Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
1998-05-27
2001-08-21
Clark, Deborah J. R. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C800S009000, C800S012000
Reexamination Certificate
active
06278039
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is Alzheimer's disease.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system involving loss of memory and cognitive function. Neuropathological hallmarks of AD extracellular amyloid plaques, whose major component is the A&bgr; peptide, a proteolytic product of the &bgr;-amyloid precursor protein (APP).
Dominant mutations in any of three mammalian genes, PS1, PS2, and APP are causative for the early-onset, familial form of AD (FAD). PS1 and PS2 encode related proteins-, while APP encodes the amyloid precursor protein (APP) (reviewed in Hardy, Trends Neurosci. 20:154-159, 1997), from which the A&bgr; peptide is generated by proteolytic processing.
Since presenilin-1 (PS1) and presenilin-2 (PS2) were first identified as genes mutated in FAD, much effort has been devoted to elucidating the biological function of presenilins in normal and disease states. Two lines of evidence support a hypothesis that presenilins affect proteolytic processing of APP First, PS(FAD) mutations are associated with an increase in a longer, more amyloidogenic form of A&bgr; known as A&bgr;42 ; and second, levels of A&bgr; are decreased several-fold in neurons derived from PS1 knockout mice relative to PS1(+) control mice. Moreover, in neurons, A&bgr; 42 may be generated in the endoplasmic reticulum, where presenilins have been localized.
C. elegans
has three presenilin genes, spe-4 (spermatogenesis defective) (L'Hernault, et al., J. Cell Biol., 119:55-68, 1992), sel-12 (suppressor and/or enhancer of lin-12) (Levitan et al., Nature 377:351-354, 1995), and hop-1 (homolog of presenilin) (Li et al., Proc. Natl. Acad. Sci USA 94:12204-12209, 1997). Rescue experiments using transgenes have shown that human PS1 and PS2 can substitute for SEL-12, demonstrating that presenilin function has been conserved from nematodes to mammals.
Construction of a nematode having mutations in nematode presenilin genes and also having phenotypes useful for screens for genes associated with the biological pathway and for screens for compounds affecting the biological pathway in which these genes function would be useful for identifying therapeutics and causative agents involved in AD.
SUMMARY OF THE INVENTION
We have discovered an improved nematode model for studying Alzheimer's disease and for screening for Alzheimer's therapeutics. Also provided is a novel method for reverse genetics.
In a first aspect, the invention features an isolated nucleic acid encoding a HOP-1 polypeptide having a mutation (preferably a deletion in or including the hop-1 gene), relative to a Hop-1 encoded by the wild-type hop-1 gene. In preferred embodiments the nucleic acid is cDNA, genomic DNA, or RNA and the mutation is a deletion.
In a third aspect, the invention features a nematode having an engineered mutation in the hop-1 gene, preferably a deletion. More preferably, the hop-1 nematode further includes a deletion or other null mutation in sel-12 or a deletion in spe-4. More preferably, the nematode has deletions or other null mutations in hop-1, sel-12, and spe-4. In a preferred embodiment, the nematode is homozygous for at least one of the aforementioned mutations.
In a fourth aspect, the invention features a method for identifying a compound capable of ameliorating diseases, including Alzheimer's disease. The method includes exposing a nematode having a hop-1 deletion or other mutation and a second deletion or other mutations in a gene selected from the group consisting of sel-12 and spe-4 to an effective amount of a compound and screening for an alteration in the phenotype of said nematode. Examples of such alterations are described herein below. An alteration in the phenotype of the nematode indicates a compound capable of ameliorating disease. Preferably, the phenotype is a phenotype conferred by the presence of hop-1, sel-12, spe-4, or the combination thereof, present in the nematode being used in the screen. In a preferred embodiment of this method the second mutation is a mutation in sel-12, the phenotype is selected from the group consisting of dead eggs or sterility, and the alteration is an alteration in either the number viable eggs or fertile offspring where such eggs or offspring, would otherwise be absent. Additional phenotypes which may be screened for to detect compounds which positively or negatively affect the genesis and progression of AD are also a part of the invention. Such screens identify both potential prophylactics and therapeutics for AD and environmental agents which may exacerbate the disease. In some of these embodiments of the invention wild-type worms or single mutations may be used (see the Detailed Description, below).
In a fifth aspect, the invention provides a method for identifying a gene involved in the development or amelioration of disease, e.g., Alzheimer's disease. The method includes selecting or screening for a reversion mutation (suppressor mutation) in a nematode having a hop-1 deletion and at least one additional second mutation in a gene selected from the group consisting of sel-12 and spe-4, and screening for an alteration in the phenotype of said nematode, an alteration in the phenotype of said nematode indicating a compound capable of ameliorating the disease. In preferred embodiments the second deletion is a deletion in sel-12 or sel-12 and spe-4, the phenotype is selected from the group consisting of dead eggs or sterility and those phenotypes provided herein, and the alteration is an alteration either of viable eggs or fertile offspring where such eggs or offspring, would otherwise be absent or one of the additional alterations provided herein. In a preferred embodiment, the method includes the additional step of screening for suppressors of the suppressor genes identified in the first selection or screen. In this embodiment, the suppressor gene is preferably placed in a genetic background in which it has a discernable phenotype.
In a related aspect, the invention provides a method for screening for genes associated with AD wherein the starting strains lack mutations in spe-4, hop-1, or sel-12. In this aspect the method includes looking for those phenotypes identified herein to be associated with the hop-1; spe-4 and hop-1; sel-12 worms or the hop-1; spe-4; sel-12 triple mutant.
In the seventh aspect, the invention features a method for constructing a nematode having a genetic deletion in a region of choice. The method includes the steps of: a) exposing said nematode to an appropriate mutagen; b) propagating said nematode for at least one generation; c) amplifying the nucleic acids of said nematode using at least two nucleic acid primers from the region of choice in the genome; and d) detecting the presence of a amplified nucleic acid which is shorter than the amplified nucleic acid from a non-mutagenized nematode. In preferred embodiments, the mutagen is selected from the group consisting of ethyl methanesulfonate, nitrosoguanidine, diethyl sulfate, N-nitroso-N-ethylurea, acetaldehyde, diepoxyoctane, diepoxybutane, trimethylpsoralen followed by UV irradiation,
32
P decay, and ionizing particles. In another embodiment the mutagen is &ggr;-irradation, ultraviolet irradiation, or X-irradiation.
In various other preferred embodiments the nematode is
C. elegans
; the method further comprises the steps of initially amplifying nucleic acid from a pool of nematodes said pool comprising at least 1000 genomes derived from distinct mutagenized nematodes; the primers have the sequences of nucleic acids within 10 kilobases of said region of choice (more preferably within 5 kilobases of said region); the nematode exposed to said mutagen is a part of a pool of at least 1000 nematodes; the detecting includes at least two rounds of amplifying, the first round of said amplifying being amplifying of nucleic acid using nucleic acid isolated from a pool of nematodes, said pool including at least 1000 nematodes genomes from the original mutagenesis.
In another preferred em
Johnson Carl
Parry Dianne
Westlund Bethany
AxyS Pharmaceuticals, Inc.
Baker Anne-Marie
Bieker-Brady Kristina
Clark Deborah J. R.
Clark & Elbing LLP
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