Patent Grant
10190128
The present invention relates to kits comprising plus-sense single stranded RNA viral vectors, as well as mixtures of these vectors and uses thereof, and methods for producing in a plant, or plant tissue, or plant cell simultaneously two or more polypeptides using the kits and vectors.
For the recombinant production of proteins in plants, the use of viral vectors represents a promising alternative to the production of transgenic plants. Viral vectors allow high yields of proteins of interest, as their genomes are present in high concentrations in plant cells due to efficient replication. Moreover, it is possible to wait with infection and subsequent production until a certain developmental stage of the host plant is accomplished and also a higher biomass becomes available. However, the production of different recombinant proteins in the same cell represents a problem when using different viral vectors derived from the same genome. This is in particular a problem when complex proteins comprising more than one polypeptide chains should be produced. When two vectors derived from one viral species with coding sequences for two different foreign proteins are co-inoculated in one plant, the two viral vectors segregate spatially in the course of infection. This dilemma is shown in
Existing approaches to overcome the problems employ non-competitive viral vectors as the Tobacco Mosaic Virus (TMV), and the Potato Virus X (PVX), aiming at very rapid and high production (US 2009/0111145 A1; EP 1 686 176 A1) (see
Therefore, it was the object of the present invention to make available kits and methods which allow recombinant production of two or more polypeptides simultaneously in plant cells, and which at the same time ensures a biosafety containment.
In one embodiment, the object is solved by a kit comprising
Surprisingly it was found that a kit comprising such a first and second vector enables systemic co-infection of single cells, and both gene products are present, as shown in Example 1.
Due to the complete deletion of the coat protein ORF and the insertion of the coat protein ORF of the respective other virus, a kit is prepared which allows for systemic co-infection and co-expression. At the same time, a containment system is obtained, as the systemic infection of a healthy plant after liberation of a single vector into the environment is not possible. Due to the combined systemic infection, the complexity regarding the devices needed for infection is much lower, as not all plant parts need to be infected individually. Preferably, the coat proteins are reciprocally exchanged; i.e. the coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived is replaced by the functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and vice versa. Such replacement is preferred, as this does not disturb the overall viral genome organization and does not generate excessive additional genome load.
Thus, in a preferred embodiment of the kit, the coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived is replaced by the functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived.
In a further preferred embodiment, the coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived is replaced by the functional coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived.
As explained above, the kits of the invention represent non-competitive viral vectors. Therefore, in a yet further preferred embodiment, the viruses from which the first and second plus-sense single-stranded viral vectors are derived belong to different virus types, preferably different virus genera, more preferably to different virus families.
It is particularly preferred to make use of the non-competitive TMV- and PVX-viruses. Such kit based on TMV- and PVX-viruses was successfully used in Example 1.
The TMV genome encodes at least four proteins: the 126 and 183-kDa replicase proteins, the 30-kDa cell-to-cell movement protein (MP), and the 17.5-kDa coat protein (CP). The MP and the CP are encoded by subgenomic RNAs, which are co-terminal with the 3′ end of genomic RNA (see
The PVX genome encodes at least five proteins: the RNA-dependent RNA-polymerase (RdRp), the triple gene block proteins 1-3 (TGBp25, TGBp12, TGBp8) and the 25 kDa coat protein (CP). The TGB proteins and the CP are encoded by subgenomic RNAs, which are co-terminal with the 3′ end of the genomic RNA (see
Potexviruses and Tobamoviruses are plant RNA viruses with a plus-sense single-stranded genome. Their genome is monopartite.
Thus, said viral vectors of the invention may be RNA being or comprising said RNA replicon or may be DNA encoding said RNA replicon.
A RNA replicon comprises the elements of viral vectors of kits of the invention. Further genetic elements will typically be present on said replicon for replication and expression. For being a RNA replicon, i.e. for autonomous replication in a plant cell, said RNA replicon encodes a RdRp or a functional derivative thereof.
Said RNA replicon preferably further has viral, in particular tobamoviral or potexviral 5′- or 3′-untranslated regions and promoter-sequences in the 5′- or 3′-untranslated regions of said RNA replicon for binding said RdRp and for replicating said RNA replicon.
Said RNA replicon further may have subgenomic promoters for generating subgenomic RNAs for the expression of the CP (coat protein), MP (movement protein(s)) or TGB (triple gene block proteins) or functional heterologous ORF proteins, where applicable.
If said viral vector is DNA, it will typically have a promoter for allowing infectious RNA production by transcription of said RNA replicon in vitro or in vivo in plants. An example of a promoter allowing for transcription of said RNA replicon from DNA in plants is the 35S promoter of Cauliflower mosaic virus that is widely used in plant biotechnology.
The RdRp used in viral vectors may be considered a functional variant of a viral RdRp if the sequence encodes a protein having a sequence identity of at least 50%, preferably at least 60%, more preferably at least 80%, and most preferably at least 90% of the native RdRp.
A list of plus-sense single stranded RNA viruses that can be used for engineering the viral vectors of the invention is presented below:
Family: Bromoviridae,
Thus, in a yet further preferred embodiment, the virus from which the first plus-sense single-stranded viral vector is derived is a Potexvirus, in particular PVX virus (potato virus X).
In a yet further preferred embodiment, the virus from which the second plus-sense single-stranded viral vector is derived is a Tobamovirus, in particular TMV virus (tobacco mosaic virus).
In Example 1, the first and second plus-sense single-stranded viral vectors comprise a heterologous ORF. Thus, it is possible to produce two different heterologous proteins in a plant cell using such first and second plus-sense single-stranded viral vectors (see FIG. 3). Preferred vectors, as used in Example 1, have the structure PVX-CP(TMV)-ORF and TMV-CP(PVX)-ORF.
