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AssigneeNone recorded
InventorsMATTHEW D. LINNIK, MARGARET M. RACKE, JOAN M. KRAKOWSKY, ARUN SUBRAMANIAM
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Novel human nerve growth factor exon 1 promoter, human nerve growth factor exon 3 promoter, fragments thereof, and modified forms thereof are described. The invention is also directed to vectors containing such promoters, cells transformed with the same, including animal models and transgenic animals containing such sequence and assay methods using these promoters.
An isolated nucleic acid comprising human nerve growth factor exon 1 promoter selected from 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A vector comprising a nucleic acid human nerve growth factor exon 1 promoter selected from 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A vector comprising pGL3-neo.
A nonhuman animal comprising human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A method of transferring a nucleic acid to a cell comprising administering to the cell a nucleic acid encoding human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A method of transferring a nucleic acid into an animal, comprising administering to the animal a nucleic acid encoding human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A transformed cell comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A method of producing a protein comprising expressing a vector comprising a human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof operably linked to a gene encoding a protein.
A method of assaying a compound comprising administering a compound to a cell, wherein the cell comprises a vector which comprises a human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A nonhuman transgenic animal, comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1-1786, or 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modified form thereof.
A method for identifying a compound capable of modifying initiation of transcription of human nerve growth factor exon 1 promoter or human nerve growth factor exon 3 promoter, comprising contacting a cell comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modification thereof, with a compound and detecting modification of initiation of transcription.
A method of characterizing a compound capable of modifying initiation of transcription of human nerve growth factor exon 1 promoter or human nerve growth factor exon 3 promoter, comprising contacting a cell comprising a nucleic acid encoding human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modification thereof, with a compound and detecting modification of initiation of transcription.
A compound capable of binding to a human nerve growth factor exon 1 promoter 1-1786, 2274-2846, human nerve growth factor exon 3 promoter 1-1877, fragment thereof, or modification thereof.
Dependencies are inferred from the claim text.
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Growth factors; Growth regulators: nerve growth factor [NGF]
Involving nucleic acids: involving reporter genes operably linked to promoters
Genetically modified animals: animals comprising random inserted nucleic acids
Peptides
Term end not estimated
USPTO file history isn’t available for this patent, so only indexed dates are shown.
Several lines of evidence point to the potential therapeutic utility of nerve growth factor in neurodegenerative diseases. NGF has been shown to prevent neurons from dying after experimentally induced injuries including ischemia (Shigeno T, et al., J Neurosci 11:2914-2919, 1991; Yamamoto S, et. al, Neurosci Lett 141:161-165, 1992; Pechan P A, et al., NeuroReport 6:669-672, 1995; Holtzman D M, et al., Ann Neurol 39:114-122, 1996), concussion (Hayes R L, et al., J Neurotrauma 12:933-41, 1995; Sinson G, et al., J Neurochem 65:2209-2216, 1995), and axotomy (Williams M and Braunwalder A., J Neurochem 47:88-97, 1986; Kromer, L. F. Science 235:214-216, 1987). NGF can also help to sustain function in aged or damaged neurons by maintaining neuronal phenotype and inducing neurite outgrowth (Fischer W, et al., Nature 329:65-68, 1987; Fischer W, et al, J Neurosci 11:1889-1906, 1991; Rylett, R. J., et al., J Neurosci 13:3956-3963, 1993; Chen K S, et al., Neuroscience 68(1): 19-27, 1995; Tuszynski M H and Gage F H, Mol Neurobiol 10:151-167, 1995).
Systemic administration of NGF is an inefficient method to achieve brain exposure due to the limited ability of NGF to cross the blood-brain barrier (Poduslo J F and Curran G L, Molec Brain Res 36:280-286, 1996). Several alternative routes of administration have proven effective, including direct intracerebroventricular administration, implantation of producer cell lines (Rosenberg M B, et al., Science 242:1575-1578, 1988), conjugation to actively transported molecules (Friden P M, et al., Science 259:373-377, 1993; Kordower J H, et al., PNAS USA September 13; 91(19): 9077-80, 1994) and transcriptional upregulation by low molecular weight compounds.
A number of small molecules have been identified that increase NGF mRNA transcription (Mocchetti I, Ann Rev Pharmacol Tioxicol 32:303-328, 1991; Carswell S, Exp Neurol 124:36-42, 1993) and some of these compounds have been demonstrated to mimic the pharmacological action of exogenous NGF in vivo (Lee, T.-H., et al., Stroke 25:1425-1432, 1994; Kaechi K, et al., JPET 264(1): 321-6, 1993; Kaechi K, et al., JPET 272:1300-1304, 1995). The majority of NGF-inducing compounds have been shown to upregulate NGF mRNA transcription via the two promoter regions which have been identified in the mouse NGF gene (Selby M J, et al., Molec Cell Biol 7:3057-3064, 1987; Nitta A., et al., Eur J Pharmacol 250:23-30, 1993). Recently, a third promoter has been suggested in the rat NGF gene (Timmusk T, et al., Soc Neurosci Absts 21:33, 1995).
Summary of Strategy to Identify Human Nerve Growth Factor Exon 1 and Exon 3 Promoters
A brief description of the cloning strategies used to develop the cell lines described in Table 1 is provided.
DNA for the human nerve growth factor exon 3 clones was originally identified by PCR screening a human P1 genomic library (clone 0095-B8, Genome Systems). A ˜6600 bp fragment containing exon 3 was cloned into a pBS SK+ vector to yield the plasmid identified as pBSEx3. A 4329 bp fragment was isolated from the insert in pBSEx3 and subcloned into a pGL2 enhancer vector (Promega) and a pGL3 basic vector (Promega) to yield clones identified as pGL2Ex3 and pGL3Ex3, respectively. The pGL2Ex3 was transfected into mouse L929 cells and the pGL3Ex3 vector was transfected into human UC11 cells to generate the data in Table 1.