WO2007095304A2 - Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux - Google Patents

Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux Download PDF

Info

Publication number
WO2007095304A2
WO2007095304A2 PCT/US2007/003942 US2007003942W WO2007095304A2 WO 2007095304 A2 WO2007095304 A2 WO 2007095304A2 US 2007003942 W US2007003942 W US 2007003942W WO 2007095304 A2 WO2007095304 A2 WO 2007095304A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
plant
polypeptide
plants
expression
Prior art date
Application number
PCT/US2007/003942
Other languages
English (en)
Other versions
WO2007095304A3 (fr
Inventor
Vidadi Yusibov
Vadim Mett
Shailaja Rabindran
Moneim Shamloul
Original Assignee
Fraunhofer Usa, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/353,905 external-priority patent/US7692063B2/en
Application filed by Fraunhofer Usa, Inc. filed Critical Fraunhofer Usa, Inc.
Priority to BRPI0707785-8A priority Critical patent/BRPI0707785A2/pt
Priority to EP07750761A priority patent/EP1984509A4/fr
Priority to CA002642138A priority patent/CA2642138A1/fr
Priority to JP2008554439A priority patent/JP2009528025A/ja
Priority to AU2007215066A priority patent/AU2007215066A1/en
Priority to CN200780007805A priority patent/CN101815784A/zh
Publication of WO2007095304A2 publication Critical patent/WO2007095304A2/fr
Priority to IL193390A priority patent/IL193390A0/en
Publication of WO2007095304A3 publication Critical patent/WO2007095304A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/14—Plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Provisional Patent Applications Numbered 60/773,255 (entitled “Bacillus Anthracis Antigens, Vaccine Compositions, and Related Methods”); 60/773,374 (entitled “HPV Antigens, Vaccine Compositions, and Related Methods”); and 60/773,378 (entitled “Influenza Antigen, Vaccine Compositions, and Related Methods”); each of which was filed on February 13, 2006. The entire contents of each of these applications is hereby incorporated by reference.
  • Patent Application number 60/944,770 filed September 15, 2006 and entitled "Influenza Antibodies, Vaccine Compositions, and Related Methods". The entire contents of this application are hereby incorporated by reference.
  • Patent Application number 60,879,450 filed January 9, 2007 and entitled "Launch Vector for the Production of Vaccine Antigens in Plants". The entire contents of each of these applications is hereby incorporated by reference.
  • Transgenic animals may provide an unlimited supply of complex proteins. Unfortunately, this system is limited by the long period of time it takes to generate new and improved products and the risk of pathogen transfer to human subjects.
  • harvesting the plant typically requires breaking the plants, for example, by removing the leaves, separating the stems from the roots, or removing the roots. Such breakage usually results in a process that initiates wilting of the plant part and apoptosis of the plant.
  • a plant undergoing apoptosis generates proteases that contribute to the degradation of the transgenically expressed protein before purification of the protein is even begun. Even if the plant is to be directly consumed, the activity of the expressed pharmaceutical protein may be reduced by harvest-induced intercellular degradation machinery.
  • Another major concern associated with producing foreign proteins in transgenic plants that are grown in open fields is the possibility of cross-pollination with plants in the wild, making it possible for the foreign protein to enter the food chain.
  • the present invention provides systems for the production of proteins or polypeptides (and/or of nucleic acids), particularly pharmaceutical proteins or polypeptides, in plants.
  • the present invention provides systems for rapid expression of proteins or polypeptides in plants.
  • the present invention provides for expression of proteins or polypeptides in young plants.
  • such young plants are sprouted seedlings.
  • the young plants e.g., sprouted seedlings
  • the plants can be consumed or harvested live.
  • the plants are grown (e.g., from a seed) in a contained, regulatable environment, e.g., indoors.
  • a protein or polypeptide to be produced in accordance with the present invention is expressed in plant cells from a nucleic acid construct that is introduced into the plant by the hand of man.
  • a protein or polypeptide to be produced in accordance with the present invention is translated in plant cells from an RNA with characteristics of a plant virus. In some such embodiments, these characteristics include or are selected from the group consisting of self-replication, cell-to- cell movement, systemic movement, and combinations thereof.
  • the RNA is self-replicating and capable of cell-to-cell movement, but is not capable of systemic movement in the plant.
  • a protein or polypeptide to be produced in accordance with the present invention is expressed in plant cells from a nucleic acid construct that replicates in Agrobacterium.
  • the present invention utilizes a construct that replicates in Agrobacterium and also contains a promoter directing expression of an RNA with characteristics of a plant virus.
  • this RNA includes sequences encoding the protein or polypeptide of interest.
  • a protein or polypeptide of interest is produced in plants by a process involving introducing into cells of a plant a vector that replicates in Agrobacterium and also contains a promoter directing expression of an RNA with characteristics of a plant virus, which RNA is not capable of systemic movement in a plant, but is capable of self- replication and which RNA further encodes the protein or polypeptide of interest.
  • a construct where a construct is utilized that replicates in Agrobacterium, the construct is introduced into plant cells by agroinfiltration.
  • nucleic acid DNA and/or RNA
  • protein of interest without limitation as to the particular use(s) of the nucleic acid or protein.
  • enzymes of use in any of a wide variety of industrial processes or bioremediation processes e.g., enzymes that degrade pollutants
  • the description of the invention, and the claims are to be considered as applying to any nucleic acid or protein of interest even if not explicitly indicated, including those with therapeutic applications and those without, hi certain embodiments the protein is not a nutritionally important protein. Any particular protein may be excluded from the present invention without being named herein.
  • the nucleic acid or protein of interest is post-transcriptionally and/or post-translationally processed in the cell in which it is expressed.
  • a protein of interest is secreted from the cell in which it is expressed.
  • the protein may naturally comprise a secretion signal sequence, or the coding region of a nucleic acid that encodes the protein may be modified to include a portion that encodes a secretion signal sequence. Secretion signal sequences are well known in the art.
  • a heterologous sequence that encodes a protein of interest may be altered to employ different codons from those present in the naturally occurring sequence, in order to improve expression in plants generally and/or in plants of a particular species.
  • the sequence may be codon optimized for expression in a particular species. Methods for performing codon optimization are known in the art.
  • the present invention also provides, among other things, plants containing expression constructs as described herein, compositions containing such plants, preparations of proteins/polypeptides isolated from such plants, compositions containing such proteins/polypeptides isolated from such plants, methods of performing such isolations, and methods of using such isolated proteins/polypeptides (or compositions containing them), including methods of treating a mammal with a pharmaceutically active protein expressed in plants.
  • administering of a pharmaceutically active protein or polypeptide to a subject in need thereof is intended as providing the pharmaceutically active protein to such subject in a manner that retains the therapeutic effectiveness of such protein for a length of time sufficient to provide a desired beneficial effect to such host.
  • the term "animal” covers all vertebrates, life forms, humans, bovines, ovines, porci ⁇ es, canines, felines, ferrets, rodents, primates, fish, birds, e.g., poultry and the like.
  • the animals are mammals.
  • the animals are humans.
  • a "characteristic portion" of a polypeptide or protein is of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide.
  • each such continuous stretch generally will contain at least 2, 5, 10, 15, 20 or more amino acids.
  • a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact protein.
  • the characteristic portion may be biologically active.
  • a "domain" of a protein or polypeptide generally refers to a segment of protein or polypeptide sequence that, when produced apart from the rest of the protein or polypeptide sequence, maintains a degree of three-dimensional integrity and/or structure. As is known in the art, many proteins and polypeptides have domain structures. In some embodiments the domain has activity (e.g., binding, catalytic, etc.). In some embodiments, a domain is immunogenic. An immunogenic domain is at least as large as a single epitope, and is typically larger; in some embodiments, an immunogenic domain contains sufficient sequence in addition to the epitope to ensure proper presentation of the epitope.
  • “Expression” refers to transcription and/or translation of an endogenous gene or a transgene in a plant, e.g., a sprout.
  • the transgene may or may not be integrated into the genomic DNA of the plant.
  • “expression” refers to transcription and/or translation in a plant of a gene present in a bacterial, plasmid, or viral nucleic acid, regardless of whether the bacterial, plasmid, or viral nucleic acid is integrated into the genomic DNA of the plant.
  • the gene may be a gene that is heterologous to the bacterium, plasmid, or virus.
  • Expression cassette or "expression vector” refers to a DNA sequence (or an
  • RNA sequence in the case of RNA viruses or RNA plasmids capable of directing expression of a particular nucleotide sequence in an appropriate host cell.
  • an expression cassette may include a promoter operably linked to a nucleotide sequence of interest, which is optionally operably linked to 3' sequences, such as 3' regulatory sequences or termination signals.
  • An expression cassette may also typically include sequences required for proper translation of the nucleotide sequence if any such sequences are needed.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example, an antisense RNA or a non-translated RNA that inhibits expression of a particular gene.
  • the expression cassette including the nucleotide sequence of interest may be chimeric, meaning that the nucleotide sequence includes more than one DNA sequence of distinct origin that are fused together by recombinant DNA techniques, resulting in a nucleotide sequence that does not occur naturally and that particularly does not occur in the plant in which it is to be expressed.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombination form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue, organ, or stage of development.
  • a nuclear expression cassette is usually inserted into the nuclear genome of a plant and is capable of directing the expression of a particular nucleotide sequence from the nuclear genome of the plant.
  • a plastid expression cassette is usually inserted into the plastid genome of a plant and is capable of directing the expression of a particularly nucleotide sequence from the plastid genome of the plant.
  • a plastid expression cassette for expression of nucleotide sequence from a plastid genome, additional elements, e.g., ribosome binding sites, or 3' stem-loop structures that impede plastid RNA polyadenylation and subsequent degradation may be required.
  • an expression cassette is utilized that comprises a promoter that is a minimal promoter such as a TATA element, and presence of a trans-activating factor may be necessary to direct expression of the nucleotide sequence, particularly to direct high level expression.
  • the expression cassette may comprise a DNA region for binding of a transcriptional activator.
  • Such expression cassettes, and the promoters therein, are referred to as being "activatable".
  • a "food” or “food product” is a liquid or solid preparation that can be ingested by humans or other animals.
  • the terms include preparations of the raw or live plants (e.g., sprouted seedlings) that may be fed live directly to humans and/or other animals.
  • Materials obtained from a plant are intended to include a whole edible plant that can be ingested by a human or other animal.
  • the term may also include any processed plant (e.g., sprouted seedling) together with a nutritional carrier that is fed to humans and other animals. Processing steps may include steps commonly used in the food or feed industry.
  • Such steps include, but are not limited to concentration or condensation of the solid matter of the plant to form, for example, a pellet, production of a paste, drying, or lyophilization, or may be produced by cutting, mashing, or grinding of the plant to various extents, or by extraction of the liquid part of the plant to produce a soup, a syrup, or a juice.
  • a processing step can also include cooking, e.g., steaming, the plant (e.g., sprouted seedling).
  • a "gene” is a sequence that encodes a protein or polypeptide (or RNA) of interest.
  • a gene may include associated regulatory sequences.
  • a gene's coding sequence may be transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
  • a “gene” may also be an RNA (e.g., a gene in a viral RNA vector).
  • regulatory sequences are promoter sequences, 5' and 3' untranslated sequences, and termination sequences.
  • introns and exons may also be included.
  • the gene is the coding sequence and the associated regulatory sequences are heterologous sequences.
  • Heterologous sequences means of different natural origin or of synthetic origin. For example, if a nucleic acid is introduced into a host cell and the host cell does not naturally contain some or all of the sequences present in the nucleic acid, then those sequences (and/or the nucleic acid) are said to be heterologous with respect to the host cell.
  • the introduced nucleic acid may include a heterologous promoter, heterologous coding sequence, or heterologous termination sequence.
  • the transforming nucleic acid may be completely heterologous or may include any possible combination of heterologous and endogenous nucleic acid sequences.
  • heterologous refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.
  • heterologous applies to cells, including plant and bacterial cells, and also to plasmids, plastids, and viruses.
  • a "host cell” is a cell into which a nucleic acid is introduced for expression.
  • an “inactive expression cassette” or “inactive expression vector” is a DNA or
  • RNA sequence that comprises an inactive or silenced foreign nucleic acid sequence which is capable of directing expression of a nucleic acid or polypeptide of interest upon its activation.
  • an inactive expression cassette has the properties of an expression cassette as described above, except that the sequence that codes for a nucleic acid or polypeptide of interest may not be operatively linked to a promoter, e.g., it may be separated from the promoter (or from another regulatory element) by an intervening nucleic acid region. Operative linkage occurs following a recombination event, so that expression then occurs.
  • expression cassette is referred to as being "activatable".
  • inducible promoter means a promoter that is turned on by the presence or absence of a particular stimulus that increases promoter activity directly or indirectly.
  • Some non-limiting examples of such stimuli include heat, light, developmental regulatory factors, wounding, hormones, and chemicals, e.g., small molecules.
  • a light-inducible promoter is the ribulose-5 -phosphate carboxylase promoter.
  • Chemically-inducible promoters also include receptor-mediated systems, e.g., those derived from other organisms, such as steroid-dependent gene expression, the Lac repressor system and the expression system utilizing the USP receptor from Drosophila mediated by juvenile growth hormone and its agonists, described in WO 97/13864, incorporated herein by reference, as well as systems utilizing combinations of receptors, e.g., as described in WO 96/27673, also incorporated herein by reference.
  • receptor-mediated systems e.g., those derived from other organisms, such as steroid-dependent gene expression, the Lac repressor system and the expression system utilizing the USP receptor from Drosophila mediated by juvenile growth hormone and its agonists, described in WO 97/13864, incorporated herein by reference, as well as systems utilizing combinations of receptors, e.g., as described in WO 96/27673, also incorporated herein by reference.
  • Additional chemically inducible promoters include elicitor-induced promoters, safener-induced promoters as well as the alcA/alcR gene activation system that is inducible by certain alcohols and ketones (WO 93/21334; Caddick et al. (1998) Nat. Biotechnol. 16:177-180, the contents of which are incorporated herein by reference).
  • Wond inducible promoters include promoters for proteinase inhibitors, e.g., proteinase inhibitor II promoter from potato, and other plant-derived promoters involved in the wound response pathway, such as promoters for polyphenyl oxidases, LAP, and TD.
  • a "marker gene” is a gene encoding a selectable or screenable trait.
  • a "medical food” includes a composition that is eaten or drunk by a subject and has a therapeutic effect on the subject.
  • a medical food includes, for example, a plant (e.g., sprouted seedling) in which a pharmaceutical protein or polypeptide has been produced, or plant matter derived therefrom.
  • a medical food may be ingested alone or may be administered in combination with a pharmaceutical composition well known in the medical arts.
  • a medical food also includes the equivalent feedstuff for non-human animals.
  • "Operably linked" refers to the association of a nucleic acid elements.
  • a promoter operably linked to a heterologous DNA which encodes a protein, promotes the production of functional mRNA corresponding to the heterologous DNA.
  • a regulatory DNA sequence is said to be "operably linked to” or “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence.
  • Oral administration of a pharmaceutically active peptide or protein means primarily administration by way of the mouth, preferably by eating, but also intends to include any administration that provides such peptides or proteins to the host's stomach or digestive track. In a preferred embodiment, oral administration results in contact of the pharmaceutically active protein with the gut mucosa.
  • a "pharmaceutically active nucleic acid” is a nucleic acid that encodes a pharmaceutically active protein or is pharmaceutically active in its own right, e.g., it has one or more pharmaceutical activities such as those described for pharmaceutically active proteins.
  • the nucleic acid may be one or more strands of an RNA interference (RNAi) agent.
  • RNAi RNA interference
  • agents include short interfering RNAs (siRNAs), or short hairpin RNAs (shRNAs), or precursor of an siRNA or microRNA-like RNA, targeted to a target transcript, e.g., a transcript of an infectious agent or an endogenous disease-related transcript of a subject.
  • a "pharmaceutically active protein or polypeptide” aids or contributes to the condition of a host in a positive manner when administered to the host in a therapeutically effective amount.
  • a pharmaceutically active protein or polypeptide has healing curative or palliative properties against a disease and may be administered to ameliorate relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder.
  • a pharmaceutically active protein or polypeptide may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition when it does emerge.
  • pharmaceutically active proteins or polypeptides includes entire proteins or polypeptides, and can also refer to " pharmaceutically active fragments thereof.
  • pharmaceutically active protein or polypeptide can also refers to a plurality of proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. It is noted that the term “pharmaceutically active protein or polypeptide” specifically includes proteins that comprise vaccine antigens, i.e., administration of the protein to a subject elicits a partially or fully protective immune response in the host. In certain embodiments of the invention the immune response protects the subject against an infectious agent, e.g., a virus, bacterial, fungal, or protozoal pathogen.
  • infectious agent e.g., a virus, bacterial, fungal, or protozoal pathogen.
  • vaccine antigens examples include hepatitis B surface antigen (HBsAg), E. coli heat-labile enterotoxin, rabies virus glycoprotein, and Norwalk virus capsid protein.
  • HBsAg hepatitis B surface antigen
  • E. coli heat-labile enterotoxin E. coli heat-labile enterotoxin
  • rabies virus glycoprotein rabies virus glycoprotein
  • Norwalk virus capsid protein hepatitis B surface antigen
  • the immune response protects the subject against a non-infectious condition or disease or lessens the severity of at least one sign or symptom of the condition or disease.
  • Diseases of interest in this regard include, but are not limited to, cancer and auto-immune diseases.
  • pharmaceutically active protein or polypeptide includes proteins that partially or fully tolerize a subject to exposure to an allergen that would otherwise elicit an allergic or anaphylactic response.
  • a “promoter,” as used herein, is a DNA sequence that initiates transcription of an associated DNA sequence.
  • the promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors.
  • Regulatory elements refer to sequences involved in conferring the expression of a nucleotide sequence. Regulatory elements include 5' regulatory sequences such as promoters that can be linked to the nucleotide sequence of interest, 3' sequences such as 3' regulatory sequences or termination signals. Regulatory elements also typically encompass sequences required for proper translation of the nucleotide sequence.
