WO2002043650A2 - Systems and methods for delivering interferon to a subject - Google Patents
Systems and methods for delivering interferon to a subject Download PDFInfo
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- WO2002043650A2 WO2002043650A2 PCT/IL2001/001099 IL0101099W WO0243650A2 WO 2002043650 A2 WO2002043650 A2 WO 2002043650A2 IL 0101099 W IL0101099 W IL 0101099W WO 0243650 A2 WO0243650 A2 WO 0243650A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
Definitions
- the present invention relates to systems and methods for providing supplemental interferon to a subject and, more particularly, to systems and methods of administering interferon via an edible plant.
- the present invention further relates to a general method for providing an orally bio-available protein to a subject.
- RNA virus vectors have been developed (Takamatsu et al., 1987; Chapman et al., 1992; Dolja et al., 1992; Kumagai et al., 1993; Rommens et al., 1995; Porta and Lomonossoff, 1996; Scholthof et al., 1996; Arazi et al., 2001).
- viruses are transmitted to other plants by their natural vectors in the field (Matthews, 1991). This issue raises serious concerns for use of plant virus vectors in the field.
- Zucchini yellow mosaic virus is one of the most devastating diseases worldwide of cucurbit species such as cucumber, squash, melon and watermelon (Desbiez and Lecoq, 1997).
- ZYMV is a member of the potyviridae family, the largest group of plant-infecting viruses (Shukla et al., 1994).
- the ZYMV genome consists of a single messenger-polarity RNA molecule of about 10 kb, encapsidated by multiple copies of a single coat protein (CP) forming a flexuous filamentous particle (Gal-On et al., 1992 J. Gen. Virol. 73: 2183-2187.).
- Viral RNA is translated into a large polyprotein that is proteolytically processed to 8-9 functional proteins by three virus-encoded proteases: PI, HC-Pro and NIa (Riechmann et al., 1992, Revers et al., 1999).
- the PI (Verchot et al., 1991) and HC-Pro (Carrington et al., 1989) proteinases are the first and second proteins located at the N' -terminus region of the polyprotein and catalyze autoproteolytic cleavage at their own C -terminus.
- the NIa protease is responsible for cis and trans proteolytic cleavages of the remainder of the viral polyprotein (Carrington et al., 1988; Riechmann et al., 1992).
- potyviruses are promising expression vectors, since their proteolytic processing strategy of gene expression requires that a foreign protein, synthesized as part of the viral polyprotein, is produced in equimolar amounts with all viral proteins (Riechmann et al., 1992; Revers et al., 1999). Moreover, taking into account the helicoidal morphology of viral particles, no packaging limitations would be expected for rather large genome insertions (Dolja et al., 1992; Scholthof et al., 1996).
- Interferon holds considerable promise as a drug in treating a number of medical conditions because of its therapeutic capabilities.
- Interferon is a naturally occurring protein with immuno-modulatory and anti- viral properties, that is produced in cultured human cells or in E. coli as a drug (reviewed by Walter et al., 1998).
- Interferon-alpha and Interferon-beta are both Type I interferons.
- Type I interferons are a large class of naturally-occurring cytokines which includes over 16 subclasses of IFN-alpha, plus IFN-beta and IFN-omega.
- the Type I interferons bind to a single cell surface receptor, and stimulate a complex sequence of signal transduction events leading ultimately to anti-viral, anti-proliferative and other immunomodulatory effects, cytokine induction, and HLA class I and class II regulation (Pestka et al., Annu. Rev. Biochem., 1987 56: 727).
- Alpha interferons are used widely for the treatment of a variety of haematological malignancies including hairy cell leukaemia, chronic myelogenous leukaemia, low grade lymphomas, cutaneous T-cell lymphomas, and solid tumours such as renal cell carcinoma, melanoma, carcinoid tumours and AIDS-related Kaposi's sarcoma (Gutterman, J.
- Interferon-beta is licensed for clinical use in treatment of relapsing-remitting multiple sclerosis and chronic viral hepatitis B and C.
- a commercial interferon alpha 2a (Roferon-A; see http://www.rocheusa.com/products/roferon) is claimed to normalize serum ALT, improve liver histology and reduce viral load in patients with chronic hepatitis C.
- the product is further indicated for the treatment of chronic hepatitis C, hairy cell leukemia and AIDS-related Kaposi's sarcoma in patients 18 years of age or older.
- chronic phase Philadelphia chromosome positive chronic myelogenous leukemia (CML) patients who are minimally pretreated (within 1 year of diagnosis).
