BRPI0707785A2 - Production of foreign nucleic acids and polypeptides in plant systems - Google Patents

Abstract

PRODUCTION OF STRANGE NUCLEIC ACIDS AND POLYPEPTIDEOS IN PLANT SYSTEMS. The present invention relates to systems and methods for producing a nucleic acid or protein in young plants. Typically, young plants are grown in a regulated, contained environment. In some embodiments, expression of a pharmaceutically active protein in young plants is controlled by an exogenously inducible promoter or a viral promoter. In some embodiments, young plants are edible and can be eaten alive or preferably harvested alive to preserve the maximum biological activity of the nucleic acid or protein. In some embodiments, the young plant is a young pea plant or a young Nicotian plant.

Description

Report of the Invention Patent for "Production of Strange Nucleic Acids and Polypeptides in Plant Systems".

Related Patent Applications

This patent application is a partial continuation of and claims the benefit of co-pending United States Patent Application No. 011 / 353,905, filed February 13, 2006, entitled "Production of Foreign Nucleic Acids and Polypeptides in Chemical Systems". Germination ". The total contents of this patent application are hereby incorporated by reference.

This patent application also claims the benefit of each of the co-pending United States Provisional Patent Applications Numbered 60 / 773,255 (entitled "Vaccine Compositions, and Related Me-thods"); 60 / 773,374 (entitled "Antigens, Vaccines Compositions, and Related Methods"); and 60 / 773,378 (entitled "Influenza Antigen, Vaccine Compositions, and Related Methods"), each of which is filed on February 13, 2006. The total contents of each of these pending applications are hereby incorporated by reference. .

This patent application further claims the benefit of co-pending United States Provisional Patent Application No. 60 / 813,955, filed June 15, 2006, entitled "Influenza Antigens, Vaccine Compositions, and Related Methods". The total contents of this patent application are hereby incorporated by reference.

This patent application further claims the benefit of co-pending United States Provisional Patent Application No. 60 / 944,770, filed September 15, 2006, entitled "Influenza Antibodies, Vaccine Compositions, and Related Methods". The overall contents of this patent application are hereby incorporated by reference.

This patent application further claims the benefit of co-pending United States Provisional Patent Application No. 60 / 879,450, filed January 9, 2007, entitled "Launch Vector for the Production of Vaccine Antigens in Plants". The total contents of each of these patent applications are hereby incorporated by reference.

Background of the Invention

The cost of pharmacists is exorbitantly high and continues to rise. Some pharmacists, in addition to their high cost, are also limited in supply, making it impossible for them to be available to every patient in need. This is particularly problematic in developing countries, where both cost and availability prevent the distribution of pharmacists to populations in need.

Several factors contribute to the high costs of pharmaceutical production, and result in the high price of pharmaceuticals for the consumer. A major contributing factor is the lack of economic means of producing the product. This is particularly true for protein and peptide based drugs. Another contributing factor to some medications is the inability to therapeutically administer effective amounts of the pharmaceutical agent orally. Many pharmacists may only be administered by injection to a particular location in the body. For example, many immunization drugs for the treatment of allergy or infectious diseases require administration by injection. Protein and peptide pharmaceuticals, such as human growth hormone and insulin, can often only be administered by injection. Another factor contributing to the cost of many pharmaceutical drugs is their distribution to the hospital or place of distribution. Particularly in hot climates, dispensing and storing pharmaceutical drugs requires costly refrigeration equipment. This is a major challenge in developing countries, where such equipment is often unavailable.

Pharmaceutical proteins and peptides have been produced in a wide variety of hosts. Many therapeutic proteins have been produced in heterologous expression systems, including procaryotes such as Escherichia coli and Bacillus subtilis, and eukaryotes such as yeast, fungus, insect cells, animal cells and transgenic animals. Bacterial expression systems are relatively easy to handle and product yield is high. However, mammalian proteins often require extensive posttranslational modification for functional activity, which may be a limiting factor in bacterial expression systems. Cell culture systems, such as mammalian, human and insect culture systems, They are most convenient for the production of complex proteins. However, long driving times, low product recovery, possible pathogenic transfer, and high capital and production costs present serious concerns. Transgenic animals can provide an unlimited supply of complex proteins. Unfortunately, this system is limited by the length of time it takes to generate new and improved products, and the risk of pathogen transfer to human subjects.

Economic and biochemical limitations for the production of protein and pharmaceutical peptides in prokaryotic and eukaryotic cells, including high production costs, low yields, segregation problems, improper protein processing modifications, scale difficulties in larger volumes , and communication, have led researchers to examine plants as new hosts for large-scale production of proteins and peptides with the expectation of reducing costs. Protein production in transgenic plants is described, for example, in United States Patent Nos. 5,750,871; 5,565,347; 5,464,763; 5,750,871; and 5,565,347. Although plants are less expensive to grow and harvest in volume than prokaryotic and eukaryotic cells, foreign gene expression in plant cells is typically low. In addition, 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 plant part formation or plant apoptosis. A plant supporting apoptosis generates proteases that contribute to the degradation of the transgenically expressed protein prior to protein purification at an early stage. Even if the plant is to be directly consumed, the activity of the expressed pharmaceutical protein can be reduced by crop-induced intercellular degradation machinery.

Another major concern associated with the production of foreign protein in transgenic plants that are grown in open fields is the possibility of cross-pollination with plants in the soil, making it possible for foreign protein to enter the food chain. The complexity of government regulations surrounding farming practices for transgenic plants makes it difficult to obtain new transgenic plants approved for agricultural use. In addition, the external environment is impossible to control, making proper foreign gene expression growth, development and regulation difficult to guarantee. For example, induction of an inducible heat, inducible light, inducible hormone, or chemically inducible promoter would be virtually impossible in an outdoor environment. Of course, the outside temperature and high levels cannot be controlled. Additionally, hormones or chemicals sprayed on a plant are similarly to be dispersed not on a plant but in the environment by wind and rain. Spraying crop fields is also very costly.

There is a need for improved systems for the production of proteins or polypeptides. There is a particular need for improved systems for the production of protein and polypeptide plants. For example, there is a need for a controlled adjustable system for pharmaceutical protein production in plants that decreases the amount of intercellular degradation of the expressed protein after harvest.

Summary of the Invention

The present invention provides systems for the production of protein or polypeptide (and / or nucleic acid), particularly protein or pharmaceutical polypeptide, in plants.

In one aspect, the present invention provides systems for rapid expression of proteins or polypeptides in plants. In some embodiments, therefore, the present invention provides expression of protein or polypeptides in young plants. In some embodiments, such young plants are sprouted shoots. In some embodiments, young plants (e.g. sprouted sprouts) may be consumed or harvested alive. In some embodiments, the plants are grown (e.g. from a seed) in an adjustable environment contained, for example, indoors.

In some embodiments, a protein or polypeptide to be produced in accordance with the present invention is expressed in plant cells of a nucleic acid construct that is introduced into the plant by human hands. In some embodiments, a protein or polypeptide to be produced in accordance with the present invention is translated into plant cells of an RNA with plant characteristics. In some embodiments, these features include or are selected from the group consisting of auto-replication, cell-to-cell movement, systemic movement, and combinations thereof. In some embodiments, RNA is self-replicating and capable of cell-to-cell motion, but is not capable of systemic motion in the plant.

In some embodiments, a protein or polypeptide to be produced according to the present invention is expressed in plant cells of an Agrobacterium-replicating nucleic acid construct. In some embodiments, the present invention utilizes an Agrobacterium-replicating construct, and also contains an expression-directing promoter of an RNA with characteristics of a plant virus. In some embodiments, this RNA includes sequences that encode the protein or polypeptide of interest. Thus, in some embodiments of the present invention, a protein or polypeptide of interest is produced in plants by a process involving introduction into cells of a plant of an Agrobacterium replicating vector, and also contains an expression targeting promoter of an RNA with characteristics of a virus. plant, whose RNA is not capable of systemic movement in a plant but is capable of self-replication, and whose RNA additionally encodes the protein or polypeptide of interest.

In some embodiments of the invention, where a construct is used that replicates in Agrobacterium, the construct is introduced into plant cells by agro infiltration.

While the present invention is described herein by reference to its use for the production of pharmaceutical proteins, the invention generally finds use for the production of essentially any nucleic acid (DNA and / or RNA) and / or protein of interest, without limitation as to -the particular nucleic acid (s) or protein. For example, enzymes used in any of a wide area of interest in industrial processes or bio-remediation processes (e.g. pollutant degrading enzymes) may be produced. Accordingly, 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 application and those without therapeutic application. In certain embodiments, the protein is not a nutritionally important protein. Any particular protein may be excluded from the present invention without being named herein.

In certain embodiments of the invention in any of its aspects, the nucleic acid or protein of interest is post-transcriptionally and / or post-translationally processed in the cell in which it is expressed. In certain embodiments of the invention, a protein of interest is secret. -da from the cell it is expressed. For example, the protein may naturally comprise a secretion signal sequence, or the decoding region of a protein-encoding nucleic acid may be modified to include a portion encoding a secretion signal sequence.

Secretion signal sequences are well known in the art.

In certain embodiments of the invention in any of its aspects, a heterologous sequence encoding a protein of interest may be altered to employ codons other than those present in the naturally occurring sequence in order to improve expression in plants generally, and / or in plants. a particular species. For example, the sequence may be codon optimized for expression in a particular species. Methods for performing codon optimization are well known in the art.

The present invention also provides, among other things, plants containing expression builders as described herein, compositions containing such plants, protein / polypeptide preparations isolated from such plants, compositions containing such protein / polypeptides isolated from such plants, methods of performing such isolations, and methods of using such isolated proteins / polypeptides (or compositions containing same), including methods of treating a mammal with a plant-expressed pharmaceutically active protein.

Definitions

"Administration" of a pharmaceutically active protein or polypeptide to an individual in need thereof is intended as the provision of the pharmaceutically active protein to such individual in a manner that retains the therapeutic efficiency of such protein for a sufficient length of time to provide a beneficial effect desired to such a host.

The term "animal" covers all life forms of vertebrates, humans, cattle, sheep, swine, canines, felines, ferrets, rodents, primates, fish, birds, for example poultry, and the like. In some embodiments, animals are mammals. In some embodiments, the animals are humans.

According to the present invention, a "characteristic portion" of a polypeptide or protein is one that contains a continuous amino acid extension, or a collection of continuous amino acid extensions, which together are characteristic of a protein or polypeptide. In some embodiments, each such continuous extension will generally contain at least 2, 5, 10, 20 or more amino acids. In general, a characteristic portion is one which, in addition to the sequence identity specified above, has in common at least one functional characteristic with the relevant intact protein. In some embodiments, the characteristic moiety may be biologically active.

A "domain" of a protein or polypeptide, as used herein, generally refers to a protein or polypeptide sequence segment that, when produced separately from the rest of the protein or polypeptide sequence, maintains a degree of integrity and / or three-dimensional structure. As is well known in the art, any 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 simple epitope, and is typically larger; In some embodiments, an immunogenic domain contains sufficient sequence in addition to the epitope to ensure correct presentation of the epitope.

"Expression" refers to the transcription and / or translation of a gene endogenous or a transgene in a plant, for example, a seedling. Otransgene may or may not be integrated into the genomic DNA of the plant. For example, "expression" refers to the transcription and / or translation in a plant of a gene present in a bacterial, plasmid, or 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 a hematologist 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 denucleotide sequence in an appropriate host cell. For example, an expression cassette may include a promoter operably linked to a nucleotide sequence of interest, which is optionally linked to 3 'sequences, such as 3' regulatory sequences, or terminal signals. An expression cassette may also typically include required sequences. for proper translation of the nucleotide sequence if any 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 an untranslated 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 sequence of source DNA. which are fused together by recombinant DNA techniques, resulting in a nucleotide sequence that is not it runs naturally, and does not occur particularly in the plant in which it is to be expressed. An expression cassette may also be one that is normally occurring, but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous to the host, that is, 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 a host cell predecessor by a transformation event. Expression of the denucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to any particular external stimulus. In the case of a multicellular organism such as a plant, the promoter may 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 expression of a particular nucleotide sequence from the nuclear genome of the plant. A plastid expression cassette is usually inserted into the genomic deplastid genome. a plant, and is capable of directing expression of a nucleotide sequence particularly from the plastid genome daplanta. In the case of a plastid expression cassette, for nucleotide sequence expression of a plastid genome, additional elements, for example ribosome binding sites, or 3 'rod-loop structures that prevent polyadenylation of plastid RNA , and subsequent degradation may be required. In certain embodiments of the invention, an expression cassette is used, which comprises a promoter that is a minimal promoter, such as a TATA element, and the presence of a trans-activating factor may be required to direct nucleotide sequence expression, particularly to direct high expression. level. For example, the expression cassette may comprise a DNA region for binding of a transcriptional activator. Such expression cassettes, and the engines thereof, are referred to as "activatable".

A "food" or "food product" is a liquid or solid preparation that can be ingested by humans or other animals. Preferably, the terms include raw or live plant preparations (e.g. germinated shoots) that can be fed live directly to humans and / or other animals. Materials obtained from a plant are intended to include a total edible plant that can be ingested by a human or other animal. The term may also include any processed plant (e.g., germinated seedlings) along with a nutritional vehicle that is fed to humans and other animals. The processing steps may include steps commonly used in the food or food industry. Such steps include, but are not limited to, concentration or condensation of the plant solid matter to form, for example, a pellet, paste, drying, or lyophilization, or may be produced by cutting, kneading, or milling the plant under vacuum. - Extensions, or by extracting the liquid part of the plant to produce a syrup, a syrup, or a juice. A processing step may include cooking, for example, plant fuming (eg sprouting seedlings).

A "gene" is a sequence that encodes a protein or polypeptide (or RNA) of interest. A gene may include associated regulatory sequences. A gene coding sequence may be transcript RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. As will be appreciated by those skilled in the art, however, a "gene" may also be an RNA (for example, a gene in a viral RNA vector). Examples of "regulatory sequences" are motor sequences, 5 "and 3 'untranslated sequences, and termination sequences. In addition, introns and exons may be included. In certain embodiments, the gene is the coding sequence and the associated regulatory sequences are regulatory sequences. "Regulatory sequences" as used herein mean different natural origin or synthetic origin. For example, if a nucleic acid is introduced into a host cell, and the host cell does not naturally contain any or all of the sequences present in nucleic acid, so these sequences (and / or nucleic acid) are said to be heterologous to the host cell.Introduced nucleic acid may include a heterologous promoter, heterologous coding sequence, or heterologous termination sequence. Alternatively, the transformation of nucleic acid may be compounded. heterologous, or may include any possible combination of heterologous or endogenous nucleic acid sequences. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same original natural cell type, but which is present in an unnatural state, for example, a different copy number, or under the same number. control of different regulatory elements. The term "heterologous" applies to cells, including plant and bacterial cells, as well as to plasmids, plastids and viruses.

A "host cell" is a cell in which a nucleic acid is introduced for expression. Typically, such an introduction requires the man's hand.

An "inactive expression cassette" or "inactive expression vector" is a DNA or RNA sequence comprising a foreign inactive or silenced nucleic acid sequence that is capable of directing expression of a nucleic acid or polypeptide of interest. after its activation. Generally, an inactive expression cassette has the properties of an expression cassette as described above, except that the sequence encoding a nucleic acid or polypeptide of interest cannot be operably linked to a promoter, for example, promoter (or other) may be separated. regulatory element) by a region of intervention nu-clicic acid. Operative binding occurs following a mismatch event, so expression then occurs. Such an expression cassette is referred to as being "activatable".

The term "inducible promoter" means a promoter that is rotated by the presence or absence of a particular stimulus that enhances promoter activity directly or indirectly. Some non-limiting examples of stimulus detail include heat, light, developmental regulatory factors, lesions, hormones and chemicals, for example, small molecules. An example of a mild inducible promoter is the ribulose-5-phosphatase carboxylase promoter. Chemically inducible promoters also include receptor mediated systems, for example those derived from other organisms, such as steroid-dependent gene expression, the Lace repressor system, or the expression system. using the juvenile growth hormone mediated Drosophilia USP receptor, described in WO97 / 13864, incorporated herein by reference, as well as systems using receptor combinations, for example, 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 ketone alcohols (WO 93/21334; Caddick et al. (1998)). Biotechnol 16: 177-180, the contents of which are incorporated herein by reference). Inducible promoters would include promoters for proteinase inhibitors, for example, potato proteinase inhibitor II promoter, and other plant-derived promoters involved in the fold response path, such as polyphenyl oxidase, LAP and TD promoters. See, for example, Gatz "Chemical Control of Gene Expression", Ann. Plant PhysioL Plant. Moi Biol. (1997) 48: 89-108), incorporated herein by reference. Other inducible promoters include plant-derived promoters, such as systemic acquired resistance targeting promoters, for example, PR promoters. It is noted that where inducible promoters are discussed herein, activatable promoters and expression cassettes may be used in one mode. similar to certain embodiments of the invention.

A "marker moan" is a gene that encodes a selectable or classifiable trait.