  • Small molecules are typically less than about one kilodalton and are biological, organic, or even inorganic compounds (e.g., cisplatin). Examples of such small molecules include nutrients such as sugars and sugar-derivatives (including phosphate derivatives), hormones (such as the phytohormones gibberellic or absisic acid), and synthetic small molecules.
  • nutrients such as sugars and sugar-derivatives (including phosphate derivatives), hormones (such as the phytohormones gibberellic or absisic acid), and synthetic small molecules.
  • hormones such as the phytohormones gibberellic or absisic acid
  • synthetic small molecules synthetic small molecules.
  • Specifically regulatable refers to the ability of a small molecule to preferentially affect transcription from one promoter or group of promoters, as opposed to non-specific effects, such as enhancement or reduction of global transcription within a cell.
  • a "sprouted seedling” or “sprout” is a young shoot from a seed or a root, preferably a recently germinated seed.
  • the sprouted seedlings of the invention are edible sprouted seedlings or sprouts (e.g., alfalfa sprouts, mung bean sprouts, radish sprouts, wheat sprouts, mustard sprouts, spinach sprouts, carrot sprouts, beet sprouts, onion sprouts, garlic sprouts, celery sprouts, rhubarb sprouts, a leaf such as cabbage sprouts, or lettuce sprouts, watercress or cress sprouts, herb sprouts such as parsley or clover sprouts, cauliflower sprouts, broccoli sprouts, soybean sprouts, lentil sprouts, edible flower sprouts such as sunflower sprouts, etc.).
  • the sprouted seedling may have developed to the two-leaf stage. Generally, the sprouts of the invention are two to fourteen days old.
  • Substantially isolated is used in several contexts and typically refers to the at least partial purification of a protein or polypeptide away from unrelated or contaminating components (for example, plant structural and metabolic proteins). Methods for isolating and purifying proteins or polypeptides are well known in the art.
  • Transformation refers to introduction of a nucleic acid into a cell, particularly the stable integration of a DNA molecule into the genome of an organism of interest.
  • Figure 1 is a schematic representation of different strategies for foreign gene expression using plant virus-based vectors.
  • Figure 2 is a schematic representation of AIMV and TMV genomes.
  • Figure 5 is a picture of a Western blot of expression of recombinant GFP in
  • Figure 4 is a picture of a Western blot of human growth hormone (hGH) production in N. benth ⁇ mi ⁇ n ⁇ plants infected with in vitro transcripts of GH.
  • Figure 5 is a schematic representation of transformation constructs for expression of recombinant proteins in Br ⁇ ssic ⁇ j ' unce ⁇ .
  • Figure 6 is a picture of an immunoblot of transgenic Br ⁇ ssic ⁇ junce ⁇ expressing human growth hormone under control of the HSPl 8.2 promoter.
  • Figure 7 depicts a schematic representation of various agrobacterial constructs containing target genes.
  • Figure 7B Vector pBIV-GUS, in which the GUS gene is inserted in pBIV.
  • Figure 7C pBIV- Virus Vector + Target, in which a viral vector or replicon (e.g., a viral genome or genome portion) carrying a target gene is inserted in to pBIV;
  • Figure 7D Vector pBIV-Virus Vector + GFP, in which GFP is the target gene carried by a viral vector or replicon inserted into pBIV.
  • the vectors depicted in Figures 7C and 7D are considered "launch" vectors because, after introduction into plant cells, they "launch” production of the viral sequences (which may be self replicating, at least in the context of the particular plant cell into which they are launched).
  • Figure 8 demonstrates GUS staining after agroinfiltration of sprouts with pBFV-GUS.
  • Figure 8 A demonstrates staining of Mung bean sprout;
  • Figure 8B demonstrates staining of Fenugreek sprouts.
  • Figure 9 demonstrates GUS staining after agroinfiltration of Brassica sprouts with pBFV-GUS.
  • Figure 9A demonstrates staining results;
  • Figure 9B is a table listing different types of sprouts tested for their ability to express protein or polypeptide delivered by means of an agrobacterial construct that includes a promoter directing expression of viral sequences carrying a gene that encodes the protein or polypeptide of interest.
  • Figure 10 demonstrates GFP expression in N. benthamiana over time after infiltration with pBI121/D4-GFPC3.
  • Figure 11 demonstrates western blot analysis of hGH expression in plants as a result of infiltration with Agrobacterium containing a virus vector containing a sequence encoding hGH.
  • FIG. 12 demonstrates expression of IA-2ic protein in in Nicotiana benthamiana plants grown in hydroponics: Lanes: 1: IA-2ic standard; 2. Magic Marker; 3.
  • Figure 13 depicts one emodiment of an agroinfiltration system utilized in accordance with the present invention to delivery an agrobacterial vector that launches viral sequences carrying a target protein of interest into young plants.
  • Figure 14 illustrates activity of lichenase detected in various pea varieties subjected to agroinfiltration using the system of Figure 13.
  • Figure 15 shows GFP expression and plant growth for speckled peas subjected to agroinfiltration using the system of Figure 13.
  • Figure 16 shows tissue specificity and optimal days post infiltration for certain speckled peas subjected to agroinfiltration using the system of Figure 13.
  • Figure 17 depicts a lichenase carrier molecule that may be used in fusions with proteins or polypeptides of interest in accordance with certain embodiments of the invention.
  • Panel A presents a schematic representation of b-1 ,3-1 ,4-glucanase (lichenase) from Clostribium thermocellum.
  • Panel B prsents a schematic representation of the circularly permuted LicKM carrier, In each panel, the vertical hatch corresponds to the N-terminal region of the catalytic doman, and the dotted box corresponds to the C-terminal region of the catalytic domain.
  • Figure 18 presents a schematic diagram of the launch vector pBID4.
  • Figure 19 is a schematic representation of each step involved in the process from target gene optimization to purification of a final product that is ready for storage according to one specific embodiment of the invention.
  • Figure 20 depicts an equipment design useful for automation of steps from plant seeding to tissue harvest.
  • the module is estimated to occupy a floor area of about 40 feet by about 70 feet and to be about 32 feet high.
  • the system shown includes the following components connected by conveyors: central shelving units where plants are maintained between steps, a seeding unit, a vacuum infiltration and rinsing unit, and a tissue harvesting unit.
  • the present invention is applicable to the production of any protein or polypeptide (or any functional DNA or RNA molecule), in plant systems.
  • the invention is directed to production of pharmaceutical proteins, but the invention generally is not limited by the particular use(s) of the nucleic acid or protein.
  • enzymes of use in any of a wide variety of industrial processes or bioremediation processes e.g., enzymes that degrade pollutants
  • the description of the invention, and the claims are to be considered as applying to any nucleic acid or protein of interest even if not explicitly indicated, including those with therapeutic applications and those without.
  • the protein is not a nutritionally important protein. Any particular protein may be excluded from the present invention without being named herein.
  • the expressed protein or polypeptide is not one that is expressed in the plant in nature. Even if the protein or polypeptide is one that is expressed in the plant in nature, according to the present invention, it is typically expressed in young plant tissue at concentrations above that which would be present in comparable tissue in nature.
  • the present invention may be utilized to produce pharmaceutical proteins of interest including, but not limited to, hormones (insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like), growth factor receptors, cytokines and immune system proteins (e.g., interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, seletins, homing receptors, T cell receptors, immunoglobulins, soluble major histos, hormones (insulin, thyroid hormone, cate
  • the present invention is utilized to produce antigenic proteins or polypeptides.
  • the present invention may be utilized to produce proteins (or portions thereof) of infectious organisms that are recognized by the immune system of an infected subject.
  • proteins or polypeptides may have particular use in the development of vaccines for protection against infection by the relevant organisms.
  • useful antigenic proteins from anthrax (Bacillus a ⁇ thracis ; e.g., LF, PA), cholera (Vibrio choler ⁇ e), cytomegalovirus, enterotoxigenic strains of E. coli , foot-and-mouth disease virus, hepatitis B (e.g.
  • hepatitis B surface antigen HBsAg
  • hepatitis C e.g., HCV core protein
  • human immunodeficiency virus e.g., Tat, Rev, Nef, gpl60, gpl20, etc.
  • human papilloma virus e.g., E7, E6
  • influenza e.g., HA, NA
  • malaria Plasmodium falciparum; e.g.
  • Pfs25, Pfs28, Pfs48/45, Pfs230 measles virus, norwalk virus, plague (Yersinia pestis; e.g., Fl, LcrV), Pseudomonas aeruginosa, rabies virus, respiratory syncytial virus (e.g., F protein, G protein), rhinovirus, rotavirus, Staphylococcus aureus, transmissible gastroenteritis virus, trypanosomes ( Trypanosoma brucei; e.g., alpha-tubulin, beta-tubulin), tuberculosis, SARS, etc. can be produced in accordance with the present inventive system.
  • Yersinia pestis e.g., Fl, LcrV
  • Pseudomonas aeruginosa rabies virus
  • respiratory syncytial virus e.g., F protein, G protein
  • rhinovirus rotavirus
  • the present invention is utilized to produce multimeric protein complexes, including, for example, immunoglobulin proteins such as antibodies (i.e., monoclonal antibodies).
  • immunoglobulin proteins such as antibodies (i.e., monoclonal antibodies).
  • anti-PA, anti-LF, anti-NA, anti-TNFa, anti-interleukin-12, etc. can be produced in accordance with the present inventive system.
  • monoclonal antibodies to antigens associated with diseases such as cancer (e.g., acute myeloid leukemia (AML), breast cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, renal cell carcinoma, solid tumors), inflammatory and autoimmune disorders (e.g., Asthma, Crohn's disease, Diabetes, Graft versus host disease (organ rejection), Inflammatory bowel disease, Lupus, Multiple sclerosis, Psoriasis, Psoriatic arthritis, Rheumatoid arthritis, Thrombosis, Vasculitis, etc), infectious diseases (e.g., anthrax (Bacillus anthracis), cholera (Vibrio cholerae), cytomegalovirus, enterotoxigenic strains of E.
  • cancer e.g., acute myeloid leukemia (AML), breast cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), non
  • coli foot-and-mouth disease virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human papilloma virus, influenza, malaria ⁇ Plasmodium falciparum;), measles virus, norwalk virus, plague (Yersinia pestis), Pseudomonas aeruginosa, rabies virus, respiratory syncytial virus, rhinovirus, rotavirus, Staphylococcus aureus, transmissible gastroenteritis virus, trypanosomes (Trypanosoma bruce ⁇ ), tuberculosis, SARS), etc can be prepared according to the present invention.
  • the present invention is utilized to produce hGH, auto-antigens for autoimmune diseases (e.g., IA-2ic for diabetes), influenza antigens, anthrax antigens, HPV antigens, influenza antibodies, etc. (see Examples).
  • autoimmune diseases e.g., IA-2ic for diabetes
  • influenza antigens e.g., anthrax antigens
  • HPV antigens e.g., HPV antigens
  • influenza antibodies e.g., etc., etc. (see Examples).
  • full-length heterologous proteins are produced. That is, a protein that occurs in nature or that has been prepared or designed in another context is produced in its entirety in young plants. In some such embodiments, the produced protein is free of any other sequences.
  • only a portion of the protein is produced, typically a portion that has a known desirable activity (e.g., an epitope of an antigenic protein; an active domain of an enzymatic protein, etc.).
  • the produced portion is a characteristic portion of the protein or polypeptide, hi some embodiments, the produced portion comprises an epitope.
  • the produced portion comprises an immunogenic domain. In some embodiments, the produced portion comprises a protein domain.
  • proteins or polypeptides are produced as fusions with other protein or polypeptide sequences.
  • fusions can be used for any of a variety of reasons.
  • proteins or polypeptides to be produced in young plants (e.g., sprouted seedlings) according to the present invention may be fused with one or more moieties to facilitate detection and/or isolation.
  • proteins or polypeptides of interest might be fused with a known antigenic epitope for which an antibody or other specific ligand is available and can be used to facilitate isolation or purification.
  • proteins or polypeptides of interest may be fused with another polypeptide of defined three-dimensional structure, for example to impart a desired three-dimensional arrangement to the protein or polypeptide of interest.
  • proteins or polypeptides of interest may be fused with one or more moieties that will direct their sub-cellular localization (or secretion) in plant cells.
  • the protein or polypeptide of interest is a protein antigen (e.g., for use in preparation of a subunit vaccine)
  • it will often be desirable to produce the protein as a fusion with a carrier molecule for example to enhance expression, stability, and/or immunogenicity, and/or to aid in isolation or purification.
  • Certain exemplary fusion partners known in the art include, for example, antibody epitopes (e.g., His tag, etc.), immunogenic carrier prteins, cholera toxin, heat labile enterotoxin, etc. (see, for example, Vella et al., Biotechnology 20:1, 1992; Kelly et al., Immuology 113:163, 2004; Buttery et al., JR Coll Phuysicians Lond 34:163, 2000; Jacobson et al, Minerva Peditr. 54:295, 2002; Dertzbaugh et al., Infect Immunol.
  • antibody epitopes e.g., His tag, etc.
  • immunogenic carrier prteins e.g., cholera toxin
  • heat labile enterotoxin e.g., etc.
  • thermostable protein such as lichenase B from Clostridium thermocellum (see, for example, United States Provisional Patent Application 60/472,495 filed May 22, 2003 and PCT Application US04/016452 filed May 24, 2004 and published as WO 2005/026375 March 24, 2005.
  • Full-length lichenase (about 35 kDa) consists of a signal peptide, a catalytic domain, a Pro-Thr-rich box, and a docking domain (see Figure 17).
  • both the Pro-Thr rich box and the docking domain can be deleted in fusion proteins.
  • the native signal peptide can be replaced with one that operates in plants.
  • the catalytic domain of this lichenase has a loop structure dividing it into two regions, the N-terminal region (N: animo acids 32-84) and the C-terminal region (C:amino acids 85-246). These two regions can be split at the loop structure and circularly permuted to make the molecule more receptive to insertions without affecting enzymatic activity.
  • a multiple cloning site (MCS) was introduced at the junction between these two regions, and a 6xHis tag and the sequence KDEL were places at the 3 '-end of the permuted carrier to facilitate purification and retention in the endoplasmic reticulum, respectively.
  • Figure 17 shows a schematic diagram of the modified lichenase (LicKM; GenBank accession number DQ776900) that has a molecular weight of about 27 kDa.
  • Target protein or polypeptide sequences can be expressed as N or C terminal fusions and/or as internal fusions into the MCS, a feature that allows for the simultaneous expression of multiple targets.
  • LicKM allows for fusions to molecules ranging in size from small peptides to full-length proteins of up to about 100 kDa or more. LicKM maintains its enzymatic activity at high temperatures (65 0 C), a property that also generally applies to fusions.
  • LicKM fusion proteins can be tracked during purification by monitoring lichenase activity.
  • the use of LicKM as a carrier molecule contributes additional advantages, including the potential for . enhanced expression, and incorporation of multiple polypeptides of interest (e.g., multiple vaccine determinants).
  • any plants that are amendable to expression of introduced constructs as described herein are useful in accordance with the present invention.
  • sprouted seedlings are utilized. As is known in the art, most sprouts are quick growing, edible plants produced from storage seeds. However, those of ordinary skill in the art will appreciate that the term "sprouted seedling" has been used herein in a more general context, to refer to young plants whether or not of a variety typically classified as "sprouts”.
  • Any plant that is grown long enough to have sufficient green biomass to allow introduction and/or expression of an expression construct as provided for herein (recognizing that the relevant time may vary depending on the mode of delivery and/or expression of the expression construct) can be considered a "sprouted seedling" herein.
  • edible plants are utilized (i.e., plants that are edible by — not toxic to — the subject to whom the protein or polypeptide is to be administered).
  • the plants of the invention may be plants of the Brassica or Arabidopsis species.
  • suitable plants that are amendable to transformation and are edible as sprouted seedlings include alfalfa, mung bean, radish, wheat, mustard, spinach, carrot, beet, onion, garlic, celery, rhubarb, a leafy plant such as cabbage or lettuce, watercress or cress, herbs such as parsley, mint, or clovers, cauliflower, broccoli, soybean, lentils, edible flowers such as the sunflower etc.
  • a wide variety of plant species have been tested for their suitability in the practice of the present invention.
  • a variety of different bean and other species including, for example, adzuki bean, alfalfa, barley, broccoli, bill jump pea, buckwheat, cabbage, cauliflower, clover, collard greens, fenugreek, flax, garbanzo bean, green pea, Japanese spinach, kale, kamut, kohlrabi, marrowfat pea, mung bean, mustard greens, pinto bean, radish, red clover, soy bean, speckled pea, sunflower, turnip, yellow trapper pea, and others were tested for their amenability to the production of heterologous proteins from viral vectors launched from an agrobacterial construct (introduced by agroinfiltration as described herein)(see, for example, Examples 5 and 8, Figure 9, etc.).
  • the present invention provides the surprising result that certain pea varieties are particularly amenable to such manipulation.
  • bill jump pea, green pea, marrowfat pea, speckled pea, and/or yellow trapper pea are particularly useful in accordance with this aspect of the invention.
  • the present invention provides production of proteins or polypeptides (e.g., antigens, antibodies, and/or other proteins) in one or more of these plants using an agrobacterial vector that launches a viral construct (i.e., an RNA with characteristics of a plant virus) encoding the relevant protein or polypeptide of interest.
  • the RNA has characteristics of (and/or includes sequences of) AlMV.
  • the RNA has characteristics of (and/or includes sequences of) TMV.
  • the present invention provides young plants (e.g., sprouted seedlings) that express a target protein or polypeptide of interest.
  • the young plants were grown from transgenic seeds; the present invention also provides seeds which can be generated and/or utilized for the methods described herein. Seeds transgenic for any gene of interest can be sprouted and optionally induced for production of a protein or polypeptide of interest.
  • seeds capable of expressing any gene of interest can be sprouted and induced through: i) virus infection, ii) agroinfiltration, or iii) bacteria that contain virus genome.
  • Seeds capable of expressing a transgene for heavy or light chain of any monoclonal antibody can be sprouted and induced for production of full-length molecule through: i) virus infection, ii) agroinfiltration, or iii) inoculation with bacteria that contain virus genome.
  • Seeds capable of expressing a transgene for one or more components of a complex molecule comprising multiple components such as slgA can be sprouted and used for producing a fully functional molecule through: i) virus infection, ii) agroinfiltration, or iii) inoculation with bacteria that contain virus genome.
  • Seeds from healthy non-transgenic plants can be sprouted and used for producing target sequences through: i) virus infection, ii) agroinfiltration, or iii) inoculation with bacteria that contain a virus genome.
  • the young plants were grown from seeds that were not transgenic. Typically, such young plants will harbor viral sequences that direct expression of the protein or polypeptide of interest. In some embodiments, the plants may also harbor agrobacterial sequences, optionally including sequences that "launched" the viral sequences.
  • any of a variety of different systems can be used to express proteins or polypeptides in young plants (e.g., sprouted seedlings).
  • transgenic cell lines or seeds are generated, which are then sprouted and grown for a period of time so that a protein or polypeptide included in the transgenic sequences is produced in young plant tissues (e.g., in sprouted seedlings).
  • Typical technologies for the production of transgenic plant cells and/or seeds include Agrobacterium tumefaciens mediated gene transfer and microprojectile bombardment or electroporation.
  • transient expression systems are utilized. Typical technologies for producing transient expression of proteins or polypeptides in plant tissues utilize plant viruses.
  • Viral transformation is a more rapid and less costly methods of transforming embryos and sprouted seedlings that can be harvested without an experimental or generational lag prior to obtaining the desired product.
  • viruses that are not attenuated can infect other plants, potentially causing environmental concerns.
  • the present invention provides expression systems having advantages of viral expression systems (e.g., rapid expression, high levels of production) and of Agrobacterium transformation (e.g., controlled administration).
