Processes using vlps with capsids resistant to hydrolases

Abstract

Novel processes and compositions are described which use viral capsid proteins resistant to hydrolases to prepare virus-like particles to enclose and subsequently isolate and purify target cargo molecules of interest including nucleic acids such as siRNA's and shRNA's, and small peptides.

What is claimed is: 
     
         1 . A virus-like particle (VLP) comprising a capsid enclosing at least one heterologous cargo molecule and a packing sequence. 
     
     
         2 . A VLP according to  claim 1 , further comprising at least one ribozyme enclosed by the capsid. 
     
     
         3 . A VLP according to  claim 2 , wherein the heterologous cargo molecule comprises an oligonucleotide. 
     
     
         4 . A VLP according to  claim 3 , wherein the heterologous cargo molecule comprises an oligoribonucleotide is a short RNA selected from siRNA, shRNA, sshRNA, lshRNA and miRNA. 
     
     
         5 . A VLP according to  claim 4 , wherein the ribozyme is flanked by the packing sequence and the oligoribonucleotide to form a nucleic acid construct. 
     
     
         6 . A VLP according to  claim 5 , comprising at least two ribozymes, wherein each ribozyme is selected to cut one end of the short RNA. 
     
     
         7 . A VLP according to  claim 4 , further comprising 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, 
     
     
         8 . A VLP according to  claim 2 , wherein the ribozyme is selected from a Hammerhead ribozyme and a Hepatitis Delta V ribozyme. 
     
     
         9 . A VLP according to  claim 2 , wherein the ribozyme is a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the oligoribonucleotide. 
     
     
         10 . A VLP according to  claim 2 , wherein the ribozyme is 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 wildtype Hepatitis Delta V ribozyme, or a mutant HDV ribozyme having a nucleic acid sequence selected from SEQ ID Nos: 10-18. 
     
     
         11 . A VLP according to  claim 1 , wherein the capsid comprises a wild type viral capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         12 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein having at least 40% 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. 
     
     
         13 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein having 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. 
     
     
         14 . A VLP according to  claim 1 , wherein the capsid comprises a wild type Enterobacteria phage MS2 capsid protein having the amino acid sequence of SEQ ID NO: 3. 
     
     
         15 . A VLP according to  claim 1 , wherein the heterologous cargo molecule comprises a peptide or a polypeptide. 
     
     
         16 . A VLP according to  claim 15 , further comprising an oligonucleotide linker coupling the heterologous cargo molecule and the viral capsid. 
     
     
         17 . A VLP according to  claim 16 , wherein the oligonucleotide linker is an oligoribonucleotide comprising a ribozyme sequence. 
     
     
         18 . A VLP according to  claim 1 , wherein the heterologous cargo molecule comprises a bi-molecular cargo molecule comprising a bifunctional polynucleotide comprising a first apatamer 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. 
     
     
         19 . A VLP according to  claim 18 , further comprising the bioactive small molecule bound to the first aptameric sequence, wherein the bioactive small molecule comprises an herbicide or a pesticide. 
     
     
         20 . A VLP according to  claim 19 , wherein the bioactive small molecule is selected from the group consisting of: atrazine, acetamipridphorate, profenofos, isocarbophos and omethoateas. 
     
     
         21 . A nucleic acid construct comprising a nucleotide sequence that encodes a short RNA, a ribozyme and a packing sequence. 
     
     
         22 . A nucleic acid construct according to  claim 21 , wherein the short RNA is an siRNA or an shRNA. 
     
     
         23 . A nucleic acid construct according to  claim 21 , further comprising a linking nucleotide sequence of 4 to 100 nucleotides of which at least 40% are A's, at least 40% are T's, or at least 40% are U's, wherein the linking nucleotide sequence is flanked by the ribozyme and the short RNA-encoding sequence 
     
     
         24 . A nucleic acid construct according to  claim 21 , wherein the ribozyme is flanked by the short RNA and the packing sequence. 
     
     
         25 . A vector comprising a nucleic acid construct according to  claim 21 . 
     
     
         26 . A host cell comprising the vector according to  claim 25 . 
     
     
         27 . A host cell according to  claim 37 , wherein the host cell is stably transfected with the vector. 
     
     
         28 . A host cell according to  claim 37 , which is selected from a bacterial cell, a plant cell, a mammalian cell, a fungal cell, and a yeast cell. 
     
     
         29 . A host cell according to  claim 27 , wherein the host cell is further 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. 
     
     
         30 . A host cell according to  claim 29 , wherein the second nucleic acid sequence encodes 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. 
     
     
         31 . A host cell according to  claim 29 , wherein the second nucleic acid sequence encodes a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). 
     
     
         32 . A nucleic acid construct according to  claim 21 , wherein the ribozyme is a Hammerhead ribozyme or a Hepatitis Delta V ribozyme. 
     
     
         33 . A nucleic acid construct according to  claim 32 , wherein the ribozyme is a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the short RNA. 
     
     
         34 . A nucleic acid construct according to  claim 32 , wherein the ribozyme is 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, or a mutant HDV ribozyme having a nucleic acid sequence selected from SEQ ID NOs: 10-18. 
     
