Translation enhancer element of the human amyloid precursor...

Details Translation enhancer element of the human amyloid precursor... Translation enhancer element of the human amyloid precursor...

1998-11-09

2001-10-30

Kunz, Gary L. (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S069100, C435S325000, C435S252300, C435S089000

Reexamination Certificate

active

06310197

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to a nucleic acid element that enhances the translation of the human amyloid precursor protein (APP) gene. This element may be ligated to other structural genes to enhance recombinant protein production. In addition, it may be ligated to reporter gene sequences and used in assays for the purpose of identifying factors that alter the expression of APP. In addition the sequence can be used as a therapeutic target for down regulating APP production.
BACKGROUND OF THE INVENTION
Alzheimer's disease develops as the result of a complex series of steps that ultimately lead to neuronal cell death and the loss of cognitive function. At present, two steps appear to be of particular importance. The first is a synthesis of the amyloid precursor protein (APP) and its processing into the A&bgr; peptides, which then polymerize and deposit as the amyloid filaments that are the hallmark of Alzheimer's disease (Selkoe,
J. Biol. Chem
. 271:18295 (1996); Scheuner, et al.,
Nature Med
. 2:864 (1996); Goate, et al.,
Nature
349:704 (199 1)). Coupled to this process is a special form of inflammation and acute phase response in the brain that leads to an increase in the production of amyloid-associated proteins, &agr;
1
-antichymotrypsin (ACT) and complement activation (Abraham, et al.,
Cell
52:487 (1989)). In vitro studies have shown that ACT and another amyloid-associated protein, apolipoprotein-E (ApoE), regulate the polymerization of A&bgr; peptides into amyloid filaments (Yee, et al.,
Nature
372:92 (1994)). The ApoE 4 and, possibly, the ACT-A alleles are inherited risk factors for Alzheimer's disease (Corder, et al.,
Science
261:921 (1992)).
Several facts suggest a direct connection between increased APP levels and the development of Alzheimer's disease and further suggest that such an increase may be linked to inflammatory mechanisms:
a) Down syndrome brains in trisomy-16 mice show increased APP protein levels beyond the 0.5-fold increase that would be expected by gene dosage (Neve, et al.,
Mol. Brain Res
. 39:185 (1996)).
b) Over-expression of APP protein in transgenic mice is necessary, even in the presence of FAD mutations, for sufficient A&bgr; peptide production to lead to the development of amyloid filament deposits and an Alzheimer's-like pathology (Quon, et al.,
Nature
352:239 (1991)). Furthermore, APP protein synthesis correlates with AP peptide production both in vitro and in vivo (Ho, et al.,
J. Biol. Chem
. 271:30929 (1996)).
c) Traumatic brain injury, a known risk factor for Alzheimer's disease, increases IL-1 as well as APP-immunoreactivity in rat brain (Nieto-Sampedro, et al.,
J. Neurosci. Res
. 17:214 (1987)).
d) IL-1 injected into the rat cerebral cortex increases the steady-state levels of APP protein at the site of the lesion (Sheng, et al.,
Neurobiol. Aging
17:761 (1996)) and primary astrocytes have been shown to be a source of secreted A&bgr; peptides (Buscigio, et al.,
Proc. Natl. Acad. Sci. U.S.A
. 90:2092 (1993)).
The identification of the mechanisms by which inflammation leads to the overproduction of APP in brain cells may lead to new therapies for controlling Alzheimer's disease. Beyond this, the discovery of new methods and elements for regulating gene expression will provide new opportunities for controlling the production of recombinant genes both in vitro and in vivo.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery of a distinct DNA element that increases the rate at which the mRNA transcribed from the amyloid precursor protein (APP) gene is translated. This element may be combined with other genes to increase recombinant protein production without increasing transcriptional activity.
In its first aspect, the invention is directed to a substantially pure DNA molecule comprising the translation enhancer element of APP operably linked to a non-homologous gene, i.e., a gene other than that encoding human APP. The translation enhancer element consists essentially of the nucleotide sequence of SEQ ID NO:1 and, in a preferred embodiment, the nonhomologous gene is located at a site between 10 and 100 nucleotides 3′ to the last 3′ nucleotide in the enhancer.
In another aspect, the present invention is directed to a vector for recombinantly expressing a peptide or protein in a eukaryotic cell. The vector contains a promoter active in the cell; a translation enhancer element having a sequence consisting essentially of that of SEQ ID NO:1 lying 3′ to the promoter; and 5′ to a DNA sequence encoding the peptide or protein for recombinant production. The sequence encoding the peptide or protein should be located 3′ to the enhancer element; be operably linked to the promoter; and be non-homologous with respect to the translation enhancer element. In a preferred embodiment, the gene undergoing recombinant expression is located at a site between 10 and 100 nucleotides 3′ to the last 3′ nucleotide in the enhancer. These vectors may be used to transform a variety of host cells, preferably eukaryotic host cells, using standard techniques for transformation. Cells transformed in this manner are also within the scope of the present invention.
The invention is also directed to a method for recombinantly producing protein in which host cells transformed with the vector discussed above are grown, in vitro or in vivo, and recombinant protein is then purified either from the host cells or from the growth medium surrounding the cells. Purification may be accomplished by standard biochemical techniques including precipitations, chromatography on various matrices, electrophoretic techniques, affinity chromatography, etc. Optionally, the method may include exposing host cells to an inducer, e.g. a cytokine such as interleukin-1&agr; and interleukin-1&bgr;, that increases the activity of the translation enhancer element. An optimal concentration of inducer can be determined by titrating it into the system and measuring the amount of recombinant protein produced at each concentration. In addition to being directed to such methods, the invention includes the recombinant peptides or proteins that are produced by these methods.
In another aspect, the present invention is directed to a method for assaying test compounds for their ability to alter the expression of human APP. This may be accomplished by preparing a vector containing a promoter, the translation enhancer element, and a non-homologous gene operably linked to the element. Preferably, the non-homologous gene will produce a product that can be quantitated with relative ease, e.g., the chloramphenicol acetyltransferase gene may be used for this purpose. Gene expression is then measured in the presence and absence of the test compound in order to determine whether there is either an enhancement or inhibition of expression. Assays may be carried out either using in vitro systems or after transforming host cells with the vector. Because over-expression of APP has been associated with Alzheimer's disease, agents that inhibit the activity of the translation enhancer element are of particular interest. Thus, the present invention includes methods in which the test compounds used are antisense agents specifically directed to the translation enhancer element. These antisense compounds should be nucleic acids complementary to a region of SEQ ID NO:1 that is at least ten bases in length. Agents of this type may undergo a variety of modifications to increase their effectiveness. Other test compounds that can be used in the assays include RNA targeting compounds that alter the enhancer function of the sequence. Such compounds may act by recognizing portions of the secondary structure assumed by different RNAs. In addition pharmacological reagents and inhibitory receptor-mediated ligands may be tested.
DEFINITIONS
The invention description provided herein uses a number of terms that refer to recombinant DNA technology. In order to provide a clear and consistent understanding

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