U.S. patent application number 16/267144 was filed with the patent office on 2019-07-18 for upconversion of light for use in optogenetic methods.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Polina Anikeeva, Karl Deisseroth.
Application Number | 20190217118 16/267144 |
Document ID | / |
Family ID | 46024837 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190217118 |
Kind Code |
A1 |
Deisseroth; Karl ; et
al. |
July 18, 2019 |
UPCONVERSION OF LIGHT FOR USE IN OPTOGENETIC METHODS
Abstract
Provided herein are compositions comprising lanthanide-doped
nanoparticles which upconvert electromagnetic radiation from
infrared or near infrared wavelengths into the visible light
spectrum. Also provided herein are methods activating
light-responsive opsin proteins expressed on plasma membranes of
neurons and selectively altering the membrane polarization state of
the neurons using the light delivered by the lanthanide-doped
nanoparticles.
Inventors: |
Deisseroth; Karl; (Stanford,
CA) ; Anikeeva; Polina; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
46024837 |
Appl. No.: |
16/267144 |
Filed: |
February 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15214403 |
Jul 19, 2016 |
10252076 |
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16267144 |
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13882703 |
Jul 16, 2013 |
9522288 |
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PCT/US11/59287 |
Nov 4, 2011 |
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15214403 |
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61410729 |
Nov 5, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61N 2005/0663 20130101;
C12N 2750/14143 20130101; A61K 9/1611 20130101; A61K 41/00
20130101; A61N 5/062 20130101; A61N 5/0613 20130101; A61K 48/0083
20130101; C12N 7/00 20130101; A61N 2005/0659 20130101; A61K 41/0038
20130101; B82Y 5/00 20130101; A61K 41/008 20130101; A61K 38/177
20130101; A61N 2005/0662 20130101; A61N 5/0622 20130101; A61D 7/00
20130101; A61K 9/0019 20130101 |
International
Class: |
A61N 5/06 20060101
A61N005/06; A61K 48/00 20060101 A61K048/00; A61K 41/00 20060101
A61K041/00; B82Y 5/00 20060101 B82Y005/00; A61K 9/16 20060101
A61K009/16; A61K 38/17 20060101 A61K038/17; C12N 7/00 20060101
C12N007/00; A61D 7/00 20060101 A61D007/00; A61K 9/00 20060101
A61K009/00 |
Claims
1.-13. (canceled)
14. A method to hyperpolarize the plasma membrane of a neural cell
in an individual comprising: (a) placing a plurality of
lanthanide-doped nanoparticles in proximity to the neural cell,
wherein the nanoparticles comprise NaYF.sub.4:Yb/X/Gd, wherein X is
erbium (Er), thulium (Tm), or Er/Tm; and (b) exposing the plurality
of nanoparticles to electromagnetic radiation in the infrared (IR)
or near infrared (NIR) spectrum, wherein the electromagnetic
radiation in the IR or NIR spectrum is upconverted into yellow,
amber or red light in the visible spectrum by the nanoparticles,
and wherein a light-responsive opsin comprising a light-responsive
chloride pump is expressed on the plasma membrane and activation of
the opsin by the light in the visible spectrum induces the
hyperpolarization of the plasma membrane.
15. A method to hyperpolarize the plasma membrane of a neural cell
in an individual comprising: (a) administering a polynucleotide
encoding a light-responsive opsin to an individual, wherein the
light-responsive opsin comprises a light-responsive chloride pump
and is expressed on the plasma membrane of a neural cell in the
individual and the opsin is capable of inducing membrane
hyperpolarization of the neural cell when illuminated with light;
(b) administering a plurality of lanthanide-doped nanoparticles in
proximity to the neural cell, wherein the nanoparticles comprise
NaYF.sub.4:Yb/X/Gd, wherein X is erbium (Er), thulium (Tm), or
Er/Tm; and (c) exposing the plurality of nanoparticles to
electromagnetic radiation in the infrared (IR) or near infrared
(NIR) spectrum, wherein the electromagnetic radiation in the IR or
NIR spectrum is upconverted into yellow, amber or red light in the
visible spectrum and the activation of the opsin by the light in
the visible spectrum induces the hyperpolarization of the plasma
membrane.
16. The method of claim 14, wherein the light-responsive opsin
comprises an amino acid sequence having at least 85% amino acid
sequence identity to SEQ ID NO:1.
17. (canceled)
18. The method of claim 14, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength of about 580 nm to about 630 nm.
19. The method of claim 14, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength of about 630 nm to about 740 nm.
20. The method of claim 14, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength corresponding to yellow or amber light.
21. The method of claim 14, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength corresponding to red light.
22. The method of claim 14, wherein the individual is a non-human
animal.
23. The method of claim 14, wherein the individual is a human.
24. The method of claim 14, wherein the neural cell is a neural
cell in the central nervous system.
25. The method of claim 14, wherein the neural cell is a neural
cell in the peripheral nervous system.
26.-32. (canceled)
33. The method of claim 14, wherein X is Er.
34. The method of claim 14, wherein X is Tm.
35. The method of claim 14, wherein X is Er/Tm.
36. The method of claim 14, wherein the neural cell is a neural
cell in the nucleus accumbens of the individual.
37. The method of claim 14, wherein the light-responsive opsin
comprises an amino acid sequence having at least 95% amino acid
sequence identity to SEQ ID NO:2 or SEQ ID NO:3.
38. The method of claim 14, wherein the light-responsive opsin
comprises a NpHR protein.
39. A method to hyperpolarize the plasma membrane of a neural cell
in an individual comprising: (a) placing a plurality of
lanthanide-doped nanoparticles in proximity to the neural cell,
wherein the nanoparticles comprise NaYF.sub.4:Yb/X/Gd, wherein X is
erbium (Er), thulium (Tm), or Er/Tm; and (b) exposing the plurality
of nanoparticles to electromagnetic radiation in the infrared (IR)
or near infrared (NIR) spectrum, wherein the electromagnetic
radiation in the IR or NIR spectrum is upconverted into green or
blue light in the visible spectrum by the nanoparticles, and
wherein a light-responsive opsin comprising a light-responsive
proton pump is expressed on the plasma membrane and activation of
the opsin by the light in the visible spectrum induces the
hyperpolarization of the plasma membrane.
40. A method to hyperpolarize the plasma membrane of a neural cell
in an individual comprising: (a) administering a polynucleotide
encoding a light-responsive opsin to an individual, wherein the
light-responsive opsin comprises a light-responsive proton pump and
is expressed on the plasma membrane of a neural cell in the
individual and the opsin is capable of inducing membrane
hyperpolarization of the neural cell when illuminated with light;
(b) administering a plurality of lanthanide-doped nanoparticles in
proximity to the neural cell, wherein the nanoparticles comprise
NaYF.sub.4:Yb/X/Gd, wherein X is erbium (Er), thulium (Tm), or
Er/Tm; and (c) exposing the plurality of nanoparticles to
electromagnetic radiation in the infrared (IR) or near infrared
(NIR) spectrum, wherein the electromagnetic radiation in the IR or
NIR spectrum is upconverted into green or blue light in the visible
spectrum and the activation of the opsin by the light in the
visible spectrum induces the hyperpolarization of the plasma
membrane.
41. The method of claim 39, wherein the light-responsive opsin
comprises an amino acid sequence having at least 85% amino acid
sequence identity to SEQ ID NO:4.
42. The method of claim 39, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength of about 450 nm to about 495 nm.
43. The method of claim 39, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength corresponding to green light.
44. The method of claim 39, wherein the electromagnetic energy in
the IR or NIR spectrum is upconverted into light having a
wavelength corresponding to blue light.
45. The method of claim 39, wherein the individual is a non-human
animal.
46. The method of claim 39, wherein the individual is a human.
47. The method of claim 39, wherein the neural cell is a neural
cell in the central nervous system.
48. The method of claim 39, wherein the neural cell is a neural
cell in the peripheral nervous system.
49. The method of claim 39, wherein X is Er.
50. The method of claim 39, wherein X is Tm.
51. The method of claim 39, wherein X is Er/Tm.
52. The method of claim 39, wherein the light-responsive opsin
comprises an amino acid sequence having at least 95% amino acid
sequence identity to SEQ ID NO:4.