A "modified food" includes a composition that is eaten or drunk by an individual, and has a therapeutic effect on the individual. A medical food includes, for example, a plant (for example, a deminer) in which a pharmaceutical protein or polypeptide has been produced, or a plant matter derived therefrom. A medical food may be tonic alone, or may be administered in combination with a pharmaceutical composition well known in the medical arts. A medical food also includes the equivalent foodstuffs for nonhuman animals.

"Operably linked" refers to the association of nucleic acid elements. For example, a promoter operably linked to a heterologous DNA encoding a protein promotes the production of functional mRNA corresponding to the heterologous DNA. A regulatory DNA sequence is referred to as "operably linked to" or "associated with" a DNA sequence encoding an RNA or a protein if both sequences are situated such that the regulatory DNA sequence affects the expression of the DNA sequence. coding

"Oral administration" of a pharmaceutically active peptide or protein means administration primarily through the mouth, preferably by food, but is also intended to include any administration that provides such peptides or proteins to the stomach of the other digestive host. In a preferred embodiment, oral administration results in contact of the pharmaceutically active protein with the intestinal mucosa.

A "pharmaceutically active nucleic acid" is a nucleic acid that encodes a pharmaceutically active protein, or is pharmaceutically active by itself, for example, has one or more pharmaceutical activities such as those described for pharmaceutically active proteins. vas. For example, the nucleic acid may be one or more RNA transfer umagent (RNAi) braids. Such agents include short-interfering RNAs (siRNAs), or short hairpin RNAs (shRNAs), or precursor of an siRNA or RNA-like microRNA, targeted for target transcription, for example, transcription of an infectious agent, or a transcript related to an individual's endogenous disease.

A "pharmaceutically active protein or polypeptide" assisted 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 curative or palliative restoration properties against a disease, and may be administered to ameliorate, alleviate, reverse, delay the onset of, or lessen the severity of one or more symptoms of a disease or illness. . A pharmaceutically active protein or polypeptide may have prophylactic properties, and may be used to delay the onset of a disease or decrease the severity of such disease or pathological condition as it arises. The term "pharmaceutically active protein or polypeptide" includes total protein or polypeptide, and may also refer to pharmaceutically active fragments thereof. It may also include pharmaceutically active protein or polypeptide analogues, or protein or polypeptide fragment analogues. The term "pharmaceutically active protein or polypeptide" may also refer 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, that is, administration of the protein to an individual induces a partially or fully protective immune response in the host. In certain embodiments of the invention, the immune response protects the individual against an infectious agent, for example, a protozoal virus, bacterial, fungus, or pathogenesis. Examples of vaccine antigens include hepatitis B surface antigen (HBsAg), heat-unstable enterotoxin from E. coli, rabies virus glycoprotein, and Nor-walk virus capsid protein. In other embodiments of the invention, the immune response protects the individual against a non-infectious condition or disease, or diminishes 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 autoimmune diseases. The term "pharmaceutically active protein or polypeptide" includes proteins that partially or fully tolerate an individual's exposure to an allergen that would otherwise induce an allergic or anaphylactic response.

A "promoter" as used herein is a DNA sequence that initiates transcription of an associated DNA sequence. The dopromotor region may also include elements that act as gene expression regulators such as activators, enhancers, and / or repressors.

"Regulatory elements" refer to sequences involved in conferring the expression of a nucleotide sequence. Regulatory elements include 3 'regulatory sequences, such as promoters that may be linked to the nucleotide sequence of interest, 3' sequences, such as 3 'regulatory sequence, or termination signals. Regulatory elements also typically involve sequences required for correct translation of the nucleotide sequence.

"Small molecules" are typically smaller than about one kilodalton, and are biological, organic, or even inorganic compounds (eg cisplatin). Examples of such small molecules include nutrients, such as sugars and sugar derivatives (including phosphate derivatives), hormones (such as gibberellic phytohormones or absiscic acid), synthetic small molecules.

"Specifically adjustable" refers to the ability of a small molecule to preferentially affect the transcription of a promoter or promoter group, as opposed to nonspecific effects such as enhancement or reduction of overall transcription within a cell.

A "sprouted seedling" or "sprout" is a young, slightly sprouted or rooted sprout, preferably a freshly sprouted seed. radish sprouts, wheat sprouts, mustard sprouts, spinach sprouts, carrot sprouts, beet sprouts, onion sprouts, dealho sprouts, celery sprouts, rhubarb sprouts, a leaf such as sprout sprouts, or sprouts sprouts. lettuce, watercress or watercress 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.). In accordance with the present invention, the germinated seedling involved the two leaf stages. Generally, the buds of the invention are from two to fourteen days old.

"Substantially isolated" is used in various contexts, and typically refers to the at least partial purification of a protein or polypeptide outside the unrelated or contaminating components (e.g., structural plant and metabolic proteins). Methods for isolating and purifying proteins or polypeptides are well known in the art.

"Transformation" refers to the introduction of a nucleic acid into a cell, particularly the stable integration of a DNA molecule into the genome of an organism of interest.

Description of Drawings

Figure 1 is a schematic representation of different strategies for foreign gene expression using virus-based vectors.

Figure 2 is a schematic representation of ALMVe TMV genomes.

Figure 3 is a representation of a Western blot of recombinant GFP expression in Nicotiana benthamiana plants isolated with Av / A4 and Av / GFP.

Figure 4 is a representation of a Western blot of human growth hormone (hGH) production in N.benthamiana plants infected with in vitro GH transcripts.

Figure 5 is a schematic representation of the transforming constructs for expression of recombinant proteins in Brassicajun-cea.

Figure 6 is a representation of a transgenic Brasuncica juncea immunomancha expressing human growth hormone under the control of the HSP18.2 promoter.

Figure 7 is a schematic representation of various agrobacterial constructs containing target genes. Figure 7A: pBIV vector, wherein a target gene is integrated between a promoted plant (IV = any plant promoter, artificial promoter, or other promoter that functions in plant cells, for example, a promoter of a plant virus such as comovirus). cauliflower mosaic) and the terminator nos; Figure 7B: pBIV-GUS vector, where the GUS gene is inserted into pBIV. Figure 7C: pBIV-Virus + Target Vector, wherein a viral vector or replicon (e.g., a genomaviral or genome portion) carrying a target gene is inserted into pBIV; FIGURE 7D: pBIV-Virus + GFP Vector, wherein GFP is a target gene driven by a pBIV inserted viral vector or replicon. The vectors depicted in Figures 7C and 7D are considered "release" vectors because, upon introduction into plant cells, they "release" the production of viral sequences (which may be self-replicating, at least in the cell context). particular plant in which they are lacy.

Figure 8 demonstrates GUS staining after agro-infiltration of pBIV-GUS shoots. Figure 8A demonstrates munguba bud staining; Figure 8B demonstrates Fenugreek sprout staining.

Figure 9 demonstrates GUS staining after agro-infiltration of Brassica shoots with pBIV-GUS. Figure 9A shows the staining results; Figure 9B is a table listing different types of probes tested for their ability to express protein or polypeptide distributed via an agrobacterial construct that includes an expression of viral sequence promoter targeting a gene encoding the protein or polypeptide of interest.

Figure 10 demonstrates GFP expression in N. benthamianas over time after pBI121 / D4-GFPC3 infiltration.

Figure 11 demonstrates Western blot analysis of hGH expression in plants as a result of Agrobacterium infiltration containing a virus vector containing a sequence encoding hGH. Routes: Route 1: Ladder; Route 2: 50 ng of hGH; Route 3: Sample of infiltrated plant with pBI121 / D4-HGH 6dpi dilution 1: 2; Route 4: Sample of infiltrated plant with pBI121 / D4-HGH 6dpi dilution 1:10; Route 5: Sample of infiltrated plant with pBI121 / D4-HGH 6dpi dilution 1:50; Route 6: Sample of infiltrated plant with pBI121 / D4-HGH 6dpi dilution 1: 100; Route 7: Sample of Healthy Plant Plant; Route 8: Plant sample of pBI121-infiltrated leaves; Route 9: Sample of infected plant D4-HGH 13dpi; Route 10: Sample of D4-HGH infected clonal root. For plant samples: a defoliation disc (~ 5 mg tissue) is ground in 100 µl of Bradley buffer with Laemmli loading buffer. 10 ul is loaded per route. Therefore, for lanes 3-6, this primary sample was diluted.

Figure 12 demonstrates protein expression IA-2ic benthamian Nicotiana plants grown in hydroponics. Routes: 1: IA-2ic standard; 2.Magic Marker; 3. Agrobacteria-injected soil growth plants; 4,5. Hydroponic growth plants 4 days after infiltration; Hydroponic growth plants 6 days after infiltration.

Figure 13 is an embodiment of an α-large infiltration system used in accordance with the present invention to deliver an agrobacterial vector that launches viral sequences that carry a target protein of interest in young plants.

Figure 14 illustrates the activity of Iichenase detected in various pea varieties subjected to agro-infiltration using the Figure 13 system.

Figure 15 shows GFP expression and plant growth for agro-infiltrated spotted peas using the Figure 13 system.

Figure 16 shows the tissue specificity and optimal days after infiltration for certain agro-infiltrated spotted peas using the system of Figure 13.

Figure 17 represents a ichenase carrier molecule that may be used in fusions with proteins or polypeptides of interest according to certain embodiments of the invention. Panel A represents a schematic representation of Clostribiumthermocellum 1,3-1,4-glucanase (Iichenase). Panel B shows a schematic representation of the LICKM circularly interchanged vehicle. In each panel, the vertical hatch corresponds to the N-terminal region of the catalytic domain, and the dashed box corresponds to the C-terminal region of the catalytic domain.

Figure 18 shows a schematic diagram of the pBID4 rollout vector.

Figure 19 is a schematic representation of each step involved in the target gene optimization process for purification of a final product that is ready for storage in accordance with a specific embodiment of the invention.

Figure 20 is a designated equipment useful for taking plant seeding steps for tissue harvesting. As depicted, the module is estimated to occupy a base area of about 12,192 m to 21,336 m (40 ft to about 70 ft), and to be fenced to 9,754 m (32 ft). The system shown includes the following components connected by the vehicles: central shelf units where plants are held between stages, a seeding unit, a vacuum and rinse infiltration unit, and a detained harvesting unit.

Description of Certain Preferred Embodiments

Proteins and Polypeptides

The present invention is applicable to the production of any protein or polypeptide (or any functional DNA or RNA molecule) in plant systems. As indicated above, in certain embodiments, the invention is directed to the production of pharmaceutical proteins, but the invention is generally not limited by the particular use (s) of nucleic acid or protein. For example, enzymes for use in any of a wide variety of industrial processes or bio-remediation processes (eg pollutant degrading enzymes) may be produced. Accordingly, 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. In certain embodiments, protein is not a nutritionally important protein. Any particular protein may be deleted from the present invention without being named herein.

Typically, 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.

To give, but a few specific examples, the present invention may be used to produce pharmaceutical proteins of interest including, but not limited to, hormones (insulin, thyroid hormone, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine). , bovine somatotropin, Ieptins and the like), growth hormones (eg, human growth hormone), growth factors (eg, epidermal growth factor, nerve growth factor, insulin-like growth factor and the like), decreasing factor receptors, cytokines and immune system proteins (eg inter-leukins, colony stimulating factor (CSF) 1 granulocyte colony stimulating factor (G-CSF), granulocyte colony stimulating factor macrophage (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, adressins, selectins, hormone receptors, T-cell receptors, imu noglobulins, higher soluble histocompatibility complex antigens, immunologically active antigens such as bacterial, parasitic, or viral antigens or allergens), autoantigens, antibodies), enzymes (tissue plasminogen activator, streptokinase, cholesterol biosyn- steriodogenic enzymes, steriodogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phosphatipases, aromatases, cytochromes, adenylate or guanilast cyclases, neura midases, and the like), receptors steroid hormone, peptide receptors), binding proteins (sterpod binding proteins, growth hormone or growth factor binding proteins, similar), transcription and translation factors, oncoproteins or proto-oncoproteins (for example , cell cycle proteins), muscle protein (myosin or tropo myosin, and the like), myeloproteins, neu-r proteins actives, tumor growth suppression proteins (angiostatin or endostatin, both of which inhibit angiogenesis), antisepsis proteins (bactericidal permeation increasing protein), structural proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin) , blood proteins (thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, Protein C, von Wile-brand factor, antithrombin III, glucocerebrosidase, colony stimulating factor erythropoietin degranulocyte (GCSF) or modified Factor VIII, anticoagulants, such as huridin), and the like.

In some particular embodiments, the present invention is used to produce antigenic proteins or polypeptides. For example, the present invention may be used to produce proteins (or portions thereof) of infectious organisms that are recognized by the immune system of an infected individual. Such proteins or polypeptides may be particularly useful in the development of vaccines to protect against infection by the relevant organisms. To give, but a few specific examples, useful antigenic proteins of anthrax (Bacillus anthracis; for example, LF, PA), cholera (Vibrio cholerae), cytomegalovirus, enterotoxigenic strains of E. coli, foot and mouth disease virus, hepatitis B (eg hepatitis B surface antigen, HBsAg), hepatitis C (eg HCV nucleus protein), human immunodeficiency virus (eg Tat, Rev, Nef, gp160, gp120, etc.), human papillomavirus (eg E7, E6), influenza (eg HA, NA), malaria (Plasmodium falciparunr, eg Pfs25, Pfs28, Pfs48 / 45, Pfs230), measles virus, norwalk virus, plague (Yersinia pestis; eg F1, LcrV), Pseudomonas aeruginosa, hail virus, respiratory syncytial virus (eg, protein F, protein G), rhinovirus, route virus, Staphylococcus aureus, transmissible gastroenteritis virus, trypanosomes (Trypanosoma brucer, for example, alpha-tubulin, beta-tubulin), tuberculosis, SARS, etc. may be produced according to the system of the present invention.

In some particular embodiments, the present invention is used to produce multimeric protein complexes, including, for example, immunoglobulin proteins, such as antibodies (i.e. monoclonal antibodies). To give, but a few specific examples, anti-PA, anti-LF, anti-NA, anti-TNFα, anti-interleukin-12, etc. can be produced according to the system of the present invention. More generally, monoclonal antibodies to disease-associated antigens such as cancer (eg, 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 (eg, Asthma, Crohn's disease, Diabetes, host disease versus Graft (organ rejection), inflammatory bowel disease, Lupus, Multiple scle-rose, Psoriasis, Psoriatic Arthritis, Rheumatoid Arthritis, Thrombosis, Vasculitis1 etc), infectious diseases (eg Bacillus anthracis), cholera (Vibrio cholerae), cytomegalovirus, enterotoxigenic strains of E. coli, foot and mouth disease virus, hepatitis B virus, hepatitis C virus, daimmunodeficiency virus human papilloma virus, influenza, malaria (P / as-modium falciparum), measles virus, norwalk virus, plague (Yersinia pestis), Pseudomonas aeruginosa, rabies virus a, respiratory syncytial virus, rhinovirus, Rota virus, Staphylococcus aureus, transmissible gastroenteritis virus, trypanosomes (Trypanosoma brucei), tuberculosis, SARS), etc. may be prepared according to the present invention.

In some particular embodiments, the present invention is used to produce hGH, autoantigens for autoimmune diseases (e.g., IA-2ic for diabetes), influenza antigens, anthrax antigens, HPV antigens, influenza antibodies, etc. (see Examples).

In some embodiments of the present invention, full length heterologous proteins are produced. That is, a protein that occurs in nature, or which has been prepared or otherwise designated is produced entirely in young plants. In some such embodiments, the protein produced is free from any other sequences. In other embodiments, 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.). In some embodiments, the portion produced is a characteristic portion of the protein or polypeptide. In some embodiments, the produced portion comprises an epitope. In some embodiments, the portion produced comprises an immunogenic domain. In some embodiments, the portion produced comprises a protein domain.

In some embodiments of the present invention, proteins or polypeptides are produced as fusions with other protein or polypeptide sequences. As is known in the art, fusions can be used for any of a variety of reasons. For example, proteins or polypeptides to be produced in young plants (e.g. germinated shoots) according to the present invention may be fused to one or more portions to facilitate detection and / or isolation. For example, proteins or polypeptides of interest should be fused to a known antigenic epitope for which an antibody or other specific ligand is available, and may be used to facilitate isolation or purification. Alternatively or additionally, proteins or polypeptides of interest may be fused to another polypeptide of defined three-dimensional structure, for example, to provide a desired three-dimensional arrangement to the protein or polypeptide of interest. As yet another example, proteins or polypeptides of interest may be fused to one or more moieties that will direct their subcellular localization (or secretion) in plant cells. Particularly when the protein or polypeptide of interest is a protein antigen (e.g., for use in preparing a subunit vaccine), it will often be desirable to produce the protein as a fusion with a carrier molecule, for example to increase expression, stability, and / or immunogenicity, and / or to aid in isolation or purification. Certain exemplary fusion patterns known in the art include, for example, antibody epitopes (e.g., His tag, etc.), immunogenic carrier proteins, cholera toxin, hot unstable enterotoxin, etc. (see, for example, Vella et al., Biotechnology 20: 1, 1992; Kelly et al., Immunology 113: 163, 2004; Buttery et al., JR Coll Physicians Lond34: 163, 2000; Jacobson et al., Minerva Peditr 54: 295, 2002; Dertzbaugh etal., Infect Immunol 61:48, 1993; Mashar et al., Vaeeine 11: 235, 1993; Liljeq-vist et al., J. Immunol Methods 210: 125, 1997; Stahl et al., Proe. Nati Aead.Sci USA 86: 6283, 1989; Ulrich et al., Adv. Virus Res. 50: 141, 1998; Smith etal., Virology 348: 475, 2006; Turpen et al., Biotechnology 13:53, 1995).