     
         35 . A plant or plant tissue transformed to contain the nucleic acid construct according to  claim 21 . 
     
     
         36 . A seed or progeny of the plant or plant tissue of  claim 35 , wherein the seed or progeny comprises the nucleic acid construct. 
     
     
         37 . 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. 
     
     
         38 . A composition according to  claim 37 , wherein the capsid is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         39 . A composition according to  claim 37 , wherein the capsid comprises a capsid protein having at least 40% 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. 
     
     
         40 . A composition according to  claim 37 , wherein the capsid comprises a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). 
     
     
         41 . A composition according to  claim 37 , wherein the heterologous cargo molecule comprises an oligonucleotide. 
     
     
         42 . A composition according to  claim 41 , wherein the heterologous cargo molecule comprises an oligoribonucleotide selected from siRNA, shRNA, sshRNA, lshRNA and miRNA. 
     
     
         43 . A composition according to  claim 42 , wherein each virus-like particle further comprises at least one ribozyme, wherein the ribozyme is flanked by the packing sequence and the oligoribonucleotide to form a nucleic acid construct. 
     
     
         44 . A composition according to  claim 42 , wherein the ribozyme is selected from a Hammerhead ribozyme and a Hepatitis Delta V ribozyme. 
     
     
         45 . A composition according to  claim 42 , wherein the ribozyme is a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the oligoribonucleotide. 
     
     
         46 . A composition according to  claim 42 , wherein the ribozyme is 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 wildtype Hepatitis Delta V ribozyme, or a mutant HDV ribozyme having a nucleic acid sequence selected from SEQ ID NOS: 10-18. 
     
     
         47 . A composition according to  claim 42 , wherein the oligoribonucleotide and the packing sequence are linked by a nucleotide sequence of 1 to 100 nucleotides in length, such linking sequence comprising more than 40% A's or more than 40% of U's. 
     
     
         48 . A composition according to  claim 41 , wherein the heterologous cargo molecule comprises a peptide or a polypeptide. 
     
     
         49 . A composition according to  claim 48 , further comprising an oligonucleotide linker coupling the heterologous cargo molecule and the viral capsid. 
     
     
         50 . A composition according to  claim 49 , wherein the oligonucleotide linker is an oligoribonucleotide comprising a ribozyme sequence. 
     
     
         51 . A 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. 
     
     
         52 . A method according to  claim 51 , wherein the capsids each comprises a viral capsid protein 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. 
     
     
         53 . A method according to  claim 51 , wherein the capsids each comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). 
     
     
         54 . A method according to  claim 51 , wherein the target cargo molecule comprises an oligonucleotide. 
     
     
         55 . A method according to  claim 51 , wherein the target cargo molecule comprises an oligoribonucleotide selected from siRNA, shRNA, sshRNA, lshRNA and miRNA. 
     
     
         56 . A method according to  claim 51 , wherein each virus-like particle further comprises a ribozyme, wherein the ribozyme is flanked by the packing sequence and the oligoribonucleotide to form a nucleic acid construct. 
     
     
         57 . A method according to  claim 51 , further comprising purification of the capsids following hydrolysis. 
     
     
         58 . A method according to  claim 51 , wherein the target cargo molecule comprises a peptide or a polypeptide. 
     
     
         59 . A composition produced by the method according to  claim 51 . 
     
     
         60 . 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. 
     
     
         61 . A process for purifying VLP's enclosing at least one heterologous cargo molecule, the process comprising: (a) obtaining a cell lysate comprising a plurality of the VLP's; (b) contacting the cell lysate with a protease for a time and under conditions sufficient to hydrolyze cell lysis products other than the VLP's to form a hydrolysate; and (c) isolating the VLP's from the hydrolsyate. 
     
     
         62 . The process according to  claim 61 , wherein step (c) comprises (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 90% by weight of the VLP's. 
     
     
         63 . The process according to  claim 61 , wherein step (c) comprises (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 90% by weight of the VLP's. 
     
     
         64 . The process according to  claim 61 , wherein step (c) comprises ultracentrifuging the hydrolysate to obtain a precipitate comprising at least about 90% by weight of the VLP's. 
     
     
         65 . The process according to  claim 61 , wherein the VLP's each comprise a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         66 . The process according to  claim 61 , wherein the VLP's each comprise a capsid which comprises a capsid protein having at least 40% 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. 
     
     
         67 . The process according to  claim 61 , wherein the VLP's each comprise a wild type Enterobacteria phage MS2 capsid protein (SEQ ID NO: 3). 
     
     
         68 . The process according to  claim 61 , wherein step (b) is performed for at least about 30 minutes at about 37° C. 
     
     
         69 . The process according to  claim 61 , further comprising before step (b), contacting the cell lysate with at least one of a nuclease, an amylase and a lypase for at least about 30 minutes at about 37° C. 
     
     
         70 . The process according to  claim 61 , wherein the protease is a peptide bond hydrolase category EC 3.4. 
     
     
         71 . The process according to  claim 61 , wherein the protease is selected from Proteinase K, Protease from  Streptomyces griseus , and Protease from  Bacillus lichenformis , pepsin and papain. 
     
