Medicago Inc.

Aramis Biotechnologies Inc.

Universitè Laval

Cellectis S.a.

Mitsubishi Tanabe Pharma Corporation

Plant Bioscience Limited

Medicago Inc. of Quebec, Canada

Centre National De La Recherche Scientifique

Cellectis

Université´de Rouen

Patrice Lerouge

Thomas Paccalet

Loiec Faye

Stephanie Aquin

Muriel Bardor

Marc-Andre D'Aoust

Pierre-Olivier Lavoie

Manon Couture

Louis-Philippe Vezina

Michele Dargis

Nathalie Landry

Daniel F Voytas

Feng Zhang

Edward Peter Rybicki

Frederic Ors

Sonia Trepanier

Aurelien Lorin

Alain Doucet

Dominique Michaud

Ko Kato

Luc Mathis

Thomas Stoddard

Dany Paquet

Nicole Bechtold

George Peter Lomonossoff

Louis-Philippe Vézina

Inga Isabel Hitzeroth

Véronique Gomord

Lucie Poulin

Marie-Claire Goulet

Luc Laurin

Loic Faye

Christophe Rihouey

Stéphanie Aquin

Carole Burel

Jin Li

Frank Sainsbury

Sonia Trépanier

Andre Gosselin

Shotaro Yamasaki

Pierre-Oliver Lavoie

Steeve Pepin

Gilbert Ethier

Linda Gaudreau

Marielle Gagne

Michele Martel

Li Liu

Masaaki Arai

Naomi Asahara

Michèle Dargis

Frédéric Ors

Normand Dubuc

Carmen Canizares

Stephanie Robert

Philippe Varennes-Jutras

David Levi Rutendo Mutepfa

Veronique Gomord

Estelle Marquet-Blouin

George Lomonossoff

Christophe Sourrouille

INFLUENZA VIRUS-LIKE PARTICLE PRODUCTION IN PLANTS

The present invention relates to the production of modified influenza vial proteins in plants. More specifically, the present invention relates to producing and increasing influenza virus-like particle (VLP) production in plants, wherein the VLPs comprise the modified influenza viral proteins, such as modified influenza hemagglutinin (HA). The HA protein may comprising an amino acid sequence comprising at least one substitution when compared to a corresponding wildtype amino acid sequence. Further provided are nucleic acid encoding the modified HA protein. Furthermore methods of producing an influenza virus like particle (VLP) and methods of increasing yield of production of an influenza virus like particle (VLP) in a plant, portion of a plant, or a plant cell, are also provided.

Influenza virus hemagglutinin mutants

An isolated expression enhancer active in a plant, portion of a plant or plant cell, the expression enhancer is provided. The isolated expression enhancer may be selected from the group consisting of nbMT78 (SEQ ID NO:1); nbATL75 (SEQ ID NO:2); nbDJ46 (SEQ ID NO:3); nbCHP79 (SEQ ID NO:4); nbEN42 (SEQ ID NO:5); atHSP69 (SEQ ID NO:6); atGRP62 (SEQ ID NO:7); atPK65 (SEQ ID NO:8); atRP46 (SEQ ID NO:9); nb30S72 (SEQ ID NO:10); nbGT61 (SEQ ID NO:11); nbPV55 (SEQ ID NO:12); nbPPI43 (SEQ ID NO:13); nbPM64 (SEQ ID NO:14); and nbH2A86 (SEQ ID NO:15). Methods for using the isolated expression enhancer are also provided.

Plant expression enhancer

An isolated expression enhancer active in a plant, portion of a plant or plant cell, the expression enhancer is provided. The isolated expression enhancer may be selected from the group consisting of nbEPI42 (SEQ ID NO:1); nbSNS46 (SEQ ID NO:2); nbCSY65 (SEQ ID NO:3); nbHEL40 (SEQ ID NO:4); and nbSEP44 (SEQ ID NO:5). Methods for using the isolated expression enhancer are also provided.

Endogenous plant expression enhancer

Production of modified influenza viral proteins in plants is described. More specifically, producing and increasing influenza virus-like particle (VLP) production in plants, wherein the VLPs comprise the modified influenza viral proteins, such as modified influenza hemagglutinin (HA) is described. The HA protein may comprise an amino acid sequence comprising at least one substitution when compared to a corresponding wildtype amino acid sequence. Further provided are nucleic acids encoding the modified HA protein. Furthermore, methods of producing an influenza virus like particle (VLP) and methods of increasing yield of production of an influenza virus like particle (VLP) in a plant, portion of a plant, or a plant cell, are also provided.