A particular fusion pattern contemplated in accordance with the present invention is a thermostable protein, such as Clostridium thermocellum Iichenase B (see, for example, United States Provisional Patent Application 60 / 472,495, filed May 22, 2003, and Requested PCT US04 / 016452, filed May 24, 2004, and published as WO 2005/026375 March 24, 2005. The full-length Iichenase (about 35 kDa) consists of a signal peptide, a domain-catalytic, a Pro-Thr rich box, and a limiting domain (see Figure 17) In accordance with the present invention, both Pro-Thr rich box and the limiting domain can be removed in fusion proteins. native can be replaced with one that operates on plants.

The catalytic domain of this Iichenase has a loop structure dividing it into two regions, the N-terminal region (N: 32-84 amino acids), and the C-terminal region (C: 85-246 amino acids). These two regions can be divided into the loop structure and circularly interchanged to produce the most receptive insert molecule 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 placed on the 3 "end of the exchanged vehicle to facilitate endoplasmic noreticular retention, respectively. Figure 7 shows a schematic diagram Modified Iichenase (LicKM; Gene Bank Accession Number DQ776900), which has a molecular weight of about 27 kDa. Consequence of target protein or polypeptide may be expressed as N or C terminal fusions, and / or as internal fusions in the MCS, a feature that allows simultaneous expression of multiple targets.

LicKM allows fusions in molecules ranging in size from small peptides to full length proteins of up to about 100kDa or more. LicKM maintains its enzymatic activity at high temperatures (65 ° C), a property that also generally applies to fusions. This feature enables easy and cost-effective recovery of protein-blends because a 10-minute heat treatment at 65 ° C removes up to 50% of plant contamination proteins. LicKM fusion proteins can be screened during purification by monitoring Iiche-nase activity. The use of LicKM as a carrier molecule contributes to additional advantages, including the potential for increased expression, and incorporation of multiple polypeptides of interest (eg, multiple vaccine determinants).

Plants

The teachings of the present invention are applicable to a wide variety of different plants. In general, any plants that are amenable to expression of introduced constructs as described herein are useful in accordance with the present invention. In many embodiments, it will be desirable to use young plants to improve the speed of protein / polypeptide production. As indicated here, in many embodiments, sprouted shoots are used. As is well known in the art, many shoots are fast growing edible plants produced from storage seeds. However, those skilled in the art will appreciate that the term "sprouting sprouts" has been used in a more general context to refer to young plants whether or not of a variety typically classified as "sprouts". Any plant that is long enough to have sufficient green biomass to permit introduction and / or expression of an expression construct as provided herein (recognizing that the relevant time may vary depending on the mode of distribution and / or expression of the expression construct). ), can be considered as a "sprouted seedling" here.

In many embodiments of the invention, edible plants are used (i.e., plants that are edible by - non-toxic - to the individual to whom the protein or polypeptide is to be administered).

In certain preferred embodiments, the plants of the invention may be plants of the species Brassica or Arabidopsis. Some suitable plants that are perfect for processing, and are edible as sprouted sprouts include alfalfa, munguba, radish, wheat, mustard, spinach, carrot, beet, onion, garlic, celery, rhubarb, a leafy plant such as cabbage. or lettuce, watercress, herbs such as parsley, mint, cauliflower, broccoli, soybeans, lentils, edible flowers, such as sunflower, etc.

A wide variety of plant species has been tested for their suitability in the practice of the present invention. A variety of different beans and other species including, for example, adzuki beans, alfalfa, barley, broccoli, bill jump pea, buckwheat, cabbage, cauliflower, clover, green cabbage, fenugreek, flax, garbanzo bean, green pea, spinach, galician cabbage, "kamut", kohlrabi, big pea, munguba, green mustard, spotted bean, radish, red clover, soybean, spotted pea, sunflower, turnip, yellow pea , and others, were tested for their receptivity to the production of heterologous proteins from advanced viral vectors of an agrobacterial construct (introduced by agro-infiltration as described herein) (see, for example, Examples 5 and 8, figure 9, etc.). . The present invention provides the surprising result that certain varieties of beans are particularly amenable to such manipulation. For example, according to the present invention, bill jump pea, green pea, big pea, speckled pea, and / or yellow pea are particularly useful in accordance with this aspect of the invention. In certain embodiments, therefore, the present invention provides for the 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 polypeptides of interest. In some embodiments, oRNA has characteristics of (and / or includes sequences from) A1MV. In some embodiments, RNA has characteristics of (and / or includes) TMV sequences.

It will be appreciated that in one aspect the present invention provides young plants (e.g. sprouts) expressing a target protein or polypeptide of interest. In some embodiments, the young plants have been grown from transgenic seeds; The present invention also provides seeds which may be generated and / or used for the methods described herein. Transgenic seeds for any gene of interest may be germinated and optionally induced to produce a protein or polypeptide of interest. For example, seeds capable of expressing any gene of interest may be germinated and induced through: i) virus infection, ii) agro-infiltration, or iii) virus-containing bacteria. Seeds capable of expressing a heavy or light chain transgene of any monoclonal antibody can be germinated and induced to produce full length molecule by: i) virus infection, ii) agro infiltration, or iii) inoculation with bacteria that contains virus genomes. Seeds capable of expressing a transgene to one or more components of a complex molecule comprising multiple components such as slgA may be germinated and used to produce a fully functional molecule by: i) virus infection, ii) agro infiltration, or iii) inoculation with bacteria contains virus genome. Seeds from healthy non-transgenic plants can be germinated and used to produce target sequences through: i) virus infection, ii) agro-infiltration, or iii) inoculation with bacteria containing virus germ.

In some embodiments, young plants have been grown from non-transgenic seeds. Typically, such young plants will harbor viral sequences that direct expression of the protein or polypeptide of interest. In some embodiments, plants may also harbor agrobacterial sequences, optionally including sequences that "release" viral sequences.

Systems for Expressing Proteins or Polypeptides in Plants

According to the present invention, any of a variety of different systems may be used to express proteins or polypeptides in young plants (e.g., sprouted shoots). In some embodiments, transgenic cell lines or seeds are generated, which are then germinated and grown over a period of time such that a protein or polypeptide included in the transgenic sequences is produced in young plant tissues (for example, in germinated shoots). Typical technologies for producing transgenic plant cells and / or seeds include Agrobacteriumtumefaciens-mediated gene transfer and microprojectile bombardment, or electroporation.

In some embodiments, transient expression systems are used. Typical technologies for the production of protein and polypeptide transient expression in plant tissues utilize plant viruses. Viral transformation is a faster and less expensive method of transforming embryos and germinated buds that can be harvested without prior experimental or generational delay. On the other hand, viruses that are not attenuated can infect other plants, potentially causing environmental problems.

The present invention provides expression systems with advantages of viral expression systems (e.g. rapid expression, high production levels), and Agrobacterium transformation (e.g. controlled administration). In particular, as discussed in detail below, the present invention provides systems wherein an agrobacterial construct (i.e. a construct that replicates in Agrobacterium and thus can be distributed to plant cells by Agrobacteri-one distribution) includes a A plant promoter that, upon being introduced into a plant, directs expression of viral sequences (e.g., including viral replication sequences) by driving a gene to a protein or polypeptide of interest. This system allows high level transient expression of proteins and polypeptides in plants.

A variety of different embodiments of expression systems, some of which produce transgenic plants and others of which provide transient expression, are discussed in further detail individually below. For any of these techniques, one skilled in the art and the present descriptive report would appreciate how to adjust and optimize protein and polypeptide expression protocols in young plant tissues (e.g., germinated shoots). Aarobacterium Transformation

Agrobacterium is a representative gene of the Rhizobiaceae gram-negative family. This species is responsible for plant tumors such as hairy root and crown bile disease. In differentiated plant tissue, which is characteristic of tumors, amino acid derivatives known as opines are produced by Agrobacterium and catabolized by the plant. The bacterial genes responsible for opine expression are a convenient source of control elements for chimeric expression cassettes. In accordance with the present invention, the Agrobacterium transformation system may be used to generate young plants (e.g., sprouted sprouts, including edible sprouted sprouts), which are harvested before the plants mature. Agrobacterium-transforming methods can easily be applied to regenerate young plants (eg germinated sprouts) that express pharmaceutical proteins.

In general, transformation of plants with Agrobacterium involves the transformation of plant cells grown in tissue culture by co-cultivation with an Agrobaeterium tumefaeiens leading to a bacterial plant / vector. The vector contains a gene that encodes a pharmaceutical protein. Agrobaeterium transfers the vector to the host cell of the plant and is then eliminated using antibiotic treatment. Transformed plant cells expressing the pharmaceutical protein are selected, differentiated, and finally regenerated into whole seedlings (Hellen et al., Plant Molecular Biology (2000) 42 (819-832); Pilon-Smits et al., Plant Physiology (Jan 1999) 119 (1): 123-132 Barfield and Pua Plant Cell Re-ports (1991) 10 (6/7): 308-314); Riva et al., Journal of Bioteehnology (December 15, 1998) 1 (3), each incorporated herein by reference.

Agrobacterial expression vectors for use in the present invention include a gene (or expression cassette) that encodes a pharmaceutical protein designed for plant operation, with upstream and downstream mating sequences of the expression cassette. The sequence sequences are usually plasmid or viral in origin, and provide characteristics necessary for the vector to transfer DNA from the bacterium to the desired plant host.

The bacterial basic construct / plant vector preferably provides a broadband host prokaryote origin of replication, a selectable prokaryote marker. Suitable selectable prokaryotic markers include resistance to antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions that are well known may also be present in the vector.

Agrobacterium T-DNA sequences are required for Agrobacterium-mediated transfer of DNA to the plant chromosome. T-DNA tumor induction genes are typically removed during the construction of an agrobacterial expression construct, and are replaced with sequences encoding the pharmaceutical protein or polypeptide. T-DNA boundary sequences are retained because they initiate integration of the T-DNA region into the plant genome. If expression of the pharmaceutical proteins is not readily accessible for detection, the bacterial-al / plant vector construct will also include a selectable marker gene suitable for determining whether a plant cell has been transformed, for example, a gene from. resistance nptll karamicin.

In the same or different bacterial / plant vector (Ti pasmid) are Ti sequences. Ti sequences include virulence genes, which encode a set of proteins responsible for T-DNA excision, transfer, and integration into the plant genome (Schell, Science (1987) 237: 1176-1183). Other sequences suitable for allowing heterologous sequence integration 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 sequence-coding pharmaceutical protein, at least the following elements: a promoter region, 5 'untranslated plant sequences, initiation codon (depending on whether or not the gene express has its recognition), and transcription termination sequences and termination. In addition, transcriptional and translational terminators may be included in the expression cassettes or chimeric genes of the present invention. Signal secretion sequences that allow protein processing and translocation, as appropriate, may also be included in the expression cassette.

A variety of transcriptional and translational promoters, signal sequences, and terminators are described, for example, in Lawton et al., Plant MoL Biol (1987) 9: 315-324, or U.S. Patent No. 5,888,789, incorporated. here by reference. In addition, structural genes for antibiotic resistance are commonly used as a deletion factor (Fraley et al., Proc. Natl. Acad. Scl. USA (1983) 80: 4803-4807), incorporated herein by reference. Unique restriction enzyme sites at the 5 'and 3' ends of the cassette allow easy insertion into a pre-existing vector.

Other binary vector systems for Agrobacterium mediated transformation, conducting at least one T-DNA limit sequence, are described in PCT / EP99 / 07414, incorporated herein by reference. The discussion of Agrobacterium-mediated transformations is found in Gelvin, SB, "Agrobacterium-Meóa and Plant Transformation: the Biologybehind the" Gene-Jockeying "Tool", Microbiology and Molecular Biology Re-views, 67 (1): 16-37 (2003), incorporated herein by reference, and references therein, all of which are incorporated herein by reference; Lo-rence A, Verpoorte R., Gene transfer and expression in plants. Methods MolBiol. (2004) 267: 329-50, incorporated herein by reference.

In certain embodiments of the invention, bacteria and others than Agrobacterium are used to introduce a nucleic acid sequence into a plant. See, for example, Broothaerts W, et al., Degene transfer to plants by diverse bacterial species, 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 collected, 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 safety to be used when necessary. Total plants are then regenerated from cultured pro-toplasts, for example, as described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: MacMillan Publishing Co. (New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants1Acad. Press, Orlando, Vol. I, 1984, and Vol. III, 1986, incorporated herein by reference.

In certain embodiments, plants are not regenerated in adult plants. For example, in some embodiments, plants are regenerated only to the sprouted sprout stage. In other embodiments, total plants are regenerated to produce seed stocks and young plants (e.g., sprouted shoots) for use in accordance with the present invention are generated from seed stock alone.

All plants from which protoplasts can be isolated and cultured to give regenerated total plants can be transformed by Agrobacteria according to the present invention so that the plantastotals are recovered, which contain a transferred gene encoding a protein or polypeptide of interest. It is well known that virtually all plants can be regenerated from culture cells or tissues, including, but not limited to, all major plant species producing edible shoots. Some suitable plants include alfalfa, munguba, radish, wheat, mustard, spinach, carrot, beet, onion, garlic, celery, rhubarb, a leafy plant such as cabbage or lettuce, watercress, herbs such as parsley, mint, or clovers. , cauliflower, broccoli, soybean, lentils, edible flowers such as sunflower, etc.

Means for regenerating transformed cell plants may vary from one plant species to the next. However, those skilled in the art will appreciate that generally a suspension of transformed prototype plants containing copies of the heterologous gene is first provided. Callus tissue is formed, and buds can be induced by callus and, subsequently, rooted. Alternatively, embryo formation may be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. Sinking the seed into water, or spraying the seed with water to increase the seed moisture content to between 35-45%, initiates germination. For agermination to proceed, the seeds are typically kept in water saturated air under controlled temperature and air flow conditions. The culture medium will usually contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and prolination to the medium, especially for species such as alfalfa. Buds and roots usually develop simultaneously. Efficient regeneration will depend on the medium, genotype, and culture history. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

Mature plants, grown from transformed plant cells, and non-segregating homozygous transgenic plants, are identified. The congenital plant produces seeds containing the transferred gene encoding a protein or polypeptide of interest. These seeds can be germinated and grown at the young plant stage (e.g., germinated sprouts) to produce the protein or polypeptide of interest.

In related embodiments, transgenic seeds (leading to the transferred gene encoding a protein or polypeptide of interest, typically integrated into the genome) can be formed into seed products, and sold with instructions on how to grow young plants at one stage. appropriate (for example, to the germinated sprout stage) for harvesting and / or administration, or formulation, as described herein. In other related embodiments, hybrids or new varieties realizing the desired traits (i.e., the traditional gene that encodes a protein or polypeptide of interest), are developed from congenital transgenic plants.

Direct Integration

Direct integration of DNA fragments into the plant cell genome by microprojectile bombardment or electroporation can also be used to introduce protein or polypeptide expression constructs of interest into plant tissues according to the present invention (see, for example, Kikkert1 JR Humiston et al., In Vitro Cellular & Developmental Biology Plant: Journal of the Tissue Culture Association (Jan / Feb 1999) 35 (1): 43-50; Bates, GW Florida State University, Tallahas-see, FL Molecular Biotechnology ( Oct 1994) 2 (2): 153-145 More particularly, vectors containing a gene encoding a protein or polypeptide of interest may be introduced into plant cells by a variety of techniques. include selectable markers for use in plant cells.Vector may also include sequences that allow selection and propagation in a secondary host such as containing a source of replication and selectable marker. Typically, secondary hosts include bacteria and yeast. In a preferred embodiment, the secondary host is Escherichia coli, the origin of replication is a replication CoIEI-like origin, and the selectable marker is a gene encoding aampicillin resistance. Such sequences are well known in the art, and are commercially available (e.g., Clontech, Palo Alto, CA or Stratagene, LaJolla, CA).

The vectors of the present invention may also be modified into intermediate plant transformation plasmids containing a region homology to an Agrobacterium tumefaciens vector, an Agrobaeterium tumefaciens T-DNA boundary region, and chimeric genes or expression cassettes described above. Additional vectors may include unarmed plant tumor including Agrobacterium tumefaci-ens plasmid.

