U.S. patent application number 15/446282 was filed with the patent office on 2017-06-22 for blue light-activated ion channel molecules and uses thereof.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is The Governors of the University of Alberta, Massachusetts Institute of Technology, President and Fellows of Harvard College. Invention is credited to Edward Boyden, Yongku Cho, Brian Y. Chow, Adam E. Cohen, Daniel R. Hochbaum, Nathan Klapoetke, Gane K.S. Wong.
Application Number | 20170174730 15/446282 |
Document ID | / |
Family ID | 53773870 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170174730 |
Kind Code |
A1 |
Klapoetke; Nathan ; et
al. |
June 22, 2017 |
BLUE LIGHT-ACTIVATED ION CHANNEL MOLECULES AND USES THEREOF
Abstract
The invention, in some aspects relates to light-activated ion
channel polypeptides and encoding nucleic acids and also relates in
part to compositions comprising light-activated ion channel
polypeptides and methods using light-activated ion channel
polypeptides to alter cell activity and function.
Inventors: |
Klapoetke; Nathan; (Ashburn,
VA) ; Boyden; Edward; (Chestnut Hill, MA) ;
Cho; Yongku; (Vernon, CT) ; Chow; Brian Y.;
(Cherry Hill, NJ) ; Wong; Gane K.S.; (Edmonton,
CA) ; Cohen; Adam E.; (Cambridge, MA) ;
Hochbaum; Daniel R.; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology
The Governors of the University of Alberta
President and Fellows of Harvard College |
Cambridge
Edmonton
Cambridge |
MA
MA |
US
CA
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
The Governors of the University of Alberta
Edmonton
MA
President and Fellows of Harvard College
Cambridge
|
Family ID: |
53773870 |
Appl. No.: |
15/446282 |
Filed: |
March 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14616228 |
Feb 6, 2015 |
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15446282 |
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61937066 |
Feb 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/00 20180101; C07K
14/405 20130101; A61K 38/00 20130101; A61P 27/02 20180101; A61N
5/062 20130101; A61P 25/00 20180101; A61B 3/10 20130101; A61K 35/30
20130101; A61B 3/0008 20130101; A61P 19/00 20180101; A61P 25/02
20180101; G01N 33/5023 20130101; A61K 41/00 20130101; A61N 5/0622
20130101; A61K 36/05 20130101; A61N 2005/0663 20130101; A61K
41/0057 20130101; A61P 27/16 20180101; A61K 35/34 20130101 |
International
Class: |
C07K 14/405 20060101
C07K014/405; G01N 33/50 20060101 G01N033/50; A61K 41/00 20060101
A61K041/00; A61N 5/06 20060101 A61N005/06 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under NSF
CBET 1053233 awarded by the National Science Foundation; NIH
1R01DA029639 and NIH 1R01NS075421 both awarded by the National
Institutes of Health; and DARPA HR0011-12-C-0068, awarded by the
Department of Defense. The government has certain rights in the
invention.
Claims
1. A light-activated ion channel polypeptide, wherein the ion
channel when expressed in a membrane and contacted with blue light
is activated, the polypeptide comprises a wild-type or modified
Scherffelia channelrhodopsin polypeptide sequence, and wherein the
light-activated ion channel polypeptide is not present in a
Scherffelia cell.
2-5. (canceled)
6. The light-activated ion channel polypeptide of claim 1, wherein
the polypeptide comprises the amino acid sequence of ChR64 (SEQ ID
NO:2).
7. The light-activated ion channel polypeptide of claim 1, wherein
the modified Scherffelia channelrhodopsin polypeptide sequence
comprises a E.fwdarw.A substitution at an amino acid residue
corresponding to amino acid 154 of the amino acid sequence of ChR64
(SEQ ID NO:2).
8. The light-activated ion channel polypeptide of claim 1, wherein
the modified Scherffelia channelrhodopsin polypeptide sequence is
the sequence set forth as SEQ ID NO:7.
9-10. (canceled)
11. A cell comprising the light-activated ion channel polypeptide
of claim 1, wherein the cell is one or more of: (i) an excitable
cell and (ii) a mammalian cell.
12-17. (canceled)
18. A nucleic acid molecule comprising the sequence encoding the
light-activated ion channel polypeptide of claim 1, wherein the
nucleic acid molecule is not in a Scherffelia cell.
19-21. (canceled)
22. A vector comprising the nucleic acid sequence of claim 18.
23-29. (canceled)
30. A method of depolarizing a cell, the method comprising,
contacting a cell with a blue light under conditions suitable to
depolarize the cell, wherein (i) the cell comprises a
light-activated ion channel polypeptide that is activated when
contacted with blue light, (ii) the light-activated ion channel
polypeptide comprises a wild-type or modified Scherffelia
channelrhodopsin polypeptide sequence, and (iii) the cell is not a
Scherffelia cell; and depolarizing the cell.
31-32. (canceled)
33. The method of claim 30, wherein the amino acid sequence of the
light-activated ion channel polypeptide sequence comprises the
sequence set forth as SEQ ID NO:2 or SEQ ID NO:7.
34-35. (canceled)
36. The method of claim 30, wherein the cell is a mammalian
cell.
37-38. (canceled)
39. The method of claim 30, wherein the cell is in a subject and
depolarizing the cell diagnoses or assists in a diagnosis of a
disorder in the subject.
40. The method of claim 30, wherein the cell is in a subject and
depolarizing the cell treats a disorder in the subject.
41. A method of assessing the effect of a candidate compound on a
cell, the method comprising, a) contacting a test cell comprising a
light-activated ion channel polypeptide that is activated when
contacted with blue light, wherein the ion channel polypeptide
comprises a wild-type or modified Scherffelia channelrhodopsin
polypeptide sequence and the cell is not a Scherifelia cell, with
blue light under conditions suitable for depolarization of the
cell; b) contacting the test cell with a candidate compound; and c)
identifying the presence or absence of a change in depolarization
or a change in a depolarization-mediated cell characteristic in the
test cell contacted with the blue light and the candidate compound
compared to depolarization or the depolarization-mediated cell
characteristic, respectively, in a control cell contacted with the
blue light and not contacted with the candidate compound; wherein a
change in depolarization or the depolarization-mediated cell
characteristic in the test cell compared to the control cell
indicates an effect of the candidate compound on the test cell.
42-46. (canceled)
47. The method of claim 41, wherein the amino acid sequence of the
light-activated ion channel polypeptide comprises an amino acid
sequence forth as SEQ ID NO:2 or SEQ ID NO:7.
48-51. (canceled)
52. The method of claim 41, wherein the cell is a mammalian
cell.
53-54. (canceled)
55. A method of treating a disorder in a subject, the method
comprising a) administering to a subject in need of such treatment,
a therapeutically effective amount of a light-activated ion channel
polypeptide that is activated when contacted with blue light,
wherein the ion channel polypeptide comprises a wild-type or
modified Scherffelia channelrhodopsin polypeptide sequence, to
treat the disorder and b) contacting the cell with blue light and
activating the light-activated ion channel in the cell under
conditions sufficient to depolarize the cell, wherein depolarizing
the cell treats the disorder in the subject.
56-65. (canceled)
66. The method of claim 55, wherein the amino acid sequence of the
blue-light-activated ion channel is set forth as SEQ ID NO:2 or SEQ
ID NO:7.
67-71. (canceled)
72. A fusion protein comprising the light-activated ion channel
polypeptide of claim 1.
73. The light-activated ion channel polypeptide of claim 1, wherein
amino acid sequence of the modified Scherffelia channelrhodopsin
polypeptide, when aligned with the polypeptide set forth as SEQ ID
NO: 2 has one or more of at least 75%, 80%, 85%, 90%, and 95%
sequence identity to the corresponding amino acids of SEQ ID
NO:2.
74. The light-activated ion channel polypeptide of claim 30,
wherein amino acid sequence of the modified Scherffelia
channelrhodopsin polypeptide, when aligned with the polypeptide set
forth as SEQ ID NO: 2 has one or more of at least 75%, 80%, 85%,
90%, and 95% sequence identity to the corresponding amino acids of
SEQ ID NO:2.
Description
RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 14/616,228, filed on Feb. 6, 2015, which claims benefit
under 35 U.S.C. .sctn.119(e) of U.S. Provisional application Ser.
No. 61/937,066 filed Feb. 7, 2014, the disclosure of each of which
is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The invention, in some aspects relates to compositions and
methods for altering cell activity and function and the use of
light-activated ion channels.
BACKGROUND OF THE INVENTION
[0004] Altering and controlling cell membrane and subcellular
region ion permeability has permitted examination of
characteristics of cells, tissues, and organisms. Light-driven
pumps and channels have been used to silence or enhance cell
activity and their use has been proposed for drug screening,
therapeutic applications, and for exploring cellular and
subcellular function.
[0005] Molecular-genetic methods for preparing cells that can be
activated (e.g., depolarized) or inactivated (e.g., hyperpolarized)
by specific wavelengths of light have been developed (see, for
example, Han, X and E. S. Boyden, 2007, PLoS ONE 2, e299). It has
been identified that the light-activated cation channel
channelrhodopsin-2 (ChR2), and the light-activated chloride pump
halorhodopsin (Halo/NpHR), when transgenically expressed in cell
such as neurons, make them sensitive to being activated by blue
light, and silenced by yellow light, respectively (Han, X. and E.
S. Boyden, 2007, PLoS ONE 2(3): e299; Boyden, E. S., et. al., 2005,
Nat Neurosci. 2005 September; 8(9):1263-8. Epub 2005 Aug. 14).
Previously identified light-activated pumps and channels have been
restricted to activation by particular wavelengths of light, thus
limiting their usefulness.
SUMMARY OF THE INVENTION
[0006] The invention, in part, relates to isolated light-activated
ion channel polypeptides and methods for their preparation and use.
The invention also includes isolated nucleic acid sequences that
encode light-driven ion channels of the invention as well as
vectors and constructs that comprise such nucleic acid sequences.
In addition, the invention in some aspects includes expression of
light-activated ion channel polypeptides in cells, tissues, and
organisms as well as methods for using the light-activated ion
channels to alter cell and tissue function and for use in diagnosis
and treatment of disorders.
[0007] The invention, in part, also relates to methods for
adjusting the voltage potential of cells, subcellular regions, or
extracellular regions. Some aspects of the invention include
methods of incorporating at least one nucleic acid sequence
encoding a light-driven ion channel into at least one target cell,
subcellular region, or extracellular region, the ion channel
functioning to change transmembrane passage of ions in response to
a specific wavelength of light. Exposing an excitable cell that
includes an expressed light-driven ion channel of the invention to
a wavelength of light that activates the channel, may result in
depolarization of the excitable cell. By contacting a cell that
includes a light-activated ion channel polypeptide of the invention
with particular wavelengths of light, the cell is depolarized. A
plurality of light-activated ion channels activated by different
wavelengths of light may be used to achieve multi-color
depolarization.
[0008] In some embodiments, the invention comprises a method for
the expression of certain classes of genes encoding for
light-driven ion channels, in genetically-targeted cells, to allow
millisecond-timescale generation of depolarizing current in
response to pulses of light. These channels can be
genetically-expressed in specific cells (e.g., using a virus) and
then used to control cells in intact organisms (including humans)
as well as cells in vitro, in response to pulses of light. Given
that these channels have different activation spectra from one
another and from the prior channels (e.g., ChR2/VChR1), they also
allow multiple colors of light to be used to depolarize different
sets of cells in the same tissue, simply by expressing channels
with different activation spectra genetically in different cells,
and then illuminating the tissue with different colors of
light.
[0009] In some aspects, the invention uses eukaryotic
channelrhodopsins, such as Scherifelia dubia and Chloromonas oogama
rhodopsin to depolarize excitable cells. These channelrhodopsins
can also be used to modify the pH of cells, or to introduce cations
as chemical transmitters.
[0010] The ability to optically perturb, modify, or control
cellular function offers many advantages over physical manipulation
mechanisms, such as speed, non-invasiveness, and the ability to
easily span vast spatial scales from the nanoscale to macroscale.
One such approach is an opto-genetic approach, in which
heterologously expressed light-activated membrane polypeptides such
as a light-activated ion channel polypeptide of the invention, are
used to move ions with various spectra of light.
[0011] According to one aspect of the invention, isolated
light-activated ion channel polypeptides are provided. The isolated
light-activated ion channel polypeptides, when expressed in a
membrane and contacted with blue light are activated, and wherein
the polypeptide comprises a wild-type or modified Scherifelia or
Chloromonas channelrhodopsin polypeptide sequence. In some
embodiments, the Scherifelia polypeptide sequence is a Scherifelia
dubia polypeptide sequence and the Chloromonas polypeptide sequence
is a Chloromonas oogama polypeptide sequence. In certain
embodiments, contacting the expressed ion channel with red light
does not activate the ion channel. In some embodiments, the
activating blue light has a wavelength in a range from about 450 nm
to about 495 nm. In some embodiments, the red light has a
wavelength of about 620 nm to about 690 nm. In some embodiments,
the polypeptide comprises the amino acid sequence of ChR64 (SEQ ID
NO:2) or ChR86 (SEQ ID NO:4). In certain embodiments, the modified
Scherifelia channelrhodopsin polypeptide sequence comprises an
E.fwdarw.A substitution at an amino acid residue corresponding to
amino acid 154 of the amino acid sequence of ChR64 (SEQ ID NO:2).
In some embodiments, the modified Scherifelia channelrhodopsin
polypeptide sequence is the sequence set forth as SEQ ID NO:7. In
some embodiments, the modified Chloromonas channelrhodopsin
polypeptide sequence comprises a D.fwdarw.A substitution at an
amino acid residue corresponding to amino acid 124 of the amino
acid sequence of ChR86 (SEQ ID NO:4). In certain embodiments, the
modified Chloromonas channelrhodopsin polypeptide sequence is the
sequence set forth as SEQ ID NO:8.
[0012] According to another aspect of the invention, a cell that
includes of any of the aforementioned embodiments of isolated
light-activated ion channel polypeptides is provided. In some
embodiments, the light-activated ion channel is activated and the
cell depolarized when the light-activated ion channel is contacted
with light under suitable conditions for depolarization of the
cell. In some embodiments, the cell is an excitable cell. In some
embodiments, the cell is a mammalian cell. In certain embodiments,
the cell is in vitro, ex vivo, or in vivo. In some embodiments, the
cell also includes one, two, three, four, or more additional
light-activated ion channel polypeptides, wherein at least one,
two, three, four, or more of the additional light-activated ion
channel polypeptides is activated by contact with light having a
non-blue light wavelength.
[0013] According to another aspect of the invention, an isolated
nucleic acid sequence that encodes any one of the aforementioned
isolated light-activated ion channel polypeptides is provided. In
certain embodiments, the sequence comprises the sequence set forth
as SEQ ID NO:1, or SEQ ID NO:3. In some embodiments, the nucleic
acid sequence is a mammalian codon-optimized DNA sequence. In some
embodiments, the light-activated ion pump encoded by the nucleic
acid sequence is expressed in the cell.
[0014] According to another aspect of the invention, a vector that
includes any of the aforementioned embodiments of an isolated
nucleic acid is provided. In some embodiments, the vector also
comprises a trafficking sequence. In some embodiments, the nucleic
acid sequence is operatively linked to a promoter sequence. In
certain embodiments, the vector also includes one, two, or more
nucleic acid signal sequences operatively linked to the nucleic
acid sequence encoding the light-activated ion channel. In some
embodiments, the vector is a plasmid vector, cosmid vector, viral
vector, or an artificial chromosome.
[0015] According to another aspect of the invention, a cell that
includes any aforementioned embodiment of a vector is provided. In
certain embodiments, the cell also includes one, two, three, four,
or more additional light-activated ion channels, wherein at least
one, two, three, four, or more of the additional light-activated
ion channels is activated by contact with light having a non-blue
light wavelength.
