Claims for Patent: 7,884,264

Summary for Patent: 7,884,264

Title:	Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in duckweed plants
Abstract:	Methods for altering the N-glycosylation pattern of proteins in higher plants are provided. In some embodiments, the methods comprise introducing into a duckweed plant a recombinant RNAi construct that provides for the inhibition of expression of .alpha.1,3-fucosyltransferase (FucT) and .beta.1,2-xylosyltransferase (XylT). 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, including duckweed 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)
Assignee:	Biolex Therapeutics, Inc. (Pittsboro, NC)
Application Number:	11/624,164
Patent Claims:	1. A duckweed plant or duckweed plant cell or nodule that expresses glycoproteins having an altered N-glycosylation pattern, wherein said altered N-glycosylation pattern is characterized by a reduction in the attachment of .alpha.1,3-fucose residues, .beta.1,2-xylose residues, or both .alpha.1,3-fucose residues and .beta.1,2-xylose residues to N-glycans of said glycoproteins when compared to the N-glycosylation pattern for said glycoproteins when expressed in a control duckweed plant or duckweed plant cell or nodule, wherein said reduction in said duckweed plant or duckweed plant cell or nodule results from inhibiting expression in said duckweed plant or duckweed plant cell or nodule of at least one .alpha.1,3-fucosyltransferase (FucT) comprising an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:3 and, at least one .beta.1,2-xylosyltransferase (XylT) comprising an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:6 or SEQ ID NO:21, wherein said control duckweed plant or duckweed plant cell or nodule has not been genetically modified to inhibit expression of said FucT or said XylT in said control duckweed plant or duckweed plant cell or nodule. 2. The duckweed plant or duckweed plant cell or nodule of claim 1, wherein said N-glycans are devoid of said fucose residues, devoid of said xylose residues, or devoid of said fucose and xylose residues. 3. The duckweed plant or duckweed plant cell or nodule of claim 1, wherein said duckweed plant or duckweed plant cell or nodule comprises a polynucleotide encoding a mammalian glycoprotein of interest. 4. The duckweed plant or duckweed plant cell or nodule of claim 3 , wherein said glycoprotein is a monoclonal antibody of interest. 5. The duckweed plant or duckweed plant cell or nodule of claim 4, wherein said monoclonal antibody has increased binding affinity for an Fc.gamma.RIII, increased antibody-dependent cellular cytotoxicity (ADCC), decreased complement-dependent cytotoxicity (CDC) activity, or any combination thereof, as a result of said altered N-glycosylation pattern of said monoclonal antibody. 6. 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) inhibiting expression of at least one .alpha.1,3-fucosyltransferase (FucT) in said a duckweed plant or duckweed plant cell or nodule, wherein said FucT comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:3; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide. 7. The method of claim 5, 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 a 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. 8. The method of claim 7, comprising 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 said .alpha.1,3-fucosyltransferase (FucT) in said duckweed plant or duckweed plant cell or nodule, wherein said first nucleotide sequence is operably linked to a promoter that is functional in a plant cell. 9. The method of claim 8, 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. 10. The method of claim 8, 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 about 200 to about 700 nucleotides; 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. 11. The method of claim 10, 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. 12. The method of claim 10, wherein said FucT forward fragment comprises nucleotides (nt) 255-985 of SEQ ID NO:1 and said spacer sequence comprises an intron. 13. The method of claim 6, further comprising inhibiting expression of at least one .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule, wherein said XyltT comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:6 or SEQ ID NO:21. 14. The method of claim 13, 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 a 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. 15. The method of claim 14, 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. 16. The method of claim 15, wherein said nucleotide sequence that is capable of inhibiting expression of said XylT comprises a sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:19, or a complement thereof; (b) the nucleotide sequence set forth in SEQ ID NO:5, 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. 17. The method of claim 15, 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 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 nucleotide sequence that is capable of inhibiting expression of said XylT is transcribed as an RNA molecule capable of forming a hairpin RNA structure. 18. The method of claim 17, 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. 19. The method of claim 17, wherein: (a) said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 and wherein said spacer sequence is an intron; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and said spacer sequence is an intron. 20. The method of claim 13, wherein a nucleotide construct comprising a fusion polynucleotide that is capable inhibiting expression of said FucT and said XylT in said duckweed plant or duckweed plant cell or nodule is introduced into 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. 21. The method of claim 20, wherein said fusion polynucleotide comprises in the 5'-to-3' orientation and operably linked: (a) a chimeric forward fragment, said chimeric forward fragment comprising in either order: (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 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; (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. 22. The method of claim 21, 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. 23. The method of claim 22, 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. 24. The method of claim 21, 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 is an intron; 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 is an intron. 25. 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) inhibiting expression of at least one a .beta.1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plant cell or nodule, wherein said XyltT comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:6 or SEQ ID NO:21; and (b) culturing said duckweed plant or duckweed plant cell or nodule under conditions suitable for expression of said heterologous polypeptide. 26. The method of claim 25, 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 a 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. 27. The method of claim 26, 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. 28. The method of claim 27, wherein said nucleotide sequence that is capable of inhibiting expression of said XylT comprises a sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:19, or a complement thereof; (b) the nucleotide sequence set forth in SEQ ID NO:5, 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. 29. The method of claim 27, 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 SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:21; (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 nucleotide sequence that is capable of inhibiting expression of said XylT is transcribed as an RNA molecule capable of forming a hairpin RNA structure. 30. The method of claim 29, 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. 31. The method of claim 29, wherein: (a) said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 and wherein said spacer sequence is an intron; or (b) said XylT forward fragment comprises nt 1-734 of SEQ ID NO:19 and wherein said spacer sequence is an intron. 32. The method of claim 6, wherein said heterologous polypeptide is a mammalian polypeptide. 33. The method of claim 32, 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. 34. 