Devices, systems and methods for optogenetic modulation of action potentials in target cells

Abstract

Aspects of the disclosure include devices, systems and methods for optogenetic modulation of action potentials in target cells. The subject devices include light-generating devices, control devices, and delivery devices for delivering vectors to target cells. The subject systems include light-activated proteins, response proteins, nucleic acids comprising nucleotide sequences encoding these proteins, as well as expression systems that facilitate expression of these proteins in target cells. Also provided are methods of using the subject devices and systems to optogenetically inhibit and intercept action potentials in target cells, e.g., to treat a neurological or psychiatric condition in a human or non-human animal subject.

What is claimed is: 
     
         1 . A system for modulating the membrane potential of a cell, the system comprising:
 a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light;   a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane; and   a device configured to illuminate a target location with a light.   
     
     
         2 . The system according to  claim 1 , wherein the light-activated protein is an ion pump. 
     
     
         3 . The system according to  claim 2 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         4 . The system according to  claim 1 , wherein the light-activated protein is an ion channel. 
     
     
         5 . The system according to  claim 4 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         6 . The system according to  claim 1 , wherein the response protein is an ion pump. 
     
     
         7 . The system according to  claim 6 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         8 . The system according to  claim 1 , wherein the response protein is an ion channel, an ion exchange protein, or an ion co-transporter. 
     
     
         9 . The system according to  claim 8 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         10 . The system according to  claim 1 , wherein the device is configured to illuminate the target location with light having a wavelength ranging from about 350 to about 750 nm. 
     
     
         11 . The system according to  claim 10 , wherein the device is configured to illuminate the target location with light having a wavelength ranging from about 530 up to about 560 nm. 
     
     
         12 . The system according to  claim 1 , wherein the device is configured to constantly illuminate the target location with a light. 
     
     
         13 . The system according to  claim 1 , wherein the device is configured to illuminate the target location with pulses of light. 
     
     
         14 . The system according to  claim 12  or  13 , wherein the device is configured to modulate the wavelength and/or the intensity of the light. 
     
     
         15 . The system according to  claim 13 , wherein the device is configured to modulate the frequency and/or the duration of the pulses of light. 
     
     
         16 . The system according to  12  or  13 , wherein the device is configured to illuminate the target location in response to a user input. 
     
     
         17 . The system according to  16 , wherein the user input comprises: the wavelength of light, the intensity of light, the duration of a light pulse, the frequency of a light pulse, and/or the target location. 
     
     
         18 . The system according to  claim 1 , wherein the device is adapted to be implanted in a subject. 
     
     
         19 . The system according to  claim 1 , wherein the target location is: a cell, a portion of a cell, a plurality of cells, a bundle of nerve fibers, a neuromuscular junction, a central nervous system (CNS) tissue, a peripheral nervous system (PNS) tissue, or an anatomical region. 
     
     
         20 . The system according to  claim 1 , wherein the first nucleic acid and the second nucleic acid are present within a single expression vector. 
     
     
         21 . The system according to  claim 1 , wherein the nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light is operably linked to a neuron-specific transcription control element. 
     
     
         22 . The system according to  claim 1 , wherein the second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane is operably linked to a neuron-specific transcription control element. 
     
     
         23 . A pharmaceutical composition comprising:
 a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light;   a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane.   
     
     
         24 . The composition according to  claim 23 , wherein the light-activated protein is an ion pump. 
     
     
         25 . The composition according to  claim 24 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         26 . The composition according to  claim 21 , wherein the light-activated protein is an ion channel. 
     
     
         27 . The composition according to  claim 26 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         28 . The composition according to  claim 23 , wherein the response protein is an ion pump. 
     
     
         29 . The composition according to  claim 28 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         30 . The composition according to  claim 23 , wherein the response protein is an ion channel, an ion-exchange protein, or an ion co-transporter. 
     
     
         31 . The composition according to  claim 30 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         32 . The composition according to  claim 23 , wherein the first nucleic acid and the second nucleic acid are present within a single expression vector. 
     
     
         33 . The composition according to  claim 23 , wherein the nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light is operably linked to a neuron-specific transcription control element. 
     