53. The method of claim 14, wherein the light-responsive opsin
comprises a GtR3 protein.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 61/410,729 filed Nov. 5, 2010, the disclosure of
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This application pertains to compositions comprising
lanthanide-doped nanoparticles which upconvert electromagnetic
radiation from infrared or near infrared wavelengths into the
visible light spectrum and methods of using lanthanide-doped
nanoparticles to deliver light to activate light-responsive opsin
proteins expressed in neurons and selectively alter the membrane
polarization state of the neurons.
BACKGROUND
[0003] Optogenetics is the combination of genetic and optical
methods used to control specific events in targeted cells of living
tissue, even within freely moving mammals and other animals, with
the temporal precision (millisecond-timescale) needed to keep pace
with functioning intact biological systems. The hallmark of
optogenetics is the introduction of fast light-responsive opsin
channel or pump proteins to the plasma membranes of target neuronal
cells that allow temporally precise manipulation of neuronal
membrane potential while maintaining cell-type resolution through
the use of specific targeting mechanisms. Among the microbial
opsins which can be used to investigate the function of neural
systems are the halorhodopsins (NpHRs), used to promote membrane
hyperpolarization when illuminated, and the channel rhodopsins,
used to depolarize membranes upon exposure to light. In just a few
short years, the field of optogenetics has furthered the
fundamental scientific understanding of how specific cell types
contribute to the function of biological tissues, such as neural
circuits, in vivo. Moreover, on the clinical side,
optogenetics-driven research has led to insights into the
neurological mechanisms underlying complex mammalian behaviors such
as anxiety, memory, fear, and addiction.
[0004] In spite of these advances, use of optogenetic methods in
animals suffers from the significant drawback of requiring the
animal to either be tethered to a light source or to have a light
source surgically implanted into the animal. Moreover, when
optogenetic methods are used to alter the function of neurons in
the brain, a light source must be placed in proximity to those
neurons. This requires drilling a hole in the animal's skull and
also presents practical difficulties when the brain region of
interest is located deep within the brain itself. Since light
poorly passes through neural tissue, this necessitates inserting a
fiber optic light source into the brain, which can result in
unintended damage to surrounding brain tissue.
[0005] What is needed, therefore, is a method to non-invasively
deliver light to neurons located within the brain and the
peripheral nervous system of animals expressing light-responsive
opsin proteins on the plasma membranes of neural cells.
[0006] Throughout this specification, references are made to
publications (e.g., scientific articles), patent applications,
patents, etc., all of which are herein incorporated by reference in
their entirety.
SUMMARY OF THE INVENTION
[0007] Provided herein are compositions and methods for
non-invasively delivering light to neurons expressing
light-responsive opsin proteins on neural plasma membranes via the
use of nanoparticles capable of upshifting electromagnetic
radiation from wavelengths associated with the infrared (IR) or
near infrared (NIR) spectrum into wavelengths associated with
visible light.
[0008] Accordingly, provided herein is a method to depolarize the
plasma membrane of a neural cell in an individual comprising: (a)
placing a plurality of lanthanide-doped nanoparticles in proximity
to the neural cell; and (b) exposing the plurality of nanoparticles
to electromagnetic radiation in the infrared (IR) or near infrared
(NIR) spectrum, wherein the electromagnetic radiation in the IR or
NIR spectrum is upconverted into light in the visible spectrum by
the nanoparticles, and wherein a light-responsive opsin is
expressed on the plasma membrane of the neural cells and activation
of the opsin by the light in the visible spectrum induces the
depolarization of the plasma membrane.
[0009] In other aspects, provided herein is a method to depolarize
the plasma membrane of a neural cell in an individual comprising:
(a) administering a polynucleotide encoding a light-responsive
opsin to an individual, wherein the light-responsive protein is
expressed on the plasma membrane of a neural cell in the
individual, and the opsin is capable of inducing membrane
depolarization of the neural cell when illuminated with light; (b)
administering a plurality of lanthanide-doped nanoparticles in
proximity to the neural cell; and (c) exposing the plurality of
nanoparticles to electromagnetic radiation in the infrared (IR) or
near infrared (NIR) spectrum, wherein the electromagnetic radiation
in the IR or NIR spectrum is upconverted into light in the visible
spectrum and the activation of the opsin by the light in the
visible spectrum induces the depolarization of the plasma
membrane.
[0010] In some aspects, provided herein is a method to
hyperpolarize the plasma membrane of a neural cell in an individual
comprising: (a) placing a plurality of lanthanide-doped
nanoparticles in proximity to the neural cell; and (b) exposing the
plurality of nanoparticles to electromagnetic radiation in the
infrared (IR) or near infrared (NIR) spectrum, wherein the
electromagnetic radiation in the IR or NIR spectrum is upconverted
into light in the visible spectrum by the nanoparticles, and
wherein a light-responsive opsin is expressed on the plasma
membrane and activation of the opsin by the light in the visible
spectrum induces the hyperpolarization of the plasma membrane.
[0011] In yet other aspects, provided herein is a method to
hyperpolarize the plasma membrane of a neural cell in an individual
comprising: (a) administering a polynucleotide encoding a
light-responsive opsin to an individual, wherein the
light-responsive protein is expressed on the plasma membrane of a
neural cell in the individual, and the opsin is capable of inducing
membrane hyperpolarization of the neural cell when illuminated with
light; (b) administering a plurality of lanthanide-doped
nanoparticles in proximity to the neural cell; and (c) exposing the
plurality of nanoparticles to electromagnetic radiation in the
infrared (IR) or near infrared (NIR) spectrum, wherein the
electromagnetic radiation in the IR or NIR spectrum is upconverted
into light in the visible spectrum and the activation of the opsin
by the light in the visible spectrum induces the hyperpolarization
of the plasma membrane.
[0012] The present disclosure is directed to apparatuses and
methods involving upconversion for deep delivery of light in vivo.
Aspects of the present disclosure relate generally to delivery of
light to tissue in vivo using upconversion of near infrared light
to the visible light spectrum and methods relating to the
applications discussed herein.
[0013] Certain aspects of the present disclosure are directed to a
light source that is implanted within living tissue. Nanoparticles
from the nanoparticle solution anchor to a target cell population
that includes cells expressing light responsive channels/opsins.
The nanoparticles are configured to respond to receipt of light of
a first wavelength by emitting light of a second, different
wavelength. For example, the nanoparticles can upconvert received
light and thereby emit light of a higher frequency.
[0014] Embodiments of the present disclosure are directed towards
injection of a site of interest with a virus, caring an opsin gene
and a nanoparticle solution. The virus causes a target cell
population at the site of interest to express the opsin gene.
Various different light sources are possible. The use of different
wavelengths can be particularly useful for facilitating the use of
different (external) light sources, e.g., as certain wavelengths
exhibit corresponding decreases in absorption by tissue of the
brain or otherwise.
[0015] Consistent with a particular embodiment of the present
disclosure, a light-emitting diode ("LED") is placed on a portion
of a skull that has been thinned. The LED is placed under the skin
near the thinned portion of the skull, and the location and/or
orientation of the LED is chosen, at least in part, based on the
location of the target cell population. For example, the LED can be
placed to reduce the distance between the LED and the target cell
population and oriented accordingly.
[0016] In certain more specific aspects of the present disclosure,
light from the LED travels through surrounding tissue to the
nanoparticles. When (near) infrared light hits the nanoparticles,
the nanoparticles absorb the infrared (IR) photons and emit visible
photons. The visible photons are then absorbed by the opsins
expressed within the target cell population causing a response
therein (e.g., triggering neural excitation or inhibition).
[0017] The LED can be powered by a battery similar to those used
for pacemakers. The LED can emit light in the infrared spectrum,
and particularly between 700 nm-1000 nm, which can travel through
the skull and intervening tissue. The light emitted from the
nanoparticles has a spectra centered between 450-550 nm. The
wavelength of the light emitted is dependent on characteristics of
the nanoparticle.
[0018] The above overview is not intended to describe each
illustrated embodiment or every implementation of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Various example embodiments may be more completely
understood in consideration of the following description and the
accompanying drawings, in which:
[0020] FIG. 1 shows a cross section of a skull, consistent with an
embodiment of the present disclosure.
