Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
1995-05-23
2001-02-13
Chambers, Jasemine C. (Department: 1819)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C435S320100, C435S173300
Reexamination Certificate
active
06187991
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a process for genetic alteration of mammalian cell lines and animals such that they express the protein encoded by the human Islet Amyloid Polypeptide (IAPP) gene. IAPP, formerly known as amylin, is the major protein component of pancreatic islet amyloid that forms in the pancreata of Non-insulin Dependent Diabetes Mellitus (NIDDM) patients. Recent studies of IAPP structural and functional characteristics suggest that IAPP, along with insulin and other hormones, plays a major role in carbohydrate metabolism. IAPP is produced, stored and secreted by pancreatic &bgr; cells in the islets of Langerhans. It can mimic the phenomenon of insulin resistance seen in NIDDM by inhibiting glucose uptake and glycogen synthesis in muscle, and liver tissue. The generation of amyloid deposits in humans is thought to be due to the ability of the center portion of the peptide (amino acids 20-29) to form a &bgr; pleated sheet structure, Rodent IAPP differs from human IAPP in that the sequences in this otherwise highly conserved protein between amino acids 20-29 are not conserved and amyloid deposits do not form in rodent pancreata. A working hypothesis is that overexpression of human IAPP leads to insulin resistance in peripheral tissues and in the formation of amyloid deposits.
Transgenic animals, especially mice, have proven to be very useful in dissecting complex systems to generate new information about human disease. Selective expression of human genes in such mice has generated novel model systems to study disease, especially when overexpression of a gene results in a disease state. With such transgenic mice, one can address issues concerning (1) tissue specificity of expression; (2) testing of hypotheses that overexpression of a particular gene leads to disease; (3) the number and identity of tissues/organs that are affected by this overexpression; and (4) effects of various treatments, including drugs, on the progression or amelioration of the disease phenotype.
The generation of transgenic mice that express human IAPP has been reported in the literature, though none of these animals developed a diabetic phenotype. Niles Fox et al. (FEBS Letters 323, 40-44 [1993]) constructed a transgene that fused the rat insulin promoter sequence to a genomic DNA fragment containing the entire human IAPP gene (exons 1-3 and introns 1 and 2). Transgene RNA expression was detected in pancreas, anterior pituitary and brain. Although plasma IAPP levels were 5-fold elevated relative to nontransgenic littermates, no metabolic consequence of this elevation was observed. C. B. Verchere et al. (Diabetologia 37, 725-729 [1994]) used a 600 bp fragment encoding the entire human proIAPP sequence. Their transgenic animals exhibited greater pancreatic content of both IAPP and insulin relative to nontransgenic littermate controls. Increased secretion of both hormones was also detected in perfused pancreas studies. No clinical manifestations of this enhanced storage and secretion were observed. Hoppener et. al. (Diabetologia 36, 1258-1265 [1993]) described the generation of multiple transgenic lines that expressed either human or rat IAPP in the mouse endocrine pancreas. Höppener's group used a 703 bp rat insulin II promoter fragment to drive expression of human or rat IAPP from genomic DNA fragments. Plasma IAPP levels were up to 15 fold elevated but no hyperglycemia nor hyperinsulinemia were observed. In a subsequent study, no amyloid plaque was seen to accumulate in vivo but intra- and extracellular amyliod fibrils did form when islets from these transgenics were cultured in vitro under conditions mimicking hyperglycemia (De Koning et al. Proc. Natl. Acad. Sci. 91, 8467-8471 [1994]).
SUMMARY OF THE INVENTION
In one embodiment, the present invention is directed to recombinant DNA comprising a non-IAPP promoter, a sequence encoding human IAPP or an active fragment thereof functionally linked to a human albumin intron I encoding sequence, a human GAPDH termination encoding sequence and a human GAPDH polyadenylaton encoding sequence, said DNA resulting in expression of a diabetic phenotype when incorporated into a suitable host.