[0016] According to another aspect of the invention, methods of
depolarizing a cell are provided. The methods include contacting a
cell that includes any aforementioned embodiment of an isolated
light-activated ion channel polypeptide, with a blue light under
conditions suitable to depolarize the cell and depolarizing the
cell. In some embodiments, the light-activated ion channel
activates in response to blue light in a range from about 450 nm to
about 495 nm. In some embodiments, the light-activated ion channel
polypeptide is encoded by the nucleic acid sequence set forth as
SEQ ID NO:1 or SEQ ID NO:3. In certain embodiments the amino acid
sequence of the light-activated ion channel polypeptide sequence
includes the sequence set forth as SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:7, or SEQ ID NO:8. In some embodiments the light-activated ion
channel is not activated in response to contact with red light. In
some embodiments, the cell is a nervous system cell, a cardiac
cell, a circulatory system cell, a visual system cell, an auditory
system cell, or a muscle cell. In certain embodiments, the cell is
a mammalian cell. In some embodiments, the cell additionally
includes one, two, three, or more additional light-activated ion
channel polypeptides, wherein at least one, two, three, four, or
more of the additional light-activated ion channel polypeptides is
activated by contact with light having a non-blue light wavelength
and is not activated by light having a blue light wavelength in a
range from about 450 nm to about 495 nm. In some embodiments, the
cell is in a subject and depolarizing the cell diagnoses or assists
in a diagnosis of a disorder in the subject. In some embodiments,
the cell is in a subject and depolarizing the cell treats a
disorder in the subject.
[0017] According to yet another aspect of the invention, methods of
assessing the effect of a candidate compound on a cell are
provided. The methods include a) contacting a test cell that
includes any aforementioned embodiment of an isolated
light-activated ion channel with blue light under conditions
suitable for depolarization of the cell; b) contacting the test
cell with a candidate compound; and c) identifying the presence or
absence of a change in depolarization or a change in a
depolarization-mediated cell characteristic in the test cell
contacted with the blue light and the candidate compound compared
to depolarization or a depolarization-mediated cell characteristic,
respectively, in a control cell contacted with the blue light and
not contacted with the candidate compound; wherein a change in
depolarization or a depolarization-mediated cell characteristic in
the test cell compared to the control indicates an effect of the
candidate compound on the test cell. In certain embodiments, the
blue light has a wavelength in a range from about 450 nm to about
495 nm. In some embodiments, the effect of the candidate compound
is an effect on the depolarization of the test cell. In some
embodiments, the effect of the candidate compound is an effect on a
depolarization-mediated cell characteristic in the test cell. In
certain embodiments, the method further includes characterizing the
change identified in the depolarization or the
depolarization-mediated cell characteristic. In some embodiments,
the light-activated ion channel is encoded by the nucleic acid
sequence set forth as SEQ ID NO:1 or SEQ ID NO:3. In some
embodiments, the amino acid sequence of the light-activated ion
channel polypeptide comprises an amino acid sequence forth as SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:8. In some
embodiments, the light-activated ion channel does not activate in
response to contact with red light. In certain embodiments, a
depolarization-mediated cell characteristic is an action potential.
In some embodiments, a depolarization-mediated cell characteristic
release of a neurotransmitter. In some embodiments, the cell is a
nervous system cell, a cardiac cell, a circulatory system cell, a
visual system cell, an auditory system cell, a muscle cell, or
another excitable cell. In some embodiments, the cell is a
mammalian cell. In certain embodiments, the cell also includes one,
two, three, or more additional light-activated ion channel
polypeptides, wherein at least one, two, three, four, or more of
the additional light-activated ion channel polypeptides is
activated by contact with light having a non-blue light wavelength
and is not activated by contact with blue light having a wavelength
in a range from about 450 nm to about 495 nm.
[0018] According to another aspect of the invention, methods of
treating a disorder in a subject are provided. The methods include
a) administering to a subject in need of such treatment, a
therapeutically effective amount of any of the aforementioned
embodiments of a blue-light-activated ion channel, to treat the
disorder and b) contacting the cell with blue light and activating
the light-activated ion channel in the cell under conditions
sufficient to depolarize the cell, wherein depolarizing the cell
treats the disorder in the subject. In some embodiments, the
light-activated ion channel is administered in the form of a cell,
wherein the cell expresses the light-activated ion channel, or in
the form of a vector, wherein the vector comprises a nucleic acid
sequence encoding the light-activated ion channel and the
administration of the vector results in expression of the
blue-light-activated ion channel in a cell in the subject. In some
embodiments, the vector also includes a signal sequence. In some
embodiments, the vector also includes a cell-specific promoter. In
certain embodiments, the disorder is a neurological disorder, a
visual system disorder, a circulatory system disorder, a
musculoskeletal system disorder, or an auditory system disorder. In
some embodiments, the method also includes administering an
additional therapeutic composition to the subject. In some
embodiments, depolarizing the cell modulates a
depolarization-mediated cell characteristic. In some embodiments, a
depolarization-mediated cell characteristic is an action potential.
In certain embodiments, a depolarization-mediated cell
characteristic release of a neurotransmitter. In some embodiments,
the blue light-activated ion channel activates in response to light
with a wavelength in a range from about 450 nm to about 495 nm. In
some embodiments, the blue-light-activated ion channel is encoded
by a nucleic acid sequence set forth as SEQ ID NO:1 or SEQ ID NO:3.
In some embodiments, the amino acid sequence of the
blue-light-activated ion channel is set forth as SEQ ID NO:2, SEQ
ID NO:4, or SEQ ID NO:7, or SEQ ID NO:8. In certain embodiments,
the blue light-activated ion channel does not activate in response
to contact with light that is not blue light. In some embodiments,
the cell is a nervous system cell, a neuron, a cardiac cell, a
circulatory system cell, a visual system cell, an auditory system
cell, or a muscle cell. In certain embodiments, the cell is a
mammalian cell. In some embodiments, the cell also includes one,
two, three, or more additional light-activated ion channel
polypeptides, wherein at least one, two, three, four, or more of
the additional light-activated ion channel polypeptides is
activated by contact with light having a non-blue light wavelength
and is not activated by contact with blue light having a wavelength
in a range from about 450 nm to about 495 nm.
[0019] The present invention is not intended to be limited to a
system or method that must satisfy one or more of any stated
objects or features of the invention. It is also important to note
that the present invention is not limited to the exemplary or
primary embodiments described herein. Modifications and
substitutions by one of ordinary skill in the art are considered to
be within the scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows action spectra recorded in HEK293 cells for
ChR2, ChR64, ChR64 E154A, and ChR86 light-activated ion channel
polypeptides when contacted with various wavelengths of light.
[0021] FIG. 2A-D shows graphs demonstrating blue light photocurrent
and kinetic comparisons in cultured hippocampal neurons in which
light-activated ion channels were expressed. Results from ChR2,
ChR64, and ChR68 are shown. FIG. 2A is a graph showing current with
contact with blue irradiance. FIGS. 2B and 2C are graphs of
t.sub.on at a 5 mW/mm.sup.2 and 4.23 mW/mm.sup.2 illumination,
respectively and provide a comparison of results for ChR2, ChR64,
and ChR68 light-activated ion channels. FIG. 2D shows the t.sub.off
results from ChR86 light activated ion channel with blue irradiance
at 4.23, 0.2, and 0.05 mW/mm.sup.2.
[0022] FIG. 3A-D provides photomicrographs and a table
demonstrating trafficking, expression and light sensitivity of
certain embodiments of light-activated ion channel polypeptides.
FIG. 3A shows a photomicrographic image of a cultured neuron
expressing wild-type SdChR. SdChR typically aggregated and formed
puncta in the soma. Scale bar 25 .mu.m. FIG. 3B shows a
photomicrographic image of a neuron expressing SdChR with an
additional trafficking sequence from Kir2.1 between the C-terminus
of SdChR and the N-terminus of eGFP. This trafficking sequence
substantially reduced intracellular puncta. Scale bar 25 .mu.m.
FIG. 3C shows a photomicrographic image of two neurons expressing
CheRiff. Inclusion of the E154A mutation reduced red light
sensitivity and reduced .tau..sub.off while maintaining excellent
membrane trafficking. Scale bar 25 .mu.m. FIG. 3D is a table
showing improvements in trafficking leading from ChR64 to CheRiff.
Scherifelia dubia Channelrhodopsin (SdChR) had promising light
sensitivity and a blue-shifted action spectrum appropriate for
pairing with QuasArs; yet it did not traffic efficiently to the
plasma membrane in rat hippocampal neurons. Of the three mutants,
CheRiff demonstrated best results for trafficking, blue
photocurrent, red photocurrent, and t.sub.off values.
[0023] FIG. 4A-B shows traces and graphs demonstrating
spectroscopic and kinetic properties of CheRiff. FIG. 4A at top
left shows components of channelrhodopsin current elicited by a
step in blue light. I.sub.pk is the difference between baseline
current and peak current. t.sub.on is the time between light onset
and peak current. .tau..sub.des is the desensitization time
constant determined by a single-exponential fit to the current
decay after the peak. I.sub.ss is steady state photocurrent.
.tau..sub.off is the channel closing time constant determined by a
single-exponential fit to the current decay after the illumination
ceases. FIG. 4A at top right shows peak (I.sub.pk) and steady state
(I.sub.ss) photocurrents in neurons expressing CheRiff (n=10
cells), ChR2 H134R (n=6 cells), and ChIEF (n=6 cells).
Photocurrents were measured in response to a 1 second 488 nm light
pulse (500 mW/cm.sup.2). CheRiff generated significantly larger
peak photocurrent than ChR2 H134R (p<0.001) or ChIEF
(p<0.001). CheRiff also had significantly larger steady state
photocurrents than ChR2 H134R (p<0.001) or ChIEF (p<0.01).
Bottom left: CheRiff had a significantly faster time to peak
(t.sub.on) when compared to ChR2 H134R (p<0.001) or ChIEF
(p<0.001). Bottom middle: CheRiff desensitized with a time
constant significantly slower than ChR2 H134R (p<0.001) or ChIEF
(p<0.001). FIG. 4A bottom right shows results when:
.tau..sub.off was measured in response to a 5 ms illumination pulse
(500 mW/cm.sup.2). CheRiff (n=9 cells) had a significantly faster
.tau..sub.off than ChR2 H134R (n=6 cells, p<0.05), and was
comparable to ChIEF (n=6 cells, p=0.94). All channelrhodopsin
comparisons were made on matched cultures, DIV 14-15. Expression
was driven by a CaMKII.alpha. promoter in identical plasmid
backbones. See Examples section for details on cell culture. FIG.
4B shows activation of CheRiff by red light used for imaging
Arch-based voltage indicators (640 nm, 900 W/cm.sup.2). FIG. 4B top
trace shows results indicating that under current-clamp (i=0) in a
neuron expressing CheRiff, pulses of red light led to a small
steady depolarization of 3.1.+-.0.2 mV (n=5 cells). FIG. 4B bottom
trace shows results indicating that under voltage-clamp (V=-65 mV),
pulses of red light led to a small inward photocurrent of
14.3.+-.3.1 pA (n=5 cells). Error bars represent s.e.m. Statistical
significance determined by one way ANOVA with Dunnett's post hoc
test. *p<0.05; **p<0.01; ***p<0.001.
[0024] FIG. 5A-E provides photomicrographic images and graphs
showing application of CheRiff in cultured hippocampal neurons.
FIG. 5A shows light micrographs (DIC) of Scherffelia dubia (strain
CCAC 0053) in side view (top) and face view (bottom). Arrows mark
eyespots (red). Scale bar 10 .mu.m. FIG. 5B shows photomicrographic
image of cultured rat hippocampal neuron expressing CheRiff-eGFP,
imaged via eGFP fluorescence. Scale bar 25 .mu.m. FIG. 5C shows
photocurrents induced by CheRiff and by Channelrhodopsin2 H134R
with illumination at 488 nm, 500 mW/cm.sup.2. FIG. 5D provides a
graph showing comparison of photocurrents as a function of
illumination intensity in matched cultures expressing CheRiff (n=5
cells) or ChR2 H134R (n=5 cells). Illumination was either over the
whole cell or confined to the soma. FIG. 5E provides a graph
showing spiking fidelity as a function of stimulation frequency and
illumination intensity in neurons expressing CheRiff (n=5 cells).
Error bars in FIG. 5D-E represent s.e.m.
BRIEF DESCRIPTION OF THE SEQUENCES
[0025] SEQ ID NO:1 is the mammalian codon-optimized DNA sequence
that encodes the wild-type Scherffelia dubia channelrhodopsin, also
referred to herein as ChR64 or SdChR:
TABLE-US-00001 atgggcggagctcctgctccagacgctcacagcgccccacctggaaacga
ttctgccggaggcagtgagtaccatgccccagctggatatcaagtgaatc
caccctaccaccccgtgcatgggtatgaggaacagtgcagctccatctac
atctactatggggccctgtgggagcaggaaacagctaggggcttccagtg
gtttgccgtgttcctgtctgccctgtttctggctttctacggctggcacg
cctataaggccagcgtgggatgggaggaagtgtacgtgtgctccgtggag
ctgatcaaagtgattctggagatctatttcgagttcaccagtcctgctat
gctgttcctgtacggagggaacattaccccatggctgagatatgccgaat
ggctgctgacatgtcccgtgatcctgattcatctgtctaacatcaccggc
ctgagtgaggaatacaataagcggacaatggctctgctggtgtccgacct
gggaactatttgcatgggagtgacagccgctctggccactgggtgggtga
agtggctgttttactgtatcggcctggtgtatggaacccagacattctac
aacgctggaatcatctacgtggagtcttactatatcatgcctgccggcgg
ctgtaagaaactggtgctggccatgactgccgtgtactattctagttggc
tgatgtttcccggcctgttcatattgggcctgaaggcatgcacaccctga
gcgtggctgggtccactattggccataccatcgccgacctgctgtccaag
aatatttggggactgctggggcacttcctgcggatcaaaattcacgagca
tatcattatgtacggcgatatcaggagaccagtgagctcccagtttctgg
gacgcaaggtggacgtgctggccttcgtgacagaggaagataaagtg.
[0026] SEQ ID NO: 2 is the amino acid sequence of the wild-type
Scherffelia dubia, also referred to herein as ChR64 or SdChR:
TABLE-US-00002 MGGAPAPDAHSAPPGNDSAGGSEYHAPAGYQVNPPYHPVHGYEEQCSSIY
IYYGALWEQETARGFQWFAVFLSALFLAFYGWHAYKASVGWEEVYVCSVE
LIKVILEIYFEFTSPAMLFLYGGNITPWLRYAEWLLTCPVILIHLSNITG
LSEEYNKRTMALLVSDLGTICMGVTAALATGWVKWLFYCIGLVYGTQTFY
NAGIIYVESYYIMPAGGCKKLVLAMTAVYYSSWLMFPGLFIFGPEGMHTL
SVAGSTIGHTIADLLSKNIWGLLGHFLRIKIHEHIIMYGDIRRPVSSQFL
GRKVDVLAFVTEEDKV.
[0027] SEQ ID NO:3 is the mammalian codon-optimized DNA sequence
that encodes wild-type Chloromonas oogama channelrhodopsin, also
referred to herein as ChR86:
TABLE-US-00003 atgctgggaaacggcagcgccattgtgcctatcgaccagtgcttttgcct
ggcttggaccgacagcctgggaagcgatacagagcagctggtggccaaca
tcctccagtggttcgccttcggcttcagcatcctgatcctgatgttctac
gcctaccagacttggagagccacttgcggttgggaggaggtctacgtctg
ttgcgtcgagctgaccaaggtcatcatcgagttcttccacgagttcgacg
accccagcatgctgtacctggctaacggacaccgagtccagtggctgaga
tacgcagagtggctgctgacttgtcccgtcatcctgatccacctgagcaa
cctgaccggcctgaaggacgactacagcaagcggaccatgaggctgctgg
tgtcagacgtgggaaccatcgtgtggggagctacaagcgccatgagcaca
ggctacgtcaaggtcatcttcttcgtgctgggttgcatctacggcgccaa
caccttcttccacgccgccaaggtgtatatcgagagctaccacgtggtgc
caaagggcagacctagaaccgtcgtgcggatcatggcttggctgttcttc
ctgtcttggggcatgttccccgtgctgttcgtcgtgggaccagaaggatt
cgacgccatcagcgtgtacggctctaccattggccacaccatcatcgacc
tcatgagcaagaattgttggggcctgctgggacactatctgagagtgctg
atccaccagcacatcatcatctacggcgacatccggaagaagaccaagat
caacgtggccggcgaggagatggaagtggagaccatggtggaccaggagg
acgaggagacagtg.
[0028] SEQ ID NO:4 is the amino acid sequence of wild-type
Chloromonas oogama channelrhodopsin, also referred to herein as
ChR86:
TABLE-US-00004 MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFY
AYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLR
YAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMST
GYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFF
LSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVL
IHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV.
[0029] SEQ ID NO:5 is the mammalian codon-optimized DNA sequence
that encodes the wild-type Channelrhodopsin-2, (see: Boyden, E. et
al., Nature Neuroscience 8, 1263-1268 (2005) and Nagel, G., et al.