The present invention relates to the production of modified influenza viral proteins in plants. More specifically, the present invention relates to producing and increasing influenza virus-like particle (VLP) production in plants, wherein the VLPs comprise the modified influenza viral proteins, such as modified influenza hemagglutinin (HA). The HA protein may comprising an amino acid sequence comprising at least one substitution when compared to a corresponding wildtype amino acid sequence. Further provided are nucleic acid encoding the modified HA protein. Furthermore methods of producing an influenza virus like particle (VLP) and methods of increasing yield of production of an influenza virus like particle (VLP) in a plant, portion of a plant, or a plant cell, are also provided.

Nucleic acids encoding modified norovirus VP1 proteins, and virus-like particles (VLPs) containing one or more of the modified norovirus VP1 proteins, and compositions containing the nucleic acids, VP1 proteins, or the VLPs are provided. Methods for producing modified norovirus VP1 protein, and norovirus VLP, production in plants, portions of the plant or a plant cell, are also provided.

Modified norovirus VP1 proteins and VLPS comprising modified norovirus VP1 proteins

Nucleic acids encoding norovirus VP1 fusion proteins and VLPs comprising the norovirus VP1 fusion proteins are provided. Methods for norovirus VP1 fusion protein and norovirus VLP production in plants are also described. The VP1 fusion protein comprises, a first sequence encoding an S domain derived from a first norovirus strain, and a second sequence encoding a P domain derived from a second norovirus strain.

Norovirus fusion proteins and VLPs comprising norovirus fusion proteins

A method for synthesizing a protein of interest within a plant or a portion of a plant is provided. The method involves treating the plant to increase secondary leaf biomass production, followed by introducing one or more than one nucleic acid sequence encoding a protein of interest operatively linked with a regulatory region that is active in the plant, into the plant. The plant is then maintained under conditions that permit the nucleic acid sequence encoding the protein of interest to be expressed in the plant or the portion of the plant. Optionally, the plant or portion of the plant may be harvested and the protein of interest extracted.

Modifying protein production in plants

An expression enhancer comprising a CPMV 5′UTR nucleotide sequence consisting of X nucleotides (CMPVX), where X=160, 155, 150, or 114 of SEQ ID NO:1, or consisting of a nucleotide sequence comprising from about 80% to 100% sequence similarity with CMPVX, where X=160, 155, 150, or 114 of SEQ ID NO:1 SEQ ID NO:1 is provided. The expression enhancer may further comprise a stuffer sequence fused to the 3′ end of the 5′UTR nucleotide sequence (CMPVX+, where X=160, 155, 150, or 114 of SEQ ID NO:1). The stuffer sequence may comprise one or more plant kozak sequences. Plants comprising the expression enhancer and methods using the expression enhancer are also described

CPMV enhancer elements

Methods of preparing plant-derived proteins or suprastructure proteins, are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized proteins, or suprastructure proteins, producing a protoplast/spheroplast fraction and apoplast fraction from the plant or plant matter, and recovering the apoplast fraction. The apoplast fraction comprises plant-derived proteins or suprastructure proteins. Alternatively, the proteins, or suprastructure proteins, may be obtained from plant or plant matter comprising plant-derived proteins or suprastructure proteins, by digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction. The digested fraction is filtered to produced a filtered fraction, and the plant-derived proteins or suprastructure proteins, are recovered from the filtered fraction.

Method of preparing plant-derived proteins

Methods of preparing plant-derived virus like particles (VLPs) are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized VLPs, producing a protoplast/spheroplast fraction and apoplast fraction from the plant or plant matter, and recovering the apoplast fraction. The apoplast fraction comprises plant-derived VLPs. Alternatively, VLPs may be obtained from plant or plant matter comprising plant-derived VLPs by digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction. The digested fraction is filtered to produced a filtered fraction, and the plant-derived VLPs are recovered from the filtered fraction.

Method of preparing plant-derived VLPs

Nucleic acids encoding modified norovirus VP1 proteins, and VLPs comprising one or more of the modified norovirus VP1 proteins are provided. Methods for modified norovirus VP1 protein, and norovirus VLP, production in plants, portions of the plant or a plant cell, are also described.

Modified norovirus VP1 proteins and VLPs comprising modified norovirus VP1 proteins

Norovirus fusion proteins and VLPS comprising norovirus fusion proteins

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CA

Marc-Andre D'Aoust