.2 kbp for primers F1201 — 1223-R1979 — 2001 in Lane 1,800 bp for primers F1201 — 1223-R1979 — 2001 in Lane 2, and 304 bp for primers F1401 — 1426-R1680 — 1705 in Lane 3), showing consistency with an intact MS2 bacteriophage genome.
  • FIG. 8 is a plot of Optical Density (OD; filled diamonds) over time, obtained with a control sample (open diamonds) and an MS2 sample following purification described for FIGS. 5 and 6 (filled squares), showing that the purified sample contained phage that retained high infectivity.
  • FIG. 9 is a gel showing results of SDS-PAGE analysis of MS2 samples following expression of MS2 capsids encapsidating RNA coding for the coat protein attached to a coat-specific 19-mer RNA hairpin.
  • FIG. 10 is a chromatograph of PCR products from PCR interrogation of an MS2 sample for presence or absence of a section of the MS2 capsid following purification, chromatographed in 2% agarose gel stained with Ethidium Bromide (304 bp in Lane 1; the leftmost Lane corresponds to 1 kb plus ladder from Life Technologies), showing consistency with an intact MS2 coat gene.
  • FIG. 12 is a gel showing results of SDS-PAGE analysis of MS2 samples following use of Proteinase K (PK) and simple precipitation with ethanol for purification of MS2 VLPs.
  • PK Proteinase K
  • FIG. 13 is a gel showing results of SDS-PAGE analysis of MS2 samples following use of constitutive hydrolases (CH), fractional precipitation with ethanol, and ultrafiltration for purification of MS2 VLPs.
  • FIG. 14 is a gel showing results of SDS-PAGE analysis of MS2 samples following use of various hydrolases, and factional precipitation with ammonium sulfate for purification of MS2 VLPs.
  • FIG. 15 is a gel showing results of PAGE analysis of RNA obtained from RNA encapsidated in MS2 capsids.
  • FIG. 16 is a gel showing results of PAGE analysis of RNA products produced following in vitro transcriptions using Hepatitis Delta Virus (HDV) ribozyme.
  • HDV Hepatitis Delta Virus
  • FIG. 17 is a gel showing results of PAGE analysis of siRNA products obtained during in vitro transcriptions using long flanking Hammerhead ribozymes.
  • FIG. 18 is a gel showing results of PAGE analysis of RNA products obtained from RNA encapsidated in VLPs, following purification of the VLP's and isolation of the RNA from the VLPs.
  • FIG. 19 is a series of gels showing results of SDS-PAGE analyses of VLP's comprising MS2 capsids, following purification and suspension of the VLPs, and exposure to various proteases for 1 hour and 4 hours of incubation.
  • FIG. 20 is an alignment of selected enterobacteria phage MS2 capsid proteins.
  • FIG. 21 is an alignment of complete leviviridae viral coat protein sequences retrieved from the UniProt database and aligned using their BLAST multiple alignment with default values for weighting array choice, gap penalties, etc.
  • FIG. 22 is a graphic illustration of backbone superposition of 1AQ3 chain B (leviviridae coat protein monomer) with 1 QBE chain C (alloleviridae coat protein monomer).
  • FIG. 23 is a graphic illustration of an alternative view of the backbone superposition of 1AQ3 chain B (leviviridae coat protein monomer) with 1 QBE chain C (alloleviridae coat protein monomer) shown in FIG. 22 .
  • FIG. 24 is a graphic illustration of another alternative view of the backbone superposition of 1AQ3 chain B (leviviridae coat protein monomer) with 1QBE chain C (alloleviridae coat protein monomer) shown in FIG. 22 .
  • FIG. 25 is a graphic illustration of another alternative view of the backbone superposition of 1AQ3 chain B (leviviridae coat protein monomer) with 1QBE chain C (alloleviridae coat protein monomer) shown in FIG. 22 .
  • FIG. 26 is a structural sequence alignment of 1AQ3, 2VTU and 1QBE.using jFATCAT rigid.
  • FIG. 27 is an alignment of complete alloleviviridae viral coat protein sequences retrieved from the UniProt database and aligned using their BLAST multiple alignment with default values for weighting array choice, gap penalties, etc.
  • FIG. 28 is a graphic illustration showing another 60 of the 180 monomers forming the isosahedral levi- and alloleviviridae capsid.
  • the backbone of each monomer of represented by a ribbon of a different color.
  • Backbone hydrogen bonds are represented by cyan lines.
  • the isosahedral three-fold axis is in the center of the figure.
  • Monomer-monomer contacts do not fill the central circle outlined by hydrogen bonds connecting the tips of flexible loops 67-81.
  • FIG. 29 is a graphic illustration showing 2 MS2 monomers (ribbons representing backbone colored dark and pale blue) surrounded by monomers in contact in the isosahedral capsid (ribbons representing monomer backbones in brown and navy).
  • the alloleviviridae Qbeta has a two residue deletion with respect to leviviridae between residues 72 & 73 (red, bottom center).
  • the central void is immediately below this deletion site ( FIG. 5 ).
  • the deletion causes it to slightly expand.
  • the Qbeta deletion at 126 removes the excursion from the segment but extensive contacts between the sheets of neighboring monomers essentially holds the monomers in place. MS2 sequence numbering is used.
  • FIG. 30 is a graphic illustration of 2 MS2 monomers (ribbons representing backbone colored dark and pale blue) surrounded by monomers in contact in the isosahedral capsid (ribbons representing monomer backbones in brown and navy).
  • the alloleviviridae Qbeta has a one residue insertion with respect to leviviridae between residues 12 & 13 (yellow, top left center), a flexible loop that extends from the outer capsid surface into solvent; a two residue insertion between residues 53 & 54 (yellow, lower left central) at the end a stand connection extending into the interior cargo; a one-residue insertion between residues 27 & 28 is also at the end of a beta-strand connector extending into the capsid cargo space. None of these insertions require movement in the monomer fold or between neighbors
  • FIG. 31 is a graphic illustration of 2 MS2 monomers (ribbons representing backbone colored dark and pale blue) surrounded by monomers in contact in the isosahedral capsid (ribbons representing monomer backbones in brown and navy).
  • the alloleviviridae Qbeta has a one residue insertion with respect to leviviridae between residues 36 & 37 (yellow, center right).
  • the loop packs against the end of the adjacent helix but inserted residues can extend into the central space above the flexible loop immediately below.
  • FIG. 32 is a graphic illustration of backbone ribbons of 3 noncovalent Enterobacteria phage MS2 noncovalent dimers packed around a symmetry point in the assembled capsid, with all of the N-termini colored green, the C-termini red.
  • the term “cargo molecule” refers to an oligonucleotide, polypeptide or peptide molecule, which is or may be enclosed by a capsid.
  • oligonucleotide refers to a short polymer of at least two, and no more than about 70 nucleotides, preferably no more than about 55 nucleotides linked by phosphodiester bonds.
  • An oligonucleotide may be an oligodeoxyribonucleotide (DNA) or a oligoribonucleotide (RNA), and encompasses short RNA molecules such as but not limited to siRNA, shRNA, sshRNA, and miRNA.
  • peptide refers to a polymeric molecule which minimally includes at least two amino acid monomers linked by peptide bond, and preferably has at least about 10, and more preferably at least about 20 amino acid monomers, and no more than about 60 amino acid monomers, preferably no more than about 50 amino acid monomers linked by peptide bonds.
  • the term encompasses polymers having about 10, about 20, about 30, about 40, about 50, or about 60 amino acid residues.
  • polypeptide refers to a polymeric molecule including at least one chain of amino acid monomers linked by peptide bonds, wherein the chain includes at least about 70 amino acid residues, preferably at least about 80, more preferably at least about 90, and still more preferably at least about 100 amino acid residues.
  • the term encompasses proteins, which may include one or more linked polypeptide chains, which may or may not be further bound to cofactors or other proteins.
  • protein as used herein is used interchangeably with the term “polypeptide.”
  • variants are a sequence that is substantially similar to the sequence of a native or wild type molecule.
  • variants include those sequences that may vary as to one or more bases, but because of the degeneracy of the genetic code, still encode the identical amino acid sequence of the native protein.
  • variants include naturally occurring alleles, and nucleotide sequences which are engineered using well-known techniques in molecular biology, such as for example site-directed mutagenesis, and which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions.
  • nucleotide sequence variants of the invention have at least 40%, at least 50%, at least 60%, at least 70% or at least 80% sequence identity to the native (endogenous) nucleotide sequence.
  • the present disclosure also encompasses nucleotide sequence variants having at least about 85% sequence identity, at least about 90% sequence identity, at least about 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • Sequence identity of amino acid sequences or nucleotide sequences, within defined regions of the molecule or across the full-length sequence, can be readily determined using conventional tools and methods known in the art and as described herein.
  • the degree of sequence identity of two amino acid sequences, or two nucleotide sequences is readily determined using alignment tools such as the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990), which are readily available from multiple online sources. Algorithms for optimal sequence alignment are well known and described in the art, including for example in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Pearson and Lipman Proc. Natl. Acad. Sci.
  • BLAST NCBI Basic Local Alignment Search Tool
  • nucleotide sequences may be also considered “substantially identical” when they hybridize to each other under stringent conditions.
  • Stringent conditions including a high hybridization temperature and low salt in hybridization buffers which permit hybridization only between nucleic acid sequences that are highly similar.
  • Stringent conditions are sequence-dependent and will be different in different circumstance, but typically include a temperature at least about 60°, which is about 10° C. to about 15° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Salt concentration is typically about 0.02 molar at pH 7.