[0021] FIG. 2 shows light delivery to target neurons, consistent
with an embodiment of the present disclosure.
[0022] FIG. 3 depicts a system that uses multiple light sources,
consistent with an embodiment of the present disclosure.
[0023] While the present disclosure is amenable to various
modifications and alternative forms, specifics thereof have been
shown by way of example in the drawings and will be described in
detail. It should be understood, however, that the intention is not
to limit the present disclosure to the particular embodiments
described. On the contrary, the intention is to cover all
modifications, equivalents, and alternatives falling within the
scope of the present disclosure including aspects defined in the
claims.
DETAILED DESCRIPTION
[0024] This invention provides, inter alia, compositions and
methods for delivering light to neural cells expressing one or more
light-responsive opsin proteins on the plasma membranes of those
neural cells. The inventors have discovered that nanoparticles
doped with a lanthanide metal (for example, Gadolinium) that
converts infrared (IR) or near infrared (NIR) electromagnetic
radiation into wavelengths corresponding to the visible light
spectrum can be used to activate light-responsive opsin proteins on
the plasma membrane of a neural cell and selectively alter the
membrane polarization state of the cell. Unlike visible light, IR
or NIR electromagnetic energy readily penetrates biological
tissues. For example, NIR can penetrate biological tissues for
distances of up to 4 centimeters (Heyward & Dale Wagner,
"Applied Body Composition Assessment", 2nd edition (2004), p. 100).
Certain equations useful for calculating light penetration in
tissue as a function of wavelength are disclosed in U.S. Pat. No.
7,043,287, the contents of which are incorporated herein by
reference. Similarly, U.S. Patent Application Publication No.
2007/0027411 discloses that near infrared Low Level Laser Treatment
light penetrates the body to a depth of between 3-5 cm. Therefore,
use of IR or NIR sources of electromagnetic radiation in
optogenetic methods can alleviate the need to place a light source
in direct proximity to neural cells. In particular, for optogenetic
techniques in the brain, use of lanthanide-doped nanoparticles in
combination with IR or NIR electromagnetic energy can permit
activation of the opsin protein without the need to puncture the
skull or insert a fiber optic light source into the brain.
Similarly, in the peripheral nervous system, opsin-expressing
nerves can be activated via IR or NM sources placed under the skin
or worn against the skin.
[0025] General Techniques
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, cell biology, biochemistry, nucleic acid chemistry,
immunology, and physiology, which are well known to those skilled
in the art. Such techniques are explained fully in the literature,
such as, Molecular Cloning: A Laboratory Manual, second edition
(Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual,
third edition (Sambrook and Russel, 2001), (jointly referred to
herein as "Sambrook"); Current Protocols in Molecular Biology (F.
M. Ausubel et al., eds., 1987, including supplements through 2001);
PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);
Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold
Spring Harbor Publications, New York; Harlow and Lane (1999), Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (jointly referred to herein as
"Harlow and Lane"), Beaucage et al. eds., Current Protocols in
Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York,
2000), Handbook of Experimental Immunology, 4th edition (D. M. Weir
& C. C. Blackwell, eds., Blackwell Science Inc., 1987), and
Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P.
Calos, eds., 1987). Other useful references include Harrison's
Principles of Internal Medicine (McGraw Hill; J. Isseleacher et
al., eds.) and Lanthanide Luminescence: Photophysical, Analytical
and Biological Aspects (Springer-Verlag, Berlin, Heidelberg;
Hanninen & Harma, eds., 2011).
Definitions
[0027] As used herein, "infrared" or "near infrared" or "infrared
light" or "near infrared light" refers to electromagnetic radiation
in the spectrum immediately above that of visible light, measured
from the nominal edge of visible red light at 0.74 .mu.m, and
extending to 300 .mu.m. These wavelengths correspond to a frequency
range of approximately 1 to 400 THz. In particular, "near infrared"
or "near infrared light" also refers to electromagnetic radiation
measuring 0.75-1.4 .mu.m in wavelength, defined by the water
absorption.
[0028] "Visible light" is defined as electromagnetic radiation with
wavelengths between 380 nm and 750 nm. In general, "electromagnetic
radiation," including light, is generated by the acceleration and
deceleration or changes in movement (vibration) of electrically
charged particles, such as parts of molecules (or adjacent atoms)
with high thermal energy, or electrons in atoms (or molecules).
[0029] The term "nanoparticles" as used herein, can also refer to
nanocrystals, nanorods, nanoclusters, clusters, particles, dots,
quantum dots, small particles, and nanostructured materials. The
term "nanoparticle" encompasses all materials with small size
(generally, though not necessarily) less than 100 nm associated
with quantum size effects.
[0030] An "individual" is a mammal including a human. Mammals
include, but are not limited to, farm animals, sport animals, pets,
primates, mice and rats. Individuals also include companion animals
including, but not limited to, dogs and cats. In some aspects, an
individual is a non-human animal, such as a mammal. In another
aspect, an individual is a human.
[0031] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0032] It is intended that every maximum numerical limitation given
throughout this specification includes every lower numerical
limitation, as if such lower numerical limitations were expressly
written herein. Every minimum numerical limitation given throughout
this specification will include every higher numerical limitation,
as if such higher numerical limitations were expressly written
herein. Every numerical range given throughout this specification
will include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0033] Lanthanide-Doped Nanoparticles
[0034] In materials science, doping is commonly used to incorporate
specific species of ions or atoms into a host lattice core
structure to produce hybrid materials with new and useful
properties. When synthesizing nanoparticles, doping can influence
not only the size and shape of the particles, but also other
properties, such as the ability to convert near infrared (NIR)
excitation into a visible emission of light.
[0035] The lanthanide metals, or lanthanoids (also known as the
"Rare Earth" metals), are elements of atomic number 57 (Lanthanum)
through 71 (Lutetium), and often include Yttrium (atomic number 39)
and Scandium (atomic number 21) because of their chemical
similarities. Lanthanide ions exhibit unique luminescent
properties, including the ability to convert near infrared
long-wavelength excitation radiation into shorter visible
wavelengths through a process known as photon upconversion.
Lanthanides usually exist as trivalent cations, in which case their
electronic configuration is (Xe) 4f, with n varying from 1
(Ce.sup.3+) to 14 (Lu.sup.3+). The transitions within the
f-manifold are responsible for many of the photo-physical
properties of the lanthanide ions, such as long-lived luminescence
and sharp absorption and emission lines. The f-electrons are
shielded from external perturbations by filled 5s and 5p orbitals,
thus giving rise to line-like spectra. Additionally, the f-f
electronic transitions of lanthanides are LaPorte forbidden,
leading to long excited state lifetimes, in the micro- to
millisecond range.
[0036] In some embodiments, any known method can be used to
synthesize lanthanide-doped nanoparticles. Such methods are well
known in the art (See, e.g., Xu & Li, 2007, Clin Chem.,
53(8):1503-10; Wang et al., 2010, Nature, 463(7284):1061-5; U.S.
Patent Application Publication Nos.: 2003/0030067 and 2010/0261263;
and U.S. Pat. No. 7,550,201, the disclosures of each of which are
incorporated herein by reference in their entireties). For example,
in some embodiments, lanthanide-doped nanorods can be synthesized
with a NaYF.sub.4 dielectric core, wherein a DI water solution (1.5
ml) of 0.3 g NaOH is mixed with 5 ml of ethanol and 5 ml of oleic
acid under stirring. To the resulting mixture is selectively added
2 ml of RECl.sub.3 (0.2 M, RE=Y, Yb, Er, Gd, Sm, Nd or La) and 1 ml
of NH.sub.4F (2 M). The solution is then transferred into an
autoclave and heated at 200.degree. C. for 2 h. Nanorods are then
obtained by centrifugation, washed with water and ethanol several
times, and finally re-dispersed in cyclohexane. In another
non-limiting example, nanoparticles can be synthesized using 2 ml
of RECl.sub.3 (0.2 M, RE=Y, Yb, Er, Gd, or Tm) in methanol added to
a flask containing 3 ml oleic acid and 7 ml of 1-octadecene. This
solution is then heated to 160.degree. C. for 30 min and cooled
down to room temperature. Thereafter, a 5 ml methanol solution of
NH.sub.4F (1.6 mmol) and NaOH (1 mmol) is added and the solution is
stirred for 30 min. After methanol evaporation, the solution is
next heated to 300.degree. C. under argon for 1.5 h and cooled down
to room temperature. The resulting nanoparticles are precipitated
by the addition of ethanol, collected by centrifugation, washed
with methanol and ethanol several times, and finally re-dispersed
in cyclohexane.