PNAS Nov. 25, 2003 vol. 100 no. 24 13940-13945), also referred to
herein as ChR2:
TABLE-US-00005 atggactatggcggcgctttgtctgccgtcggacgcgaacttttgttcgt
tactaatcctgtggtggtgaacgggtccgtcctggtccctgaggatcaat
gttactgtgccggatggattgaatctcgcggcacgaacggcgctcagacc
gcgtcaaatgtcctgcagtggcttgcagcaggattcagcattttgctgct
gatgttctatgcctaccaaacctggaaatctacatgcggctgggaggaga
tctatgtgtgcgccattgaaatggttaaggtgattctcgagttctttttt
gagtttaagaatccctctatgctctaccttgccacaggacaccgggtgca
gtggctgcgctatgcagagtggctgctcacttgtcctgtcatccttatcc
acctgagcaacctcaccggcctgagcaacgactacagcaggagaaccatg
ggactccttgtctcagacatcgggactatcgtgtggggggctaccagcgc
catggcaaccggctatgttaaagtcatcttcttttgtcttggattgtgct
atggcgcgaacacattttttcacgccgccaaagcatatatcgagggttat
catactgtgccaaagggtcggtgccgccaggtcgtgaccggcatggcatg
gctgtttttcgtgagctggggtatgttcccaattctcttcattttggggc
ccgaaggttttggcgtcctgagcgtctatggctccaccgtaggtcacacg
attattgatctgatgagtaaaaattgttgggggttgttgggacactacct
gcgcgtcctgatccacgagcacatattgattcacggagatatccgcaaaa
ccaccaaactgaacatcggcggaacggagatcgaggtcgagactctcgtc
gaagacgaagccgaggccggagccgtg.
[0030] SEQ ID NO: 6 is the amino acid sequence of the wild-type
Channelrhodopsin-2, (see: Boyden, E. et al., Nature Neuroscience 8,
1263-1268 (2005) and Nagel, G., et al. PNAS Nov. 25, 2003 vol 100
no. 24 13940-13945), also referred to herein as ChR2:
TABLE-US-00006 MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQT
ASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFF
EFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIFILSNLTGLSNDYSRRT
MGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEG
YHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGH
TIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETL VEDEAEAGAV.
[0031] SEQ ID NO:7 is the amino acid sequence of ChR64 with an
E.fwdarw.A substitution at amino acid position 154:
TABLE-US-00007 MGGAPAPDAHSAPPGNDSAGGSEYHAPAGYQVNPPYHPVHGYEEQCSSIY
IYYGALWEQETARGFQWFAVFLSALFLAFYGWHAYKASVGWEEVYVCSVE
LIKVILEIYFEFTSPAMLFLYGGNITPWLRYAEWLLTCPVILIHLSNITG
LSEAYNKRTMALLVSDLGTICMGVTAALATGWVKWLFYCIGLVYGTQTFY
NAGIIYVESYYIMPAGGCKKLVLAMTAVYYSSWLMFPGLFIFGPEGMHTL
SVAGSTIGHTIADLLSKNIWGLLGHFLRIKIHEHIIMYGDIRRPVSSQFL
GRKVDVLAFVTEEDKV.
[0032] SEQ ID NO:8 is the amino acid sequence of ChR86 with a
D.fwdarw.A substitution at amino acid position 124:
TABLE-US-00008 MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFY
AYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLR
YAEWLLTCPVILIHLSNLTGLKDAYSKRTMRLLVSDVGTIVWGATSAMST
GYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFF
LSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVL
IHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV.
[0033] SEQ ID NO:9 is the DNA sequence of the ER export sequence
(also referred to herein as "ER2":
TABLE-US-00009 ttctgctacgagaatgaagtg.
[0034] SEQ ID NO: 10 is the amino acid sequence of the ER export
sequence (also referred to herein as "ER2":
TABLE-US-00010 FCYENEV.
[0035] SEQ ID NO:11 is the DNA sequence of KGC, which is a C
terminal export sequence, (also referred to as a "trafficking
sequence") from the potassium channel Kir2.1. It is also referred
to herein as "TS":
TABLE-US-00011 aaatccagaattacttctgaaggggagtatatccctctggatcaaataga
catcaatgtt.
[0036] SEQ ID NO:12 is the amino acid sequence of KGC, which is a C
terminal export sequence, (also referred to as a "trafficking
sequence") from the potassium channel Kir2.1. It is also referred
to herein as "TS":
TABLE-US-00012 KSRITSEGEYIPLDQIDINV.
DETAILED DESCRIPTION
[0037] The invention in some aspects relates to the expression in
cells of light-driven ion channel polypeptides that can be
activated by contact with one or more pulses of light, which
results in strong depolarization of the cell. Light-activated
channels of the invention, also referred to herein as
light-activated ion channels can be expressed in specific cells,
tissues, and/or organisms and used to control cells in vivo, ex
vivo, and in vitro in response to pulses of light of a suitable
wavelength. This invention, in part, includes genes, DNA, mRNA, and
proteins for light-gated ion channels, also referred to herein as
light-activated channels. Expression of light-activated channels of
the invention in genetically targeted cells enables
millisecond-timescale generation of depolarizing current in
response to pulses of light. Light-activated channels of the
invention can be genetically expressed in specific cells (for
example, through use of a virus) and then used to control
electrical activity in cells in intact organisms, including, but
not limited to humans, as well as cells in vitro, in response to
pulses of light.
[0038] Channelrhodopsins are well known in the art as tools for
optical control of membrane potential in electrically excitable
cells. Light-activated channel polypeptides in some embodiments of
the invention differ from prior channelrhodopsin tools in a number
of ways, such as, but not limited to higher maximal photocurrents
possible under saturating illumination and lower illumination
intensity needed to trigger precisely timed neuronal action
potentials. The ability to activate neurons at lower intensity
allows use of light-activated ion channel polypeptides of the
invention to be used in combination with one or more red-shifted
light-activated pumps and channels (for example, but not limited
to: Halo/NpHR, Arch, VChR1) without spurious activation of the
red-shifted species by the blue light used to activate the
light-activated ion channel polypeptides of the invention. In
addition, in some embodiments, light-activated ion channel
polypeptides of the invention may be expressed in combination with
one or more other red-shifted reporters (e.g. Arch-based voltage
indicators, R-GECO calcium indicator) without spurious activation
of the reporter by the blue light used to activate the
light-activated ion channel polypeptides of the invention. Certain
embodiments of light-activated ion channel polypeptides of the
invention can be used to activate neurons over larger regions,
using lower power light sources, and with less risk of
phototoxicity than was previously possible using alternative
channelrhodopsins. An additional advantage of a light-activated ion
channel polypeptide of the invention is its ability and use to
trigger neuronal action potentials when only a sub-cellular region
is illuminated. This capability permits studies of sub-cellular
electrical dynamics to be performed using embodiments of
light-activated ion channel polypeptides of the invention. In
addition, light-activated ion channel polypeptides of the invention
are not activated by, or are only minimally activated by contact
with red light, further facilitating combination with red-shifted
optical actuators and reporters.
[0039] In certain embodiments, the invention includes use of
eukaryotic rhodopsins, such as from the genus Scherffelia or
Chloromonas, including but not limited to rhodopsin from
Scherifelia dubia ("ChR64", "SdChR") or Chloromonas oogama
("ChR86"), and variants thereof, to depolarize excitable cells. In
certain embodiments of the invention, these light-activated ion
channel polypeptides of the invention may be used to modify the pH
of cells, or to introduce cations as chemical transmitters.
[0040] In some embodiments, light-activated ion channel
polypeptides of the invention may be variants of ChR64 or ChR86
polypeptides. Thus, in part, the invention also includes targeted
site-directed mutagenesis at specific amino acid residue(s) of
channelrhodopsins to alter efficacy and kinetics of light-activated
ion channel polypeptides of the invention. One mutation, which
corresponds to D144 of ChR2 sequence, is demonstrated herein as
improving channel turn-off kinetics while preserving photocurrent
amplitude. Certain embodiments of light-activated ion channel
polypeptides of the invention include specific amino acid changes,
for example substitution. For example, an E154A mutation to ChR64
speeds up turn-off kinetics and preserves photocurrent amplitude.
Similarly, a D124A single point mutation in the amino acid sequence
of ChR86 also alters performance of this light-activated ion
channel polypeptide of the invention when expressed and contacted
with suitable light to activate the ion channel. A non-limiting
example of an embodiment of a construct of a light-activated ion
channel polypeptide is referred to herein as CheRiff. CheRiff
includes the ChR64 sequence that has an E154A mutation and also
includes the "TS" trafficking sequence. The construct is defined as
ChR64(E154A)-TS-fluorophore. As used herein, the term "TS" is also
referred to as the "KGC" sequence, which is set forth as:
KSRITSEGEYIPLDQIDINV (SEQ ID NO:12).
[0041] It has been identified that not all channelrhodopsins can be
expressed in cells and utilized to alter ion conductance through
the channel, because many channelrhodopsins have been found to not
traffick properly and/or function in mammalian cells. Many
channelrhodopsins have now been examined and the light-activated
ion channel polypeptides ChR64 and ChR86 have now been identified
as functioning more effectively and better in mammalian cells than
other classes of channelrhodopsins.
[0042] Light-activated ion channel polypeptides of the invention
have been genetically express in excitable cells and the cells
illuminated with light, which resulted in the rapid depolarization
and optically evoked spiking of these cells in response to light.
Thus, the light-activated ion channel polypeptides of invention may
be utilized for light-control of cellular functions in vivo
(including, but not limited to in human and non-human primates) and
in vitro, and accordingly has broad-ranging impact on prosthetics,
drug screening, and other biotechnological areas, non-limiting
examples of which are discussed herein.
[0043] Light-activated ion channel polypeptides derived from
Scherifelia dubia and Chloromonas oogama rhodopsin sequences, have
now been identified.
[0044] Light-activated ion channel polypeptides of the invention
are ion channels and may be expressed in a membrane of a cell. An
ion channel is an integral membrane protein that forms a pore
through a membrane and assist in establishing and modulating the
small voltage gradient that exists across the plasma membrane of
all cells and are also found in subcellular membranes of organelles
such as the endoplasmic reticulum (ER), mitochondria, etc. When a
light-activated ion channel polypeptide of the invention is
activated by contacting the cell with appropriate light, the
channel pore opens and permits conductance of ions such as sodium,
potassium, calcium, etc. through the pore. It has been identified
that light-activated ion channel polypeptides of the invention, are
activated by contact with blue light. In some embodiments, a
light-activated ion channel polypeptide of the invention is not
activated by one or both of yellow light or red light. Certain
embodiments of the invention may include a light-activated ion
channel polypeptide that is minimally or not at all activated by at
least one of red or yellow light. For example when contacted with a
red or yellow light, a light-activated ion channel polypeptide may
be activated not at all, or at least less than 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or
20% of the level of activation of the light-activated ion channel
polypeptide when contacted with blue light. Similarly, ion
conduction through a light-activated ion channel polypeptide of the
invention, when contacted with a red light may be at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% less than the level of ion
conduction of the light-activated ion channel polypeptide that is
detected with the same method when the ion channel polypeptide is
contacted with a blue light.
[0045] Similarly, ion conduction through a light-activated ion
channel polypeptide of the invention, when contacted with a yellow
light may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less
than the level of ion conduction of the light-activated ion channel
polypeptide that is detected with the same method when the ion
channel polypeptide is contacted with a blue light.
[0046] In some embodiments of the invention, light-activated
channels may be used to modify the transmembrane potential (and/or
ionic composition) of cells (and/or their sub-cellular regions, and
their local environment). For example, the use of inwardly
rectifying cationic channels will depolarize cells by moving
positively charged ions from the extracellular environment to the
cytoplasm. Under certain conditions, their use can decrease the
intracellular pH (and/or cation concentration) or increase the
extracellular pH (and/or cation concentration). In some
embodiments, the presence of light-activated ion channel
polypeptides of the invention in one, two, three, or more (e.g. a
plurality) of cells in a tissue or organism, can result in
depolarization of the single cell or the plurality of cells by
contacting the light-activated ion channel polypeptides of the
invention with light of suitable wavelength.
[0047] When expressed in a cell, some light-activated ion channel
polypeptides of the invention can be activated by contacting the
cell with blue light having a wavelength between about 450 nm to
495 nm. The light-activated ion channel polypeptides of the
invention may also be activated when contacted with wavelengths of
light that are outside this range, for example, contact with
violet, green, yellow, orange, or red light may activate a
light-activated ion channel polypeptide of the invention at some
level. Thus, activation of a light-activated ion channel
polypeptide of the invention, when contacted with a violet, green,
yellow, or orange light may be at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% less than the level of
activation (e.g., which may in some embodiments be measured as ion
conduction) of the light-activated ion channel polypeptide that is
detected with the same method when the ion channel polypeptide is
contacted with a blue light. Most effective and efficient level of
activation of a light-activated ion channel polypeptide of the
invention may occur when contacted with light in the range of 450
nm to 495 nm, 455 nm to 490 nm, 460 nm to 485 nm, 465 nm to 480 nm,
or 455 nm to 485 nm. Thus, light-activated ion channel polypeptides
of the invention are activated by contact with blue light, but may
also be activated, at a less effective lower level of activation
when contacted with other colors of light.
[0048] Contacting an excitable cell that includes a
blue-light-activated ion channel polypeptide of the invention with
a light in the blue spectrum strongly depolarizes the cell. For
example, contact with light in a wavelength range such as between
450 nm and 495 nm, 455 nm and 490 nm, 460 nm and 485 nm, 465 nm and
480 nm, or 455 nm and 485 nm depolarizes the cell. Light-activated
ion channel polypeptides of the invention have a peak wavelength
sensitivity in the blue range and thus demonstrate a higher
photocurrent at blue wavelengths than previously identified
light-activated channels, for example, ChR2.
[0049] Light-activated ion channel polypeptides of the invention
permit ion conductance and depolarization when contacted under
suitable conditions with an appropriate wavelength of light. As
will be understood by those in the art, the term "depolarized" used
in the context of cells means an upward change in the cell voltage.
For example, in an excitable cell at a baseline voltage of about
-65 mV, a positive change in voltage, e.g., up to 5, 10, 15, 20,
30, 40, or more millivolts (mV) is a depolarization of that cell.
When the change in voltage is sufficient to reach the cell's spike
initiation voltage threshold an action potential (e.g. a spike)
results. When a cell is depolarized by activating a light-activated
ion channel polypeptide of the invention with an appropriate
wavelength of light, the cell voltage becomes more positive than
the baseline level, and an incoming signal may more easily raise
the cell's voltage sufficiently to reach the threshold and trigger
an action potential in the cell. It has been discovered that by
contacting a cell expressing a light-activated ion channel
polypeptide of the invention with light in the range between about
455 nm to about 485 nm, the voltage of the cell becomes less
negative and may rise by at least about 20, 30, 40, 50, 60, 70, 80,
90, 100 mV (depending on the cell type) thus, depolarizing the
cell.
[0050] Specific ranges of wavelengths of light useful to activate
ion channels of the invention are provided and described herein. It
will be understood that a light of appropriate wavelength for
activation and will have a power and intensity appropriate for
activation. It is well known in the art that light pulse duration,
intensity, and power are parameters that can be altered when
activating a channel with light. Thus, one skilled in the art will
be able to adjust power, intensity appropriately when using a
wavelength taught herein to activate a light-activated ion channel
polypeptide of the invention. A benefit of a light-activated ion
channel polypeptide of the invention, may be the ability to "tune"
its response using an appropriate illumination variables (e.g.,
wavelength, intensity, duration, etc.) to activate the channel.
Methods of adjusting illumination variables are well-known in the
art and representative methods can be found in publications such
as: Lin, J., et al., Biophys. J. 2009 Mar. 4; 96(5):1803-14; Wang,
H., et al., 2007 Proc Natl Acad Sci USA. 2007 May 8;
104(19):8143-8. Epub 2007 May 1, each of which is incorporated
herein by reference. Thus, it is possible to utilize a narrow range
of one or more illumination characteristics to activate a
light-activated ion channel polypeptide of the invention. This may
be useful to illuminate a light-activated ion channel polypeptide
that is co-expressed with one or more other light activated
channels that can be illuminated with a different set of
illumination parameters (for example, though not intended to be
limiting, different wavelengths) for their activation, thus
permitting controlled activation of a mixed population of
light-activated channels. A light-activated ion channel polypeptide
of the invention responds strongly to blue light and is activated,
and therefore, because there are other channelrhodopsins that
depolarize cells respond to green, red, or yellow light, in certain
embodiments of the invention, a light-activated ion channel
polypeptide of the invention can be expressed in a separate
population of cells from a population of cells expressing one of
these other opsins, allowing multiple colors of light to be used to
excite these two populations of cells or neuronal projections from
one site, at different times.
