Methods for the delivery of toxins or enzymatically active portions thereof

Abstract

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.

What is claimed is: 
     
         1 . A method of intoxicating a cell with a toxin or an enzymatically active fragment thereof, the method comprising:
 a. mixing the toxin or the enzymatically active fragment thereof with a lipid or polymeric carrier, thereby forming a mixture; and   b. exposing the mixture to one or more cells;   
       wherein the cell becomes intoxicated with said toxin or said enzymatically active fragment. 
     
     
         2 . The method of  claim 1 , wherein the toxin or fragment thereof comprises at least an enzymatic portion of botulinum neurotoxin, tetanospasmin, tetrodotoxin,  Clostridium difficile  toxin Tcd A,  Clostridium difficile  toxin Tcd B,  Clostridium  Lethal Toxin, Anthrax Lethal Factor, Anthrax Edema Factor, Ricin, Exotoxin A, Diphtheria toxin, Cholera toxin, Tetanus toxins, Shiga toxin, latrotoxin, or a combination thereof. 
     
     
         3 . The method of  claim 2 , wherein the enzymatically active fragment of the toxin comprises the light chain or chain A of said toxin. 
     
     
         4 . The method of  claim 3 , wherein the light chain or chain A includes a mutation or deletion. 
     
     
         5 . The method of  claim 4 , wherein the botulinum neurotoxin or light chain portion is derived from a serotype with a sequence similarity of at least about 40% to a sequence, wherein the serotype is selected from the group consisting of serotype A, B, C, D, E, F, and G, and wherein the sequence is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. 
     
     
         6 . The method of  claim 1 , wherein the lipid or polymeric carrier comprises one or more lipid or polymeric carrier type DNA transfection reagents. 
     
     
         7 . The method of  claim 6 , wherein the DNA transfection reagent comprises polylysine, polyethylenimine (PEI), a polymeric carrier, a cationic lipid reagent, polycationic polymers, or any combination thereof. 
     
     
         8 . The method of  claim 6 , wherein the DNA transfection reagent is in an amount between about 0.1 pM and about 1 μM. 
     
     
         9 . The method of  claim 8 , wherein the DNA transfection reagent is in an amount between about 1 nM and about 10 nM. 
     
     
         10 . The method of  claim 6 , wherein the toxin or the enzymatically active fragment thereof is mixed with the DNA transfection reagent for a length of time between about 5 minutes and about 72 hours. 
     
     
         11 . The method of  claim 10 , wherein the toxin or the enzymatically active fragment thereof is mixed with the DNA transfection reagent for a length of time between about 1 hour and about 6 hours. 
     
     
         12 . A method of delivering a botulinum neurotoxin or an enzymatically active fragment thereof to the inside of one or more cells in vitro, the method comprising:
 a. contacting the botulinum neurotoxin or an enzymatically active fragment of the toxin with a lipid or polymeric carrier to thereby obtain a mixture; and   b. exposing the one or more cells to the mixture in an amount to allow the botulinum neurotoxin or enzymatically active fragment thereof to enter the cell;   
       wherein the cells are intoxicated with the botulinum neurotoxin or the enzymatically active fragment thereof. 
     
     
         13 . The method of  claim 12 , wherein the botulinum neurotoxin comprises a serotype selected from the group consisting of serotype A, B, C, D, E, F, and G. 
     
     
         14 . The method of  claim 12 , wherein the enzymatically active fragment comprises the light chain or portion thereof from a serotype with a sequence similarity of at least about 40% to a sequence, wherein the serotype is selected from the group consisting of serotype A, B, C, D, E, F, and G, and wherein the sequence is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. 
     
     
         15 . The method of  claim 12 , further including assessing the level of one or more SNARE proteins to determine a level of cell intoxication, wherein the SNARE proteins comprise synaptobrevin 2, syntaxin, SNAP 25, or a combination thereof. 
     
     
         16 . A method of preparing one or more cells for a cell-based assay, wherein the cell is intoxicated with a toxin or an enzymatically active fragment thereof, the method comprising:
 a. contacting the botulinum neurotoxin or an enzymatically active fragment of the toxin with a lipid or polymeric carrier to thereby obtain a mixture; and   b. exposing the one or more cells to the mixture in an amount to allow the botulinum neurotoxin or the enzymatically active fragment thereof to enter the one or more cells;   
       wherein the one or more cells are intoxicated with the toxin or the enzymatically active fragment thereof. 
     
     
         17 . A system or kit for delivering a toxin, an enzymatically active fragment thereof, or a recombinant SNARE endoprotease to inside of a cell in vitro, wherein the system or kit comprises:
 a. one or more toxins, enzymatically active fragments thereof, or recombinant SNARE endoproteases; and   b. one or more lipid or polymeric carriers.   
     
     
         18 . The system or kit of  claim 17 , further including one or more cells. 
     
     
         19 . The system or kit of  claim 18 , wherein the one or more cells are transfected with a nucleic acid molecule having a sequence that comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or combination thereof. 
     
     
         20 . An isolated cell that comprises a toxin, an enzymatically active fragment thereof, or a recombinant SNARE endoprotease, wherein the cell comprises HEK293, HIT-T15, a neuroendocrine derived cell line, an immortalized cell line, or a tumor derived cell line.