[0037] In one embodiment, the materials for the lanthanide-doped
nanoparticle core can include a wide variety of dielectric
materials. In various embodiments, the dielectric core can include
lanthanide-doped oxide materials. Lanthanides include lanthanum
(La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium
(Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb),
dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium
(Yb), and lutetium (Lu). Other suitable dielectric core materials
include non-lanthanide elements such as yttrium (Y) and scandium
(Sc). Hence, suitable dielectric core materials include, but are
not limited to, Y.sub.2O.sub.3, Y.sub.2O.sub.2S, NaYF.sub.4,
NaYbF4, Na doped YbF.sub.3, YAG, YAP, Nd.sub.2O.sub.3, LaF.sub.3,
LaCl.sub.3, La.sub.2O.sub.3, TiO.sub.2, LuPO.sub.4, YVO.sub.4,
YbF.sub.3, YF.sub.3, or SiO.sub.2. In one embodiment, the
dielectric nanoparticle core is NaYF.sub.4. These dielectric cores
can be doped with one or more Er, Eu, Yb, Tm, Nd, Tb, Ce, Y, U, Pr,
La, Gd and other rare-earth species or a combination thereof. In
one embodiment, the dielectric core material is doped with Gd. In
another embodiment, the lanthanide-doped nanoparticle comprises
NaYF.sub.4:Yb/X/Gd, wherein X is Er, Tm, or Er/Tm. In some
embodiments, the lanthanide-doped nanoparticles comprise a
NaYF.sub.4:Yb/Er (18/2 mol %) dielectric core doped with any of
about 0 mol %, about 5 mol %, about 10 mol %, about 15 mol %, about
20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40
mol %, about 45 mol %, about 50 mol %, about 55 mol %, about or 60
mol % Gd.sup.3+ ions, inclusive, including any mol % in between
these values. In other embodiments, the lanthanide-doped
nanoparticles comprise a NaYF.sub.4:Yb/Er (18/2 mol %) dielectric
core doped with any of about 0 mol %, about 5 mol %, about 10 mol
%, about 15 mol %, about 20 mol %, about 25 mol %, or about 30 mol
% Yb.sup.3+ ions, inclusive, including any mol % in between these
values. In yet other embodiments, the lanthanide-doped
nanoparticles comprise a NaYF.sub.4:Yb/Er (18/2 mol %) dielectric
core doped with any of about 0 mol %, about 5 mol %, about 10 mol
%, about 15 mol %, about 20 mol %, about 25 mol %, or about 30 mol
% Er.sup.3+ ions, inclusive, including any mol % in between these
values. In other embodiments, the lanthanide-doped nanoparticles
comprise a NaYF.sub.4:Yb/Er (18/2 mol %) dielectric core doped with
any of about 0 mol %, about 5 mol %, about 10 mol %, about 15 mol
%, about 20 mol %, about 25 mol %, or about 30 mol % Tm.sup.3+
ions, inclusive, including any mol % in between these values. In
another embodiment, the lanthanide-doped nanoparticle is selected
from the group consisting of NaYF.sub.4:Yb/Er/Gd (18/2/5 mol %),
NaYF.sub.4:Yb/Tm/Er/Gd (20/0.2/0.1/5 mol %), NaYF.sub.4:Yb/Tm/Er/Gd
(20/0.2/0.05/5 mol %), and NaYF.sub.4:Yb/Tm/Gd (20/0.2/5 mol
%).
[0038] In some aspects, the lanthanide-doped nanoparticles
disclosed herein are conjugated to one or more delivery molecules
to target them to one or more molecules expressed on the surface of
a neural cell of interest (such as a neural cell expressing one or
more light-responsive opsin proteins on its plasma membrane). These
can include, without limitation, antibodies or fragments thereof,
small molecules, as well as lectins or any other carbohydrate
motif. The delivery molecules ensure that the lanthanide-doped
nanoparticles remain in close proximity to the opsin proteins to
permit activation upon upconversion of IR or NIR electromagnetic
radiation. Antibody conjugation to nanoparticles is well-known in
the art (See, e.g., U.S. Patent Application Publication No.:
2010/0209352 and 2008/0267876, the contents of each of which are
incorporated by reference herein in their entireties).
[0039] In another aspect, lanthanide-doped nanoparticles can be
embedded or trapped within a biocompatible material which is
surgically placed proximal to (such as adjacent to or around) the
neural cell of interest (such as a neural cell expressing one or
more light-responsive opsin proteins on its plasma membrane). In
some embodiments, the biocompatible material is transparent, so
that visible light produced by the upconversion of IR or NIR
electromagnetic radiation by the lanthanide-doped nanoparticles can
reach the light-responsive opsin proteins expressed on the surface
of the neural cell of interest. The biocompatible materials used to
embed or trap the lanthanide-doped nanoparticles can include, but
are not limited to, Ioplex materials and other hydrogels such as
those based on 2-hydroxyethyl methacrylate or acrylamide, and poly
ether polyurethane ureas (PEUU) including Biomer (Ethicon Corp.),
Avcothane (Avco-Everrett Laboratories), polyethylene,
polypropylene, polytetrafluoroethylene (Gore-Tex.TM.),
poly(vinylchloride), polydimethylsiloxane, an ethylene-acrylic acid
copolymer, knitted or woven Dacron, polyester-polyurethane,
polyurethane, polycarbonatepolyurethane (Corethane.TM.), polyamide
(Nylon) and polystyrene. In one embodiment, the biocompatible
material can be polydimethylsiloxane (PDMS). Additional compounds
that may be used for embedding and/or trapping the lanthanide-doped
nanoparticles disclosed herein are described in Kirk-Othmer,
Encyclopedia of Chemical Technology, 3rd Edition 1982 (Vol. 19, pp.
275-313, and Vol. 18, pp. 219-2220), van der Giessen et al., 1996,
Circulation, 94:1690-1997 (1996), U.S. Patent Application
Publication No.: 2011/0054305, and U.S. Pat. No. 6,491,965, the
contents of each which are incorporated herein by reference in
their entireties.
[0040] Light-Responsive Opsin Proteins
[0041] Provided herein are optogenetic-based compositions for
selectively hyperpolarizing or depolarizing neurons of the central
or peripheral nervous system. Optogenetics refers to the
combination of genetic and optical methods used to control specific
events in targeted cells of living tissue, even within freely
moving mammals and other animals, with the temporal precision
(millisecond-timescale) needed to keep pace with functioning intact
biological systems. Optogenetics requires the introduction of fast
light-responsive channel or pump proteins to the plasma membranes
of target neuronal cells that allow temporally precise manipulation
of neuronal membrane potential while maintaining cell-type
resolution through the use of specific targeting mechanisms.
[0042] Light-responsive opsins that may be used in the present
invention include opsins that induce hyperpolarization in neurons
by light and opsins that induce depolarization in neurons by light.
Examples of opsins are shown in Tables 1 and 2 below.