     
         34 . The composition according to  claim 23 , wherein the second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane is operably linked to a neuron-specific transcription control element. 
     
     
         35 . A cell comprising:
 a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light;   a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane.   
     
     
         36 . The cell according to  claim 35 , wherein the light-activated protein is an ion pump. 
     
     
         37 . The cell according to  claim 36 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         38 . The cell according to  claim 35 , wherein the light-activated protein is an ion channel. 
     
     
         39 . The cell according to  claim 38 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         40 . The cell according to  claim 35 , wherein the response protein is an ion pump. 
     
     
         41 . The cell according to  claim 40 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         42 . The cell according to  claim 35 , wherein the response protein is an ion channel, an ion exchange protein, or an ion co-transporter. 
     
     
         43 . The cell according to  claim 42 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         44 . The cell according to  claim 35 , wherein the first nucleic acid and the second nucleic acid are present within a single expression vector. 
     
     
         45 . The cell according to  claim 35 , wherein the nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light is operably linked to a neuron-specific transcription control element. 
     
     
         46 . The cell according to  claim 35 , wherein the second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane is operably linked to a neuron-specific transcription control element. 
     
     
         47 . A method for modulating the membrane potential of a cell in response to light, the method comprising exposing a cell to light of an activating wavelength, wherein the cell is genetically modified with:
 a) a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light; and   b) a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane.   
     
     
         48 . The method according to  claim 47 , wherein the light-activated protein is an ion pump. 
     
     
         49 . The method according to  claim 48 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         50 . The method according to  claim 47 , wherein the light-activated protein is an ion channel. 
     
     
         51 . The method according to  claim 50 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         52 . The method according to  claim 47 , wherein the response protein is an ion pump. 
     
     
         53 . The method according to  claim 52 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         54 . The method according to  claim 47 , wherein the response protein is an ion channel, an ion exchange protein, or an ion co-transporter. 
     
     
         55 . The method according to  claim 54 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         56 . The method according to  claim 47 , wherein exposing the cell to light of an activating wavelength inhibits retrograde propagating action potentials within the cell. 
     
     
         57 . A method for inhibiting the activity of a voltage-gated sodium channel in a cell in response to light, the method comprising exposing the cell to light of an activating wavelength, wherein the cell is genetically modified with:
 a) a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a plurality of hydrogen ions to pass through a cell membrane in an outward direction in response to light; and   b) a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the presence of the hydrogen ions on or near the external surface of the cell membrane by allowing a plurality of sodium ions to pass through the cell membrane in an inward direction, causing sustained depolarization of the cell membrane, thereby inactivating the voltage-gated sodium ion channel and inhibiting its activity.   
     
     
         58 . The method according to  claim 57 , wherein the light-activated protein is an ion pump. 
     
     
         59 . The method according to  claim 58 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         60 . The method according to  claim 57 , wherein the light-activated protein is an ion channel. 
     
     
         61 . The method according to  claim 60 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         62 . The method according to  claim 57 , wherein the response protein is an ion pump. 
     
     
         63 . The method according to  claim 62 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         64 . The method according to  claim 57 , wherein the response protein is an ion channel, an ion exchange protein, or an ion co-transporter. 
     
     
         65 . The method according to  claim 64 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         66 . The method according to  claim 57 , wherein exposing the cell to light of an activating wavelength inhibits retrograde or anterograde propagating action potentials within the cell. 
     
     
         67 . A method of treating a condition in a subject, the method comprising:
 genetically modifying a target cell of the subject with:
 a) a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a membrane of the target cell in response to light; and 
 b) a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the membrane of the target cell by allowing a second ion to pass through the membrane, wherein passage of the second ion through the membrane treats the condition; and 
   exposing the target cell to light of an activating wavelength to treat the subject for the condition.   
     
     
         68 . The method according to  claim 67 , wherein the light-activated protein is an ion pump. 
     
     
         69 . The method according to  claim 68 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         70 . The method according to  claim 67 , wherein the light-activated protein is an ion channel. 
     