- the interferon used was human interferon-alpha prepared by the method of Cantell, administered in phosphate buffered saline, at a dose of 0.01 to 5 IU per pound body weight. While these specifications suggest that such low doses of interferon administered to the oropharyngeal mucosa, preferably in a form adapted for prolonged contact with the oral mucosa, may be efficacious for treatment of a wide variety of conditions including cancer, the experimental evidence for conditions other than shipping fever, feline leukaemia, canine parvovirus and theileriosis is largely anecdotal. In particular, no properly controlled trials of this treatment in any animal model for human cancers are presented.
- Carrington specifically teaches "A method for expressing at least one protein in a plant or plant cell, said method comprising infecting a plant or plant cell susceptible to a polyprotein-producing potyvirus with said potyvirus, expressing said potyvirus to produce said polyprotein, wherein said potyvirus codes for at least one protein non-native to the potyvirus and wherein said non-native protein is released from said polyprotein by proteolytic processing.”
- these teachings contain neither a hint nor a suggestion that such a non-native protein would be orally bio-available.
- the teachings of Carrington include hypothetical production of insulin, hGH, interleukin, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor
- a system for providing supplemental interferon to a subject includes: (a) a viral vector, the vector designed and constructed to be capable of infecting a plant and expressing at least a portion of an interferon gene therein and (b) the plant, at least a portion of the plant being edible by the subject.
- the gene product of the at least a portion of an interferon gene is bio-available to the subject consuming the at least a portion of said plant.
- a system for providing supplemental interferon to a subject includes: (a) a DNA sequence designed and constructed to be capable of expressing at least a portion of an interferon gene in a plant; and (b) the plant, at least a portion of the plant being edible by the subject and the plant susceptible to transformation by the DNA sequence.
- the gene product of the at least a portion of an interferon gene is bioavailable to the subject consuming 5 the at least a portion of said plant.
- a method for providing supplemental interferon to a subject includes the steps of: (a) causing a plant to express at least a portion of an interferon gene in at least some cells thereof; and (b) feeding at least a l o portion of the plant to the subj ect.
- the method includes the steps of: (a) causing a plant to express at least a portion of the orally bio-available protein in at least some cells thereof; and (b)
- the viral vector is a potyvirus vector.
- the potyvirus is zucchini yellow mosaic virus (ZYMV).
- ZYMV zucchini yellow mosaic virus
- the ZYMV is an attenuated strain containing a mutation as listed in SEQ ID NOs. : 7 and 8.
- an interferon gene includes a mammalian 5 interferon gene sequence.
- the mammalian interferon gene sequence includes at least a portion of a human interferon gene sequence.
- the human interferon gene sequence is selected from the group consisting of interferon alpha 2a (SEQ ID NO.: 1) and any gene at least 85% homologous thereto as analyzed by the FastA program.
- the FASTA program family (FastA, TFastA, FastX, TFastX, and SSearch) was written by Professor William Pearson of the University of Virginia Department of Biochemistry (Pearson and Lipman, Proc. Natl. Acad. Sci, USA 85; 2444-2448 (1988)). In collaboration with Dr.
- the vector expresses at least a portion of a protein selected from the group consisting of the interferon alpha 2a gene product (SEQ ID NO.: 2) and any protein at least 85% homologous thereto as analyzed by the FastA program.
- transmissibility of the viral vector from the plant to a second plant is prevented by a mutation in the viral vector.
- system further includes a means for introducing the DNA sequence into at least one cell of the plant, thereby transforming the cell.
- the DNA sequence includes a left border and a right border of the agrobacterium T-DNA.
- the step of causing is accomplished by an action selected from the group consisting of: (i) infecting at least one cell of the plant with a viral vector, the viral vector designed and constructed to be capable of expressing at least a portion of an interferon gene therein; and (ii) transforming at least one cell of the plant with a DNA sequence designed and constructed to be capable of expressing at least a portion of an interferon gene therein.
- the step of causing is accomplished by an action selected from the group consisting of: (i) infecting at least one cell of the plant with a viral vector, the viral vector designed and constructed to be capable of expressing at least a portion of a gene encoding the orally bio-available protein therein; and (ii) transforming at least one cell of the plant with a DNA sequence designed and constructed to be capable of expressing at least a portion of a gene encoding the orally bio-available protein therein.
- the human interferon gene sequence is selected from the group consisting of interferon beta (SEQ ID NO.: 11) of interferon gamma (SEQ ID NO.: 13) and any gene at least 85% homologous to either of the interferon genes as analyzed by the FastA program.
- the vector expresses at least a portion of a protein selected from the group consisting of the interferon beta gene product (SEQ ID NO.: 12), the interferon gamma gene product (SEQ ID NO.: 14) and any protein at least 85% homologous to either of the interferon gene products as analyzed by the FastA program.
- the present invention successfully addresses the shortcomings of the presently known configurations by providing systems and methods of providing supplemental interferon, or other orally bio-available proteins, to a subject.