However, it is not required, that these vectors comprise such heterologous ORFs. Rather, the plus-sense single-stranded viral vectors may be used in one preferred embodiment only as complementing vectors. In such embodiment either of the vectors or both do not comprise a heterologous ORF. Preferred viral vector combinations are vector combinations having the following structures: (a) PVX-CP(TMV) and TMV-CP(PVX); (b) PVX-CP(TMV)-ORF and TMV-CP(PVX); (c) PVX-CP(TMV) and TMV-CP(PVX)-ORF and PVX-CP(TMV)-ORF and TMV-CP(PVX)-ORF (as shown in
Thus in another preferred embodiment of the invention, the first plus-sense single-stranded viral vector and/or the second plus-sense single-stranded viral vector comprise a functional heterologous ORF. The ORF may be the same or different, preferably different. In such embodiment, the vectors are used as complementing vectors and in addition are used for expressing a heterologous ORF of interest.
For being expressible from the viral vectors in a plant or in plant tissue, said viral vectors comprising a functional heterologous ORF comprise one or more subgenomic promoters and other sequences required for expression such as ribosome binding site and/or an internal ribosome entry site (IRES).
Thus in another preferred embodiment of the invention, the first plus-sense single-stranded viral vector and/or the second plus-sense single-stranded viral vector do not comprise a functional heterologous ORF. In this embodiment, the kit is used as complementing viral vector kit, which can be used to add further viral vector(s) comprising functional heterologous ORF(s), as described below which do not comprise a coat protein.
It was surprisingly found that using the complementing vector pair PVX-CP(TMV)-ORF and TMV-CP(PVX)-ORF, and the coat-protein-deficient vector PVX-ΔCP-ORF, it is possible to achieve expression of all ORFs, as described in Example 1. Therefore, it was surprisingly found that further heterologous ORFs may be expressed in the same plant cells based on the complementing vectors and one or more further vectors carrying heterologous ORF(s), wherein the further vectors have a complete deletion of the coat protein. Due to the complete deletion, the genomic load of virus is considerably lower and the coat protein deficiency of the further vectors comprise additional containment (
“PVX-CP(TMV)-ORF” is understood as viral vector based on PVX, wherein the native coat protein is replaced by the coat protein of TMV, and wherein the vector comprises a functional heterologous ORF.
“TMV-CP(PVX)-ORF” is understood as viral vector based on TMV, wherein the native coat protein is replaced by the coat protein of PVX, and wherein the vector comprises a functional heterologous ORF.
“PVX-ΔCP-ORF” is understood as viral vector based on PVX, wherein the native coat protein ORF is completely deleted, and wherein the vector comprises a functional heterologous ORF (see also
“PVX-ΔCP-ΔMPs-ORF” is understood as viral vector based on PVX, wherein the native coat protein ORF is completely deleted, and wherein the viral vector is devoid of functional movement proteins, and wherein the vector comprises a functional heterologous ORF (see also
“PVX-ORF” is understood as viral vector based on PVX, wherein the vector comprises a functional heterologous ORF.
“TMV-ORF” is understood as viral vector based on TMV, wherein the vector comprises a functional heterologous ORF.
A “functional heterologous ORF” is understood as open reading frame (ORF), which is not present in the respective unmodified virus, and which can be expressed to yield a functional gene product. Such gene product may be RNA or a protein, preferably a protein. Preferably, the functional heterologous ORF is heterologous for all viral vectors used. Typically, the functional heterologous ORF is under control of suitable subgenomic promoters or ribosome-binding sites or IRES.
Therefore, in one particularly preferred embodiment, this further viral vector is a third plus-sense single stranded RNA viral vector with the following features:
In a particularly preferred embodiment, the kit further comprises
Moreover, it is possible to additionally delete the movement protein(s) of the third plus-sense single-stranded viral vector(s), thereby further reducing viral genomic size.
Thus, in a particularly preferred embodiment, the at least one third plus-sense single stranded RNA viral vector(s) is/are devoid of the functional movement protein ORF(s) of the virus from which the at least one third plus-sense single-stranded viral vector(s) are derived, preferably the functional movement protein ORF(s) of the virus from which the at least one third plus-sense single-stranded viral vector(s) are derived are completely deleted (see also
Thus in a preferred embodiment, the virus from which the third plus-sense single-stranded viral vector is derived is a Potexvirus, in particular PVX virus (potato virus X) and the third plus-sense single-stranded viral vector is devoid of the functional movement protein ORF(s) of the Potexvirus.
As shown in the Example 1, the complementation was achieved using PVX-ΔCP-ORF as third viral vector. Such a vector is a preferred vector for a kit according to the invention.
In a further embodiment, the movement proteins of PVX may be deleted. Such vector has the structure PVX-ΔCP-ΔTGB-ORF.
The generation of such deletion is described in Example 1.
Thus, in a particularly preferred embodiment, the virus from which the third plus-sense single-stranded viral vector is derived is a Potexvirus, in particular PVX virus (potato virus X). Even more preferred are vectors of the structure PVX-ΔCP-ΔTGB-ORF and/or PVX-ΔCP-ORF.
Therefore, in a particularly preferred embodiment of the invention, the virus from which the first plus-sense single-stranded viral vector is derived is a Potexvirus, in particular PVX virus (potato virus X), and the virus from which the second plus-sense single-stranded viral vector is derived is a Tobamovirus, in particular TMV virus (tobacco mosaic virus), and the third plus-sense single-stranded viral vector is derived is a Potexvirus, in particular PVX virus (potato virus X).
