DNA molecules comprising a promoter capable of conferring...

Details DNA molecules comprising a promoter capable of conferring... DNA molecules comprising a promoter capable of conferring...

1997-04-04

2002-04-23

Crouch, Deborah (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S325000, C435S252300, C435S254110, C435S320100, C536S023100, C536S023500, C536S024100

Reexamination Certificate

active

06376239

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to recombinant DNA molecules comprising the promoter region of the sel-12 gene of
Caenorhabditis elegans
(
C. elegans
) or promoter regions of genes homologous to the sel-12 gene, being capable of conferring expression of a heterologous DNA sequence in neural cells, advantageously all neural cells, preferably at all stages of development. The present invention also provides vectors comprising the DNA molecules. The present invention also relates to pharmaceutical and diagnostic compositions comprising such recombinant DNA molecules and vectors. Furthermore, the present invention relates to transgenic animals, e.g., in species ranging from the fruit fly to mammals, such as non-human animals, comprising the aforementioned recombinant DNA molecules or vectors stably integrated into their genome, as well as their use for the identification of substances capable of complementing a neuronal disorder. The present invention also relates to the use of the inventive recombinant DNA molecules and vectors for the preparation of pharmaceutical compositions for treating, preventing, and/or delaying a neuronal disorder in a subject. Furthermore, the recombinant DNA molecules and vectors of the invention also can be used for the preparation of pharmaceutical compositions for inducing a neuronal disorder in a non-human animal.
Several documents are cited throughout the text of this specification with a References section provided prior to the claims. Citations can be in the form of a numerical designation or designations in brackets, with the numerical designation or designations corresponding to a document or documents similarly numbered in the Reference section. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
BACKGROUND OF THE INVENTION
In the field of neuroscience and medical therapy, there is a great demand for test systems to study the function and interaction of gene products, the malfunction or expression of which cause neuronal diseases. Such systems would also be suitable for drug development against neuronal diseases. A prominent example for a neuronal disease is familial Alzheimer's disease (FAD). The majority of cases with FAD are linked to mutations of the presenilin (PS) genes. These genes are homologous to the sel-12 gene of
C. elegans
, which has been postulated to function in the facilitated signaling by lin-12 and glp-1 [15].
Of the genes involved in FAD, mutations within the PS genes are most frequently associated with the early onset of the disease [1]. These genes encode two highly homologous proteins (PS-1, PS-2) [1-5] which appear to adopt a seven to nine transmembrane (TM) domain structure [1-5]. Most of the mutations which co-segregate with FAD patients occur within the PS-1 gene whereas only two mutations have been identified within the PS-2 gene so far [1-5]. Both PS proteins are predominantly located within the endoplasmic reticulum and the early Golgi [6-8], where they co-localize with the &bgr;-amyloid precursor protein (&bgr;APP) [7]. Mutations in the PS genes cause elevated levels of the long form of amyloid &bgr;-peptide terminating at amino acid 42/43 [5, 9, 10], the key player in sporadic Alzheimer's disease (AD) as well as in FAD [11]. It was shown recently that PS-1 is proteolytically cleaved [12, 13] within the large loop between TM6 and TM7[13]. Since endogenous full-length PS proteins were not detected [7, 12, 13], it was suggested that the proteolytic fragments of PS proteins exhibit their biological function. This is further supported by the finding that the FAD associates PS-1 &Dgr;exon9 mutant [14] which lacks the cleavage site, accumulated as full-length protein [12].
The biological functions of the PS proteins are yet unknown. However, sequence similarities to the
C. elegans
sel-12 [15] (42%) and to a lesser extent to the SPC-4 [1, 16] proteins suggest a functional conservation of these proteins. The conservation of topology of sel-12 and PS-1 further supports this assumption [17, 18]. Interestingly, the FAD linked mutations within the human PS genes all occur at positions which are conserved with the two homologous
C. elegans
genes [1-5, 15, 16] suggesting that these mutations are located at functionally important positions. Mutations in sel-12 suppress the Multivulva phenotype of an hyper-active lin-12/Notch mutant. sel-12 mutants themselves have a strong egg-laying (Egl) defect which is reminiscent of the Egl phenotype caused by reducing lin-12 activity [15]. These results suggested that sel-12 is either directly involved in lin-12 signaling or in the receptor transport or recycling [15].
Recently, Levitan [20] developed a model system to study PS structure and function in
C. elegans
. In their system, PS-1 was expressed from an engineered lin-12 promoter variant described in Wilkinson and Greenwald [23]. This promoter is expressed in the vulva precursor cells, most likely the only cells where sel-12 function is required for the correct execution of egg-laying behavior. It was shown that PS-1 expression in the vulva precursor cells can functionally replace sel-12. For studying all aspects of genes involved in neuronal diseases such as FAD, in particular their effect on the psycho/motoric system however, the system described by Levitan [20] suffers from several drawbacks.
For example, although the PS genes in humans are ubiquitously expressed [2, 3], their neural expression is a prerequisite for understanding their role in the pathophysiology of FAD [6]. The promoter employed in Levitan [20], however, is not expressed in most neural cells, and, therefore, defects of sel-12 mutants like an uncoordinated (kinker) phenotype and a general lethargy of the animals cannot be complemented by their promoter constructs. Furthermore, the lin-12 promoter only functions efficiently in the genetic background of smg-1 mutants [23]. However, smg-1 mutants significantly suppress the sel-12 phenotype [20].
Thus, a technical problem present in the art and believed not heretofore provided is providing promoter regions capable of conferring expression of a heterologous DNA sequence in all neural cells, preferably at all stages of development.
OBJECTS AND SUMMARY OF THE INVENTION
Since Alzheimer is a disease of the nervous system, the inventor decided to study the function and interaction of proteins involved in this disease in neural cells since otherwise non-informative or even false positive results may be obtained.
Thus, an object of the present invention can be to provide promoter regions capable of conferring the expression of a heterologous DNA sequence in neural cells, advantageously all neural cells, and preferably at all stages of development. Accordingly, it is an object of the invention to address, and preferably provide a solution, to the technical problem in the art, by providing embodiments of the invention.
Further objects of the invention can include providing, inter alia:
an isolated nucleic acid molecule, e.g., a nucleotide sequence such as an isolated DNA molecule or a recombinant DNA molecule comprising the promoter of the sel-12 gene from
C. elegans
or the promoter region of a gene homologous to the sel-12 gene being-capable of conferring expression in neural cells, advantageously all neural cells, and preferably at all stages of development, and optionally operatively linked thereto at least one isolated nucleic acid molecule or nucleotide-sequence, e.g., a heterologous DNA sequence;
a vector comprising at least one inventive isolated nucleic acid molecule, e.g., recombinant DNA molecule;
a phar

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