- Figures 1 A and B depict a viral vector for use in conjunction with a system according to the present invention
- Figures 2 A and B illustrate stability and accumulation of recombinant AGII in plants by means of an immunoblot and histogram
- FIGS 3 A-D illustrate that AGII-interferon alpha-2a (AGII-IFN) does not affect cucumber development or yield, and is stable in planta by means of photographs, histograms and an RT PCR analysis;
- AGII-IFN AGII-interferon alpha-2a
- Figures 4 A-C illustrate AGII-IFN-mediated synthesis of IFN in squash and cucumber leaves by means of histograms and an immunoblot;
- Figures 5 A-D illustrate AGII-IFN mediated synthesis of IFN in squash and cucumber fruits and fruit parts as histograms
- FIGS 6 A-H illustrate expression of foreign proteins in various plant parts via AGII vector.
- the present invention is of systems and methods for providing supplemental interferon to a subject.
- the present invention can be used to deliver interferon orally as a portion of an edible plant, for example a cucurbit fruit such as cucumber, squash or melon.
- the present invention further relates to a general method for providing an orally bio-available protein to a subject.
- Figures 1 A and B illustrate a viral vector for use as part of a system according to the present invention. Specifically, the AGII strain of ZYMV with IFN gene inserted into its genome is illustrated.
- Figure 1A is a schematic presentation of the AGII genome.
- AGII non-coding (hatched shading), and coding (open boxes) regions including the inserted foreign gene (FG) are shown.
- Arrows indicate NIa protease involved in proteolysis of foreign gene product.
- NIa cleavage sites are indicated by /.
- Restriction enzyme sites used for sub-cloning are indicated.
- Nucleotides specifying restriction endonuclease recognition sites, inserted to create the polylinker and their encoded amino acid residues are indicated in bold in Figure IB. Insertion of interferon gene occurs between the Nib and CP genes. Amino acid sequence is indicated by italics.
- the viral vector of the system is designed and constructed to be capable of infecting a plant, expressing at least a portion of an interferon gene therein. Therefore, the gene product of the at least a portion of an interferon gene is bio-available to the subject consuming the at least a portion of said plant. Delivery may be effected, for example, using what is commonly referred to as a "gene gun" by those ordinarily skilled in the art.
- the viral vector is a potyvirus vector, more preferably the potyvirus is zucchini yellow mosaic virus (ZYMV), more preferably still the ZYMV is an attenuated strain, for example one containing a mutation as listed in SEQ ID NOs.: 7 and 8, the ZYMV-AGII engineered strain.
- the at least a portion of an interferon gene may include a mammalian interferon gene sequence or a recombinant interferon gene derived from a combination of naturally occurring interferon genes.
- the mammalian interferon gene sequence may include, for example, at least a portion of a human interferon gene sequence including, but not limited to, interferon alpha 2a (SEQ ID NO.: 1).
- the mammalian interferon gene may include at least a portion of a gene at least 85% homologous to the interferon 2 alpha gene as analyzed by the FastA program.
- the human interferon gene sequence may be an interferon beta, for example SEQ ID NO.: 11 or an interferon gamma, for example, SEQ ID NO.: 13 or any gene at least 85% homologous to either of these interferon genes as analyzed by the FastA program.
- the vector may express at least a portion of an interferon beta gene product, for example, SEQ ID NO.: 12, or an interferon gamma gene product, for example, SEQ ID NO.: 14 or any protein at least 85% homologous to either of these interferon gene products as analyzed by the FastA program.
- an interferon beta gene product for example, SEQ ID NO.: 12
- an interferon gamma gene product for example, SEQ ID NO.: 14 or any protein at least 85% homologous to either of these interferon gene products as analyzed by the FastA program.
- the vector expresses at least a portion of a protein including, but not limited to, the interferon alpha 2a gene product (SEQ ID NO.: 2) or any protein at least 85% homologous thereto as analyzed by the FastA program.
- FastA may be implemented, for example, as part of the BLAST or GCG program packages.
- BLAST and FastA are services offered by the NCBI of the National library of Medicine of the National Institutes of Health. Both are accessible via the Internet, and one ordinarily skilled in the art of molecular biology will be familiar with access and use thereof.
- transmissibility of the viral vector from the plant to a second plant is prevented by a mutation therein.
- the system of the present invention further includes the plant, at least a portion of which is edible by the subject.
- the present invention is further embodied by a system for providing supplemental interferon to a subject.
- the system includes a DNA sequence designed and constructed to be capable of expressing at least a portion of an interferon gene in a plant.
- the interferon gene is as described hereinabove.
- the system further includes the plant, at least a portion of which is edible by the subject.
- the plant is susceptible to transformation by the DNA sequence.
- the system further includes a means for introducing the DNA sequence into at least one cell of the plant, thereby transforming the cell.