In an even more preferred embodiment, the kit comprises or contains
(A)
In Example 1, also the complementing vectors comprise a heterologous ORF. It is however also possible that only the at least one third plus-sense single-stranded viral vector(s) comprise a heterologous ORF in order to reduce viral vector size of the complementing vectors. Also, it is preferred that one viral vector comprises only one heterologous ORF, in order to reduce excessive viral genomic size.
In yet another preferred embodiment, the at least one additional plus-sense single stranded RNA viral vector(s) comprises the features of a first or second plus-sense single-stranded viral vector described above, and comprises at least one functional heterologous ORF, wherein the at least one functional heterologous ORF is different from the functional heterologous ORF(s) of the first or second plus-sense single-stranded viral vector, in case one or both of the vectors comprise functional heterologous ORF(s).
Such additional plus-sense single stranded RNA viral vector(s) is obtainable by replacing (in case a functional heterologous ORF is present) or introducing (in case no functional heterologous ORF is present) a functional heterologous ORF in the first or second plus-sense single stranded RNA viral vector.
Thus, in a preferred embodiment preferably the at least one additional plus-sense single stranded RNA viral vector(s) differs from the first or second plus-sense single-stranded viral vector by a functional heterologous ORF.
In a yet further preferred embodiment, the at least one additional plus-sense single stranded RNA viral vector(s) is/are derived from the same virus from which the first plus-sense single-stranded viral vector or the second plus-sense single-stranded viral vector is derived, and comprises at least one functional heterologous ORF. In particularly preferred embodiment, a wt viral vector may be used to introduce at least one functional heterologous ORF in order to obtain such additional plus-sense single stranded RNA viral vector. In another particularly preferred embodiment, the at least one additional plus-sense single stranded RNA viral vector(s) comprises the coat protein, the movement protein(s) and RNA-dependent RNA polymerase of the virus from which the first plus-sense single-stranded viral vector or the second plus-sense single-stranded viral vector is derived, and at least one functional heterologous ORF. Such at least one functional heterologous ORF may be the same or different functional heterologous ORF as in the first plus-sense single-stranded viral vector or the second plus-sense single-stranded viral vector (in case they comprise such functional heterologous ORF), preferably different.
Thus, preferably, two or more different third and/or additional plus-sense single stranded RNA viral vectors comprise different functional heterologous ORF(s).
In a further preferred embodiment, at least one third and/or additional plus-sense single stranded RNA viral vector(s) comprise one functional heterologous ORF.
Said viral vectors may be built on a natural plus-sense single-stranded virus e.g. by using genetic components from such a virus, in particular Tobamovirus or Potexvirus. Said viral vectors of the invention or of kits of the invention may be obtainable by inserting said heterologous ORF sequences into viral vectors encoding a virus and by replacing the sequence encoding a CP as described above. However, various modifications may be made to the various genetic components of a natural virus, such as to the RdRp gene of the Potexvirus, or to the 5′ or 3′ non-translated regions of a Potexvirus.
The viral vectors of the kits of the invention may be mixed, to obtain mixtures of such viral vectors. Such mixtures are e.g. in solid form, such as a dried or freeze-dried solid, or in a liquid, e.g. as aqueous solution, suspension or dispersion, or as gels. Such mixtures can be used to infect plants, plant cells or plant tissues.
Therefore, the present invention also relates to a mixture comprising 2, 3, or more different vectors of the following vectors a) to c):
In Example 1, a mixture of or PVX-ΔCP-ORF, PVX-CP(TMV)-ORF and TMV-CP(PVX)-ORF was used successfully for infection.
In a preferred embodiment, the mixture comprises
In a further preferred embodiment, the mixture comprises
The kits and mixtures of the invention may be used for successfully infecting plants or plant cells with the vectors as described above.
Thus the present invention also relates to a plant or plant cell comprising at least one first plus-sense single stranded RNA viral vector and at least one second plus-sense single stranded RNA viral vector as defined above, and optionally at least one third and/or additional plus-sense single stranded RNA viral vector(s) as defined above.
For the first time, it is now possible to obtain simultaneous co-expression of the different heterologous ORFs of the vectors systemically in more than one plant cell in more than one tissue using plant RNA viral vectors.
Therefore, preferably more than one plant cell in more than one tissue comprises at least one first plus-sense single stranded RNA viral vector and at least one second plus-sense single stranded RNA vector, and optionally at least one third and/or additional plus-sense single stranded RNA viral vector(s), as described above.
Therefore, in another preferred embodiment, more than one plant cell in more than one tissue comprises the heterologous polypeptides encoded by the ORFs of at least two different third and/or additional plus-sense single stranded RNA viral vector(s) as defined herein.
The kits and mixtures of the invention can be used for expression of heterologous proteins. In particular, the kits allow for the first time the contained production of heterooligomeric proteins in systemically infected plants using viral vectors for commercial applications.
Therefore, the invention relates in one embodiment to a method for producing in a plant, or plant tissue, or plant cell a heterooligomeric polypeptide and/or two or more polypeptides, comprising providing to at least one plant cell
In a particularly preferred embodiment, the method is followed by isolating the heterooligomeric polypeptide and/or two or more polypeptides from the plant, plant tissue, or plant cell.
Methods for isolating proteins from plants or plant cells are known in the art. In one method, a protein of interest may be isolated from a plant apoplast as described in WO 03/020938.
Surprisingly, systemic infection with all vectors can be achieved using the vectors of the kits. Therefore, in a preferred embodiment, systemic infection of the plant is achieved.
The method is in particular useful for producing heterooligomeric proteins, which should be produced in one cell and should be assembled in such cell to yield a functional protein. Examples of such heterooligomeric proteins are immunoglobulins, in particular an antibody or antibody fragment, or certain interleukins, such as IL-12.