According to the present embodiment, direct transformation vectors of the invention involve microinjection of the vectors directly from the plant cells by the use of micropipettes to mechanically transfer recombinant DNA (see, for example, Crossway, Mol. Gen. Genet., 202: 179-185, 1985, incorporated herein by reference). Genetic material can also be transferred into the plant cell by the use of polyethylene glycols (see, for example, Krens et al., Nature (1982) 296: 72-74). Another method of introducing nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid, either within the matrix, or small particles, or on the surface (see, for example, Klein et al. Nature (1987) 327: 70-73; Knudsen and Muller Plant (1991) 185: 330-336)). Still another method of introduction is fusion of protoplasts with other entities, or fused lipid mini-cells, cells, lysosomes, or other surface bodies (see, for example, 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, for example, Fromm et al., Proc. Natl. Acad. Sci. USA (1985) 82: 5824). According to this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. High field resistance reversibility electrical impulses permeate biomembranes allowing the introduction of plasmids. Electroporated plant protoplasts reshape the cell, split the wall, and form callus from the plant, which can be regenerated to form the germinated shoots of the invention. Those skilled in the art would appreciate how to use these methods to transform plant cells which can be used to generate edible germinated shoots.

Viral Transformation

In accordance with the present invention, plant virus vectors used to infect and produce foreign protein in seeds, embryos, germinated shoots. In this regard, 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-action nucleic acid elements required for replication. Expression of high levels of foreign genes that encode short peptides as well as large complex proteins (eg, by tobamoviral vectors) is described, for example, by McCormick etal. (Proc. Nati Acad. Sci. USA (1999) 96: 703-708; Kumagai et al. (Gene (2000) 245: 169-174 and Verch et al.) (J. Immunol. Methods (1998) 220, 69-75, each incorporated herein by reference.) Thus, virus vector vectors have a demonstrated ability to express short peptides as well as large complex proteins.

In certain embodiments, young plants (e.g., shoots) expressing pharmaceutical proteins, such as insulin, GAD, and IA-2 associated with type 1 diabetes, are generated using host / virus system. Young plants produced by viral infection provide a source of protein or polypeptide of interest that has already been shown to be safe. For example, shoots are free from contamination with animal pathogens. Unlike, for example, tobacco, edible bud proteins could at least in theory be used in purely non-purifying applications, significantly reducing costs.

In addition, a virus / young plant system (eg bro-to) also offers a much simpler, less expensive route to scale and fabrication, since the relevant genes (encoding the protein or polypeptide of interest) are produced on the virus, which can be developed on a commercial scale within a few days. In contrast, transgenic plants may require up to 5-7 years before sufficient seed or plant material is available for large-scale or commercial trials.

In accordance with the present invention, plantat RNA viruses have certain advantages, while making 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 sequence. Many plant RNA viruses have small genomes, and in vitro fecal cDNA clones are available to facilitate genetic manipulation. Once infectious virus material enters the susceptible host cell, it replicates at high levels and spreads rapidly throughout the plant (one days after inoculation). Virus particles are easily and economically recovered from infected tissue. Viruses have a wide host range, enabling the use of a simple construct to infect various susceptible species. These features are easily transferable to the shoots.

Figure 1 illustrates various strategies for expressing foreign genes using plant viruses. Strange sequences can be expressed by replacing one of the desired sequence viral genes by inserting foreign sequences into the virus genome at a suitable position, or by fusing foreign peptides to the structural proteins of a virus. In addition, any of these approaches can be combined to express foreign sequences by trans-complementing vital functions of a virus. A number of different strategies exist as tools for expressing foreign sequences in virus-infected plants using tobacco mosaic virus (TMV), alfalfa mosaic virus (AIMV), and chimeras thereof.

The AIMV genome is a representative of the Bromovirus family of viruses, and consists of three genomic RNAs (RNAs1-3) and subgenomic RNA (RNA4) (Figure 2). Genomic RNAs 1 and 2 encode virus replication proteins P1 and P2, respectively. Genomic RNA3 encodes the P3 cell-to-cell movement protein and the sheath protein (CP). ACP is translated from subgenomic RNA4, which is synthesized from genomic RNA3, and is required to initiate infection. Studies have shown the involvement of CP in multiple functions, including genome activation, replication, RNA stability, symptom formation, and RNA encapsidation (see, for example, Bole et al., Virology (1971)). : 73-85; Van Der Vossen et al., Virology (1994) 202: 891-903; Yusibov et al., Virology 208: 405-407; Yu-sibov et al., Virology (1998) 242: 1-5; Bole et al., (Review, 100 refs.) J. Gen.Viroi (1990) 80: 1089-1102; De Graaff1 Virology (1995) 208: 583-589; Jasparset al., Adv. Virus Res (1974) 19, 37-149; Loesch-Fries, Virology (1985) 146: 177-187; Neeleman et al., Virology (1991) 181: 687-693; Neeleman et al., Virology (1993) 196: 883-887 Van Der Kuyl et al., Virology (1991) 183: 731-738; Van Der Kuyl et al., Virology (1991) 185: 496-499).

Encapsidation of viral particles is essential for long-distance movement of viruses from inoculated to uninoculated parts of the seed, embryo, or young plant (eg, germinated buds) and for systemic infection. In accordance with the present invention, inoculation may occur at any stage of plant development. In embryos and embryos, the spread of the inoculated virus should be very rapid. AIMV virions are encapsulated by a single CP (24 kD), forming more than one particle type. The size (30 to 60 mm in length and 18 nm in diameter) and shape (spherical, ellipsoidal, or bacilliform) of the particle depend on the size of the encapsidated RNA. After assembly, AIMVCP N-terminal is to be located on the surface of virus particles, and does not appear to interfere with virus assembly (Bole et al., Virology (1971) 6: 73-85). AIMV CP with an additional 38 N-terminal amino acid peptide forms particles in vitro and retains biological activity (Yusi-bov et al., J. Gen. ViroL (1995) 77: 567-573).

The AIMV has a wide host range, which includes a number of agriculturally valuable crop plants, including seedlings, embryos, and sprouts. Together, these characteristics make AIMV CPum an excellent candidate as a carrier molecule and AIMV an attractive candidate vector for the expression of foreign sequences for the expression of foreign sequences in the plant at the bud development stage. In addition, after expression of a vector heterologous, such as TMV, AIMVCP encapsulates the TMV genome without interfering with virus infectivity (Yusibov et al., Proc. NatL Acad. Sci. USA (1997) 94: 5784-5788, incorporated herein by reference). This allows the use of TMV as a virus to convey it to AIMV CP fused to foreign sequences. TMV, the tobamovirus prototype, has a genome consisting of a simple sense RNA encapsidated with a 17.0kD CP, which results in particles rod-shaped (300 nm long) (Figure 2). CP is the only structural protein of TMV1 and is required for encapsidation and long-range movement of the virus in an infected host (Saito et al., Virology (1990) 176: 329-336). The 183 and 128 kD of proteins are translated from genomic RNA, and are required for virus replication (Ishikawa et al., Nucleic Acids Res. (1986) 14: 8291-8308). The 30 kD protein is the virus cell-to-cell movement protein (Meshi et al., EMBO J. (1987) 6: 2557-2563). Movement and coat proteins are translated from subgenomic RNAs (Hunter et al., Nature (1976) 260: 759-760; Bruening et al., Virology (1976) 71: 498-517; Beachy etal., Virology (1976) 73: 498-507, each incorporated herein by reference).

The schematic representation of AIMV and TMV genomes is shown in Figure 2. AIMV RNAs 1 and 2 encode replicaseP1 and P2 proteins, respectively; genomic RNA3 encodes the P3 cell-to-cell moving protein, and the viral coat protein (CP). PC is translated from subgenomic RNA4 synthesized from genomic RNA3. TMV 126 kD and 183 kD proteins are required for replication; the 30 kD protein is the cell to viral cell movement protein; and the 17 kD protein is the virus CP. CP and 30 kD protein are translated from subgenomic RNAs. Arrows indicate the position of subgenomic promoters.

Flower Transformation

Other methods that can be used to introduce a gene encoding a protein or polypeptide of interest into plant cells include transformation of the plant flower. The transformation of Arabi-dopsis thaliana can be achieved by immersing the plant flowers in a solution of Agrobaeterium tumefaeiens (Curtis and Nam, TransgenieReseraeh (August 2001) 10 4: 363-371; Qing et al., Molecular Breeding · .New Strategies in Plant Improvement (Feb 2000). (1): 67-72). Transformed plants are formed in the seed population generated by "dipped plants". At a specific point during flower development, a poro exists in the ovary wall through which Agrobacterium tumefaciens gains access to the interior of the ovary. Once inside the ovary, Agrobacterium tumefaciens proliferates and transforms individual eggs (Desfeux et al., Plant Physiology (July 2000) 123 (3): 895-904). Transformed eggs follow the typical trajectory of seed formation within the ovary. Transient Expression Mediated by Aarobacterium

As indicated herein, in many embodiments of the present invention, systems for rapid (e.g., transient) expression of proteins or polypeptides in plants are desirable. Among other things, the present invention provides a powerful system for achieving such rapid expression in plants (particularly young plants, for example germinated sprouts) that utilizes an agrobacterial construct to deliver a viral expression system encoding the protein or polypeptide of inte Come back.

Specifically, according to the present invention, a "release vector" is prepared to contain agrobacterial sequences including replication sequences and also to contain plant viral sequences (including self-replicating sequences) that carry a gene encoding protein or polypeptide. interest. The release vector is introduced into plant tissue, preferably by agro-infiltration, which allows for substantially systemic distribution. For transient transformation, unintegrated T-DNA copies of the launch vector remain transiently present in the nucleus, and are transcribed leading to expression of the driving genes (Kapila et al., 1997). Agro-bacterium-mediated transient expression, unlike viral vectors, cannot lead to systemic diffusion of gene expression of interest. An advantage of this system is the ability to clone genes larger than 2kb to generate constructs that would be impossible to obtain with viral vectors (Voinnet et al., 2003). In addition, using such a technique, it is possible to transform the plant with more than one gene. transgene, such that multimeric proteins (e.g., antibody subunits of complexed proteins) may be expressed and counted. Furthermore, the possibility of co-expression of multiple transgenes by co-infiltrating with different Agrobacterium may be advantageous of either by separate infiltration or by using mixed cultures.

In certain embodiments, the release vector includes sequences that allow selection (or at least detection) in Agrobacterium, and also selection / detection in infiltrated tissues. In addition, the launch vector typically includes sequences that are transcribed into the plant to produce viral RNA production, followed by generation of viral proteins.

In addition, production of viral proteins and viral RNA produces rapid production of multiple copies of RNA encoding the pharmaceutically active protein of interest. Such production results in the production of fast target protein of interest in a relatively short period of time. In this way a highly efficient protein production system can be generated.

The agro-infiltration technique using viral expression vectors can be used to produce limited amount of protein of interest so as to check expression levels before deciding whether it is worthwhile to generate transgenic plants. Alternatively or additionally, the infiltration technique using viral expression vectors is useful for rapid generation of plants capable of producing vast amounts of protein as a primary production platform. This transient expression system can therefore be used on an industrial scale.

Additionally provided are any of a variety of different Agrobacterial plasmids, binary plasmids, or derivatives thereof, such as pBIV, pBI1221, pGreen, etc., which may be used in these other aspects of the invention. Numerous suitable vectors are known in the art, and may be directed and / or modified according to methods known in the art, or those described herein for use in the methods described herein.

Figure 18 shows a schematic diagram of a particular exemplary deletion vector, pBID4. This vector contains the cauliflower mosaic virus 35S promoter (a DNA plant virus) that triggers initial transcription of the recombinant viral genome following introduction into plants, and the nos terminator, the Agrobaeteri-a napolin synthase transcriptional terminator. The vector additionally contains tobacco mosaic virus genome sequences including genes for virus replication (126 / 183K) and cell to cell movement (MP). The vector further contains a gene encoding a polypeptide of interest, inserted into a single cloning site within the tobacco demosaic virus genome sequences, and under the transcriptional control of the subgenomic coat protein 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 resulting vector is viral self-replicating exposed RNA that is less subjected. recombination than vectors containing CP, and which cannot be effectively spread and survive in the environment. Left and right boundary sequences (LB and RB) delimit the region of the release vector that is transferred into plant cells following infiltration of plants with recombinant Agrobacterium that drives the vector. Following introduction of agrobacteria that carries this vector into plant tissue (typically by agro-infiltration, but alternatively by injection, or other means), multiple single stranded (ssDNA) DNA copies of sequence between LB and RB are generated and released about. minutes These introduced sequences are then amplified by viral replication. Target gene translation results in the accumulation of large quantities of target protein or polypeptide over a short period of time.

In some embodiments of the invention, Agrobacterium transientemediated 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 target protein, or about 50-200, or about 50, 60.70, 80, 90, 100, 110, 120, 130 , 140, 150, 160, 170, 180, 190, or about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1500, 1750 , 2000, 2500, 3000 mg, or more protein per kg plant detained, is produced.

In some embodiments of the invention, these expression levels are achieved within about 6, 5, 4, 3 or 2 weeks of infiltration. In some embodiments, these expression levels are achieved within about 10, 7, 5, 4, 3, 2 days, or even 1 day of expression construct introduction. Thus, the introduction time (e.g., infiltration) for harvesting is typically less than about 2 weeks, 10 days, 1 week, or less. In addition, a very attractive feature of the invention is that it allows protein production within about 8 weeks or less from amino acid sequence selection (even including time for "preliminary" expression studies). Also, each batch of Protein can typically be produced within about 8 weeks, 6 weeks, 5 weeks, or less. Those skilled in the art will appreciate that these numbers may vary somewhat depending on the type of plant used. Many shoots, including peas, will fall into the numbers given. Benthamian Nicotiana, however, may grow longer, particularly before infiltration, as they develop more slowly (from a very small seed). Other expected adjustments will become clear to those skilled in the art based on the biology of the particular plants used.

The inventors have also found that various Nicotian plants are particularly useful in practicing this aspect of the invention, including in particular Nicotian benthamian. It will be understood by those skilled in the art that Nicotian plants are not generally considered to be "sprouts". Nevertheless, the present invention teaches that Nieotiana seedlings (particularly Benthamian Nieotiana seedlings) are useful in the practice of the invention. In general, in the practice of this embodiment of the invention, Nieotiana benthamiana plants are grown long enough to allow the development of an appropriate amount of biomass prior to infiltration (i.e. for distribution of agrobacteria containing the releasing vector). Typically, 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.

The present inventors have surprisingly additionally found that while both TMV and AIMV sequences may prove effective in such release vector constructs, in some embodiments, AIMV sequences are particularly efficient in ensuring high level protein production or distributed polypeptides. .

Thus, in certain particular embodiments of the present invention, proteins or polypeptides of interest are produced in young pea plants, or young Nicotian (e.g., Nicotianabenthamian) plants of a release vector that directs production of AIMV sequences leading to the gene of interest.

Expression Constructs

Many features of useful expression constructs according to the present invention will be specific to the particular expression system used as discussed above. However, certain aspects that may be applicable through different expression systems are discussed in further detail herein.

To give, but an example, in many embodiments of the present invention, it will be desirable for the expression of the protein or polypeptide (or nucleic acid) of interest to be inducible. In many such embodiments, the production of an RNA encoding the protein or polypeptide of interest (and / or production of an antisense RNA) is under the control of an inducible (e.g., exogenously inducible) promoter. Exogenously inducible promoters are likely to increase or decrease the expression of a transcription in response to an external stimulus, rather than an internal stimulus. A number of environmental factors can act as such ex-stimulus. In certain embodiments of the invention, transcription is controlled by an inducible heat promoter, such as a heat shock promoter.

Externally inducible promoters may be particularly useful in the context of controlled adjustable growth adjustments. For example, using a heat shock promoter, the temperature of a contained environment may simply be elevated to induce relevant transcriptional expression. It will be appreciated, of course, that an inducible heat promoter can never be used outdoors because the temperature outside cannot be controlled. The promoter would be turned on anytime the outside temperature rose above a certain level. Similarly, the engine would be turned off every time the outside temperature dropped. Such temperature changes can occur in a single day, for example by turning the expression on during the day and turning off at night. A heat-inducible promoter such as that described herein would likewise not even be practical for use in a greenhouse, which is susceptible to climate change to almost the same degree as outdoors. Growing generally designed plants in a greenhouse is very costly. In contrast, in the present system, many variables can be controlled so that the maximum amount of expression can be achieved with every harvest.

Other externally inducible promoters that may be used in accordance with the present invention include deluz unspoken promoters. Inducible light promoters may be maintained as conductive promoters if light in the contained dimmable environment is always on. Alternatively, the expression of the relevant transcription may be linked at a particular time during development by simply turning on the light.

In still other embodiments, a chemically inducible promoter is used to induce expression of the relevant transcription. According to these embodiments, the chemist may simply be mixed or sprayed on a seed, embryo, or seedling (e.g., sprout) to induce expression of the relevant transcription. Spraying and fogging can be precisely controlled and directed on a particular particular, embryo, or seedling (e.g., sprout) as desired. A contained environment is devoid of wind or air currents, which can disperse the chemical out of the intended container, so that the chemical remains in the intended container.