- These means may include, for example, what is commonly referred to as permanent or transient "agrobacterium mediated transformation” or use of what is commonly referred to as a "gene gun” by those ordinarily skilled in the art of plant transformation.
- DNA sequence itself may include portions designed to facilitate genetic transformation of plant cells. These portions may include, for example, a left border and a right border of the agrobacterium T plasmid.
- the present invention is further embodied by a method for providing supplemental interferon to a subject.
- the method includes the step of causing a plant to express at least a portion of an interferon gene in at least some cells thereof.
- the phrase "at least some cells thereof refers to cells found within a plant, seeds thereof, and tissue culture cells derived therefrom.
- the method further includes the step of feeding at least a portion of the plant to the subject.
- the interferon is as described hereinabove. It will be appreciated that the step of causing may be accomplished in a wide variety of ways. For example, "causing" may include infecting at least one cell of the plant with a viral vector.
- the viral vector is designed and constructed to be capable of expressing at least a portion of an interferon gene within the infected cell.
- the vector is further designed and constructed to cause assembly of virions, which infect adjacent cells. More preferably, delivery to a single cell of the plant results in systemic infection of the plant.
- causing may include transforming at least one cell of the plant with a DNA sequence designed and constructed to be capable of expressing at least a portion of an interferon gene therein.
- a transformation may be either a somatic cell transformation or a germ line transformation.
- the present invention is further embodied by a method for providing an orally bio-available protein to a subject.
- the method includes the step of causing a plant to express at least a portion of the orally bio-available protein in at least some cells thereof.
- the method further includes the step of feeding at least a portion of the plant to the subject.
- the step of causing may be affected in a variety of ways, as detailed hereinabove for interferon, which is an example of an orally bio-available protein.
- Feeding may involve, for example, administration of fresh plant parts, dried plant parts, lyophilized plant parts, ground plant parts, powdered plant parts, juice extracted from plant parts, preserved (e.g. pickled or jellied) plant parts or plant parts subjected to any combination of processes including one of these processes.
- AGII-IFN AGII-interferon alpha 2a
- Figure 3A Consistent with GFP expression in the fruits, IFN-2a activity measured in squash and cucumber was concentrated mainly in fruit embryonic tissue. Accumulation of AGII-IFN virions in fruits is a result of foreign gene expression mediated by viral replication and spread.
- IFN-2a in cucumber leaves varied in accordance with the leaf developmental stage. In fully expanded leaves, weighing more than 10 g, the IFN-2a activity had declined while virus accumulation remained stable. This miscorrelation between AGII-IFN virion accumulation and foreign gene expression levels was probably due to a decrease of virus replication in mature tissue, together with a relatively turnover of interferon alpha-2a compared with the stability of the virion.
- the addition of seven amino acids at the carboxyl terminus of the IFN-2a in the AGII expression system did not affect its activity as, confirming the earlier observation of Petska that addition of amino acid residues to the termini of interferon did not affect its activity (Pestka et al, 1987). It is noteworthy that no IFN activity was lost when plant tissue was lyophilized.
- interferon which is expressed in cucurbit fruit, may be administered orally to treat patients.
- the present invention demonstrates the feasibility of using a potyvirus, for example the engineered attenuated AGII strain of ZYMV as an expression vector in cucurbits.
- the primary advantage of the present invention with respect to prior art is that the disclosed invention allows a significant reduction in the cost of production of interferon by eliminating the need for purification.
- edible plant parts may be subjected to simple processes such as grinding and drying to produce, for example, freeze dried fruit powder
- the simplest embodiment of the invention involves giving the subject fresh produce to eat.
- distribution of plants to patients is within the scope of the claimed invention.
- the present invention eliminates not only purification costs, but greatly reduces distribution, storage, shipping and packaging costs as well.
- system and method of the present invention serve, to a large degree to eliminate concerns regarding toxic contaminants in the interferon preparation. This stems from the fact that, since the interferon is not prepared in bacteria, it is unlikely that bacterial toxins will be introduced during the manufacturing process. Similarly, there is no danger of introduction of human pathogens during the manufacturing process because human cell cultures are not employed. Thus, concerns about residual antibiotics, artificial preservatives and cell culture additives are also eliminated by practice of the present invention.
- the present invention has all the inherent advantages of prior art oral administration methods including ease and comfort of administration. These factors make self-administration more acceptable to patients.
- the plant cell wall can provide a slow release effect in vivo (Walmsley and Arntzen, 2000), perhaps making the present invention more suitable for use in certain clinical applications, for example Hepatitis C .
- the plant cell wall makes the present invention "saliva insoluble", thereby differentiating it from the prior art. It is believed that the interferon of the present invention is protected from protease activity in the digestion system. As a result, the interferon is available for subsequent absorption in the gut wall, a possibility which is typically ruled out by prior art teachings.