TABLE-US-00001 Opsin Biological Wavelength Type Origin Sensitivity
Defined action NpHR Natronomonas 589 nm max Inhibition pharaonis
(hyperpolarization) BR Halobacterium 570 nm max Inhibition helobium
(hyperpolarization) AR Acetabulaira 518 nm max Inhibition
acetabulum (hyperpolarization) GtR3 Guillardia 472 nm max
Inhibition theta (hyperpolarization) Mac Leptosphaeria 470-500 nm
max Inhibition maculans (hyperpolarization) NpHr3.0 Natronomonas
680 nm utility Inhibition pharaonis 589 nm max (hyperpolarization)
NpHR3.1 Natronomonas 680 nm utility Inhibition pharaonis 589 nm max
(hyperpolarization)
TABLE-US-00002 Wavelength Opsin Type Biological Origin Sensitivity
Defined action VChR1 Volvox carteri 589 nm utility Excitation 535
nm max (depolarization) DChR Dunaliella salina 500 nm max
Excitation (depolarization) ChR2 Chlamydomonas 470 nm max
Excitation reinhardtii 380-405 nm utility (depolarization) ChETA
Chlamydomonas 470 nm max Excitation reinhardtii 380-405 nm utility
(depolarization) SFO Chlamydomonas 470 nm max Excitation
reinhardtii 530 nm max (depolarization) Inactivation SSFO
Chlamydomonas 445 nm max Step-like activation reinhardtii 590 nm;
390-400 nm (depolarization) Inactivation C1V1 Volvox carteri and
542 nm max Excitation Chlamydomonas (depolarization) reinhardtii
C1V1 E122 Volvox carteri and 546 nm max Excitation Chlamydomonas
(depolarization) reinhardtii C1V1 E162 Volvox carteri and 542 nm
max Excitation Chlamydomonas (depolarization) reinhardtii C1V1
E122/E162 Volvox carteri and 546 nm max Excitation Chlamydomonas
(depolarization) reinhardtii
[0043] As used herein, a light-responsive opsin (such as NpHR, BR,
AR, GtR3, Mac, ChR2, VChR1, DChR, and ChETA) includes naturally
occurring protein and functional variants, fragments, fusion
proteins comprising the fragments, or the full length protein. For
example, the signal peptide may be deleted. A variant may have an
amino acid sequence at least about any of 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the naturally
occurring protein sequence. A functional variant may have the same
or similar hyperpolarization function or depolarization function as
the naturally occurring protein.
[0044] Enhanced Intracellular Transport Amino Acid Motifs
[0045] The present disclosure provides for the modification of
light-responsive opsin proteins expressed in a cell by the addition
of one or more amino acid sequence motifs which enhance transport
to the plasma membranes of mammalian cells. Light-responsive opsin
proteins having components derived from evolutionarily simpler
organisms may not be expressed or tolerated by mammalian cells or
may exhibit impaired subcellular localization when expressed at
high levels in mammalian cells. Consequently, in some embodiments,
the light-responsive opsin proteins expressed in a cell can be
fused to one or more amino acid sequence motifs selected from the
group consisting of a signal peptide, an endoplasmic reticulum (ER)
export signal, a membrane trafficking signal, and/or an N-terminal
golgi export signal. The one or more amino acid sequence motifs
which enhance light-responsive opsin protein transport to the
plasma membranes of mammalian cells can be fused to the N-terminus,
the C-terminus, or to both the N- and C-terminal ends of the
light-responsive opsin protein. Optionally, the light-responsive
opsin protein and the one or more amino acid sequence motifs may be
separated by a linker. In some embodiments, the light-responsive
opsin protein can be modified by the addition of a trafficking
signal (ts) which enhances transport of the protein to the cell
plasma membrane. In some embodiments, the trafficking signal can be
derived from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In other embodiments, the trafficking
signal can comprise the amino acid sequence
KSRITSEGEYIPLDQIDINV.
[0046] Additional protein motifs which can enhance light-responsive
opsin protein transport to the plasma membrane of a cell are
described in U.S. Patent Application Publication No. 2009/0093403,
which is incorporated herein by reference in its entirety. In some
embodiments, the signal peptide sequence in the protein can be
deleted or substituted with a signal peptide sequence from a
different protein.
[0047] Light-Responsive Chloride Pumps
[0048] In some aspects, the light-responsive opsin proteins
described herein are light-responsive chloride pumps. In some
aspects of the methods provided herein, one or more members of the
Halorhodopsin family of light-responsive chloride pumps are
expressed on the plasma membranes of neurons of the central and
peripheral nervous systems.
[0049] In some aspects, said one or more light-responsive chloride
pump proteins expressed on the plasma membranes of nerve cells of
the central or peripheral nervous systems can be derived from
Natronomonas pharaonic. In some embodiments, the light-responsive
chloride pump proteins can be responsive to amber light as well as
red light and can mediate a hyperpolarizing current in the
interneuron when the light-responsive chloride pump proteins are
illuminated with amber or red light. The wavelength of light which
can activate the light-responsive chloride pumps can be between
about 580 and about 630 nm. In some embodiments, the light can be
at a wavelength of about 590 nm or the light can have a wavelength
greater than about 630 nm (e.g. less than about 740 nm). In another
embodiment, the light has a wavelength of around 630 nm. In some
embodiments, the light-responsive chloride pump protein can
hyperpolarize a neural membrane for at least about 90 minutes when
exposed to a continuous pulse of light. In some embodiments, the
light-responsive chloride pump protein can comprise an amino acid
sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1.
Additionally, the light-responsive chloride pump protein can
comprise substitutions, deletions, and/or insertions introduced
into a native amino acid sequence to increase or decrease
sensitivity to light, increase or decrease sensitivity to
particular wavelengths of light, and/or increase or decrease the
ability of the light-responsive protein to regulate the
polarization state of the plasma membrane of the cell. In some
embodiments, the light-responsive chloride pump protein contains
one or more conservative amino acid substitutions. In some
embodiments, the light-responsive protein contains one or more
non-conservative amino acid substitutions. The light-responsive
protein comprising substitutions, deletions, and/or insertions
introduced into the native amino acid sequence suitably retains the
ability to hyperpolarize the plasma membrane of a neuronal cell in
response to light.
[0050] Additionally, in other aspects, the light-responsive
chloride pump protein can comprise a core amino acid sequence at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% identical to the sequence shown in SEQ ID NO: 1 and an
endoplasmic reticulum (ER) export signal. This ER export signal can
be fused to the C-terminus of the core amino acid sequence or can
be fused to the N-terminus of the core amino acid sequence. In some
embodiments, the ER export signal is linked to the core amino acid
sequence by a linker. The linker can comprise any of about 5, 10,
20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,
400, or 500 amino acids in length. The linker may further comprise
a fluorescent protein, for example, but not limited to, a yellow
fluorescent protein, a red fluorescent protein, a green fluorescent
protein, or a cyan fluorescent protein. In some embodiments, the ER
export signal can comprise the amino acid sequence FXYENE, where X
can be any amino acid. In another embodiment, the ER export signal
can comprise the amino acid sequence VXXSL, where X can be any
amino acid. In some embodiments, the ER export signal can comprise
the amino acid sequence FCYENEV.
[0051] In other aspects, the light-responsive chloride pump
proteins provided herein can comprise a light-responsive protein
expressed on the cell membrane, wherein the protein comprises a
core amino acid sequence at least about 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 1 and a trafficking signal (e.g., which can enhance
transport of the light-responsive chloride pump protein to the
plasma membrane). The trafficking signal may be fused to the
C-terminus of the core amino acid sequence or may be fused to the
N-terminus of the core amino acid sequence. In some embodiments,
the trafficking signal can be linked to the core amino acid
sequence by a linker which can comprise any of about 5, 10, 20, 30,
40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or
500 amino acids in length. The linker may further comprise a
fluorescent protein, for example, but not limited to, a yellow
fluorescent protein, a red fluorescent protein, a green fluorescent
protein, or a cyan fluorescent protein. In some embodiments, the
trafficking signal can be derived from the amino acid sequence of
the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal can comprise the amino acid
sequence KSRITSEGEYIPLDQIDINV.
[0052] In some aspects, the light-responsive chloride pump protein
can comprise a core amino acid sequence at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
sequence shown in SEQ ID NO: 1 and at least one (such as one, two,
three, or more) amino acid sequence motifs which enhance transport
to the plasma membranes of mammalian cells selected from the group
consisting of an ER export signal, a signal peptide, and a membrane
trafficking signal. In some embodiments, the light-responsive
chloride pump protein comprises an N-terminal signal peptide, a
C-terminal ER Export signal, and a C-terminal trafficking signal.
In some embodiments, the C-terminal ER Export signal and the
C-terminal trafficking signal can be linked by a linker. The linker
can comprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,
175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length.
