Systems, methods and compositions for optical stimulation of target cells

Abstract

Stimulation of target cells using light, e.g., in vivo or in vitro, is implemented using a variety of methods and devices. One example involves a vector for delivering a light-activated NpHR-based molecule comprising a nucleic acid sequence that codes for light-activated NpHR-based molecule and a promoter. Either a high expression of the molecule manifests a toxicity level that is less than about 75%, or the light-activated NpHR-based proteins are expressed using at least two NpHR-based molecular variants. Each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron. Other aspects and embodiments are directed to systems, methods, kits, compositions of matter and molecules for ion pumps or for controlling inhibitory currents in a cell (e.g., in in vivo and in vitro environments).

1 . A method for generating an inhibitory neuron-current flow, the method comprising:
 in a neuron, using a NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron, wherein a high expression of the molecule manifests a toxicity level that is less than about 75%.   
     
     
         2 . The method of  claim 1 , wherein the molecule is expressed using at least one of the NpHR molecular variants from Table 2, namely, Non-Humanized NpHR, SP nChR-S -NpHR, NpHR-ETQV, NpHR-actin, NpHR-ERexport. 
     
     
         3 . The method of  claim 1 , wherein the toxicity is less than about 40%. 
     
     
         4 . The method of  claim 1 , wherein the toxicity is less than about 10%. 
     
     
         5 . The method of  claim 1 , wherein the molecule uses an endogenous cofactor to produce the inhibitory current, and wherein the inhibitory current substantially dissuades depolarization of the neuron in less than 500 milliseconds after being exposing to light. 
     
     
         6 . The method of  claim 1 , wherein the inhibitory molecule does not have the sequence as set forth in GenBank accession number EF474018 and uses an endogenous cofactor to produce the inhibitory current. 
     
     
         7 . The method of  claim 1 , wherein the step of using a NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron, includes using at least two NpHR-based molecular variants. 
     
     
         8 . The method of  claim 1 , wherein the step of using a NpHR-based molecule includes causing the cell to express light-activated NpHR-based molecule and exposing the cell to light to activate the NpHR-based protein. 
     
     
         9 . The method of  claim 8 , wherein the step of causing the cell to express light-activated NpHR-based molecule includes using at least two NpHR-based constructs. 
     
     
         10 . The method of  claim 9 , wherein the two NpHR-based constructs are independently capable of causing the cell to express light-activated NpHR-based protein, wherein the two NpHR-based constructs are either part of the same sequence or independently delivered. 
     
     
         11 . The method of  claim 10 , wherein the step of using an NpHR-based molecule includes using a ChR2-based molecule to control transmission of the neuron. 
     
     
         12 . A method of optically controlling cell properties, comprising:
 causing the cell to express light-activated NpHR-based proteins using at least two NpHR-based molecular variants, each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron; and   exposing the cell to light to activate the NpHR-based protein.   
     
     
         13 . The method of  claim 12 , wherein the molecule is expressed using at least one of the NpHR molecular variants from Table 2, namely, Non-Humanized NpHR, SP nAChR-S -NpHR, NpHR-ETQV, NpHR-actin, NpHR-ERexport. 
     
     
         14 . A method of optically controlling cell properties, comprising:
 causing the cell to express light-activated NpHR-based protein; and   exposing the cell to light to activate the NpHR-based protein, wherein the NpHR-based molecule is characterized in that a high expression of the molecule manifests a toxicity level that is less than about 75%.   
     
     
         15 . The method of  claim 14 , wherein the NpHR-based molecule is characterized in that the high expression of the molecule corresponds to about 3×10e7 infectious units per milliliter. 
     
     
         16 . A nucleic acid sequence comprising a gene for a light-activated NpHR-based molecule and a promoter, wherein the molecule responds to light by producing an inhibitory current to dissuade depolarization of a neuron, wherein a high expression of the molecule manifests a toxicity level that is less than about 75%. 
     
     
         17 . The nucleic acid sequence of  claim 16 , wherein the molecule uses an endogenous cofactor to produce the inhibitory current, wherein the endogenous cofactor is all-trans-retinal (ATR). 
     
     
         18 . The method of  claim 16 , wherein the inhibitory current substantially dissuades depolarization of the neuron in less than 500 milliseconds after being exposed to light. 
     
     
         19 . A composition comprising: a light-activated NpHR-based molecule that is expressed in a cell wherein the cell is selected from the group consisting of mammalian cells, neuronal cells and stem cells, wherein a high expression of the molecule manifests a toxicity level that is less than about 75%. 
     
     
         20 . The composition of  claim 19 , wherein the molecule is expressed using at least one of the NpHR molecular variants from Table 2, namely, Non-Humanized NpHR, SP nAChR-s -NpHR, NpHR-ETQV, NpHR-actin, NpHR-ERexport. 
     
     
         21 . A method of controlling synaptic transmissions, comprising:
 causing a neuron that ends in a synapse to express light-activated NpHR-based molecules, and   exposing the cell to light to activate the NpHR-based molecules, wherein the step of exposing to light dissuades a synaptic event,   wherein either
 a high expression of the molecules manifests a toxicity level that is less than about 75%, or 
 the light-activated NpHR-based molecules are expressed using at least two NpHR-based molecular variants, each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron. 
   
     
     
         22 . A vector for delivering a nucleic acid sequence that codes for light-activated NpHR-based molecules and a promoter, wherein either
 a high expression of the molecules manifests a toxicity level that is less than about 75%, or   the light-activated NpHR-based molecules are expressed using at least two NpHR-based molecular variants, each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron.   
     
     
         23 . A NpHR-based molecule for use in therapy wherein the molecule is capable of responding to light by producing an inhibitory current to dissuade depolarization of a neuron and wherein the molecule is capable of producing the inhibitory current, and wherein a high expression of the molecule manifests a toxicity level that is less than about 75%. 
     
     
         24 . A nucleic acid molecule comprising a nucleotide sequence encoding a NpHR based protein for use in the treatment of CNS disorders wherein the protein is capable of responding to light by producing an inhibitory current to dissuade depolarization of a neuron, and wherein a high expression of the molecule manifests a toxicity level that is less than about 75%. 
     
     
         25 . Use of a NpHR-based molecule in the manufacture of a medicament for the treatment of neurological or CNS disorders wherein the said molecule is capable of responding to light by producing an inhibitory current to dissuade depolarization of a neuron, and wherein a high expression of the molecule manifests a toxicity level that is less than about 75%. 
     
     
         26 . An assembly or kit of parts, comprising: a product containing an NpHR-based molecular variant and another opsin-based molecule as a combined preparation for use in the treatment of disease of a neurological or CNS disorder, wherein at least the NpHR-based molecular variant is useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of a cell, and wherein a high expression of the molecule manifests a toxicity level that is less than about 75%.