Claims for Patent: 8,716,557

Summary for Patent: 8,716,557

Title:	Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in plants
Abstract:	Methods for altering the N-glycosylation pattern of proteins in higher plants are provided. The methods comprise introducing into the plant a recombinant RNAi construct that provides for the inhibition of expression of .alpha.1,3-fucosyltransferase (FucT) and .beta.1,2-xylosyltransferase (XylT) in a plant. Use of these RNAi constructs to inhibit or suppress expression of both of these enzymes, and isoforms thereof, advantageously provides for the production of endogenous and heterologous proteins having a \"humanized\" N-glycosylation pattern without impacting plant growth and development. Stably transformed higher plants having this protein N-glycosylation pattern are provided. Glycoprotein compositions, including monoclonal antibody compositions, having substantially homogeneous glycosylation profiles, and which are substantially homogeneous for the G0 glycoform, are also provided.
Inventor(s):	Dickey; Lynn F. (Cary, NC), Cox; Kevin M. (Raleigh, NC), Peele; Charles G. (Apex, NC), Wang; Ming-Bo (Canberra, AU)
Assignee:	Synthon Biopharmaceuticals B.V. (Nijmegen, NL)
Application Number:	11/624,158
Patent Claims:	1. A nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in a duckweed plant, and a second nucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said first nucleotide sequence is operably linked to a first promoter that is functional in a plant cell, and wherein said second nucleotide sequence is operably linked to a second promoter that is functional in a plant cell, wherein said first nucleotide sequence comprises in the 5'-to-3' orientation and operably linked: (a) a FucT forward fragment, said FucT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2; (b) a spacer sequence comprising a nucleotide fragment of the sequence set forth in SEQ ID NO:1; and (c) a FucT reverse fragment, said FucT reverse fragment having sufficient length and sufficient complementarity to said FucT forward fragment such that said first nucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure. 2. The nucleotide construct of claim 1, wherein said FucT reverse fragment comprises the complement of said FucT forward fragment or a sequence having at least 90% sequence identity to the complement of said FucT forward fragment. 3. The nucleotide construct of claim 1, wherein said FucT forward fragment comprises nucleotides (nt) 255-985 of SEQ ID NO: 1. 4. The nucleotide construct of claim 1, wherein said nucleotide fragment of the sequence set forth in SEQ ID NO:1 comprises about 200 to 700 nucleotides immediately downstream of said FucT forward fragment. 5. The nucleotide construct of claim 4, wherein said spacer sequence comprises (nt) 986-1444 of SEQ ID NO:1. 6. A nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in a duckweed plant, and a second nucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said first nucleotide sequence is operably linked to a first promoter that is functional in a plant cell, and wherein said second nucleotide sequence is operably linked to a second promoter that is functional in a plant cell, wherein said first nucleotide sequence comprises in the 5'-to-3' direction and operably linked: (a) a sense nucleotide sequence comprising at least 19 contiguous nucleotides of SEQ ID NO:1; and (b) an antisense nucleotide sequence comprising at least 19 contiguous nucleotides of said sense nucleotide sequence; wherein said first nucleotide sequence is transcribed as a small hairpin RNA having a base-paired stem length shorter than about 200 base pairs. 7. The nucleotide construct of claim 6, wherein said promoter operably linked to said first nucleotide sequence is a type 3 RNA polymerase III promoter. 8. The nucleotide construct of claim 1, wherein said XylT is a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:21; and (b) an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:21. 9. The nucleotide construct of claim 8, wherein said second nucleotide sequence comprises a sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:19, or a complement thereof; (b) the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:20, or a complement thereof; (c) a nucleotide sequence having at least 90% sequence identity to the sequence of preceding item (a) or (b); and (d) a fragment of the nucleotide sequence of any one of preceding items (a) through (c), wherein said fragment comprises at least 75 contiguous nucleotides of said nucleotide sequence. 10. The nucleotide construct of claim 8, wherein said second nucleotide sequence comprises in the 5'-to-3' orientation and operably linked: (a) a XylT forward fragment, said XylT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (b) a spacer sequence comprising about 200 to about 700 nucleotides; and (c) a XylT reverse fragment, said XylT reverse fragment having sufficient length and sufficient complementarity to said XylT forward fragment such that said second nucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure. 11. The nucleotide construct of claim 10, wherein said XylT reverse fragment comprises the complement of said XylT forward fragment or a sequence having at least 90% sequence identity to the complement of said XylT forward fragment. 