Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-10-08
2002-10-08
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S285000, C800S286000, C800S288000, C800S317300, C435S069400, C435S069500, C435S091210, C435S235100, C435S320100, C435S468000, C536S023720
Reexamination Certificate
active
06462255
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the field of genetically engineering transgenic plants. More specifically, the invention relates to the use of viral RNA to achieve high level expression of foreign genes in plants.
The use of transgenic plants for high level expression of foreign genes has been targeted as an inexpensive means for mass producing desired products. All higher plants are photoautotrophic, requiring only CO
2
, H
2
O, NO
3
−1
, SO
4
−2
, PO
4
−3
and trace amounts of other elements for growth. From these inexpensive starting materials, plants are capable of synthesizing a variety of valuable products. Progress in utilizing transgenic plants as low cost factories will depend on both the characterization of biosynthetic pathways and on the further development of gene expression technologies.
In the past decade, a number of techniques have been developed to transfer genes into plants (Potrykus, I.,
Annual Rev. Plant Physiol. Plant Mol. Biol.
42:205-225 (1991)). For example, chromosomally integrated transgenes have been expressed by a variety of promoters offering developmental control of gene expression. (Walden and Schell,
Eur. J. Biochem.
192:563-576 (1990)). This technology has been used primarily to improve certain agronomic traits such as disease resistance or food quality. (Joshi and Joshi,
Febs. Lett.
281:1-8 (1991)). However, the utility of known transgene methodology is limited by 1) the difficulty of obtaining high level expression of individual transgenes 2) the lack of means necessary for coordinating control of several transgenes in an individual plant 3) the lack of means to enable precise temporal control of gene expression and 4) the lack of adequate means to enable shutting off introduced genes in the uninduced state (Walden and Schell,
Eur. J. Biochem
192:563-576 (1990)).
The most highly expressed genes in plants are encoded in plant RNA viral genomes. Many RNA viruses have gene expression levels or host ranges that make them useful for development as commercial vectors. (Ahlquist, P., and Pacha, R. F.,
Physiol. Plant.
79:163-167 (1990), Joshi, R. L., and Joshi, V.,
FEBS Lett.
281:1-8 (1991), Turpen, T. H., and Dawson, W. O., Amplification, movement and expression of genes in plants by viral-based vectors, Transgenic plants: fundamentals and applications (A. Hiatt, ed.), Marcel Dekker, Inc., New York, pp. 195-217. (1992)). For example, tobacco (
Nicotiana tabacum
) accumulates approximately 10 mg of tobacco mosaic tombamovirus (TMV) per gram of fresh-weight tissue 7-14 days after inoculation. TMV coat protein synthesis can represent 70% of the total cellular protein synthesis and can constitute 10% of the total leaf dry weight. A single specific RNA transcript can accumulate to 10% of the total leaf mRNA. This transcript level is over two orders of magnitude higher than the transcription level observed for chromosomally integrated genes using conventional plant genetic engineering technology. This level of foreign gene expression has not yet been obtained using the prior art viral vectors in plants.
Most plant viruses contain genomes of plus sense RNA (messenger RNA polarity) (Zaitlin and Hull,
Ann. Rev. Plant Physiol.
38:291-315 (1987)). Plus sense plant viruses are a very versatile class of viruses to develop as gene expression vectors since there are a large number of strains from some 22 plus sense viral groups which are compatible with a wide number of host plant species. (Martelli, G. P.,
Plant Disease
76:436 (1992)). In addition, an evolutionarily related RNA-dependent RNA polymerase is encoded by each of these strains. This enzyme is responsible for genome replication and mRNA synthesis resulting in some of the highest levels of gene expression known in plants.
In order to develop a plant virus as a gene vector, one must be able to manipulate molecular clones of viral genomes and retain the ability to generate infectious recombinants. The techniques required to genetically engineer RNA viruses have progressed rapidly. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is used to make all of the constructions. The genome of many plus sense RNA viruses can be manipulated as plasmid DNA copies and then transcribed in vitro to produce infectious RNA molecules (reviewed in Turpen and Dawson, Transgenic Plants, Fundamentals and Applications, Marcel Dekker, New York, pp. 195-217 (1992)).