  • an agrobacterial construct i.e., a construct that replicates in Agrobacterium and therefore can be delivered to plant cells by delivery of Agrobacterium
  • a plant promoter that, after being introduced into a plant, directs expression of viral sequences (e.g., including viral replication sequences) carrying a gene for a protein or polypeptide of interest.
  • viral sequences e.g., including viral replication sequences
  • This system allows controlled, high level transient expression of proteins or polypeptides in plants.
  • a variety of different embodiments of expression systems, some of which produce transgenic plants and others of which provide for transient expression are discussed in further detail individually below. For any of these techniques, the skilled artisan reading the present specification would appreciate how to adjust and optimize protocols for expression of proteins or polypeptides in young plant tissues (e.g., sprouted seedlings).
  • Agrobacterium is a representative genus of the gram-negative family
  • Rhizobiaceae This species is responsible for plant tumors such as crown gall and hairy root disease.
  • amino acid derivatives known as opines are produced by the Agrobacterium and catabolized by the plant.
  • the bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes.
  • the Agrobacterium transformation system may be used to generate young plants (e.g., sprouted seedlings, including edible sprouted seedlings), which are merely harvested earlier than the mature plants.
  • Agrobacterium transformation methods can easily be applied to regenerate young plants (e.g., sprouted seedlings) expressing pharmaceutical proteins.
  • transforming plants with Agrobacterium involves the transformation of plant cells grown in tissue culture by co-cultivation with an Agrobacterium tumefaciens carrying a plant/bacterial vector.
  • the vector contains a gene encoding a pharmaceutical protein.
  • the Agrobacterium transfers the vector to the plant host cell and is then eliminated using antibiotic treatment.
  • Transformed plant cells expressing the pharmaceutical protein are selected, differentiated, and finally regenerated into complete plantlets (Hellens et al., Plant Molecular Biology (2000) 42(819-832); Pilon-Smits et al, Plant Physiolog.
  • Agrobacterial expression vectors for use in the present invention include a gene (or expression cassette) encoding a pharmaceutical protein designed for operation in plants, with companion sequences upstream and downstream of the expression cassette.
  • the companion sequences are generally of plasmid or viral origin and provide necessary characteristics to the vector to transfer DNA from bacteria to the desired plant host.
  • the basic bacterial/plant vector construct preferably provides a broad host range prokaryote replication origin,' a prokaryote selectable marker. Suitable prokaryotic selectable markers include resistance toward antibiotics such as ampicillin or tetracycline.
  • Other DNA sequences encoding additional functions that are well known in the art may also be present in the vector.
  • Agrobacterium T-DNA sequences are required for Agrobacterium mediated transfer of DNA to the plant chromosome.
  • the tumor-inducing genes of the T-DNA are typically removed during construction of an agrobacterial expression construct, and are replaced with sequences encoding the pharmaceutical protein or polypeptide.
  • the T-DNA border sequences are retained because they initiate integration of the T-DNA region into the plant genome. If expression of the pharmaceutical protein is not readily amenable to detection, the bacterial/plant vector construct will also include a selectable marker gene suitable for determining if a plant cell has been transformed, e.g., the nptll kanamycin resistance gene.
  • Ti plasmid On the same or different bacterial/plant vector (Ti plasmid) are Ti sequences.
  • Ti sequences include the virulence genes, which encode a set of proteins responsible for the excision, transfer and integration of the T-DNA into the plant genome (Schell, Science (1987) 237: 1176-1183). Other sequences suitable for permitting integration of the heterologous sequence into the plant genome may also include transposon sequences, and the like, for homologous recombination.
  • Certain constructs will include the expression cassette encoding the protein of interest.
  • One, two, or more expression cassettes may be used in a given transformation.
  • the recombinant expression cassette contains, in addition to the pharmaceutical protein encoding sequence, at least the following elements: a promoter region, plant 5' untranslated sequences, initiation codon (depending upon whether or not the expressed gene has its own), and transcription and translation termination sequences.
  • transcription and translation terminators may be included in the expression cassettes or chimeric genes of the present invention.
  • Signal secretion sequences that allow processing and translocation of the protein, as appropriate, may also be included in the expression cassette.
  • bacteria other than Agrobacteria are used to introduce a nucleic acid sequence into a plant. See, e.g., Broothaerts W, et al., Gene transfer to plants by diverse species of bacteria, Nature (2005), 433(7026):629-633, which is incorporated herein by reference.
  • Seeds are prepared from plants that have been infected with Agrobacteria (or other bacteria) such that the desired heterologous gene encoding a protein or polypeptide of interest is introduced. Such seeds are harvested, dried, cleaned, and tested for viability and for the presence and expression of a desired gene product. Once this has been determined, seed stock is stored under appropriate conditions of temperature, humidity, sanitation, and security to be used when necessary. Whole plants are then regenerated from cultured protoplasts, e.g., as described in Evans et al., Handbook of Plant Cell Cultures, Vol. l:MacMillan Publishing Co. New York, 1983); and Vasil LR. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. Ill, 1986, incorporated herein by reference.
  • the plants are not regenerated into adult plants.
  • plants are regenerated only to the sprouted seedling stage.
  • whole plants are regenerated to produce seed stocks and young plants (e.g., sprouted seedlings) for use in accordance with the present invention are generated from the seeds of the seed stock.
  • All plants from which protoplasts can be isolated and cultured to give whole, regenerated plants can be transformed by Agrobacteria according to the present invention so that whole plants are recovered that contain a transferred gene encoding a protein or polypeptide of interest. It is known that practically all plants can be regenerated from cultured cells or tissues, including, but not limited to, all major species of plants that produce edible sprouts.
  • Some suitable plants include alfalfa, mung bean, radish, wheat, mustard, spinach, carrot, beet, onion, garlic, celery, rhubarb, a leafy plant such as cabbage or lettuce, watercress or cress, herbs such as parsley, mint, or clovers, cauliflower, broccoli, soybean, lentils, edible flowers such as the sunflower etc.
  • Means for regeneration of plants from transformed cells may vary from one species of plants to the next. However, those skilled in the art will appreciate that generally a suspension of transformed protoplants containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. Steeping the seed in water or spraying the seed with water to increase the moisture content of the seed to between 35-45% initiates germination. For germination to proceed, the seeds are typically maintained in air saturated with water under controlled temperature and airflow conditions. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins.
  • glutamic acid and proline are also advantageous to add glutamic acid and proline to the medium, especially for such species as alfalfa.
  • Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, the genotype, and the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
  • Mature plants, grown from the transformed plant cells, are selfed, and non- segregating, homozygous transgenic plants are identified.
  • the inbred plant produces seeds containing the transferred gene encoding a protein or polypeptide of interest. These seeds can be germinated and grown to the young plant (e.g., sprouted seedling) stage to produce the protein or polypeptide of interest.
  • transgenic seeds (carrying the transferred gene encoding a protein or polypeptide of interest, typically integrated into the genome) may be formed into seed products and sold with instructions on how to grow young plants to an appropriate stage (e.g., to the sprouted seedling stage) for harvesting and/or or administration or formulation as described herein.
  • hybrids or novel varieties embodying the desired traits i.e., the transferred gene encoding a protein or polypeptide of interest
  • Direct integration of DNA fragments into the genome of plant cells by microprojectile bombardment or electroporation may also be used to introduce expression constructs encoding proteins or polypeptides of interest into plant tissues according to the present invention (see, e.g., Kikkert, J.R. Humiston et al., In Vitro Cellular & Developmental Biology. Plant: Journal of the Tissue Culture Association. (Jan/Feb 1999) 35 (l):43-50; Bates, G. W. Florida State University, Tallahassee, FL. Molecular Biotechnology (Oct 1994) 2(2): 135-145). More particularly, vectors containing a gene encoding a protein or polypeptide of interest can be introduced into plant cells by a variety of techniques.
  • the vectors may include selectable markers for use in plant cells.
  • the vectors may also include sequences that allow their selection and propagation in a secondary host, such as sequences containing an origin of replication and selectable marker.
  • secondary hosts include bacteria and yeast.
  • the secondary host is Escherichia coli
  • the origin of replication is a colEl-type origin of replication
  • the selectable marker is a gene encoding ampicillin resistance.
  • sequences are well known in the art and are commercially available (e.g., Clontech, Palo Alto, CA or Stratagene, La Jolla, CA).
  • the vectors of the present invention may also be modified to intermediate plant transformation plasmids that contain a region of homology to an Agrobacterium tumefaciens vector, a T-DNA border region from Agrobacterium tumefaciens, and chimeric genes or expression cassettes described above. Further vectors may include a disarmed plant tumor inducing plasmid of Agrobacterium tumefaciens.
  • direct transformation of the vectors invention involves microinjecting the vectors directly into plant cells by the use of micropipettes to mechanically transfer the recombinant DNA (see, e.g., Crossway, MoI. Gen. Genet., 202:179-185, 1985, incorporated herein by reference).
  • the genetic material may also be transferred into the plant cell by using polyethylene glycols (see, e.g., Krens et al., Nature (1982) 296:72-74).
  • nucleic acid segments Another method of introducing nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (see, e.g., Klein et al., Nature (1987) 327:70- 73; Knudsen and Muller Planta (1991) 185:330-336)). Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (see, e.g., Fraley et al., Proc. Natl. Acad. Sci. USA (1982) 79:1859- 1863).
  • Vectors of the invention may also be introduced into plant cells by electroporation (see, e.g., Fromm et al. Proc. Natl. Acad. Sci. USA (1985) 82:5824).
  • plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the pasmids. Electroporated plant protoplasts reform the cell wall divide and form plant callus, which can be regenerated to form the sprouted seedlings of the invention.
  • Those skilled in the art would appreciate how to utilize these methods to transform plants cells that can be used to generate edible sprouted seedlings.
  • plant virus vectors are used to infect and produce foreign protein in seeds, embryos, sprouted seedlings.
  • infection includes any method of introducing a viral genome, or portion thereof, into a cell, including, but not limited to, the natural infectious process of a virus, abrasion, inoculation, etc.
  • the term includes introducing a genomic RNA transcript, or a cDNA copy thereof, into a cell.
  • the viral genome need not be a complete genome but will typically contain sufficient sequences to allow replication.
  • the genome may encode a viral replicase and may contain any cis-acting nucleic acid elements necessary for replication.
  • young plants which express pharmaceutical proteins such as insulin, GAD, and IA-2 associated with type 1 diabetes
  • Young plants produced by viral infection provide a source of protein or polypeptide of interest that has already been demonstrated to be safe.
  • sprouts are free of contamination with animal pathogens.
  • proteins from an edible sprout could at least in theory be used in oral applications without purification, thus significantly reducing costs. ' . . .
  • a virus/young plant e.g., sprout
  • a virus/young plant also offers a much simpler, less expensive route for scale-up and manufacturing, since the relevant genes (encoding the protein or polypeptide of interest) are introduced into the virus, which can be grown up to a commercial scale within a few days.
  • transgenic plants can require up to 5-7 years before sufficient seeds or plant material are available for large-scale trials or commercialization.
  • plant RNA viruses have certain advantages, which make them attractive as vectors for foreign protein expression.
  • the molecular biology and pathology of a number of plant RNA viruses are well characterized and there is considerable knowledge of virus biology, genetics, and regulatory sequences.
  • Most plant RNA viruses have small genomes and infectious cDNA clones are available to facilitate genetic manipulation. Once the infectious virus material enters the susceptible host cell, it replicates to high levels and spreads rapidly throughout the entire plant (one to ten days post inoculation). Virus particles are easily and economically recovered from infected tissue. Viruses have a wide host range, enabling the use of a single construct for infection of several susceptible species. These characteristics are easily transferable to sprouts.
  • FIG. 1 illustrates several different strategies for expressing foreign genes using plant viruses.
  • Foreign sequences can be expressed by replacing one of the viral genes with desired sequence, by inserting foreign sequences into the virus genome at an appropriate position, or by fusing foreign peptides to the structural proteins of a virus.
  • any of these approaches can be combined to express foreign sequences by trans- complementation of vital functions of a virus.
  • a number of different strategies exist as tools to express foreign sequences in virus-infected plants using tobacco mosaic virus (TMV), alfalfa mosaic virus (AlMV), and chimeras thereof.
  • TMV tobacco mosaic virus
  • AlMV alfalfa mosaic virus
  • the genome of AlMV is a representative of the Bromoviridae family of viruses and consists of three genomic RNAs (RNAs 1-3) and subgenomic RNA (RNA4) ( Figure 2).
  • Genomic RNAsI and 2 encode virus replicase proteins Pl and 2, respectively.
  • Genomic RNA3 encodes the cell-to-cell movement protein P3 and the coat protein (CP).
  • the CP is translated from subgenomic RNA4, which is synthesized from genomic RN A3, and is required to start the infection.
  • Encapsidation of viral particles is essential for long distance movement of virus from inoculated to un-inoculated parts of the seed, embryo, or young plant (e.g., sprouted seedling) and for systemic infection.
  • inoculation can occur at any stage of plant development. In embryos and sprouts, spread of the inoculated virus should be very rapid.
  • Virions of AlMV are encapsidated by a unique CP (24 kD), forming more than one type of particle. The size (30- to 60-nm in length and 18 nm in diameter) and shape (spherical, ellipsoidal, or bacilliform) of the particle depends on the size of the encapsidated RNA.
  • the N-terminus of the AIMV CP Upon assembly, the N-terminus of the AIMV CP is thought to be located on the surface of the virus particles and does not appear to interfere with virus assembly (BoI et al., Virology (1971) 6: 73-85). Additionally, the AIMV CP with an additional 38-amino acid peptide at its N-terminus forms particles in vitro and retains biological activity (Yusibov et al., J. Gen. Virol. (1995) 77: 567-573). [00113] AlMV has a wide host range, which includes a number of agriculturally valuable crop plants, including plant seeds, embryos, and sprouts.
  • the AIMV CP an excellent candidate as a carrier molecule and AlMV an attractive candidate vector for the expression of foreign sequences in the plant at the sprout stage of development.
  • the AlMV CP encapsidates TMV genome without interfering with virus infectivity (Yusibov et al., Proc. N ⁇ tl. Ac ⁇ d. Sd. USA (1997) 94: 5784-5788, incorporated herein by reference). This allows the use of TMV as a carrier virus for AlMV CP fused to foreign sequences.
  • TMV the prototype of the tobamoviruses
  • the CP is the only structural protein of TMV and is required for encapsidation and long distance movement of the virus in an infected host(Saito et al., Virology (1990) 176: 329-336).
  • the 183 and 126 kD proteins are translated from genomic RNA and are requifed ' for virus replication (Ishikawa et al., Nucleic Acids Res. ' (1986) 14: 8291-8308).
  • the 30 kD protein is the cell-to-cell movement protein of virus (Meshi et al., EMBOJ. (1987) 6: 2557-2563). Movement and coat proteins are translated from subgenomic mRNAs (Hunter et al., Nature (1976) 260: 759-760; Bruening et al., Virology (1976) 71: 498-517; Beachy et al., Virology (1976) 73: 498-507, each incorporated herein by reference).
  • RNAsI and 2 of AlMV encode replicase proteins Pl and P2, respectively; genomic RN A3 encodes cell-to-cell movement protein P3 and the viral coat protein (CP).
  • the CP is translated from subgenomic RNA4 synthesized from genomic RNA3.
  • the 126 kD and 183 kD proteins of TMV are required for replication; the 30 kD protein is the viral cell-to-cell movement protein; and the 17 kD protein is the CP of virus.
  • the CP and the 30 kD protein are translated from subgenomic RNAs. Arrows indicate position of subgenomic promoters.
  • Transformation of Arabidopsis thaliana can be achieved by dipping the plant flowers into a solution of Agrobacterium tumefaciens (Curtis and Nam, Transgenic Research (Aug 2001) 10 4:363-371; Qing et al., Molecular Breeding: New Strategies in Plant Improvement (Feb 2000). (l):67-72). Transformed plants are formed in the population of seeds generated by the "dipped" plants.
  • the Agrobacterium tumefaciens proliferates and transforms individual ovules (Desfeux et al., Plant Physiology (July 2000) 123 (3): 895-904).
  • the transformed ovules follow the typical pathway of seed formation within the ovary.
  • systems for rapid (e.g., transient) expression of proteins or polypeptides in plants are desirable.
  • the present invention provides a powerful system for achieving such rapid expression in plants (particularly in young plants, e.g., sprouted seedlings) that utilizes an agrobacterial construct to deliver a viral expression system encoding the protein or polypeptide of interest.
  • a "launch vector” is prepared that contains agrobacterial sequences including replication sequences and also contains plant viral sequences (including self-replication sequences) that carry a gene encoding the protein or polypeptide of interest
  • the launch vector is introduced into plant tissue, preferably by agroinfiltration, which allows substantially systemic delivery.
  • plant tissue preferably by agroinfiltration, which allows substantially systemic delivery.
  • non-integrated T-DNA copies of the launch vector remain transiently present in the nucleous and are transcribed leading to the expression of the carrying genes (Kapila et al., 1997).
  • Agrobacterium-mediated transient expression differently from viral vectors, cannot lead to the systemic spreading of the expression of the gene of interest.
  • One advantage of this system is the possibility to clone genes larger than 2kb to generate constructs that would be impossible to obtain with viral vectors (Voinnet et ⁇ l., 2003). Furthermore, using such technique, it is possible to transform the plant with more than one transgene, such that multimeric proteins (e.g., antibodies subunits of complexed proteins) can be expressed and assembled. Furthermore, the possibility of co-expression of multiple transgenes by means of co-infiltration with different Agrob ⁇ cterium can be taken advantage of, either by separate infiltration or using mixed cultures.
  • the launch vector includes sequences that allow for selection (or at least detection) in Agrob ⁇ cteri ⁇ and also for selection/detection in infiltrated tissues. Furthermore, the launch vector typically includes sequences that are transcribed in the plant to yield viral RNA production, followed by generation of viral proteins. Furthermore, production of viral proteins and viral RNA yields rapid production of multiple copies of RNA encoding the pharmaceutically active protein of interest. Such production results in rapid protein production of the target of interest in a relatively short period of time. Thus, a highly efficient system for protein production can be generated.
  • the agroinfiltration technique utilizing viral expression vectors can be used to produce limited quantity of protein of interest in order to verify the expression levels before deciding if it is worth generating transgenic plants.
  • the agroinfiltration technique utilizing viral expression vectors is useful for rapid generation of plants capable of producing huge amounts of protein as a primary production platform.
  • this transient expression system can be used on industrial scale.
  • Agrobacterial plasmids, binary plasmids, or derivatives thereof such as pBI V, pBI 1221 , pGreen, etc., which can be used in these and other aspects of the invention.
  • Figure 18 presents a schematic diagram of a particular exemplary launch vector, pBID4.