  • the term “conservative variant” refers to a nucleotide sequence that encodes an identical or essentially identical amino acid sequence as that of a reference sequence. Due to the degeneracy of the genetic code, whereby almost always more than one codon may code for each amino acid, nucleotide sequences encoding very closely related proteins may not share a high level of sequence identity. Moreover, different organisms have preferred codons for many amino acids, and different organisms or even different strains of the same organism, e.g., E. coli strains, can have different preferred codons for the same amino acid.
  • a first nucleotide acid sequence which encodes essentially the same polypeptide as a second nucleotide acid sequence is considered substantially identical to the second nucleotide sequence, even if they do not share a minimum percentage sequence identity, or would not hybridize to one another under stringent conditions. Additionally, it should be understood that with the limited exception of ATG, which is usually the sole codon for methionine, any sequence can be modified to yield a functionally identical molecule by standard techniques, and such modifications are encompassed by the present disclosure.
  • the present disclosure specifically contemplates protein variants of a native protein, which have amino acid sequences having at least 15%, at least 16%, at least 21%, at least 40%, at least 41%, at least 52%, at least 53%, at least 56%, at least 59% or at least 86% sequence identity to a native nucleotide sequence.
  • the degree of sequence identity between two amino acid sequences may be determined using the BLASTp algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which an identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • polypeptides may be “substantially similar” in that an amino acid may be substituted with a similar amino acid residue without affecting the function of the mature protein.
  • Polypeptide sequences which are “substantially similar” share sequences as noted above except that residue positions, which are not identical, may have conservative amino acid changes.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acid substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • a nucleic acid encoding a peptide, polypeptide or protein may be obtained by screening selected cDNA or genomic libraries using a deduced amino acid sequence for a given protein. Conventional procedures using primer extension procedures, as described for example in Sambrook et al., can be used to detect precursors and processing intermediates.
  • VLP'S VIRUS-LIKE PARTICLES COMPOSED OF A CAPSID ENCLOSING A CARGO MOLECULE
  • compositions described herein are the result in part of the appreciation that certain viral capsids can be prepared and/or used in novel manufacturing and purification methods to improve commercialization procedures for nucleic acids.
  • the methods described herein use recombinant viral capsids which are resistant to readily available hydrolases, to enclose heterologous cargo molecules such as nucleic acids, peptides, or polypeptides including proteins.
  • the capsid may be a wild type capsid or a mutant capsid derived from a wild type capsid, provided that the capsid exhibits resistance to hydrolysis catalyzed by at least one hydrolase acting on peptide bonds when the capsids are contacted with the hydrolase.
  • the phrases “resistance to hydrolysis” and “hydrolase resistant” refer to any capsid which, when present in a whole cell lysate also containing polypeptides which are cell lysis products and not enclosed in the capsids, and subjected to hydrolysis using a peptide bond hydrolase category EC 3.4 for a time and under conditions sufficient for at least 60, at least 70, at least 80, or at least 90 of every 100 individual polypeptides present in the lysate (which are cell lysis products and not enclosed in the capsids) to be cleaved (i.e.
  • Hydrolysis may be conducted for a period of time and under conditions sufficient for the average molecular weight of cell proteins remaining from the cell line following hydrolysis is less than about two thirds, less than about one half, less than about one third, less than about one fourth, or less than about one fifth, of the average molecular weight of the cell proteins before the hydrolysis is conducted.
  • Methods may further comprise purifying the intact capsid remaining after hydrolysis, and measuring the weight of capsids and the weight of total dry cell matter before and after hydrolysis and purification, wherein the weight of capsids divided by the weight of total dry cell matter after hydrolysis and purification is at least twice the weight of capsids divided by the weight of total dry cell matter measured before the hydrolysis and purification.
  • the weight of capsids divided by the weight of total dry cell matter after hydrolysis and purification may be at least 10 times more than, preferably 100 times more than, more preferably 1,000 times more than, and most preferably 10,000 times more than the weight of capsids divided by the weight of total dry cell matter measured before such hydrolysis and purification.
  • Hydrolases are enzymes that catalyze hydrolysis reactions classified under the identity number EC 3 by the European Commission. For example, enzymes that catalyze hydrolysis of ester bonds have identity numbers starting with EC 3.1. Enzymes that catalyze hydrolysis of glycosidic bonds have identity numbers starting with EC 3.2. Enzymes that catalyze hydrolysis of peptide bonds have identity numbers starting with EC 3.4. Proteases, which are enzymes that catalyze hydrolysis of proteins, are classified using identity numbers starting with EC 3.4, including but not limited to Proteinase K and subtilisin. For example, Proteinase K has identity number EC 3.4.21.64.
  • VLP's which are resistant, in non-limiting example, Proteinase K, Protease from Streptomyces griseus , Protease from Bacillus lichenformis , pepsin and papain, and methods and processes of using such VLP's.
  • the Nomenclature Committee of the International Union of biochemistry and Molecular Biology also recommends naming and classification of enzymes by the reactions they catalyze. Their complete recommendations are freely and widely available, and for example can be accessed online at http://enzyme.expasy.org and, www.chem.qmul.ac.uk/iubmb/enzyme/, among others.
  • the IUBMB developed a shorthand for describing what sites each enzyme is active against. Enzymes that indescriminately cut are referred to as broadly specific.
  • Cleavage patterns for the other enzymes are described as Xaa
  • Some enzymes have more binding requirements than this so the description can become more complicated. For an enzyme that catalyzes a very specific reaction, for example an enzyme that processes prothrombin to active thrombin, then that activity is the basis of the cleavage description. In certain instances the precise activity of an enzyme may not be clear, and in such cases, cleavage results against standard test proteins like B-chain insulin are reported.
  • enzymes that catalyze hydrolysis of peptide bonds which have identity numbers starting with EC 3.4 broadly specific enzymes can be used which have Xaa
  • the capsids can be further selected and/or prepared such that they can be isolated and purified using simple isolation and purification procedures, as described in further detail herein.
  • the capsids can be selected or genetically modified to have significantly higher hydrophobicity than a surrounding matrix as described herein, so as to selectively partition into a non-polar water-immiscible phase into which they are simply extracted.
  • a capsid may be selected or genetically modified for improved ability to selectively crystallize from solution.
  • capsids Use of simple and effective purification processes using the capsids is enabled by the choice of certain wild type capsids, or modifications to the amino acid sequence of proteins comprising the wild type capsids, such that the capsid exhibits resistance to hydrolysis catalyzed by at least one hydrolase acting on peptide bonds as described herein above.
  • Such wild type capsids such as the wild type MS2 capsid, can be used in a purification process in which certain inexpensive enzymes such as Proteinase K or subtilisin are used for proteolysis.
  • a non-limiting example is the Enterobacteria phage MS2 (SEQ ID NO: 1, whole MS2 wild type genome; SEQ ID NO: 2, MS2 wild type coat protein, DNA sequence; and SEQ ID NO: 3, MS2 wild type coat protein, amino acid sequence.
  • MS2 wild type coat protein amino acid sequence: MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVR QSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNS DCELIVKAMQGLLKDGNPIPSAIAANSGIY
  • the unmodified, wild type MS2 capsid though lacking an envelope is resistant to a variety of category EC 3.4 hydrolases, including but not limited to Proteinase K and subtilisin, such that a highly purified composition of the capsid, which may contain a cargo molecule, can be prepared from a whole cell lysate.
  • the present disclosure provides VLPs comprising viral capsids comprising the wild type MS2 capsid protein, and/or capsid proteins sharing homology with wild type MS2 capsid proteins, which viral capsids encapsidate the cargo molecule.
  • the cargo molecule may comprise one or more heterologous nucleic acids, peptides, polypeptides or proteins.
  • VLP's can then be isolated and purified from a whole cell lysate after a hydrolysis step using a category EC 3.4 hydrolase, to produce a composition of VLP's of high purity, for example at least 60%, at least 70%, a least 80%, or at least 85% by weight VLP's.
  • Compositions having a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and 98% by weight of VLP's are expressly contemplated.
  • the present disclosure encompasses a composition
  • a composition comprising: a) a plurality of virus-like particles each comprising a wild type viral capsid and at least one target heterologous cargo molecule enclosed in the wild type viral capsid; and b) one or more cell lysis products present in an amount of less than 40 grams, less than 30 grams, less than 20 grams, less than 15 grams, less than 10 grams, and preferably less than 9, 8, 7, 6, 5, 4, 3, more preferably less than 2 grams, and still more preferably less than 1 gram, for every 100 grams of capsid present in the composition, wherein the cell lysis products are selected from proteins, polypeptides, peptides and any combination thereof. Subsequently the cargo molecules can be readily harvested from the capsids. Accordingly, such compositions are highly desirable for all applications where high purity and/or high production efficiency is required.
  • Hydrolase resistant capsids as described herein may be used to enclose different types of cargo molecules to form a virus-like particle.
  • the cargo molecule can be but is not limited to any one or more oligonucleotide or oligoribonucleotide (DNA, RNA, LNA, PNA, siRNA, shRNA, sshRNA, lshRNA or miRNA, or any oligonucleotide comprising any type of non-naturally occurring nucleic acid), any peptide, polypeptide or protein.
  • a cargo molecule which is an oligonucleotide or oligoribonucleotide may be enclosed in a capsid with or without the use of a linker.
  • a capsid can be triggered for example to self-assemble from capsid protein in the presence of nucleotide cargo, such as an oligoribonucleotide.
  • a capsid as described herein may enclose a target heterologous RNA strand, such as for example a target heterologous RNA strand containing a total of between 1,800 and 2,248 ribonucleotides, including the 19-mer pack site from Enterobacteria phage MS2, such RNA strand transcribed from a plasmid separate from a plasmid coding for the capsid proteins, as described by Wei, Y. et al. (2008) J. Clin. Microbiol. 46:1734-1740.