- lyophilized plant material should be stable at room temperature without degradation of interferon contained therein. This serves to break the "cold chain" of transportation and storage, further reducing the final cost of each unit of delivered interferon. Further, this capacity for distribution without refrigeration makes practice of the present invention more feasible in less developed areas of the world. Such a consideration is crucial, for example in treatment of HCV and HTV.
- Interferon alpha-2a has proven to be exceptionally stable.
- the present invention offers several additional advantages relative to known plant bio-reactor systems. Yield is good because the vector is benign with respect to the host plant. Non-transmissibility by the natural aphid vector is easily achieved. The foreign gene, because it is not incorporated into the germ line of the plant, is not transmissible in seeds or pollen of the infected plant. In addition, transgenic plants require a significant development time due to requirements for screening and propagation. The present invention is free of this limit. Further, the present invention does not require delivery of viral RNA, relying instead upon delivery of a cDNA vector. This serves to significantly reduce the chance of accidental delivery to a plant because the cDNA expression vector is not an infectious virus .
- the aphid non-transmissible mutation was introduced in two steps. First, a Pstl site was introduced in the NIa protease motif (DTVMLQ) within the Nib gene, between the encoding sequences of Leu and Glu (LQ), by site-directed mutagenesis on AG (Gal-On, 2000), with the partial clone pKS? sacI22 (7515-9591) used as a template. The resulting mutant clone was designated pKS? Sacl-Pstl. A nucleotide change, altering coat protein (CP) residue Ala 9 to Thr, was then introduced by PCR on pKS? Sacl-Pstl as a template with an appropriate sense oligonucleotide
- GFP jellyfish green fluorescent protein
- uidA beta-glucuronidase
- GUS glycosylcholine
- the coding region of GFP was amplified by PCR, using sense and antisense oligonucleotides (SEQ ID Nos.; 17 and 18) that were both flanked by P-stl sites.
- the amplified fragments were digested by Pstl and cloned into the partial clone pKS ⁇ SacI-Pstl-poly.
- a similar cloning strategy was used for uidA (SEQ ID NO.: 16) using sense and antisense oligonucleotides (SEQ ID Nos.; 19 and 20), except that the antisense primer contained a flanking SaR site instead of -E .
- Amplified PCR fragments were then digested by Pstl and Sail and cloned into pKS ⁇ SacI-Pstl-poly.
- pKS ⁇ SacI-Pstl-poly clones were double-digested by Sacl Mlul, and the resulting fragment containing the foreign gene was cloned into AGII genome to create AGII-GFP and AGII-GUS . Insertion of human interferon-alpha 2a dFN-2a) genes into the AGII genome
- the coding region of IFN (SEQ ID NO.: 1) and CMV-CP were amplified by PCR, using sense and antisense oligonucleotides (SEQ ID Nos.: 3 ands 4) that were both flanked by Sail sites.
- the amplified fragments were digested by Sail and cloned into the partial clone pKS? Sad- Sail -poly.
- Amplified PCR fragments were then digested by Sail and cloned into pKS? Sad- Sail -poly.
- pKS? Sad- Sail -poly clones were double-digested by Sacl/Mlul, and the resulting fragment containing the IFN gene was cloned into AGII genome to create AGII-IFN.
- RT-PCR of viral progeny was conducted in a one-tube single-step method modified from Sellner et al. (1992). A 50-microliter volume was used containing the polylinker flanking primers
- Infected plant material was subjected to enzyme-linked immunosorbent assay (ELISA) with anti-ZYMV CP polyclonal antibody, as described previously by Antignus et al. (1989).
- ELISA enzyme-linked immunosorbent assay
- the quantity of AGII-IFN was estimated by checking against a known amount of purified AGII virion in the ELISA plate.
- Plant tissue was collected, frozen in liquid N 2 and lyophilized for 24 h. Lyophilized tissue was ground by pestle and mortar and extracted in PBS with a ratio of 1:1-1.5 (dry weight tissue/per unit volume of PBS). One milliter of the homogenate was centrifuged for 10 min at 10,000 g in an Eppendorf minifuge, and the supernatant was used for ELISA, immunoblot analysis and interferon activity assay. IFN activity was assayed in 96-well microtiter plates by the inhibition of vesicular stomatitis virus cytopathic effect on human Wish (ATCC CCL-25) cells, as described previously (Rubinstein et al, 1981). Calibration standards of IFN were included in every plate.
- IFN activity was expressed in international units per milliliter (IU/ml), 2x10 8 IU are equivalent to 1 mg IFN.
- IU/ml international units per milliliter
- 2x10 8 IU are equivalent to 1 mg IFN.