  • This vector contains the 35S promoter of cauliflower mosaic virus (a DNA plant virus) that drives initial transcription of the recombinant viral genome following introduction into plants, and the nos terminator, the transcriptional terminator of Agrobacterium nopaline sunthase.
  • the vector further contains sequences of the tobacco mosaic virus genome including genes for virus replication (126/183K) and cell-t-cell movement (MP).
  • the vector further contains a gene encoding a polypeptide of interest, inserted into a unique cloning site within the tobacco mosaic virus genome sequences and under the transcriptional control of the coat protein subgenomic mRNA promoter. Because this "target gene" (i.e., gene encoding a protein or polypeptide of interest) replaces coding sequences for the TMV coat protein, the resultant viral vector is naked self-replicating RNA that is less subject to recombination than CP-contaiing vectors, and that cannot effectively spread and survive in the environment. Left and right border sequences (LB and RB) delimit the region of the launch vector that is transferred into plant cells following infiltration of plants with recombinant Agrobacterium carrying the vector.
  • LB and RB border sequences
  • ssDNA single-stranded DNA
  • Agrobacterium-mediated transient expression produces up to about 5 g or more of target protein per kg of plant tissue. For example, in some embodiments, up to about 4, 3, 2, 1, or 0.5 g of target protein is produced per kg of plant tissue. In some embodiments, at least about 20-500 mg, or about 50-500 of target protein, or about 50-200, or about 50, 60, 70, 80, 90, 100, 110.
  • these expression levels are achieved within about 6, 5, 4, 3, or 2 weeks from infiltration. In some embodiments, these expression levels are achieved within about 10, 7, 5, 4, 3, 2 days, or even 1 day, from introduction of the expression construct.
  • the time from introduction (e.g., infiltration) to harvest is typically less than about 2 weeks, 10 days, 1 week or less.
  • one very attractive aspect of this embodiment of the invention is that it allows production of protein within about 8 weeks or less from the selection of amino acid sequence (even including time for "preliminary" expression studies).
  • each batch of protein can typically be produced within about 8 weeks, 6, weeks, 5 weeks, or less. Those of ordinary skill in the art will appreciate that these numbers may vary somewhat depending on the type of plant used.
  • Nicotiana benthamiana may be grown longer, particularly prior to infiltration, as they are slower growing (from a much smaller seed). Other expected adjustments will be clear to those of ordinary skill in the art based on biology of the particular plants utilized.
  • the present inventors have used a launch vector system to produce a variety of target proteins and polypeptides in a variety of different young plants.
  • the inventors have surprisingly found that certain pea varieties including for example, marrowfat pea, bill jump pea, yellow trapper pea, speckled pea, and green pea are particularly useful in the practice of this aspect of the invention (see, for example, Example 7).
  • certain Nicotiana plants are particularly useful in the practice of this aspect of the invention, including in particular Nicotiana benthamiana. It will be understood by those of ordinary skill in the art that Nicotiana plants are generally not considered to be "sprouts”.
  • Nicotiana benthamiana plants are useful in the practice of the invention, hi general, in the practice of this embodiment of the invention, Nicotiana benthamiana plants are grown for a time sufficient to allow development of an appropriate amount of biomass prior to infiltration (i.e., to delivery of agrobacteria containing the launch vector). Typically, the plants are grown for a period of more than about 3 weeks, more typically more than about 4 weeks, or between about 5-6 weeks to accumulate biomass prior to infiltration. [00127] The present inventors have further surprisingly found that, although both
  • TMV and AlMV sequences can prove effective in such launch vector constructs, in some ' " ' embodiments, AlMV sequences are particularly efficient at ensuring high level production of delivered protein or polypeptides.
  • proteins or polypeptides of interest are produced in young pea plants or young Nicotania plants (e.g., Nicotiana benthamiana) from a launch vector that directs production of AlMV sequences carrying the gene of interest.
  • Nicotania plants e.g., Nicotiana benthamiana
  • RNA encoding the protein or polypeptide of interest is under the control of an inducible (e.g. exogenously inducible) promoter.
  • inducible promoters are caused to increase or decrease expression of a transcript in response to an external, rather than an internal stimulus. A number of environmental factors can act as such an external stimulus.
  • transcription is controlled by a heat- inducible promoter, such as a heat-shock promoter.
  • Externally inducible promoters may be particularly useful in the context of controlled, regulatable growth settings. For example, using a heat-shock promoter the temperature of a contained environment may simply be raised to induce expression of the relevant transcript.
  • a heat inducible promoter could never be used in the outdoors because the outdoor temperature cannot be controlled. The promoter would be turned on any time the outdoor temperature rose above a certain level. Similarly, the promoter would be turned off every time the outdoor temperature dropped. Such temperature shifts could occur in a single day, for example, turning expression on in the daytime and off at night.
  • a heat inducible promoter such as those described herein, would likely not even be practical for use in a greenhouse, which is susceptible to climatic shifts to almost the same degree as the outdoors. Growth of genetically engineered plants in a greenhouse is quite costly. In contrast, in the present system, every variable can be controlled so that the maximum amount of expression can be achieved with every harvest.
  • Other externally-inducible promoters include light inducible promoters. Light-inducible promoters can be maintained as constitutive promoters if the light in the contained regulatable environment is always on. Alternatively, expression of the relevant transcript can be turned on at a particular time during development by simply turning on the light.
  • a chemically inducible promoter is used to induce expression of the relevant transcript.
  • the chemical could simply be misted or sprayed onto a seed, embryo, or young plant (e.g., seedling) to induce expression of the relevant transcript. Spraying and misting can be precisely controlled and directed onto a paricular seed, embryo, or young plant (e.g., seedling) as desired.
  • a contained environment is devoid of wind or air currents, which could disperse the chemical away from the intended recipient, so that the chemical stays on the recipient for which it was intended.
  • One aspect of the present invention is the production of proteins or polypeptides (and/or nucleic acids) in plants under controlled, regulated growth conditions.
  • one attractive feature associated with producing proteins or polypeptides in young plants e.g., sprouted seedlings
  • reduced growth times are required than would be utilized if the same plant type were grown to adulthood.
  • smaller spaces can be utilized than would be required if plants were grown to their full adult size.
  • growing plants that express pharmaceutical proteins or polypeptides in a controlled, regulatable environment provides a pharmaceutical product faster (because the plants are harvested younger) and with less effort, risk, and regulatory considerations than growing genetically engineered plants.
  • a contained, regulatable environment used in the present invention reduces or eliminates the risk of cross-pollinating plants in the nature.
  • proteins or polypeptides are expressed in plants grown in an enclosed system. Such a system avoids risk of environmental contamination and also provides a regulatable, reproducible environment capable of achieving repeated comparable results. Furthermore, such a system allows controlled induction of protein or polypeptide expression where desired, for example through the use of an inducible promoter or other regulatable feature as described herein.
  • plants are grown in a contained, regulatable environment.
  • the contained, regulatable environment is a housing unit or room in which the seeds can be grown indoors. All environmental factors of the contained, regulatable environment may be controlled. Since sprouts do not require light to grow, and lighting can be expensive, in one particular embodiment, plants are grown (e.g., to the sprouted seedling stage) indoors in the absence of light.
  • Other environmental factors that can be regulated in a contained, regulatable environment of the present invention include temperature, humidity, water, nutrients, gas (e.g., O 2 or CO 2 content or air circulation), chemicals (small molecules such as sugars and sugar derivatives or hormones such as such as the phytohormones gibberellic or absisic acid, etc.) and the like.
  • gas e.g., O 2 or CO 2 content or air circulation
  • chemicals small molecules such as sugars and sugar derivatives or hormones such as such as the phytohormones gibberellic or absisic acid, etc.
  • plants are grown hydroponically.
  • Hydroponic growth systems offer a number of advantages. For example, hydroponic growth systems often require less space than soil-based systems to grow the same number of plants. Nutrients and other agents can typically be flowed through a hydroponic system, making controlled growth easier to accomplish than in soil-based systems. Many aspects of hydroponic growth may be automated. Furthermore, harvesting of hydroponically grown plants is trivial. Plants can be harvested simply by being lifted from their growth solution. No cleansing is required at the time of harvest. Being able to harvest young plants (e.g., sprouted seedlings) directly from a hydroponic environment without washing or scrubbing minimizes breakage of the harvested material. Breakage and wilting of plants induces apoptosis.
  • Nutrients and other agents can typically be flowed through a hydroponic system, making controlled growth easier to accomplish than in soil-based systems. Many aspects of hydroponic growth may be automated.
  • harvesting of hydroponically grown plants is trivial. Plants can be harvested simply by being lifted from their
  • apoptosis During apoptosis, certain proteolytic enzymes become active, which can degrade a pharmaceutical protein or polypeptide expressed in the sprouted seedling, resulting in decreased therapeutic activity of the protein. Apoptosis-induced proteolysis can significantly decrease the yield of protein from mature plants. Using the methods of the present invention, apoptosis is preferably never induced (i.e., apoptosis is avoided), for example because no harvesting takes place until the moment the proteins are extracted from the plant. [001391 In. certain embodiments of the present invention, plants (e.g., sprouted seedlings) are grown in trays that can be watered, sprayed, or misted at any time during development.
  • the tray may be fitted with one or more watering, spraying, misting, and draining apparatus that can deliver and/or remove water, nutrients, chemicals etc. at specific time and at precise quantities during development of the sprouted seedling.
  • seeds require sufficient moisture to keep them damp. Excess moisture drains through holes in the trays into drains in the floor of the room.
  • drainage water is treated as appropriate for removal of harmful chemicals before discharge back into the environment.
  • trays are contained within a very small space. Since often no light is required for young plants (e.g., sprouted seedlings) to grow, the trays containing seeds, embryos, or young plants (e.g., sprouted seedlings) may be tightly stacked vertically on top of one another, providing a large quantity of biomass per unit floor space in a housing facility constructed specifically for these purposes. In addition, the stacks of trays can be arranged in horizontal rows within the housing unit.
  • seedlings Once the seedlings have grown to a stage appropriate for harvest (e.g., about two to fourteen days or longer depending on the type of plant, the protein or polypeptide being produced, etc) individual trays are moved into a processing facility, either manually or by automatic means, such as a conveyor belt.
  • stage appropriate for harvest e.g., about two to fourteen days or longer depending on the type of plant, the protein or polypeptide being produced, etc.
  • the time at which expression is induced may desirably be selected to maximize expression of the protein or polypeptide or interest in the young plant by the time of harvest.
  • Inducing expression in an embryo at a particular stage of growth e.g., at a particular number of days after germination
  • inducing expression from the promoter 4 days after germination may result in more protein synthesis than inducing expression from the promoter after 3 days or after 5 days.
  • young plants are harvested at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days after germination.
  • young plants particularly Nicotania plants, are grown for several weeks after germination.
  • young plants may be harvested at a certain time after introduction of encoding sequences.
  • the sprouted seedlings may be harvested at a time when expression is at its maximum post-transformation, e.g., at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days post-transformation. It could also be that young plants (e.g., sprouts) develop one, two, three or more months post-transformation, depending on the germination of the seed.
  • the seeds, embryos, or young plants are allowed to grow until sufficient levels of the pharmaceutical protein or polypeptide of interest are expressed.
  • sufficient levels are levels that would provide a therapeutic benefit to a patient if the harvested biomass were eaten raw.
  • sufficient levels are levels from which the pharmaceutical protein or polypeptide can be concentrated or purified from the biomass and formulated into a pharmaceutical composition that provides a therapeutic benefit to a subject upon administration.
  • sprouted seedling stage at which time the sprouted seedlings are harvested.
  • the sprouted seedlings are harvested live. Harvesting live sprouted seedlings has several advantages including minimal effort and breakage.
  • the present invention provides a unique system that in turns provides young plant biomass containing high levels of expressed protein or polypeptide (and/or nucleic acid) of interest. Whether consumed directly or processed into the form of a pharmaceutical composition, when young plants (e.g., sprouted seedlings) are grown in a contained, regulatable environment, the plant biomass and/or pharmaceutical composition derived from the biomass can be provided to a consumer at low cost. In addition, the fact that the conditions for growth of the young plants can be controlled makes the quality and purity of the product consistent.
  • the contained, regulatable environment of the invention also obviates many safety regulations of the EPA that can prevent scientists from growing genetically engineered agricultural products out of doors.
  • the present invention provides various preparations and formulations of proteins or polypeptides (and/or active nucleic acids) produced in young plants.
  • produced proteins or polypeptides are not isolated from plant tissue but rather are provided in the context of live young plants (e.g., sprouted seedlings).
  • plant tissue containing expressed protein or polypeptide is provided directly for consumption.
  • the present invention provides edible young plant biomass (e.g., edible sprouted seedlings) containing expressed protein or polypeptide.
  • edible young plants express sufficient levels of pharmaceutical proteins or polypeptides and are consumed live
  • absolutely no harvesting occurs before the sprouted seedlings are consumed.
  • young plants e.g., sprouted seedlings
  • genetically engineered seeds or embryos are delivered to a patient in need of treatment and grown to the sprouted seedling stage by the patient.
  • a supply of genetically engineered sprouted seedlings is provided to a patient, or to a doctor who will be treating patients, so that a continual stock of sprouted seedlings expressing certain desirable pharmaceutical proteins may be cultivated. This may be particularly valuable for populations in developing countries, where expensive pharmaceuticals are not affordable or deliverable. The ease with which the sprouted seedlings of the invention can be grown makes the sprouted seedlings of the present invention particularly desirable for such developing populations.
  • plant biomass is processed prior to consumption or formulation, for example, by homogenizing, crushing, drying, or extracting.
  • the expressed protein or polypeptide is isolated or purified from the biomass and formulated into a pharmaceutical composition.
  • live young plants may be ground, crushed, or blended to produce a slurry of biomass, in a buffer containing protease inhibitors.
  • the buffer is at about 4 0 C.
  • the biomass is air-dried, spray dried, frozen, or freeze-dried.
  • some of these methods, such as air- drying may result in a loss of activity of the pharmaceutical protein or polypeptide.
  • the plants utilized e.g., sprouted seedlings
  • are very small and typically have a large surface area to volume ratio this is much less likely to occur.
  • many techniques for harvesting the biomass that minimize proteolysis of the pharmaceutical protein or polypeptide are available and could be applied to the present invention.
  • the present invention provides young plants expressing a pharmaceutically active protein or polypeptide that maintains its pharmaceutical activity when administered to a host in need thereof.
  • Preferred hosts include vertebrates, preferably mammals, more preferably humans.
  • the hosts include veterinary subjects such as bovines, ovines, canines, felines, etc.
  • an edible sprout is administered orally to a subject in a therapeutically effective amount.
  • the pharmaceutically active protein is provided in a pharmaceutical preparation, as described herein.
  • compositions of the present invention can be administered in a wide variety of ways to the host, such as, for example, orally enterally, nasally, parenterally, intramuscularly or intravenously, rectally, vaginally, topically, ocularly, pulmonarily, or by contact application.
  • a pharmaceutical protein expressed in a young plant e.g., a sprout
  • a pharmaceutically active protein expressed in a young plant e.g., in a sprout
  • Proteins are isolated and purified in accordance with conventional conditions and techniques known in the art. These include methods such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, and the like.
  • compositions of the present invention typically include an effective amount of a pharmaceutically active protein or polypeptide together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials.
  • a pharmaceutically active protein produced according to the present invention may be employed in dosage forms such as tablets, capsules, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, powder packets, liquid solutions, solvents, diluents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid bindings as long as the biological activity of the protein is not destroyed by such dosage form).
  • Examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other nontoxic compatible lubricants such
  • the protein may be provided as a pharmaceutical composition by means of conventional mixing granulating dragee-making, dissolving, lyophilizing, or similar processes.
  • it may be desirable to prolong the effect of a pharmaceutical preparation by slowing the absorption of the pharmaceutical protein or polypeptide that is subcutaneously or intramuscularly injected. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the protein or polypeptide then depends upon its rate of dissolution, which in turn, may depend upon size and form.
  • delayed absorption of a parenterally administered protein is accomplished by dissolving or suspending the protein in an oil vehicle.
  • injectable depot forms are made by forming microencapsule matrices of the protein in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of protein to polymer and the nature of the particular polymer employed, the rate of release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the protein in liposomes or microemulsions, which are compatible with body tissues.
  • Enterally administered protein or polypeptide preparations may be introduced in solid, semi-solid, suspension or emulsion form and may be compounded with any pharmaceutically acceptable carriers, such as water, suspending agents, and emulsifying agents.
  • the proteins or polypeptides of the invention may also be administered by means of pumps or sustained-release forms, especially when administered as a preventive measure, so as to prevent the development of disease in a subject or to ameliorate or delay an already established disease.
  • Proteins or polypeptides produced according to the present invention are particularly well suited for oral administration as pharmaceutical compositions.
  • Harvested young plants e.g., seedlings
  • such compositions as described above are ingested orally alone or ingested together with food or feed or a beverage.
  • Compositions for oral administration include sprouted seedlings; extractions of the young plants (e.g., sprouted seedlings), and proteins or polypeptides purified from young plants provided as dry powders, foodstuffs, aqueous or non-aqueous solvents, suspensions, or emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
  • Aqueous carriers include water, water- alcohol solutions, emulsions or suspensions, including saline and buffered medial parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose or fixed oils.
  • dry powders include any young plant (e.g., sprouted seedling) biomass that has been dried, for example, freeze dried, air dried, or spray dried.