12. The nucleotide construct of claim 10 wherein said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ ID NO:19. 13. The nucleotide construct of claim 10, wherein said spacer sequence within said second nucleotide sequence comprises a nucleotide fragment of the sequence set forth in SEQ ID NO:4 or SEQ ID NO:19. 14. The nucleotide construct of claim 13, wherein said nucleotide fragment of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 19 comprises about 200 to 700 nucleotides immediately downstream of said XylT forward fragment. 15. The nucleotide construct of claim 14, wherein: (a) said XylT forward fragment comprises nt 318-1052 of SEQ ID NO:4 and said spacer sequence comprises nt 1053-1599 of SEQ ID NO:4; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and said spacer sequence comprises nt 735-1282 of SEQ ID NO:19. 16. The nucleotide construct of claim 10, wherein said spacer sequence within said second nucleotide sequence comprises an intron. 17. The nucleotide construct of claim 8, wherein said second nucleotide sequence comprises in the 5'-to-3' direction and operably linked: (a) a sense nucleotide sequence comprising at least 19 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:19; and (b) an antisense nucleotide sequence comprising at least 19 contiguous nucleotides of the complement of said sense nucleotide sequence of preceding item (a); wherein said second nucleotide sequence is transcribed as a small hairpin RNA having a base-paired stem length shorter than about 200 base pairs. 18. The nucleotide construct of claim 17, wherein said promoter operably linked to said second nucleotide sequence is a type 3 RNA polymerase III promoter. 19. A nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in a duckweed plant, and a second nucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said first nucleotide sequence is operably linked to a first promoter that is functional in a plant cell, and wherein said second nucleotide sequence is operably linked to a second promoter that is functional in a plant cell, wherein said second nucleotide sequence comprises in the 5'-to-3' orientation and operably linked: (a) a XylT forward fragment, said XylT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (b) a spacer sequence comprising a nucleotide fragment of the sequence set forth in SEQ ID NO:4 or SEQ ID NO:19; and (c) a XylT reverse fragment, said XylT reverse fragment having sufficient length and sufficient complementarity to said XylT forward fragment such that said second nucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure. 20. The nucleotide construct of claim 19, wherein said XylT reverse fragment comprises the complement of said XylT forward fragment or a sequence having at least 90% sequence identity to the complement of said XylT forward fragment. 21. The nucleotide construct of claim 19, wherein said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ ID NO:19. 22. The nucleotide construct of claim 19, wherein said nucleotide fragment of the sequence set forth in SEQ ID NO:4 or SEQ ID NO: 19 comprises about 200 to 700 nucleotides immediately downstream of said XylT forward fragment. 23. The nucleotide construct of claim 22 wherein: (a) said XylT forward fragment comprises nt 318-1052 of SEQ ID NO:4 and said spacer sequence comprises nt 1053-1599 of SEQ ID NO:4; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and said spacer sequence comprises nt 735-1282 of SEQ ID NO:19. 24. A nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in a duckweed plant, and a second nucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said first nucleotide sequence is operably linked to a first promoter that is functional in a plant cell, and wherein said second nucleotide sequence is operably linked to a second promoter that is functional in a plant cell, wherein said second nucleotide sequence comprises in the 5'-to-3' direction and operably linked: (a) a sense nucleotide sequence comprising at least 19 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:19; and (b) an antisense nucleotide sequence comprising at least 19 contiguous nucleotides of the complement of said sense nucleotide sequence of preceding item (a); wherein said second nucleotide sequence is transcribed as a small hairpin RNA having a base-paired stem length shorter than about 200 base pairs. 25. The nucleotide construct of claim 24, wherein said promoter operably linked to said second nucleotide sequence is a type 3 RNA polymerase III promoter. 26. The nucleotide construct of claim 19, wherein said FucT is a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth in SEQ ID NO:3; and (b) an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3. 