Recombinant human CLN2 protein and methods of its production and use
Inactive Publication Date: 2002-01-17
UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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Benefits of technology
[0006] It is the object of the invention to provide a therapy by which a patient having disorder characterized by a deficient amount of functional CLN2 protein in the affected cells can be treated by administering to the patient an amount of CLN2 protein effective to reduce or eliminate the symptoms caused by the deficiency in CLN2 protein.
[0020] The subject invention is directed to a method for treating a patient having disorder characterized by a deficient amount of functional CLN2 protein in the affected cells by administering to the patient an amount of CLN2 protein effective to reduce or eliminate the symptoms caused by the deficiency in CLN2 protein. Alternatively, the amount of CLN2 protein administered may be such that normal levels of CLN2 protein in the cell are restored.
[0027] A composition of this invention preferably includes an uptake inhibitor which decreases local clearance of CLN2 protein by cell surface receptors. This helps ensure that the CLN2 protein is administered evenly, such that more cells get some CLN2 protein, rather than the few cells close to the site of administration getting most of the CLN2 protein. Clearance mechanisms include endocytosis by cell surface receptors such as the mannose receptor, the asialoglycoprotein receptor, and the mannose-6-phospate receptor. Thus a preferred uptake inhibitor is mannose-6-phosphate. The uptake inhibitor can be administered in a composition with CLN2 protein, or can be separately but simultaneously administered, or the two can be administered at different times as long as the uptake inhibitor is able to have the desired effect.
[0030] CLN2 protein or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid). To reduce its systemic side effects and increase cellular penetration, this may be a preferred method for introducing CLN2.
Problems solved by technology
Progression is reflected by a decline in mental abilities, increasingly severe and untreatable seizures, blindness and loss of motor skills while further progression can result in dementia or a vegetative state.
There is no effective treatment for NCL and all childhood forms are eventually fatal.
Method used
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Plasmid Construction, Cell Selection, and Gene Amplification
[0033] CHO cells were transfected with PvuI linearized pMSXND1 CLN2 (a fragment corresponding to nucleotides 1-1707 of human CLN-2 cDNA (Genbank Accession No. AF017456, 175 Arg variant) in the expression vector pMXNS [8]) using the lipofectamine procedure (Gibco). Stable transfectants were isolated by selection with 700 microgram / ml G418 and individual colonies isolated using cloning cylinders. Select colonies were treated with 0.2 micromolar MTX in .alpha.-MEM without nucleotides / 10% dialyzed FBS to select for cells that had undergone gene amplification. When cells became resistant, the MTX concentration was increased and the procedure repeated. After twelve cycles of selection, cells resistant to 400 micromolar methotrexate were obtained.
[0034] The TPP-I activity assay was conducted using a modification of the method of Vines and Warburton (Biochem. Biphys. Acta. 1998 1384 pp233-42). Unless indicated otherwise...
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The present invention relates to a method for treating a patient having disorder characterized by a deficient amount of functional CLN2 protein in the affected cells, which comprises administering to the patient an amount of CLN2 protein effective to reduce or eliminate the symptoms caused by the deficiency in CLN2 protein.
Description
[0001] This application claims priority of U.S. Ser. No. 60 / 203,407, filed May 11, 2000. This application is related to U.S. Ser. No. 08 / 931,608, filed Sep. 16, 1997, which is incorporated by reference in its entirety.[0002] The research leading to the present invention was supported, at least in part, by NIDDK grant number D K 45992. Accordingly, the government may have certain rights in this invention.[0003] The neuronal ceroid lipofuscinoses (NCLs) are a group of closely related hereditary neurodegenerative disorders which affect infants, children and adults, and which occur at a frequency of between 2 and 4 in 100,000 live births. Most forms of NCL afflict children and their early symptoms and disease progression tend to be similar. Initial diagnosis is frequently based upon visual problems, behavioral changes and seizures. Progression is reflected by a decline in mental abilities, increasingly severe and untreatable seizures, blindness and loss of motor skills while further pro...
Claims
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Login to View More IPC IPC(8): C12N15/09A61K38/46A61K38/48A61K47/24A61P25/00
CPCA61K38/4813A61K38/43C12Y304/14009A61P25/00A61P25/28
InventorLOBEL, PETER
OwnerUNIV OF MEDICINE & DENTISTRY OF NEW JERSEY