  • young plants may be air dried by placing them in a commercial air dryer at about 120 degrees Fahrenheit until the biomass contains less than 5% moisture by weight. The dried plants are stored for further processing as bulk solids or further processed by grinding to a desired mesh sized powder.
  • Herbal preparations are well known in the art. Herbal preparations that may be used to administer young plants of the present invention include liquid and solid herbal preparations. Some examples of herbal preparations include tinctures, extracts (e.g., aqueous extracts, alcohol extracts), decoctions, dried preparations (e.g., air-dried, spray dried, frozen, or freeze-dried), powders (e.g., lyophilized powder), and liquid. Herbal preparations can be provided in any standard delivery vehicle, such as a capsule, tablet, suppository, liquid dosage, etc. Those skilled in the art will appreciate the various formulations and modalities of delivery of herbal preparations that may be applied to the present invention.
  • a particularly preferred method of obtaining the desired pharmaceutically active protein or polypeptide is by extraction.
  • Fresh young plants e.g., seedlings
  • Young plants may also be extracted in a buffered solution.
  • the fresh harvested plants may be transferred into an amount of ice-cold water at a ratio of one to one by weight that has been buffered with, e.g., phosphate buffer.
  • Protease inhibitors can also be added as required.
  • Plant tissue can be disrupted by vigorous blending or grinding while suspended in the buffer solution and the extracted biomass removed by filtration or centrifugation.
  • the protein product carried in solution can be further purified by additional steps or converted to a dry powder by freeze-drying or precipitation.
  • Extraction can also be carried out by pressing.
  • Live plants can be extracted by pressing in a press or by being crushed as they are passed through closely spaced rollers.
  • the fluids expressed from the crushed plants are collected and processed according to methods well known in the art. Extraction by pressing allows the release of the products in a more concentrated form. However, the overall yield of the product may be lower than if the product were extracted in solution.
  • the young plants e.g., sprouted seedlings
  • extractions powders, dried preparations and purified protein products, etc.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active pharmaceutical protein may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • additional substances other than inert diluents e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions examples include polymeric substances and waxes; - - - [00167]
  • a young plant expressing a pharmaceutically active protein of the present invention or biomass of such a young plant is administered orally as medicinal food.
  • Such edible compositions are consumed by eating raw, if in a solid form, or by drinking, if in liquid form.
  • the plant material is directly ingested without a prior processing step or after minimal culinary preparation.
  • the pharmaceutically active protein or polypeptide is expressed in a sprout of which can be eaten directly.
  • the protein is expressed in an alfalfa sprout, mung bean sprout, or spinach or lettuce leaf sprout, a young pea plant, etc.
  • the sprouted seedling biomass is processed and the material recovered after the processing step is ingested.
  • Processing methods preferably used in the present invention are methods commonly used in the food or feed industry.
  • the final products of such methods still include a substantial amount of the expressed pharmaceutically active protein or polypeptide and are preferably conveniently eaten or drunk.
  • the final product may also be mixed with other food or feed forms, such as salts, carriers, favor enhancers, antibiotics, and the like, and consumed in solid, semi-solid, suspension, emulsion, or liquid form.
  • such methods include a conservation step, such as, e.g., pasteurization, cooking, or addition of conservation and preservation agents.
  • Any plant is used and processed in the present invention to produce edible or drinkable plant matter.
  • the amount of pharmaceutically active protein in an edible or drinkable sprout preparation may be tested by methods standard in the art, e.g., gel electrophoresis, Elisa, or Western blot analysis, using an antibody specific for the protein. This determination can be used to standardize the amount of protein ingested. For example, the amount of therapeutically active protein in a sprout juice determined and regulated, for example, by mixing batches of product having different levels of protein so that the quantity of juice to be drunk to ingest a single dose can be standardized. Use of a contained, regulatable environment in accordance with the present invention, however, should minimize the need to carry out such standardization procedures.
  • compositions of the present invention can be administered therapeutically or prophylactically. In certain preferred embodiments, the compositions may be used to treat or prevent (e.g., to delay onset of) a disease.
  • any individual who suffers from a disease or who is at risk of developing a disease may be treated. It will be appreciated that an individual can be considered at risk for developing a disease without having been diagnosed with any symptoms of the disease. For example, if the individual has a particular genetic marker identified as being associated with increased risk for developing a particular disease, that individual will be considered at risk for developing the disease. Similarly, if members of an individual's family have been diagnosed with a particular disease, e.g., cancer, the individual may be considered to be at risk for developing that disease.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsif ⁇ ers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents such as, for example, water or other solvents, solubil
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compositions of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active protein.
  • suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active protein.
  • Dosage forms for topical or transdermal administration of a pharmaceutical composition of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active protein, or preparation thereof is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention.
  • the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a pharmaceutically active protein to the body.
  • Such dosage forms can be made by suspending or dispensing the pharmaceutically active protein in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the pharmaceutically active protein across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the pharmaceutically active protein in a polymer matrix or gel.
  • compositions are administered in such amounts and for such time as is necessary to achieve the desired result.
  • a “therapeutically effective amount” of a pharmaceutical composition is that amount effective for treating, attenuating, or preventing a disease in a host.
  • the "amount effective to treat, attenuate, or prevent disease” refers to a nontoxic but sufficient amount of the pharmaceutical composition to treat, attenuate, or prevent disease in any host.
  • the "therapeutically effective amount” can be an amount to treat, attenuate, or prevent diabetes.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the stage of the disease, the particular pharmaceutical mixture, its mode of administration, and the like. Young plants (e.g., sprouted seedlings) of the invention and/or protein preparations thereof are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of pharmaceutically active protein appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention are preferably decided by an attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex of the patient, diet of the patient, pharmacokinetic condition of the patient, the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental' with the specific compound employed; and like factors well known in the medical arts.
  • compositions of the present invention can be employed in combination therapies, that is, the pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved.
  • the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another anti-cancer agent), or they may achieve different effects.
  • the present invention also provides a pharmaceutical pack or kit including live young plants (e.g., sprouted seedlings) of the present invention or preparations, extracts, or pharmaceutical compositions containing the pharmaceutically active protein or polypeptide expressed by the young plants in one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • the pharmaceutical pack or kit includes an additional approved therapeutic agent for use as a combination therapy.
  • Optionally associated with such containers can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.
  • kits are provided that include each protein in a diagnostic kit for genetic and immunologic diseases.
  • the present invention provides kits having test samples of biological proteins and reagents for testing for the presence of antibodies in a patient's serum to those biological proteins.
  • the kit may provide IA-2 and GAD and reagents to test for the presence of excess amounts of antibodies to these proteins in a patient's serum, a clear indication of the presence or development of type 1 diabetes.
  • the present invention can be extended to provide diagnostic reagents in the form of a kit.
  • the proteins insulin, GAD, and 1A-2, associated with the autoimmune reaction in diabetes can be provided as oral formulations and administered to induce oral tolerance to these proteins.
  • one or more therapeutic protein can be provided in an injectable formulation or vaccine for administration.
  • pharmaceutical doses or instructions therefor are provided in the kit for administration to an individual diagnosed with a disease, e.g., a diabetic individual, or an individual at risk for developing a disease, e.g., type 1 diabetes.
  • Seeds of plants are transformed by Agrobacterium are harvested, dried, cleaned, and tested for viability and presence of desired genetic material. Seed stock is stored under appropriate conditions until use. At the time of use, appropriate amounts of seeds are soaked in water containing an amount of surface sterilizing agent (e.g., Clorox) for 20 minutes to 4 hours. Seeds are spread onto a flat of trays, which contain provisions for sustenance of growth and drainage of water. Trays containing the seeds are put on racks in the contained, regulatable environment under controlled temperature, lighting, access, air circulation, water supply, and drainage. Trays are misted with water from misters equipped with automatic timers for one to 30 minutes at intervals of 30 minutes to four hours, sufficient to keep seeds damp. Excess moisture drains through holes in the trays into drains in the floor of the room.
  • surface sterilizing agent e.g., Clorox
  • Seeds are incubated for about two to fourteen days before harvest and processing. At some point during the incubation process, from four hours to seven days prior to harvest, seeds are exposed to environmental conditions that cause the induction of an introduced or indigenous DNA promoter sequence that causes an increase in the synthesis of one or more desired proteins in the tissues of the sprouting seedling. A transient increase in the incubation temperature from about 30 0 C to about 37 0 C to cause induction of a heat shock promoter. [00184] After incubation of the seedlings for two to fourteen days, the seedlings are harvested by moving the individual trays into a processing facility on a conveyor belt. The harvested seedlings are processed by extraction in phosphate buffered solution containing protease inhibitors. The seedlings are disrupted by vigorous blending or grinding while suspended in the buffer solution and the extracted biomass removed by centrifugation.
  • Seeds of desired plants are obtained from a contract of commercial grower as wild-type seeds.
  • the seed stock is stored under appropriate conditions of temperature, humidity, sanitation, and security until use. At the time of use, appropriate amounts of seeds are soaked in water and incubated on trays as described above under controlled conditions.
  • the germinated seedlings are sprayed with a solution containing a virus harboring a transgene, and further or simultaneously treated with a material that causes mechanical abrasion of the plant leaf tissue. In this example, the leaves are abraded with a spray of air containing abrasive particles.
  • the virus is allowed to systemically infect the plants for an appropriate period; from about one to about ten days and expression of the desired heterologous protein is monitored. [00187] After infection of the seedlings for one to ten days, the seedlings are harvested as described in Example 1 above.
  • Example 3 Expression of Diabetes Associated Proteins and Human Growth Hormone from Viral vectors (Av A4) in Nicotiana Benthamiana Plants
  • Type 1 or juvenile diabetes is a disease that affects children, adolescents, and young adults.
  • Beta islet cells of the pancreatic endocrine system produce insulin in response to the metabolic signal of high blood glucose.
  • the underlying etiology of the disease is the attack and destruction of the beta islet cells by the body's own immune system.
  • Autoantigens such as insulin, GAD and IA-2 are useful in inducing at least a degree of oral tolerance to these proteins in susceptible individuals and prevent or reduce the damage to islet cells that results in the development of diabetes.
  • Non-obese diabetic mice spontaneously develop an autoimmune form of diabetes (Zahang, et al., Proc. Natl. Acad. Sci. USA (1991) 88:10252-10256).
  • the gene encoding human insulin has been fused with the cholera toxin B subunit gene and the resulting construct expressed in transgenic potato plants.
  • GAD and IA-2 proteins are purified using the His-tag method. Production of raw material and proteins takes place in two stages: 1) small scale (mg quantities) for preliminary studies in animals; and 2) medium scale (gram quantities for clinical trials. - - - - • •
  • each construct contains a T7 or SP6 RNA polymerase promoter fused to the exact 5' terminus of viral genomic RNA and a unique restriction site at the 3' end that is used to linearize the plasmid prior to in vitro transcription.
  • the T7 or SP6 RNA polymerase then generates run-off transcripts, which are used to inoculate plants. Plants are inoculated mechanically at two-leaf stage, by gently rubbing the inoculum onto the leaf surface in the presence of an abrasive agent, such as carborundum powder (320-grit; Fisher, Pittsburgh, PA).
  • an abrasive agent such as carborundum powder (320-grit; Fisher, Pittsburgh, PA).
  • RNA transcript Five to ten plants are inoculated per construct and 1-2 tg of each RNA transcript is used per inoculation. The plants are monitored for severity of symptoms, spread of virus throughout entire plant, and product recovery. At 10-15 days post infection (dpi) leaf samples from infected leaf samples are harvested to assess the presence of full-size recombinant IA-2 and GAD. A portion of the harvested material (10 leaves) is frozen in _80-C and retained as a seed inoculum for the subsequent production scale-up of selected constructs. The rest of the tissue is processed immediately.
  • the procedure is optimized to recover optimum quantities of high purity product (90-95% purity). Once products with the expected sizes are recovered and serological identity (recognized by specific antibodies using Western blot and ELISA) determined, the stability of the constructs is tested by three passages on healthy plants. Problems with assembly, recovery or stability of recombinant virus with proteins in the size range employed are manage at the level of nucleotide sequence or amino acid sequence by changing the conditions of infection or by using an alternative host plant.
  • stage 1 a small quantity (100 ul) of in vitro synthesized transcripts of the recombinant constructs is prepared and used to inoculate 10 plants. Within 10-12 days after inoculation the leaves are harvested, tested for the presence of GAD or IA-2 by Western blot, and stored at _70°C as seed material. A portion of this material (3-4) is used to inoculate 150-200 plants (1-2 kg of fresh tissue). Fifteen to twenty days after inoculation, recombinant protein is recovered and used for functional studies. An average of 60 mg of product per batch is expected:
  • Alfalfa mosaic virus CP is unique in its ability to encapsidate the genomic RNAs of unrelated viruses into infectious particles in the infected host. This unique ability of Alfalfa mosaic virus CP is exploited to engineer hybrid vectors that specifically target selected crop species.
  • GFP-specific antibodies did not react with any proteins in plants inoculated with only Av/A4. Neither Alfalfa mosaic virus CP- nor GFP-specific antibodies reacted with any protein in extracts from plants inoculated with Av/GFP only, suggesting the lack of systemic movement. [00198] We have also engineered and expressed human growth hormone using this
  • FIG. 4 is a Western Blot of hGH produced in N. benthamiana plants infected with in vitro transcripts of 125C/hGH. Samples were analyzed 24 hours post inoculation. 1 ⁇ g of purified hGH was loaded as standard. MWM is molecular weight marker. The arrow in Figure 4 points toward the hGH band on the blot detected by hGH-specific antibodies.
  • the virulence of anthrax is due to at least two major virulence factors. These factors are a polyglutamate capsule that helps protect the bacterium in the host and a three- part circulating toxin.
  • Anthrax toxin is encoded by a 184 kb plasmid named pXOl and consists of a receptor-binding protein named protective antigen (PA), and two enzymatically active proteins named edema factor and lethal factor (Bhatnagar and Batra. Crit. Rev. Mirobiol. (2001) 27(3): 167-200).
  • PA protective antigen
  • PA lethal factor
  • LF lethal factor
  • Transformation vector The binary vector pGREENII 0229 (Hellens et al.,
  • Plant Molecular Biology (Apr 2000) 42(6):819-832) is used for plant transformation (see Figure S).
  • This plasmid includes the following components. 1) The pGREENII plasmid backbone includes sequences necessary for replication in Escherichia coli and Agrobacterium tumefaciens. 2) The Agrobacterium tumefaciens T-DNA left and right border (LB and RB) sequences are necessary for integration of all sequences between LB and RB into the plant genome. 3) The npt gene encoding kanamycin resistance for selection of Escherichia coli and Agrobacterium tumefacients tranformants.
  • the nos-bar gene which encodes resistance to the herbicide Bialaphos (resistance to Bialophos is used to select transgenic plants.
  • the nos-bar gene is transcribed from the Cauliflower mosaic virus (CAMV) 35S promoter with constitutive activity in plants, and transcription of the gene is terminated using the CaMV terminator.
  • AMV Cauliflower mosaic virus
  • vir genes PA and LF are driven either by the CAMV 35S promoter as shown in Figure 5, or by the HSPl 8.2 promoter (GenBank accession # Xl 7295, locus At5g59720) for the Arabidopsis thaliana low molecular weight heat shock protein (Matsuhara et al., The Plant Journal: for Cell & Molecular Biolog (Apr 2000) 22(l):79-86). Transcriptional termination is mediated by the terminator of the nopaline synthase gene (nos).
  • the 35S and HSP18.2 promoters where chosen for their differing activities.
  • the 35 S promoter is constitutively active in most plant tissues. In many cases the 35S promoter drives high-level expression of the protein of interest.
  • the HSPl 8.2 promoter is nearly inactive unless the plants are challenged by heat shock. High-level expression of some proteins can only be achieved using an inducible promoter system.
  • Vector construction and plant transformatioa The vectors are constructed using DH5, a laboratory strain of Escherichia coli. The construction is analyzed by restriction endonuclease mapping and sequencing. After construction is confirmed the vector is used to transform Agrobacterium tumefaciens strain GV3101, lacking a native Ti plasmid and suitable for transfer of the TDNA of binary vectors into plants. Brassica j ' uncea plants are transformed by introduction of GV3101 carrying the transformation vector into flowers by any available transformation method.
  • PA and LF genes Modifications introduced into PA and LF genes.
  • the nucleotide sequences of both PA and LF are modified for expression in plants.
  • the coding sequence of native PA is 2295 bp and native LF is 2430 bp. These sequences are modified as follows: 1) Mutations are introduced that render PA and LF inactive as toxins. Phe314 and Phe315 are deleted from PA and His686 and His690 are mutated to Ala. 2) Codon usage is optimized for expression in Brassica juncea. Since the codon usage and GC/AT ratio of Brassica juncea is neutral the changes introduced into PA and LF are not extensive. Four codons are changed in PA and seven in LF.
  • the intention of the second construct is to optimize the accumulation for PA and LF by targeting and retaining the proteins in the endoplasmic reticulum 40 .
  • Targeting and retention in the ER has the potential to produce high-level accumulation of a recombinant protein (Haseloff, et al., Proceedings of the National Academy of Sciences USA. (Mar 18, 1997) 94(6):2122-2127).
  • an amino-terminal signal sequence and a carboxyl terminal ER retention sequence is added.
  • the signal sequence includes 22 codons derived from the Arabidopsis thaliana chitinase gene (Haseloff, et al., supra).
  • the ER retention sequence is the tetrapeptide HisAspGluLeu (Gomord et al, The Plant Journal: for Cell & Molecular Biology. (Feb 1997) 11(2):313-325).
  • the signal sequence is added by PCR stitching using the synthetic genes produced by Entelechon as templates (Tomme et al., J Bacteriol. (1995) 177:4356-4363).
  • the ER retention sequence is added by PCR amplification of the PA and LF genes using a 3' primer into which the HisAspGluLeu coding sequence has been incorporated.