27. The nucleotide construct of claim 26, wherein said first nucleotide sequence comprises a sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:1 or a complement thereof; (b) the nucleotide sequence set forth in SEQ ID NO:2 or a complement thereof; (c) a nucleotide sequence having at least 90% sequence identity to the sequence of preceding item (a) or (b); and (d) a fragment of the nucleotide sequence of any one of preceding items (a) through (c), wherein said fragment comprises at least 75 contiguous nucleotides of said nucleotide sequence. 28. A nucleotide construct comprising a fusion polynucleotide that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) and a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said fusion polynucleotide is operably linked to a promoter that is functional in a plant cell, said fusion polynucleotide comprising in the 5'-to-3' orientation and operably linked: (a) a chimeric forward fragment, said chimeric forward fragment comprising: (i) a first fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2; and (ii) a second fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of a polynucleotide encoding said XylT; (b) a spacer sequence comprising a fragment of said polynucleotide encoding said XylT, said fragment being about 200 to about 700 nucleotides in length; and (c) a reverse fragment, said reverse fragment having sufficient length and sufficient complementarity to said chimeric forward fragment such that said fusion polynucleotide is transcribed as an RNA molecule capable of forming a hairpin RNA structure; wherein said polynucleotide encoding said XylT is selected from the group consisting of: (1) a polynucleotide comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (2) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:6 or SEQ ID NO:21; and (3) a polynucleotide encoding a polypeptide having at least 90% sequence identity to the polypeptide set forth in SEQ ID NO:6 or SEQ ID NO:21. 29. The nucleotide construct of claim 28, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment. 30. The nucleotide construct of claim 28, wherein said spacer sequence comprises about 200 to 700 nucleotides immediately downstream of the second fragment of said chimeric forward fragment. 31. The nucleotide construct of claim 28, wherein said FucT is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3. 32. The nucleotide construct of claim 28, wherein said XylT is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:21. 33. The nucleotide construct of claim 28, wherein said first fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO: 1; and said second fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:19. 34. The nucleotide construct of claim 33, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment. 35. The nucleotide construct of claim 33, wherein said chimeric forward fragment comprises: (a) nucleotides (nt) 254-855 of SEQ ID NO:1 and nt 318-943 of SEQ ID NO:4; or (b) nucleotides (nt) 254-855 of SEQ ID NO:1 and nt 1-626 of SEQ ID NO:19. 36. The nucleotide construct of claim 33, wherein said spacer sequence comprises about 200 to 700 nucleotides immediately downstream of the second fragment of said chimeric forward fragment. 37. The nucleotide construct of claim 36, wherein: (a) said chimeric forward fragment comprises nt 254-855 of SEQ ID NO:1 and nt 318-943 of SEQ ID NO:4 and said spacer sequence comprises nt 944-1443 of SEQ ID NO:4; or (b) said chimeric forward fragment comprises nt 254-855 of SEQ ID NO:1 and nt 1-626 of SEQ ID NO: 19 and said spacer sequence comprises nt 627-1126 of SEQ ID NO:19. 38. A nucleotide construct comprising a fusion polynucleotide that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) and a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said fusion polynucleotide is operably linked to a promoter that is functional in a plant cell, said fusion polynucleotide comprising in the 5'-to-3' orientation and operably linked: (a) a chimeric forward fragment, said chimeric forward fragment comprising: (i) a first fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; and (ii) a second fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of a polynucleotide encoding said FucT; (b) a spacer sequence comprising a fragment of said polynucleotide encoding said FucT, said fragment being about 200 to about 700 nucleotides in length; and c) a reverse fragment, said reverse fragment having sufficient length and sufficient complementarity to said chimeric forward fragment such that said fusion polynucleotide is transcribed as an RNA molecule capable of forming a hairpin RNA structure; wherein said polynucleotide encoding said FucT is selected from the group consisting of: (1) a polynucleotide comprising SEQ ID NO:1 or SEQ ID NO:2; (2) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:3; and (3) a polynucleotide encoding a polypeptide having at least 90% sequence identity to the polypeptide set forth in SEQ ID NO:3. 39. The nucleotide construct of claim 38, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment. 40. The nucleotide construct of claim 38, wherein said spacer sequence comprises about 200 to 700 nucleotides immediately downstream of the second fragment of said chimeric forward fragment. 