Agrobacterium-mediated delivery of viral vectors in plant cells may be used for the provision of viral vectors. Various other methods usually used for stable transformation of plants may also be used for the provision of viral vectors into plant cells such as direct introduction of a nucleic acid sequence into cells by means of microprojectile bombardment, electroporation or PEG-mediated transformation of protoplasts. Agrobacterium-mediated plant transformation is preferred. Thus, a heterologous nucleotide sequence may be transformed into plant cells by various technologies such as by a Ti-plasmid vector carried by Agrobacterium (U.S. Pat. Nos. 5,591,616; 4,940,838; 5,464,763), particle or microprojectile bombardment (U.S. Pat. No. 5,100,792; EP00444882 B1; EP 00434616 B1). In principle, other plant transformation methods can also be used e.g. microinjection (WO 09/209696; WO 09/400583A1; EP 175966 B1), electroporation (EP00564595 B1; EP00290395 B1; WO 08/706614 A1), etc. The choice of the transformation method depends inter alia on the plant species to be transformed. For example, microprojectile bombardment may be preferred for monocot transformation, while for dicots, Agrobacterium-mediated transformation gives generally better results.
The present invention is preferably carried out with higher, multi-cellular plants. Preferred plants for the use in this invention include any plant species with preference given to agronomically and horticulturally important species. Common crop plants for the use in present invention include alfalfa, barley, beans, canola, cowpeas, cotton, corn, clover, lotus, lentils, lupine, millet, oats, peas, peanuts, rice, rye, sweet clover, sunflower, sweetpea, soybean, sorghum triticale, yam beans, velvet beans, vetch, wheat, wisteria, and nut plants. The plant species preferred for practicing of this invention are including but not restricted to: Representatives of Graminae, Compositae, Solanacea and Rosaceae. Additionally, preferred species for use in the invention, as well as those specified above, plants from the genera: Arabidopsis, Agrostis, Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena, Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza, Panicum, Pelargonium, Pennisetum, Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum, Vicia, Vigna, Vitis, Zea, and the Olyreae, the Pharoideae and many others.
Preferred plants are Nicotiana species like Nicotiana benthamiana and Nicotiana tabacum; preferred plant species other than Nicotiana species are Petunia hybrida, Brassica campestris, B. juncea, cress, arugula, mustard, Strawberry, spinach, Chenopodium capitatum, alfalfa, lettuce, sunflower, potato and cucumber.
Preferred Potexviruses are PVX, bamboo mosaic virus, papaya mosaic virus, alternanthera mosaic virus, clover yellow mosaic virus, plantain virus X, white clover mosaic virus and potato aucuba mosaic virus, in particular potato virus X (PVX).
The major application of the present invention is the production of a protein of interest in plants, plant leaves or plant tissue or cell culture. If the process of the invention is performed in plants, plants that do not enter the human or animal food chain are preferred, like Nicotiana species. Plants that do not enter the human or animal food chain can be cultivated in an open field and harvested within certain period after infection with said viral vectors. Preferably, whole plants or plant parts shall be confined to a contained environment, e.g. a glasshouse or a designed chamber for the incubation period necessary to provide for desired level of expression.
In a further embodiment, the present invention relates to a PVX virus vector, wherein
It was surprisingly found that complete deletion of the PVX coat protein allows successful co-infection. In the state of the art, deletions of the coat protein resulted in vectors wherein the 3′ end of the coat protein was still present. Such vectors are described in Komarova et al. (2006, Biochemistry (Mosc); 71: 846-850) and Tyulkina et al. (2011, Acta naturae; 3: 73-82). However, such partial deletion of the ORF is insufficient for achieving coinfection. Therefore, the novel PVX vectors of the invention are surprisingly useful for coinfection, in particular with the complementing vectors of the kits of the invention above.
In another embodiment of the invention, it is also possible to make use of coat-protein deficient viral vectors for infection of plants without the danger of a contamination of the environment. In this embodiment, at least two viral vectors with the same backbone are used, which lack their native coat protein. Due to the coat protein deficiency, a systemic infection and/or contamination of the environment are not possible. Using the same backbone for expression of two or more different heterologous ORFs allows simple construction of the vectors. Moreover, the deletion of coat protein allows introduction of a heterologous ORF without excessive increase of viral genome size. Moreover, as the vectors can replicate within the plant cells, higher protein expression yields can be achieved compared to a simple transient expression using A. tumefaciens cells (Example 2).
Therefore, in another embodiment, the present invention relates to a method for producing in a plant, or plant tissue, or plant cell a heterooligomeric polypeptide and/or two or more polypeptides, comprising providing to a plant, or plant tissue, or plant cell at least two plus-sense single stranded RNA viral vectors,
In a particularly preferred embodiment, the coat proteins are completely deleted. Therefore, in a preferred embodiment, the coat protein ORF of the virus from which the at least two plus-sense single stranded RNA viral vectors are derived, is completely deleted in the at least two plus-sense single stranded RNA viral vectors.
In a further preferred embodiment, the virus from which the at least two plus-sense single stranded RNA viral vectors are derived is a Potexvirus, in particular PVX virus (potato virus X).
In an even more preferred embodiment, the coat protein ORF of the virus from which the at least two plus-sense single stranded RNA viral vectors are derived, is completely deleted in the at least two plus-sense single stranded RNA viral vectors, and the virus from which the at least two plus-sense single stranded RNA viral vectors are derived is a Potexvirus, in particular PVX virus (potato virus X).
A variety of methods are known to provide the vectors to the plant, or plant tissue, or plant cell. In particular, they may be provided by agroinfection, by transfection with DNA, in particular DNA comprising cDNA of the viral genome, by transfection with RNA, in particular RNA corresponding to the virus genome. Such methods are for example described in Fischer, R., et al. (2004; Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7, 152-158); Komarova, T. V. et al. (2010; Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines 9, 859-876) and Lico, C. et al. (2012; The use of plants for the production of therapeutic human peptides. Plant Cell Rep 31, 439-451).