  • Modified PA and LF sequences are cloned into 35S and HSP18.2 expression vectors. Two modified version of each PA and LF (a total of 4) are cloned. In total, 8 different constructions are tested.
  • Polymerase chain reaction using oligonucleotide primers specific for the construct are used to confirm the presence of the TDNA in putative transgenic plants.
  • the presence of the TDNA is then be examined by blotting of genomic DNA using a specific DNA probe, the modified PA or LF genes.
  • expression of the protein of interest is examined by SDS-PAGE followed by "staining with Coomassie Blue or immunoblotting.
  • the protocol for analysis of protein expression differs depending upon the construct used to produce the transgenic plant. Plants carrying 35S promoter constructions are analyzed directly since expression from this promoter is constitutive. Plants carrying the HSPl 8.2 promoter are heat shocked before analysis. The kinetics of recombinant protein expression might differ in different transgenic plants.
  • Figure 6 shows an immunoblot of transgenic Brassica juncea. expressing human growth hormone (hGH) under control of the HSPl 8.,2 promoter.
  • hGH human growth hormone
  • Transgenic Brassica juncea were grown in potting mix. As shown in Figure 6, a leaf weighing 1 to 3 grams fresh weight was detached from the plant and placed in a petri dish containing a filter paper moistened with water. The petri dish is covered and placed in a high humidity, 37 0 C incubator for 1.5 hours.
  • the petri dish containing the leaf was then removed from the incubator and placed for 5 hours in a 24 0 C growth chamber under fluorescent lights at an intensity of 100 ⁇ mol photons m ⁇ 2 s "1 .
  • the plant material was then harvested and analyzed by immunoblotting using a monoclonal antibody against hGH (Sigma Chemical Co., Product #G-8523).
  • the lower band is the 16 kDa recombinant hGH. This band is not observed before heat shock.
  • Lane 8 shows the results from a transgenic plant transformed with the vector alone.
  • the higher molecular weight band is a non-specific reaction with the horseradish peroxidase-linked secondary antibody used to detect immune-complexes.
  • PA is predicted to be approximately 88 kDa and LF 93 kDa.
  • Example 5 Expression of a GUS Reporter in Sprouts From an Agrobacterial Construct Containing Viral Sequences
  • pBI121 containing a GUS reporter gene was transformed into Agrobacterium tumefaciens LBA 4404. Bacterial cultures were grown overnight in YEB medium containing 50 ⁇ g/ml kanamycin, 20 ⁇ M acetosyringone and 10 mM MES pH 5.6. Overnight cultures were centrifuged, resuspended in MMA medium (MS salts, 10 mM MES pH 5.6, 200 ⁇ M acetosyringone and 2% sucrose) at OD ⁇ oo 2.0 and used for vacuum infiltration of sprouts.
  • MMA medium MS salts, 10 mM MES pH 5.6, 200 ⁇ M acetosyringone and 2% sucrose
  • Example 6 Expression of Diabetes Associated Proteins and Human Growth Hormone from an Agrobacterial Construct Containing Viral Sequences
  • D4-hGH and D4-GFP The parental vector, D4, and vectors derived therefrom, are described in Shivprasad et al., Virology, 255(2):312-23, 1999.
  • the resultant plasmid is referred to as pBID4.
  • the 35S promoter directs transcription of the TMV sequence.
  • viral transcripts are produced.
  • components needed for viral replication and spread throughout the plant are also produced. These components include, e.g., replicase, movement protein, and coat protein, which may be from TMV or from another virus such as alfalfa mosaic virus in various embodiments.
  • These components may be encoded within the TMV sequence, the plasmid sequence, provided in a separate viral vector or plasmid, or the plant may be a transgenic plant comprising a transgene that encodes the components. See, for example USSN 10/770,600 filed February 3, 2004, which is incorporated herein by reference.
  • a subgenomic TMV promoter within the TMV sequence directs transcription of the hGH sequence.
  • a hammerhead ribozyme was introduced 3' of the TMV sequence.
  • the ribozyme is not required for the present invention.
  • the nos terminator (well known in the art) is 3' of the ribozyme sequence.
  • the final vector contained D4-hGH or D4-GFP.
  • D4-hGH or D4-GFP constructs in pBI121 were then used to transform A. tumefaciens and plants infiltrated with transformed Agrobacterium. Since a replicating virus is present we incubated the plants for 2 weeks.
  • Leaf discs were analysed for hGH production by Western blots. GFP expression was monitored by illuminating the plants with long wave ultraviolet light and photographed. The results shown in Figure 10 demonstrates GFP expression throughout the infiltrated leaves.
  • Western blot analysis shown in Figure 11 using antibody directed to hGH confirmed that the construct was functional in plants, and hGH could be detected at high levels.
  • IA-2ic was engineered into pBID4 plasmid to generate pBID4-IA-
  • AMV viral based vector carrying GFP was generated. Resulting plasmids was used to transform Agrobacterium and hydroponically grown Nicotiana benthamiana was infiltrated with transformed Agrobacterium. Briefly, Nicotiana benthamiana seeds were sown on a Rockwool slab (18 X 8 X1) pre- wetted in Vz strength Hoagland solution as a nutrient in hydroponic conditions. The hydroponics plants were kept in the same solution for four weeks. Four week-old plants were then vacuum infiltrated in Agrobacterium suspension (OD ⁇ oo 0.1) carrying pBID4-IA-2ic or AMV viral based vector carrying GFP.
  • MMA medium MS salts, 10 mM MES pH 5.6, 20 g/1 sucrose, 200 ⁇ M acetosyringone
  • OD ⁇ oo 0.1
  • Re-suspended culture was incubated at room temperature for 2-3 h with gentle shaking, and hydroponics Nicotiana benthamiana plants were placed in the bacterial suspension, then vacuum was applied for 30-60 second at room temperature.
  • Figure 12 demonstrates western blot analysis of plants expressing IA-2ic. Analysis of plants infiltrated with AMV based GFP constructs under light demonstrated expression of GFP throughout leaves of infiltrated plants.
  • Example 7 Expression of Proteins (Green Fluorescence Protein; Lichenase) in Pea Varieties from a an Agrobacterial Construct Containing Viral Sequences
  • the present Example describes agroinfiltration of certain pea varieties with agrobacterial constructs that carry a viral expression vector.
  • Figure 13 depicts the overall strategy utilized for agrobacterial constructs carrying AlMV sequences.
  • AlMV has a segmented genome (as discussed above)
  • three different constructs were used: one (pMOG-Rl&2) that carried AlMV sequences encoding replicases 1 and 2; one (pBI-RNA3) that carried AlMV RNA3 sequences (optionally including a gene of interest to be expressed, which typically replaces the natural AJMV coat protein sequences in RNA3); and one (pBI-CP) that carried AlMV sequences encoding the AlMV coat protein.
  • pMOG-Rl&2 that carried AlMV sequences encoding replicases 1 and 2
  • pBI-RNA3 that carried AlMV RNA3 sequences (optionally including a gene of interest to be expressed, which typically replaces the natural AJMV coat protein sequences in RNA3)
  • pBI-CP that carried AlMV sequences encoding the AlMV coat protein.
  • inclusion of the construct encoding the AlMV coat protein is not, strictly
  • bean sprouts were grown hydroponically with a non-soil support (in this case cheesecloth) as is known in the art. Sprouts were submerged in the combined agrobacterium mixture, and vacuum was applied for 90 sec at -28 psi ot achieve agroinfiltration. In some cases, vaccum was applied more than once.
  • a non-soil support in this case cheesecloth
  • sprouts were rinsed once in tap water after agroinfiltration, and then were returned to the growth space and watered. Sprouts were later harvested, pooled, and extracted.
  • Figures 14-16 present the results of various experiments performed according to the strategy illustrated in Figure 13.
  • Lichenase- producing constructs were agroinfiltrated into speckled pea (SP), yellow pea (YP), or bill jump pea (BP) srpouts.
  • SP speckled pea
  • YP yellow pea
  • BP bill jump pea
  • the sprouts Prior to agroinfiltration, the sprouts were grown for 2 days in the dark (to allow germination) and 8 days in the light (as indicated by the "2(8)" notation). These 10 days were then agroinf ⁇ ltrated as described above, and were grown for 5 days post inoculation (dpi) prior to detection of produced protein.
  • dpi post inoculation
  • lichenase protein was produced by all three pea varieties, with yellow pea having somewhat higher production levels than speckled pea, and bill jump pea having somewhat lower production levels.
  • Such variability is well within the realm of experience of those of ordinary skill in expressing proteins in plants; in light of these results, no more than routine experimentation would be required to select a preferred plant variety for production of any particular protein.
  • expression levels observed in these pea sprout systems were approximately 1/3 of those observed when expressing the relevant proteins (e.g., Lich-PA) in Nicotiana benthamiana (not shown).
  • Figure 15 shows successful production of GFP in speckled pea and yellow pea grown for different periods of time prior to agorinfiltration.
  • vaccuum was applied three times for 90 seconds each.
  • the particular resuspension medium utilized was "full MMA".
  • Figure 16 shows expression of lichenase in speckled pea leaves (L), whole sprouts (W), roots (R), or hypocotyl (H) at different periods of time post-infiltration. As can be seen, most expression is in the leaves. Moreover, expression is best in this particular system approximately 5-7 days after infiltration. Some variation should be expected based on the age and variety of plant being utilized, as well as the protein(s) being expressed.
  • the present Example describes a technology platform that will revolutionize the manufacture of biopharmaceuticals, reducing lot production time, in some cases to five weeks.
  • the particular production system described in this Example utilizes an agrobacterial vector to deliver very large copy numbers of plant viral RNAs that carry the coding sequence of the protein or polypeptide of interest into young plants within a few minutes. High levels of transient expression of the protein or polypeptide of interest can thus be achieved in bulk plant biomass within a week of introducing the agrobacterial vector into the plant.
  • the technology platform described in this Example is applicable for a broad range of monomelic or multimeric proteins, including vaccine antigens, monoclonal antibodies and other therapeutics (among other things), and can be performed in a variety of plant species. Expression in plants is more likely to provide correct folding and solubility than some altearnative systems and has the added advantage of being free from animal pathogens. In this particular Example, we focus on engineering and producing vaccine antigens and monoclonal antibodies in seedlings. [00235] As described in this Example, genes encoding the proteins or polypeptides of interest are cloned into a launch vector that combines elements of agrobacterial and plant RNA virus sequences.
  • Agrobacterium is then used to introduce millions of copies of the launch vector into plants by vacuum infiltration.
  • the vector sequences are further massively amplified through viral replication.
  • Target proteins are then produced from these viral transcripts in less than a week following introduction of the vector. It will be appreciated that it is not required (though it is also not prohibited) for the agrobacterial vector to integrate in the plant cells. Rather, the agrobacterial elements of the vector merely facilitate rapid systemic delivery (via agroinfiltration) of the viral RNA from which protein is ultimately produced.
  • This system can be used to produce very large amounts (e.g., hundred milligram quantities) of target protein per kg of fresh plant tissue. Furthermore, very high capacity can be achieved as large quantities of seeds (which, as noted, need not be transgenic) can be readily made available.
  • target protein or polypeptide is produced as a fusion with a carrier molecule, for example to stabilize the protein and/or to facilitate downstream processing.
  • a carrier molecule for example to stabilize the protein and/or to facilitate downstream processing.
  • Such carriers may be particularly useful in the production of antigen proteins.
  • Carriers of particular interest include, for example, thermostable proteins such as those described in, for example, USSN 60/472,495 filed May 22, 2003 and PCT/US04/016452, filed May 24, 2004 and published as WO 05/026375 on March 24, 2005).
  • thermostable carrier molecule is an engineered version of ⁇ -l,2-l,4-gucanase (LicKM) from Clostridium thermocellum.
  • Target sequences can be rapidly engineered as N-terminal, C-terminal or internal fusions to LicKM.
  • the presence of foreign sequences in LicKM does not result in loss of the molecule's thermostability. This property facilitates easy and cost efficient recovery of target proteins.
  • a simple heat treatment step e.g., up to 10 minutes at 65 0 C results in removal of most of the host proteins.
  • more than one target protein can be fused to each molecule of LicKM but using multiple insertion sites simultaneously.
  • seeds from non-genetically modified plants are germinated in hydroponic conditions and maintained for a period of time (e.g., typically up to about 10 days for sprouts; sometimes 2-6 weeks for Nicotiana benthamani ⁇ ) in contained indoor plant facilities prior to vacuum infiltration. Under these conditions, the yield of plant biomass per square foot is typically 4-5 fold higher than that achieved with plants grown in " soil.
  • young plants e.g., sprouted seedlings
  • Very high capacity can be achieved by stacking racks of young plants (e.g., seedlings) above one another on shelving with fitted light units (a 1000 sq. ft. growth room will provide up to four metric tons of biomass in less than two weeks). All aerial parts of plants are infiltrated with agrobacteria harboring the launch vector by applying, and then quickly releasing, a vacuum. This forces agrobacteria into the whole canopy of plant tissue with near complete leaf coverage. Target protein then accumulates over a period of a few days (e.g., about 2 to about 14 days, in some embodiments about 2 to 10, 3 to 7, 4 to 6, etc. days).
  • a few days e.g., about 2 to about 14 days, in some embodiments about 2 to 10, 3 to 7, 4 to 6, etc. days).
  • seed material is non-transgenic, seed generation and storage are inexpensive and straightforward and there is effectively no limitation of scale-up. Thus, very large quantities of recombinant protein can potentially be generated in a matter of weeks.
  • Produced target proteins can be isolated, if desired.
  • a heat step can be useful in the isolation of targets fused to a thermostable carrier.
  • Other purifications can involve various separation and/or chromatography steps. For example, steps such as ammonium sulfate precipitation, pH-dependent separation, size exclusion chromatorgraphy, ion exchange chromatography, and affinity chromatography may be employed.
  • the equipment design depicted in Figure 20 uses a tower concept such that the entire module will fit within an approximately 5000 sq. ft. space.
  • the central shelving unit provides the necessary space, light and water supply to grow 1.5 metic tons of plant biomass. Humidity, temperature and light are automatically monitored and controlled.
  • plants will be grown hydroponically in trays that are 1 ' x 2' in size. Thus, approximately 6000 trays will be used.
  • the module can be constructed from 36 individual storage unis that are stackable. Each storage unit will hold 6 x 7 x 4 trays.
  • the modularity of the design allows for scalable production. There are two robots, one on each side of the shelves, that get the trays in and out of the shelves.
  • Automated seeding is done on a material suitable for hydroponic growth. Once the plants have reached an appropriate maturity, a conveyor transports them to the infiltration unit, which has a vacuum chamber that has the capacity to infiltrate 1.5 metric tons of plant biomass in about 8 hours. A rinsing unit removes excess agrobacterial culture. Once the plants are rinsed they are conveyed back to the central shelving unit for target accumulation for several days. Subsequently, the trays are conveyed to the harvesting unit where the plants are harvested and homogenized. The homogenized plant extract is then transported to a downstream processing unit.
  • Example 9 Expression of HPV Antigen from a an Agrobacterial Construct Containing Viral Sequences
  • Agrob ⁇ cterium infiltration can be utilized (Turpen et ⁇ l., 1993, J. Virol. Methods, 42:227). Healthy leaves of N. benth ⁇ mi ⁇ n ⁇ were infiltrated with A. rhizogenes containing viral vectors engineered to express LicKM-E7 or LicKM-E7GGG.
  • A. rhizogenes strain A4 ATCC 43057
  • A. tumef ⁇ ciens GV3103
  • the constructs pBI-D4- PRACS-LicKM-E7-KDEL, pBI-D4-PRACS- LicKM-E7VAC, pBI-D4-PRACS-LicKM-E7GGG-KDEL and P BI-D4-PRACS-LicKM- E7GGG-VAC Agrob ⁇ cterium cultures were grown and induced as described (Kapila et ⁇ l., 1997, Plant ScL, 122:101).
  • a 2 ml starter-culture (picked from a fresh colony) was grown overnight in YEB (5 g/1 beef extract, 1 g/1 yeast extract, 5 g/1 peptone, 5 g/1 sucrose, 2 mM MgSO4) with 25 ⁇ g/ml kanamycin at 28 0 C.
  • the starter culture was diluted' 1:500 into 500 ml of YEB with 25 ⁇ g/ml kanamycin, 10 mM 2-4(-morpholino)ethanesulfonic acid (MES) pH 5.6, 2 mM additional MgSO4 and 20 ⁇ M acetosyringone.
  • the diluted culture was then grown overnight to an O.D.600 of ⁇ 1.7 at 28 0 C.
  • the cells were centrifuged at 3,000 x g for 15 minutes and re-suspended in MMA medium (MS salts, 10 mM MES pH 5.6, 20 g/1 sucrose, 200 ⁇ M acetosyringone) to an O.D.600 of 2.4, kept for 1 hour at room temperature, and used for Agrobacterium- ⁇ nf ⁇ tration.
  • MMA medium MS salts, 10 mM MES pH 5.6, 20 g/1 sucrose, 200 ⁇ M acetosyringone
  • Example 10 Expression of Anthrax Antigen from a an Agrobacterial Construct Containing Viral Sequences
  • Agrob ⁇ cterium infiltration can be utilized to produce anthrax antigen(s). Healthy leaves of N. benth ⁇ mi ⁇ n ⁇ were infiltrated with A. rhizogenes containing viral vectors engineered to express LicKM or LicKM-PAD4.
  • the vector used was pBI-D4, a version of the viral expression vector D4 introduced into the Agrob ⁇ cterium vector pBI121. (Chen et ⁇ l., 2003, MoI. Breed., 11 :287).
  • the 35S promoter is fused at the 5' end of the viral sequence.
  • the vector sequence is positioned between the BamHI and Sad sites of pBI121.
  • the hammerhead ribozyme is placed 3' of the viral sequence (Turpen et ⁇ l., 1993, J. Virol. Methods, 42:227). These constructs include fusions of sequences encoding LicKM-PAD4 or LicKM, to sequences encoding the signal peptide from tobacco PR- Ia protein, a 6x His tag and the ER-retention anchor sequence KDEL (see SEQ ID NO.: 10).
  • the A. rhizogenes strain A4 (ATCC 43057) was transformed with the constructs pBI-D4-PRLicKM-PAD4K and pBI-D4-PRLicKMK. Agrob ⁇ cterium cultures were grown and induced as described (Kapila et ⁇ l., 1997, Plant ScL, 122:101). A 2 ml starter-culture (picked from a fresh colony) was grown overnight in YEB (5 g/1 beef extract, 1 g/1 yeast extract, 5 g/1 peptone, 5 g/1 sucrose, 2 mM MgSO 4 ) with 25 ⁇ g/ml kanamycin at 28 0 C.
  • YEB 5 g/1 beef extract, 1 g/1 yeast extract, 5 g/1 peptone, 5 g/1 sucrose, 2 mM MgSO 4
  • the starter culture was diluted 1 :500 into 500 ml of YEB with 25 ⁇ g/ml kanamycin, 10 mM 2-4(-mo ⁇ holino)ethanesulfonic acid (MES) pH 5.6, 2 mM additional MgSO 4 and 20 ⁇ M acetosyringone.
  • the diluted culture was then grown overnight to an O.D. ⁇ oo of ⁇ 1.7 at 28 0 C.
  • the cells were centrifuged at 3,000 x g for 15 minutes and re-suspended in MMA medium (MS salts, 10 mM MES pH 5.6, 20 g/1 sucrose, 200 ⁇ M acetosyringone) to an "O.D.
  • Example 11 Expression of Influenza Antigen from a an Agrobacterial Construct Containing Viral Sequences
  • Agrob ⁇ cterium infiltration can be utilized to express influenza antigen(s).
  • Transfection of whole plants from wounds inoculated with Agrob ⁇ cterium tumef ⁇ ciens containing cDNA of tobacco mosaic virus J. Virol. Methods, 42:227) Healthy leaves of N. benth ⁇ mi ⁇ n ⁇ were infiltrated with A. rhizogenes containing viral vectors engineered to express LicKM-HA or LicKM-NA.
  • the A. rhizogenes strain A4 (ATCC 43057) was transformed with the constructs pBI-D4- PRACS-LicKM-HA-KDEL, pBI-D4-PRACS-LicKM-HA-VAC, pBI- D4-PRACS-LicKM-NA-KDEL and pBI-D4-PRACS-LicKM-NA-VAC. Agrob ⁇ cterium cultures were grown and induced as described by Kapila et ⁇ l. (Kapila J., De Rycke R., Van Montagu M. and Angenon G. (1997) An Agrob ⁇ cterium- ⁇ nediated transient gene expression system for intact leaves. Plant ScL 122, 101-108.).
  • a 2 ml starter-culture (picked from a fresh colony) was grown overnight in YEB (5 g/1 beef extract, 1 g/1 yeast extract, 5 g/1 peptone, 5 g/1 sucrose, 2 mM MgSO-O with 25 ⁇ g/ml kanamycin at 28 0 C.
  • the starter culture was diluted 1:500 into 500 ml of YEB with 25 ⁇ g/ml kanamycin, 10 mM 2-4(- morpholino)ethanesulfonic acid (MES) pH 5.6, 2 mM additional MgSO4 and 20 ⁇ M acetosyringone.
  • the diluted culture was then grown overnight to an O.D. 6 ooof —1.7 at 28 0 C.
  • the cells were centrifuged at 3,000 x g for 15 minutes and re-suspended in MMA medium (MS salts, 10 mM MES pH 5.6, 20 g/1 sucrose, 200 ⁇ M acetosyringone) to an O.D.600 of 2.4, kept for 1-3 hour at room temperature, and used for Agrobacterium- inf ⁇ ltration.
  • JV. benthamiana leaves were injected with the Agrobacterium-suspension using a disposable syringe without a needle. Infiltrated leaves were harvested 6 days post- infiltration. Plants can be screened for the presence of target antigen expression by assessment of lichenase activity assay and immunoblot analysis. [00250] Zymogram analysis revealed the expression of both HA and NA chimeric
  • the activity band related to the fusion proteins show a higher molecular weight than the lichenase control and the same molecular weight of the product expressed by plants after agro-infection, confirming the presence of whole fusion product.
  • Example 12 Expression of Influenza Antibody from a an Agrobacterial Construct Containing Viral Sequences