41. The nucleotide construct of claim 38, wherein said FucT is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3. 42. The nucleotide construct of claim 38, wherein said XylT is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:21. 43. The nucleotide construct of claim 38, wherein said first fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO: 19; and said second fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO:1. 44. The nucleotide construct of claim 43, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment. 45. The nucleotide construct of claim 43, wherein said spacer sequence comprises about 200 to 700 nucleotides immediately downstream of the second fragment of said chimeric forward fragment. 46. A nucleotide construct comprising a first polynucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in a duckweed plant, wherein said first polynucleotide sequence comprises in the 5'-to-3' orientation and operably linked: (a) a FucT forward fragment, said FucT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2; (b) a spacer sequence comprising a fragment of said polynucleotide encoding said FucT, said fragment being about 200 to about 700 nucleotides in length; and (c) a FucT reverse fragment, said FucT reverse fragment having sufficient length and sufficient complementarity to said FucT forward fragment such that said first polynucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure; wherein said first polynucleotide sequence is operably linked to a promoter that is functional in a plant cell. 47. The nucleotide construct of claim 46, wherein said FucT reverse fragment comprises the complement of said FucT forward fragment or a sequence having at least 90% sequence identity to the complement of said FucT forward fragment. 48. The nucleotide construct of claim 46, wherein said spacer sequence within said first polynucleotide sequence comprises about 200 to 700 nucleotides immediately downstream of said FucT forward fragment. 49. The nucleotide construct of claim 46, wherein said FucT is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3. 50. The nucleotide construct of claim 49, wherein said FucT forward fragment comprises about 500 to about 800 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2. 51. The nucleotide construct of claim 50, wherein said FucT reverse fragment comprises the complement of said FucT forward fragment or a sequence having at least 90% sequence identity to the complement of said FucT forward fragment. 52. The nucleotide construct of claim 50, wherein said FucT forward fragment comprises nucleotides (nt) 255-985 of SEQ ID NO: 1. 53. The nucleotide construct of claim 50, wherein said spacer sequence comprises about 200 to 700 nucleotides immediately downstream of said FucT forward fragment. 54. The nucleotide construct of claim 53, wherein said spacer sequence comprises nt 986-1444 of SEQ ID NO:1. 55. A nucleotide construct comprising a first polynucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said first polynucleotide sequence comprises in the 5'-to-3' orientation and operably linked: (a) a XylT forward fragment, said XylT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (b) a spacer sequence comprising a fragment of said polynucleotide encoding said XylT, said fragment being about 200 to about 700 nucleotides in length; and (c) a XylT reverse fragment, said XylT reverse fragment having sufficient length and sufficient complementarity to said XylT forward fragment such that said first polynucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure; wherein said first polynucleotide sequence is operably linked to a promoter that is functional in a plant cell. 56. The nucleotide construct of claim 55, wherein said XylT reverse fragment comprises the complement of said XylT forward fragment or a sequence having at least 90% sequence identity to the complement of said XylT forward fragment. 57. The nucleotide construct of claim 55, wherein said spacer sequence within said first polynucleotide sequence comprises about 200 to 700 nucleotides immediately downstream of said XylT forward fragment. 58. The nucleotide construct of claim 55, wherein said XylT is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:21. 59. The nucleotide construct of claim 58, wherein said XylT forward fragment comprises about 500 to about 800 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20. 60. The nucleotide construct of claim 59, wherein said XylT reverse fragment comprises the complement of said XylT forward fragment or a sequence having at least 90% sequence identity to the complement of said XylT forward fragment. 61. The nucleotide construct of claim 59, wherein said XylT forward fragment comprises nucleotides nt 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ ID NO:19. 62. The nucleotide construct of claim 59, wherein said spacer sequence comprises about 200 to 700 nucleotides immediately downstream of said XylT forward fragment. 63. The nucleotide construct of claim 62, wherein: (a) said XylT forward fragment comprises nt 318-1052 of SEQ ID NO:4 and said spacer sequence comprises nt 1053-1599 of SEQ ID NO:4; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and said spacer sequence comprises nt 735-1282 of SEQ ID NO:19. 