Alternatively, the cloned cDNAs of the viral genomes may be used for transcribing the RNA in vitro, which is then used for infection.
Alternatively, the cloned cDNAs of the viral genomes may be cloned into suitable vector constructs, such as plasmids or DNA fragment, which allow transcription of the genome. In particular, such construct comprises suitable promoter and terminator sequences.
In case of agroinfection, the cDNA of the viral genome is introduced into T-DNA, which is then provided to the plants. Again such T-DNA comprises further regulatory DNA components, which allow transcription of the genome. In particular, such T-DNA construct comprises suitable promoter and terminator sequences.
The different Agrobacterium cell cultures, which comprise the respective viral vectors comprising the different functional heterologous ORFs may be mixed and may be administered to plants for infection.
Therefore, in one embodiment, the present invention relates to a mixture of Agrobacterium cells, in particular Agrobacterium tumefaciens cells,
In a preferred embodiment of the present invention, at least two, three, four, five or more Agrobacterium cells comprise two, three, four, five or more two, three, four, five or more, even more preferably two Agrobacterium cells in the mixture comprise different plus-sense single stranded RNA vectors. Even more preferably, the two Agrobacterium cells in the mixture each contain one plus-sense single stranded RNA vector.
In a further preferred embodiment, the virus from which the plus-sense single stranded RNA viral vectors are derived is a Potexvirus, in particular PVX virus (potato virus X).
In a further preferred embodiment, the coat protein ORF of the virus from which the plus-sense single stranded RNA viral vectors are derived, is completely deleted in the plus-sense single stranded RNA viral vectors.
In a yet further preferred embodiment of the invention, the virus from which the plus-sense single stranded RNA viral vectors are derived is a Potexvirus, in particular PVX virus (potato virus X) and the coat protein ORF of the virus from which the plus-sense single stranded RNA viral vectors are derived, is completely deleted in the plus-sense single stranded RNA viral vectors.
Proteins of interest, or fragments thereof, that can be expressed, in sense or antisense orientation, using the invention include: starch modifying enzymes (starch synthase, starch phosphorylation enzyme, debranching enzyme, starch branching enzyme, starch branching enzyme II, granule bound starch synthase), sucrose phosphate synthase, sucrose phosphorylase, polygalacturonase, polyfructan sucrase, ADP glucose pyrophosphorylase, cyclodextrin glycosyltransferase, fructosyl transferase, glycogen synthase, pectin esterase, aprotinin, avidin, bacterial levansucrase, E. coli glgA protein, MAPK4 and orthologues, nitrogen assimilation/methabolism enzyme, glutamine synthase, plant osmotin, 2S albumin, thaumatin, site-specific recombinase/integrase (FLP, Cre, R recombinase, Int, SSVI Integrase R, Integrase phiC31, or an active fragment or variant thereof), isopentenyl transferase, Sca M5 (soybean calmodulin), coleopteran type toxin or an insecticidally active fragment, ubiquitin conjugating enzyme (E2) fusion proteins, enzymes that metabolize lipids, amino acids, sugars, nucleic acids and polysaccharides, superoxide dismutase, inactive proenzyme form of a protease, plant protein toxins, traits altering fiber in fiber producing plants, Coleopteran active toxin from Bacillus thuringiensis (Bt2 toxin, insecticidal crystal protein (ICP), CrylC toxin, delta endotoxin, polyopeptide toxin, protoxin etc.), insect specific toxin AalT, cellulose degrading enzymes, E1 cellulase from Acidothermus celluloticus, lignin modifying enzymes, cinnamoyl alcohol dehydrogenase, trehalose-6-phosphate synthase, enzymes of cytokinin metabolic pathway, HMG-CoA reductase, E. coli inorganic pyrophosphatase, seed storage protein, Erwinia herbicola lycopen synthase, ACC oxidase, pTOM36 encoded protein, phytase, ketohydrolase, acetoacetyl CoA reductase, PHB (polyhydroxybutanoate) synthase, acyl carrier protein, napin, EA9, non-higher plant phytoene synthase, pTOM5 encoded protein, ETR (ethylene receptor), plastidic pyruvate phosphate dikinase, nematode-inducible transmembrane pore protein, trait enhancing photosynthetic or plastid function of the plant cell, stilbene synthase, an enzyme capable of hydroxylating phenols, catechol dioxygenase, catechol 2,3-dioxygenase, chloromuconate cycloisomerase, anthranilate synthase, Brassica AGL15 protein, fructose 1,6-biphosphatase (FBPase), AMV RNA3, PVY replicase, PLRV replicase, potyvirus coat protein, CMV coat protein, TMV coat protein, luteovirus replicase, MDMV messenger RNA, mutant geminiviral replicase, Umbellularia californica C12:0 preferring acyl-ACP thioesterase, plant C10 or C12:0 preferring acyl-ACP thioesterase, C14:0 preferring acyl-ACP thioesterase (luxD), plant synthase factor A, plant synthase factor B, 6-desaturase, protein having an enzymatic activity in the peroxysomal-oxidation of fatty acids in plant cells, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, lipase, maize acetyl-CoA-carboxylase, 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), phosphinothricin acetyl transferase (BAR, PAT), CP4 protein, ACC deaminase, ribozyme, protein having posttranslational cleavage site, protein fusion consisting of a DNA-binding domain of Gal4 transcriptional activator and a transcriptional activation domain, a translational fusion of oleosin protein with protein of interest capable of targeting the fusion protein into the lipid phase, DHPS gene conferring sulfonamide resistance, bacterial nitrilase, 2,4-D monooxygenase, acetolactate synthase or acetohydroxyacid synthase (ALS, AHAS), polygalacturonase, bacterial nitrilase, fusion of amino terminal hydrophobic region of a mature phosphate translocator protein residing in the inner envelope membrane of the plastid with protein of interest to be targeted into said membrane etc.).