     
         71 . The method according to  claim 70 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         72 . The method according to  claim 67 , wherein the response protein is an ion pump. 
     
     
         73 . The method according to  claim 72 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         74 . The method according to  claim 67 , wherein the response protein is an ion channel, an ion exchange protein, or an ion co-transporter. 
     
     
         75 . The method according to  claim 74 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         76 . The method according to  claim 67 , wherein exposing the cell to light of an activating wavelength inhibits retrograde or anterograde propagating action potentials within the cell. 
     
     
         77 . The method according to  claim 67 , wherein the condition is a cardiac condition, a gastrointestinal condition, an endocrine condition, a neurological condition, or a psychiatric condition. 
     
     
         78 . A method for treating a condition in a subject, the method comprising:
 genetically modifying a nerve cell of the subject with:
 a) a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a plurality of hydrogen ions to pass through a membrane of the nerve cell in an outward direction in response to light; and 
 b) a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the plurality of hydrogen ions through the membrane of the nerve cell by allowing a plurality of sodium ions to pass through the membrane in an inward direction, wherein passage of the plurality of sodium ions through the membrane depolarizes the membrane and inactivates a voltage-gated sodium channel in the membrane of the nerve cell, and thereby treats the neurological condition; and 
   exposing the nerve cell to light of an activating wavelength to treat the subject for the condition.   
     
     
         79 . The method according to  claim 78 , wherein the light-activated protein is a hydrogen ion pump. 
     
     
         80 . The method according to  claim 78 , wherein the light-activated protein is a hydrogen ion channel. 
     
     
         81 . The method according to  claim 78 , wherein the response protein is a sodium ion pump. 
     
     
         82 . The method according to  claim 78 , wherein the response protein is a sodium ion channel, a sodium ion exchange protein, or a sodium ion-cotransporter. 
     
     
         83 . The method according to  claim 78 , wherein exposing the cell to light of an activating wavelength inhibits retrograde or anterograde propagating action potentials within the cell. 
     
     
         84 . The method according to  claim 78 , wherein the condition is a cardiac condition, a gastrointestinal condition, or a neurological condition. 
     
     
         85 . A method of selectively inhibiting retrograde propagating action potentials within a nerve cell or a portion thereof, the method comprising:
 genetically modifying the nerve cell with:
 a) a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a plurality of hydrogen ions to pass through a membrane of the nerve cell in response to light; and 
 b) a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the hydrogen ions through the membrane of the nerve cell by allowing a plurality of sodium ions to pass through the membrane, wherein passage of the sodium ions through the membrane depolarizes the membrane and inactivates a plurality of voltage-gated sodium channels in the membrane of the nerve cell that cause retrograde propagating action potentials; and 
   exposing the nerve cell, or a portion thereof, to light of an activating wavelength to inhibit retrograde propagating action potentials therein.   
     
     
         86 . The method according to  claim 85 , wherein the light-activated protein is a hydrogen ion pump. 
     
     
         87 . The method according to  claim 85 , wherein the light-activated protein is a hydrogen ion channel. 
     
     
         88 . The method according to  claim 85 , wherein the response protein is a sodium ion pump. 
     
     
         89 . The method according to  claim 85 , wherein the response protein is a sodium ion channel, a sodium ion exchange protein, or a sodium ion co-transporter. 
     
     
         90 . A kit comprising:
 a first nucleic acid comprising a nucleotide sequence encoding a light-activated protein that is adapted to allow a first ion to pass through a cell membrane in response to light; and   a second nucleic acid comprising a nucleotide sequence encoding a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane.   
     
     
         91 . The kit according to  claim 90 , wherein the light-activated protein is an ion pump. 
     
     
         92 . The kit according to  claim 91 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         93 . The kit according to  claim 90 , wherein the light-activated protein is an ion channel, an ion exchange protein, or an ion co-transporter. 
     
     
         94 . The kit according to  claim 93 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, a calcium ion channel, or a cation channel. 
     
     
         95 . The kit according to  claim 90 , wherein the response protein is an ion pump. 
     