64. The nucleotide construct of claim 28, further comprising at least one polynucleotide encoding a polypeptide of interest, wherein said polynucleotide encoding said polypeptide of interest is operably linked to a promoter that is functional in a plant cell. 65. The nucleotide construct of claim 64, wherein said polypeptide of interest is a mammalian polypeptide. 66. The nucleotide construct of claim 65, wherein said mammalian polypeptide is a monoclonal antibody. 67. The nucleotide construct of claim 65, wherein said mammalian polypeptide is selected from the group consisting of an interferon, erythropoietin (EPO), tissue plasminogen activator (tPA), plasminogen, blood coagulation factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and therapeutic immunoglobulins. 68. A vector comprising a nucleotide construct according to claim 28. 69. A duckweed plant or duckweed plant cell or nodule comprising a nucleotide construct according to claim 28 stably integrated into its genome. 70. The duckweed plant or duckweed plant cell or nodule of claim 69, wherein said duckweed is from a genus selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella, genus Landoltia, and genus Lemna. 71. The duckweed plant or duckweed plant cell or nodule of claim 70, wherein said duckweed is a member of a species selected from the group consisting of Lemna minor, Lemna miniscula, Lemna aequinoctialis, and Lemna gibba. 72. A method for stably transforming a duckweed plant to express glycoproteins having an altered N-glycosylation pattern, said method comprising introducing into said duckweed plant the nucleotide construct according to claim 28. 73. The method of claim 72, wherein said duckweed plant expresses at least one heterologous polypeptide of interest. 74. The method of claim 73, wherein said heterologous polypeptide of interest is a mammalian polypeptide or biologically active variant thereof. 75. The method of claim 74, wherein said mammalian polypeptide is selected from the group consisting of an interferon, erythropoietin (EPO), tissue plasminogen activator (tPA), plasminogen, blood coagulation factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and therapeutic immunoglobulins. 76. A method for stably transforming a duckweed plant to express a heterologous polypeptide of interest having an altered N-glycosylation pattern, said method comprising introducing into said duckweed plant the nucleotide construct according to claim 64. 77. The method of claim 76, wherein the N-glycosylation pattern is characterized by a reduction in the attachment of .alpha.1,3-fucose residues to N-glycans attached to said heterologous polypeptide, a reduction in the attachment of .beta.1,2-xylose residues to N-glycans attached to said heterologous polypeptide, or a reduction in the attachment of .alpha.1,3-fucose residues and .beta.1,2-xylose residues to N-glycans attached to said heterologous polypeptide. 78. A method for producing a heterologous mammalian glycoprotein in a duckweed plant, wherein said heterologous mammalian glycoprotein has a reduction in the attachment of .alpha.1,3-fucose and .beta.1,2-xylose residues to N-glycans of said glycoprotein compared to said glycoprotein when produced in a control duckweed plant that has not been genetically modified to inhibit expression of .alpha.1,3-fucosyltransferase (FucT) and .beta.1,2-xylosyltransferase (XylT) in said control duckweed plant, said method comprising: (a) introducing into said duckweed plant an expression cassette comprising a sequence encoding a mammalian polypeptide that is post-translationally processed as said glycoprotein, and a polynucleotide comprising the nucleotide construct of claim 28; and (b) culturing said duckweed plant under conditions suitable for expression of said glycoprotein. 79. The method of claim 78, wherein said mammalian polypeptide is selected from the group consisting of an interferon, erythropoietin (EPO), tissue plasminogen activator (tPA), plasminogen, blood coagulation factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and therapeutic immunoglobulins. 80. A method for reducing heterogeneity of the N-glycosylation profile of a glycoprotein produced in a duckweed plant when compared to the heterogeneity of the N-glycosylation profile of said glycoprotein when produced in a control duckweed plant that has not been genetically modified to inhibit expression of .alpha.1,3-fucosyltransferase (FucT) and .beta.1,2-xylosyltransferase (XylT) in said control duckweed plant, said method comprising introducing into said duckweed plant the nucleotide construct according to claim 28, and culturing said duckweed plant under conditions suitable for expression of said glycoprotein. 81. The method of claim 80, wherein said glycoprotein is an endogenous glycoprotein. 82. The method of claim 80, wherein said glycoprotein is a heterologous glycoprotein. 83. The method of claim 82, wherein said heterologous glycoprotein is a mammalian glycoprotein. 