Especially preferred is recombinant DNA wherein the non-IAPP promoter is selected from the group consisting of promoters for the genes for rat insulin I, rat insulin II, human insulin, mouse IAPP, rat beta cell-specific gluocokinase, glucose transporter 2, human tyrosine amino transferase, human albumin, mouse albumin, rat liver specific glucokinase, and mouse metallothionein.
Also preferred is recombinant DNA wherein said promoter is the rat insulin II promoter.
Especially also preferred is recombinant DNA wherein said sequence encoding human IAPP or an active fragment thereof has the characteristics of cDNA.
Further preferred is recombinant DNA wherein said sequence encoding human IAPP or an active fragment thereof has the characteristics of genomic DNA.
Also further preferred is recombinant DNA wherein said sequence is that of SEQ ID NO: 4.
Also especially further preferred is recombinant DNA wherein said sequence of cDNA is that of SEQ ID NO: 5.
In another embodiment, the DNA sequence encoding human IAPP is replaced by a DNA sequence encoding mouse IAPP or an active fragment thereof, with said mouse DNA preferably having the characteristics of cDNA.
The present invention is also directed to vectors comprising recombinant DNA of the present invention (SEQ. ID NO: 1).
The present invention is also directed to an eukaryotic cell line comprising recombinant DNA of the present invention with preferred cell lines selected from the group consisting of rat insulinoma (RIN) cells, hamster insulinoma (HIT) cells and &bgr;-TC3 mouse insulinoma cells.
The present invention is also directed to transgenic non-human mammals comprising recombinant DNA of the present invention with especially preferred transgenic mammals being mice and rats, said transgenic mammals exhibiting a diabetic phenotype.
In another embodiment, the present invention is directed to a method for treating an animal having disease characterized by an over expression of an IAPP gene product comprising,
administering a therapeutically-effective amount of an inhibitor of the over expression of said IAPP gene product to said mammal.
In yet another embodiment, the present invention is directed to a method of evaluating the effect of a treatment comprising administering said treatment and evaluating the effect of said treatment on the product of over expression of a gene encoding IAPP.
Preferred is the method wherein said treatment is administered to an animal with an especially preferred animal being a human.
The present invention is also directed to a method for determining if a subject is at risk for diabetes or obesity comprising examining said subject for the over expression of an IAPP gene product, said over expression being indicative of risk.
In still yet another embodiment, the present invention is directed to a method of evaluating an animal model for a disorder or disease state comprising determining if an IAPP gene in said animal model is expressed at a predetermined level with a preferred method being wherein said level is higher than the level in a wild type or normal animal.
REFERENCES:
patent: 4736866 (1988-04-01), Leder et al.
patent: 5175383 (1992-12-01), Leder et al.
patent: 5175384 (1992-12-01), Krimpenfort et al.
patent: 5221778 (1993-06-01), Byrne et al.
patent: 5387742 (1995-02-01), Cordell
patent: WO9314200 (1993-07-01), None
patent: WO9400558 (1994-01-01), None
Venchere et al., “Transgenic Mice Overproducing Islet Amyloid Polypeptide Have Increased Insulin Storage and Secretion In Vitro”;Diabetologia,(1994) 37: pp. 725 -728.
Fox et al., Human Islet Amyloid Polypeptide Transgenic Mice As A Model Of Non-Insulin-Dependent Diabetes Mellitus (NIDDM); FEBS, vol. 323, No. 1.2, pp. 40 -44, May, 1993.
Hoppener et al., “Molecular Physiology Of The Islet Amyloid Polypeptide (IAPP)/Amylin Gene in Man, Rate, And Transgenic Mice”;J.
Carty Maynard D.
Kreutter David K.
Soeller Walter C.
Benson Gregg C.
Chambers Jasemine C.
Hauda Karen M.
Pfizer Inc
Richardson Peter C.
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