Methods for the delivery of toxins or enzymatically active portions thereof
The present invention relates to methods, systems, and kits for intoxicating cells, neuronal and non-neuronal cells, with a toxin or fragment thereof. This is done by subjecting toxin substrate and a lipid or polymeric carrier (e.g., DNA uptake facilitating agent) to one or more cells for use in cell based assays. In an aspect, the methods of the present invention allow for high throughput assays and, as such, for the evaluation of drug candidates.
1. A method of assessing in vitro an effect of a molecule or compound on a cell intoxicated with a toxin or an enzymatically active fragment thereof; the method comprises:
a. contacting a botulinum neurotoxin or an enzymatically active fragment of the botulinum neurotoxin with a lipid or polymeric carrier to thereby obtain a mixture;
b. exposing cells to the mixture in an amount to allow the botulinum neurotoxin or enzymatically active fragment thereof to enter the cell, to thereby obtain intoxicated cells;
c. subjecting the intoxicated cells to the molecule or compound to be assessed; and
d. assessing the effect of said molecule or compound on the intoxicated cell by assessing the percent cleavage of SNAP25, a level of one or more SNARE proteins, or a combination thereof.
2. The method of claim 1 , wherein assessing the effect of the molecule or compound is assessing an amount of endoprotease activity or an amount of endosome release of the toxin.
3. The method of claim 2 , wherein the molecule or compound being assessed is an antagonist or an agonist thereof.
4. The method of claim 2 , wherein assessing the level of one or more SNARE proteins consists of assessing synaptobrevin 1, synaptobrevin 2, syntaxin, and a combination thereof.
5. The method of claim 2 , wherein the cell is an insulinoma or neuroendocrine cell, the method further comprises:
a. subjecting the intoxicated cell to glucose under conditions that allow for insulin secretion to occur; or subjecting the intoxicated cell to potassium solution mediated depolarization; and
b. assessing release of the botulinum neurotoxin, the enzymatically active fragment thereof, or the cleavage of recombinant SNARE proteins by assessing a level of insulin secretion;
wherein an increase of insulin secretion is indicative of an increased inhibition of the botulinum neurotoxin, the enzymatically active fragment thereof, or the cleavage of recombinant SNARE proteins; and a decrease of insulin secretion is indicative of a decreased inhibition thereof.
6. The method of claim 2 , wherein the cell is an insulinoma or other neuroendocrine cell, the method further comprises:
a. transfecting the cell with a plasmid encoding a neuropeptide reporter or vesicle fusion indicator;
b. subjecting the mixture to glucose under conditions that allow for insulin secretion to occur; or subjecting the cell to potassium solution mediated depolarization;
c. assessing protease activity of the botulinum neurotoxin, the enzymatically active fragment thereof, or the cleavage of recombinant SNARE proteins by assessing a level of insulin secretion; wherein an increase of insulin secretion is indicative of an increased inhibition of the botulinum neurotoxin, the enzymatically active fragment thereof, or the cleavage of recombinant SNARE proteins; and a decrease of insulin secretion is indicative of a decreased inhibition thereof.
7. The method of claim 1 , further comprising:
a. labeling a toxin substrate with a donor fluorophore and an accepter separated by a SNARE sequence containing the endoprotease cleavage site, wherein the toxin substrate is a recombinant SNARE protein, to thereby obtain a treated toxin substrate under the condition of the intoxicated cells subjecting to the molecule or compound,
b. exciting said donor fluorophore; and
c. determining resonance energy transfer in said treated toxin substrate to a control substrate with the absence of the molecule or compound, wherein a difference in resonance energy transfer in said treated toxin substrate as compared to said control substrate is indicative of toxin protease activity as the effect of the molecule or compound on the intoxicated cell.
8. The method of claim 1 , wherein the lipid or polymeric carrier is one or more lipid or polymeric carrier type DNA transfection reagents.
9. The method of claim 8 , wherein the DNA transfection reagent consist of: polylysine, polyethylenimine (PEI), a polymeric carrier, a cationic lipid reagent, polycationic polymers, and any combination thereof.
10. The method of claim 8 , wherein the botulinum neurotoxin is contacted with the one or more lipid or polymeric carrier type DNA transfection reagents in an amount between about 0.1 pM and about 1 μM.
11. The method of claim 10 , wherein the botulinum neurotoxin is contacted with the one or more lipid or polymeric carrier type DNA transfection reagents in an amount between about 1 nM and about 10 nM.
12. The method of claim 8 , wherein the botulinum neurotoxin is contacted with the one or more lipid or polymeric carrier type DNA transfection reagents for a length of time between about 5 minutes and about 72 hours.
13. The method of claim 12 , wherein the botulinum neurotoxin is contacted with the one or more lipid or polymeric carrier type DNA transfection reagents for a length of time between about 1 hour and about 6 hours.