- immunoblot ECL, Amersham-Pharmacia Biotech, UK
- extracts were separated on 15% SDS-PAGE and immunoblotted with an anti-IFN polyclonal antibody at 1 :1000 dilution.
- Squash plants (cv. Guliver) were inoculated with a ZYMV-AGII-IFN cDNA at the seedling stage (4 days post emergence). Verification of infection was determined for each plant two weeks post infection by a DAS-ELISA with specific anti-ZYMV antibodies. Each plant was tested for interferon alpha biological activity 3 weeks post infection by a standard interferon alpha assay. Fruits were collected from AGII-IFN infected plants and AGII infected plants as a negative control 38 days after planting. Picked fruit was washed carefully, sliced, freeze-dried, and ground to a homogeneous powder.
- Powder was then extracted with phosphate saline buffer in a ratio of 1/7.6 (w/v) and the soluble fraction was collected and tested for its interferon alpha biological activity. Activity of 120,000 IU/ml interferon alpha was obtained. Similar procedure was done for negative control fruit. All interferon assays employed the National Institute of Health interferon alpha as a standard for activity.
- Cytokines were measured in the serum by ELISA for IL4, IL10, IL12, and IFN gamma using Genzyme Diagnostics kits (Genzyme Diagnostics, Boston, MA, USA) according to manufacturer's instructions. Serum levels were measured in all mice from all groups 14 days after starting the oral administration.
- Example 1 Engineering AG to be an aphid non-transmissible virus
- ZYMV like other potyviruses, is naturally transmitted by aphids in a non-persistent manner (Desbiez and Lecoq, 1997). It has been shown that the CP As ⁇ 8 Ala 9 Gly 10 (DAG) motif is involved in transmission of ZYMV by aphids, and that mutation of alanine to threonine abolishes ZYMV transmission by aphids (Gal-On et al, 1992). A site-directed mutagenesis was performed to switch Ala 9 residue to Thr (SEQ ID NOs.: 9 and 10) in the DAG motif of the AG CP, and the resultant mutant virus was designated AGI .
- DAG CP As ⁇ 8 Ala 9 Gly 10
- AGI Inoculation of AGI cDNA to squash plants resulted in infection indistinguishable from that caused by AG.
- An aphid transmission assay (Antignus et al, 1989) demonstrated that the AGI could not be transmitted by aphids, and this characteristic remained stable for prolonged propagation and several plant-to-plant mechanical inoculation passages. Based upon these encouraging results, AGI became the basis for further manipulation as detailed in hereinabove and used in example 2.
- Example 2 Expression of reporter genes via AGII vector in various cucurbits tissues including the edible fruit
- the bacterial uidA and jellyfish GFP genes were inserted into the NIb-CP site (Fig. IB).
- Fig. IB Essentially 100% of squash plants inoculated by particle bombardment with the recombinant cDNA corresponding to AGII-GFP and AGII-GUS became infected.
- Typical vein clearing and mild mosaic symptoms appeared in AGII-GFP infected squash 5-7 dpi.
- AGII-GUS a 4-d delay of symptom appearance was observed.
- AGII-GUS-infected squash was analyzed for GUS activity 15 dpi, and GUS staining was observed in leaves, stems and roots (Fig. 6A-D). Distribution of GUS staining was not uniform in infected leaves, and staining concentrated around the major veins and neighboring cell clusters (Fig. 6A). Stems showed uniform staining, concentrated around the vascular tissue (Fig. 6B-C). Interestingly, strong GUS staining was detected in adventives (Fig. 6C) and lateral roots (Fig. 6D).
- AGII-GFP infected cucumbers were analyzed for GFP by visualization under UV light. Green fluorescence was observed in AGII-GFP infected leaves, stems, flowers and fruit (Fig. 6E, F-right, G, H-left), indicating GFP expression in these organs. Similar fluorescence was not observed in identically developed organs infected with AGII (Fig. 6F-left, 6H-right); a non-uniform fluorescence was seen in leaves (Fig. 6E) and male flowers (Fig. 6G). In fruits, fluorescence was located mainly in the embryonic tissue and to a lesser degree in the peel layer or mesocarp (Fig. 6H-left).
- Example 3 Expression of a biologically active human interferon-alpha 2a via AGII in cucurbits
- IFN coding sequence into the NIb-CP insertion site (Figs. 1A and IB). Plasmids containing AGII-IFN cDNA were inoculated on squash and cucumber plants yielding full infectivity. Symptoms similar to those elicited by the parental virus AGII were observed within 5-7 dpi. The presence of the IFN-2a gene within the AGII genome was verified by RT-PCR analysis of the progeny virus containing IFN-2a gene between Nib and CP.
- Figure 2A is an RT-PCR analysis of progeny viral RNA.