[0051] Light-activated ion channel polypeptides of the invention
can be used either alone, using a selective light spectrum for
activation and depolarization and can also be used in combination
with other light-activated ion channels that utilize different
wavelength of light for activation and depolarization, thus
allowing two, three, four, or more different wavelengths of light
to be used to depolarize different sets of cells in a tissue or
organism by expressing channels with different activation spectra
in different cells and then illuminating the tissue and/or organism
with the appropriate wavelengths of light to activate the channels
and depolarize the cells. In some embodiments of the invention, a
light-activated ion channel of the invention is not activated by
either or both of red or yellow light. This feature permits their
use in clean non-perturbative imaging with another ion channel that
is activated by red and/or yellow light (for example, R-GECO, Arch
voltage imaging, etc.).
[0052] Thus, the invention, in some embodiments may include the
expression of different types of ion channels, some of which are
light activated ion channel polypeptides of the invention, and some
that are not. Methods of the invention, in some embodiments, may
include expression of light activated ion channels that are
activated by different (e.g., non-overlapping) wavelengths of
light. This permits simultaneous activation of one or more
light-activated ion channel polypeptides of the invention using
blue light range illumination, and imaging with a voltage/ion
sensor channel polypeptide that is activated when contacted with
light in the red and/or yellow range. As a non-limiting example,
blue-light-activated ion channel polypeptides of the invention may
be expressed in a cell and used in conjunction with ion channels
that are activated by yellow and/or red light. Such use provides
for activation of the light-activated ion channels of the invention
by contact with light in the blue light range, and simultaneous
monitoring of cell signal (e.g., calcium, voltage, etc.) using the
ion channels activated by contact/illumination with red and/or
yellow light.
[0053] In exemplary implementations, the invention comprises
methods for preparing and using genes encoding light-activated ion
channel polypeptides of the invention that have now been
identified. The invention, in part, also includes isolated nucleic
acids comprising sequences that encode light-activated ion channel
polypeptides of the invention as well as vectors and constructs
that comprise such nucleic acid sequences. In some embodiments the
invention includes expression of polypeptides encoded by the
nucleic acid sequences, in cells, tissues, and organisms.
Taxonomy and Sequence Sources
[0054] In particular, the present invention includes, in part,
novel light-activated ion channel polypeptides and their use to
depolarize cells. In some non-limiting embodiments of the invention
one or more newly identified light-activated ion channel
polypeptides may be expressed in cells. Some light-activated ion
channel polypeptides of the invention have amino acid sequences
derived from Scherffelia dubia or Chloromonas oogama rhodopsins
that are naturally expressed. In certain aspects of the invention,
the amino acid or encoding nucleic acid sequence of a polypeptide
that is a variant of a Scherffelia dubia or Chloromonas oogama
polypeptide or encoding nucleic acid sequence, may be referred to
herein as being "derived" from the Scherffelia dubia or Chloromonas
oogama amino acid sequence or nucleic acid sequence, respectively.
Some embodiments of the invention include isolated wild-type or
modified nucleic acid and/or amino acid rhodopsin sequences from
Scherffelia dubia or Chloromonas oogama, and in some aspects, the
invention also includes methods for their use. One skilled in the
art will understand that a light-activated ion channel polypeptides
of the invention can be identified based on sequence homology to a
light-activated ion channel polypeptide sequence disclosed
herein.
[0055] Light-activated ion channel polypeptides of the invention
are transmembrane channel polypeptides that use light energy to
open permitting ion conductance through their pore, thus altering
the potential of the membrane in which they are expressed. A
non-limiting example of an ion that can be moved through a pore of
the invention includes a sodium ion, a potassium ion, a calcium
ion, a proton, etc. Light-activated ion channel polypeptides of the
invention can be activated by sustained light and/or by light
pulses and by permitting ion conductance upon activation.
Activation of light-activated ion channel polypeptides of the
invention can depolarize cells and alter the voltage in cells and
organelles in which they are expressed.
[0056] The wild-type and modified Scherffelia dubia or Chloromonas
oogama rhodopsin nucleic acid and amino acid sequences used in
aspects and methods of the invention are "isolated" sequences. As
used herein, the term "isolated" used in reference to a
polynucleotide, nucleic acid sequence or polypeptide sequence of a
rhodopsin, it means a polynucleotide, nucleic acid sequence, or
polypeptide sequence that is separate from its native environment
and present in sufficient quantity to permit its identification or
use. Thus, an isolated polynucleotide, nucleic acid sequence, or
polypeptide sequence of the invention is a polynucleotide, nucleic
acid sequence, or polypeptide sequence that is not part of, or
included in its native host. For example, a nucleic acid or
polypeptide sequence may be naturally expressed in a cell or
organism of a member of the Scherffelia or Chloromonas genus, but
when the sequence is not part of or included in a Scherffelia or
Chloromonas cell or organism, it is considered to be isolated.
Thus, a nucleic acid or polypeptide sequence of a Scherffelia or
Chloromonas or other channelrhodopsin that is present in a vector,
in a heterologous cell, tissue, or organism, etc., is an isolated
sequence. The term "heterologous" as used herein, means a cell,
tissue or organism that is not the native cell, tissue, or
organism. The terms, "protein", "polypeptides", and "peptides" are
used interchangeably herein. As used herein, the term
"polynucleotide", "nucleic acid sequence" used in reference to
sequences that encode a light-activated channel polypeptide of the
invention may be used interchangeably.
Light-Activated Ion Channel Sequences Including Modified
Sequences
[0057] A light-activated ion channel polypeptide of the invention
may comprise a wild-type polypeptide sequence or may be a modified
polypeptide sequence. As used herein the term "modified" or
"modification" in reference to a nucleic acid or polypeptide
sequence refers to a change of one, two, three, four, five, six, or
more amino acids in the sequence as compared to the wild-type
sequence from which it was derived. For example, a modified
polypeptide sequence may be identical to a wild-type polypeptide
sequence except that it has one, two, three, four, five, or more
amino acid substitutions, deletions, insertions, or combinations
thereof. In some embodiments of the invention a modified sequence
may include one, two, three, four, or more amino acid substitutions
in a wild-type channelrhodopsin sequence.
[0058] It will be understood that sequences of light-activated ion
channel polypeptides of the invention may be derived from various
members of the Scherffelia genus or the Chloromonas genus or
homologs thereof. Using standard methods for determining sequence
homology one of ordinary skill in the art is able to identify
additional channelrhodopsin sequences (including, but not limited
to other Scherffelia or Chloromonas sequences) to identify
homologous polypeptides that also function as light-activated ion
channel polypeptides of the invention.
[0059] The invention, in some aspects also includes light-activated
ion channel polypeptides having one or more substitutions or other
modifications from those described herein. For example, sequences
of light-activated ion channel polypeptides can be modified with
one or more substitutions, deletions, insertions, or other
modifications and can be tested using methods described herein for
characteristics including, but not limited to: expression, cell
localization, activation and depolarization in response to contact
with light using methods disclosed herein. Exemplary modifications
include, but are not limited to conservative amino acid
substitutions, which will produce molecules having functional
characteristics similar to those of the molecule from which such
modifications are made. "Conservative amino acid substitutions" are
substitutions that do not result in a significant change in the
activity or tertiary structure of a selected polypeptide or
protein. Such substitutions typically involve replacing a selected
amino acid residue with a different residue having similar
physico-chemical properties. For example, substitution of Glu for
Asp is considered a conservative substitution because both are
similarly-sized negatively-charged amino acids. Groupings of amino
acids by physico-chemical properties are known to those of skill in
the art. The following groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) [see, for example, Creighton, Proteins
(1984)]. Light-activated ion channel polypeptides of the invention
that include modifications, including but not limited to one, two,
three, four, or more conservative amino acid substitutions can be
identified and tested for characteristics including, but not
limited to: expression, cell localization, activation and
depolarization and depolarization-effects in response to contact
with light using methods disclosed herein.
[0060] A light-activated ion channel polypeptide of the invention
may include amino acid variants (e.g., polypeptides having a
modified sequence) of a sequence set forth herein or another
rhodopsin sequence. Modified light-activated ion channel
polypeptide sequences may have at least about 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence similarity
(also referred to as sequence identity) to the polypeptide sequence
of a light-activated ion channel polypeptide disclosed herein, such
as ChR64, ChR86, or variants thereof, etc. Similarity in this
context means sequence similarity or identity. Such sequence
similarity can be determined using standard techniques known in the
art. Light-activated ion channel polypeptides of the present
invention include light-activated ion channel polypeptide and
nucleic acid sequences provided herein and variants that have at
least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
similarity to a provided sequence.
[0061] To determine the percent identity (similarity) of two amino
acid sequences the sequences are aligned for optimal comparison
purposes (e.g., gaps may be introduced in the sequence of one
protein for optimal alignment with the other protein). The amino
acid residues at corresponding amino acid positions are then
compared. When a position in one sequence is occupied by the same
amino acid residue as the corresponding position in the other
sequence, then the molecules have identity/similarity at that
position. The percent identity or percent similarity between the
two sequences is a function of the number of identical positions
shared by the sequences (i.e., % identity or % similarity=number of
identical positions/total number of positions.times.100). Such an
alignment can be performed using any one of a number of well-known
computer algorithms designed and used in the art for such a
purpose. Similarly, percent identity/similarity of polynucleotide
sequences encoding a light-activated channel polypeptide of the
invention can be determined using art-known alignment and
comparison methods for nucleic acids.
[0062] Light-activated ion channel polypeptides of the invention
may be shorter or longer than the light-activated ion channel
polypeptide sequences set forth herein. Thus, in some embodiments
of the invention, included within the definition of light-activated
ion channel polypeptides of the invention are full-length
polypeptides or functional fragments thereof. In addition, nucleic
acids of the invention may be used to obtain additional coding
regions, and thus additional polypeptide sequences, using
techniques known in the art.
[0063] In some aspects of the invention, substantially similar
light-activated ion channel polypeptide sequences may have at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or
100% similarity to a light-activated ion channel polypeptide
sequence disclosed herein, non-limiting examples of which include
ChR64, ChR86, and variants thereof. Art-known alignment methods and
tools can be used to align substantially similar sequences
permitting positional identification of amino acids that may be
modified as described herein to prepare a light-activated ion
channel polypeptide of the invention. Non-limiting examples of
variants of SEQ ID 2 and SEQ ID 4, include SEQ ID NO:7 and SEQ ID
NO:8, respectively.
Sequence modifications can be in one or more of three classes:
substitutions, insertions, or deletions. These modified sequences,
(which may also be referred to as variants) ordinarily are prepared
by site specific mutagenesis of nucleic acids in the DNA encoding a
light-activated ion channel polypeptide of the invention, using
cassette or PCR mutagenesis or other techniques known in the art,
to produce DNA encoding the modified light-activated ion channel
polypeptide, and thereafter expressing the DNA in recombinant cell
culture. Where amino acid substitutions are made to a small
fragment of a polypeptide, the substitutions can be made by
directly synthesizing the polypeptide. In certain embodiments of
the invention, activity of variant or fragment of a light-activated
channel polypeptide or a variant of a light-activated channel
polypeptide can be tested by cloning the gene encoding the altered
polypeptide into a bacterial or mammalian expression vector,
introducing the vector into an appropriate host cell, expressing
the altered polypeptide, and testing for a functional capability of
the polypeptide as disclosed herein.
[0064] Amino acid sequence variants may be characterized by the
predetermined nature of the variation, a feature that sets them
apart from naturally occurring allelic or interspecies variation of
the light-activated ion channel polypeptides of the invention.
Modified light-activated ion channel polypeptides of the invention
generally exhibit the same qualitative biological activity as the
naturally occurring analogue, although variants can also be
selected that have modified characteristics.
[0065] A site or region for introducing an amino acid sequence
modification may be predetermined, and the mutation per se need not
be predetermined. For example, to optimize the performance of a
mutation at a given site, random mutagenesis may be conducted at
the target codon or region and the expressed modified
light-activated ion channel polypeptide screened for the optimal
combination of desired activity. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, for example, M13 primer mutagenesis and PCR
mutagenesis.
[0066] Amino acid substitutions are typically of single residues
and in certain embodiments of the invention, 1, 2, 3, 4, 5, 6, 7 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitutions
can be made in the amino acid sequence of a light-activated ion
channel polypeptide of the invention, for example, though not
intended to be limiting, in a sequence set forth here as SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:8. Amino acid
insertions in the amino acid sequence of a light-activated ion
channel polypeptide of the invention, for example, though not
intended to be limiting, in a sequence set forth here as SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:8 and may include
insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 amino acids, although larger insertions may be
tolerated. Amino Acid deletions in the sequence of a
light-activated ion channel polypeptide of the invention, for
example, though not intended to be limiting, in a sequence set
forth here as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:8
may include deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 amino acids, although larger
insertions may be tolerated.
[0067] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final modified light-activated
ion channel polypeptide of the invention. Generally these changes
are done on a few amino acids to minimize the alteration of the
molecule. However, larger changes may be tolerated in certain
circumstances.
[0068] Variants of light-activated ion channel polypeptides set
forth herein, may exhibit the same qualitative light-activated ion
channel activity as one or more of the sequences set forth herein,
such as ChR64, ChR86, or variants thereof, but may show some
altered characteristics such as altered photocurrent, stability,
speed, compatibility, and toxicity, or a combination thereof. For
example, the polypeptide can be modified such that it has an
increased photocurrent and/or has less toxicity than another
light-activated ion channel polypeptide.
[0069] A modified light-activated ion channel polypeptide of the
invention can incorporate unnatural amino acids as well as natural
amino acids. An unnatural amino acid can be included in a
light-activated ion channel polypeptide of the invention to enhance
a characteristic such as photocurrent, stability, speed,
compatibility, or to lower toxicity, etc.
[0070] According to principles of this invention, the performance
of light-activated ion channel polypeptides can be tuned for
optimal use, including in the context of their use in conjunction
with other molecules or optical apparatus. For example, in order to
achieve optimal contrast for multiple-color stimulation, one may
desire to either improve or decrease the performance of one
molecule with respect to one another, by the appendage of
trafficking enhancing sequences or creation of genetic variants by
site-directed mutagenesis, directed evolution, gene shuffling, or
altering codon usage. Light-activated ion channel polypeptides may
have inherently varying spectral sensitivity. This may be used to
advantage in vivo (where scattering and absorption will vary with
respect to wavelength, coherence, and polarization), by tuning the
linearity or non-linearity of response to optical illumination with
respect to time, power, and illumination history.
[0071] In some embodiments, the invention includes the use of
targeted site-directed mutagenesis at specific amino acid residues
of channelrhodopsins including but not limited to residues of
rhodopsins of Scherffelia and Chloromonas. Specific locations for
single mutations can be identified and alone, or in combination
with two or more additional mutations can be placed into a
channelrhodopsin sequence and tested with respect to their
activation and photocurrent amplitude. Thus, sequences of
light-activated ion channel polypeptides of the invention, and/or
similar channelrhodopsin sequences can be modified and the
resulting polypeptides tested using methods disclosed herein.
[0072] Another aspect of the invention provides nucleic acid
sequences that code for a light-activated ion channel polypeptide
of the invention. It would be understood by a person of skill in
the art that the light-activated ion channel polypeptides of the
present invention can be coded for by various nucleic acids. Each
amino acid in the protein is represented by one or more sets of 3
nucleic acids (codons). Because many amino acids are represented by
more than one codon, there is not a unique nucleic acid sequence
that codes for a given protein. It is well understood by those of
skill in the art how to make a nucleic acid that can code for
light-activated ion channel polypeptides of the invention by
knowing the amino acid sequence of the protein. A nucleic acid
sequence that codes for a polypeptide or protein is the "gene" of
that polypeptide or protein. A gene can be RNA, DNA, or other
nucleic acid than will code for the polypeptide or protein.