84. The method of claim 83, wherein said mammalian glycoprotein is selected from the group consisting of an interferon, erythropoietin (EPO), tissue plasminogen activator (tPA), plasminogen, blood coagulation factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and therapeutic immunoglobulins. 85. The method of claim 80, wherein at least 90% of the N-glycans within said profile are GlcNAc2Man3GlcNAc2 (G0), and wherein said profile is devoid of N-glycan species having fucose, xylose, or both fucose and xylose attached thereto. 86. The method of claim 85, wherein at least 95% of the N-glycans within said profile are G0. 87. The method according to claim 80, wherein the reduced heterogeneity of said N-glycosylation profile is maintained with scale-up in production of said duckweed plant, wherein production scale is increased by at least 6,500-fold. 88. The method according to claim 80, wherein the reduced heterogeneity of said N-glycosylation profile is maintained with continuous clonal culture of said duckweed plant. 89. The method of claim 88, wherein the reduced heterogeneity of said N-glycosylation profile is maintained with continuous clonal culture of said duckweed plant for at least 8 months. 90. A method for altering the N-glycosylation pattern of a heterologous polypeptide produced in a duckweed plant or duckweed plant cell or nodule, said method comprising: (a) introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in said duckweed plant or duckweed plant cell or nodule, and wherein said first nucleotide sequence is operably linked to a promoter that is functional in a plant cell, said first nucleotide sequence comprising in the 5'-to-3' orientation and operably linked: (i) a FucT forward fragment, said FucT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2; (ii) a spacer sequence comprising about 200 to about 700 nucleotides immediately downstream of said FucT forward fragment; and (iii) a FucT reverse fragment, said FucT reverse fragment having sufficient length and sufficient complementarity to said FucT forward fragment such that said first nucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide. 91. The method of claim 90, wherein said FucT reverse fragment comprises the complement of said FucT forward fragment or a sequence having at least 90% sequence identity to the complement of said FucT forward fragment. 92. The method of claim 90, wherein said FucT forward fragment comprises nucleotides (nt) 255-985 of SEQ ID NO:1 and said spacer sequence comprises nt 986-1444 of SEQ ID NO:1. 93. A method for altering the N-glycosylation pattern of a heterologous polypeptide produced in a duckweed plant or duckweed plant cell or nodule, said method comprising: (a) introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in said duckweed plant or duckweed plant cell or nodule, and wherein said first nucleotide sequence is operably linked to a promoter that is functional in a plant cell, said first nucleotide sequence comprising in the 5'-to-3' orientation and operably linked: (i) a sense nucleotide sequence comprising at least 19 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2; and (ii) an antisense nucleotide sequence comprising at least 19 contiguous nucleotides of the complement of said sense nucleotide sequence; wherein said first nucleotide sequence is transcribed as a small hairpin RNA having a base-paired stem length shorter than about 200 base pairs; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide. 94. The method of claim 93, wherein said promoter operably linked to said first nucleotide sequence is a type 3 RNA polymerase III promoter. 95. The method of claim 90, further comprising inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule. 96. The method of claim 95, wherein the expression of said XylT is inhibited by a method selected from the group consisting of: (a) introducing a polynucleotide into said duckweed plant or duckweed plant cell or nodule, wherein said polynucleotide inhibits expression of said XylT in said duckweed plant or duckweed plant cell or nodule; (b) eliminating a gene in said duckweed plant or duckweed plant cell or nodule, wherein said gene encodes said XylT; and (c) mutating a gene in said duckweed plant or duckweed plant cell or nodule, wherein said gene encodes said XylT. 97. The method of claim 96, comprising introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a nucleotide sequence that is capable of inhibiting expression of said XylT in said duckweed plant or duckweed plant cell or nodule, wherein said nucleotide sequence that is capable of inhibiting expression of said XylT is operably linked to a promoter that is functional in a plant cell. 98. The method of claim 97, wherein said nucleotide sequence that is capable of inhibiting expression of said XylT comprises in the 5'-to-3' orientation and operably linked: (a) a XylT forward fragment, said XylT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of a polynucleotide encoding said XylT; (b) a spacer sequence comprising about 200 to about 700 nucleotides immediately downstream of said XylT forward fragment; and (c) a XylT reverse fragment, said XylT reverse fragment having sufficient length and sufficient complementarity to said XylT forward fragment such that said nucleotide sequence that is capable of inhibiting expression of said XylT is transcribed as an RNA molecule capable of forming a hairpin RNA structure; wherein said polynucleotide encoding said XylT is selected from the group consisting of: (1) a polynucleotide comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (2) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:6 or SEQ ID NO:21; and (3) a polynucleotide encoding a polypeptide having at least 90% sequence identity to the polypeptide set forth in SEQ ID NO:6 or SEQ ID NO:21. 