Any human or animal protein can be expressed using the system of the invention. Examples of such proteins of interest include inter alia the following proteins of pharmaceutical interest: immune response proteins (antibodies, single chain antibodies, T cell receptors etc.), antigens, colony stimulating factors, relaxins, polypeptide hormones, cytokines and their receptors, interferons, growth factors and coagulation factors, enzymatically active lysosomal enzyme, fibrinolytic polypeptides, blood clotting factors, trypsinogen, 1-antitrypsin (AAT), as well as function-conservative proteins like fusions, mutant versions and synthetic derivatives of the above proteins.
As shown by the Example and as explained above, the kits, mixtures, vectors, plants and plant cells are in particular useful for expression of polypeptides, in particular heterooligomeric polypeptides in plant.
Therefore, the invention relates in one embodiment to the use of a kit of the invention, or of a mixture of the invention, or of a PVX virus vector of the invention, or of a plant or plant cell of the invention, or of a plant of the invention, or of a mixture of Agrobacterium cells of the invention, for producing polypeptides, in particular heterooligomeric polypeptides.
The terms “movement protein(s)” or “MP(s)” is known to the skilled person and is understood as protein(s) required for cell-to-cell movement like the MP ORF in tobamoviruses.
The term “triple gene block proteins” or “TGB” is known to the skilled person and is understood as proteins required for cell-to-cell movement like the TGB ORFs in potexviruses.
The term “coat protein” is known to the skilled person and is understood as protein(s) building the virus coat.
The complementation vectors were generated with help of the gene splicing by overlap extension (SOE) PCR method. For the construction of a PVX with the coat protein of TMV three PCR products were created. In PCR1 the mCherry gene was inserted into the PVX genome (PVX-mCherry) and the subgenomic promoter of the PVX coat protein was amplified with primers mCherry-ClaI and SOE-TMVCP rv including an overlapping sequence for the fusion. PCR2 amplified a part of the subgenomic promoter and the ORF of the TMV coat protein from the vector pJL24 (US 2010/0071085 A1) with primers SOE-TMVCP fw and SOE-TMVCP2 rv including two overlapping sequences, the 5′-end complementary to PCR product 1 and the 3′-end complementary to PCR product 3. With PCR 3 the 3′ part of the PVX genome including parts of the plasmid backbone was created with primers SOE-TMVCP2-fw and M13 universe from the vector PVX-mCherry. In a fourth PCR lacking primers all PCR products were fused due to the overlapping sequences and again amplified in PCR 5 with the primers mCherry-ClaI and M13 universe. The final PCR product was then cut with ClaI and SalI and ligated into the PVX vector which was cut with the same enzymes and dephosporylated with a calf intestinal phosphatase (CIP). The PVX vector also consists of the plasmid backbone of the binary pTRAc vector (Mclean, 2007, J Gen Virol 88, 1460-1469). The PVX genome of the UK3 strain is integrated between left border and right border of the T-DNA. The subgenomic promoter of the coat protein is duplicated and a multiple cloning site with the restriction enzymes NheI, ClaI and SmaI is integrated (pPVX201 Patent WO96/12027). In the final vector construct pPVX-mCherry-CPTMV codes for the potexviral RNA-dependent RNA-polymerase and the triple gene block proteins, as well as the heterologous genes for the fluorescent protein mCherry and the coat protein of TMV. The subgenomic promoters for the expression of mCherry and the TMV coat protein are the duplicated sg promoters of the PVX coat protein.
For the construction of a TMV vector two PCR products were created. In PCR 1 the sequence of the green fluorescent protein was amplified adding a PacI restriction site at the 5′-end and a part of coat protein subgenomic promoter of TMV with the primers PacI-GFP-TMV and SOE2-CP rv (Table 1) on the plasmid pJL24. In the second PCR the coat protein sequence of PVX was amplified with the primers SOE2-CP fw and SOE2-CP-NotI creating a construct with a part of the subgenomic promoter of TMV at the 5′-end and a NotI restriction site at the 3′-end. The two PCR products were fused in a third PCR without primers and amplified in a fourth PCR with the primers PacI-GFP-TMV and SOE2-CP NotI. The final PCR product was cut with the enzymes PacI and NotI and purified over an agarose gel. The target vector pTRBOG (US 2010/0071085 A1) was treated with the same restriction enzymes and dephosphorylated with CIP. The PCR fragment was ligated into the TMV vector and resulted in the plasmid pTMV-GFP-CPPVX.
For the construction of PVX vectors with complete coat protein fusions existing PVX vectors with an N-terminal mBanana coat protein fusions with the 2A sequence were used. The PVX-mBanana-2A-CP for example is a PVX vector compatible for Agroinfection. The backbone of the plasmid is the binary pTRAc vector. The PVX genome of the UK3 strain is integrated between left border and right border of the T-DNA. The chosen vectors have a coat protein fusion of different fluorescent proteins with the 2A sequence of the Food and Mouth Disease Virus (FMDV), e.g. the yellow fluorescent protein mBanana. The 3′ part of the PVX genome was amplified with a PCR and the restriction site BspEI which is also at the beginning of the 2A sequence in the fusion vectors was added to the 5′ part with the primers 2ADelCPfw (5′-AATCCGGATAACTACGTCTACATAACCG-3′ (SEQ ID No. 11)) and M13 universe (5′-GTTGTAAAACGACGGCCAGT-3′ (SEQ ID No. 1)). The primer M13 universe binds inside the vector backbone outside the PVX genome and the primer 2ADelCpfw binds directly downstream of the coat protein coding sequence and adds the BspEI site. The product was subcloned into the pCR2.1-Topo vector (Lifetechnologies, Carlsbad, USA) amplified in E. coli SCS110 to create non methylated plasmid DNA. The pCR2.1 vector was cut with BspEI and XhoI. The target vectors with the coat protein fusions were treated with the same enzymes so the 2A sequence and the 3′-end of the PVX genome was deleted. The 3′-end without the complete coat protein sequence was then ligated into the PVX genome and confirmed by sequencing.