[0073] It is understood in the art that the codon systems in
different organisms can be slightly different, and that therefore
where the expression of a given protein from a given organism is
desired, the nucleic acid sequence can be modified for expression
within that organism. Thus, in some embodiments, a light-activated
ion channel polypeptide of the invention is encoded by a
mammalian-codon-optimized nucleic acid sequence, which may in some
embodiments be a human-codon optimized nucleic acid sequence. An
aspect of the invention provides a nucleic acid sequence that
encodes a light-activated ion channel polypeptide that is optimized
for expression with a mammalian cell. In certain aspects of the
invention, a nucleic acid sequence is optimized for expression in a
human cell.
Delivery of Light-Activated Ion Channel Polypeptides
[0074] Delivery of a light-activated ion channel polypeptide to a
cell and/or expression of a light-activated ion channel polypeptide
in a cell can be done using art-known delivery means.
[0075] In some embodiments of the invention a light-activated ion
channel polypeptide of the invention is included in a fusion
protein. It is well known in the art how to prepare and utilize
fusion proteins that comprise a polypeptide sequence. In certain
embodiments of the invention, a fusion protein can be used to
deliver a light-activated ion channel polypeptide to a cell and can
also in some embodiments be used to target a light-activated ion
channel polypeptide of the invention to specific cells or to
specific cells, tissues, or regions in a subject. Targeting and
suitable targeting sequences for delivery to a desired cell, tissue
or region can be performed using art-known procedures.
[0076] It is an aspect of the invention to provide a
light-activated ion channel polypeptide of the invention that is
non-toxic, or substantially non-toxic in cells in which it is
expressed. In the absence of light, a light-activated ion channel
polypeptide of the invention does not significantly alter cell
health or ongoing electrical activity in the cell in which it is
expressed.
[0077] In some embodiments of the invention, a light-activated ion
channel polypeptide of the invention is genetically introduced into
a cellular membrane, and reagents and methods are provided for
genetically targeted expression of light-activated ion channel
polypeptides, including ChR64, ChR86, and variants thereof, etc.
Genetic targeting can be used to deliver light-activated ion
channel polypeptides to specific cell types, to specific cell
subtypes, to specific spatial regions within an organism, and to
sub-cellular regions within a cell. Genetic targeting also relates
to the control of the amount of light-activated ion channel
polypeptide expressed, and the timing of the expression.
[0078] Some embodiments of the invention include a reagent for
genetically targeted expression of a light-activated ion channel
polypeptide, wherein the reagent comprises a vector that contains
the gene for the light-activated ion channel polypeptide.
[0079] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting between different genetic
environments another nucleic acid to which it has been operatively
linked. The term "vector" also refers to a virus or organism that
is capable of transporting the nucleic acid molecule. One type of
vector is an episome, i.e., a nucleic acid molecule capable of
extra-chromosomal replication. Some useful vectors are those
capable of autonomous replication and/or expression of nucleic
acids to which they are linked. Vectors capable of directing the
expression of genes to which they are operatively linked are
referred to herein as "expression vectors". Other useful vectors,
include, but are not limited to viruses such as lentiviruses,
retroviruses, adenoviruses, and phages. Vectors useful in some
methods of the invention can genetically insert light-activated ion
channel polypeptides into dividing and non-dividing cells and can
insert light-activated ion channel polypeptides to cells that are
in vivo, in vitro, or ex vivo cells.
[0080] Vectors useful in methods of the invention may include
additional sequences including, but not limited to one or more
signal sequences and/or promoter sequences, or a combination
thereof. Expression vectors and methods of their use are well known
in the art. Non-limiting examples of suitable expression vectors
and methods for their use are provided herein.
[0081] In certain embodiments of the invention, a vector may be a
lentivirus comprising the gene for a light-activated ion channel
polypeptide of the invention, such as ChR64, ChR86, or a variant
thereof. A lentivirus is a non-limiting example of a vector that
may be used to create stable cell line. The term "cell line" as
used herein is an established cell culture that will continue to
proliferate given the appropriate medium.
[0082] Promoters that may be used in methods and vectors of the
invention include, but are not limited to, cell-specific promoters
or general promoters. Methods for selecting and using cell-specific
promoters and general promoters are well known in the art. A
non-limiting example of a general purpose promoter that allows
expression of a light-activated ion channel polypeptide in a wide
variety of cell types--thus a promoter for a gene that is widely
expressed in a variety of cell types, for example a "housekeeping
gene" can be used to express a light-activated ion channel
polypeptide in a variety of cell types. Non-limiting examples of
general promoters are provided elsewhere herein and suitable
alternative promoters are well known in the art.
[0083] In certain embodiments of the invention, a promoter may be
an inducible promoter, examples of which include, but are not
limited to tetracycline-on or tetracycline-off, or
tamoxifen-inducible Cre-ER.
Methods of Use of Light-activated Ion Channel Polypeptides of the
Invention
[0084] Light-activated ion channel polypeptides of the invention
are well suited for targeting cells and specifically altering
voltage-associated cell activities. In some embodiments of the
invention, light-activated ion channel polypeptides of the
invention can utilized to introduce cations into cells, thus
activating endogenous signaling pathways (such as calcium dependent
signaling), and then drugs are applied that modulate the response
of the cell (using a calcium or voltage-sensitive dye). This allows
new kinds of drug screening using just light to activate the
channels of interest, and using just light to read out the effects
of a drug on the channels of interest.
[0085] In certain aspects of the invention, a wild-type or modified
Scherffelia or Chloromonas light-activated ion channel polypeptide
of the invention may be used to sensitize cells to blue light. Such
methods may be used to treat blindness and introduce visual
perception to blue light.
[0086] In another aspect of the invention, a light-activated ion
channel polypeptide of the invention may be used to decrease the pH
of a cell in which it is expressed. Such a technique may be used to
treat alkalosis.
[0087] Another aspect of the invention includes methods of using
one or more light-activated proton pumps in conjunction with the
use of light-activated ion channel polypeptides of the invention
for the coupled effect of hyperpolarization and intracellular
alkalinization. For example, both phenomena can induce spontaneous
spiking in neurons by triggering hyperpolarization-induced cation
currents or pH-dependent hyper-excitability. Another aspect of the
invention is to utilize a light-activated ion channel polypeptide
to generate sub-cellular voltage or pH gradients, particularly at
synapses and in synaptic vesicles to alter synaptic transmission,
and mitochondria to improve ATP synthesis.
[0088] Working operation of a prototype of this invention was
demonstrated by genetically expressing light-activated ion channel
molecules of the invention in excitable cells, illuminating the
cells with suitable wavelengths of light, and demonstrating rapid
depolarization of the cells in response to the light, as well as
rapid release from depolarization upon cessation of light.
Depending on the particular implementation, methods of the
invention allow light control of cellular functions in vivo, ex
vivo, and in vitro.
[0089] In non-limiting examples of methods of the invention,
microbial channelrhodopsins are used in mammalian cells without
need for any kind of chemical supplement, and in normal cellular
environmental conditions and ionic concentrations. For example,
genes encoding channelrhodopsins of Scherffelia and Chloromonas
have been used in exemplary implementations of the invention. These
sequences in humanized or mouse-optimized form allow depolarization
at blue light wavelengths (e.g., light-activated ion channel
polypeptides of the invention).
[0090] As used herein, the term "ion channel" means a transmembrane
polypeptide that forms a pore, which when activated opens,
permitting ion conductance through the pore across the membrane.
Many ion channels do not express well in a cell and/or their
expression may be toxic to the cell and reduce cell health. Thus it
was necessary to prepare and screen numerous channelrhodopsin
light-activated ion channel polypeptides to identify
light-activated ion channels of the invention that can be expressed
in cells without significantly reducing cell health and
viability.
[0091] Light-activated ion channels of the invention have been
found to be suitable for expression and use in mammalian cells
without need for any kind of chemical supplement, and in normal
cellular environmental conditions and ionic concentrations.
Light-activated ion channel polypeptides of the invention have been
found to differ from previously identified channels in that the
light-activated ion channel polypeptides of the invention activate
most efficiently at a wavelengths of light in the blue light
range.
Cells and Subjects
[0092] A cell used in methods and with sequences of the invention
may be an excitable cell or a non-excitable cell. A cell in which a
light-activated ion channel polypeptide of the invention may be
expressed and may be used in methods of the invention include
prokaryotic and eukaryotic cells. Useful cells include but are not
limited to mammalian cells. Examples of cells in which a
light-activated ion channel polypeptide of the invention may be
expressed are excitable cells, which include cells able to produce
and respond to electrical signals. Examples of excitable cell types
include, but are not limited to neurons, muscles, cardiac cells,
and secretory cells (such as pancreatic cells, adrenal medulla
cells, pituitary cells, etc.).
[0093] Non-limiting examples of cells that may be used in methods
of the invention include: nervous system cells, cardiac cells,
circulatory system cells, visual system cells, auditory system
cells, secretory cells, endocrine cells, or muscle cells. In some
embodiments, a cell used in conjunction with the invention may be a
healthy normal cell, which is not known to have a disease, disorder
or abnormal condition. In some embodiments, a cell used in
conjunction with methods and channels of the invention may be an
abnormal cell, for example, a cell that has been diagnosed as
having a disorder, disease, or condition, including, but not
limited to a degenerative cell, a neurological disease-bearing
cell, a cell model of a disease or condition, an injured cell, etc.
In some embodiments of the invention, a cell may be a control
cell.
[0094] Light-activated ion channel polypeptides of the invention
may be expressed in cells from culture, cells in solution, cells
obtained from subjects, and/or cells in a subject (in vivo cells).
Light-activated ion channel polypeptides of the invention may be
expressed and activated in cultured cells, cultured tissues (e.g.,
brain slice preparations, etc.), and in living subjects, etc. As
used herein, a the term "subject" may refer to a human, non-human
primate, cow, horse, pig, sheep, goat, dog, cat, rodent, fly or any
other vertebrate or invertebrate organism.
Controls and Candidate Compound Testing
[0095] Light-activated ion channel polypeptides of the invention
and methods using light-activated ion channel polypeptides of the
invention can be utilized to assess changes in cells, tissues, and
subjects in which they are expressed. Some embodiments of the
invention include use of light-activated ion channel polypeptides
of the invention to identify effects of candidate compounds on
cells, tissues, and subjects. Results of testing a light-activated
ion channel polypeptide of the invention can be advantageously
compared to a control.
[0096] As used herein a control may be a predetermined value, which
can take a variety of forms. It can be a single cut-off value, such
as a median or mean. It can be established based upon comparative
groups, such as cells or tissues that include the light-activated
ion channel polypeptide of the invention and are contacted with
light, but are not contacted with the candidate compound and the
same type of cells or tissues that under the same testing condition
are contacted with the candidate compound. Another example of
comparative groups may include cells or tissues that have a
disorder or condition and groups without the disorder or condition.
Another comparative group may be cells from a group with a family
history of a disease or condition and cells from a group without
such a family history. A predetermined value can be arranged, for
example, where a tested population is divided equally (or
unequally) into groups based on results of testing. Those skilled
in the art are able to select appropriate control groups and values
for use in comparative methods of the invention.
[0097] As a non-limiting example of use of a light-activated ion
channel polypeptide to identify a candidate therapeutic agent or
compound, a light-activated ion channel polypeptide of the
invention may be expressed in an excitable cell in culture or in a
subject and the excitable cell may be contacted with a light that
activates the light-activated ion channel polypeptide and with a
candidate therapeutic compound. In one embodiment, a test cell that
includes a light-activated ion channel polypeptide of the invention
can be contacted with a light that depolarizes the cell and also
contacted with a candidate compound. The cell, tissue, and/or
subject that include the cell can be monitored for the presence or
absence of a change that occurs in the test conditions versus the
control conditions. For example, in a cell, a change may be a
change in the depolarization or in a depolarization-mediated cell
characteristic in the test cell versus a control cell, and a change
in depolarization or the depolarization-mediated cell
characteristic in the test cell compared to the control may
indicate that the candidate compound has an effect on the test cell
or tissue that includes the cell. In some embodiments of the
invention, a depolarization-mediated cell characteristic may be an
action potential, pH change in a cell, release of a
neurotransmitter, etc. and may in come embodiments, include a
downstream effect on one or more additional cells, which occurs due
to the depolarization of the cell that includes the light-activated
ion channel polypeptide. Art-known methods can be used to assess
depolarization and depolarization-mediated cell characteristics and
changes to the depolarization or depolarization-mediated cell
characteristics upon excitation of a light-activated ion channel
polypeptide of the invention, with or without additional contact
with a candidate compound.
[0098] Candidate-compound identification methods of the invention
that are performed in a subject or in cultured or in vitro cells.
Candidate-compound identification methods of the invention that are
performed in a subject, may include expressing a light-activated
ion channel polypeptide in the subject, contacting the subject with
a light under suitable conditions to activate the light-activated
ion channel polypeptide and depolarize the cell, and administering
to the subject a candidate compound. The subject is then monitored
to determine whether any change occurs that differs from a control
effect in a subject.
[0099] Candidate-compound identification methods of the invention
that are performed in vitro may include expressing a
light-activated ion channel polypeptide in a cell, which may or may
not be a cultured cell, contacting the cell with a light under
suitable conditions to activate the light-activated ion channel
polypeptide and depolarize the cell, and contacting the cell with a
candidate compound. The cell is then monitored to determine whether
any change occurs that differs from a control effect in a cell.
Thus, for example, a cell expressing the light-activated ion
channel polypeptide can, in the presence of a candidate compound,
be contacted with a light appropriate to activate the
light-activated ion channel polypeptide. Contact of the
light-activated ion channel polypeptide with the candidate compound
may also occur at one or more time points prior to, at the same
time as, or subsequent to contact with the light appropriate to
activate the light-activated ion channel polypeptide. A result of
such contact with the candidate compound can be measured and
compared to a control value as a determination of the presence or
absence of an effect of the candidate compound.
[0100] Methods of identifying effects of candidate compounds using
light-activated ion channel polypeptides of the invention may also
include additional steps and assays to further characterizing an
identified change in the cell, tissue, or subject when the cell is
contacted with the candidate compound. In some embodiments, testing
in a cell, tissue, or subject can also include one or more cells
that has a light-activated ion channel polypeptide of the
invention, and that also has one, two, three, or more additional
different light-activated ion channels, wherein at least one, two,
three, four, or more of the additional light-activated ion channels
is activated by contact with light having a different wavelength
than used to activate the blue-light-activated ion channel
polypeptide of the invention.
[0101] In a non-limiting example of a candidate drug identification
method of the invention, cells that include a light-activated ion
channel polypeptide of the invention are depolarized, thus
triggering release of a neurotransmitter from the cell, and then
drugs are applied that modulate the response of the cell to
depolarization (determined for example using patch clamping methods
or other suitable art-known means). Such methods enable new kinds
of drug screening using just light to activate the channels of
interest, and using just light to read out the effects of a drug on
the channels and channel-containing cells of interest.
[0102] In some embodiments, light-activated ion channel
polypeptides of the invention can be used in test systems and
assays for assessing membrane protein trafficking and physiological
function in heterologously expressed systems and the use of use of
light-activated channels to depolarize a cell.
Methods of Treating
[0103] Some aspects of the invention include methods of treating a
disorder or condition in a cell, tissue, or subject using
light-activated ion channel polypeptides of the invention.
Treatment methods of the invention may include administering to a
subject in need of such treatment, a therapeutically effective
amount of a light-activated ion channel polypeptide of the
invention to treat the disorder. It will be understood that a
treatment may be a prophylactic treatment or may be a treatment
administered following the diagnosis of a disease or condition. A
treatment of the invention may reduce or eliminate a symptom or
characteristic of a disorder, disease, or condition or may
eliminate the disorder, disease, or condition itself. It will be
understood that a treatment of the invention may reduce or
eliminate progression of a disease, disorder or condition and may
in some instances result in the regression of the disease,
disorder, or condition. A treatment need not entirely eliminate the
disease, disorder, or condition to be effective.
[0104] Administration of a light-activated ion channel polypeptide
of the invention may include administration of a pharmaceutical
composition that includes a cell, wherein the cell expresses the
light-activated ion channel. Administration of a light-activated
ion channel polypeptide of the invention may include administration
of a pharmaceutical composition that includes a vector, wherein the
vector comprises a nucleic acid sequence encoding the
light-activated ion channel and the administration of the vector
results in expression of the light-activated ion channel in a cell
in the subject.