The linker can also further comprise a fluorescent protein, for
example, but not limited to, a yellow fluorescent protein, a red
fluorescent protein, a green fluorescent protein, or a cyan
fluorescent protein. In some embodiments the ER Export signal can
be more C-terminally located than the trafficking signal. In other
embodiments the trafficking signal is more C-terminally located
than the ER Export signal. In some embodiments, the signal peptide
comprises the amino acid sequence MTETLPPVTESAVALQAE. In another
embodiment, the light-responsive chloride pump protein comprises an
amino acid sequence at least 95% identical to SEQ ID NO:2.
[0053] Moreover, in other aspects, the light-responsive chloride
pump proteins can comprise a core amino acid sequence at least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequence shown in SEQ ID NO: 1, wherein the
N-terminal signal peptide of SEQ ID NO:1 is deleted or substituted.
In some embodiments, other signal peptides (such as signal peptides
from other opsins) can be used. The light-responsive protein can
further comprise an ER transport signal and/or a membrane
trafficking signal described herein. In some embodiments, the
light-responsive chloride pump protein comprises an amino acid
sequence at least 95% identical to SEQ ID NO:3.
[0054] In some embodiments, the light-responsive opsin protein is a
NpHR opsin protein comprising an amino acid sequence at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
identical to the sequence shown in SEQ ID NO:1. In some
embodiments, the NpHR opsin protein further comprises an
endoplasmic reticulum (ER) export signal and/or a membrane
trafficking signal. For example, the NpHR opsin protein comprises
an amino acid sequence at least 95% identical to the sequence shown
in SEQ ID NO:1 and an endoplasmic reticulum (ER) export signal. In
some embodiments, the amino acid sequence at least 95% identical to
the sequence shown in SEQ ID NO:1 is linked to the ER export signal
through a linker. In some embodiments, the ER export signal
comprises the amino acid sequence FXYENE, where X can be any amino
acid. In another embodiment, the ER export signal comprises the
amino acid sequence VXXSL, where X can be any amino acid. In some
embodiments, the ER export signal comprises the amino acid sequence
FCYENEV. In some embodiments, the NpHR opsin protein comprises an
amino acid sequence at least 95% identical to the sequence shown in
SEQ ID NO:1, an ER export signal, and a membrane trafficking
signal. In other embodiments, the NpHR opsin protein comprises,
from the N-terminus to the C-terminus, the amino acid sequence at
least 95% identical to the sequence shown in SEQ ID NO:1, the ER
export signal, and the membrane trafficking signal. In other
embodiments, the NpHR opsin protein comprises, from the N-terminus
to the C-terminus, the amino acid sequence at least 95% identical
to the sequence shown in SEQ ID NO:1, the membrane trafficking
signal, and the ER export signal. In some embodiments, the membrane
trafficking signal is derived from the amino acid sequence of the
human inward rectifier potassium channel Kir2.1. In some
embodiments, the membrane trafficking signal comprises the amino
acid sequence KSRITSEGEYIPLDQIDINV. In some embodiments, the
membrane trafficking signal is linked to the amino acid sequence at
least 95% identical to the sequence shown in SEQ ID NO:1 by a
linker. In some embodiments, the membrane trafficking signal is
linked to the ER export signal through a linker. The linker may
comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200,
225, 250, 275, 300, 400, or 500 amino acids in length. The linker
may further comprise a fluorescent protein, for example, but not
limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments, the light-responsive opsin protein
further comprises an N-terminal signal peptide. In some
embodiments, the light-responsive opsin protein comprises the amino
acid sequence of SEQ ID NO:2. In some embodiments, the
light-responsive opsin protein comprises the amino acid sequence of
SEQ ID NO:3.
[0055] Also provided herein are polynucleotides encoding any of the
light-responsive chloride ion pump proteins described herein, such
as a light-responsive protein comprising a core amino acid sequence
at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% identical to the sequence shown in SEQ ID NO:1, an ER export
signal, and a membrane trafficking signal. In another embodiment,
the polynucleotides comprise a sequence which encodes an amino acid
at least 95% identical to SEQ ID NO:2 and/or SEQ ID NO:3. The
polynucleotides may be in an expression vector (such as, but not
limited to, a viral vector described herein). The polynucleotides
may be used for expression of the light-responsive chloride ion
pump proteins in neurons of the central or peripheral nervous
systems.
[0056] Further disclosure related to light-responsive chloride pump
proteins can be found in U.S. Patent Application Publication Nos:
2009/0093403 and 2010/0145418 as well as in International Patent
Application No: PCT/US2011/028893, the disclosures of each of which
are hereby incorporated by reference in their entireties.
[0057] Light-Responsive Proton Pumps
[0058] In some aspects, the light-responsive opsin proteins
described herein are light-responsive proton pumps. In some aspects
of the compositions and methods provided herein, one or more
light-responsive proton pumps are expressed on the plasma membranes
of neurons of the central or peripheral nervous systems.
[0059] In some embodiments, the light-responsive proton pump
protein can be responsive to blue light and can be derived from
Guillardia theta, wherein the proton pump protein can be capable of
mediating a hyperpolarizing current in the cell when the cell is
illuminated with blue light. The light can have a wavelength
between about 450 and about 495 nm or can have a wavelength of
about 490 nm. In another embodiment, the light-responsive proton
pump protein can comprise an amino acid sequence at least about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to the sequence shown in SEQ ID NO:4. The light-responsive proton
pump protein can additionally comprise substitutions, deletions,
and/or insertions introduced into a native amino acid sequence to
increase or decrease sensitivity to light, increase or decrease
sensitivity to particular wavelengths of light, and/or increase or
decrease the ability of the light-responsive proton pump protein to
regulate the polarization state of the plasma membrane of the cell.
Additionally, the light-responsive proton pump protein can contain
one or more conservative amino acid substitutions and/or one or
more non-conservative amino acid substitutions. The
light-responsive proton pump protein comprising substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to hyperpolarize the plasma
membrane of a neuronal cell in response to light.
[0060] In other aspects of the methods disclosed herein, the
light-responsive proton pump protein can comprise a core amino acid
sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:4
and at least one (such as one, two, three, or more) amino acid
sequence motifs which enhance transport to the plasma membranes of
mammalian cells selected from the group consisting of a signal
peptide, an ER export signal, and a membrane trafficking signal. In
some embodiments, the light-responsive proton pump protein
comprises an N-terminal signal peptide and a C-terminal ER export
signal. In some embodiments, the light-responsive proton pump
protein comprises an N-terminal signal peptide and a C-terminal
trafficking signal. In some embodiments, the light-responsive
proton pump protein comprises an N-terminal signal peptide, a
C-terminal ER Export signal, and a C-terminal trafficking signal.
In some embodiments, the light-responsive proton pump protein
comprises a C-terminal ER Export signal and a C-terminal
trafficking signal. In some embodiments, the C-terminal ER Export
signal and the C-terminal trafficking signal are linked by a
linker. The linker can comprise any of about 5, 10, 20, 30, 40, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The linker may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER Export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0061] Also provided herein are isolated polynucleotides encoding
any of the light-responsive proton pump proteins described herein,
such as a light-responsive proton pump protein comprising a core
amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO:4. Also provided herein are expression vectors (such as a
viral vector described herein) comprising a polynucleotide encoding
the proteins described herein, such as a light-responsive proton
pump protein comprising a core amino acid sequence at least about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to the sequence shown in SEQ ID NO:4. The polynucleotides may be
used for expression of the light-responsive proton pumps in neural
cells of the central or peripheral nervous systems.
[0062] Further disclosure related to light-responsive proton pump
proteins can be found in International Patent Application No.
PCT/US2011/028893, the disclosure of which is hereby incorporated
by reference in its entirety.
[0063] Light-Activated Cation Channel Proteins
[0064] In some aspects, the light-responsive opsin proteins
described herein are light-activated cation channel proteins. In
some aspects of the methods provided herein, one or more
light-activated cation channels can be expressed on the plasma
membranes of the neural cells of the central or peripheral nervous
systems.