Growth Environments One aspect of the present invention is the production of proteins or polypeptides (and / or nucleic acids) in plants under controlled controlled growth conditions. According to the present invention, an attractive feature associated with protein or polypeptide production in young plants (e.g., germinated shoots) is that reduced growth times are required than would be used if the same type of plant were grown at adulthood. In addition, in many instances smaller spaces may be used than would be required if plants were developed to their full adult size. In general, growing plants expressing pharmaceutical proteins or polypeptides in a controlled regulated environment provides a faster pharmaceutical product (because plants are harvested younger), and with less effort, risk, and regulatory considerations than development. genetically engineered plants. A contained adjustable environment used in the present invention reduces or eliminates the risk of nature cross-pollinating plants.

In many embodiments of the invention, proteins or polypeptides are expressed in plants grown in an enclosed system. This system avoids the risk of environmental contamination, and also provides adjustable reproducible environment capable of achieving comparable repeat results. In addition, such a system allows controlled induction of protein or polypeptide expression where desired, for example, through the use of an inducible promoter, or other adjustable feature as described herein.

In certain embodiments of the present invention, plants are grown in a contained adjustable environment. In some embodiments, the adjustable environment contained is a housing or environment unit which seeds can be grown internally. All environmental factors of the contained adjustable environment can be controlled. Since buds do not require light to grow, and lighting can be costly, in a particular embodiment, plants are grown (for example, to germinated bud stage) internally in the absence of light.

Other environmental factors that may be regulated in a contained environment of the present invention include temperature, humidity, water, nutrients, gas (e.g., O2 or CO2 content, or circulation), chemicals (small molecules, such as such as sugars and sugar derivatives, or hormones, such as gibberellic phytohormones or absiscic acid, etc.), and the like.

In many embodiments of the invention, plants are grown hydroponically. Hydroponic growth systems offer a number of advantages. For example, hydroponic growing systems require less space than soil-based systems to develop the same number of plants. Nutrients or other agents may typically be drained through a hydroponic system, making controlled growth easier to follow than in soil-based systems. Many aspects of hydroponic growth can be automatic. In addition, harvesting hydroponically growing plants is trivial. Plants can be harvested simply by raising their growth solution. No cleaning is required at harvest time. Being able to harvest young plants (eg germinated shoots) directly from a hydroponic environment without washing or scrubbing, minimizing breakage of harvested material. Plant breakage and wasting induce apoptosis. During apoptosis, certain proteolytic enzymes become active, which can degrade a protein or pharmaceutical polypeptide expressed in the germinated sprout, resulting in decreased protein activity of the protein. Apoptosis-induced proteolysis could significantly decreased protein production of mature plants. Using the methods of the present invention, apoptosis is preferably never induced (i.e. apoptosis is avoided), for example, because no harvesting occurs until proteins are extracted from the plant.

In certain embodiments of the present invention, plants (e.g., sprouted shoots) are grown in trays that can be irrigated, sprayed, or misted at any time during development. For example, the tray may be seated with one or more irrigation, spraying, fogging and draining apparatus which may dispense and / or remove water, nutrients, chemicals, etc., at a specified time, and in precise amounts during development. of sprouted sprout. For example, seeds require sufficient moisture to keep them moist.

Excess moisture drains through holes in the trays in the ambient soil drains. Preferably, the drainage water is treated as appropriate for removal of harmful chemicals prior to discharge back into the environment.

Another advantage of trays is that they can be contained within a very small space. Since no light is required for young plants (eg germinated sprouts) to grow, trays containing seeds, embryos or young plants (eg germinated sprouts) can be tightly stacked vertically over one another, providing a large amount of biomass per plant. ground space unit in a housing facility built specifically for this purpose. In addition, the stacks of trays may be arranged in horizontal series within a housing unit. Once the shoots have grown to an appropriate harvesting stage (for example, about two to fourteen days or more, depending on the type of plant, protein or polypeptide to be produced, etc.), individual trays are moved at a facility. either manually or by automatic means such as a conveyor belt.

A variety of aspects of growing conditions and techniques applicable to the practice of the present invention will be readily apparent to those skilled in the art. For example, when an inducible expression system is used, the time at which expression is induced may desirably be selected to maximize expression of the protein or polypeptide of interest in the young plant by harvest time. Induction of expression in an embryo at a particular stage of growth (for example, a particular number of days after germination) may result in maximum production of the protein or polypeptide of interest at harvest time. For example, induction of expression from the promoter 4 days after germination may result in more protein synthesis than induction of expression from the promoter after 3 days or after 5 days. Those skilled in the art will appreciate that maximizing expression can be achieved by routine experimentation. In preferred embodiments, young plants, particularly sprouted shoots, are harvested at about 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days after germination. In other embodiments, particularly Nicotian plants, they are grown for several weeks after germination.

When a constitutive expression system rather than an inducible expression system is used, young plants may be harvested for some time after introduction of coding sequences. For example, if a germinated sprout were virally transformed into an earlier stage of development (eg, the embryo stage), germinated sprouts can be harvested at a time when pressure is at its maximum post-transformation, for example. , at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days post-transformation. It may also be that young plants (eg sprouts) develop one, two, three or more months after transformation, depending on the germinating germination.

Generally, once pharmaceutical protein expression begins, seeds, embryos or young plants (e.g. germinated shoots) are allowed to grow until sufficient levels of pharmaceutical protein or polypeptide of interest are expressed. In certain embodiments, sufficient levels are levels that would provide a therapeutic benefit to a patient if the harvested biomass was raw material. Alternatively, sufficient levels are levels at 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 an individual upon administration.

In certain embodiments, since expression of pharmaceutical protein is induced, growth is allowed to continue until the germinated shoot stage, at which time the germinated shoots are harvested. In a particularly preferred embodiment, the germinated shoots are harvested alive. Sprouted sprouting sprouts have several advantages, including minimal effort and breakage.

Among other things, the present invention provides a unique system which in turn provides young plant biomass containing high levels of expressed protein or polypeptide (and / or nucleic acid) of interest. If directly consumed or processed in the form of a pharmaceutical composition, when young plants (eg germinated sprouts) are grown in a contained controlled environment, plant biomass and / or pharmaceutical composition derived from biomass may be provided. to a low cost consumer. In addition, the fact that the conditions for young plant growth can be controlled makes the product quality and purity consistent. The contained adjustable environment of the invention also makes obvious many safe EPA regulations that may prevent scientists from developing genetically engineered outdoor agricultural products.

Protein / Polypeptide Preparations

The present invention provides various preparations and formulations of proteins or polypeptides (and / or active nucleic acids) produced in young plants.

In some embodiments of the invention, proteins or polypeptides produced are not isolated from plant tissue, but are preferably provided in the context of live young plants (e.g. germinated shoots). In some embodiments, where the plant is edible, the plant tissue containing expressed protein or polypeptide is provided directly for consumption. Accordingly, the present invention provides young edible seedling biomass (e.g., edible germinated shoots) containing expressed protein or polypeptide.

Where edible young plants (eg germinated sprouts) express sufficient levels of pharmaceutical proteins or polypeptides, and are eaten alive, in some embodiments absolutely no harvest occurs before sprouted sprouts are consumed. This ensures that there is no harvest-induced proteolytic breakdown of the pharmaceutical protein prior to administration of the pharmaceutical protein to a patient in need of treatment. For example, young plants (eg sprouted shoots) that are ready to be consumed can be distributed directly to a patient. Alternatively, genetically engineered seeds or embryos are distributed to a patient in need of treatment and developed to the bud-germinated stage. In one embodiment, a supply of genetically engineered germinated shoots is provided to a patient, or a physician who will be treating the patients, so that a continuous stock of germinated shoots expressing certain desirable pharmaceutical proteins may be grown. This can be particularly valuable for populations in developing countries where costly pharmacists are offered or distributed. The ease with which germinated sprouts of the invention can be grown makes the germinated sprouts of the present invention particularly desirable for such a developing population.

In some embodiments, plant biomass is processed prior to consumption or formulation, for example by homogenization, trituration, drying, or extraction. In some embodiments, the expressed protein or polypeptide is isolated or purified from biomass, and formed into a pharmaceutical composition.

For example, live young plants (e.g. sprouts) may be ground, ground or mixed to produce a slurry of biomass in buffer-containing protease inhibitors. Preferably the tampon is at about 4 ° C. In certain embodiments, the biomass is air dried, spray dried, frozen, or frozen frozen. As with immune plants, some of these methods, such as air drying, may result in a loss of activity of the protein or pharmaceutical polypeptide. However, due to the fact that the plants used (eg sprouted shoots) are very small and typically have a large surface area due to volume, this is much less likely to occur. Those skilled in the art will appreciate that many biomass harvesting techniques that minimize protein proteolysis or pharmaceutical polypeptide are available and may be applied to the present invention.

Pharmaceutical Administration and Compositions

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. In accordance with the present invention, hosts include veterinary individuals such as cattle, sheep, canines, felines, etc. In preferred embodiments, an edible sprout is orally administered to an individual in a therapeutically effective amount. In other preferred embodiments, the pharmaceutically active protein is provided in a pharmaceutical preparation as described herein.

The pharmaceutical preparations of the present invention may be administered in a wide variety of ways to the host, such as, for example, oral, enteral, nasal, parenteral, intramuscular or intravenously, rectally, vaginal, topical, ocular, pulmonary, or by application. contact. In a preferred embodiment, a pharmaceutical protein expressed in a young plant (e.g., a sprout) is administered to a host orally. In another preferred embodiment, a pharmaceutically active protein expressed in a young plant (e.g., a bud) is extracted and / or purified, and used for the preparation of a pharmaceutical composition. Proteins are isolated and purified according to conventional conditions and techniques known in the art. These include methods such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, and the like.

The compositions of the present invention typically include an effective amount of a pharmaceutically active protein or polypeptide together with one or more pharmaceutically suitable, organic or inorganic, liquid or solid carrier materials.

A pharmaceutically active protein produced in accordance with the present invention may be employed in dosage forms such as tablets, capsules, lozenges, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, powder packets, liquid solutions, solvents, diluents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid bonds provided that the biological activity of the protein is not destroyed by such a dosage form.

Examples of materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as comolactose, glucose and sucrose, starches such as cornstarch and debatata starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt, gelatin; baby powder; exipients such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; Sunflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; solution isotonic saline solution; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, eagents fragrances, preservatives, and antioxidants may also be present in the composition, according to the judgment of the formulator (also Remington's Pharmaceutical Sciences, Fifteenth Edition, EW martin (Mack Publishing Co., Easton PA, 1975). It is provided as a pharmaceutical composition by conventional granulation production processes by mixing, dissolving, lyophilizing, or the like.

In certain embodiments it may be desirable to prolong the effect of a pharmaceutical preparation by lowering the absorption of the protein or pharmaceutical polypeptide that is subcutaneously or intramuscularly injected. This may be accompanied by the use of a crystalline or amorphous liquid suspension with poor water solubility. The rate of absorption of the protein or polypeptide depends on its dissolution rate which, in turn, may depend on size and shape.

Alternatively, delayed absorption of a parenterally administered protein is accompanied by the dissolution or suspension of the protein in an oil vehicle. Injectable depot forms are produced by forming microencapsulated protein matrices in biodegradable polymers such as polylactide-polyglycolide. Depending on the protein-polymer ratio and the nature of the particular polymer employed, the release rate may be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by retaining 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 and polypeptides of the invention may also be administered by means of pumps or sustained release forms, especially when administered as a preventative measure, in order to prevent the development of a disease in an individual, or to improve or prevent it. delay an 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. sprouts) can be administered live, or can be processed in a variety of ways, for example, air drying, freeze drying, extraction, etc., depending on the properties of the protein or polypeptide product. desired shape of the final product.

In certain embodiments, such compositions as described above are orally ingested only, or ingested together with food or feed, or with a drink. Compositions for oral administration include sprouted sprouts; extraction of young plants (eg, sprouted shoots), and purified young plant proteins or polypeptides provided as dry powders, food products, non-aqueous aqueous solvents, suspensions or emulsions.

Examples of 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 medial parenteral vehicles. ponates including sodium chloride solution, Rin-ger dextrose solution, dextrose plus sodium chloride solution, lactose-containing Ringer's solution, or fixed oils.

Examples of dry powders include any young plant biomass (e.g., sprouted sprouts) that has been dried, e.g. freeze dried, air dried, or spray dried. For example, young plants may be 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 plants are then stored for further processing as mass solids, or further processed by grinding to a desired sized mesh powder. Alternatively, freeze drying may be used for products that are sensitive to air drying. Products may be freeze-dried by placing them in a vacuum dryer, and dried frozen under a vacuum until the biomass contains less than 5% moisture by weight. The dried material may be further processed as described herein.

Herbaceous preparations are well known in the art. Herbaceous preparations which may be used for administering young plants of the present invention include liquid and solid herbaceous preparations. Some examples of herbaceous preparations include tinctures, extracts (e.g., aqueous extracts, alcohol extracts), decoction, dry preparations (e.g., air dried, spray dried, or freeze dried), powders (e.g. freeze dried powder) , and liquid. Herbaceous preparations may 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 modes of delivery of herbaceous preparations which may be applied to the present invention.

One skilled in the art will appreciate that a particularly preferred method of obtaining the desired pharmaceutically active protein or polypeptide is by extraction. Fresh young plants (eg sprouts) can be extracted to remove desired protein products from residual biomass, thereby increasing the concentration and product purity. Young plants can also be extracted in a buffered solution. For example, 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, for example, phosphate buffer. Protease inhibitors may also be added as required. Plant tissue can be disrupted by mixing or milling vigo-roses while suspended in the buffer solution, and external biomass removed by filtration or centrifugation. The protein product conducted in solution may be further purified by further steps, or converted to a dry powder by freeze drying or precipitation.

Extraction may also be performed by pressing. Living plants can be extracted by pressing on a press, or being tripled as they are passed through closely spaced rollers. Fluids expressed from crushed plants are collected and processed according to methods well known in the art. Press extraction allows the release of products in a more concentrated form. However, the total yield of the product may be lower than if the product was extracted in solution.

Young plants (eg sprouted shoots), extractions, powders, dried preparations and purified protein products, etc. may also be in encapsulated form with or without one or more excipients as noted above. Solid dosage forms of tablets, pills, capsules, pills and granules may be prepared with coatings and shells, such as enteric coatings, controlled release coatings, and other coatings well known in the art of pharmaceutical formulation. In such solid dosage forms the active pharmaceutical protein may be admixed with at least one inert diluent such as saccharose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert ingredients, for example tablet lubricants and other tablet adjuvants such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, dosage forms may also comprise buffering agents. They may optionally contain opacity agents, and may also be of a composition that they release the active ingredient (s) only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of encrusting compositions that may be used include polymeric substances and waxes.

In other embodiments, a young plant expressing a pharmaceutically active protein of the present invention or young plant biomass is administered orally as a medicinal food. Such edible compositions are consumed as raw foodstuffs, in solid form, or per drink if in liquid form. In a preferred embodiment, the plant material is directly ingested without prior processing step, or after minimal culinary preparation. For example, the pharmaceutically active protein or polypeptide is expressed in a bud from which it can be eaten directly. For example, the protein is expressed in an alfalfa sprout, munguba sprout, or spinach sprout, or lettuce leaf sprout, a young pea plant, etc. In an alternate embodiment, the germinated sprout 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 and feed industry. The end products of such methods further 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, flavor enhancers, antibiotics, and the like, in solid, semi-solid, suspension, emulsion, or liquid form. In another preferred embodiment, such methods include a preservation step, such as, for example, pasteurizing, baking, or adding preserving and preserving 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 can be tested by standard methods in the art, for example, gel electrophoresis, Elisa, or Western blot analysis using a protein-specific antibody. This determination can be used to standardize the amount of protein ingested. For example, the amount of therapeutically active protein in a determined shoot juice is regulated, for example, by mixing batches of product having efficient protein levels so that the amount of juice to be drunk to ingest a single dose may be be standardized. Use of an adjustable environment contained in accordance with the present invention, however, should minimize the need to perform such standardization procedures.

A pharmaceutically active protein or polypeptide produced in a young plant (for example in a seedling) and eaten by a host is absorbed by the digestive system. An advantage of ingesting a sprouted sprout or preparation of sprouted sprout, particularly intact sprouts or sprout biomass that has only been minimally processed, is to provide protein encapsulation or sequestration in plant cells. Thus, the protein may receive at least some digestive protection in the upper digestive tract before reaching the intestine, and a higher proportion of active would be available for perception.

The pharmaceutical compositions of the present invention may be administered therapeutically or prophylactically. In certain preferred embodiments, the compositions may be used to treat or prevent (e.g. to delay the onset of) a disease. For example, any individual suffering from a disease or at risk of developing a disease can be treated. It will be appreciated that an individual may be considered at risk of 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 an increased risk of developing a particular disease, that individual will be considered at risk of developing the disease. Similarly, if members of a family of individuals have been diagnosed with a particular disease, for example cancer, the individual may be considered to be at risk of developing this disease.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the proteins or polypeptide of interest, the liquid dosage forms may be inert conditilents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, acetate. ethylene, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular cottonseed oil, deamendoim oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfume agents.

Compositions for rectal or vaginal administration are preferably suppositories which may 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 to at room temperature, but liquids at body temperature and therefore melt in the rectum or vaginal cavity and release the proteinative.

