Targeted lysosomal enzyme compounds

A technology of lysosomal enzymes and compounds, applied in the field of targeted lysosomal enzyme compounds, can solve problems such as inability to improve central nervous system defects and the like

Inactive Publication Date: 2014-11-12
ANGLACHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Like bone marrow transplantation, this approach does not improve CNS deficits associated with MPS-II because the enzyme does not cross the blood-brain barrier as expected (BBB; Wraith et al., Eur. J. Pediatr. 1676: 267-7, 2008)

Method used

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  • Targeted lysosomal enzyme compounds
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  • Targeted lysosomal enzyme compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0259] Design of IDS-Angiopep-2 Fusion Protein

[0260] A series of IDS-Angiopep-2 constructs were designed. IDS cDNA was obtained from Origene (cat. no. RC219187). Three basic configurations were used: N-terminal fusions (An2-IDS and An2-IDS-His), C-terminal fusions (IDS-An2 and IDS-An2-His) and N- and C-terminal fusions (An2- IDS-An2 and An2-IDS-An2-His), both with and without 8x His tag ( figure 1 ). Controls without angiopep-2 (IDS and IDS-His) were also generated.

Embodiment 2

[0262] Expression and activity of recombinant hIDS protein in CHO-S cells

[0263] These constructs were subsequently expressed in CHO-S cells grown in suspension. The IDS construct was expressed in FreeStyle CHO-S cells (Invitrogen) by transient transfection using linear 25 kDa polyethyleneimine (PEI, Polyscience) as the transfection reagent. In one example, DNA (1 mg) was mixed with 70 mL of FreeStyle CHO Expression Medium (Invitrogen) and incubated at room temperature for 15 min. PEI (2 mg) alone was incubated in 70 mL of medium for 15 minutes, and then the DNA and PEI solutions were mixed and incubated for an additional 15 minutes. DNA / PEI complex mixture was added to 360mL containing 1×10 9 Medium for CHO-S cells. At 37°C, 8% CO 2 After four hours of incubation with moderate agitation, 500 mL of warm medium was added. CHO-S cells were further incubated under the same conditions for 5 days before harvesting.

[0264] To determine whether cells express and secrete IDS...

Embodiment 3

[0269] Characterization and optimization of expression

[0270] To further characterize expression, time-course evaluation of IDS expression and activity in suspension-grown CHO-S cells was determined for IDS-His and IDS-An2-His fusion proteins, as Figure 5A and Figure 5B shown in . According to these data, maximal IDS expression and activity was observed five days after transfection. No recapture of IDS-An2-His by CHO-S cells was observed in these experiments.

[0271] To further optimize the transfection conditions, two different numbers of cells (1.25 × 10 7 cells or 2.5 x 10 7 cells) for transfection. Three different ratios of DNA to polyethyleneimine (PEI) were used (1:1, 1:2, 1:3 and 1:4).

[0272] According to these experiments, such as by IDS activity ( Figure 5A ) and through expression analysis ( Figure 5B ), the best results were obtained using a DNA:PEI ratio of 1:2.

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Abstract

The present invention is related to a compound that includes a lysosomal enzyme and a targeting moiety, for example, where compound is a fusion protein including iduronate-2-sulfatase and Angiopep-2. In certain embodiments, these compounds, owning to the presence of the targeting moiety can crossing the blood-brain barrier or accumulate in the lysosome more effectively than the enzyme alone. The invention also features methods for treating lysosomal storage disorders (e.g., mucopolysaccharidosis Type II) using such compounds.

Description

technical field [0001] The present invention relates to compounds comprising a lysosomal enzyme and a targeting moiety and the use of such combinations in the treatment of disorders resulting from the absence of such enzymes. Background technique [0002] Lysosomal storage disorders are a group of approximately 50 rare genetic conditions in which a subject has deficiencies in lysosomal enzymes required for normal metabolism. These disorders are usually caused by autosomal or X-linked recessive genes. As a group, the incidence of these conditions is approximately 1:5000 to 1:10,000. [0003] Hunter syndrome or mucopolysaccharidosis type II (MPS-II) is caused by a deficiency of iduronate-2-sulfatase (iduronate-2-sulfatase) ( IDS; also known as idursulfatase), an enzyme required for the lysosomal degradation of heparin sulfate and dermatan sulfate. Because the disease is X-linked recessive, it primarily affects males. Those with this condition are unable to break down and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16A61K47/48A61K9/127A61K9/14A61P3/00C07K14/81C07K19/00C12N9/14C12N9/96
CPCA61K47/48246A61K38/00C12N9/16C07K2319/01A61K47/64C12Y301/06013A61K38/465A61P3/00A61P25/00A61P43/00
Inventor 多米尼克·波依温让-保罗·卡斯泰恩米歇尔·德默勒萨斯米塔·塔里帕西让-克里斯托夫·柯里西蒙·洛德-杜富尔
Owner ANGLACHEM INC
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