  • RNA interference is a phenomenon mediated by short RNA molecules such as siRNA molecules, which can be used for selective suppression of a target gene of interest, and has multiple applications in biotechnology and medicine.
  • short RNA molecules can be employed to target a specific gene of interest in an organism to obtain a desirable phenotype.
  • Short RNA molecules, including siRNA are however easily degraded by ubiquitous enzymes called RNAses. Capsids, such as those described herein, protect encapsidated RNA from enzymatic degradation.
  • a capsid as described herein may however enclose an RNA strand containing one or more ribozymes, either self-cleaving ribozymes (cis-acting), or in certain cases capable of cleaving bonds in other RNA (trans-acting).
  • One or more ribozymes may be included for example to specifically cut RNA sequence(s) to produce a specifically tailored RNA molecule, such as for example but not limited to an siRNA molecule.
  • variants of Hammerhead and Hepatitis Delta Virus ribozymes are known and can be used to cut long RNA sequences.
  • novel VLPs comprising a capsid encapsidating one or more ribozymes attached to pack sequences as described above (i.e., RNA sequences with strong affinity to the interior wall of a capsid), and the ribozymes used to cut short RNA sequences from packing sequences attached to the ribozymes.
  • RNA sequences such as siRNA, shRNA, sshRNA, and miRNA sequences from the packing sequence(s) used to encapsidate them.
  • short RNA encompasses short single stranded and short hairpin (stem loop) RNA sequences having a double stranded stem and a single-stranded loop or hairpin.
  • One or more short RNA sequences can also be encapsidated into a viral capsid, either wild type or genetically modified, which has been modified to insert an external peptide tag, to deliver a protein or drug molecule to a specific class of cell.
  • Wild type capsids may also be genetically modified to insert external peptide sequences acting as ligands for certain surface protein cell receptors can be advantageously used to encapsidate short RNA sequences aimed at inducing RNAi in specific target cells.
  • Such compositions are much simpler, less expensive and more reliably manufactured than current alternatives for short RNA delivery.
  • VLP's which include one or more ribozymes the disclosure further contemplates VLPs containing the resulting products after the ribozymes have cut the RNA.
  • VLP's which include a transcription terminator can use for example the T7 transcription terminator as described in Studier F W, Moffatt B A. (1986) J. Mol. Biol. May 5; 189(1):113-30; and R Sousa, D Patra, E M Lafer (1992) J. Mol. Biol. 224:319-334.
  • T7 RNA polymerase the following two sequences are suitable transcription terminators for T7 RNA polymerase:
  • VLPs as described herein may alternatively enclose at least one target peptide, polypeptide or protein.
  • an oligonucleotide linker can be used to couple the target heterologous cargo molecule and the viral capsid.
  • a cargo molecule which is a peptide, polypeptide or protein preferably is packaged in a capsid using a linker. The packaging process is promoted by the linker, consisting of a short RNA aptamer sequence, which forms a link between the coat protein and a peptide tag fused to the target cargo molecule. (See Fiedler, J.
  • the oligonucleotide linker may consist of DNA, RNA, LNA, PNA, and the like.
  • the linker is for example a 50- to 100-mer having a short sequence, for example about 20 nt long, at a first end with binding specificity for the inside of the capsid coat, and another sequence, for example about 70 nt long, at the second, opposite end which has a binding specificity for the cargo peptide, polypeptide or protein.
  • a slow ribozyme may be incorporated into a linker consisting of RNA.
  • a slow ribozyme can be incorporated between the packing sequence (binding to the coat protein) and the aptamer (binding to the tag of target protein). Upon activation, the ribozyme will separate the coat protein from the target protein.
  • a capsid as described herein may enclose at least one target protein N-terminally tagged with a peptide able to non-covalently bind to an aptamer- and capsid pack sequence-containing RNA strand, for example an N-terminal tag and aptamer- and pack sequence-containing RNA strand as described by Fiedler, J. et al. (2010).
  • a cargo molecule can be a bi-molecular cargo molecule, and capsids described herein may also encapsidate a bi-molecular cargo molecule, which may or may not include one or more ribozymes.
  • a bi-molecular cargo molecule may comprise an aptamer linked to a bifunctional polynucleotide. The aptamer may have a sequence specifically selected using SELEX to exhibit specific binding to a bioactive small molecule, i.e., a molecule having a low molecular weight, preferably lower than 1,500 Da.
  • the bifunctional polynucleotide has both a first aptameric activity for binding the low-molecular weight bioactive cargo molecule, and a second aptameric activity for binding a packing sequence of a capsid.
  • the bifunctional polynucleotide linked to the bioactive cargo molecule forms the bi-molecular cargo molecule which can then be linked to the capsid.
  • Such a cargo molecule can be used to bind the bioactive small molecule, and thus load the VLP with the small molecule.
  • the present disclosure thus also encompasses a VLP comprising a capsid linked to such a synthetic bi-molecular cargo molecule.
  • Examples of low molecular weight bioactives which can be loaded into a VLP by binding to an RNA aptamer include herbicides such as 2,4-D (2,4-Dichlorophenoxyacetic acid), December ((3,6-dichloro-2-methoxybenzoic acid), Paraquat (N,N′-dimethyl-4,4′-bipyridinium dichloride), Oryzalin (4-(dipropylamino)-3,5-dinitrobenzenesulfonamide), DCMU (3,4-dichlorophenyl)-1,1-dimethylurea), Trifluralin (2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline), Imazapic (-methyl-2-[4-methyl-5-oxo-4-(propan-2-yl)-4,5-dihydro-1H-imidazol-2-yl]pyridine-3-carboxylic acid), Aminopyralid (4-a
  • RNA aptamers can also be used to bind insecticides such as, Propargite (2-(4-tert-butylphenoxy)cyclohexyl prop-2-yne-1-sulfonate), Chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate), Cypermethrin, Phosmet (2-Dimethoxyphosphinothioylthiomethyl)isoindoline-1,3-dione), Permethrin (3-Phenoxybenzyl (1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate), Diazinon (O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl]phosphorothioate), Methylparathion (O,O-Dimethyl O-(4-nitrophen
  • aptamers have been described to bind acetamiprid, phorate, profenofos, isocarbophos and omethoate, as exemplified by Sett et al. (2012) Open Journal of Applied Biosensor, 1:p. 9-19 using DNA aptamers built in a similar manner as RNA aptamers are built using SELEX.
  • These herbicides, insecticides or fungicides are bioactive small molecules, i.e., molecules having a low molecular weight, preferably lower than 1,500 Da. Due to their small size they can permeate capsids forming VLPs of the current disclosure, as exemplified by Wu et al. (2005) Delivery of antisense oligonucleotides to leukemia cells by RNA bacteriophage capsids, Nanomedicine: Nanotechnology, Biology and Medicine, 1:p. 67-76. These small bioactive molecules are added to VLPs of the current disclosure which encapsidate aptamers designed using SELEX to bind the small bioactive molecules, after such VLPs have been formed, either before or after purification.
  • Suitable solvents used for loading the small bioactive molecules into the VLPs range from polar such as water and water-ethanol blends to non-polar such as, for example, isooctane, toluene, dichloromethane, or chloroform. Using non-polar solvents for the dissolution of VLPs is done, for example, as described by Johnson et al.
  • VLPs encapsidating both siRNA and small bioactive molecules are preferred in applications where a synergistic effect is achieved between the two bioactive ingredients, for example in those cases where the targeted plant, insect or fungus is resistant to the small bioactive molecule.
  • the siRNA is designed to target the biologic pathway that confers the plant, insect or fungus resistance to the small bioactive molecule, as exemplified by Sammons et al., Polynucleotide molecules for gene regulation in plants, US 2011/0296556.
  • the bi-functional polynucleotide may encode at least one siRNA, shRNA, sshRNA, lshRNA or miRNA, and the cargo molecule can be a small (low molecular weight) protein or peptide. Accordingly, a bi-molecular cargo molecule can be capable of binding a low molecular bioactive protein or peptide.
  • Such a bi-molecular cargo molecule may comprise a biologically active protein or peptide, coupled to a polynucleotide encoding at least one siRNA or shRNA or sshRNA or lshRNA of miRNA, and having a first aptameric activity for binding the bioactive protein or peptide cargo molecule and a second aptameric activity for binding a packing sequence of a capsid.
  • the polynucleotide is linked to the protein or peptide cargo molecule and is capable of linking to packing sequence of a capsid.
  • a bifunctional polynucleotide as described above may optionally include one or more ribozyme sequences.
  • a VLP including a bi-molecular cargo molecule including a bifunctional polynucleotide as described above may optionally include one or more ribozymes.
  • the present disclosure also encompasses a VLP comprising a capsid and reaction products of the bi-molecular cargo molecule after at least one ribozyme has reacted with bimolecular cargo molecule to cut the cargo molecule into constituent parts including the aptamer.
  • VLPs as described herein may be assembled by any available method(s) which produces a VLP with an assembled, hydrolase resistant capsid encapsidating one or more cargo molecule(s), and optionally any linker, packing sequence, one or more ribozymes, or tags.
  • capsids and cargo molecules may be co-expressed in any expression system.
  • Recombinant DNA encoding one or more capsid proteins, one or more cargo molecule(s), and optionally any linker, packing sequence, ribozyme(s) or tags can be readily introduced into the host cells, e.g., bacterial cells, plant cells, yeast cells, fungal cells, and animal cells (including insect and mammalian) by transfection with one or more expression vectors by any procedure useful for introducing such a vector into a particular cell, and stably transfecting the cell to yield a cell which expresses the recombinant sequence(s).