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Diabetes (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des systèmes et des procédés permettant de produire un acide nucléique ou une protéine dans de jeunes plantes. Généralement, les jeunes plantes sont cultivées dans un environnement clos pouvant être régulé. Dans certains modes de réalisation, l'expression d'une protéine active d'un point de vue pharmaceutique chez les jeunes plantes est régulée par un promoteur inductible de manière exogène ou un promoteur viral. Dans d'autres modes de réalisation, les jeunes plantes sont comestibles et peuvent être mangées vivantes ou de préférence récoltées vivantes afin de préserver l'activité biologique maximale de l'acide nucléique ou de la protéine. Dans des modes de réalisation encore différents, la jeune plante est un jeune pois ou une jeune plante Nicotiana.
PCT/US2007/003942 2006-02-13 2007-02-13 Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux WO2007095304A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
BRPI0707785-8A BRPI0707785A2 (pt) 2006-02-13 2007-02-13 produÇço de Ácidos nuclÉicos estranhos e polipeptÍdeos em sistemas de planta
EP07750761A EP1984509A4 (fr) 2006-02-13 2007-02-13 Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux
CA002642138A CA2642138A1 (fr) 2006-02-13 2007-02-13 Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux
JP2008554439A JP2009528025A (ja) 2006-02-13 2007-02-13 植物系における外来核酸およびポリペプチドの産生
AU2007215066A AU2007215066A1 (en) 2006-02-13 2007-02-13 Production of foreign nucleic acids and polypeptides in plant systems
CN200780007805A CN101815784A (zh) 2006-02-13 2007-02-13 植物系统中外来核酸和多肽的产生
IL193390A IL193390A0 (en) 2006-02-13 2008-08-12 Production of foreign nucleic acids and polypeptides in plant systems