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 plasters. The active protein, or preparation thereof, is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers as may be required. Phthalmic formulation, ear drops and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the traditional advantage of providing controlled distribution of a pharmaceutically active protein to the body. Such dosage forms may be produced by suspending or dispensing the pharmaceutically active protein in the meiocorrect. Absorption enhancers may also be used to increase the flow of pharmaceutically active protein through the skin. The rate may be controlled by either providing a detachable control membrane or by dispersing the pharmaceutically active protein into a polymer matrix or gel.

The compositions are administered in such amounts and such time as is necessary to achieve the desired result. As described above, in certain embodiments of the present invention, a "therapeutically effective amount" of a pharmaceutical composition is that amount effective for treating, attenuating, or preventing a disease in a host. Thus, the "effective amount to treat, attenuate or prevent disease" as used herein refers to a non-toxic but sufficient amount of the pharmaceutical composition to treat, alleviate or prevent disease in any host. As an example, the "therapeutically effective amount" may be an amount to treat, alleviate or prevent diabetes.

The exact amount required will vary from individual to individual, depending on the individual's species, age and general condition, disease stage, particular pharmaceutical mixture, mode of administration, and the like. Young plants (e.g. germinated shoots) of the invention and / or protein preparations thereof are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The term "dosage unit form" as used herein refers to a physically distinct unit of pharmaceutically active protein appropriate for the patient to be treated. It will be understood, however, that the total daily use of the compositions of the present invention is preferably decided by a attending physician within the scope of medical judgment. The therapeutically effective dose level specific to any particular patient or organism will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the specific compound employed; the specific composition employed; age, body weight, general health, patient sex, patient diet, patient pharmacokinetic conditions, time of administration, route of administration, and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination or coincidental with the specific compound employed; and similar factors well known in the medical arts.

It will also be appreciated that the pharmaceutical compositions of the present invention may be employed in combination therapies, that is, the pharmaceutical compositions may be administered concurrently with, prior to or subsequent to one or more other desired therapies, or medical procedures. The particular combination of therapies (therapies or procedures) to employ in a combination regimen will take into account compatibility of the desired therapies and / or procedures, and the desired therapeutic effect to be achieved. It will also be appreciated that asterapies employed may achieve a desired effect for the same disease (for example, a compound of the invention may be administered concurrently with another anticancer agent), or they may achieve different effects.

In yet another aspect, the present invention also provides a wrapper or kit including live young plants (e.g. germinated shoots) of the present invention, or pharmaceutical preparations, extracts, or 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. In certain embodiments, the pharmaceutical wrap or kit includes an additional approved therapeutic agent for use as a combination therapy. Optionally associated with such container (s) may be a notice in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceutical products, with notices reflecting approval by the manufacturing, use, or sale agency for human administration.

The present invention involves the purification and scale provided of the production of pharmaceutical protein from young plants (e.g. germinated shoots) using any of a variety of plant expression systems, more preferably plantaviral expression systems (i.e. , systems including a viral component). Kits are provided that include each protein in a diagnostic kit for genetic and immunological diseases. In a preferred embodiment, the present invention provides kits having biological protein assay samples and reagents for testing the presence of antibodies in a patient serum for those biological proteins. For example, the kit may provide IA-2 and GAD and reagents for testing 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 may be extended to provide diagnostic reagents in the form of a kit. As a non-limiting example, the insulin proteins GAD and IA-2, associated with the autoimmune reaction in diabetes, may be provided as oral formulations and administered to induce oral tolerance to these proteins. Alternatively, one or more therapeutic proteins may be provided in an injectable formulation, Ouvacin for administration. According to preferred embodiments, pharmaceutical doses or instructions thereof are provided in the kit for administration to an individual diagnosed with a disease, for example, a diabetic individual, or an individual at risk of developing a disease, for example, type 1 diabetes.

Equivalents

The following representative examples are intended to help illustrate the invention, and are not intended to, nor should be construed to limit the scope of the invention. Indeed, various modifications of the invention and many additional embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the following examples. and references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art. The following examples contain important additional information, exemplification, and guidance that may be adapted to the practice of this invention in its various embodiments, and the equivalents thereof.

Examples

Example 1: Production of Pharmaceutical Proteins or Polypeptides in Transgenic Broilers Prepared by Agrobacterium Transformation

Agrobacterium-transformed plant seeds are harvested, dried, cleaned and tested for viability and the presence of desired genetic material. Seed stock is stored under proper conditions until use. At time of use, appropriate amounts of seeds are immersed in water containing an amount of surface sterilizing agent (e.g., Chlorox) for 20 minutes to 4 hours. The seeds are scattered on a flat surface of trays, which contain growth support and water drainage provisions. Seed trays are placed on shelves in the adjustable environment contained controlled temperature, lighting access, air circulation, water supply, and drainage. The trays are misted with water from foggers equipped with automatic regulators for one to 30 minutes at intervals of 30 minutes to four hours, enough to keep the seeds moist. Excess moisture drains through the holes in the trays and drains at the base of the room.

The seeds are allowed to germinate and develop under controlled conditions. The seeds are incubated for about two fourteen days before harvesting and processing. At some point during the incubation process, from four hours to seven days of harvesting, seeds are exposed to environmental conditions that induce an inherent or introduced DNA promoter sequence that causes an increase in the synthesis of one or more desired proteins. in the tissues of the germinating bud. A transient increase in incubation temperature from about 30 ° C to about 37 ° C to cause induction of a heat shock promoter.

After incubation of the shoots for two to fourteen days, the shoots are harvested by moving the individual trays in a ease of processing on a conveyor belt. Harvested shoots are processed by extraction into protease inhibitors containing phosphate buffered solution. The shoots are broken by vigorous mixing or grinding while suspended in the buffer solution and the extracted biomass removed by centrifugation.

Example 2: Production of Pharmaceutical Proteins or Polypeptides in Transiently Germinated Broaches Infected by a Plant Virus

Desired plant seeds are obtained from a commercial cultivator contract as wild type seeds. Seed stock is stored under appropriate conditions of temperature, humidity, sanitation, and safety until use. At time of use, appropriate amounts of seed are immersed in water and incubated in trays as described above under controlled conditions.

After incubation for two to fourteen days, the germinated shoots are sprayed with a solution containing a transgene-harboring virus, and additionally or simultaneously treated with a material which causes mechanical abrasion of the plant leaf tissue. In this example, the sheets are closed with a spray of abrasive particles containing air. The virus is allowed to systemically infect plants for an appropriate period; from about one to about ten days, and expression of the desired heterologous protein is monitored.

After infection of the shoots for one to ten days, the shoots are harvested as described in Example 1 above.

Example 3: Expression of Protein Associated with Diabetes and Human Viral Vector Growth Hormone (AvA4) in NicotianaBenthamian Plants

Juvenile or type 1 diabetes that begins early is a disease that affects children, adolescents and young adults. Beta-island cells from 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 beta island cells by the body's own immune system.

Individuals with type 1 diabetes produce antibodies against at least three proteins that are normally found in all subjects, namely insulin, glutamic acid decarboxylase (GAD), and protein tyrosine phosphatase IA-2 (Leslie et al., Diabetologia (1999). ) 42: 30-14). IA-21 autoantibodies and GAD proteins are found in 50% to 75% of type 1 diabetes patients before disease onset. In addition, autoantibodies against these apparently normal proteins appear 7 to 8 years after the onset of classic symptoms associated with type 1 diabetes.

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 damage to island cells, which results. in the development of diabetes. Nonobese diabetic mice (NOD) spontaneously develop an autoimmune form of diabetes (Zahang, et al., Proc. Nati Acad. Sci. USA (1991) 88: 10252-10256). The gene encoding human insulin was fused to the cholera detoxin B subunit gene and the resulting construct expressed in batatatransgenic plants. When these plants were fed to NOD1 mice the animals showed a substantial reduction in pancreatic island inflammation and a delay in the progression of diabetes (Arakawa et al., Nat.Biotecnoi (1998) 16: 934-936. GAD protein was also expressed in plants These transgenic potatoes, when fed to NOD1 mice also either prevent or significantly delay the onset of diabetes after 40 weeks (Ma et al., Nature Medicine (1997) 3: 793-796).

In this example, certain non-limiting conditions for expression and recovery of GAD and IA-2 from plants are described. The production of raw materials and proteins occurs in two stages: 1) small scale (mg quantities) for preliminary animal studies; and 2) medium scale (quantities in grams) for clinical trials.

Test and Movement Stability: To conduct viral stability and movement tests, small amounts of each construct are synthesized. Each construct contains a T7 or SP6 RNA polymerase promoter fused to the exact 5 'terminus of viral genomic RNA, and a site unique 3 'end restriction restriction which is used to deinitiate the plasmid prior to in vitro transcription. The T7 or SP6 RNA polymerase then generates escape transcripts, which are used to inoculate plants. Plants are mechanically inoculated into two leaf stages by gently rubbing the inoculum on the leaf surface in the presence of an abrasive agent such as decarborundum powder (320-grain; Fisher, Pittsburgh, PA). Five to ten plants are inoculated per construct and 1-2 1g of each RNA transcription is used per inoculation. Plants are monitored for symptom severity, virus diffusion throughout the plant, and product recovery. At 0-15 days post infection (dpi), leaf samples from infected leaf samples are collected to assess the presence of full-size recombinant IA-2 and GAD. A portion of the harvested material (10 leaves) is frozen at -80 ° C and retained as a seed inoculum for the subsequent production scale of selected constructs. The rest of the tissue is processed immediately.

At 15-20 days post infection (dpi), IA-2 and recombinant GAD are recovered. The procedure is optimized to recover optimal amounts of high purity product (90-95% purity). Once products of the expected size are recovered and serological identity (known by specific antibodies using Westernblot and ELISA) is determined, the stability of the constructs is tested by three passages in audible plants. Problems with assembly, retrieval, or stability of recombinant protein viruses in the size range employed are controlled at the nucleotide sequence or amino acid sequence level by changing infection conditions, or by using an alternate host plant.

Seed Batch Establishment and Medium Scale Production Procedure: When stage 1 is completed, a small amount (100 nl) of in vitro synthesized transcripts of the recombinant constructs are prepared and used to inoculate 10 plants. Within 10-12 days after inoculation, the leaves are harvested, tested for GAD or IA-2 by Westernblot, 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 fresh detained). 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.

Plant Inoculation and Product Recovery: In vitro recombinant transcripts containing GAD and IA-2 are synthesized using T7 RNA polymerase and purified plasmid DNA. Transcriptions are covered using the m7G (5) ppp (5) G. RNA cover structure analog. For inoculation, a mixture of in vitro transcription products is applied to the leaves of the target host plants after surface abradation. leaf with carborundum and rubbing gently on the leaf surface to spread the inoculum and additionally flatten the surface. The purity and activity of the plant that produced IA-1 and GAD are tested. The antibody deletion capacity of plant-produced antigens is tested by ELI-SA during and after protein purification.

Nicotiana Benthamiana Protein Expression: To express full-length proteins in virus-infected plants, we used a functional complementation approximation to express green fluorescent jellyfish protein (GFP) in N. benthamiana plants (Figure 4). During plant-by-plant passages, the amount of Alfalfa (Av) / GFP mosaic virus in the infected tissue gradually decreases and after the third transfer, only Av / A4 was detectable. (This is an advantage of a point-of-view environmental safety) Using this approach, we can express an average of 100 pg of GFP per gram of fresh tissue. An important component of this system, CP Alfalfa Mosaic Virus, is unique in its ability to encapsulate unrelated virus genomic RNAs into infectious particles in the infected host. This unique capacity of the CP Alfalfa mosaic virus is exploited to design hybrid vectors that specifically target select-nothing crop species.

Recombinant GFP expression in Av / A4 and Av / A4GFP-inoculated Nicotiana ben-thamiana plants was analyzed by Manchamen-to Western (see Figure 3). Protein extracts from systematically infected leaves as described herein were electrophoresed on 12% SDS-polyacrylamide gel, transferred to a nitrocellular membrane, and reacted with protein-specific antibodies. Alfalfa mosaic viruses of specific CP-antibodies recognized the expected size protein (24.0 kD) in plants inoculated with Av / A4, or with the mixture of Av / A4 and Av / A4GFP. GFP-specific antibodies recognize proteins only in plant extracts inoculated with the Av / A4 and Av / A4GFP mixture. GFP-specific antibodies do not react with any proteins in plants inoculated with Av / A4 alone. Neither CP Alfalfa mosaic virus nor GFP-specific antibodies react with any protein in plant extracts inoculated with Av / GFP alone, suggesting a lack of systemic movement.

Also projected and expressed human growth hormone using this Av / A4 vector system. Nicotiana benthamia-na plants were inoculated with in vitro transcriptions, and the plants monitored for hGH production. No protein specific signal could be detected at 5 dpi (days after inoculation), although at 11 dpi we could detect a signal for hGH in the inoculated plants. Figure 4 is a Western blot of hGH produced on N. benthamiana plants infected with 125C / hGH in vitro transcription. Samples were analyzed 24 hours after inoculation. 1 pg of purified hGH was loaded as standard. MWM is molecular weight marker. The arrow in Figure 4 points to the hGH range in the spot detected by hGH specific antibodies.

Example 4: Expression of Human Growth-Associated Anthrax and Hormone Proteins in Transgenic Plants Brassica Juncea

The anthrax virulence 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. The antrhax toxin is encoded by a 184 kb plasmid called pX01, and consists of a receptor binding protein called a protective antigen (PA), and two enzymatically active proteins called edema factor and lethal factor ( Bhatnagar and Batra, Crit Rev. Mi-robiol (2001) 27 (3): 167-200).

In this example, two components of inactivated anthrax toxin, protective antigen (PA) and lethal factor (LF) are produced in Brassica juncea leaves at a concentration of at least 0.1 mg / gm dry weight. Codon use has been optimized. of these genes into plants and we have changed amino acid sequences to minimize toxicity for both toxin molecules. It was proposed to insert the synthesized DNA into vectors capable of regulating gene expression in Brassiea juncea. The PA gene has been altered to remove phenylalanine residues at positions 314 and 315. The LF gene has an alanine substituted by a histidine at position 686 or position 690 (see, for example, Singh et al. J. Bioi Chem. (1994) 269: 29039-29046; Klimpel et al., Mol. Mierobioi (1994) 13: 1093-1097).

Transformation Vector: The binary vector pGREENII 0229 (Hellen et al., Plant Molecular Biology (April 2000) 42 (6): 819-832) is used for plant transformation (see figure 5). This plasmid includes the following components: 1)

The pGREENII plasmid support includes sequences necessary for replication in Escherichia coli and Agrobacterium tumefaciens. 2) The left and right boundary sequences of agrobacterium tumefaciens T-DNA (LB and RB) are required for integration of all sequences between LBe RB into the plant genome. 3) The npt gene encoding kana-micin resistance for selection of Escherichia coli and Agrobacterium tumors transformants. 4) The nos-bar gene, which encodes resistance to the herbicide Bialophos (Bioalophos resistance is used to select transgenic plants). The nos-bar gene is transcribed from Cauliflower demosaic virus (CAMV) 35S promoter with constitutive activity in plants, and gene transcription is terminated using the CaMV terminator. 5) Vir PA and LF gene expression is triggered by either the CAMV 35S promoter as shown in Figure 5 or by the HSP18.2 promoter (GeneN5 Bank Access X17295 site At5g59720) for the molecular low heat shock protein Arabidopsis thaliana (Matsuhara et al., The Plant Journal: ForCell & MolecuIarBioIog (April 2000) 22 (1): 79-86). Transcriptional termination is mediated by the nopaline synthase gene terminator (nos). Promoters35S and HSP18.2 where chosen for their different activities. The 35S promoter is constitutively active in many plant tissues. In many case 35S promoter triggers high level expression of the protein of interest. By contrast, the HSP18.2 promoter is almost inactive unless the plants are hit by heat shock. High level expression of some proteins can only be achieved using an inducible promoter system.

Vector Construct and Plant Transformation: Vectors are constructed using DH5, a laboratory strain of Escherichia coli. The construct is analyzed by restriction endonuclease mapping and sequencing.

After the construct is confirmed, the vector is used to transform Agrobacterium tumefaciens strain cepaGV3101, lacking a native plasmidTi and suitable for TDNA transfer of binary vectors into plants. Brassica juncea plants are transformed by introducing GV3101 leading the transformation vector in flowers by any available transformation method.

Modifications Introduced into PA and LF Genes: The nucleotide sequences of both PA and LF are modified for expression in plants. The native PA coding sequence is 2295 bp and native LF is 2430 bp. Sequences are modified as follows: 1) Mutations are introduced that render PA and LF inactive as toxins. Phe314 and Phe315 are taken from PA and His686 and His690 are changed to Ala. 2) Codon usage is optimized for expression in Brassica juncea. Since the use of Brassica juncea's GC / AT codon and reason is neutral, the changes introduced in PA and LF are not extensive. Four codons are changed in PA and seven in LF. 3) Unique restriction sites are added to the 5 'and 3' ends of the genes for cloning into the plant expression vector. [4] A consensus sequence for translational initiation will be added (Joshi et al., PlantMolecuIarBioIogy. (Dec. 1997) 35 (993-1001). The optimized translational initiation locus restriction sites are illustrated in Figure 5. The 5 'end shows the added XbaI restriction site, and the consensus translational initiation site for plant genes, which includes the start codon followed by an Ala codon. Sac1 restriction restriction Modified PA and LF genes are chemically synthesized by Entelechon, Inc. (Regensburg, Germany).