  • the host cell is preferably of eukaryotic origin, e.g., plant, mammalian, insect, yeast or fungal sources, but non-eukaryotic host cells may also be used.
  • Suitable expression systems include but are not limited to microorganisms such as bacteria (e.g., E. coli ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequences for the VLP elements.
  • E. coli e.g., E. coli
  • expression in E. coli is a suitable expression system.
  • the present disclosure expressly contemplates plant cells which have been transformed using a nucleic acid construct as described herein, and which expresses a capsid coat protein, cargo molecule and a and optionally any linker, packing sequence, one or more ribozymes, or tags.
  • Means for transforming cells including plant cells and preparing transgenic cells are well known in the art.
  • Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments can be used to transform cells and will as generally recognized include promoters, enhancers, and/or polylinkers.
  • Transgenic cells specifically contemplated include transgenic plant cells including but not limited to cells obtained from corn, soybean, wheat, vegetables, grains, legumes, fruit trees, and so on, or any plant which would benefit from introduction of a VLP as described herein. Also contemplated are plants, plant tissue obtained from cells transformed as described herein, and the seed or progeny of the plant or plant tissue.
  • Expression of assembled VLPs can be obtained for example by constructing at least one expression vector including sequences encoding all elements of the VLP. Sometimes two vectors are used, a first which includes a sequence encoding the cargo molecule(s) and optionally any linker, packing sequence, one or more ribozymes, or tags; and a second vector which includes a sequence encoding the capsid protein. In an exemplary process for generating exemplary VLPs including siRNA, two vectors may be co-expressed in the host cell for generation of the VLP, as further detailed in the Examples. Methods and tools for constructing such expression vectors containing the coding sequences and transcriptional and translational control sequences are well known in the art.
  • Vector(s) once constructed are transferred to the host cells also using techniques well known in the art, and the cells then maintained under culture conditions for a time sufficient for expression and assembling of the VLP's to occur, all using conventional techniques.
  • the present disclosure thus encompasses host cells containing any such vectors, and cells which have been transformed by such vectors, as well as cells containing the VLP's.
  • the VLP's When the VLP's have been expressed and assembled in the host cells, they may be isolated and purified using any method known in the art for virus purification.
  • the cells can be lysed using conventional cell lysis techniques and agents, and the cell lysate subjected to hydrolysis using at least one peptide bond hydrolase category EC 3.4 such as but not limited to Proteinase K or subtilisin. Intact capsids remaining in the cell lysate following hydrolysis can be removed and purified using conventional protein isolation techniques.
  • Purification of capsids, VLPs or proteins may also include methods generally known in the art.
  • the resulting lysate can be subjected to one or more isolation or purification steps.
  • Such steps may include for example enzymatic lipolysis, DNA hydrolysis, and proteolysis steps.
  • a proteolysis step may be performed for example using a blend of endo- and exo-proteases.
  • capsids with their cargo molecules can be separated from surrounding matrix by extraction, for example into a suitable non-polar water-immiscible solvent, or by crystallization from a suitable solvent.
  • hydrolysis and/or proteolysis steps transform contaminants from the capsid that are contained in the lysate matrix into small, water soluble molecules.
  • Hydrophobic capsids may then be extracted into an organic phase such as 1,3-bis(trifluoromethyl)benzene.
  • Purification of capsids, VLPs or proteins may include for example at least one liquid-liquid extraction step, at least one fractional precipitation step, at least one ultrafiltration step, or at least one crystallization step.
  • a liquid-liquid extraction may comprise for example use of an immiscible non-aqueous non-polar solvent, such as but not limited to benzene, toluene, hexane, heptane, octane, chloroform, dichloromethane, or carbon tetrachloride.
  • Purifying may include at least one crystallization step.
  • Use of one or more hydrolytic steps, and especially of one or more proteolytic steps eliminates certain problems observed with current separation processes used for cargo molecules, which are mainly result from the large number and varying degree of binding interactions which take place between cargo molecules and components derived from the cell culture in which they are produced.
  • the capsids described herein resist hydrolytic steps such that the matrix which results after hydrolysis includes intact capsids which safely partition any cargo molecules from the surrounding matrix, thereby interrupting the troublesome binding interactions which interfere with current purification processes.
  • the capsid can be opened to obtain the cargo molecule, which maybe a protein or polypeptide, a peptide, or a nucleic acid molecule as described herein.
  • Capsids can be opened using any one of several possible procedures known in the art, including for example heating in an aqueous solution above 50° C.; repeated freeze-thawing; incubating with denaturing agents such as formamide; by incubating with one or more proteases; or by a combination of any of these procedures.
  • Capsid proteins which are resistant to hydrolases and useful in the VLPs and methods according to the present disclosure can also be variants of, or derived from the wild type MS2 capsid protein.
  • Capsid proteins may comprise, for example, at least one substitution, deletion or insertion of an amino acid residue relative to the wild type MS2 capsid amino acid sequence.
  • Such capsid proteins may be naturally occurring variants or can be obtained by genetically modifying the MS2 capsid protein using conventional techniques, provided that the variant or modified capsid protein forms a non-enveloped capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4 as described herein.
  • MS2 capsid proteins which can assemble into capsids which are resistant to hydrolysis as described herein can be engineered by making select modifications in the amino acid sequence according to conventional and well-known principles in physical chemistry and biochemistry to produce a protein which retains resistance to hydrolysis as described herein and in the Examples herein below.
  • the shape or global fold of a functional protein is determined by the amino acid sequence of the protein, and that the fold defines the protein's function.
  • the global fold is comprised of one or more folding domains. When more than one folding domain exists in the global fold, the domains generally bind together, loosely or tightly along a domain interface.
  • the domain fold can be broken down into a folding core of tightly packed, well-defined secondary structure elements which is primarily responsible for the domain's shape and a more mobile outer layer typically comprised of turns and loops whose conformations are influenced by interactions with the folding core as well as interactions with nearby domains and other molecules, including solvent and other proteins.
  • VLPs according to the present disclosure and as used in any of the methods and processes, thus encompass those comprising a capsid protein having at least 15%, 16%, 21%, 40%, 41%, 52%, 53%, 56%, 59% or at least 86% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • Such VLPs include for example a VLP comprising a capsid protein having at least 52% sequence identity with SEQ ID NO: 3) as described above.
  • VLP comprising a capsid protein having at least 53% sequence identity to SEQ ID NO:3, which can be obtained substantially as described above but not disregarding the FR capsid sequence, representing 53% sequence identity to wild-type enterobacteria phage MS2 capsid protein (SEQ ID NO:3). Also included is a VLP comprising a capsid protein having at least 56% sequence identity to SEQ ID NO:3, when it is considered that when the structures identified as 1AQ3 (van den Worm, S. H., Stonehouse, N. J., Valegard, K., Murray, J. B., Walton, C., Fridborg, K., Stockley, P. G., Liljas, L. (1998) Nucleic Acids Res.
  • VLP comprising a capsid protein having at least 59% sequence identity to SEQ ID NO:3, when it is considered that the sequence of the MS2 viral capsid protein compared to that of the GA viral capsid protein is 59%. Also included is a VLP comprising a capsid protein having at least 86% sequence identity to SEQ ID NO:3, when it is considered that the sequence of the MS2 viral capsid protein compared to that of the FR capsid protein is 86%.
  • VLPs according to the present disclosure thus encompass those comprising a capsid protein having at least 15%, 16%, or 21% sequence identity with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) based on a valid structure anchored alignment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a VLP may thus comprise any of the MS2 capsid protein variants as described herein.
  • Genetically modified capsid proteins consistent with those described herein can be produced for example by constructing at least one DNA plasmid encoding at least one capsid protein having at least one amino acid substitution, deletion or insertion relative to the amino acid sequence of the wild type MS2 capsid protein, making multiple copies of each plasmid, transforming a cell line with the plasmids; maintaining the cells for a time and under conditions sufficient for the transformed cells to express and assemble capsids encapsidating nucleic acids; lysing the cells to form a cell lysate; subjecting the cell lysate to hydrolysis using at least one peptide bond hydrolase, category EC 3.4; and removing intact capsids remaining in the cell lysate following hydrolysis to obtain capsids having increased resistance to at least one hydrolase relative to the wild type capsid protein. Following purification of the resulting, intact capsids,
  • the specialized capsids described herein can be used in research and development and in industrial manufacturing facilities to provide improved yields, since the purification processes used in both settings have the same matrix composition. Having such same composition mainly depends on using the same cell line in both research and development and manufacturing processes. However, differences in matrix composition due to using different cell lines are greatly reduced after proteolytic steps used in both research and development and manufacturing stages. This feature enables use of different cell lines in both stages with a minimal manufacturing yield penalty.
  • MS2 bacteriophage ATCC No. 15597-B1, from American Type Culture Collection, Rockville, Md.
  • E. Coli host ATCC No. 15669
  • OD Optical Density
  • ODi represents OD immediately after inoculation with host. Infection was done at 2.3 hours.
  • Ln(OD/ODi) was plotted on the left axis (full diamonds) and pH was plotted on the right axis (open squares). This experiment was ended 5.3 hours after inoculation with host. Lysate obtained was centrifuged at 2,000 g and filtered through a 0.2 ⁇ m membrane to eliminate remaining bacteria and bacterial debris.
  • MS2 bacteriophage Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 2 .
  • MS2 bacteriophage Treatment of MS2 bacteriophage was conducted as follows. Samples were taken during treatment and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 3 . 4 mL lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54. A sample of the aqueous solution after extraction with Freon 11 was taken and analyzed (sample in Lane 1, FIG. 3 ). To the partially purified phage solution (130 ⁇ L) 370 ⁇ L of 20 mMCaCl 2 aqueous solution was added.