[0065] In some aspects, the light-activated cation channel protein
can be derived from Chlamydomonas reinhardtii, wherein the cation
channel protein can be capable of mediating a depolarizing current
in the cell when the cell is illuminated with light. In another
embodiment, the light-activated cation channel protein can comprise
an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO:5. The light used to activate the light-activated cation
channel protein derived from Chlamydomonas reinhardtii can have a
wavelength between about 460 and about 495 nm or can have a
wavelength of about 480 nm. Additionally, the light can have an
intensity of at least about 100 Hz. In some embodiments, activation
of the light-activated cation channel derived from Chlamydomonas
reinhardtii with light having an intensity of 100 Hz can cause
depolarization-induced synaptic depletion of the neurons expressing
the light-activated cation channel. The light-activated cation
channel protein can additionally comprise substitutions, deletions,
and/or insertions introduced into a native amino acid sequence to
increase or decrease sensitivity to light, increase or decrease
sensitivity to particular wavelengths of light, and/or increase or
decrease the ability of the light-activated cation channel protein
to regulate the polarization state of the plasma membrane of the
cell.
[0066] Additionally, the light-activated cation channel protein can
contain one or more conservative amino acid substitutions and/or
one or more non-conservative amino acid substitutions. The
light-activated proton pump protein comprising substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to depolarize the plasma
membrane of a neuronal cell in response to light.
[0067] Further disclosure related to light-activated cation channel
proteins can be found in U.S. Patent Application Publication No.
2007/0054319 and International Patent Application Publication Nos.
WO 2009/131837 and WO 2007/024391, the disclosures of each of which
are hereby incorporated by reference in their entireties.
[0068] Step Function Opsins and Stabilized Step Function Opsins
[0069] In other embodiments, the light-activated cation channel
protein can be a step function opsin (SFO) protein or a stabilized
step function opsin (SSFO) protein that can have specific amino
acid substitutions at key positions throughout the retinal binding
pocket of the protein. In some embodiments, the SFO protein can
have a mutation at amino acid residue C128 of SEQ ID NO:5. In other
embodiments, the SFO protein has a C128A mutation in SEQ ID NO:5.
In other embodiments, the SFO protein has a C128S mutation in SEQ
ID NO:5. In another embodiment, the SFO protein has a C128T
mutation in SEQ ID NO:5. In some embodiments, the SFO protein can
comprise an amino acid sequence at least about 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence
shown in SEQ ID NO:6.
[0070] In some embodiments, the SSFO protein can have a mutation at
amino acid residue D156 of SEQ ID NO:5. In other embodiments, the
SSFO protein can have a mutation at both amino acid residues C128
and D156 of SEQ ID NO:5. In one embodiment, the SSFO protein has an
C128S and a D156A mutation in SEQ ID NO:5. In another embodiment,
the SSFO protein can comprise an amino acid sequence at least about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to the sequence shown in SEQ ID NO:7.
[0071] In some embodiments the SFO or SSFO proteins provided herein
can be capable of mediating a depolarizing current in the cell when
the cell is illuminated with blue light. In other embodiments, the
light can have a wavelength of about 445 nm. Additionally, the
light can have an intensity of about 100 Hz. In some embodiments,
activation of the SFO or SSFO protein with light having an
intensity of 100 Hz can cause depolarization-induced synaptic
depletion of the neurons expressing the SFO or SSFO protein. In
some embodiments, each of the disclosed step function opsin and
stabilized step function opsin proteins can have specific
properties and characteristics for use in depolarizing the membrane
of a neuronal cell in response to light.
[0072] Further disclosure related to SFO or SSFO proteins can be
found in International Patent Application Publication No. WO
2010/056970 and U.S. Provisional Patent Application Nos. 61/410,704
and 61/511,905, the disclosures of each of which are hereby
incorporated by reference in their entireties.
[0073] C1V1 Chimeric Cation Channels
[0074] In other embodiments, the light-activated cation channel
protein can be a C1V1 chimeric protein derived from the VChR1
protein of Volvox carteri and the ChR1 protein from Chlamydomonas
reinhardti, wherein the protein comprises the amino acid sequence
of VChR1 having at least the first and second transmembrane helices
replaced by the first and second transmembrane helices of ChR1; is
responsive to light; and is capable of mediating a depolarizing
current in the cell when the cell is illuminated with light. In
some embodiments, the C1V1 protein can further comprise a
replacement within the intracellular loop domain located between
the second and third transmembrane helices of the chimeric light
responsive protein, wherein at least a portion of the intracellular
loop domain is replaced by the corresponding portion from ChR1. In
another embodiment, the portion of the intracellular loop domain of
the C1V1 chimeric protein can be replaced with the corresponding
portion from ChR1 extending to amino acid residue A145 of the ChR1.
In other embodiments, the C1V1 chimeric protein can further
comprise a replacement within the third transmembrane helix of the
chimeric light responsive protein, wherein at least a portion of
the third transmembrane helix is replaced by the corresponding
sequence of ChR1. In yet another embodiment, the portion of the
intracellular loop domain of the C1V1 chimeric protein can be
replaced with the corresponding portion from ChR1 extending to
amino acid residue W163 of the ChR1. In other embodiments, the C1V1
chimeric protein can comprise an amino acid sequence at least about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to the sequence shown in SEQ ID NO:8.
[0075] In some embodiments, the C1V1 protein can mediate a
depolarizing current in the cell when the cell is illuminated with
green light. In other embodiments, the light can have a wavelength
of between about 540 nm to about 560 nm. In some embodiments, the
light can have a wavelength of about 542 nm. In some embodiments,
the C1V1 chimeric protein is not capable of mediating a
depolarizing current in the cell when the cell is illuminated with
violet light. In some embodiments, the chimeric protein is not
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light having a wavelength of about 405 nm.
Additionally, the light can have an intensity of about 100 Hz. In
some embodiments, activation of the C1V1 chimeric protein with
light having an intensity of 100 Hz can cause
depolarization-induced synaptic depletion of the neurons expressing
the C1V1 chimeric protein. In some embodiments, the disclosed C1V1
chimeric protein can have specific properties and characteristics
for use in depolarizing the membrane of a neuronal cell in response
to light.
[0076] C1V1 Chimeric Mutant Variants
[0077] In some aspects, the invention can include polypeptides
comprising substituted or mutated amino acid sequences, wherein the
mutant polypeptide retains the characteristic light-responsive
nature of the precursor C1V1 chimeric polypeptide but may also
possess altered properties in some specific aspects. For example,
the mutant light-activated C1V1 chimeric proteins described herein
can exhibit an increased level of expression both within an animal
cell or on the animal cell plasma membrane; an altered
responsiveness when exposed to different wavelengths of light,
particularly red light; and/or a combination of traits whereby the
chimeric C1V1 polypeptide possess the properties of low
desensitization, fast deactivation, low violet-light activation for
minimal cross-activation with other light-activated cation
channels, and/or strong expression in animal cells.
[0078] Accordingly, provided herein are C1V1 chimeric
light-activated proteins that can have specific amino acid
substitutions at key positions throughout the retinal binding
pocket of the VChR1 portion of the chimeric polypeptide. In some
embodiments, the C1V1 protein can have a mutation at amino acid
residue E122 of SEQ ID NO:7. In some embodiments, the C1V1 protein
can have a mutation at amino acid residue E162 of SEQ ID NO:7. In
other embodiments, the C1V1 protein can have a mutation at both
amino acid residues E162 and E122 of SEQ ID NO:7. In other
embodiments, the C1V1 protein can comprise an amino acid sequence
at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% identical to the sequence shown in SEQ ID NO:9, SEQ ID NO:10,
or SEQ ID NO:11. In some embodiments, each of the disclosed mutant
C1V1 chimeric proteins can have specific properties and
characteristics for use in depolarizing the membrane of an animal
cell in response to light.
[0079] In some aspects, the C1V1-E122 mutant chimeric protein is
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light. In some embodiments the light can
be green light. In other embodiments, the light can have a
wavelength of between about 540 nm to about 560 nm. In some
embodiments, the light can have a wavelength of about 546 nm. In
other embodiments, the C1V1-E122 mutant chimeric protein can
mediate a depolarizing current in the cell when the cell is
illuminated with red light. In some embodiments, the red light can
have a wavelength of about 630 nm. In some embodiments, the
C1V1-E122 mutant chimeric protein does not mediate a depolarizing
current in the cell when the cell is illuminated with violet light.