A second construction is also produced for PA and LF. The intention of the second construct is to optimize the accumulation of PA and LF by objectifying and retaining proteins in the endoplasmic reticulum40. Objectivation and retention in ER have the potential to produce high level accumulation of a recombinant protein (Haseloff, et al., Proceedings of the National Aca-Demy of Sciences USA. (March 18, 1997) 94 (6): 2122-2127 ). To achieve this, an amino terminal signal sequence and a carboxyl terminal RE retention sequence is added. The signal sequence includes 22 codons derived from the Arabidopsis thaliana chitinase gene (Haseloff, etal., Supra). The ER retention sequence is the HisAspGIu-Leu tetrapeptide (Gomord et al., The Plant Journal: for Cell & Molecular Biology. (Feb1997) 11 (2): 313-325). The signal sequence is added by PCR saturation using the synthetic genes produced by Entelechon as templates (Tomme et al., J. BacterioL (1995) 177: 4356-4363). The RE retention sequence is added by PCR amplification of the PA and LF genes using a 3 'primer in which the HisAspGIu Leufoi coding sequence is incorporated. Modified PA and LF sequences are cloned into 35S and HSP18.2 expression vectors. Two modified versions of each eLF PA (a total of 4) are cloned. In total 8 different buildings are tested.

Selection and Analysis of Plant Expression Transaens: After transformation of Brassica juncea with Agrobacterium tumefaciens GV3101 strain leading to the transformation construct, the plants are allowed to progress through their normal developmental program. Within 25 days, mature seeds are produced, dried and harvested. Seeds are sown in pot soil and sprouting plants are grown for 15 days. The leaves of each plant are then sprayed with a 0.0058% (w / v) Biolophos solution (FINALE, Farnam, Inc., Phoenix, AZ). Sensitive non-transgenic plants are typically killed within 5 to 7 days. Bialophos-resistant plants are evident by their green, hearty appearance. Putative transgenic plants are confirmed and analyzed using a series of protocols. Polymerase chain reaction using oligonucleotide primers for the construct is used to confirm the presence of TDNA in putative transgenic plants. The presence of TDNA is then to be examined by genomic DNA staining using a specific DNA probe, the modified PA or LF genes. Finally, expression of the protein of interest is examined by SDS-PAGE, followed by Coomissie Blue staining, or immunoblotting. The protocol for protein expression analysis differs depending on the construct used to produce the transgenic plant. Plants driving the 35S pro-motor are analyzed directly, as expression from this engine is constitutive. Plants conducting the HSP18.2 promoter are heat shocked prior to analysis. Recombinant protein expression kinetics may differ in different transgenic plants. Ultimately, induction conditions are optimized for each standard method in use. However, for initial analysis it is possible to perform a standardized heat shock protocol that is established for expression of other recombinant proteins in Brassica juncea.

Figure 6 shows a transgenic Brassica juncea immunoblot expressing human growth hormone (hGH) under the HSP18.2 promoter control. Transgenic brassica juncea were grown in a pot mixture. As shown in Figure 6, a leaf weighing 1 to 3 grams of fresh weight was detached from the plant and placed on a Petri dish containing a water dampened filter paper. The petri dish is covered in a high humidity incubator at 37 ° C for 1.5 hours. The Petri dish containing the leaf was then removed from the incubator and placed for 5 hours in a growth chamber at 24 ° C under fluorescent lights at an intensity of 100 μηιοΙ photons m "2 s'1. The plant material was then harvested and analyzed. by immunomanulking using a monoclonal antibody against hHG (Sigma Chemical Co., Product No. G-8523) .The lower range is 16 kDa recombinant hGH. This range is not observed before the heat shock. Route 8 shows the results of a transgenic plant transformed with the vector alone.The highest molecular weight range is a non-specific reaction with the horseradish peroxidase-linked secondary antibody used to detect immunocomplexes.AP is predicted to be approximately 88 kDa and LF 93 kDa.

Following an initial classification for anthrax toxin expression, a more detailed expression analysis is performed. An ELISA method is used to quantify the level of expression. Additional analysis is performed on subsequent plant management. In order to avoid the need to select transgenic plants in subsequent generations, it is ultimately necessary to isolate transgenic lines that are non-aggregating from the TDNA construct. This is accompanied by self-pollination of the primary transgenic plants, raising the secondary generation plants to maturity, and testing the tertiary generation for resistance segregation of Bialophos. Second-generation individuals are identified to be non-segregating. Next, the progeny of non-segregating plants is increased and used for analysis of production scale conditions (eg 1,600 kg per month of dry biomass). ). For production scale, the decreasing and induction conditions are optimized for growing of growing plants. At this point it is also desirable to characterize the insertion site and TDNA copy number of elite lines and to characterize mRNA expression level expression.

Example 5: Expression of a GUS Rapporteur on Buds of 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 pg / ml kanamicin, 20μΜ acetosiringone and 10 mM MES pH 5.6. Overnight cultures were centrifuged, resuspended in MMA medium (MS salts, 10 mM deMES pH 5.6, 200 μΜ acetosiringone and 2% sucrose) in OD60O 2.0, used for bud vacuum filtration. .

Seeds from various plants were soaked in water for 24 hours at room temperature on a rocking platform, transferred into plastic containers with wet paper filter, and incubated for 4 to 6 days (depending on plant species) for 12 hours at 21 ° C.

After vacuum infiltration, the shoots were incubated for an additional 48-60 hours for 12 hours in daylight at 21 ° C, and GUS activity was detected in situ using X-glue histochemical substrate. Staining was performed overnight at 37 ° C and plant samples were devoid of staining in ethanol.

The system has been tested using a wide variety of broaches. See Figure 8A and 8B, and Figure 9.Example 6: Expression of Human Growth-Related Diabetes and Hormone-Associated Proteins of an Agrobacterial Construct Containing Viral SequencesA TMV-based vector has been created in an agrobacterial vector: D4-hGH or D4-GFP, for this , first one has to create multiple cloning sites in pBI121-Xba1-BanH1 -SaM -Pac1-BsiW1-Stu1 -Xho1 -Spe1-Kpn1-Sac1-pBI121. Using appropriate primers and PCR, local edible pBI121 was created. After confirmation of the sequence, we then proceeded to introduce genomic sequence of TMV in this plasmid. See also U.S.S.N. 10 / 770,600, filed February 3, 2004, and United States Provisional Patent Application No. 60 / 444,615, filed February 3, 2003, incorporated herein by reference, and U.S.S.N. 10 / 832,603 and U.S. Provisional Patent Application 60 / 546,339, incorporated herein by reference, for further discussion of D4-hGH and D4-GFP. Parenteral vector, D4, and vectors derived therefrom, are described in Shivprasad etal., Virology, 255 (2): 312-23, 1999. The resulting plasmid is referred to as pBID4.

The cauliflower mosaic virus 35S promoter was fused following TMV. In this way, the S35 promoter directs TMV sequence transcription. Upon successful incorporation into a cell, viral transcripts are produced. In certain embodiments of the invention, components required for viral replication and diffusion throughout the entire plant are also produced. These components include, for example, replicate, motion protein, and coat protein, which may be from TMV or another virus, such as alfalfa mosaic virus in various embodiments. These components may be coated 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 encoding the components. See, for example, USSN10 / 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 'from the TMV sequence. 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 either D4-hGH or D4-GFP. These empBI121 constructs were then used to transform A. tumefaciens and plants infiltrated with transformed Agrobacterium. As long as a replicating virus is present, the plants are incubated for 2 weeks. Unmanaged leaf discs for Western blot hGH production. GFP expression was monitored by long wave ultraviolet light plant illumination and photographed. The results shown in Figure 10 demonstrate GFP expression across all infiltrated leaves. Western blot analysis shown in Figure 11 using hGH-directed antibody confirmed that the construct was functional in plants, and hGH could be detected at high levels.

Similarly, IA-2ic was engineered into pBID4 plasmid to pBID4-IA-2ic. Additionally, viral AMV-based vector leadingGFP was generated. The resulting plasmid was used to transform Agrobac-terium and hydroponically grown benthamian Nicotiana was infiltrated with transformed Agrobaeterium. Briefly, Nieotiana bentha-miana seeds were sown in a Rockwool (18 X 18 X1) plate pre-moistened in resistance Hoagland Vz solution as a nutrient in hydroponic conditions. Hydroponic plants were kept in the same solution for four weeks. Four-week-old plants were then infiltrated with suspension vacuum of Agrobaeterium (OD60 x 0.1) driving pBID4-IA-2ie or viral AMV-based vector driving GFP.

Bacterial cultures were grown and induced overnight, then cells were resuspended in MMA medium (MS salts, 10 mM MES, pH 5.6, sucrose 20 g / l, acetosiringone 200 μΜ) in OD600 of 0 ,1. The resuspended culture was incubated at room temperature for 2-3 hours with gentle shaking, and Nieotiana ben-thamiana hydroponic plants were placed in the bacterial suspension, then vacuum was applied for 30-60 seconds at room temperature. The vacuum was rapidly released to facilitate efficient infusion of the bacteria into the tissue, and infiltrated plants were maintained in 1/2 resistance Hoagland solution nutrient for 5-7 days. Alternatively, the bacterial suspension was forced under the epidermis of fully expanded leaves using a 10 cm needle-free syringe. Leaves were harvested 3-6 days post-infiltration and stored at -80 ° C or analyzed for expression analysis. Figure 12 shows western blot analysis of plants expressing IA-2ic. Analysis of infiltrated plants with AMV-based GFP constructs under light demonstrated GFP expression across all infiltrated plant leaves.

Example 7: Protein Expression (Green Fluorescent Protein; Lychee) in Pea Varieties of an Agrobacterial Construct Containing Viral Sequences

The present Example describes agro-infiltration of certain pea varieties with agrobacterial constructs leading to a viral expression vector.

Figure 13 represents the total strategy used for bacterial constructs conducting A1MV sequences. Since A1MV has a segmented genome (as discussed above), three different constructs were used: one (pMOG-R1 & 2) that leads A1MV sequences that encode replicases 1 and 2; one (pBI-RNA3) that drives A1MVRNA3 sequences (optionally including a gene of interest to be expressed, which typically replaces the A1MVnatural coat protein sequences in RNA3); and one (pBI-CP) that drives A1MV sequences that co-difficult the A1MV coat protein. Those skilled in the art will appreciate that inclusion of the construct encoding the A1MV coat protein is not, strictly speaking, required. However, it may be useful to include such a construct, as the coat protein may facilitate replication and / or systemic diffusion of viral sequences (and hence of the gene of interest they carry).

Even where it is desirable to ensure expression of the A1MV coat protein, it is not essential that three constructs be used. For example, an alternative may be to use plants that are already transgenic to the A1MV coat protein so that they do not need to be separately supplied. . Alternatively or additionally, plants may be used which are transgenic to one or both of the A1MV repli-case proteins. In such plants (optionally additionally transgenic for the A1MV coat protein), a simple construct may be used, conducting only the RNA3 and sequence gene of interest, to achieve similar results.

As indicated in Figure 13, in these particular experiments constructs expressing three different proteins, green fluorescence protein (GFP), Iichenase (Lich), and a delichenase-Anthrax protective antigen fusion (Li-PA) were used.

In the particular experiments shown in Figure 13, at least one liter of each agrobacterial strain (ie, each strain carrying one of the three pMOG-R1 & R2, pBI-RNA3 / Protein of Interest; pLI-CP) constructs was produced, transformed into pellets. , and resuspended in MMA to an OD of 1.0. Those skilled in the art will appreciate that different D.Es may be desirable for different plants. For peas, higher D.Es, for example, in the range of 1.5 are often desirable if technically achievable.

In the particular experiments provided in Figure 13, α-bacterial strains leading replicase (pMOG-R1 & R2), coat protein (pMOG-CP), and RNA3 / Target Protein (pBI-RNA3 / GFP, Lich, or Li-AO) were combined at a ratio of 1: 1: 2 to a total volume of 1.5 L. Those skilled in the art will appreciate that these ratios and volumes are not critical, the particular ratio used has been selected to reflect that the A1MV life cycle typically involves higher RNA3 levels than other genome components. Also, there is a desire for higher levels of RNA that drive the gene of interest (ie, GFP, Lichenase, Lichenase Protective Antigen ougene).

In the particular experiments provided in Figure 13, defective shoots were grown hydroponically with a soilless support (nest, coarse cotton fabric) as is known in the art. The broils were submerged in the combined agrobacterium mixture, and vacuum applied for 90 seconds at -28 psi to achieve agro infiltration. In some cases, vacuum has been applied more than once.

In the particular experiments provided in Figure 13, the shoots were once immersed in tap water after agro-infiltration, and then returned to the growth space and irrigated. The shoots were later harvested, collected, and extracted.

Figures 14-16 show the results of various experiments performed according to the strategy illustrated in Figure 13. For example, as shown in Figure 14, Liche-nase-producing constructs were infiltrated into speckled pea (SP) shoots, yellow pea (YP), or bill jump pea (BP). Prior to agro-infiltration, the shoots were grown for 2 days in the dark (to allow germination), and 8 days in light (as indicated by the notation "2 (8)". These 10 days were then infiltrated as described above, and were 5 days post-inoculation (dpi) prior to detection of protein produced As shown in Figure 14, Iichenase protein was produced by all three pea varieties, with yellow pea having somewhat higher production levels than speckled peas, and bill jump peas having somewhat lower levels of production, such variability is well within the experience of those skilled in the protein expression technique, and in light of these results, no more than routine experiments are required to select variety of plant to produce any particular protein. In general, expression levels observed in these pea sprout approximately 1/3 of those observed upon expression of the relevant proteins (eg Lich-PA) in Nicotiana benthamiana (not shown). These shoots have the advantage over benthamian nicotiana that they are edible, which should simplify any required isolation of pharmaceutically reactive protein (s) produced as all plant components are known to be harmful to humans at least. . Also, they grow fast and are cheap. On the other hand, seed storage is easier for Nicotiana benthamiana, as the seeds are much smaller than those of sprouts generally and peas in particular.

As can be seen with reference to Figure 14, in these particular experiments, the above described A1MV production system was successful in producing protein than it was in an analogous TMV system (as discussed herein). Those skilled in the art will appreciate that a TMV system will be preferable for production of certain particular proteins in certain particular plant species, whereby A1MV is preferable in other circumstances including these.

Figure 15 shows successful production of PFM in light pea and yellow pea grown for different periods of time prior to agro infiltration. In the particular experiments presented in figure 15, vacuum was applied three times for 90 seconds each. Also, the particular suspending medium used was "full MMA". Those skilled in the art will appreciate that any of a variety of other infiltration media, including minimal MMA, AB, etc., may alternatively be used, and may increase or decrease the ultimate efficiency of agro-infiltration and thus protein or protein production. polypeptide.

Figure 16 shows Iichenase expression in leafy pea leaves (L), whole shoots (W), roots (R), or hypocotyl (H) at different periods of post-infiltration time. As can be seen, more pressure is on the leaves. In addition, expression is better in this particular system approximately 5-7 days after infiltration. Some variation should be expected based on the age and variety of plant being used, as well as the protein (s) being expressed.

Other similar experiments demonstrate, for example, that use of a pBI-CP construct lacks the increased production of 5/3 'RTU of the protein of interest, presumably by the increased coat protein (CP) production. . Also, in this system, 9-12 day old plants were great for expression. In general, higher protein levels were expressed in younger, chewed older tissue had more biomass. Still other similar experiments generated that 24 hour light exposure of the plants rather than a 12/12 light / dark cycle materially decreased the levels. of the protein of interest.

Still other similar experiments have shown that exposure of pea shoots to weak detergent during agro-infiltration may increase protein production levels of interest, presumably by increased efficiency and / or extension of successful agro-infiltration.

Example 8: Molecular System for Large-Scale Production of Proteins and Polypeptides in Plants

The present Example describes a technology platform that will evolve biopharmaceutical manufacturing, greatly reducing production time, in some cases five weeks ago. The particle production system described in this Example utilizes an agrobacterial vector to distribute very large copy numbers of plant viral RNAs that drive the coding sequence of the protein or polypeptide of interest in young plants within a few minutes. High levels of transient expression of the protein or polypeptide of interest may thus be achieved in large plant biomass within one week of introduction of the agrobacterial vector into the plant.

The technology platform described in this Example is applicable to a wide range of monomeric or dimeric proteins, including vaccine antigens, monoclonal antibodies, and other therapeutics (among other things), and may be performed on a variety of plant species. Plant expression is more likely to provide correct folding and solubility than alternative systems, and has the added advantage of being free of animal pathogens. In this particular Example, the design and production of vaccine antigens and monoclonal antibodies in shoots was focused.