     
         72 . The process according to  claim 61 , wherein the heterologous cargo molecule comprises an oligonucleotide. 
     
     
         73 . The process according to  claim 61 , wherein the heterologous cargo molecule comprises an oligoribonucleotide selected from siRNA, shRNA, sshRNA, lshRNA and miRNA. 
     
     
         74 . The process according to  claim 61 , wherein the VLP's each further comprise a ribozyme flanked by a packing sequence and the oligoribonucleotide to form a nucleic acid construct. 
     
     
         75 . The process according to  claim 74 , wherein the ribozyme is selected from a Hammerhead ribozyme and a Hepatitis Delta V ribozyme. 
     
     
         76 . The process according to  claim 74 , wherein the ribozyme is a Hammerhead ribozyme variant having a contiguous set of nucleotides complementary to at least 6 contiguous nucleotides of the oligoribonucleotide. 
     
     
         77 . The process according to  claim 74 , wherein the ribozyme is 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 wildtype Hepatitis Delta V ribozyme, or a mutant HDV ribozyme having a nucleic acid sequence selected from SEQ ID Nos: 10-18. 
     
     
         78 . The process according to  claim 74 , wherein the oligoribonucleotide and the packing sequence are linked by a linker sequence of at least 1 to 100 nucleotides, and comprising more than 40% A's or more than 40% of U's. 
     
     
         79 . The process according to  claim 61 , wherein the heterologous cargo molecule comprises a peptide or a polypeptide. 
     
     
         80 . The process according to  claim 79 , wherein the VLP's each further comprise an oligonucleotide linker coupling the heterologous cargo molecule and the viral capsid. 
     
     
         81 . The process according to  claim 80 , wherein the oligonucleotide linker is an oligoribonucleotide comprising a ribozyme sequence. 
     
     
         82 . A VLP according to  claim 1 , wherein the capsid comprises 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 is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         83 . A VLP according to  claim 1 , wherein the capsid comprises 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, and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         84 . A VLP according to  claim 1 , wherein the capsid comprises 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, the S residue at position 2 is deleted and the N residue at position 3 is deleted, and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         85 . A VLP according to  claim 1 , wherein the capsid comprises 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, and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         86 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a single (1) amino acid deletion in the 112-117 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         87 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a single (1) amino acid deletion in the 112-117 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         88 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) 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. 
     
     
         89 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) having a 1-2 residue insertion in the 44-55 segment and is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         90 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) 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. 
     
     
         91 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) 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. 
     
     
         92 . A VLP according to  claim 1 , wherein the capsid comprises a capsid protein with the amino acid sequence of wild type Enterobacteria phage MS2 capsid (SEQ ID NO:3) 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. 
     
     
         93 . A VLP according to  claim 1 , wherein the capsid comprises 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. 
     
     
         94 . A VLP according to  claim 1 , wherein the capsid comprises 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. 
     
     
         95 . A VLP according to claim  109 , wherein the linker sequence is -(Gly) x -, wherein x=0-6, or a Gly-Ser linker selected from -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly-. 
     
     
         96 . A VLP according to claim  109 , wherein the capsid comprises a 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. 
     
     
         97 . A VLP according to claim  109 , wherein the capsid comprises a capsid protein wherein the C-terminus is extended with a 0-6 residue linker segment whose C-terminus s 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. 
     
     
         98 . A VLP according to claim  109 , wherein the capsid comprises a capsid protein in which one or both linker sequences is -(Gly) x -, wherein x=0-6, or a Gly-Ser linker selected from -Gly-Gly-Ser-Gly-Gly-, -Gly-Gly-Ser and -Gly-Ser-Gly- which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         99 . A VLP according to claim  109 , wherein the capsid comprises a capsid protein of in which one or both linker sequences is -(Gly) x -, x=1. which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         100 . A VLP according to claim  136 , wherein the capsid comprises a capsid protein of in which one or both linker sequences is -(Gly) x -, x=2. which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         101 . A VLP according to claim  109 , wherein the capsid comprises a capsid protein of in which one or both linker sequences is -(Gly) x -, x=3. which assembles into a capsid which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4. 
     
     
         102 . A VLP according  claim 1 , wherein one or more coat protein sequences is N-terminally truncated by 1-3 residues and a linker sequence is lengthened by the number of residues deleted, and which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4, wherein the linker sequence is -(Gly) x -, wherein x=0-6. 
     
     
         103 . A VLP according  claim 1 , wherein one or more coat protein sequences is C-terminally truncated by 1 residue and a linker sequence is lengthened by the one residue and which is resistant to hydrolysis catalyzed by a peptide bond hydrolase category EC 3.4, wherein the linker sequence is -(Gly) x -, wherein x=0-6. 
     
     
         104 . A VLP according  claim 1 , wherein the first coat protein sequence in a concatenated dimer is C-terminally truncated by 1 residue and a linker sequence is 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, wherein the linker sequence is -(Gly) x -, wherein x=0-6. 
     
     
         105 . A VLP according  claim 1 , containing N- and C-terminal truncations and which is resistant to hydrolysis catalyzed by peptide bond hydrolase category EC 3.4.