In the present invention a plant virus expression system with novel containment features was developed. In this system two different viral vectors complement a defective function of each other, by reciprocal coat protein exchange. By these means no viral vector alone can systemically infect a plant. In this example a TMV vector with GFP as functional heterologous ORF and the coat protein of PVX (representing the “second vector”) and a PVX vector with mCherry as functional heterologous ORF and the CP of TMV (representing the “first vector”) and a PVX vector with mBanana as functional heterologous ORF and with a complete deletion of the CP (representing a “third vector”) were created.
The viral vectors were transformed into Agrobacterium tumefaciens strain GV3101:pMP90RK for PVX based vectors, and GV2260 for TMV based vectors. The Agrobacteria were grown at 26° C. in YEB media (0.5% beef extract, 0.1% yeast extract, 0.5% peptone, 0.5% sucrose, 2 mM MgSO4) with the antibiotics carbenicillin (100 mg/1), rifampicin (50 mg/1) and kanamycin (50 mg/l for GV3101 and 25 mg/l for GV2260). After 24 hours the cultures were supplemented with 10 μM MES (pH5.6), 10 μM glucose and 20 μM acetosyringone and incubated for another day. The cultures were then set to an OD600=1 with 2×infiltration media (100 g/l sucrose, 3.6 g/l glucose, 8.6 g/l Murashige and Skoog (MS) salts, pH 5.6), supplemented with 200 μM acetosyringone and incubated for 30 minutes at room temperature. For co-inoculation of two or more different constructs the cultures were mixed so each culture would have an OD600 of 1. The mixtures were inoculated into ca. 4 weeks old N. benthamiana leaves with a syringe without needle. The plants were further incubated in a phytochamber with constant light (25000-30000 lux) at 26° C. for 12 h and 12 h 20° C. in the dark.
The plants were monitored each day under the specific conditions for the chosen fluorescent protein (Table 2). For GFP visualization a handheld UV lamp (7000 μW, Novodirect, Kehl/Rhein, Germany), for mCherry a green LCD lamp (KL2500, Leica, Wetzlar, Germany) and for mBanana a high intensity blue LED lamp (Optimax™450 Spectroline, Spectronics corporation, New York, USA) was used. Pictures were taken with a Nikon Coolpix 5400 camera (Nikon Deutschland, Dusseldorf, Germany).
In
After 26 dpi the plants were systemically infected with the two or even three different viral vectors. This demonstrates the ability of the viral vectors to complement each others innate coat protein deficiencies and secondly support systemic movement of further defective viral vectors.
In
For the isolation of total soluble proteins the leaves were harvested at different time points and homogenized with two volumes PBS (pH 7.4). Insoluble plant parts were separated during centrifugation at 13000 rpm for 10 min at 4° C. The total amount of soluble proteins was measured with a Bradford assay using Roti®Quant reagent (Roth, Karlsruhe, Germany).
For the quantification of fluorescence isolated total soluble protein was used. 50 μg of total protein were diluted in 100 μl of PBS and fluorescence profiles were measured in a microtiter plate reader (ELISA-Reader Infinite M200, TECAN Group Ltd, Männedorf, Switzerland). Fluorescent proteins were also visualized in SDS gels before Coomassie Brilliant Blue staining.
For protein analysis discontinuous SDS-PAGE and Western blotting was used. The plant sap was supplemented with 5× reducing sample buffer (62.5 mM Tris-HCl pH 6.8, 30% glycin, 4% SDS, 10% 2-mercaptoethanol, 0.05% bromophenol blue) and directly loaded on the gels for visualization of fluorescence in the gels or boiled for 5-10 minutes for Western blotting. The probes were then loaded onto a 12% SDS gel and after electrophoresis either stained with Coomassie Brilliant Blue or blotted on a nitrocellulose membrane for Western blot analysis. The membranes were blocked for 1 h with 5% skimmed milk in PBS and then incubated with a polyclonal antibody against the coat protein of PVX (DSMZ, Braunschweig, Germany) or TMV (Bioreba A G, Reinach, Switzerland) for at least 2 h at room temperature. As secondary antibody a monoclonal alkaline phosphatase-conjugated goat anti-rabbit antibody (Dianova, Hamburg, Germany) was used and the signal was visualized with nitroblue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (NBT/BCIP) (Roth, Karlsruhe, Germany).
In SDS-PAGEs the successful co-expression of the heterologous proteins was confirmed again (
These data confirm the lack of the own coat protein in the viruses and the expression of the foreign coat protein as well as a heterologous complementation of the coat protein function in co-infections.
To confirm the co-expression of mCherry and mBanana by two different PVX based vectors in the same cells, the infected leaves were analyzed with the fluorescence microscope Biorevo BZ-9000 (Keyence, Neu-Isenburg, Germany). The leaves infected with the complementing pair showed a clear co-expression of mCherry and GFP in the same cells (
We further analyzed if also a second TMV vector with a deficient CP gene can co-infect the plants with our complementing pair. However, the infection only showed a co-expression of two different PVX-based vectors in infected leaves (
These findings were confirmed in SDS-PAGE and Western blot analysis (
In summary we could confirm the co-expression of the complementing pair with an additional PVX-based vector lacking the coat protein, whereas a second TMV vector was not able to co-infect the plants with the complementing pair. With these vectors we are the first to show a co-expression of two viral vectors based on the same RNA virus in systemic infected leaves.