The interaction of plants with viruses presents unique opportunities for the production of complex molecules as typified by the TMV/tobacco system (Dawson, W. O.,
Virology
186:359-367 (1992)). Extremely high levels of viral nucleic acids and/or proteins accumulate in infected cells in a brief period of time. The virus catalyzes rapid cell-to-cell movement of its genome throughout the plant, with no significant tissue tropism. The infection is maintained throughout the life of the plant. The plants are not significantly adversely affected by the viral infection since the virus causes little or no general cytotoxicity or specific suppression of host gene expression.
The tobacco mosaic tobamovirus is of particular interest to the instant invention in light of its ability to express genes at high levels in plants. TMV is a member of the tobamovirus group. TMV virions are 300 nm×18 nm tubes with a 4 nm-diameter hollow canal, and consist of 2140 units of a single structural protein helically wound around a single RNA molecule. The genome is a 6395 base plus-sense RNA. The 5′-end is capped and the 3′-end contains a series of pseudoknots and a tRNA-like structure that will specifically accept histidine. The genomic RNA functions as mRNA for the production of proteins involved in viral replication: a 126-kDa protein that initiates 68 nucleotides from the 5′-terminus and a 183-kDa protein synthesized by readthrough of an amber termination codon approximately 10% of the time (FIG.
1
). Only the 183-kDa and 126-kDa viral proteins are required for TMV replication in trans. (Ogawa, T., Watanabe, Y., Meshi, T., and Okada, Y.,
Virology
185:580-584 (1991)). Additional proteins are translated from subgenomic size mRNA produced during replication (reviewed in Dawson, W. O.,
Adv. Virus Res.
38:307-342 (1990)). The 30-kDa protein is required for cell-to-cell movement; the 17.5-kDa capsid protein is the single viral structural protein. The function of the predicted 54-kDa protein is unknown.
The minimal sequences required in cis for TMV replication are located at the extreme 5′ and 3′ noncoding regions (replication origins), as determined by analysis of deletion mutants in plant protoplasts (Takamatsu, N., et al.,
J. Virol.
64:3686-3693 (1990), Takamatsu, N., et al.,
J. Virol.
65:1619-1622 (1991)). In whole plants, helper-dependent RNA replicons, constructed by deletion of most of the 126/183-kDa replication protein sequence and most of the 30-kDa movement protein sequence, are replicated and spread systemically in the presence of wild type TMV (Raffo A. J., and Dawson W. O.,
Virology
184:277-289 (1991)).
Turpen, et al. discloses a simple and reliable gene transfer method wherein cDNA of TMV is engineered into A. tumefaciens for expression in plant cells (Turpen, T. H., Ph.D. Dissertation, University of California, Riverside, pp. 88-105 (1992)). This method provides an alternative to the use of synthetic infectious transcripts to inoculate plants based on host transcription of viral cDNA in vivo. Turpen showed successful transfection of tobacco (
N. tabacum
cv. Xanthi and Xanthi
c) with wild type and defective viral genomes using this methodology.
Transfection also occurs spontaneously in transgenic lines containing defective or wild type cDNA of TMV integrated chromosomally (Turpen, T. H., Ph.D. Dissertation, University of California, Riverside, pp. 106-132 (1992), Yamaya, J., et al.,
Mol. Gen. Genet.
211:520-525 (1988)). Thus, once chromosomally integrated, viral replication can be derived from the process of ho
Chiang Robin C.
Fox David T.
Gallegos Thomas
Halluin Albert P.
Large Scale Biology Corporation
LandOfFree
Viral amplification of recombinant messenger RNA in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Viral amplification of recombinant messenger RNA in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Viral amplification of recombinant messenger RNA in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2976905