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Methods and materials for treatment of pompe's disease

An amino acid, molecular complex technology, applied in chemical instruments and methods, biochemical equipment and methods, pharmaceutical formulations, etc., can solve problems such as expensive

Active Publication Date: 2015-02-25
OXYRANE UK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, or Both are very expensive, costing well over $300,000 per year

Method used

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  • Methods and materials for treatment of pompe's disease
  • Methods and materials for treatment of pompe's disease
  • Methods and materials for treatment of pompe's disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Decapping and demannosylation of recombinant huGAA by CcMan5 and Concanavalis α-mannosidase

[0131] Recombinant human GAA (rhGAA) was produced as described in WO2011 / 039634 using the Yarrowia lipolytica producer strain OXYY1589, which contains three copies of the human alpha glucosidase gene (also known as acid alpha glucosidase or acid maltase EC 3.2.1.3) and two copies of the Yarrowia lipolytica MNN4 gene. The amino acid sequence of human GAA is in figure 1 listed in . The genotype of strain OXY1589 is as follows:

[0132] MatA, leu2-958, ura3-302, xpr2-322,

[0133] gut2-744, ade2-844

[0134] POX2-Lip2pre-huGAA:URA3Ex::zeta

[0135] POX2-Lip2pre-huGAA:LEU2Ex::zeta

[0136] POX2-Lip2pre-hGM-CSF:GUTEx::zeta

[0137] YlMNN4-POX2-hp4d-YLMNN4:ADE2::PT targeted

[0138] RhGAA was decapped and demannosylated with Cellulobacterium cellulosus mannosidase (CcMan5) and Concanavalia alpha mannosidase (JbMan) (Sigma Product M7257, 3.0 M ammonium phosphate suspension)....

Embodiment 2

[0144] Purification of 110kDa rhGAA

[0145] The 110 kDa form of rhGAA was isolated from strain OXYY1589 as follows. After harvesting, the broth was centrifuged and filtered using a Durapore membrane (Merck Millipore). Ammonium sulfate (AMS) was added to a concentration of 1M and solutes were filtered prior to loading on a hydrophobic interaction chromatography (HIC) column equilibrated in 20 mM sodium phosphate pH 6, 1M ammonium sulfate. The product was eluted with 20 mM sodium phosphate pH 6.

[0146] The product was first concentrated via tangential flow filtration (TFF) on a regenerated cellulose membrane followed by buffer exchange to 20 mM sodium acetate pH 4.5 before loading on the second chromatography column. This material was loaded on a cation exchange chromatography (CEX) column equilibrated with 20 mM sodium acetate pH 4.5. After the column was loaded, it was washed with equilibration buffer until the UV absorbance signal reached baseline, then washed with 20...

Embodiment 3

[0149] Purification of 110kDa rhGAA

[0150] The 110 kDa form of rhGAA was isolated from strain OXYY1589 as follows. After harvest, the material was centrifuged and filtered, after which the concentration of AMS was increased to 1M. The solutes were filtered again and the product was captured on a HIC column equilibrated with 20 mM sodium phosphate pH 6, 1 M AMS and released in a step gradient from 1 to 0 M AMS in 20 mM sodium phosphate pH 6 buffer.

[0151] The eluate was concentrated and buffer exchanged to 10 mM BIS-TRIS, pH 6 via TFF on a Vivaflow 200 model (PES membrane, 10 kD MWCO, Sartorius). The desalted material was introduced onto an anion exchange chromatography (AEC) column. After the column was cleaned until the UV signal had almost reached baseline, a two-phase continuous salt gradient was applied; the first gradient was from 0 to 0.3M NaCl and the second from 0.3 to 1M NaCl. Fractions were collected during the gradient and screened for GAA via qualitative 4...

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Abstract

This document relates to molecular complexes having acid alpha glucosidase activity and at least one modification that results in enhanced ability of the molecular complex to be transported to the interior of a mammalian cell.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application Serial No. 61 / 611,485, filed March 15, 2012. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of the present application. field of invention [0003] The present invention relates to isolated molecular complexes having acid alpha glucosidase activity, and more particularly to molecular complexes comprising at least two polypeptides derived from precursor molecules by proteolysis, wherein the molecular complexes comprise at least one A modification that results in an increased ability of the molecular complex to be transported inside the mammalian cell. Background of the invention [0004] Pompe's disease (also known as glycogen-storage disease type II or acid maltase deficiency) is a rare autosomal recessive disorder in which acid alpha glucosidase (GAA ) deficiency leads to the accumu...

Claims

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Application Information

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IPC IPC(8): A61K38/47C12N9/26
CPCC12Y302/0102C12N9/2408A61K38/00C12N9/2451A61K47/64C12N9/62A61K38/43A61K38/47C12Y302/01003A61P21/00A61P43/00A61K47/50A61K38/17C12N1/14C12N1/16C12P21/02C12P21/06C07K2319/70C07K14/65
Inventor W.沃维肯K.C.T.A.M.皮恩斯J.R.L.斯陶特G.N.派纳尔特
Owner OXYRANE UK
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