Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Nonplant protein is expressed from the polynucleotide
Reexamination Certificate
2001-03-16
2004-08-03
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Nonplant protein is expressed from the polynucleotide
C800S317400, C435S423000, C536S023100
Reexamination Certificate
active
06770799
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention pertains to the field of transgenic plants. More particularly, the invention pertains to the expression of a recombinant form of human acetylcholinesterase in transgenic plants.
2. Description of Related Art
Acetylcholine (ACh) is one of the major signaling molecules in metazoans, functioning mostly as a neurotransmitter in chemical synapses between neurons and in neuromuscular junctions. To ensure a discrete “all-or-none” response across the synapse, the release of ACh is tightly controlled and the neurotransmitter is efficiently removed by the hydrolyzing enzyme, acetylcholinesterase (AChE). In humans, AChE is encoded by a single gene which yields, through alternative splicing of its pre-mRNA, three polypeptide isoforms having distinct C-termini. See Soreq et al.,
Proc. Nat. Acad. Sci. U.S.A.
87: 9688-9692 (1990); Ben Aziz-Aloya et al.,
Proc. Natl. Acad. Sci. U.S.A.
90: 2471-5 (1993); GenBank Accession No. M55040; and U.S. Pat. No. 5,595,903. The complete disclosure of each of the foregoing references is hereby incorporated herein by reference.
Various compounds are well known to inhibit the hydrolyzing activity of AChE. Exposure to such anti-AChE agents leads to over-stimulation of cholinergic pathways, causing muscular tetany, autonomous dysfunction and potentially death. While some naturally occurring AChE inhibitors are very potent, human exposure to them is rare. However, man-made anti-AChE compounds, especially organophosphates (OPs), are widely used as pesticides and pose a substantial occupational and environmental risk. Even more ominous is the fear of deliberate use of OPs as chemical warfare agents against individuals or populations.
Current medical interventions, in the case of acute exposure to anticholinesterase agents, include use of the muscarinic receptor antagonist, atropine, and oximes to reactivate the OP-modified AChE. The reversible carbamate, pyridostigmine bromide, is also used as a prophylactic. However, these conventional treatments have limited effectiveness and have serious short and long-term side effects. In fact, the routine treatments, while successfully decreasing anticholinesterase-induced lethality, rarely alleviate post-exposure delayed toxicity, which may result in significant performance deficits, and even permanent brain damage.
A different approach in treatment and prevention of anti-AChE toxicity seeks to mimic one of the physiological lines of defense against such agents present in mammals. Butyrylcholinesterase (BuChE) is a serum cholinesterase with a broad hydrolytic spectrum that provides protection against a variety of AChE inhibitors. A similar end may be achieved by a variant of AChE found on the membranes of erythrocytes. Both enzymes are believed to serve as circulating scavengers for anti-AChE agents in protection of the vital synaptic AChE. Therefore, administration of cholinesterases could boost their natural potential to counter-act the toxic effects of anti-cholinergic agents. The efficacy of this treatment to protect against a challenge of OPs was tested in a variety of animal models such as mice, rats, guinea pigs, and primates, and was found to be comparable to or better than the currently-used drug regimens in preventing OP-induced mortality without any detrimental side-effects.
Enzyme therapy has the additional benefit of the relatively long half-life time (several days) of the injected enzymes in the blood stream, making it especially useful for prophylaxis. In the foregoing experiments, cholinesterases purified from human or animal blood were used. To be effective, the stoichiometry of cholinesterase to inhibitor must be close to unity. Hence, large amounts of pure, properly folded, stable enzymatic preparations that are free of mammalian pathogens are needed, if enzyme therapy is to be feasible.
Genetically engineered plants have recently been recognized as one of the most cost-effective means for the production of useful recombinant proteins and pharmaceuticals. Therefore, we examined the use of transgenic plants as a cost-effective and safe alternative to the production of human acetylcholinesterase (hAChE) from blood or cell cultures, herein providing the first demonstration of the expression in plants of a key protein component of the nervous system of humans.
SUMMARY OF THE INVENTION
Briefly stated, the invention includes one or more plant cells comprising a polynucleotide that encodes a human acetylcholinesterase.
An embodiment of the invention includes a method of making a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase, comprising the steps of introducing into at least one plant cell a polynucleotide that encodes a human acetylcholinesterase, and regenerating from the plant cell a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase in at least one tissue type of the transgenic plant.
Another embodiment of the invention includes a method of making a physiologically active human acetylcholinesterase, comprising the steps of introducing into at least one plant cell a polynucleotide that encodes a human acetylcholinesterase, regenerating from the plant cell a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase in at least one tissue type of the transgenic plant, and isolating or purifying from the transgenic plant or a part thereof a physiologically active human acetylcholinesterase.
Another embodiment of the invention includes a method of treating a victim of acetylcholinesterase poisoning, comprising the step of administering a therapeutic amount of a physiologically active human acetylcholinesterase expressed in plant tissue.
REFERENCES:
patent: 4956282 (1990-09-01), Goodman et al.
patent: 5595903 (1997-01-01), Seroq et al.
patent: 5891725 (1999-04-01), Seroq et al.
Meisssner, R. et al, 1997, “A new model system for tomato genetics”, the Plant Journal, 12(6), 1465-1472.
Haq, T. et al, 1995, “Oral Immunization with a Recombinant Bacterial Antigen Produced in Transgenic Plants”, Science, vol. 268, pp 714-716.
Aziz-Aloya, R. et al, 1993, “Expression of a human acetylcholinesterase promoter-reporter construct in developing neuromuscular junctions of Xenopus embryos”, Proc. Natl. Acad. Sci., vol. 90, pp 2471-2475.
Sroeq, H. et al, 1990, “Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G+C-rich attenuating structure”, Proc. Natl. Acad. Sci., vol. 87, pp. 9688-9692.
Arntzen Charles J.
Mason Hugh S.
Mor Tsafrir S.
Soreq Hermona
Brown & Michaels PC
Helmer Georgia L
McElwain Elizabeth F.
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