[0105] An effective amount of a light-activated ion channel
polypeptide of the invention is an amount that increases the level
of the light-activated ion channel polypeptide in a cell, tissue or
subject to a level that is beneficial for the subject. An effective
amount may also be determined by assessing physiological effects of
administration on a cell or subject, such as a decrease in symptoms
following administration. Other assays will be known to one of
ordinary skill in the art and can be employed for measuring the
level of the response to a treatment. The amount of a treatment may
be varied for example by increasing or decreasing the amount of the
light-activated ion channel polypeptide administered, by changing
the therapeutic composition in which the light-activated ion
channel polypeptide is administered, by changing the route of
administration, by changing the dosage timing, by changing the
activation amounts and parameters of a light-activated ion channel
polypeptide of the invention, and so on. The effective amount will
vary with the particular condition being treated, the age and
physical condition of the subject being treated; the severity of
the condition, the duration of the treatment, the nature of the
concurrent therapy (if any), the specific route of administration,
and the like factors within the knowledge and expertise of the
health practitioner. For example, an effective amount may depend
upon the location and number of cells in the subject in which the
light-activated ion channel polypeptide is to be expressed. An
effective amount may also depend on the location of the tissue to
be treated.
[0106] Effective amounts will also depend, of course, on the
particular condition being treated, the severity of the condition,
the individual patient parameters including age, physical
condition, size and weight, the duration of the treatment, the
nature of concurrent therapy (if any), the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose of a composition to increase the level of a light-activated
ion channel polypeptide, and/or to alter the length or timing of
activation of a light-activated ion channel polypeptide in a
subject (alone or in combination with other therapeutic agents) be
used, that is, the highest safe dose or amount according to sound
medical judgment. It will be understood by those of ordinary skill
in the art, however, that a patient may insist upon a lower dose or
tolerable dose for medical reasons, psychological reasons or for
virtually any other reasons.
[0107] A light-activated ion channel polypeptide of the invention
may be administered using art-known methods. The manner and dosage
administered may be adjusted by the individual physician or
veterinarian, particularly in the event of any complication. The
absolute amount administered will depend upon a variety of factors,
including the material selected for administration, whether the
administration is in single or multiple doses, and individual
subject parameters including age, physical condition, size, weight,
and the stage of the disease or condition. These factors are well
known to those of ordinary skill in the art and can be addressed
with no more than routine experimentation.
[0108] Pharmaceutical compositions that deliver light-activated ion
channel polypeptides of the invention may be administered alone, in
combination with each other, and/or in combination with other drug
therapies, or other treatment regimens that are administered to
subjects. A pharmaceutical composition used in the foregoing
methods may contain an effective amount of a therapeutic compound
that will increase the level of a light-activated ion channel
polypeptide to a level that produces the desired response in a unit
of weight or volume suitable for administration to a subject. In
some embodiments of the invention, a pharmaceutical composition of
the invention may include a pharmaceutically acceptable
carrier.
[0109] Pharmaceutically acceptable carriers include diluents,
fillers, salts, buffers, stabilizers, solubilizers and other
materials that are well-known in the art. Exemplary
pharmaceutically acceptable carriers are described in U.S. Pat. No.
5,211,657 and others are known by those skilled in the art. In
certain embodiments of the invention, such preparations may contain
salt, buffering agents, preservatives, compatible carriers, aqueous
solutions, water, etc. When used in medicine, the salts may be
pharmaceutically acceptable, but non-pharmaceutically acceptable
salts may conveniently be used to prepare
pharmaceutically-acceptable salts thereof and are not excluded from
the scope of the invention. Such pharmacologically and
pharmaceutically-acceptable salts include, but are not limited to,
those prepared from the following acids: hydrochloric, hydrobromic,
sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric,
formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0110] One or more of a light-activated ion channel polypeptide or
encoding polynucleotide of the invention, or a cell or vector
comprising a light-activated ion channel polypeptide or encoding
nucleic acid of the invention, may be administered, for example in
a pharmaceutical composition, directly to a tissue. Direct tissue
administration may be achieved by direct injection, and such
administration may be done once, or alternatively a plurality of
times. If administered multiple times, the polypeptides,
polynucleotides, cells, and/or vectors may be administered via
different routes. For example, the first (or the first few)
administrations may be made directly into the affected tissue while
later administrations may be systemic.
[0111] The dose of a pharmaceutical composition that is
administered to a subject to increase the level of light-activated
ion channel polypeptide in cells of the subject can be chosen in
accordance with different parameters, in particular in accordance
with the mode of administration used and the state of the subject.
Other factors include the desired period of treatment. In the event
that a response in a subject is insufficient at the initial doses
applied, higher doses (or effectively higher doses by a different,
more localized delivery route) may be employed to the extent that
patient tolerance permits. The amount and timing of activation of a
light-activated ion channel polypeptide of the invention (e.g.,
light wavelength, length of light contact, etc.) that has been
administered to a subject can also be adjusted based on efficacy of
the treatment in a particular subject. Parameters for illumination
and activation of light-activated ion channel polypeptides of the
invention that have been administered to a subject can be
determined using art-known methods and without requiring undue
experimentation.
[0112] Various modes of administration will be known to one of
ordinary skill in the art that can be used to effectively deliver a
pharmaceutical composition to increase the level of a
light-activated ion channel polypeptide in a desired cell, tissue
or body region of a subject. Methods for administering such a
composition or other pharmaceutical compound of the invention may
be topical, intravenous, oral, intracavity, intrathecal,
intrasynovial, buccal, sublingual, intranasal, transdermal,
intravitreal, subcutaneous, intramuscular and intradermal
administration. The invention is not limited by the particular
modes of administration disclosed herein. Standard references in
the art (e.g., Remington's Pharmaceutical Sciences, 18th edition,
1990) provide modes of administration and formulations for delivery
of various pharmaceutical preparations and formulations in
pharmaceutical carriers. Other protocols which are useful for the
administration of a therapeutic compound of the invention will be
known to one of ordinary skill in the art, in which the dose
amount, schedule of administration, sites of administration, mode
of administration (e.g., intra-organ) and the like vary from those
presented herein.
[0113] Administration of a cell or vector to increase
light-activated ion channel polypeptide levels in a mammal other
than a human; and administration and use of light-activated ion
channel polypeptides of the invention, e.g. for testing purposes or
veterinary therapeutic purposes, is carried out under substantially
the same conditions as described above. It will be understood by
one of ordinary skill in the art that this invention is applicable
to both human and animals. Thus this invention is intended to be
used in husbandry and veterinary medicine as well as in human
therapeutics.
[0114] In some aspects of the invention, methods of treatment using
a light-activated ion channel polypeptide of the invention are
applied to cells including but not limited to a nervous system
cell, a neuron, a cardiac cell, a circulatory system cell, a visual
system cell, an auditory system cell, a muscle cell, or an
endocrine cell, etc. Disorders and conditions that may be treated
using methods of the invention include, injury, brain damage,
degenerative neurological conditions (e.g., Parkinson's disease,
Alzheimer's disease, seizure, vision loss, hearing loss, etc.
Disorders, Diseases and Conditions
[0115] Light-activated ion channel polypeptides of the invention
may be used to target cells and membranes, and to alter
voltage-associated cell activities. In some embodiments, a
blue-light-activated ion channel polypeptides of the invention may
be used to sensitize cells to blue light. Such methods may be used
to treat blindness.
[0116] In another aspect of the invention, a light-activated ion
channel polypeptide may be used to decrease the pH of a cell in
which it is expressed. Such a technique may be used to treat
alkalosis.
[0117] Another aspect of the invention includes methods of using
light-activated proton pumps in conjunction with the use of
light-activated ion channel polypeptides of the invention for the
coupled effect of hyperpolarization and intracellular
alkalinization. For example, both phenomena can induce spontaneous
spiking in neurons by triggering hyperpolarization-induced cation
currents or pH-dependent hyper-excitability.
[0118] In some embodiments, methods and light-activated ion channel
polypeptides of the invention may be used for the treatment of
visual system disorders, for example to treat vision reduction or
loss. A light-activated ion channel polypeptide of the invention
may be administered to a subject who has a vision reduction or loss
and the cell that expresses the light-activated ion channel
polypeptide of the invention can function as light-sensitive cells
in the visual system, thereby permitting a gain of visual function
in the subject.
[0119] The present invention in some aspects, includes preparing
nucleic acid sequences and polynucleotide sequences; expressing in
cells and membranes polypeptides encoded by the prepared nucleic
acid and polynucleotide sequences; illuminating the cells and/or
membranes with suitable light, and demonstrating rapid
depolarization of the cells and/or a change in conductance across
the membrane in response to light, as well as rapid release from
depolarization upon cessation of light. The ability to controllably
alter voltage across membranes and cell depolarization with light
has been demonstrated. The present invention enables light-control
of cellular functions in vivo, ex vivo, and in vitro, and the light
activated ion channels of the invention and their use, have
broad-ranging applications for drug screening, treatments, and
research applications, some of which are describe herein.
[0120] In illustrative implementations of this invention, the
ability to optically perturb, modify, or control cellular function
offers many advantages over physical manipulation mechanisms. These
advantages comprise speed, non-invasiveness, and the ability to
easily span vast spatial scales from the nanoscale to
macroscale.
[0121] The reagents use in the present invention (and the class of
molecules that they represent), allow, at least: currents activated
by blue light wavelengths which may differ in spectra from older
molecules (opening up multi-color control of cells).
EXAMPLES
Example 1
Introduction
[0122] The present invention describes the use of light-gated
channels to modify the transmembrane potential (and/or ionic
composition) of cells (and/or their sub-cellular regions, and their
local environment). In particular, the use of inwardly rectifying
cationic channels will depolarize cells by moving positively
charged ions from the extracellular environment to the cytoplasm.
Under certain conditions, their use can decrease the intracellular
pH (and/or increase the intracellular cation concentration) or
increase the extracellular pH (and/or decrease the extracellular
cation concentration). Compared to the currently reported natural
gene sequences used to depolarize neurons in the prior art [see for
example, Zhang, F. et al. Nature 446, 633-639, (2007) and Han, X
& Boyden, E. S. PloS one 2, e299, (2007), the content of each
of which is incorporated herein by reference] (this disclosure
notwithstanding), ChR64 and ChR86 have demonstrably improved
photocurrent generation in response to blue light.
[0123] Experiments were performed in which the gene derived from
either Scherifelia dubia or Chloromonas oogama was expressed in a
cell. The gene derived from Scherifelia dubia encoded the amino
acid sequence set forth herein as SEQ ID NO:2, and is referred to
herein as ChR64, which is encoded by mammalian codon-optimized DNA
sequence set forth herein as SEQ ID NO: 1. The gene derived from
Chloromonas oogama encoded the amino acid sequence set forth herein
as SEQ ID NO:4, and is referred to herein as ChR86, which is
encoded by the mammalian codon-optimized DNA sequence set forth
herein as SEQ ID NO:3. In the experiments, ChR64 and ChR86 were
expressed in cells as described as follows. [Also, for descriptions
and examples of experimental methods and procedures see Chow, B. Y.
et al. Nature 463, 98-102, (2010), the content of which is
incorporated by reference herein].
Methods
[0124] (1) The opsin gene was cloned into a lentiviral or
adeno-associated virus (AAV) packaging plasmid, or another desired
expression plasmid, and then GFP was cloned downstream of the
preferred gene, eliminating the stop codon of the opsin gene, thus
creating a fusion protein.
[0125] (2) The viral or expression plasmid contained either a
strong ubiquitous promoter, a cell-specific promoter, or a strong
general promoter followed by one more logical elements (such as a
lox-stop-lox sequence, which would be removed by Cre recombinase
selectively expressed in cells in a transgenic animal, or in a
second virus, thus enabling the strong general promoter to then
drive the gene [for descriptions and examples of experimental
methods and procedures see for example, Atasoy, D., et al., J.
Neurosci 28, 7025-7030, (2008) and Kuhlman, S. J. & Huang, Z.
J. PLoS ONE 3, e2005, (2008), the content of each of which is
incorporated by reference herein].
[0126] (3) If using a viral plasmid, the viral vector was
synthesized using the viral plasmid, using standard techniques [for
descriptions and examples of experimental methods and procedures
see for example, Sena-Esteves, M., et al., J Virol Methods 122,
131-139, (2004), the content of which is incorporated by reference
herein].
[0127] (4) If using a virus, as appropriate for gene therapy (over
600 people have been treated with AAV carrying various genetic
payloads to date, in 48 separate clinical trials, without a single
adverse event), the virus is injected using a small needle or
cannula into the area of interest, thus delivering the gene
encoding the opsin fusion protein into the cells of interest. If
using another expression vector, directly electroporate or inject
that vector into the cell or organism (for acutely expressing the
opsin, or making a cell line, or a transgenic mouse or other
animal).
[0128] (5) Illuminate with light. For ChR64 and ChR86, peak
illumination wavelengths with which the expressed light-activated
ion channel polypeptides were contacted were 470 nm.+-.15 nm.
[0129] (6) The above wavelengths illustrate typical modes of
operation, but are not meant to constrain the protocols that can be
used. Either narrower or broader wavelengths, or
differently-centered illumination spectra, are used. For prosthetic
uses, the devices used to deliver light may be implanted for
examples using LED and fiber arrays using standard procedures [for
descriptions and examples of experimental methods and procedures
see for example Campagnola, L., et al., J Neurosci Methods 169,
27-33, (2008), the content of which is incorporated by reference
herein.]. For drug screening, a xenon lamp or LED can be used to
deliver the light.
[0130] The performance of the above example may be changed by
expressing a light-activated ion channel polypeptide that is
altered from either ChR64 or ChR86 by site-directed mutagenesis,
such as the E154A single mutation to ChR64 and the D124A single
point mutation to ChR86. The performance of a light-activated ion
channel polypeptide of the invention may also be improved by
appending C-terminal peptide sequences to affect cellular
trafficking, such as the C terminal Kir2.1 signal sequence (denoted
as "KGC") [see Munoz-Jordan, J. L. et al. J. Virol. 79, 8004-8013,
(2005), the content of which is incorporated by reference herein]
(amino acid sequence: KSRITSEGEYIPLDQIDINV SEQ ID NO: 12; DNA
sequence:
aaatccagaattacttctgaaggggagtatatccctctggatcaaatagacatcaatgtt (SEQ
ID NO:11).
Methods of Use of Light Activated Ion Channel Polypeptides
[0131] It has now been demonstrate that ChR64 and ChR86 can be
activated with low blue light powers and have no red-light
sensitivity. By using these blue-peaked channelrhodopsins together
with red-shifted fluorescent sensors, it is possible to
simultaneously image physiological response (e.g. voltage, ion,
etc.) and optically depolarize cells using low blue light powers,
without interference in the imaging channel. This simultaneous
imaging and optical depolarization is particularly useful for
feedback control and interrogation of cellular and network
physiology.
[0132] The performance of the above said molecules or classes of
molecules can be tuned for optimal use, particularly in context of
their use in conjunction with other molecules or optical apparatus.
For example, to achieve optimal contrast for simultaneous optical
depolarization and imaging, one may desire to either improve or
decrease the performance of one molecule with respect to another,
by the appendage of trafficking enhancing sequences or creation of
genetic variants by site-directed mutagenesis, directed evolution,
gene shuffling, or altering codon usage. Molecules or classes of
molecules may have inherently varying spectral sensitivity that may
be functionally advantageous in vivo (where scattering and
absorption will vary with respect to wavelength, coherence, and
polarization), by tuning the linearity or non-linearity of response
to optical illumination with respect to time, power, and
illumination history.
[0133] The ability to introduce cations into cells, thus activating
endogenous signaling pathways (such as calcium dependent
signaling), and then applying drugs that modulate the response of
the cell (using a calcium or voltage-sensitive dye as the readout
of cellular electrophysiology), is also enabled by this disclosure.
This enables new kinds of drug screening using just light to
activate the channels of interest, and using just light to read out
the effects of a drug on the channels of interest.
[0134] Another aspect of the invention is the use of
light-activated channel to decrease the pH of the cell. Such a
technique may be used to treat alkalosis.
[0135] Another aspect of the invention is to generate sub-cellular
voltage or pH gradients, particularly at synapses and in synaptic
vesicles to alter synaptic transmission; and in mitochondria to
modulate ATP synthesis.
[0136] Another aspect of the invention is the various compositions
of matter that have been prepared including, but not limited to:
(1) plasmids encoding for the above genes; (2) lentiviruses
carrying payloads encoding for the above described genes; (3)
adeno-associated viruses carrying payloads encoding for the
above-described genes; (4) cells expressing the above-described
genes; and (5) animals expressing the above-described genes.
Example 2
[0137] Studies were performed to prepare sequences and to express
light-activated ion channels in cells, tissues, and subjects.