  • the mixture was incubated at 37° C. and after 1 hour it was placed in an ice-water bath. A sample was then taken and analyzed: sample in Lane 2, FIG. 3 .
  • the incubation product was diluted to 2 mL with deionized (DI) water and filtered through 100 kDa membrane.
  • the retentate 150 ⁇ L was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time.
  • a sample of the retentate was then taken and analyzed: sample in Lane 3, FIG. 3 . Only weak bands at lower than 10 kDa were observed, indicating complete degradation of phage.
  • MS2 bacteriophage Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 3 . 4 mL lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54. The aqueous solution containing partially purified phage was diluted to 2 mL with deionized water, filtered through a 300 kDa membrane and the filtrate was filtered through a 100 kDa membrane, from which 1504, of retentate was obtained.
  • trichlorofluoromethane Reon 11
  • the retentate was then diluted to 2 mL with deionized (DI) water and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (150 ⁇ L) was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 4, FIG. 3.370 ⁇ L of 20 mMCaCl 2 aqueous solution was added to the retentate (130 ⁇ L). The mixture was incubated at 37° C. and after 1 hour it was placed in an ice-water bath. A sample was then taken and analyzed: sample in Lane 5, FIG. 3 .
  • DI deionized
  • the product was then diluted to 2 mL with deionized (DI) water and filtered through a 100 kDa membrane.
  • the retentate 150 ⁇ L was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time.
  • a sample of the retentate was then taken and analyzed: sample in Lane 6, FIG. 3 .
  • the product obtained contained phage with purity higher than 99%.
  • MS2 bacteriophage Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 4 .
  • Example A 4 mL lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54.
  • the aqueous solution containing partially purified phage was diluted to 2 mL with deionized water, filtered through a 100 kDa membrane, from which 150 ⁇ L of retentate was obtained.
  • the retentate was then diluted to 2 mL with deionized (DI) water and filtered through the same 100 kDa membrane.
  • DI deionized
  • Dilution and ultrafiltration of the retentate was repeated one more time.
  • a sample of the retentate was then taken and analyzed: sample in Lane 1, FIG. 4 .
  • 0.15 mg of Proteinase K dissolved in 370 ⁇ L of 20 mMCaCl 2 aqueous solution was added to the retentate (130 ⁇ L).
  • the mixture was incubated at 37° C. and after 1 hour it was placed in an ice-water bath.
  • a sample was then taken and analyzed: sample in Lane 2, FIG. 4 .
  • the product was then diluted to 2 mL with deionized (DI) water and filtered through a 100 kDa membrane.
  • DI deionized
  • the retentate (150 ⁇ L) was diluted to 2 mL with DI water and filtered again through the same membrane. Dilution and ultrafiltration of the retentate was repeated one more time. A sample of the retentate was then taken and analyzed: sample in Lane 3, FIG. 4 .
  • the product obtained contained phage with purity higher than 99%.
  • MS2 bacteriophage Purification of MS2 bacteriophage was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 5 . 50 mL lysate obtained at end of Example A was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54. A sample of the aqueous solution after extraction with Freon 11 was taken and analyzed (sample in Lane 1, FIG. 5 ).
  • the liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 30 min. The supernatant was allowed to reach room temperature and 130 ⁇ L of 1% NaOH was added to bring the pH of the liquid to 8. 0.81 mL of ethanol at room temperature was slowly added with vigorous agitation to bring the ethanol concentration in the liquid to 20%. The liquid was kept at room temperature for 30 min and centrifuged at 16,000 g at room temperature for 30 min. The supernatant was placed in an ice/water bath for 15 min and 1.3 mL of 1% acetic acid was slowly added at 0° C. with vigorous agitation to bring the pH of the liquid to 4. 1.5 mL of ethanol at 0° C.
  • MS2's coat protein of 14 kDa, retained by a membrane through which proteins with less than 100 kDa molecular weight are able to permeate, is clearly visible, consistent with the presence of intact MS2 capsids.
  • a UV spectrum on the same retentate is shown in FIG. 6 , which is consistent with results published by G. F. Rohrmann and R. G. Krueger, (1970) J. Virol., 6(3):26 for pure MS2 phage.
  • F forward
  • R reverse
  • OD(600 nm) was 0.621 hour after of infection and dropped to 0.21 after 2 additional hours, while during the same time a control sample attained OD(600 nm) of 0.82 1 hour after infection and 1.2 after 2 additional hours, as shown in FIG. 7 .
  • This test showed a highly infectious phage in the retentate and therefore demonstrated that the purification processes used to isolate it did not compromise its integrity.
  • the product obtained contained MS2 bacteriophage with purity higher than 99%.
  • MS2 bacteriophage Purification of MS2 bacteriophage using different exogenous proteases was attempted substantially as described in Example E, with the exception that proteases other than Proteinase K were used.
  • MS2 bacteriophage was successfully purified after proteolysis promoted by Protease from Bacillus licheniformis (P5380, Sigma Aldrich).
  • Protease from Bacillus licheniformis
  • P6887 porcine gastric mucosa
  • proteolysis reactions using Papain from papaya latex P3125, Sigma Aldrich
  • MS2 capsids were conducted as follows. Samples were taken during the course of expression and SDS PAGE analysis was run on the samples to monitor capsid production. Results obtained are shown in FIG. 8 .
  • the following DNA sequence, encoding MS2's coat protein and its specific RNA 19-mer PAC site was cloned into pDEST14 A252 plasmid (Life Technologies):
  • coli (Life Technologies) cells were transformed using such plasmid.
  • BL21(DE3) containing the plasmid were grown in 750 mL of LB medium containing ampicillin at 37° C., to OD(600 nm) equal to 0.8.
  • a pre-induction sample was then taken and analyzed: sample in Lane 1, FIG. 8 .
  • Isopropyl ⁇ -D-1-thiogalactopyranoside (Sigma-Aldrich) was then added to a final concentration of 1 mM.
  • Four hours post-induction cells were harvested by centrifugation at 3,000 g and 4° C. for 40 min. A sample was then taken and analyzed: sample in Lane 2, FIG. 8 .
  • MS2 capsids Purification of MS2 capsids was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 8 .
  • a fraction of the pellet from Example H equivalent to 115 mL of culture was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g.
  • the cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54.
  • the retentate was then diluted to 2 mL with phosphate buffered saline and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (150 ⁇ L) was repeated four more times. A sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 8 . MS2's coat protein, of 14 kDa, retained by a membrane through which proteins with less than 100 kDa molecular weight are able to permeate, is clearly visible, consistent with the presence of intact MS2 capsids.
  • the PCR product chromatographed in 2% agarose gel stained with Ethidium Bromide, as shown in FIG. 10 ( 304 by in Lane 1; the leftmost Lane corresponds to 1 kb plus ladder from Life Technologies), was consistent with an intact MS2 coat gene.
  • the product obtained contained MS2 capsids with purity higher than 99%.
  • MS2 VLPs Purification of MS2 VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 11 .
  • One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mMMgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54.
  • the diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 ⁇ L of retentate was obtained.
  • the retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 ⁇ L) was repeated four more times.
  • a sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 12 . Impurities in this sample represented about 5.1% of the sample weight.
  • the product obtained contained MS2 VLPs with purity of about 95%.
  • MS2 VLPs Purification of MS2 VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 13 .
  • One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54.
  • the diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 200 ⁇ L of retentate was obtained.
  • the retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 ⁇ L) was repeated four more times.
  • a sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 3, FIG. 13 . Impurities in this sample represented about 4.7% of the sample weight.
  • the product obtained contained MS2 VLPs with purity higher than about 95%.
  • MS2 VLPs Purification of MS2 VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 13 .
  • One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. The cell lysate obtained was partially purified by precipitation using ammonium sulfate and extraction using trichlorofluoromethane (Freon 11) as described by Strauss & Sinsheimer (1963) J. Mol. Biol. 7:43-54.
  • the liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. About 100 ⁇ L of 10% acetic acid aqueous solution was added to the supernatant in an ice/water bath to bring the pH of the liquid to 4.01. Then, at the same temperature and with vigorous agitation, 1.3 mL of ethanol was slowly added. The liquid was kept at 0° C. for 30 minutes and centrifuged at 16,000 g at 4° C. for 20 min. The pellet was suspended in 2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl 2 adjusted to pH 7.5.
  • the diluted sample was filtered through a Vivaspin 2 (Sartorius) 100 kDa membrane from which 2004, of retentate was obtained.
  • the retentate was then diluted to 2 mL with the same buffer and filtered through the same 100 kDa membrane. Dilution and ultrafiltration of the retentate (200 ⁇ L) was repeated four more times.
  • a sample of the retentate was then taken and analyzed by SDS PAGE: sample in Lane 4, FIG. 13 . Impurities in this sample represented about 0.9% of the sample weight.
  • the product obtained contained MS2 VLPs with purity higher than about 99%.
  • MS2 VLPs Purification of MS2 VLPs was conducted as follows. Samples were taken during purification and SDS PAGE analysis was run on the samples. Results obtained are shown in FIG. 14 .
  • One sixth of the pellet obtained from an experiment identical to Example H was resuspended in 20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and sonicated to lyse cells. Cell debris was removed by centrifugation at 16,000 g. A sample of the supernatant was taken and analyzed by SDS PAGE: sample in Lane 1, FIG. 14 . Impurities in this sample represented about 70% of the sample weight.
  • Four other identical fractions of the pellet obtained from such experiment identical to Example H were processed in the same manner.
  • the first centrifuged cell lysate was placed in an ice-water bath for 15 minutes and 0.1 grams of ammonium sulfate was added. The mixture was vortexed until complete dissolution of ammonium sulfate was achieved.