- Total RNA was extracted from AGII-IFN systemically infected leaves, at 14 or 24 dpi, and subjected to RT-PCR with primers flanking the NIb-CP insertion site. Plasmids harboring cDNA of AGII-IFN (pAGII-IFN) were subjected to PCR as a control. Amplified products were then analyzed on an EtBr agarose gel (image negative is shown) The expected size (bp) of amplified fragment, containing the inserted gene and flanking 476 bp of AGII, is marked by an arrow. ind ⁇ ll-Eco ⁇ I digested Lambda DNA was used as molecular weight marker
- Figure 2B illustrates accumulation AGII-IFN in squash plants. Accumulation is expressed as the percentage of AGII accumulation (100%). The level of the virus was determined by DAS-ELISA and is the average of three independent samples taken from three independent plants. All samples were collected from developmentally equivalent leaves at the indicated dpi.
- the IFN gene was maintained intact in the AGII genome at least 24 dpi. (Fig. 2A) and accumulated to similar levels as AGII (Fig. 2B). Moreover, stability of the IFN gene was maintained after six serial passages (at 3-week intervals) from plant to plant.
- FIG. 3A includes photographs of AGII-IFN-infected and virus-free plants, which were taken 45 days after seedling inoculation. No difference is apparent. Plant infection was verified by DAS-ELISA.
- AGII-IFN infection was evaluated by monitoring the plant phenotype and symptom expression, and by estimating the crop yield.
- cucumber plants infected with AGII-IFN developed normally.
- AGII-IFN plants did not show any visible symptoms on their leaves or fruit, and were phenotypically indistinguishable from virus-free plants (Fig. 3A).
- Infected squash plants also developed normally, showing only mild diffused mosaic symptoms on their leaves, and no symptoms on their fruits (not pictured).
- Crop yield was measured by collecting marketable cucumber fruits (about 60 g each) for a period of 1 month, beginning 3 weeks post inoculation.
- Figure 3B is a histogram comparing cucumber yield among virus-free plants, and AGII-and AGII-IFN-infected plants. Fruits (average size of 60 g) were collected from plants during 1 month. Data are given as the mean ⁇ SD of three or four independent plants. A yield of about 2 kg of fruit per plant was obtained in virus-free plants (Fig. 3B), and a comparable yield was obtained in AGII-IFN and AGII inoculated plants (Fig. 3B).
- Figure 3C is a histogram showing accumulation of AGII and AGII-IFN viruses in cucumber plants. The level of virus was determined by DAS-ELISA in four samples from independent plants. All samples were collected from developmentally equivalent leaves at 45 dpi. Similar levels of virus accumulation were measured in the leaves of these plants (Fig. 3C), demonstrating that virus infection did not affect fruit production.
- Figure 3D is an RT-PCR analysis of progeny viral RNA. Total RNA was extracted from leaves of recombinant virus (as indicated) infected plants or from virus-free plants, and subjected to RT-PCR with primers flanking the IFN insertion point.
- a plasmid harboring AGII-IFN cDNA (pAGII-IFN) was subjected to PCR as a control.
- the expected size (bp) of the fragment with (995) or without (476) the IFN is marked by an arrow.
- Htn ⁇ II-iscoRI-digested Lambda DNA was used as a molecular weight marker (M); it is noteworthy that the IFN gene within AGII-IFN remained intact in tested plants (plants numbers 17 and 20 are shown), even 2 months post inoculation, as confirmed by RT-PCR (Fig. 3D).
- Figure 4A is a histogram of IFN activity measured in leaves of AGII-IFN-inoculated cucumber at 60 dpi.
- Figure 4B is an Immunoblot analysis of samples tested in figure 4A. Soluble protein extracts (70 ⁇ g) were analyzed by using anti-IFN polyclonal antibody. Recombinant IFN (Rec, 4 ng) was used as a control for gel mobility. Immunoblot analysis of samples which had been analyzed for interferon revealed the presence of a protein band that reacted with an anti-IFN antibody. Moreover, band intensity correlated with the level of IFN activity, indicating that this band represented IFN (Fig. 4B). As predicted, this band exhibited a slightly slower gel mobility than that of recombinant hIFN-2a due to the addition of eight amino acid residues to the IFN sequence (Fig. IB).
- Figure 4C illustrates IFN activity measured in leaves of AGII-IFN inoculated squash at 30 dpi. The values obtained after subtracting the background activity (of AGII-infected squash). Data are given as the mean ⁇ SD of three independent measurements.
- IFN activity in young leaves (4 th from the top, Fig. 4C) was comparable with that in those of cucumber (Fig. 4A).