99. The method of claim 98, wherein said XylT reverse fragment comprises the complement of said XylT forward fragment or a sequence having at least 90% sequence identity to the complement of said XylT forward fragment. 100. The method of claim 98, wherein: (a) said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 and wherein said spacer sequence comprises nt 1053-1599 of SEQ ID NO:4; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and said spacer sequence comprises nt 735-1282 of SEQ ID NO: 19. 101. The method of claim 97, wherein said nucleotide sequence that is capable of inhibiting expression of said XylT comprises in the 5'-to-3' direction and operably linked: (a) a sense nucleotide sequence comprising at least 19 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:19; and (b) an antisense nucleotide sequence comprising at least 19 contiguous nucleotides of the complement of said sense nucleotide sequence of preceding item (a); wherein said nucleotide sequence that is capable of inhibiting expression of said XylT is transcribed as a small hairpin RNA having a base-paired stem length shorter than about 200 base pairs. 102. The method of claim 101, wherein said promoter operably linked to said nucleotide sequence that is capable of inhibiting expression of said XylT is a type 3 RNA polymerase III promoter. 103. A method for altering the N-glycosylation pattern of a heterologous polypeptide produced in a duckweed plant or duckweed plant cell or nodule, said method comprising: (a) introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule, and wherein said first nucleotide sequence is operably linked to a promoter that is functional in a plant cell, said first nucleotide sequence comprising in the 5'-to-3' orientation and operably linked: (i) a XylT forward fragment, said XylT forward fragment comprising about 500 to about 800 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (ii) a spacer sequence comprising about 200 to about 700 nucleotides immediately downstream of said XylT forward fragment; and (iii) a XylT reverse fragment, said XylT reverse fragment having sufficient length and sufficient complementarity to said XylT forward fragment such that said first nucleotide sequence is transcribed as an RNA molecule capable of forming a hairpin RNA structure; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide. 104. The method of claim 103, wherein said XylT reverse fragment comprises the complement of said XylT forward fragment or a sequence having at least 90% sequence identity to the complement of said XylT forward fragment. 105. The method of claim 103, wherein: (a) said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 and wherein said spacer sequence comprises nt 1053-1599 of SEQ ID NO:4; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and wherein said spacer sequence comprises nt 735-1282 of SEQ ID NO: 19. 106. A method for altering the N-glycosylation pattern of a heterologous polypeptide produced in a duckweed plant or duckweed plant cell or nodule, said method comprising: (a) introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a first nucleotide sequence that is capable of inhibiting expression of a .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule, and wherein said first nucleotide sequence is operably linked to a promoter that is functional in a plant cell, said first nucleotide sequence comprising in the 5'-to-3' orientation and operably linked: (i) a sense nucleotide sequence comprising at least 19 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO: 19; and (ii) an antisense nucleotide sequence comprising at least 19 contiguous nucleotides of the complement of said sense nucleotide sequence of preceding item (a); wherein said nucleotide sequence that is capable of inhibiting expression of said XylT is transcribed as a small hairpin RNA having a base-paired stem length shorter than about 200 base pairs; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide. 107. The method of claim 106, wherein said promoter operably linked to said first nucleotide sequence is a type 3 RNA polymerase III promoter. 108. The method of claim 103, further comprising inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) in said duckweed plant or duckweed plant cell or nodule. 