  • the liquid was kept at 0° C. for 2 hours and centrifuged at 16,000 g at 4° C. for 30 min. 0.4 grams of ammonium sulfate was added to the supernatant and vortexed until complete dissolution of ammonium sulfate was achieved. The liquid was kept at 0° C.
  • the purified MS2 VLPs pellet was suspended in 0.2 mL of an aqueous buffer consisting of 20 mM Tris-HCl and 10 mM MgCl2 adjusted to pH 7.5.
  • the second centrifuged cell lysate was incubated at 37° C. for five hours, placed in an ice-water bath for the same amount of time as the first centrifuged cell lysate and subsequently processed in identical manner as the first centrifuged cell lysate. 0.15 mg of Proteinase K (Sigma Aldrich, St. Louis, Mo.) was added to the third centrifuged cell lysate.
  • the product obtained contained MS2 VLPs with purity of about 75%. Protein concentration of this sample was 25.4 mg/mL.
  • Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.784 and OD-280 nm was 0.453. These measurements are consistent with RNA yield of about 11 mg per liter of culture.
  • the product obtained contained MS2 VLPs with purity of about 94.3%. Protein concentration of this sample was 21.0 mg/mL.
  • Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.632 and OD-280 nm was 0.321. These measurements are consistent with RNA yield of about 10 mg per liter of culture.
  • the product obtained contained MS2 VLPs with purity of about 95.6%. Protein concentration of this sample was 19.4 mg/mL.
  • Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.666 and OD-280 nm was 0.353. These measurements are consistent with RNA yield of about 11 mg per liter of culture.
  • the product obtained contained MS2 VLPs with purity of about 96%. Protein concentration of this sample was 19.8 mg/mL.
  • Optical density measured in a 1 cm cell at 260 nm (OD-260 nm) of a 200:1 dilution of this sample was 0.661 and OD-280 nm was 0.354. These measurements are consistent with RNA yield of about 11 mg per liter of culture.
  • MS2 Capsids Encapsidating shRNA Targeting Green Fluorescent Protein (GFP) and HDV Ribozyme Attached to MS2 19-Mer RNA Hairpin
  • MS2 capsids Production of MS2 capsids is conducted as follows.
  • the following DNA sequence Sequence A (SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence B (SEQ ID NO: 8) was cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of Sequence B (SEQ ID NO: 8)(not shown). Sequence B (SEQ ID NO: 8) encodes, shRNA hairpin, Hepatitis Delta Virus (HDV) ribozyme designed to cleave the 3′ end of the siRNA hairpin, and MS2's specific RNA 19-mer, was cloned into plasmid pACYC184:
  • HDV Hepatitis Delta Virus
  • MS2 capsids Production of MS2 capsids is conducted as follows.
  • the following DNA sequence Sequence A (SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence C (SEQ ID NO: 9) is cloned into plasmid pACYC184.
  • a transcription terminator is also cloned at the 3′ end of Sequence C (SEQ ID NO: 9)(not shown).
  • Sequence C (SEQ ID NO: 9) encodes T7 promoter, Hammerhead ribozyme designed to cleave the 5′ end of a siRNA hairpin, siRNA hairpin, Hepatitis Delta Virus (HDV) ribozyme designed to cleave the 3′ end of the siRNA hairpin, and MS2's specific RNA 19-mer, and another HDV ribozyme is cloned into plasmid pACYC184:
  • Sequence G76U (SEQ ID NO: 10) Sequence G40U: (SEQ ID NO: 11) Sequence Al6G: (SEQ ID NO: 12) TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCGGCCTCCTCGCTGGCGCC GGCTGGGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG Sequence G39U: (SEQ ID NO: 13) TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC TTCAGGGTCAGCTTGTCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCC GGCTGTGCAACATTCGTGGCGAATGGGACCATATATATATACATGAGGAT TACCCATGTCCATGG Sequence A78G: (SEQ ID NO: 14) TAATACGACTCACTATAGCAAGCTGACCCTGAAGTTCATCAAGAGTGAAC T
  • MS2 capsids Production of MS2 capsids is conducted as follows. The following DNA sequence, Sequence A (SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence D (SEQ ID NO: 19) is cloned into plasmid pACYC184. A transcription terminator is also cloned at the 3′ end of Sequence D (SEQ ID NO: 19)(not shown). Sequence D (SEQ ID NO: 19) encodes T7 promoter, Hammerhead ribozyme designed to cleave the 5′ end of a siRNA hairpin, siRNA hairpin, Hammerhead ribozyme designed to cleave the 3′ end of the siRNA hairpin, MS2's specific RNA 19-mer and an HDV ribozyme:
  • coli (Life Technologies) cells are transformed with the 2 plasmids one containing Sequence A (SEQ ID NO: 7) and the Sequence D (SEQ ID NO: 19) selecting for chloramphenicol and ampicillin resistant transformants.
  • these transformants are grown at 37° in 750 mL LB medium containing both ampicillin and chloramphenicol.
  • Cells are harvested 4 hours post-induction by centrifugation at 3,000 g and 4° C. for 40 min. A sample is taken prior to induction and at the time of harvest for analysis.
  • Sequence 10MNT (doesn't cut well): (SEQ ID NO: 20) Sequence 15MNT (doesn't cut well): (SEQ ID NO: 21) Sequence 20MNT (cuts well): (SEQ ID NO: 22) Sequence 22MNT (cuts well): (SEQ ID NO: 23) Sequence 25MNT (cuts well): (SEQ ID NO: 24) Sequence 27MNT (cuts well): (SEQ ID NO: 25)
  • RNA strands which cut well are those which include a Hammerhead ribozyme sequence for which proper folding of the ribozyme sequence is thermodynamically favored over the folding of the shRNA (“hairpin”) section of the whole strand. Thermodynamic parameters for each of the above sequences were calculated to determine which sequences cut well and which do not.
  • MS2 capsids Production of MS2 capsids is conducted as follows. The following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence E (SEQ ID NO: 26) is cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of Sequence E (SEQ ID NO: 26)(not shown).
  • Sequence E encodes BamHI restriction site, T7 promoter and start sequence, HDV ribozyme designed to cleave in trans mode the 3′ end of the siRNA against Enhanced Green Fluorescent Protein (siEGPF), ATAT spacer, long Hammerhead ribozyme designed to cleave the 5′ end of the siEGFP hairpin, siEGFP hairpin, complementary sequence to the HDV ribozyme cleaving in trans mode the 3′ end of the siEGFP hairpin, MS2's specific RNA 19-mer, and Pad restriction site.:
  • BamHI and HindIII site are added at the 5′ and 3′ end respectively to facilitate cloning into pACYC184 (not shown).
  • MS2 capsids Production of MS2 capsids is conducted as follows. The following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence F (SEQ ID NO: 27) is cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of Sequence F (SEQ ID NO: 27)(not shown). Sequence F (SEQ ID NO: 27) encodes T7 promoter and start sequence, 3 Hammerhead ribozymes designed to cleave the 5′ ends of the siRNAs against Enhanced Green Fluorescent Protein (siEGPF), 3 different siEGFP hairpins, 3 long Hammerhead ribozymes designed to cleave the 3′ ends of the siEGFP hairpins, MS2's specific RNA 19-mer:
  • MS2 Capsids Using a Transcript Coding for 3 Different siRNAs Targeting GFP Flanked by Spacers Located at their 5′ Ends and HDV Ribozymes at their 3′ Ends, Attached to MS2 19-Mer RNA Hairpin
  • MS2 capsids Production of MS2 capsids is conducted as follows. The following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence G (SEQ ID NO: 28) is cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of Sequence G (SEQ ID NO: 28) (not shown). Sequence G (SEQ ID NO: 28) encodes BamHI restriction site, T7 promoter and start sequence, 3 spacers at the 5′ ends of the siRNAs against Enhanced Green Fluorescent Protein (siEGPF), 3 different siEGFP hairpins, 3 HDV ribozymes designed to cleave the 3′ ends of the siEGFP hairpins, MS2's specific RNA 19-mer, and Pad restriction site.:
  • MS2 capsids Production of MS2 capsids is conducted as follows. The following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's coat protein is cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence H (SEQ ID NO: 29) was cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of sequence H (not shown).
  • MS2 Capsids Using a Transcript Coding for 2 Different siRNAs Targeting Green Fluorescent Protein (GFP) Flanked by Spacers Located at their 3′ Ends and 5′ Ends, Attached to MS2 19-Mer RNA Hairpin
  • GFP Green Fluorescent Protein
  • MS2 capsids Production of MS2 capsids is conducted as follows. The following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's coat protein was cloned into pDEST14 (Life Technologies) plasmid:
  • Sequence I (SEQ ID NO: 30) was cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of Sequence I (SEQ ID NO: 30)(not shown). Sequence I (SEQ ID NO: 30) coded for T7 promoter and start sequence, 3 spacers separating the ends of the siRNAs against Green Fluorescent Protein (siGPF), 2 different siGFP hairpins, MS2's specific RNA 19-mer:
  • MS2 capsids obtained in Examples O through X are purified using procedures outlined in Example N.
  • RNA encapsidated in MS2 capsids purified as described in Example N was extracted from each experiment using TRIzol® reagent according to the protocol supplied by the manufacturer (Life Technologies, Grand Island, N.Y.). RNA obtained was denatured by heating for 5 min at 95° C. in formamide and analyzed by electrophoresis in 17.6 cm ⁇ 38 cm ⁇ 0.04 cm (W, L, T) gels composed of 8% polyacrylamide, 8 molar urea, 1.08% Tris base, 0.55% Boric acid, and 0.093% EDTA. The running buffer had the same concentrations of Tris base, Boric acid and EDTA as the gel. Power was delivered at about 40 W.
