Compositions and methods of using differentiated cells sensitized to botulinum neurotoxin

• US 8,778,623 B2
• Filed: 02/28/2013
• Issued: 07/15/2014
• Est. Priority Date: 09/14/2012
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Johnson, Eric Arthur, Whitemarsh, Regina, Pellett, Sabine
• Primary Examiner(s): Navarro, Albert
• Assistant Examiner(s): Portner, Ginny
• Application Number:
  US13/779,972
• Publication Number:
  US 20140080142A1
• Time in Patent Office: 502 Days
• Field of Search: 435/70.3, 435/7.4, 435/326
• US Class Current: 435/7.4
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/96486 Metalloendopeptidases (3.4.24)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Differentiated cholinergic cells having motor neuron-like morphology and increased sensitivity to botulinum neurotoxin are provided herein. Methods of using such differentiated cells for detecting neurotoxin are also provided.

Claims

1. An isolated cell population having increased sensitivity to botulinum neurotoxin, wherein the cell population comprises differentiated NG 108-15 cells obtained by culturing undifferentiated NG108-15 cells in a serum-free culture medium comprising retinoic acid and an agonist of sonic hedgehog (SHH) signaling for a time sufficient for the cells to exhibit a differentiated, motor neuron-like morphology, and wherein the differentiated NG108-15 cells are cholinergic and exhibit increased sensitivity to botulinum neurotoxin relative to undifferentiated NG108-15 cells or NG108-15 cells differentiated in the absence of retinoic acid and the SHH signaling agonist.

2. The cell population of claim 1, wherein culturing further comprises culturing the undifferentiated NG108-15 cells in the presence of a myotube.

3. The cell population of claim 2, wherein the myotube is a differentiated C2C12 cell.

4. The cell population of claim 2, wherein the undifferentiated NG108-15 cells are cultured in the presence of the myotube for at least 5 days.

5. The cell population of claim 1, wherein culturing further comprises culturing the undifferentiated NG108-15 cells in the presence of a ganglioside.

6. The cell population of claim 5, wherein the ganglioside is GT 1 b.

7. The cell population of claim 1, wherein differentiated NG108-15 cells synthesize choline acetyltransferase (ChAT).

8. The cell population of claim 1, wherein the botulinum neurotoxin is selected from the group consisting of botulinum neurotoxin serotype A (BoNT/A), botulinum neurotoxin serotype B (BoNT/B), botulinum neurotoxin serotype C (BoNT/C), botulinum neurotoxin serotype D (BoNT/D), botulinum neurotoxin serotype E (BoNT/E), botulinum neurotoxin serotype F (BoNT/F), and botulinum neurotoxin serotype G (BoNT/G), or a subtype thereof.

9. The cell population of claim 1, wherein the agonist of SHH signaling is purmorphamine or SHH.

10. The cell population of claim 9, wherein the agonist is purmorphamine.

11. A method of detecting botulinum neurotoxin in a sample, where the method comprises

(a) contacting a differentiated NG108-15 cell of claim 1 sensitized to botulinum neurotoxin to the sample;

and

(b) detecting cleavage of a neurotoxin-specific cleavage substrate in the contacted cell, wherein cleavage of the cleavage substrate is indicative of biologically active botulinum neurotoxin in the sample.

12. The method of claim 11, wherein the neurotoxin-specific cleavage substrate comprises a polypeptide selected from the group consisting of SNAP-25, VAMP/synaptobrevin, and syntaxin.

13. The method of claim 12, wherein cleavage of SNAP-25 is indicative of a toxin selected from BoNT/A, BoNT/C, and BoNT/E in the sample.

14. The method of claim 12, wherein cleavage of VAMP/synaptobrevin is indicative of BoNT/B, BoNT/D, BoNT/F, or BoNT/G in the sample.

15. The method of claim 12, wherein cleavage of syntaxin is indicative of BoNT/C in the sample.

16. The method of claim 11, wherein the sample is a biological sample.

17. The method of claim 16, wherein the biological sample comprises a material selected from serum, blood, plasma, and tissue.

18. The method of claim 11, wherein the sample is selected from a food, a medicament, and a product for human consumption.