- the amount of AGII CP in the tested leaves was measured by quantitative DAS-ELISA (Fig. 4A, below histogram). An increase in the amount of AGII CP was measured as the leaf matured. No correlation was obtained between CP accumulation and the biological activity of IFN. This was especially prominent in fully expanded leaves that contained the greatest amount of AGII CP and exhibited the lowest IFN activity (Fig. 4A).
- Figures 5A and B depict IFN activity found in fruit extracts from AGII-IFN inoculated cucumber (Fig. 5A) or squash (Fig. 5B) plants, 60 or 30 dpi, respectively.
- the values obtained after subtracting the background activity (of AGII-infected plants). Data are given as the mean ⁇ SD of three independent measurements. Tested fruit developmental stage (weight) and AGII-IFN virus amount are presented below the histogram. n.d. not determined.
- the IFN activity measured in fruits from the same cucumber and squash plants was two-to fourfold lower than activity in leaves (Figs. 4A and 4C) of the same plants.
- a twofold greater increase in IFN activity was measured in squash fruits than in those of cucumber (Figs. 5A and 5B).
- Accumulation of AGII CP in cucumber fruits was two orders of magnitude less than in leaves, which is consistent with the IFN activity difference between the two organs.
- Figures 5 C and D depict IFN activity found in fruit parts from AGII-IFN inoculated cucumber plants 20 (Fig. 5C) or squash (Fig. 5D) 60 or 30 dpi, respectively.
- Example 5 Effect of oral administration of human interferon alpha 2a produced in squash on experimental colitis in mice
- mice were normal inbred females mice maintained on standard laboratory chow and kept in 12 hr light/dark cycles. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBs). Treated mice were dosed orally with extract of squash fruit expressing interferon alpha 2a for 14 days following colitis induction.
- TNBs trinitrobenzene sulfonic acid
- colitis induced mice received either similar amounts of extract from squash fruit not expressing interferon alpha 2a or bovine serum albumin. Colitis was assessed in each group by standard clinical, macroscopic and microscopic scores. Serum cytokine secretion was determined by ELISA.
- this experiment demonstrates that oral administration of squash extract from fruit expressing human interferon alpha 2a exerted a positive impact on the intestine of colitis induced mice. This indicates that the interferon was absorbed in the digestive tract after swallowing in contrast to prior art teachings. Whether the observed effect is systemic or local, it represents a significant improvement in the applicability of oral interferon treatment to clinical medicine.
- Table 2 Effect of oral administration of extracts from squash fruit expressing interferon alpha 2a in a mouse colitis model
- Antignus Y, Raccah, B, Gal-On, A, Cohen, S, 1989.
- HC-Pro gene alters symptom expression in cucurbits and exhibits protection against the severe homologous virus.
- Microbe Interact. 13, 316-324 Gopinath, K, Wellink, J, Porta, C, Taylor, K. M, Lomonossoff, G. P, van Kammen, A, 2000. Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants. Virology 267, 159-173. Gotsman I, Shlomai A, Alper R, Rabbani E, Engelhardt D, Ilan Y, 2001.
- Rapid, high-level expression of biologically active a-trichosanthin in transfected plants by an RNA viral vector Proc. Natl. Acad. Sci. USA 90, 427-430. Kumagai, M. H, Donson, J, della-Cioppa, G, Grill, L. K, 2000. Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector. Gene. 245, 169-174.
- Virology 8 15-23. Verchot, J, Koonin, E. V, Carrington, J. C, 1991. The 35-kDa protein from the N-terminus of a potyviral polyprotein functions as a third virus-encoded proteinase.
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WO2005035767A1 (en) * | 2003-09-30 | 2005-04-21 | Biolex, Inc. | Alpha interferon variants |
US7915483B2 (en) | 2004-07-29 | 2011-03-29 | Biolex Therapeutics, Inc. | C-terminally truncated interferon |
US7959910B2 (en) | 2000-07-31 | 2011-06-14 | Biolex Therapeutics, Inc. | C-terminally truncated interferon alpha variants |
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US5866787A (en) * | 1993-03-08 | 1999-02-02 | Cleveland Clinic Foundation | Transgenic plants co-expressing a functional human 2-5A system |
US6096547A (en) * | 1985-07-29 | 2000-08-01 | Calgene, Llc | Method and transgenic plant for producing mammalian peptides |
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US7959910B2 (en) | 2000-07-31 | 2011-06-14 | Biolex Therapeutics, Inc. | C-terminally truncated interferon alpha variants |
US8182803B2 (en) | 2000-07-31 | 2012-05-22 | Biolex Therapeutics, Inc. | C-terminally truncated interferon alpha variants |
WO2005035767A1 (en) * | 2003-09-30 | 2005-04-21 | Biolex, Inc. | Alpha interferon variants |
US7915483B2 (en) | 2004-07-29 | 2011-03-29 | Biolex Therapeutics, Inc. | C-terminally truncated interferon |
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