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Biglycan variant polypeptides and methods of use

a polypeptide and variant technology, applied in the field of proteoglycans, can solve the problems of negative influence of biglycan on the m form and the m form, and achieve the effect of preventing or treating a disorder in the subject and low bone mass

Inactive Publication Date: 2019-01-31
TIVORSAN PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

T2-rhBGN effectively improves muscle health, bone formation, and neuromuscular junction stability, offering a promising therapeutic approach for various musculoskeletal and neurological disorders by potentiating agrin-induced AChR clustering and stabilizing plasma membranes.

Problems solved by technology

While not wanting to be bound by a particular theory it is believed that the D form of biglycan negatively affects the M form (when in a mixture).
It merely negatively affects the M form.

Method used

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  • Biglycan variant polypeptides and methods of use
  • Biglycan variant polypeptides and methods of use
  • Biglycan variant polypeptides and methods of use

Examples

Experimental program
Comparison scheme
Effect test

example 2

M- and D-rhBGN are Chemically Distinct

[0138]The M and D forms purified from CHO clone 57 HIC pool over two size exclusion columns were further analyzed by Capillary Electrophoresis (FIG. 3) was conducted using an Agilent Bioanalyzer 2100 and Agilent Protein 80 assay kit under non-reducing conditions according to manufacturer's protocols. The apparent molecular weight and mobility of D-rhBGN (65.5 kd, 31.00, respectively) was greater than that of M-rhBGN (60 kd, 30.44, respectively), indicating differential posttranslational modification of the two forms.

[0139]The M and D forms were characterized by N-linked oligosaccaride profiling analysis (FIG. 4). Approximately 250 μg of M- and D-rhBGN were digested into glycopeptides with trypsin at 37° C. overnight. The glycopeptides were purified with a C18 cartridge, eluted in sequential elutions of 20%, 40%, and 100% isopropanol, combined, and dried. N-glycans were released by enzymatic cleavage with PNGase F, and then purified of contaminan...

example 3

M-rhBGN is Highly Active in Bioassays

[0140]M-rhBGN purified from CHO clone 171 by two rounds of SEC was analyzed (FIG. 5) by an in-vitro bioassay for biglycan activity where the ability of biglycan to potentiate agrin-induced AChR clustering in cultured H2K myotubes is measured based on the method described in (Amenta et al., J. Neuroscience, 2012). M-rhBGN showed a robust and broad dose-response. Activity was observed in a 30-fold range of >50% of maximal activity between 0.016-0.512 μg / ml. (FIG. 5.)

example 4

D-rhBGN Shows Low Bioactivity

[0141]The SEC fractions containing D-rhBGN were analyzed by the bioassay describe above. As shown in FIG. 5, there was only low activity (<25% of maximal) across the entire concentration range tested (0.004-0.512 μg / ml).

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Abstract

The invention provides compositions and methods for treating, preventing, and diagnosing diseases or conditions associated with an abnormal level or activity of biglycan; disorders associated with an unstable cytoplasmic membrane, due, e.g., to an unstable dystrophin associated protein complex (DAPC); disorders associated with abnormal synapses or neuromuscular junctions, including those resulting from an abnormal MuSK activation or acetylcholine receptor (AChR) aggregation. Examples of diseases include Amyotrophic Lateral Sclerosis (ALS), as well as muscular dystrophies, such as Duchenne's Muscular Dystrophy, Becker's Muscular Dystrophy, neuromuscular disorders and neurological disorders.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. application Ser. No. 14 / 050,225 filed Oct. 9, 2013, now pending; which claims the benefit under 35 USC § 119(e) to U.S. Application Ser. No. 61 / 712,177 filed Oct. 10, 2012, now expired. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with government support under grants UO1NS064295 (NIH), R21AR055878 (NIAM) and HD023924 (DHHS) awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION OF SEQUENCE LISTING[0003]The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, named TIVOR1100-2_ST25.txt was created on Oct. 9, 2018 and is 13 KB. The file can be assessed...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/47G01N33/68A61K38/17
CPCA61K38/17C07K14/47C07K14/4725G01N33/6896G01N2400/00A61P9/04A61P19/02A61P19/08A61P19/10A61P21/00A61P21/02A61P21/04A61P25/00
Inventor FALLON, JUSTINJOHN, ELIZABETH
Owner TIVORSAN PHARMA