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Methods for treating pompe disease

A Pompe disease and fusion protein technology, applied in the field of treatment of Pompe disease, can solve problems such as no significant success, and achieve the effect of cost-effectiveness, potential and simple method

Inactive Publication Date: 2010-01-27
BIOMARIN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Drug therapy strategies, diet control, and bone marrow transplantation have been employed as treatments for Pompe disease, but without significant success

Method used

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  • Methods for treating pompe disease
  • Methods for treating pompe disease
  • Methods for treating pompe disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Production of recombinant wild-type GAA and GILT-tagged GAA

[0092] plasmid

[0093] DNA encoding full-length wild-type human GAA was isolated and inserted into an expression vector for production of recombinant human GAA. The DNA cassette encoding amino acids 1-952 of fully human GAA (hereafter referred to as "cassette 635") was derived from IMAGE clone 4374238 (OpenBiosystems) and was derivatized using the following PCR primers:

[0094] GAA13: 5'-GGAATTCCAACCATGGGAGTGAGGCACCCGCCC (SEQ ID NO: 1) and

[0095] GAA27: 5'-GCTCTAGACTAACACCAGCTGACGAGAAACTGC (SEQ ID NO: 2).

[0096] Cassette 635 was digested with EcoRI and Xbal, inactivated by treatment with Klenow DNA polymerase, and ligated into the Klenow-treated HindIII site of the expression vector pCEP4 (Invitrogen), resulting in plasmid p635. Hereinafter, ZC-635 refers to wild-type untagged GAA protein.

[0097] A DNA cassette for the production of recombinant GILT-tagged GAA ZC-701 (hereinafter...

Embodiment 2

[0110] Example 2: Affinity of GILT-tagged GAA for CI-MPR

[0111] Binding affinity utilization of GILT-tagged GAA ZC-701 for CI-MPR Surface plasmon resonance assay (surface plasmon resonance assay) to determine. Two biotinylated and His-tagged recombinant proteins (containing wild-type CI-MPR domain 10-13 and one point mutation variant) were formed, respectively, according to standard molecular techniques. Schematic representation of the two recombinant proteins as Figure 3A shown. Plasmid p1288 contains the IGF-II signal peptide followed by: a poly-His (poly-His) tag; a biotin AS domain; and a sequence encoding wild-type CI-MPR domains 10-13. Plasmid p1355 contains the IGF-II signal peptide followed by: a poly-His (poly-His) tag; a biotin AS domain; and a CI-MPR encoding a point mutation I1572T that effectively reduces the affinity of the receptor for IGF-II Sequences of domains 10-13. The specific DNA and amino acid sequences associated with these two recombinant pr...

Embodiment 3

[0130] Example 3: Analysis of N-linked oligosaccharides shows that ZC-701 lacks M6P

[0131] N-linked oligosaccharide analysis was performed to determine the oligosaccharide profile of ZC-701 using PNGase deglycosylation followed by a combination of HPLC analysis with fluorescence detection (Blue Stream Laboratories).

[0132] Cleavage of N-linked sugars (carbohydrates) from glycoprotein samples was performed by N-glycanase at a ratio of 1:100 (enzyme to substrate) using approximately 100 μg of protein per sample. Once released, glycans were extracted using cold ethanol and dried by centrifugation. The recovered oligosaccharides were labeled with 2-aminobenzamide (2-AB) under acidic conditions in the presence of sodium cyanoborohydride. After the derivatization step, by The S sample filter catheter (Prozyme) removes excess dye and other reactive reagents remaining in the sample.

[0133] Analysis of N-linked oligosaccharides by HPLC-FLD used the following conditions: mob...

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Abstract

The present invention provides methods for treating Pompe disease in a subject by administering to the subject a therapeutically effective amount of a fusion protein which includes human acid alpha-glucosidase (GAA), or a fragment thereof, and a lysosomal targeting domain. The lysosomal targeting domain binds the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.

Description

[0001] References to related applications [0002] This application claims U.S. Provisional Patent Application No. 60 / 900,187, filed February 7, 2007, U.S. Provisional Patent Application No. 60 / 879,255, filed January 5, 2007, filed November 13, 2006 Priority to US Provisional Patent Application No. 60 / 858,514, the contents of each of which are hereby incorporated in their entirety. This application is also related to US Patent Application No. 11 / 057,058, filed February 10, 2005, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to methods and compositions for the treatment of Pompe disease. More specifically, the present invention relates to a therapy for the treatment of Pompe disease by targeting acid alpha-glucosidase to lysosomes in a mannose-6-phosphate-independent manner method. Background technique [0004] Pompe disease is an autosomal recessive genetic disorder (gene disorder) caused by deficiency or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P3/00A61K47/48
Inventor 乔纳森·勒博茨约翰·马加
Owner BIOMARIN PHARMA INC
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