Biolex Therapeutics, Inc.

Synthon Biopharmaceuticals B.v.

Biolex, Inc.

Medarex Inc.

Synthon B.v.

Charles G Peele

David B Passmore

David Spencer

Amelia Nancy Black

Lynn F Dickey

Kevin Cox

John Gasdaska

Xiaowei Wang

Ming-Bo Wang

Kevin M Cox

Lynn Dickey

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 &#x3b1;1,3-fucosyltransferase (FucT) and &#x3b2;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.

Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in plants

Compositions and methods for regulating expression of nucleotide sequences of interest in a plant are provided. Compositions include novel nucleic acid molecules, and variants and fragments thereof, for expression control elements isolated from theubiquitin, r-histone and chitinase genes. A method for expressing a nucleotide sequence of interest in a plant using the expression control elements disclosed herein is further provided. The method includes introducing into a plant or plant cell or nodule an expression construct comprising an expression control element of the present invention operably linked to a nucleotide sequence of interest. In particular, the compositions and methods find use in enhancing expression of nucleotide sequences of interest in duckweed. Also provided is a novelsignal peptide-encoding sequence and the signal peptide encoded thereby. Where an expression construct of the invention is designed to express a polypeptide of interest, this novel signal peptide-encoding sequence can be included within the expression construct of the invention to provide for extracellular secretion of the encoded polypeptide of interest.

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The present invention provides methods and compositions for the production of recombinant plasminogen, microplasminogen, and fragments thereof in a duckweed expression system. It is the novel finding of the present invention that a duckweed expression system may be used to produce high levels of plasminogen and microplasminogen. The duckweed-produced plasminogen and microplasminogen can be activated to produce a polypeptide having protease activity. Thus, the invention encompasses methods for the expression of plasminogen, microplasminogen, and fragments thereof in duckweed, duckweed plants that are transformed with expression cassettes for the expression of plasminogen, microplasminogen, and fragments thereof, and nucleic acids comprising nucleotide sequences encoding plasminogen, microplasminogen, and fragments thereof, where these nucleotide sequences are modified to enhance their expression in duckweed.

Expression of plasminogen and microplasminogen in duckweed

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 &#x3b1;1,3-fucosyltransferase (FucT) and &#x3b2;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.

Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in duckweed plants

Expression of plasminogen and microplasminogen in duck weed

Methods, nucleic acid sequences, and transformed duckweed plant or duckweed nodule cultures for the expression and the secretion of biologically active polypeptides from genetically engineered duckweed are provided. Expression of recombinant polypeptides in duckweed is improved by modifying the nucleotide sequence of the expression cassette encoding the polypeptide for improved expression in duckweed. Recovery of biologically active polypeptides from duckweed is improved by linking the biologically active polypeptide to a signal peptide that directs the secretion of the polypeptide into the culture medium. Methods of producing recombinant monoclonal antibodies having effector function, and antibodies produced by these methods are provided.

Expression of monoclonal antibodies in duckweed

Compositions and methods for regulating expression of nucleotide sequences of interest in a plant are provided. Compositions include novel nucleic acid molecules, and variants and fragments thereof, for expression control elements isolated from the Lemnaceae ubiquitin, r-histone and chitinase genes. A method for expressing a nucleotide sequence of interest in a plant using the expression control elements disclosed herein is further provided. The method includes introducing into a plant or plant cell or nodule an expression construct comprising an expression control element of the present invention operably linked to a nucleotide sequence of interest. In particular, the compositions and methods find use in enhancing expression of nucleotide sequences of interest in duckweed. Also provided is a novel Lemnaceae signal peptide-encoding sequence and the signal peptide encoded thereby. Where an expression construct of the invention is designed to express a polypeptide of interest, this novel signal peptide-encoding sequence can be included within the expression construct of the invention to provide for extracellular secretion of the encoded polypeptide of interest.

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Apex, NC, United States of America

Charles G Peele