The capability of coat protein complementation enabling systemic movement and the simultaneous production of recombinant proteins and at the same time ensuring a biosafety containment clearly show the benefit of the invention.
For the expression of two or more recombinant proteins with viral vectors derived from one virus species, Potato Virus X (PVX) vectors were constructed which are lacking the coat protein. In these vectors the entire coat protein open reading frame was deleted, in contrast to known vectors were a part of the C-terminal coding region of the coat protein was preserved.
The viral vectors were transformed into Agrobacterium tumefaciens strain GV3101:pMP90RK and grown at 26° C. in YEB media (0.5% beef extract, 0.1% yeast extract, 0.5% peptone, 0.5% sucrose, 2 mM MgSO4) with the antibiotics carbenicillin (100 mg/1), rifampicin (50 mg/1) and kanamycin (50 mg/1). After 24 hours the cultures were supplemented with 10 μM MES (pH5.6), 10 μM glucose and 20 μM acetosyringone and incubated for another day. The cultures were then set to an OD600=1 with 2×infiltration media (100 g/l sucrose, 3.6 g/l glucose, 8.6 g/l Murashige and Skoog (MS) salts, pH 5.6), supplemented with 200 μM acetosyringone and incubated for 30 minutes at room temperature. For co-inoculation of two or more different constructs the cultures were mixed so each culture would have an OD600 of 1. The mixtures were inoculated into ca. 4 weeks old N. benthamiana leaves with a syringe without needle. The plants were further incubated in a phytochamber with constant light (25000-30000 lux) at 26° C. for 12 h and 12 h 20° C. in the dark.
The plants were monitored each day under the specific conditions for the chosen fluorescent protein (Table 3). For GFP visualization a handheld UV lamp (7000 μW, Novodirect, Kehl/Rhein, Germany), for mCherry a green LCD lamp (KL2500, Leica, Wetzlar, Germany) was used. Pictures were taken with a Nikon Coolpix 5400 camera (Nikon Deutschland, Dusseldorf, Germany).
The results clearly show that the C-terminal coding region of the coat protein is not strictly required for the replication of the PVX vector. GFP and mCherry are produced in the inoculated plant cells (
At 4 days post inoculation a good co-expression of both fluorescent proteins was visible and the expression reached the best co-expression levels at 6 dpi for this enzyme combination (
For the isolation of total soluble proteins the leaves were harvested at different time points and homogenized with two volumes PBS (pH 7.4). Insoluble plant parts were separated during centrifugation at 13000 rpm for 10 min at 4° C. The total amount of soluble proteins was measured with a Bradford assay using Roti®Quant reagent (Roth, Karlsruhe, Germany).
For the quantification of fluorescence isolated total soluble protein was used. 50 μg of total proteins were diluted in 100 μl of PBS and fluorescence profiles were measured in a microtiter plate reader (ELISA-Reader Infinite M200, TECAN Group Ltd, Männedorf, Switzerland). Fluorescent proteins were also visualized in SDS gels before Coomassie Brilliant Blue staining.
For protein analysis discontinuous SDS-PAGE and Western blotting was used (Laemmli, 1970). The plant sap was supplemented with 5× reducing sample buffer (62.5 mM Tris-HCl pH 6.8, 30% glycin, 4% SDS, 10% 2-mercaptoethanol, 0.05% bromophenol blue) and directly loaded on the gels for visualization of fluorescence in the gels or boiled for 5-10 minutes for Western blotting. The probes were then loaded onto a 12% SDS gel and after electrophoresis either stained with Coomassie Brilliant Blue or blotted on a nitrocellulose membrane for Western blot analysis. The membranes were blocked for 1 h with 5% skimmed milk in PBS and then incubated with a polyclonal antibody against the coat protein of PVX (DSMZ, Braunschweig, Germany) for at least 2 h at room temperature. As second antibody a monoclonal alkaline phosphatase-conjugated goat anti-rabbit antibody (Dianova, Hamburg, Germany) was used and the signal was visualized with nitroblue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (NBT/BCIP) (Roth, Karlsruhe, Germany).
With SDS-PAGE and Western blot analysis it could be proven, that the PVX vectors express no coat protein (data not shown). Although former publications indicate the demand for the C-terminal coding region of the coat protein for the replication of the PVX vector, here it could be shown that the coat protein coding sequence can be completely deleted. On the contrary a good co-expression of two different and potentially more PVXΔCP vectors could be shown.
To confirm the co-expression of two different PVX based vectors in the same cells the infected leaves were analyzed with the fluorescence microscope Biorevo BZ-9000 (Keyence, Neu-Isenburg, Germany). The pictures confirmed no signals for the non-infected N. benthamiana plant (
In summary, we could confirm the co-expression of different PVX-based vectors lacking its CP in agroinfiltrated leaves. This findings show that PVX vectors are capable of co-infections after knockout of CP functions. This is in contrast to the aforementioned known PVX-based vectors and also to TMV-based vectors, which are not capable of co-infections even after deletion of the MP and CP genes in the vectors (Julve et al., 2013).
Due to the coat protein deletion, containment is ensured. Moreover, the present invention overcomes the incompatibility of two or more vectors in one cell and therefore allows simultaneous expression of two or more heterologous ORFs located on different viral vectors in the same plant cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13160627 | Mar 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/055730 | 3/21/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/147235 | 9/25/2014 | WO | A |
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| 20160145635 A1 | May 2016 | US |
