Fusion proteins for delivery of gdnf and bdnf to the central nervous system

A compound, A-X-B technology, applied in neuregulin, nervous system diseases, DNA/RNA fragments, etc., can solve cumbersome and difficult problems

Inactive Publication Date: 2012-05-16
ANGLACHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are tedious and difficult

Method used

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  • Fusion proteins for delivery of gdnf and bdnf to the central nervous system
  • Fusion proteins for delivery of gdnf and bdnf to the central nervous system
  • Fusion proteins for delivery of gdnf and bdnf to the central nervous system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Angiopep-2 / GDNF construct

[0157] Generate sequences including Angiopep-2 and hGDNF (hGDNF 78-211 ) construct. These constructs include the N-terminus (His) 6 Tag, thrombin cleavage site, Angiopep-2 sequence and GDNF sequence. A control peptide was also generated without the Angiopep-2 sequence ( figure 2 ). The amino acid sequences of the N-terminal portions of these sequences are shown in image 3 . The strategy used to clone these constructs is described in Figure 4-7 middle. Similar strategies can be used to generate BDNF constructs. The sequences of these constructs are shown in Figure 8-12 middle. A schematic showing the Angiopep-2-GDNF compound bound to GFRα1 is shown in Figure 13 middle.

[0158] Generate additional GDNF constructs such as Figure 14 shown in. These include hGDNF in which Angiopep-2 is attached to its N-terminus 78-211 (An2-hGDNF); hGDNF in which Angiopep-2 is attached to its C-terminus 78-211 (hGDN F-An2); hGDNF with reverse...

Embodiment 2

[0160] Receptor binding of GDNF construct conjugates

[0161] To detect receptor binding of GDNF conjugates, a double-sandwich Elisa was used. Briefly, mouse anti-human IgG antibodies were bound to one plate ( Figure 15 ). A GFRα1 receptor / IgG Fc fusion was added, which binds to these antibodies. To detect ligand binding, GDNF, GDNF conjugates, or Angiopep-2 were each added to the plates. The plates were then treated sequentially with goat anti-GDNF antibody and alkaline phosphatase-conjugated rabbit anti-goat IgG antibody. These samples were then treated with p-nitrophenyl phosphate (p-NPP), an alkaline phosphatase (AP) substrate that changes from colorless to yellow after AP treatment. Binding of proteins is detected based on this color change.

[0162] In this assay, all fusion proteins tested were able to bind the GDNF receptor at levels similar to GDNF itself ( Figure 16 ). Angiopep-2 (a negative control) was not observed to bind to this receptor. Individual bindi...

Embodiment 3

[0165] homodimer formation

[0166] As mentioned above, GDNF is known to form homodimers via disulfide bonds ( Figure 17 ). Such dimer formation was also observed with the An2-GDNF protein ( Figure 18 ). These disulfide bonds can be reduced by treatment with reducing agents such as dithiothreitol (DTT).

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Abstract

The present invention relates to a compound that includes a peptide vector, such as angiopep-2 which acts as a carrier across the blood-brain barrier, linked to glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), or a related molecule, such as an analog or a fragment thereof. The compounds of the invention may be used to treat any disease where increased neuronal survival or growth is desired, e.g., neurodegenerative diseases, such as Parkinson's disease or amyotrophic lateral sclerosis. Other diseases can be treated using the compounds include schizophrenia and depression.

Description

technical field [0001] The present invention relates to a peptide carrier and glial cell-derived neurotrophic factor. (GDNF) or brain-derived neurotrophic factor (BDNF) conjugates (conjugates or conjugates), and uses thereof. Background technique [0002] Diseases associated with neuronal loss or damage are serious conditions and affect millions of people worldwide. Although therapies such as GDNF hold promise in the treatment of neurodegenerative diseases such as Parkinson's disease, prior to the present invention, the delivery of such therapies to the brain has been complicated by the inability of the active agent to cross (cross or cross) the blood-brain barrier . In fact, previous clinical trials involving GDNF therapy for Parkinson's disease required the use of direct injection of the agent into the brain, and BDNF trials for the treatment of amyotrophic lateral sclerosis involved intrathoracic injection of the agent. These methods are cumbersome and difficult. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/00A61K38/16A61K47/48A61P25/18A61P25/24A61P25/28C07K14/475C07K19/00C12N15/62C07K14/81
CPCA61K47/48246C07K14/48A61K38/00C07K2319/03C07K14/4756A61K47/64A61P25/00A61P25/14A61P25/16A61P25/18A61P25/24A61P25/28
Inventor 米歇尔·德默勒多米尼克·波依温让-保罗·卡斯泰恩
Owner ANGLACHEM INC
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