Non-limiting exemplary methods are set forth Example 1. General
methods also applicable to light-activated channel molecules and
methods for their use are disclosed in publications such as US
Published Application No. 2010/0234273, US Published Application
No. 20110165681, Chow B Y, et. al. Methods Enzymol. 2011;
497:425-43; Chow, B Y, et al. Nature 2010 Jan. 7; 463(7277):98-102,
the content of each of which is incorporated by reference
herein.
[0138] Studies were performed to prepare sequences and to express
light-activated ion channels in cells, tissues, and subjects.
Non-limiting exemplary methods are set forth below.
Plasmid Construction and Site Directed Mutagenesis.
[0139] Opsins were mammalian codon-optimized, and synthesized by
Genscript (Genscript Corp., NJ). Opsins were fused in frame,
without stop codons, ahead of GFP (using BamHI and AgeI) in a
lentiviral vector containing the CaMKII promoter, enabling direct
neuron transfection, HEK cell transfection (expression in HEK cells
is enabled by a ubiquitous promoter upstream of the lentiviral
cassette), and lentivirus production and transfection.
[0140] Amino acid sequences of various opsins were as follows:
ChR64 (SEQ ID NO:2); ChR86 (SEQ ID NO:4); ChR64 with E154A
substitution (SEQ ID NO:7); ChR86 with D124A substitution (SEQ ID
NO:8).
[0141] The `ER2` ER export sequence corresponded to amino acid
sequence FCYENEV, DNA sequence ttctgctacgagaatgaagtg. The `KGC`
signal sequence corresponded to amino acid sequence
KSRITSEGEYIPLDQIDINV (SEQ ID NO:12), DNA sequence of KGC signal
sequence:
aaatccagaattacttctgaaggggagtatatccctctggatcaaatagacatcaatgtt (SEQ
ID NO: 11).
Neuron Culture, Transfection, Infection, and Imaging
[0142] All procedures involving animals were in accordance with the
National Institutes of Health Guide for the care and use of
laboratory animals and approved by the Massachusetts Institute of
Technology Animal Care and Use Committee. Swiss Webster or C57 mice
(Taconic, Hudson, N.Y. or The Jackson Laboratory, Bar Harbor,
Mass.) were used. For hippocampal cultures, hippocampal regions of
postnatal day 0 or day 1 mice were isolated and digested with
trypsin (1 mg/ml) for .about.12 min, and then treated with Hanks
solution supplemented with 10-20% fetal bovine serum and trypsin
inhibitor (Sigma-Aldrich, St. Louis, Mo.). Tissue was then
mechanically dissociated with Pasteur pipettes, and centrifuged at
1000 rpm at 4.degree. C. for 10 min. Dissociated neurons were
plated at a density of approximately four hippocampi per 20 glass
coverslips, coated with Matrigel (BD Biosciences, Sparks, Md.). For
cortical cultures, dissociated mouse cortical neurons (postnatal
day 0 or 1) were prepared as previously described, and plated at a
density of 100-200 k per glass coverslip coated with Matrigel (BD
Biosciences, Sparks, Md.). Cultures were maintained in Neurobasal
Medium supplemented with B27 [Invitrogen, (Life Technologies
Corporation, Carlsbad, Calif.)] and glutamine. Hippocampal and
cortical cultures were used interchangeably; no differences in
reagent performance were noted.
[0143] Neurons were transfected at 3-5 days in vitro using calcium
phosphate [Invitrogen, (Life Technologies Corporation, Carlsbad,
Calif.)]. GFP fluorescence was used to identify successfully
transfected neurons. Alternatively, neurons were infected with
0.1-3 .mu.l of lentivirus or adeno-associated virus (AAV) per well
at 3-5 days in vitro.
HEK 293FT Cell Culture and Transfection
[0144] HEK 293FT cells [Invitrogen, (Life Technologies Corporation,
Carlsbad, Calif.)] were maintained between 10-70% confluence in D10
medium (Cellgro, Manassas, Va.) supplemented with 10% fetal bovine
serum [Invitrogen, (Life Technologies Corporation, Carlsbad,
Calif.)], 1% penicillin/streptomycin (Cellgro, Manassas, Va.), and
1% sodium pyruvate (Biowhittaker, Walkersville, Md.)). For
recording, cells were plated at 5-20% confluence on glass
coverslips coated with Matrigel (BD Biosciences, Sparks, Md.).
Adherent cells were transfected approximately 24 hours post-plating
either with TransLT 293 lipofectamine transfection kits (Mirus,
Madison, Wis.) or with calcium phosphate transfection kits
[Invitrogen, (Life Technologies Corporation, Carlsbad, Calif.)],
and recorded via whole-cell patch clamp between 36-72 hours
post-transfection.
Lentivirus Preparation
[0145] HEK293FT cells [Invitrogen, (Life Technologies Corporation,
Carlsbad, Calif.)] were transfected with the lentiviral plasmid,
the viral helper plasmid pA8.74, and the pseudotyping plasmid
pMD2.G. The supernatant of transfected HEK cells containing virus
was then collected 48 hours after transfection, purified, and then
pelleted through ultracentrifugation. Lentivirus pellet was
resuspended in phosphate buffered saline (PBS) and stored at
-80.degree. C. until further usage in vitro or in vivo. The
estimated final titer is approximately 10.sup.9 infectious
units/mL.
In Vitro Whole Cell Patch Clamp Recording & Optical
Stimulation
[0146] Whole cell patch clamp recordings were made using a
Multiclamp 700B amplifier, a Digidata 1440 digitizer, and a PC
running pClamp (Molecular Devices, Sunnyvale, Calif.). Neurons were
bathed in room temperature Tyrode containing 125 mM NaCl, 2 mM KCl,
3 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES, 30 mM glucose, 0.01
mM NBQX and 0.01 mM GABAzine. The Tyrode pH was adjusted to 7.3
with NaOH and the osmolarity was adjusted to 300 mOsm with sucrose.
HEK cells were bathed in a Tyrode bath solution identical to that
for neurons, but lacking GABAzine and NBQX. Borosilicate glass
pipettes (Warner Instruments, Hamden, Conn.) with an outer diameter
of 1.2 mm and a wall thickness of 0.255 mm were pulled to a
resistance of 3-9 M.OMEGA. with a P-97 Flaming/Brown micropipette
puller (Sutter Instruments, Novato, Calif.) and filled with a
solution containing 125 mM K-gluconate, 8 mM NaCl, 0.1 mM
CaCl.sub.2, 0.6 mM MgCl2, 1 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, and
0.4 mM Na-GTP. The pipette solution pH was adjusted to 7.3 with KOH
and the osmolarity was adjusted to 298 mOsm with sucrose. Access
resistance was 5-30 M.OMEGA., monitored throughout the
voltage-clamp recording. Resting membrane potential was .about.-60
mV for neurons and .about.-30 mV for HEK 293FT cells in
current-clamp recording.
[0147] Photocurrents were measured with 500 ms light pulses in
neurons voltage-clamped at -60 mV, and in HEK 293FT cells
voltage-clamped at -30 mV. Light-induced membrane
hyperpolarizations were measured with 500 ms light pulses in cells
current-clamped at their resting membrane potential. Light pulses
for all wavelengths except 660 nm and action spectrum
characterization experiments were delivered with a DG-4 optical
switch with 300 W xenon lamp (Sutter Instruments, Novato, Calif.),
controlled via TTL pulses generated through a Digidata signal
generator. Green light was delivered with a 575.+-.25 nm bandpass
filter (Chroma, Bellows Falls, Vt.) and a 575.+-.7.5 nm bandpass
filter (Chroma, Bellows Falls, Vt.). Action spectra were taken with
a Till Photonics Polychrome V, 150 W Xenon lamp, 15 nm
monochromator bandwidth.
[0148] Data was analyzed using Clampfit (Molecular Devices,
Sunnyvale, Calif.) and MATLAB (Mathworks, Inc., Natick, Mass.).
Example 3
[0149] ChR64 (SEQ ID NO:2); ChR86 (SEQ ID NO:4); ChR64 with E154A
substitution (SEQ ID NO:7); ChR86 with D124A substitution (SEQ ID
NO:8) and ChR2 including a ChR2 H134R substitution mutant were
expressed in HEK293 cells using methods described in Examples.
Normalized action spectrum were recorded in the cells under
physiological conditions with the voltage clamped to -65 mV. Equal
photon flux was used at each wavelength.
[0150] FIG. 1 shows action spectra recorded in HEK293 cells.
[0151] FIG. 2A-D shows blue light photocurrent and kinetic
comparisons in cultured hippocampal neurons.
[0152] FIG. 3A-D shows improvements in trafficking leading from
ChR64 to CheRiff FIG. 3A shows photomicrographic image of a
cultured neuron expressing wild-type SdChR.
[0153] SdChR typically aggregated and formed puncta in the soma.
FIG. 3B shows photomicrographic image of a neuron expressing SdChR
with an additional trafficking sequence from Kir2.1 between the
C-terminus of SdChR and the N-terminus of eGFP. This trafficking
sequence substantially reduced intracellular puncta. FIG. 3C shows
photomicrographic image of two neurons expressing CheRiff.
Inclusion of the E154A mutation reduced red light sensitivity and
reduced .tau..sub.off while maintaining excellent membrane
trafficking. FIG. 3D shows data demonstrating that there were
improvements in trafficking leading from ChR64 to CheRiff.
Scherffelia dubia Channelrhodopsin (SdChR) had promising light
sensitivity and a blue-shifted action spectrum appropriate for
pairing with QuasArs; yet it did not traffic efficiently to the
plasma membrane in rat hippocampal neurons.
[0154] FIG. 4A-B shows spectroscopic and kinetic properties of
CheRiff. FIG. 4A at top left shows Components of channelrhodopsin
current elicited by a step in blue light. I.sub.pk is the
difference between baseline current and peak current. t.sub.on is
the time between light onset and peak current. .tau..sub.des is the
desensitization time constant determined by a single-exponential
fit to the current decay after the peak. I.sub.ss is steady state
photocurrent. .DELTA..sub.off is the channel closing time constant
determined by a single-exponential fit to the current decay after
the illumination ceases. FIG. 4A at top right shows peak (I.sub.pk)
and steady state (I.sub.ss) photocurrents in neurons expressing
CheRiff (n=10 cells), ChR2 H134R (n=6 cells), and ChIEF (n=6
cells). Photocurrents were measured in response to a 1 second 488
nm light pulse (500 mW/cm.sup.2). CheRiff generated significantly
larger peak photocurrent than ChR2 H134R (p<0.001) or ChIEF
(p<0.001). CheRiff also had significantly larger steady state
photocurrents than ChR2 H134R (p<0.001) or ChIEF (p<0.01).
Bottom left: CheRiff had a significantly faster time to peak
(t.sub.on) when compared to ChR2 H134R (p<0.001) or ChIEF
(p<0.001). Bottom middle: CheRiff desensitized with a time
constant significantly slower than ChR2 H134R (p<0.001) or ChIEF
(p<0.001). FIG. 4A bottom right shows results when:
.tau..sub.off was measured in response to a 5 ms illumination pulse
(500 mW/cm.sup.2). CheRiff (n=9 cells) had a significantly faster
.tau..sub.off than ChR2 H134R (n=6 cells, p<0.05), and was
comparable to ChIEF (n=6 cells, p=0.94). All channelrhodopsin
comparisons were made on matched cultures, DIV 14-15. Expression
was driven by a CaMKII.alpha. promoter in identical plasmid
backbones. See Examples section for details on cell culture. FIG.
4B shows activation of CheRiff by red light used for imaging
Arch-based voltage indicators (640 nm, 900 W/cm.sup.2). FIG. 4B top
shows results indicating that under current-clamp (i=0) in a neuron
expressing CheRiff, pulses of red light led to a small steady
depolarization of 3.1.+-.0.2 mV (n=5 cells). FIG. 4B bottom shows
results indicating that under voltage-clamp (V=-65 mV), pulses of
red light led to a small inward photocurrent of 14.3.+-.3.1 pA (n=5
cells).
[0155] Table 1 contains a summary of the comparisons between
CheRiff, ChR2 H134R, and ChIEF. [For additional information on
ChIEF, see Lin, J. Y., et al., Biophysical Journal (2009) Vol, 96,
Issue 5, 4 March, pp 1803-1814, the content of which is
incorporated by reference herein].
TABLE-US-00013 TABLE 1 Comparison of CheRiff, ChIEF, and ChR2
H134R. Red I.sub.max (nA; 488 nm, photocurrent Red light ChR 0.5
W/cm.sup.2) Steady t.sub.on .tau..sub.des .tau..sub.off (pA; 640
nm, depolarization variant Peak state (ms) (ms) (ms) 300
W/cm.sup.2) (mV), 300 W/cm.sup.2 CheRiff 2.0 .+-. 0.1 1.33 .+-.
0.08 4.5 .+-. 0.3 400 .+-. 40 16 .+-. 0.5 10.5 .+-. 2.8 2.3 .+-.
0.3 ChIEF 0.9 .+-. 0.1 0.81 .+-. 0.10 18 .+-. 1.8 51 .+-. 10 15
.+-. 2 15.0 .+-. 2.5 2.1 .+-. 0.15 ChR2 1.1 .+-. 0.1 0.65 .+-. 0.09
9.1 .+-. 0.7 40 .+-. 5 25 .+-. 4 2.2 .+-. 0.9 1.0 H134R
[0156] FIG. 5A-E shows application of CheRiff in cultured
hippocampal neurons. FIG. 5A shows light micrographs (DIC) of
Scherffelia dubia (strain CCAC 0053) in side view (top) and face
view (bottom). Arrows mark eyespots (red). Strain and micrographs
courtesy of CCAC [www.ccac.uni-koeln.de/] and Sebastian Hess
(Cologne Biocenter), respectively. FIG. 5B shows photomicrographic
image of cultured rat hippocampal neuron expressing CheRiff-eGFP,
imaged via eGFP fluorescence. FIG. 5C shows photocurrents induced
by CheRiff and by Channelrhodopsin2 H134R with illumination at 488
nm, 500 mW/cm.sup.2. FIG. 5D provides a graph showing comparison of
photocurrents as a function of illumination intensity in matched
cultures expressing CheRiff (n=5 cells) or ChR2 H134R (n=5 cells).
Illumination was either over the whole cell or confined to the
soma. FIG. 5E provides a graph showing spiking fidelity as a
function of stimulation frequency and illumination intensity in
neurons expressing CheRiff (n=5 cells).
EQUIVALENTS
[0157] Although several embodiments of the present invention have
been described and illustrated herein, those of ordinary skill in
the art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto; the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, and/or method described herein.
In addition, any combination of two or more such features, systems,
articles, materials, and/or methods, if such features, systems,
articles, materials, and/or methods are not mutually inconsistent,
is included within the scope of the present invention.
[0158] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0159] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one." The phrase
"and/or," as used herein in the specification and in the claims,
should be understood to mean "either or both" of the elements so
conjoined, i.e., elements that are conjunctively present in some
cases and disjunctively present in other cases. Other elements may
optionally be present other than the elements specifically
identified by the "and/or" clause, whether related or unrelated to
those elements specifically identified, unless clearly indicated to
the contrary.
[0160] All references, patents and patent applications and
publications that are cited or referred to in this application are
incorporated by reference in their entirety herein.