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 and said second nucleotide sequence are operably linked to at least one promoter that is functional in a plant cell; 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; and 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. 35. The nucleotide construct of claim 34, wherein said first nucleotide sequence is operably linked to a first promoter, and wherein said second nucleotide sequence is operably linked to a second promoter. 36. The nucleotide construct of claim 34, 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. 37. The nucleotide construct of claim 34, 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 about 200 to about 700 nucleotides; 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. 38. The nucleotide construct of claim 37, 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. 39. The nucleotide construct of claim 37, wherein said FucT forward fragment comprises nucleotides (nt) 255-985 of SEQ ID NO:1. 40. The nucleotide construct of claim 37, wherein said spacer sequence of said first nucleotide sequence comprises an intron. 41. The nucleotide construct of claim 34, 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. 42. The nucleotide construct of claim 34, 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:2; (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. 43. The nucleotide construct of claim 42, 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. 44. The nucleotide construct of claim 42, wherein said XylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ ID NO:19. 45. The nucleotide construct of claim 42, wherein said spacer sequence within said second nucleotide sequence comprises an intron. 46. 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, and wherein said fusion polynucleotide comprises in the 5'-to-3' orientation and operably linked: (a) a chimeric forward fragment, said chimeric forward fragment comprising in either order: (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 SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:2; (b) a spacer sequence comprising about 200 to about 700 nucleotides; 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. 47. The nucleotide construct of claim 46, 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. 48. The nucleotide construct of claim 46, wherein said spacer sequence comprises an intron. 49. The nucleotide construct of claim 46, 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. 50. The nucleotide construct of claim 49, 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. 51. The nucleotide construct of claim 49, 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. 52. The nucleotide construct of claim 49, wherein said spacer sequence comprises an intron. 53. 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 about 200 to about 700 nucleotides; 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. 54. The nucleotide construct of claim 53, 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. 55. The nucleotide construct of claim 53, wherein said spacer sequence of said first polynucleotide comprises an intron. 56. The nucleotide construct of claim 53, wherein said FucT forward fragment comprises about 500 to about 800 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2. 57. The nucleotide construct of claim 56, 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. 58. The nucleotide construct of claim 56, wherein said FucT forward fragment comprises nucleotides (nt) 255-985 of SEQ ID NO:1. 59. The nucleotide construct of claim 56, wherein said spacer sequence of said first polynucleotide sequence comprises an intron. 60. 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 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 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. 61. The nucleotide construct of claim 60, 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. 62. The nucleotide construct of claim 60, wherein said spacer sequence within said first polynucleotide sequence comprises an intron. 63. The nucleotide construct of claim 60, 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. 64. The nucleotide construct of claim 63, 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. 65. The nucleotide construct of claim 63, wherein said XylT forward fragment comprises nucleotides nt 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ ID NO:19. 66. The nucleotide construct of claim 63, wherein said spacer sequence within said first polynucleotide sequence comprises an intron. 67. The nucleotide construct of claim 46, 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. 68. The nucleotide construct of claim 67, wherein said polypeptide of interest is a mammalian polypeptide. 69. The nucleotide construct of claim 68, 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. 70. A vector comprising the nucleotide construct according to claim 46. 71. A duckweed plant or duckweed plant cell or nodule comprising the nucleotide construct according to claim 46 stably integrated into its genome. 72. The duckweed plant or duckweed plant cell or nodule of claim 71, 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. 73. The duckweed plant or plant cell of claim 72, wherein said duckweed is a member of a species selected from the group consisting of Lemna minor, Lemna miniscula, Lemna aequinoctialis, and Lemna gibba. 74. 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 46. 75. The method of claim 74, wherein said duckweed plant expresses at least one heterologous polypeptide of interest. 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 67. 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 both 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_when compared to the N-glycosylation pattern of said heterologous polypeptide when expressed in a control duckweed plant that has not been genetically modified to inhibit expression of said FucT and said XylT in said control duckweed plant. 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 when produced in said duckweed plant when compared to N-glycans 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: (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 46; 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, said method comprising introducing into said duckweed plant the nucleotide construct according to claim 46, 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 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. 84. 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. 85. The method of claim 84, wherein the reduced heterogeneity of said N-glycosylation profile is maintained with continuous clonal culture of said duckweed plant for at least 8 months. 86. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2; (b) the nucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO: 19, or SEQ ID NO:20; (c) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:6; or SEQ ID NO:21; (d) a nucleotide sequence comprising at least 90% sequence identity to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, wherein said polynucleotide encodes a polypeptide having .alpha.1,3-fucosyltransferase (FucT) activity; (e) a nucleotide sequence comprising at least 90% sequence identity to the sequence set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:2, wherein said polynucleotide encodes a polypeptide having .beta.1,2-xylosyltransferase (XylT) activity; (f) a nucleotide sequence comprising at least 50 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20, or a complement thereof; (g) a nucleotide sequence comprising at least 50contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20 and a complement thereof; (h) a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:3, wherein said polynucleotide encodes a polypeptide having FucT activity; (i) a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:6 or SEQ ID NO:2, wherein said polynucleotide encodes a polypeptide having XylT activity; and (j) the complement of the nucleotide sequence of any one of preceding items (a) through (i).

Details for Patent 7,884,264

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		2026-01-17
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2026-01-17
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2026-01-17