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US77337406P 2006-02-13 2006-02-13
US77325506P 2006-02-13 2006-02-13
US77337806P 2006-02-13 2006-02-13
US60/773,374 2006-02-13
US11/353,905 US7692063B2 (en) 2002-11-12 2006-02-13 Production of foreign nucleic acids and polypeptides in sprout systems
US11/353,905 2006-02-13
US60/773,255 2006-02-13
US60/773,378 2006-02-13
US87945007P 2007-01-09 2007-01-09
US60/879,450 2007-01-09

Publications (2)

Publication Number Publication Date
WO2007095304A2 true WO2007095304A2 (fr) 2007-08-23
WO2007095304A3 WO2007095304A3 (fr) 2010-01-21

Family

ID=38372119

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/003942 WO2007095304A2 (fr) 2006-02-13 2007-02-13 Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux

Country Status (8)

Country Link
EP (1) EP1984509A4 (fr)
JP (1) JP2009528025A (fr)
KR (1) KR20080113372A (fr)
CN (1) CN101815784A (fr)
AU (1) AU2007215066A1 (fr)
BR (1) BRPI0707785A2 (fr)
CA (1) CA2642138A1 (fr)
WO (1) WO2007095304A2 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010037063A3 (fr) * 2008-09-28 2010-07-22 Fraunhofer Usa, Inc. Vaccins au plasmodium, antigènes, compositions et procédés
EP2306807A1 (fr) * 2008-06-30 2011-04-13 ORF Liftaekni HF. Production industrielle, à base de plantes, de protéines recombinantes non animales dans un environnement défini
US8951791B2 (en) 2003-02-03 2015-02-10 Ibio, Inc. System for expression of genes in plants
US9012199B2 (en) 2003-05-22 2015-04-21 Ibio, Inc. Recombinant carrier molecule for expression, delivery and purification of target polypeptides
WO2015156340A1 (fr) * 2014-04-10 2015-10-15 三菱化学株式会社 Procédé de production d'une protéine utile à l'aide d'une plante
CN107249627A (zh) * 2015-01-30 2017-10-13 国立大学法人东京大学 含有乳酸菌的组合物、hpv感染症及hpv相关肿瘤中的至少任一种疾病的治疗用口服医药组合物及粘膜免疫诱导剂
US9809644B2 (en) 2009-09-29 2017-11-07 Ibio Inc. Influenza hemagglutinin antibodies, compositions and related methods
US10125370B2 (en) 2013-09-06 2018-11-13 Mitsubishi Chemical Corporation Method for producing protein in plants using lighting with at least 50% red light
WO2018232406A1 (fr) * 2017-06-16 2018-12-20 Arizona Board Of Regents On Behalf Of The University Of Arizona Procédés, systèmes et compositions pour la production à partir de légumineuses de protéines thérapeutiques et de matériaux médicaux thérapeutiques
WO2020074704A1 (fr) * 2018-10-11 2020-04-16 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédés de détection d'allergènes de soja
EP3550028A4 (fr) * 2016-11-30 2020-09-02 Kirin Holdings Kabushiki Kaisha Procédé de production de protéine utile à l'aide d'une plante
US10889826B2 (en) 2017-06-16 2021-01-12 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for producing epidermal growth factor (EGF) in soybeans
US11098320B2 (en) 2017-02-03 2021-08-24 Arizona Board Of Regents On Behalf Of The University Of Arizona Engineering high-protein-content soybeans

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1919504B1 (fr) 2005-08-03 2013-10-16 iBio, Inc. Anticorps diriges contre l'antigene protecteur de bacillus anthracis
CA2692933C (fr) 2007-07-11 2016-10-18 Fraunhofer Usa, Inc. Antigenes yersinia pestis, compositions de vaccins, et methodes associees
US8734803B2 (en) 2008-09-28 2014-05-27 Ibio Inc. Humanized neuraminidase antibody and methods of use thereof
CN103146736A (zh) * 2013-03-28 2013-06-12 福建农林大学 一种植物表达载体pGⅡ0229-GUS的构建及应用
JP6578681B2 (ja) * 2015-03-11 2019-09-25 三菱ケミカル株式会社 植物栽培方法及びそれを用いる有用タンパク質の製造
CN110423746B (zh) * 2019-07-16 2021-03-12 北京林业大学 编码小肽pfer的基因序列、小肽pfer和应用及诱导植物生根的方法
CN114736920A (zh) * 2022-03-01 2022-07-12 中国计量大学 农杆菌侵染菜豆叶片实现目的基因瞬时过表达或瞬时沉默的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5693506A (en) * 1993-11-16 1997-12-02 The Regents Of The University Of California Process for protein production in plants
US6700040B2 (en) * 1998-01-16 2004-03-02 Large Scale Biology Corporation Cytoplasmic gene inhibition or gene expression in transfected plants by a tobraviral vector
US7683238B2 (en) * 2002-11-12 2010-03-23 iBio, Inc. and Fraunhofer USA, Inc. Production of pharmaceutically active proteins in sprouted seedlings
CA2526720C (fr) * 2003-05-22 2013-10-22 Fraunhofer Usa, Inc. Molecule support recombinee pour l'expression, l'administration et la purification de polypeptides cibles
ATE469231T1 (de) * 2003-11-10 2010-06-15 Icon Genetics Gmbh Von rna-virus abgeleitetes pflanzenexpressionssystem
WO2005081905A2 (fr) * 2004-02-20 2005-09-09 Fraunhofer Usa Inc. Systemes et methodes d'expression clonale dans des plantes
EP1616959A1 (fr) * 2004-07-07 2006-01-18 Icon Genetics AG Procédé d'expression transitoire de protéines dans les plantes biologiquement sur

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP1984509A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8951791B2 (en) 2003-02-03 2015-02-10 Ibio, Inc. System for expression of genes in plants
US9551001B2 (en) 2003-02-03 2017-01-24 Ibio, Inc. System for expression of genes in plants
US9765349B2 (en) 2003-02-03 2017-09-19 Ibio, Inc. System for expression of genes in plants
US9012199B2 (en) 2003-05-22 2015-04-21 Ibio, Inc. Recombinant carrier molecule for expression, delivery and purification of target polypeptides
EP2306807A1 (fr) * 2008-06-30 2011-04-13 ORF Liftaekni HF. Production industrielle, à base de plantes, de protéines recombinantes non animales dans un environnement défini
WO2010037063A3 (fr) * 2008-09-28 2010-07-22 Fraunhofer Usa, Inc. Vaccins au plasmodium, antigènes, compositions et procédés
US9809644B2 (en) 2009-09-29 2017-11-07 Ibio Inc. Influenza hemagglutinin antibodies, compositions and related methods
US10125370B2 (en) 2013-09-06 2018-11-13 Mitsubishi Chemical Corporation Method for producing protein in plants using lighting with at least 50% red light
WO2015156340A1 (fr) * 2014-04-10 2015-10-15 三菱化学株式会社 Procédé de production d'une protéine utile à l'aide d'une plante
CN107249627A (zh) * 2015-01-30 2017-10-13 国立大学法人东京大学 含有乳酸菌的组合物、hpv感染症及hpv相关肿瘤中的至少任一种疾病的治疗用口服医药组合物及粘膜免疫诱导剂
CN107249627B (zh) * 2015-01-30 2021-06-29 国立大学法人东京大学 含有来自hpv e7蛋白质的多肽的乳酸菌的组合物
EP3550028A4 (fr) * 2016-11-30 2020-09-02 Kirin Holdings Kabushiki Kaisha Procédé de production de protéine utile à l'aide d'une plante
US11098320B2 (en) 2017-02-03 2021-08-24 Arizona Board Of Regents On Behalf Of The University Of Arizona Engineering high-protein-content soybeans
WO2018232406A1 (fr) * 2017-06-16 2018-12-20 Arizona Board Of Regents On Behalf Of The University Of Arizona Procédés, systèmes et compositions pour la production à partir de légumineuses de protéines thérapeutiques et de matériaux médicaux thérapeutiques
US10889826B2 (en) 2017-06-16 2021-01-12 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for producing epidermal growth factor (EGF) in soybeans
WO2020074704A1 (fr) * 2018-10-11 2020-04-16 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédés de détection d'allergènes de soja

Also Published As

Publication number Publication date
BRPI0707785A2 (pt) 2011-05-10
CN101815784A (zh) 2010-08-25
EP1984509A4 (fr) 2010-07-14
JP2009528025A (ja) 2009-08-06
WO2007095304A3 (fr) 2010-01-21
CA2642138A1 (fr) 2007-08-23
KR20080113372A (ko) 2008-12-30
AU2007215066A1 (en) 2007-08-23
EP1984509A2 (fr) 2008-10-29

Similar Documents

Publication Publication Date Title
WO2007095304A2 (fr) Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux
US7683238B2 (en) Production of pharmaceutically active proteins in sprouted seedlings
Molina et al. High‐yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts
Birch‐Machin et al. Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability
US20110070609A1 (en) Production of Foreign Nucleic Acids and Polypeptides in Sprout Systems
Ma et al. Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products
CA2824152C (fr) Methode et systeme de production d'un polypeptide heterologue dans un plant de nicotiana tabacum
KR101540496B1 (ko) 세균 독소 백신
WO2007095318A2 (fr) antigenes de la grippe, compositions de vaccins et procedes associes
KR20080038131A (ko) 생물학적으로 활성인 거대분자의 점막 또는 장관 투여
KR20010085901A (ko) 식물에서의 치료학적 활성 단백질
US8277816B2 (en) Bacillus anthracis antigens, vaccine compositions, and related methods
TW200914611A (en) Pig edema disease vaccine
Rosales-Mendoza et al. Transgenic carrot tap roots expressing an immunogenic F1–V fusion protein from Yersinia pestis are immunogenic in mice
US20070178148A1 (en) Preparations of growth hormone
Zhang et al. Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli
CN111565747B (zh) 与猪fc片段融合的抗原，以及包含该抗原的疫苗组合物
Cheung et al. Expression and subcellular targeting of human insulin-like growth factor binding protein-3 in transgenic tobacco plants
Wiktorek-Smagur et al. Green way of biomedicine–how to force plants to produce new important proteins
Unni et al. Transgenic Cucumis sativus expressing the hepatitis B surface antigen
Alli et al. Pharming vaccines for hepatitis and cytomegalovirus: towards the development of multivalent and subunit vaccines for oral delivery of antigens
Mor et al. Expression of rotavirus proteins in transgenic plants
Erickson et al. Edible vaccines in plants for livestock pathogens
WO2004105466A2 (fr) Transformation de fleurs

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780007805.7

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 2007215066

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2642138

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 193390

Country of ref document: IL

Ref document number: 2008554439

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 3313/KOLNP/2008

Country of ref document: IN

Ref document number: 2007750761

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 1020087022552

Country of ref document: KR

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07750761

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: PI0707785

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20080813