     
         96 . The kit according to  claim 95 , wherein the ion pump is a hydrogen ion pump, a sodium ion pump, a potassium ion pump, a chloride ion pump, or a calcium ion pump. 
     
     
         97 . The kit according to  claim 90 , wherein the response protein is an ion channel. 
     
     
         98 . The kit according to  claim 97 , wherein the ion channel is a hydrogen ion channel, a sodium ion channel, a potassium ion channel, a chloride ion channel, or a calcium ion channel. 
     
     
         99 . The kit according to  claim 90 , further comprising a device configured to illuminate a target location with a light. 
     
     
         100 . The kit according to  claim 99 , wherein the device is configured to illuminate the target location with light having a wavelength ranging from about 350 to about 750 nm. 
     
     
         101 . The kit according to  claim 99 , wherein the device is configured to illuminate the target location with light having a wavelength ranging from about 530 up to about 560 nm. 
     
     
         102 . The kit according to  claim 99 , wherein the device is configured to constantly illuminate the target location with a light. 
     
     
         103 . The kit according to  claim 99 , wherein the device is configured to illuminate the target location with pulses of light. 
     
     
         104 . The kit according to  claim 102  or  103 , wherein the device is configured to modulate the wavelength and/or the intensity of the light. 
     
     
         105 . The kit according to  claim 103 , wherein the device is configured to modulate the frequency and/or duration of the pulses of light. 
     
     
         106 . The kit according to  claim 102  or  103 , wherein the device is configured to illuminate the target location in response to a user input. 
     
     
         107 . The kit according to  claim 106 , wherein the user input comprises: the wavelength of light, the intensity of light, the duration of a light pulse, the frequency of a light pulse, and/or the target location. 
     
     
         108 . The kit according to  claim 90 , wherein the device is adapted to be implanted in a subject. 
     
     
         109 . The kit according to  claim 90 , wherein the target location is: a cell, a portion of a cell, a plurality of cells, a bundle of nerve fibers, a neuromuscular junction, a central nervous system (CNS) tissue, a peripheral nervous system (PNS) tissue, or an anatomical region. 
     
     
         110 . A fusion polypeptide comprising:
 a) a light-responsive protein that is adapted to allow a first ion to pass through a cell membrane in response to light; and   b) a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane.   
     
     
         111 . The fusion polypeptide according to  claim 110 , wherein the fusion polypeptide comprises, in order from amino terminus to carboxyl terminus:
 a) a light-responsive protein that is adapted to allow a first ion to pass through a cell membrane in response to light;   b) a membrane trafficking signal;   c) a response protein that responds to the passage of the first ion through the cell membrane by allowing a second ion to pass through the cell membrane; and   d) a membrane trafficking signal.   
     
     
         112 . The fusion polypeptide according to  claim 110  or  111 , wherein the response protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ASIC2a polypeptide amino acid sequence set forth in SEQ ID NO:19. 
     
     
         113 . The fusion polypeptide according to  claim 111 , wherein the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:37). 
     
     
         114 . The fusion polypeptide according to any one of  claims 110 - 113 , further comprising an endoplasmic reticulum (ER) export signal. 
     
     
         115 . The fusion polypeptide according to  claim 114 , wherein the ER export signal comprises the amino acid sequence FCYENEV (SEQ ID NO:47) 
     
     
         116 . The fusion polypeptide according to any one of  claims 110 - 115 , wherein the fusion polypeptide comprises, interposed between the light-responsive polypeptide and the response protein, a self-cleaving polypeptide. 
     
     
         117 . The fusion polypeptide according to  claim 116 , wherein the self-cleaving polypeptide comprises the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO:49). 
     
     
         118 . A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide according to any one of  claims 110 - 117 . 
     
     
         119 . A recombinant expression vector comprising the nucleic acid according to  claim 118 . 
     
     
         120 . The recombinant expression vector according to  claim 119 , wherein the nucleotide sequence encoding the fusion polypeptide is operably linked to a neuron-specific transcriptional control element. 
     
     
         121 . A cell genetically modified with the recombinant expression vector according to  claim 119 . 
     
     
         122 . The cell according to  claim 121 , wherein the cell is a neuron.