As described in this Example, genes encoding the proteins or polypeptides of interest are cloned into a launch vector that combines elements of agrobacterial sequences and plant RNA viruses. Agrobacterium is then used to introduce millions of copies of the release vector into plants by vacuum infiltration. In tissue deplants, vector sequences are further massively amplified through viral replication. Target proteins are then produced from these viral transcripts in less than one week following introduction of the vector. It will be appreciated that it is not required (although also non-prohibitive) for the agrobacterial vector to integrate into the plant cells. Preferably1 the agrobacterial elements of the vector merely facilitate rapid systemic distribution (via agro-infiltration) of viral RNA of which protein is lately produced.

This system can be used to produce very large amounts (e.g., hundreds of milligram amounts) of target protein per kg of fresh plant tissue. In addition, very high capacity can be achieved as large amounts of seeds (which, as noted, do not need to be transgenic) can be readily made available.

As described herein, in some examples, the 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. 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, for example, in Russell N 60 / 472,495, filed May 22, 2003, ePCT / US04 / 016452, filed May 24, 2004, and published as WO 05/026375, filed March 24, 2005. We note that thermostable carrier talmolecule is a designed version of Clostridium termocellum β-1,2-1,4-gucanase (LicKM). Target sequences can be readily designed as N-terminal, C-terminal, or internal fusions for LicKM. The presence of foreign sequences in LicKM does not result in loss of the term stability of the molecule. This property facilitates easy and cost effective recovery of target proteins. A simple heat-treating step (e.g., up to 10 minutes at 65 ° C) results in the removal of much of the host proteins. In addition, more than one target protein may be fused to each LicKM molecule, but using multiple insertion sites simultaneously.

Specifically, seeds of non-genetically modified plants are germinated under hydroponic conditions and maintained for a period of time (for example, typically up to about 10 sprout days; sometimes 2-6 weeks for Nicotiana benthamania) in growing facilities. contained inside plants prior to vacuum infiltration. Under these conditions, the plant biomass yield per square foot is typically 4-5 times higher than that achieved with soil-grown plants. By using young plants (eg, sprouted shoots), a large cover can be generated with a large amount (for example, about 100-1000 kg) of green tissue per square foot. To give a few examples, a cover with about 700 g of green tissue / square foot can typically be generated with shoots such as peas; a cover of about 200 g of green tissue / square foot is typically generated with Nicotiana benthamiana. Those skilled in the art will appreciate that other plants are likely to be in such amounts.

Very high capacity can be achieved by stacking young plant shelves (eg sprouts) one above the other shelf with settled light units (a growth environment of 92,903 m2 (1000 square feet) will provide up to four metric tons of biomass in less than two weeks). All aerial parts of the plants are infiltrated with agrobacterium that houses the release vector by application, and then rapidly release from a vacuum. This forces the agrobacterium into full coverage of almost complete leaf cover plant tissue. . The 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).

The total seed-to-tissue process typically harvested was a few weeks (for example, less than 6, 5, 4, 3, or two weeks; often about 2 weeks for shoots). Since seed material is non-transgenic, seed generation and storage is inexpensive and straightforward, and there is no effective scale limitation. Thus, very large amounts of recombinant protein can potentially be generated in a matter of weeks.

The target proteins produced may be isolated if desired.

A heat step may be useful in isolating targets fused to a thermostable vehicle. Further purifications may involve various steps of separation and / or chromatography. For example, steps such as ammonium sulfate precipitation, pH-dependent separation, size exclusion chromatography, ion exchange chromatography, and affinity chromatography may be employed.

The total process contemplated in this Example involves 1) gene optimization and synthesis; 2) cloning to the release vector; 3) preparation of plant material and agrobacterial crops; 4) vacuum infiltration of germinated shoots; 5) target expression; 6) tissue harvesting; 7) and target recovery. To enable high production, much of this process has been automated. Specifically, all steps of non-transgenic seed sowing and fermentation of agrobacterial crops through homogenization, bleaching and volume reduction of homogenized plant material (see Figure 19) will be automated. A module capable of producing 1.5 metric tonnes of plant biomes in a batch will be designated and built. Specifically, the equipment conceptually shown in Figure 20 will be constructed.

The design of the equipment depicted in Figure 20 uses a tower concept such that the total modulus will rest within a space of approximately 464,515 m2 (5000 square feet). The central rack unit provides the necessary space, light and water supply to grow (1.5 metric tons) of plant biomass. Humidity, temperature and light are automatically monitored and controlled. In the drawing depicted in Figure 20, the plants will be hydroponically grown in bunches that are 0.305m χ 0.61 Om (11 χ 2 ') in size. Thus, approximately 6000 trays will be used. The module can be constructed of 36 individual storage units which are stackable. Each storage unit will hold 6 χ 7 χ 4 trays. The modularity of the design allows for scale production. There are two robots, one on each shelf, that place the trays on and off the shelves. Automatic sowing is made of a material suitable for hydroponic growth. Once the plants have reached an appropriate maturity, a vehicle 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 flood unit removes excess agrobacterial culture. Once the plants are flooded, they are transported back to the central shelf unit for target accumulation for several days. Subsequently, the trays are transported 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: HPV Antigen Expression of an Abrobacterial Construct Containing Viral Sequences

Agrobacterium-mediated transient expression system driven by Agrobacterium infiltration can be used (Turpen et al., 1993, J. Virol. Methods, 42: 227). Healthy N. benthamiana leaves were infiltrated with A. rhizogenes containing viral vectors designed to express LicKM-E7 or LicKM-0E7GGG.

The A. rhizogenes A4 (ATCC 43057) or A. tumefaciens (GV3103) strain was transformed with the pBI-D4-PRACS-LiKM-E7-KDEL, pBI-D4-PRACS-LicKM-E7VAC, pBI-D4-PRACS constructs. -LicKM-E7GGG-KDELe pBI-D4-PRACS-LicKM-E7GGG-VAC. Agrobacterium cultures were grown and induced as described (Kapila et al., 1997, Plant Sci., 122: 101). A 2 ml starter culture (picked from a fresh colony) was grown overnight in YEB (5 g / l beef extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose, 2 mM MgSO 4 with 25 pg / ml kanamicin at 28 ° C. The starter culture was diluted 1: 500 in 500 ml YEB with 25 mg / ml kanamicin, 10 mM 2-4 (-morpholino) ethanesulfonic acid ( MES) pH 5.6, additional 2 mM MgSO4 and 20 pg / ml acetosiringone The diluted culture was then grown overnight at an O.Deo of -1.7 at 28 ° C. were centrifuged at 3,000 χ g for 15 minutes and resuspended in MMA medium (MS salts, 10 mM MES pH5.6, sucrose 20 g / l, 200 μΜ acetosiringone) to an O.Deoo 2.4, maintained. for 1 hour at room temperature and used for agro-bacterium infiltration N. benthamiana leaves were injected with the Agrobacterium suspension using an available syringe without a needle Infiltrated leaves were collected 6 days post-infiltration dog.

Example 10: Anthrax Antigen Expression of a Bacterial Construct Containing Viral Sequences

An Agrobacterium-mediated transient expression system driven by Agrobacterium infiltration may be used to produce anthrax antigen (s). Healthy N. benthamiana leaves were infiltrated with A. rhizogenes containing viral vectors designed to express LyKM-E7 or LicKM-PAD4. The vector used was pBI-D4, a version of the viral expression vector D4 introduced into the Agrobaeterium pBI121 vector. (Chen. Etal., 2003, Mol. Breed., 11: 287). The 35S promoter is fused to the 5 'end of the viral sequence. The vector sequence is positioned between the PBI121BamHI and Sacl sites. Hammerhead ribozyme is placed 3 'viral sequence (Turpei et al., 1993, J. Virol. Methods, 42: 227). These constructs include fusions of sequences coding for LicKM-PAD4 or LicKM, sequences encoding the protein signal peptide PR-1a protein, um6x His target, and the retention anchor sequence of RE KDEL (see SEQ IDN0: 10). ).

The A. rhizogenes A4 strain (ATCC 43057) was transformed with the pBI-D4-PRLicKM-PAD4K and pBI-D4-PRLicKMK constructs. Agobaeterium cultures were grown and induced as described (Kapila et al., 1997, PIantSei., 122: 101). A 2 ml starter culture (picked from a fresh colony) was grown overnight in YEB (5 g / l carnebovine extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose, 2mM MgSO4 with 25 pg / ml kanamicin at 28 ° C. Starter culture was 1: 500 in 500 ml YEB with 25 pg / ml kanamicin, 10 mM 2-4 (-morpholino) ethanesulfonic acid ( MES) pH 5.6, 2 mM additional MgSO4 and 20 pg / ml acetosiringone The diluted culture was then grown overnight at an O.D.6 of -1.7 at 28 ° C. were centrifuged at 3,000 χ g for 15 minutes and resuspended in MMA medium (MS salts, 10mM MES pH 5.6, sucrose 20 g / l, 200 μΜ acetosiringone) at an O.D.60 of 2.4, kept for 1 hour at room temperature and used for Agrobacterium infiltration N. benthamiana leaves were injected with the Agrobacterium suspension using a syringe available without a needle Infiltrated leaves were harvested 6 days post-infiltration. to.

Example 11: Influenza Antigen Expression of a Bacterial Construct Containing Viral Sequences

An Agrobacterium-mediated transient expression system triggered by Agrobacterium infiltration may be used to produce influenza antigen (s). (Turpey et al., (1993). Transfection of total plants of Agrobacterium tumefaciens-inoculated coils containing tobacco mosaic virus cDNA (J. Virol. Methods, 42: 227). Healthy N. benthamiana leaves were infiltrated with A. rhizogenes containing viral vectors designed to express LicKM-HA or LicKM-NA.

A. rhizogenes A4 strain (ATCC 43057) was transformed with the pBI-D4-PRACS-LicKM-HA-KDEL, pBI-D4-PRACS-LicKM-HA-VAC constructs, pBI-D4-PRACS-LicKM-NA-KDEL and pBI-D4-PRACS-LicKM-NA-VAC. Agrobacterium cultures were grown and induced as described by Kapila., De Rycke R., Van Montagu M and Angenon G. (1997). An Agrobacterium-mediated transient gene expression for intact leaves.Plant Sci. 122, 101-108). A 2 ml starter culture (picked from a fresh colony) was grown overnight in YEB (5 g / l carnebovine extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose, 2mM MgSO 4) with 25 pg / ml kanamycin at 28 ° C. The starter culture was 1: 500 in 500 ml YEB with 25 pg / ml kanamicin, 10 mM ethanesulfonic acid 2-4 (-morpholine) pH 5.6, 2 mM additional MgSO4 and 20 pg / ml acetosiringone. The diluted culture was then grown overnight at an O.D.60 of -1.7 at 28 ° C. The cells were centrifuged at 3,000 χ g for 15 minutes and resuspended in MMA medium (MS salts, 10mM MES pH 5.6, sucrose 20 g / l, 200 μΜ acetosiringone) at an O.Deoo of 2, 4, kept for 1-3 hours at room temperature, and used for Agrobacterium infiltration. N. benthamiana leaves were injected with the Agrobacterium suspension using an available syringe without a needle. The infiltrated leaves were harvested 6 days after infiltration. Plants can be classified for presence of target antigen expression by Iichenase activity assay evaluation and smear analysis.

Zymogram analysis revealed the expression of both chimeric HA and NA proteins in the benthamian-tested Nicotiana transgenic roots. The expression is associated with lichenase activity. The reactivity range for fusion proteins shows a higher molecular weight than lichenase control, and the same molecular weight of plant-expressed product after agro-infection, confirming the presence of total fusion product.

Example 12: Expression of Influenza Antibody of an Agrobacterial Construct Containing Viral Sequences

Other Embodiments

Those skilled in the art will appreciate that the foregoing represents certain preferred embodiments of the present invention, and should not be construed to limit the spirit or scope of the invention as defined by the following claims:

Claims (45)

A method for producing pharmaceutically active protein or polypeptide in young plants, comprising the steps of: infusing a young pea plant with an agro-bacterial construct that includes a viral sequence promoter control transcript, whose viral sequences carry coding sequences. which encode a protein or polypeptide of interest, incubate the infused young pea plant under conditions and for a time sufficient to permit transcription from the promoter such that a viral transcript is produced, whose viral transcript includes sequences that encode a protein or polypeptide of interest; of the young pea plant infused under conditions and for a time sufficient to permit production of the protein or polypeptide. A method according to claim 1, wherein the bacterial construct comprises viral sequences from tobacco mosaic virus, alfalfa mosaic virus, cauliflower mosaic virus, or any combination of the foregoing. A method according to claim 1, wherein the bacterial construct comprises tobacco mosaic virus viral sequences. A method according to claim 1, wherein the bacterial construct comprises alfalfa mosaic virus viral sequences. The method of claim 1, wherein the bacterial construct comprises cauliflower mosaic virus viral sequences. A method according to claim 1, wherein the viral sequences are under the control of the 35S promoter of the decouve-flower mosaic virus. The method of claim 6, wherein the cauliflower mosaic virus promoter-35S triggers initial transcription of a recombinant viral genome following infusion into the young pea plant. The method of claim 1, wherein the transcriptional control of viral sequence promoter is a constitutive promoter. The method of claim 1, wherein the viral sequence promoter control transcription is an inducible promoter. The method of claim 9, wherein the inducible promoter is a heat shock promoter. The method of claim 9, wherein the inducible promoter is an inducible light promoter. The method of claim 9, wherein the inducible promoter is a chemically inducible promoter. The method of claim 1, wherein the agrobacterial construct comprises a transcriptional terminator. The method of claim 13, wherein the transcriptional terminator is the transcriptional terminator of Agrobacterium napolinasintase. The method of claim 1, wherein the agrobacterial construct comprises one or more genes for virus replication. The method of claim 1, wherein the agrobacterial construct comprises one or more genes for cell cell movement. The method of claim 1, wherein the protein or polypeptide of interest is under the transcriptional control of a viral mRNA promoter. The method of claim 17, wherein the viral mRNA promoter is the subgenomic coat protein mRNA promoter. The method of claim 1, wherein the agrobacterial construct comprises left and right edge sequences. The method of claim 19, wherein the left and right edge sequences delimit the region of the agro-bacterial construct that is transferred into young pea plant cells following infusion of the young pea plant into the agrobacterial construct. The method according to claim 1, wherein the young pea plant is infused with multiple constructs. The method according to claim 1, wherein the young cannabis plant is selected from the group consisting of large pea, bill jump pea, yellow pea, spotted pea, and green pea. The method of claim 1, wherein the protein or polypeptide of interest is insulin. The method of claim 1, wherein the protein or polypeptide of interest is glutamic acid decarboxylase. The method of claim 1, wherein the protein or polypeptide of interest is protein tyrosine phosphatase similar to protein IA-2. The method of claim 1, wherein the protein or polypeptide of interest is green fluorescent protein. A method according to claim 1, wherein the protein or polypeptide of interest is human growth hormone. The method of claim 1, wherein the protein or polypeptide of interest is one or more anthrax toxin components. The method of claim 28, wherein the anthrax toxin component is protective antigen. The method of claim 29, wherein a mutation has been introduced which renders the protective antigen inactive as a toxin. The method of claim 28, wherein the anthrax toxin component is a lethal factor. A method according to claim 31, wherein a mutation has been introduced which renders the lethal factor inactive as a toxin. A method according to claim 1, wherein the protein or polypeptide of interest comprises lichenase sequences. The method of claim 1, wherein the infused young pea plant is incubated under hydroponic growing conditions. The method of claim 1, wherein at least 5 grams of the protein or polypeptide is produced per kilogram of plant. The method of claim 1, wherein at least about 1 gram of protein or polypeptide is produced per kilogram of plant tissue. A method according to claim 1, wherein at least about 0.5 gram of protein or polypeptide is produced per kilogram of plant tissue. A method according to claim 1, wherein at least about 20-500 mg of the protein or polypeptide is produced per plant tissue chiogram. The method of claim 1, wherein the time sufficient to permit protein or polypeptide production is at least about 6 weeks of infusion of the agrobacterial construct. A method according to claim 1, wherein the time sufficient to permit protein or polypeptide production is at least about 3 weeks of infusion of the agrobacterial construct. The method of claim 1, wherein the time sufficient to permit protein or polypeptide production is at least about 2 weeks of infusion of the agrobacterial construct. The method of claim 1, wherein the time sufficient to permit protein or polypeptide production is at least about 1 week of infusion of the agrobacterial construct. A method according to claim 1, wherein the time sufficient to permit protein or polypeptide production is at least about 1 day of infusion of the agrobacterial construct. The method of claim 1, wherein the young plants are young pea plants. 45. A method for producing pharmaceutically active protein or polypeptide in Nicotian plants, comprising the steps of: infusing a young Nicotiana plant with an agro-bacterial construct that includes a viral sequence promoter control transcript, whose viral sequences carry coding sequences that encode a protein or polypeptide of interest; incubation of the infused young Nicotiana plant under conditions and for a time sufficient to permit transcription from the promoter such that a viral transcript is produced whose viral transcript includes sequences that encode a protein or polypeptide of interest; Young Nicotiana infused under conditions and long enough to allow production of protein or polypeptide.