Sequence CWU 1
1
121948DNAArtificial SequenceSynthetic Oligonucleotide,
Mammalian-condon optimized DNA 1atgggcggag ctcctgctcc agacgctcac
agcgccccac ctggaaacga ttctgccgga 60ggcagtgagt accatgcccc agctggatat
caagtgaatc caccctacca ccccgtgcat 120gggtatgagg aacagtgcag
ctccatctac atctactatg gggccctgtg ggagcaggaa 180acagctaggg
gcttccagtg gtttgccgtg ttcctgtctg ccctgtttct ggctttctac
240ggctggcacg cctataaggc cagcgtggga tgggaggaag tgtacgtgtg
ctccgtggag 300ctgatcaaag tgattctgga gatctatttc gagttcacca
gtcctgctat gctgttcctg 360tacggaggga acattacccc atggctgaga
tatgccgaat ggctgctgac atgtcccgtg 420atcctgattc atctgtctaa
catcaccggc ctgagtgagg aatacaataa gcggacaatg 480gctctgctgg
tgtccgacct gggaactatt tgcatgggag tgacagccgc tctggccact
540gggtgggtga agtggctgtt ttactgtatc ggcctggtgt atggaaccca
gacattctac 600aacgctggaa tcatctacgt ggagtcttac tatatcatgc
ctgccggcgg ctgtaagaaa 660ctggtgctgg ccatgactgc cgtgtactat
tctagttggc tgatgtttcc cggcctgttc 720atctttgggc ctgaaggcat
gcacaccctg agcgtggctg ggtccactat tggccatacc 780atcgccgacc
tgctgtccaa gaatatttgg ggactgctgg ggcacttcct gcggatcaaa
840attcacgagc atatcattat gtacggcgat atcaggagac cagtgagctc
ccagtttctg 900ggacgcaagg tggacgtgct ggccttcgtg acagaggaag ataaagtg
9482316PRTScherffelia dubia 2Met Gly Gly Ala Pro Ala Pro Asp Ala
His Ser Ala Pro Pro Gly Asn 1 5 10 15 Asp Ser Ala Gly Gly Ser Glu
Tyr His Ala Pro Ala Gly Tyr Gln Val 20 25 30 Asn Pro Pro Tyr His
Pro Val His Gly Tyr Glu Glu Gln Cys Ser Ser 35 40 45 Ile Tyr Ile
Tyr Tyr Gly Ala Leu Trp Glu Gln Glu Thr Ala Arg Gly 50 55 60 Phe
Gln Trp Phe Ala Val Phe Leu Ser Ala Leu Phe Leu Ala Phe Tyr 65 70
75 80 Gly Trp His Ala Tyr Lys Ala Ser Val Gly Trp Glu Glu Val Tyr
Val 85 90 95 Cys Ser Val Glu Leu Ile Lys Val Ile Leu Glu Ile Tyr
Phe Glu Phe 100 105 110 Thr Ser Pro Ala Met Leu Phe Leu Tyr Gly Gly
Asn Ile Thr Pro Trp 115 120 125 Leu Arg Tyr Ala Glu Trp Leu Leu Thr
Cys Pro Val Ile Leu Ile His 130 135 140 Leu Ser Asn Ile Thr Gly Leu
Ser Glu Glu Tyr Asn Lys Arg Thr Met 145 150 155 160 Ala Leu Leu Val
Ser Asp Leu Gly Thr Ile Cys Met Gly Val Thr Ala 165 170 175 Ala Leu
Ala Thr Gly Trp Val Lys Trp Leu Phe Tyr Cys Ile Gly Leu 180 185 190
Val Tyr Gly Thr Gln Thr Phe Tyr Asn Ala Gly Ile Ile Tyr Val Glu 195
200 205 Ser Tyr Tyr Ile Met Pro Ala Gly Gly Cys Lys Lys Leu Val Leu
Ala 210 215 220 Met Thr Ala Val Tyr Tyr Ser Ser Trp Leu Met Phe Pro
Gly Leu Phe 225 230 235 240 Ile Phe Gly Pro Glu Gly Met His Thr Leu
Ser Val Ala Gly Ser Thr 245 250 255 Ile Gly His Thr Ile Ala Asp Leu
Leu Ser Lys Asn Ile Trp Gly Leu 260 265 270 Leu Gly His Phe Leu Arg
Ile Lys Ile His Glu His Ile Ile Met Tyr 275 280 285 Gly Asp Ile Arg
Arg Pro Val Ser Ser Gln Phe Leu Gly Arg Lys Val 290 295 300 Asp Val
Leu Ala Phe Val Thr Glu Glu Asp Lys Val 305 310 315
3864DNAArtificial SequenceSynthetic Oligonucleotide,
Mammalian-condon optimized DNA 3atgctgggaa acggcagcgc cattgtgcct
atcgaccagt gcttttgcct ggcttggacc 60gacagcctgg gaagcgatac agagcagctg
gtggccaaca tcctccagtg gttcgccttc 120ggcttcagca tcctgatcct
gatgttctac gcctaccaga cttggagagc cacttgcggt 180tgggaggagg
tctacgtctg ttgcgtcgag ctgaccaagg tcatcatcga gttcttccac
240gagttcgacg accccagcat gctgtacctg gctaacggac accgagtcca
gtggctgaga 300tacgcagagt ggctgctgac ttgtcccgtc atcctgatcc
acctgagcaa cctgaccggc 360ctgaaggacg actacagcaa gcggaccatg
aggctgctgg tgtcagacgt gggaaccatc 420gtgtggggag ctacaagcgc
catgagcaca ggctacgtca aggtcatctt cttcgtgctg 480ggttgcatct
acggcgccaa caccttcttc cacgccgcca aggtgtatat cgagagctac
540cacgtggtgc caaagggcag acctagaacc gtcgtgcgga tcatggcttg
gctgttcttc 600ctgtcttggg gcatgttccc cgtgctgttc gtcgtgggac
cagaaggatt cgacgccatc 660agcgtgtacg gctctaccat tggccacacc
atcatcgacc tcatgagcaa gaattgttgg 720ggcctgctgg gacactatct
gagagtgctg atccaccagc acatcatcat ctacggcgac 780atccggaaga
agaccaagat caacgtggcc ggcgaggaga tggaagtgga gaccatggtg
840gaccaggagg acgaggagac agtg 8644288PRTChloromonas oogama 4Met Leu
Gly Asn Gly Ser Ala Ile Val Pro Ile Asp Gln Cys Phe Cys 1 5 10 15
Leu Ala Trp Thr Asp Ser Leu Gly Ser Asp Thr Glu Gln Leu Val Ala 20
25 30 Asn Ile Leu Gln Trp Phe Ala Phe Gly Phe Ser Ile Leu Ile Leu
Met 35 40 45 Phe Tyr Ala Tyr Gln Thr Trp Arg Ala Thr Cys Gly Trp
Glu Glu Val 50 55 60 Tyr Val Cys Cys Val Glu Leu Thr Lys Val Ile
Ile Glu Phe Phe His 65 70 75 80 Glu Phe Asp Asp Pro Ser Met Leu Tyr
Leu Ala Asn Gly His Arg Val 85 90 95 Gln Trp Leu Arg Tyr Ala Glu
Trp Leu Leu Thr Cys Pro Val Ile Leu 100 105 110 Ile His Leu Ser Asn
Leu Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg 115 120 125 Thr Met Arg
Leu Leu Val Ser Asp Val Gly Thr Ile Val Trp Gly Ala 130 135 140 Thr
Ser Ala Met Ser Thr Gly Tyr Val Lys Val Ile Phe Phe Val Leu 145 150
155 160 Gly Cys Ile Tyr Gly Ala Asn Thr Phe Phe His Ala Ala Lys Val
Tyr 165 170 175 Ile Glu Ser Tyr His Val Val Pro Lys Gly Arg Pro Arg
Thr Val Val 180 185 190 Arg Ile Met Ala Trp Leu Phe Phe Leu Ser Trp
Gly Met Phe Pro Val 195 200 205 Leu Phe Val Val Gly Pro Glu Gly Phe
Asp Ala Ile Ser Val Tyr Gly 210 215 220 Ser Thr Ile Gly His Thr Ile
Ile Asp Leu Met Ser Lys Asn Cys Trp 225 230 235 240 Gly Leu Leu Gly
His Tyr Leu Arg Val Leu Ile His Gln His Ile Ile 245 250 255 Ile Tyr
Gly Asp Ile Arg Lys Lys Thr Lys Ile Asn Val Ala Gly Glu 260 265 270
Glu Met Glu Val Glu Thr Met Val Asp Gln Glu Asp Glu Glu Thr Val 275
280 285 5927DNAArtificial SequenceSynthetic Oligonucleotide,
mammalian-codon optimized DNA 5atggactatg gcggcgcttt gtctgccgtc
ggacgcgaac ttttgttcgt tactaatcct 60gtggtggtga acgggtccgt cctggtccct
gaggatcaat gttactgtgc cggatggatt 120gaatctcgcg gcacgaacgg
cgctcagacc gcgtcaaatg tcctgcagtg gcttgcagca 180ggattcagca
ttttgctgct gatgttctat gcctaccaaa cctggaaatc tacatgcggc
240tgggaggaga tctatgtgtg cgccattgaa atggttaagg tgattctcga
gttctttttt 300gagtttaaga atccctctat gctctacctt gccacaggac
accgggtgca gtggctgcgc 360tatgcagagt ggctgctcac ttgtcctgtc
atccttatcc acctgagcaa cctcaccggc 420ctgagcaacg actacagcag
gagaaccatg ggactccttg tctcagacat cgggactatc 480gtgtgggggg
ctaccagcgc catggcaacc ggctatgtta aagtcatctt cttttgtctt
540ggattgtgct atggcgcgaa cacatttttt cacgccgcca aagcatatat
cgagggttat 600catactgtgc caaagggtcg gtgccgccag gtcgtgaccg
gcatggcatg gctgtttttc 660gtgagctggg gtatgttccc aattctcttc
attttggggc ccgaaggttt tggcgtcctg 720agcgtctatg gctccaccgt
aggtcacacg attattgatc tgatgagtaa aaattgttgg 780gggttgttgg
gacactacct gcgcgtcctg atccacgagc acatattgat tcacggagat
840atccgcaaaa ccaccaaact gaacatcggc ggaacggaga tcgaggtcga
gactctcgtc 900gaagacgaag ccgaggccgg agccgtg 9276309PRTChlamydomonas
reinhardtii 6Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu
Leu Leu Phe 1 5 10 15 Val Thr Asn Pro Val Val Val Asn Gly Ser Val
Leu Val Pro Glu Asp 20 25 30 Gln Cys Tyr Cys Ala Gly Trp Ile Glu
Ser Arg Gly Thr Asn Gly Ala 35 40 45 Gln Thr Ala Ser Asn Val Leu
Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60 Leu Leu Leu Met Phe
Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly 65 70 75 80 Trp Glu Glu
Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu 85 90 95 Glu
Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala Thr 100 105
110 Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu Leu Thr Cys
115 120 125 Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr Gly Leu Ser
Asn Asp 130 135 140 Tyr Ser Arg Arg Thr Met Gly Leu Leu Val Ser Asp
Ile Gly Thr Ile 145 150 155 160 Val Trp Gly Ala Thr Ser Ala Met Ala
Thr Gly Tyr Val Lys Val Ile 165 170 175 Phe Phe Cys Leu Gly Leu Cys
Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190 Ala Lys Ala Tyr Ile
Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200 205 Arg Gln Val
Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly 210 215 220 Met
Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly Val Leu 225 230
235 240 Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile Ile Asp Leu Met
Ser 245 250 255 Lys Asn Cys Trp Gly Leu Leu Gly His Tyr Leu Arg Val
Leu Ile His 260 265 270 Glu His Ile Leu Ile His Gly Asp Ile Arg Lys
Thr Thr Lys Leu Asn 275 280 285 Ile Gly Gly Thr Glu Ile Glu Val Glu
Thr Leu Val Glu Asp Glu Ala 290 295 300 Glu Ala Gly Ala Val 305
7316PRTArtificial SequenceSynthetic amino acid sequence 7Met Gly
Gly Ala Pro Ala Pro Asp Ala His Ser Ala Pro Pro Gly Asn 1 5 10 15
Asp Ser Ala Gly Gly Ser Glu Tyr His Ala Pro Ala Gly Tyr Gln Val 20
25 30 Asn Pro Pro Tyr His Pro Val His Gly Tyr Glu Glu Gln Cys Ser
Ser 35 40 45 Ile Tyr Ile Tyr Tyr Gly Ala Leu Trp Glu Gln Glu Thr
Ala Arg Gly 50 55 60 Phe Gln Trp Phe Ala Val Phe Leu Ser Ala Leu
Phe Leu Ala Phe Tyr 65 70 75 80 Gly Trp His Ala Tyr Lys Ala Ser Val
Gly Trp Glu Glu Val Tyr Val 85 90 95 Cys Ser Val Glu Leu Ile Lys
Val Ile Leu Glu Ile Tyr Phe Glu Phe 100 105 110 Thr Ser Pro Ala Met
Leu Phe Leu Tyr Gly Gly Asn Ile Thr Pro Trp 115 120 125 Leu Arg Tyr
Ala Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile His 130 135 140 Leu
Ser Asn Ile Thr Gly Leu Ser Glu Ala Tyr Asn Lys Arg Thr Met 145 150
155 160 Ala Leu Leu Val Ser Asp Leu Gly Thr Ile Cys Met Gly Val Thr
Ala 165 170 175 Ala Leu Ala Thr Gly Trp Val Lys Trp Leu Phe Tyr Cys
Ile Gly Leu 180 185 190 Val Tyr Gly Thr Gln Thr Phe Tyr Asn Ala Gly
Ile Ile Tyr Val Glu 195 200 205 Ser Tyr Tyr Ile Met Pro Ala Gly Gly
Cys Lys Lys Leu Val Leu Ala 210 215 220 Met Thr Ala Val Tyr Tyr Ser
Ser Trp Leu Met Phe Pro Gly Leu Phe 225 230 235 240 Ile Phe Gly Pro
Glu Gly Met His Thr Leu Ser Val Ala Gly Ser Thr 245 250 255 Ile Gly
His Thr Ile Ala Asp Leu Leu Ser Lys Asn Ile Trp Gly Leu 260 265 270
Leu Gly His Phe Leu Arg Ile Lys Ile His Glu His Ile Ile Met Tyr 275
280 285 Gly Asp Ile Arg Arg Pro Val Ser Ser Gln Phe Leu Gly Arg Lys
Val 290 295 300 Asp Val Leu Ala Phe Val Thr Glu Glu Asp Lys Val 305
310 315 8288PRTArtificial SequenceSynthetic amino acid sequence
8Met Leu Gly Asn Gly Ser Ala Ile Val Pro Ile Asp Gln Cys Phe Cys 1
5 10 15 Leu Ala Trp Thr Asp Ser Leu Gly Ser Asp Thr Glu Gln Leu Val
Ala 20 25 30 Asn Ile Leu Gln Trp Phe Ala Phe Gly Phe Ser Ile Leu
Ile Leu Met 35 40 45 Phe Tyr Ala Tyr Gln Thr Trp Arg Ala Thr Cys
Gly Trp Glu Glu Val 50 55 60 Tyr Val Cys Cys Val Glu Leu Thr Lys
Val Ile Ile Glu Phe Phe His 65 70 75 80 Glu Phe Asp Asp Pro Ser Met
Leu Tyr Leu Ala Asn Gly His Arg Val 85 90 95 Gln Trp Leu Arg Tyr
Ala Glu Trp Leu Leu Thr Cys Pro Val Ile Leu 100 105 110 Ile His Leu
Ser Asn Leu Thr Gly Leu Lys Asp Ala Tyr Ser Lys Arg 115 120 125 Thr
Met Arg Leu Leu Val Ser Asp Val Gly Thr Ile Val Trp Gly Ala 130 135
140 Thr Ser Ala Met Ser Thr Gly Tyr Val Lys Val Ile Phe Phe Val Leu
145 150 155 160 Gly Cys Ile Tyr Gly Ala Asn Thr Phe Phe His Ala Ala
Lys Val Tyr 165 170 175 Ile Glu Ser Tyr His Val Val Pro Lys Gly Arg
Pro Arg Thr Val Val 180 185 190 Arg Ile Met Ala Trp Leu Phe Phe Leu
Ser Trp Gly Met Phe Pro Val 195 200 205 Leu Phe Val Val Gly Pro Glu
Gly Phe Asp Ala Ile Ser Val Tyr Gly 210 215 220 Ser Thr Ile Gly His
Thr Ile Ile Asp Leu Met Ser Lys Asn Cys Trp 225 230 235 240 Gly Leu
Leu Gly His Tyr Leu Arg Val Leu Ile His Gln His Ile Ile 245 250 255
Ile Tyr Gly Asp Ile Arg Lys Lys Thr Lys Ile Asn Val Ala Gly Glu 260
265 270 Glu Met Glu Val Glu Thr Met Val Asp Gln Glu Asp Glu Glu Thr
Val 275 280 285 921DNAArtificial SequenceSynthetic Oligonucleotide
9ttctgctacg agaatgaagt g 21107PRTArtificial SequenceSynthetic
polypeptide 10Phe Cys Tyr Glu Asn Glu Val 1 5 1159DNAArtificial
SequenceSynthetic oligonucleotide 11aaatccagaa ttacttctga
aggggagtat atccctctgg atcaaataga catcaatgt 591220PRTArtificial
SequenceSynthetic polypeptide 12Lys Ser Arg Ile Thr Ser Glu Gly Glu
Tyr Ile Pro Leu Asp Gln Ile 1 5 10 15 Asp Ile Asn Val 20
* * * * *