  • RNA electrophoresis in FIG. 15 refers to the same lane numbers for protein electrophoresis in FIG. 14 .
  • a single RNA band can be observed in each lane, consistent with high purity RNA recovered in each case.
  • T7-Rz2 was used for in-vitro transcriptions. This construct was cloned into pACYC184 plasmid (New England Biolabs). One Shot BL21(DE3) Chemically Competent E. coli (Life Technologies) cells were transformed using this plasmid. BL21(DE3) containing the plasmid were grown in LB medium containing ampicillin at 37° C., to OD(600 nm) equal to 0.8. Plasmids were isolated using QIAprep® Spin Miniprep Kit (Qiagen) following manufacturer's instructions. NcoI (New England Biolabs) was used to cut isolated plasmids at the restrictions sites introduced into this construct.
  • Template T7-Rz2 encodes T7 promoter, shRNA hairpin, HDV ribozyme designed to cleave the 3′ end of the shRNA hairpin, ATATATATAT spacer, and NcoI restriction site as follows:
  • the results show that the HDV ribozyme cut.
  • the top band was the uncut transcript and the two lower bands were the expected cut pieces of RNA.
  • the gel in FIG. 16 shows a set of RNA markers in the leftmost lane, a 49 nucleotide shRNA marker in the following lane, in-vitro transcription and cutting of T7-Rz2 construct run for 1 hour at 37° C. in the third lane and in-vitro transcription and cutting of T7-Rz2 construct run for 1 hour at 37° C. and incubated one additional hour at 42° C. in the fourth, rightmost lane.
  • the slowest of the three most intense bands had a higher intensity at 37 than at 37/42.
  • the two faster bands had higher intensity at 37/42 than at 37.
  • the lowest molecular weight band in the last lane run slightly slower than the 49 nt shRNA standard, as expected for the resulting 51 nucleotide shRNA in T7Rz2.
  • the results as shown in FIG. 16 are consistent with the hypothesis that HDV ribozyme cut in T7-Rz2 .
  • T7-Rz1 and T7-Rz4 were used for in-vitro transcriptions.
  • T7-Rz1 comprises common ribozymes, i.e., with stems hybridizing the siRNA being cut of less than 6 pairs of hybridizing nucleotides.
  • T7-Rz4 comprises flanking rybozymes with long-length stems that hybridize the siRNA being cut, i.e. stems with more than 6 pairs of hybridizing nucleotides.
  • T7-Rz1 encoded T7 promoter Hammerhead (HH) ribozyme designed to cleave the 5′ end of a siRNA hairpin with 5 nucleotides complementary to siRNA, siRNA hairpin, HH ribozyme designed to cleave the 3′ end of the siRNA hairpin with 5 nucleotides complementary to siRNA, and NcoI restriction site:
  • results obtained with construct T7-Rz1 were consistent with a hypothesis that ribozymes cut to a very low extent.
  • results obtained with construct T7-Rz4 shown in FIG. 17 were consistent with a hypothesis that ribozymes cut to a significant extent.
  • MS2 capsids were produced as follows. The following DNA sequence (Sequence A; SEQ ID NO: 7), encoding MS2's coat protein was cloned into pDEST14 (Life Technologies) plasmid:
  • construct T7-Rz4 The following DNA sequence (construct T7-Rz4) was cloned into plasmid pACYC184. A transcription terminator was also cloned at the 3′ end of construct T7-Rz4 (not shown).
  • Example CC Purification of Virus Like Particles produced in Example CC. was conducted as in Example N.
  • RNA encapsidated in Virus Like Particles purified as described in Example DD were extracted using TRIzol® reagent according to the protocol supplied by the manufacturer (Life Technologies, Grand Island, N.Y.). RNA obtained was denatured by heating for 5 min at 95° C. in formamide and analyzed by electrophoresis in Novex® denaturing 15% polyacrylamide TBE-Urea gels (Life Technologies) run at 70° C. RNA bands were visualized using 0.5 ⁇ g of Ethidium Bromide (Sigma-Aldrich, St. Louis, Mo.) per mL of aqueous solution. Results obtained are shown in lane 3, FIG. 18 . Lane 1 shows a set of molecular standards. Lane 2 shows a chemically synthesized shRNA 49 nucleotides long.
  • Virus Like Particles Comprising MS2 Capsids Obtained in Example DD are Resistant to Proteinase K from Engyodontium album, licheniformis , Pepsin from Porcine Gastric Mucosa, and Papain from Papaya Latex
  • 2 ⁇ g of Protease from Bacillus licheniformis was then added to this suspension and was incubated at 37° C. Samples were taken for protein concentration and SDS PAGE analyses after 1 hour, and 4 hours of incubation. Protein concentration in these 2 samples was 1769, and 1785 mg/L respectively.
  • the MS2 viral capsid protein (SEQ.ID No. 3) has a single folding domain and belongs to fold family d.85.1 (RNA bacteriophage capsid protein) of superfamily d.85, which includes leviviridae and alloleviviridae capsid proteins.
  • Each capsid monomer in this family is made up of a 6-stranded beta sheet followed by the two helices (sometimes described as a long helix with a kink) 180 monomers assemble noncovalently to form an icosahedral (roughly spherical) viral capsid with a continuous beta-sheet layer facing the capsid interior and the alpha-helices on the capsid exterior.
  • X-ray crystal structures have been solved and placed in the public domain for the enterobacteriophage MS2, GA (UniProt sequence identifier P07234) and FR (UniProt sequence identifier P03614) viral capsids and the capsid of MS2 formed from an MS2 dimer in which one C-terminus of one MS2 has been fused to the N-terminus of another, all d.85.1 family leviviridae coat proteins.
  • the Protein Data Bank identifiers for these structures are 1AQ3 (SEQ ID NO: 34), 1GAV (SEQ ID NO: 35), 1FRS (SEQ ID NO: 36) and 2VTU (SEQ ID NO: 37), respectively, and alignment of these is shown FIG. 20 .
  • the residue numbering is sequential residue numbering, for example SEQ ID 3 starting with 0 for the lead Met (M) residue which is removed by the cell, as used for most PDB structures.
  • the sequences of MS2 viral capsid protein vs the GA and FR viral capsid proteins are 59% and 87% identical respectively. Only 56% of the sequence positions have identical sequence and topologically equivalent positions with respect to the backbone overlays when all three sequences are considered together.
  • the rms deviation of the backbone conformations of MS2 viral capsid protein vs the GA and FR viral capsid monomers are under 1 A.
  • the backbone rms deviation of 1AQ3 monomer A vs 1GAV monomer 0 is 0.89 Angstroms.
  • the backbone rms deviation of 1AQ3 monomer A vs 1FRS monomer A is 0.37 Angstroms.
  • compositionally identical proteins within an asymmetric unit generally backbone rms deviations of 1 Angstrom or greater although topologically equivalent Calpha atoms of the core tend to differ by less, about 0.45 Anstroms (Cyrus Chothia & Arthur M Lesk (1986) EMBO J. 5, 823-826).
  • 1AQ3 monomer A and 1AQ3 monomer B have rms deviation of 1.72 A (jFATCAT rigid) primarily because of conformational differences in the Lys66-Trp82 region.
  • ef108465 came from GenBank (www.ncbi.nlm.nih.gov/genbank).
  • asterisk (*) indicates conserved residues
  • x is calculated to be substitutable based on sidechain solvent accessibility, hydrogen bonding requirements and backbone conformational constraints.
  • Fifty-seven (57) residues in the sequences of these family members are conserved, or 45% of the sequences are identical to on another. Some of these sequences have an additional residue following the C-terminal Tyr129 residue of SEQ ID NO: 3, others have 1-2 residues removed from the N-terminus with respect to SEQ ID NO: 3. There are no insertions or deletions within the fold.
  • Glycines can stably fold into backbone conformations disallowed to other amino acids because its sidechain consists of a single hydrogen atom.
  • the proline sidechain is cyclized into a stiff ring which is covalently bound to its backbone nitrogen through elimination of its amide hydrogen, constraining proline to a small subset of backbone conformations with respect to the other amino acids and eliminating its ability to be a hydrogen bond donor.
  • the domain fold and domain association for assembly into capsids is stabilized by the backbone hydrogen bonding patterns that define its secondary structural units, hydrogen bonds between sidechain and backbone atoms that stabilize local structure or bind neighboring secondary structure units (e.g. helices, strands, coil, loops, turns and flexible termini) together, hydrogen bonds between the atoms of different sidechains that stabilize local structure or bind neighboring secondary structure units (e.g. helices, strands, coil, loops, turns and flexible termini) together and the close packing of hydrophobic sidechain atoms that serves to both energetically stabilize the fold through van der Waals interactions and to prevent solvent penetration into the fold which might lead to destabilization and local unfolding.
  • backbone hydrogen bonding patterns that define its secondary structural units, hydrogen bonds between sidechain and backbone atoms that stabilize local structure or bind neighboring secondary structure units (e.g. helices, strands, coil, loops, turns and flexible termini) together, hydrogen bonds between the atoms of different sidechains that
  • residues do not participate in domain fold maintenance or in domain-domain interactions. So long as their backbone conformations do not have special requirements satisfied only by Gly or cis-Pro in order to participate in the domain fold, these residues can be mutated, singly or as a group, without substantially affecting the final domain fold or the overall topology of its surface, and can be identified as a class unequivocally by surface accessibility calculations performed on known structures (See, e.g., Fraczkiewicz & Braun, J M B; Meth Enzym; J Comp Chem 19, 319 (1998)), followed by hydrogen bond analysis of known structures, all conventional techniques in the study of protein structure and function.
