Genetically engineered cells sensitive to clostridial neurotoxins

A neurotoxin and genetic engineering technology, applied in the field of cells, can solve problems affecting the cleavage properties of SNARE proteins

Pending Publication Date: 2022-05-27
IPSEN BIOPHARM LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] However, differences between modified and recombinant Clostridial neurotoxins and their wild-type counterparts may affect the desired SNARE protein cleavage properties of neurotoxins

Method used

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  • Genetically engineered cells sensitive to clostridial neurotoxins
  • Genetically engineered cells sensitive to clostridial neurotoxins
  • Genetically engineered cells sensitive to clostridial neurotoxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0213] Example 1 - Selection of the Best Parental Cell Lines for Formation of Indicator Cell Lines

[0214] Neuro2A (N2a; ATCC CCL-131), BE(2)-M17 (M17; ATCC CRL-2267), IMR-32 (ATCC CCL-127) and NG 108-15 [108CC15] (ATCC HB-12317) were studied cells with the aim of selecting the best parental cell line for the development of stably transfected cell lines.

[0215] After delivery, cells were allowed to recover and grow. Cell stocks were then frozen and stored in liquid nitrogen. Once sufficient cell stock vials have been prepared, cells are assayed for sensitivity to BoNT / A.

[0216] Cells were cultured in medium containing BoNT / A (Metabiologics, Inc.) for 8 or 24 hours. Cells cultured for 8 hours were cultured in medium containing 0.1 nM, 1 nM or 10 nM BoNT / A. Cells cultured for 24 hours were cultured in medium containing 1 nM, 0.1 nM or 0.01 nM BoNT / A.

[0217] Cleavage of endogenous SNAP-25 was analyzed by Western blot using an anti-SNAP-25 antibody (Sigma #S9684) usi...

Embodiment 2

[0218] Example 2 - Transfection of cells with plasmids containing the indicated constructs

[0219] The sensitivity of NG108 cells and M17 cells to puromycin (InvivoGen #ANT-PR) and G418 (VWR #97064-358) was determined. Cells were grown to ~50% confluence and then incubated with various concentrations of puromycin and G418. Both cell lines showed similar sensitivity to puromycin and G418.

[0220] A plasmid (pD2500; Atum) was engineered to contain nucleic acid sequences encoding puromycin-N-acetyltransferase (PuroR), a chimeric protein and a 2A self-cleaving peptide. In the expressed product, the 2A self-cleaving peptide is located between the PuroR and the chimeric protein. The chimeric protein contains SNAP-25 and luciferase (at the C-terminus) between the N-terminal and C-terminal fluorescent proteins. PuroR confers resistance to puromycin. In addition to fluorescence-based measurements of degradation facilitated by fluorescent proteins, luciferase allows luminescence...

Embodiment 3

[0227] Example 3 - Confirmation of cleavage of indicator proteins

[0228] The plasmid (pcDNA3.1) was engineered to contain SEQ ID NO: 3 encoding the BoNT / A light chain, CFP and N-terminal SBP tag. The nucleic acid encoding the BoNT / A light chain was synthesized using DNA2.0 (Atum).

[0229] Cells from Example 2 stably transfected with the indicated constructs (mScarlet-SNAP25-GenLuc or mScarlet-SNAP25-CyanNluc) were transiently transfected with an expression vector containing the CFP-BoNT / A construct. Numerous red rather than green or cyan cells were confirmed 24 and 48 post-transfection, indicating that the indicator protein was cleaved and the C-terminal fragment was rapidly degraded.

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Abstract

A cell has been genetically engineered to be highly sensitive to clostridial neurotoxins, such as botulinum neurotoxins and tetanus neurotoxins. Methods of making such genetically engineered cells and methods of determining the activity of modified or recombinant clostridial neurotoxins using such cells.

Description

[0001] Field of Invention [0002] The present invention generally relates to a cell that has been genetically engineered to have increased sensitivity to Clostridial neurotoxins, such as botulinum neurotoxin and tetanus neurotoxin. The present invention also relates to methods of making such cells and methods of using such cells to assay the activity of polypeptides derived from such neurotoxins, such as modified and recombinant forms of such Clostridial neurotoxins. Background technique [0003] The anaerobic gram-positive bacterium Clostridium botuliuum produces a variety of different types of neurotoxins, including botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). [0004] BoNT is the most potent toxin known, with median lethal dose (LD50) values ​​in mice ranging from 0.5 to 5 ng / kg, depending on the serotype. BoNT is adsorbed in the gastrointestinal tract and, after entering the systemic circulation, binds to the presynaptic membrane of cholinergic nerve termin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02
CPCC12N5/0618C12N5/0693C07K14/33G01N33/5014C12N2510/02G01N2333/33C12N2510/00Y02A50/30C12N15/85
Inventor G·A·奥勒B·格尔兹
Owner IPSEN BIOPHARM LTD
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