109. The method of claim 108, wherein the expression of said FucT is inhibited by a method selected from the group consisting of: (a) introducing a polynucleotide into said duckweed plant or duckweed plant cell or nodule, wherein said polynucleotide inhibits expression of said FucT in said duckweed plant or duckweed plant cell or nodule; (b) eliminating a gene in said duckweed plant or duckweed plant cell or nodule, wherein said gene encodes said FucT; and (c) mutating a gene in said duckweed plant or duckweed plant cell or nodule, wherein said gene encodes said FucT. 110. A method for altering the N-glycosylation pattern of a heterologous polypeptide produced in a duckweed plant or duckweed plant cell or nodule, said method comprising: (a) introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a fusion polynucleotide that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) and a .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule, wherein said fusion polynucleotide is operably linked to a promoter that is functional in a plant cell, said fusion polynucleotide comprising in the 5'-to-3' orientation and operably linked: (i) a chimeric forward fragment, said chimeric forward fragment comprising: (a) a first fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO:2; and (b) a second fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of a polynucleotide encoding said XylT; (ii) a spacer sequence comprising about 200 to about 700 nucleotides immediately downstream of said second fragment of said chimeric forward fragment; and (iii) a reverse fragment, said reverse fragment having sufficient length and sufficient complementarity to said chimeric forward fragment such that said fusion polynucleotide is transcribed as an RNA molecule capable of forming a hairpin RNA structure; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide; wherein said polynucleotide encoding said XylT is selected from the group consisting of: (1) a polynucleotide comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; (2) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:6 or SEQ ID NO:21; and (3) a polynucleotide encoding a polypeptide having at least 90% sequence identity to the polypeptide set forth in SEQ ID NO:6 or SEQ ID NO:21. 111. The method of claim 110, wherein said first fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO: 1; and said second fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:19. 112. The method of claim 111, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment. 113. The method of claim 111, wherein: (a) said chimeric forward fragment comprises nucleotides (nt) 254-855 of SEQ ID NO:1 and nt 318-943 of SEQ ID NO:4, and wherein said spacer sequence comprises nt 944-1443 of SEQ ID NO:4; or (b) said chimeric forward fragment comprises nucleotides (nt) 254-855 of SEQ ID NO:1 and nt 1-626 of SEQ ID NO: 19, and wherein said spacer sequence comprises nt 627-1126 of SEQ ID NO:19. 114. A method for altering the N-glycosylation pattern of a heterologous polypeptide produced in a duckweed plant or duckweed plant cell or nodule, said method comprising: (a) introducing into said duckweed plant or duckweed plant cell or nodule a nucleotide construct comprising a fusion polynucleotide that is capable of inhibiting expression of an .alpha.1,3-fucosyltransferase (FucT) and a .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule, wherein said fusion polynucleotide is operably linked to a promoter that is functional in a plant cell, said fusion polynucleotide comprising in the 5'-to-3' orientation and operably linked: (i) a chimeric forward fragment, said chimeric forward fragment comprising: (a) a first fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20; and (b) a second fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of a polynucleotide encoding said FucT; (ii) a spacer sequence comprising about 200 to about 700 nucleotides immediately downstream of said second fragment of said chimeric forward fragment; and (iii) a reverse fragment, said reverse fragment having sufficient length and sufficient complementarity to said chimeric forward fragment such that said fusion polynucleotide is transcribed as an RNA molecule capable of forming a hairpin RNA structure; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide; wherein said polynucleotide encoding said FucT is selected from the group consisting of: (1) a polynucleotide comprising SEQ ID NO:1 or SEQ ID NO:2; (2) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:3; and (3) a polynucleotide encoding a polypeptide having at least 90% sequence identity to the polypeptide set forth in SEQ ID NO:3. 115. The method of claim 114, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment. 116. The method of claim 114, wherein said first fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO: 19; and said second fragment comprises about 500 to about 650 contiguous nucleotides of SEQ ID NO:1. 117. The method of claim 116, wherein said reverse fragment comprises the complement of said chimeric forward fragment or a sequence having at least 90% sequence identity to the complement of said chimeric forward fragment.

Details for Patent 8,716,557

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26