  • PETTERSEN THOMAS D. GODDARD, CONRAD C. HUANG, GREGORY S. COUCH, DANIEL M. GREENBLATT, ELAINE C. MENG, THOMAS E. FERRIN (2004 J Comp Chem 25, 1605-1612) with hydrogen bond criteria relaxed by 0.5 A and 30 deg); and which backbone conformations allowed by all amino acid residues except proline.
  • unstructured loops, random coils and N- & C-termini which have surface exposure but do not provide critical stabilization to the rest of the protein fold (frequently via the packing of sidechains against structured elements or the shielding of interacting faces of adjacent structured elements from solvent or in the case of capsids, cargo) are excellent candidates for (1) residue deletion if significant repositioning of the joined structured elements is not required, (2) insertion of amino acid residues if the addition of residues will not significantly alter the relative disposition of structured elements in the fold or screen surface exposed residues from satisfying their hydrogen bonding capacity with hydrogen bond donors or acceptors in the protein's environment or (3) the incorporation of naturally-occurring amino acid mutation(s) or mutation(s) to normative residues which can be covalently linked to useful moieties, e.g.
  • the simplest multiple alignment algorithms are usually available to the general public at the public domain sequence and structure data bases. These algorithms can correctly align sequences that share a very low % identity if the sequence space is populated by a continuous spectrum of sequences from a high % identity, for example 90%, to a low % identity, for example 20%. These algorithms tend to fail to correctly align clusters of sequences with the same fold when those cluster share a low % identity; however, such clusters can be successfully and unequivocally aligned if the x-ray crystal structure of one or more members of each cluster has been solved and well refined.
  • the alloleviviridae coat proteins belong to the same fold family as the leviviridae coat proteins (fold family d.85.1) and also assemble into isosahedral capsids comprised of 180 monomers.
  • the multiple alignments of the sequences of alloleviviridae coat proteins deposited in UniProt are shown in the alignment table in FIG. 27 . Sixty percent (60%) of the alloleviviridae coat protein sequence is conserved.
  • the coat proteins of levi- and alloleviviridae are both about 130 amino acid residues long but because the percent of identical residues is low, about 20%, multiple sequence alignment algorithms typically fail to correctly alignment the allolevi- against the leviviridae sequences.
  • the rms deviation is in the range 2.33-2.76 Angstroms depending upon which of the independently refined monomers is compared, primarily due to differences in the backbone disposition of N-terminal residues 1-3 and segments 8-18, 26-28, 50-55 and 67-76 (numbering references the topologically equivalent residues in the MS2 structure 1AQ3) which connect secondary structure elements, as shown in FIGS. 22-25 and described in the accompanying figure descriptions.
  • the backbone rms deviation measured by jFATCAT for independently refined monomers in 1AQ3 is 1.72 A due to conformational differences in the same regions.
  • topological alignment is shown in the table, secondary structure assignment by hydrogen bonding pattern (DSSP, W Wolfgang Kabsch & Christian Sander (1983), Biopolymers 22, 2577-2636) is indicated for 1AQ3 and segments that show the greatest deviation either because the refined backbone conformations are substantially different are because the segments were too mobile to be localized in electron density during refinement are provided in lower case. Regions which show backbone flexibility in the crystal environment are also excellent candidates for insertion/and or deletion as if the interactions between these residues and the rest of the fold was important for fold stabilization, their electron density would be localized. Appending the same information for 2VTU provides further insight into segments best adapted to accommodate change. These comparisons are captured symbolically in FIG. 26 which shows alignment of 1AQ3 vs 2VTU vs 1QBE.
  • FIG. 32 shows backbone ribbon diagrams of 3 noncovalent Enterobacteria phage MS2 noncovalent dimers packed around a symmetry point in the assembled capsid. Three noncovalent MS2 coat protein dimers are packed around a symmetry point in the assembled isosahedral capsid (dimer one right, tan & brown chains; dimer two bottom, dark and medium blue chains; dimer three upper right, medium gray and dark blue chains).
  • All chain N-termini are colored green, all C-termini are colored red.
  • the proximity of the termini mean that that the sequences of the monomers can be fused into a single chain to form a covalent dimer, either as done for 2VTU by appending one monomer after the other, i.e., creating a single protein chain that consists of (monomer residues 1-129-monomer residues 1-129) or by adding additional linking residues between the monomer sequences (monomer 1-129-linker residues-monomer 1-129) as long as the relative chain directions (from N- to C-terminus) allow a continuous peptide chain to be formed from the concatenated monomers.
  • the geometry of a beta sheet can be defined by the curvature of the sheet (Cyrus Chothia, Jiri Novotny, Robert Bruccoleri, Martin Karplus (1985) J Mol Biol 186, 651-663).
  • the tight coupling in 2VTU constrains the beta sheet to a lower curvature giving rise to an octahedral rather than an isosahedral capsid.
  • the incorporation of a linker between monomers of 0-6 residues would provide enough flexibility to allow the covalent dimer to relax into the same required for an isosahedral capsid, with physical properties likely to be more closely related to the isosahedral noncovalent capsid structure.
  • the linker will be 1-6 residues, however, for example, the covalent dimer of 2VTU actually has Ser2 deleted in the second copy. In such cases, the linker length can be 0.
  • Residues chosen for the linker should have small sidechains to avoid steric strain which can be caused by a large number of atoms packing into a relatively small volume. Strain can also be minimized by avoiding the choice of amino acid residues with smaller backbone conformational space, for example Pro. Avoiding strain can translate into a protein which folds more quickly or more efficiently. Bulkier and charged sidechains, particularly in the middle section of longer loops tend to be binding targets for proteases. Gly-containing linkers are preferred.
  • N-terminal residues 1-3 can satisfy their hydrogen bonding potential with the C-terminal residue 129 and water and vice versa; therefore, it should be possible to delete some or all of these residues and form stable VLPs with the truncated proteins or alternatively with the corresponding potential linker lengths extended by the number of deletions in concatenated proteins.
  • VLPs comprising a capsid comprising a capsid protein which is a variant of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the A residue at position 1 is deleted.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the A residue at position 1 is deleted and the S residue at position 2 is deleted.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that that the A residue at position 1 is deleted, the S residue at position 2 is deleted and the N residue at position 3 is deleted.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) except that the Y reside at position 129 is deleted.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single (1) amino acid deletion in the 112-117 segment.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single (1) amino acid deletion in the 112-117 segment.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a 1-2 residue insertion in the 65-83 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but NO:3) having a 1-2 residue insertion in the 44-55 segment.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single (1) residue insertion in the 33-43 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a 1-2 residue insertion in the 24-30 segment.
  • a VLP may comprise a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) but having a single (1) residue insertion in the 10-18 segment.
  • a VLP may comprise a capsid protein monomer sequence concatenated with a second capsid monomer sequence which assembles into a capsid which resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • a VLP may comprise a capsid protein monomer sequence whose C-terminus is extended with a 0-6 residue linker segment whose C-terminus is concatenated with a second capsid monomer sequence, all of which assembles into a capsid which resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • Suitable linker sequences include but are not limited to -(Gly) x -, where x is 0-6, or a Gly-Ser linker such as but not limited to -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly-.
  • a VLP may further comprise a capsid protein monomer sequence concatenated with a third capsid monomer sequence which assembles into a capsid which resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the C-terminus can be extended with a 0-6 residue linker segment whose C-terminus is concatenated with a third capsid monomer sequence, all of which assembles into a capsid which resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4.
  • the linker is -(Gly) x -, and x is 1, 2 or 3.

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US9822361B2 (en) 2013-06-19 2017-11-21 Apse, Inc. Compositions and methods using capsids resistant to hydrolases
US10428329B2 (en) * 2013-06-19 2019-10-01 Apse, Inc. Compositions and methods using capsids resistant to hydrolases
US20160194613A1 (en) * 2013-09-11 2016-07-07 Georgia Tech Research Corporation Compositions and methods for inhibiting gene expressions
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WO2015038915A3 (en) * 2013-09-12 2015-05-07 Apse, Llc Compositions and methods using capsids resistant to hydrolases
WO2017066484A3 (en) * 2015-10-13 2017-05-26 Carter Daniel C Nsp10 self-assembling fusion proteins for vaccines, therapeutics, diagnostics and other nanomaterial applications
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US11718851B2 (en) 2016-03-15 2023-08-08 Rnaissance Ag Llc Methods and compositions for increased double stranded RNA production
CN108795961A (zh) * 2018-05-31 2018-11-13 中华人民共和国大榭出入境检验检疫局 肠道病毒pcr质控品装甲rna及制备方法
WO2020254876A1 (en) 2019-06-18 2020-12-24 Janssen Sciences Ireland Unlimited Company Virus-like particle delivery of hepatitis b virus (hbv) vaccines

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AU2017261590B2 (en) 2019-12-12
BR112014012162A2 (pt) 2019-08-13
CA2860310A1 (en) 2013-06-27
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AU2012358267A1 (en) 2014-06-05
IL232662B (en) 2019-05-30
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CN104114696B (zh) 2017-05-03
AR089440A1 (es) 2014-08-20
SG11201402657WA (en) 2014-09-26
JP6332879B2 (ja) 2018-05-30
HK1203082A1 (zh) 2015-10-16
MX358762B (es) 2018-09-03
EP4019636A1 (en) 2022-06-29
WO2013096866A2 (en) 2013-06-27
CN104114696A (zh) 2014-10-22
JP2018038437A (ja) 2018-03-15
AU2017261590A1 (en) 2017-12-07
BR112014012162B1 (pt) 2021-09-08

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