In some embodiments, the chimeric protein does not mediate a
depolarizing current in the cell when the cell is illuminated with
light having a wavelength of about 405 nm. Additionally, the light
can have an intensity of about 100 Hz. In some embodiments,
activation of the C1V1-E122 mutant chimeric protein with light
having an intensity of 100 Hz can cause depolarization-induced
synaptic depletion of the neurons expressing the C1V1-E122 mutant
chimeric protein. In some embodiments, the disclosed C1V1-E122
mutant chimeric protein can have specific properties and
characteristics for use in depolarizing the membrane of a neuronal
cell in response to light.
[0080] In other aspects, the C1V1-E162 mutant chimeric protein is
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light. In some embodiments the light can
be green light. In other embodiments, the light can have a
wavelength of between about 540 nm to about 535 nm. In some
embodiments, the light can have a wavelength of about 542 nm. In
other embodiments, the light can have a wavelength of about 530 nm.
In some embodiments, the C1V1-E162 mutant chimeric protein does not
mediate a depolarizing current in the cell when the cell is
illuminated with violet light. In some embodiments, the chimeric
protein does not mediate a depolarizing current in the cell when
the cell is illuminated with light having a wavelength of about 405
nm. Additionally, the light can have an intensity of about 100 Hz.
In some embodiments, activation of the C1V1-E162 mutant chimeric
protein with light having an intensity of 100 Hz can cause
depolarization-induced synaptic depletion of the neurons expressing
the C1V1-E162 mutant chimeric protein. In some embodiments, the
disclosed C1V1-E162 mutant chimeric protein can have specific
properties and characteristics for use in depolarizing the membrane
of a neuronal cell in response to light.
[0081] In yet other aspects, the C1V1-E122/E162 mutant chimeric
protein is capable of mediating a depolarizing current in the cell
when the cell is illuminated with light. In some embodiments the
light can be green light. In other embodiments, the light can have
a wavelength of between about 540 nm to about 560 nm. In some
embodiments, the light can have a wavelength of about 546 nm. In
some embodiments, the C1V1-E122/E162 mutant chimeric protein does
not mediate a depolarizing current in the cell when the cell is
illuminated with violet light. In some embodiments, the chimeric
protein does not mediate a depolarizing current in the cell when
the cell is illuminated with light having a wavelength of about 405
nm. In some embodiments, the C1V1-E122/E162 mutant chimeric protein
can exhibit less activation when exposed to violet light relative
to C1V1 chimeric proteins lacking mutations at E122/E162 or
relative to other light-activated cation channel proteins.
Additionally, the light can have an intensity of about 100 Hz. In
some embodiments, activation of the C1V1-E122/E162 mutant chimeric
protein with light having an intensity of 100 Hz can cause
depolarization-induced synaptic depletion of the neurons expressing
the C1V1-E122/E162 mutant chimeric protein. In some embodiments,
the disclosed C1V1-E122/E162 mutant chimeric protein can have
specific properties and characteristics for use in depolarizing the
membrane of a neuronal cell in response to light.
[0082] Further disclosure related to C1V1 chimeric cation channels
as well as mutant variants of the same can be found in U.S.
Provisional Patent Application Nos. 61/410,736, 61/410,744, and
61/511,912, the disclosures of each of which are hereby
incorporated by reference in their entireties.
[0083] Polynucleotides
[0084] The disclosure also provides polynucleotides comprising a
nucleotide sequence encoding a light-responsive opsin protein
described herein. In some embodiments, the polynucleotide comprises
an expression cassette. In some embodiments, the polynucleotide is
a vector comprising the above-described nucleic acid(s). In some
embodiments, the nucleic acid encoding a light-activated protein of
the disclosure is operably linked to a promoter. Promoters are well
known in the art. Any promoter that functions in the host cell can
be used for expression of the light-responsive opsin proteins
and/or any variant thereof of the present disclosure. In one
embodiment, the promoter used to drive expression of the
light-responsive opsin proteins is a promoter that is specific to
motor neurons. In another embodiment, the promoter used to drive
expression of the light-responsive opsin proteins is a promoter
that is specific to central nervous system neurons. In other
embodiments, the promoter is capable of driving expression of the
light-responsive opsin proteins in neurons of both the sympathetic
and/or the parasympathetic nervous systems. Initiation control
regions or promoters, which are useful to drive expression of the
light-responsive opsin proteins or variant thereof in a specific
animal cell are numerous and familiar to those skilled in the art.
Virtually any promoter capable of driving these nucleic acids can
be used. Examples of motor neuron-specific genes can be found, for
example, in Kudo, et al., Human Mol. Genetics, 2010, 19(16):
3233-3253, the contents of which are hereby incorporated by
reference in their entirety. In some embodiments, the promoter used
to drive expression of the light-activated protein can be the Thy1
promoter, which is capable of driving robust expression of
transgenes in neurons of both the central and peripheral nervous
systems (See, e.g., Llewellyn, et al., 2010, Nat. Med.,
16(10):1161-1166). In other embodiments, the promoter used to drive
expression of the light-responsive opsin protein can be the
EF1.alpha. promoter, a cytomegalovirus (CMV) promoter, the CAG
promoter, the sinapsin promoter, or any other ubiquitous promoter
capable of driving expression of the light-responsive opsin
proteins in the peripheral and/or central nervous system neurons of
mammals.
[0085] Also provided herein are vectors comprising a nucleotide
sequence encoding a light-responsive opsin protein or any variant
thereof described herein. The vectors that can be administered
according to the present invention also include vectors comprising
a nucleotide sequence which encodes an RNA (e.g., an mRNA) that
when transcribed from the polynucleotides of the vector will result
in the accumulation of light-responsive opsin proteins on the
plasma membranes of target animal cells. Vectors which may be used,
include, without limitation, lentiviral, HSV, adenoviral, and
andeno-associated viral (AAV) vectors. Lentiviruses include, but
are not limited to HW-1, HIV-2, SW, FW and EIAV. Lentiviruses may
be pseudotyped with the envelope proteins of other viruses,
including, but not limited to VSV, rabies, Mo-MLV, baculovirus and
Ebola. Such vectors may be prepared using standard methods in the
art.
[0086] In some embodiments, the vector is a recombinant AAV vector.
AAV vectors are DNA viruses of relatively small size that can
integrate, in a stable and site-specific manner, into the genome of
the cells that they infect. They are able to infect a wide spectrum
of cells without inducing any effects on cellular growth,
morphology or differentiation, and they do not appear to be
involved in human pathologies. The AAV genome has been cloned,
sequenced and characterized. It encompasses approximately 4700
bases and contains an inverted terminal repeat (ITR) region of
approximately 145 bases at each end, which serves as an origin of
replication for the virus. The remainder of the genome is divided
into two essential regions that carry the encapsidation functions:
the left-hand part of the genome, that contains the rep gene
involved in viral replication and expression of the viral genes;
and the right-hand part of the genome, that contains the cap gene
encoding the capsid proteins of the virus.
[0087] AAV vectors may be prepared using standard methods in the
art. Adeno-associated viruses of any serotype are suitable (See,
e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J.
R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.
Tattersall "The Evolution of Parvovirus Taxonomy" in Parvoviruses
(J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p
5-14, Hudder Arnold, London, U K (2006); and D E Bowles, J E
Rabinowitz, R T Samulski "The Genus Dependovirus" (J R Kerr, S F
Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 15-23, Hudder
Arnold, London, UK (2006), the disclosures of each of which are
hereby incorporated by reference herein in their entireties).
Methods for purifying for vectors may be found in, for example,
U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 and
International Patent Application Publication No.: WO/1999/011764
titled "Methods for Generating High Titer Helper-free Preparation
of Recombinant AAV Vectors", the disclosures of which are herein
incorporated by reference in their entirety. Preparation of hybrid
vectors is described in, for example, PCT Application No.
PCT/US2005/027091, the disclosure of which is herein incorporated
by reference in its entirety. The use of vectors derived from the
AAVs for transferring genes in vitro and in vivo has been described
(See e.g., International Patent Application Publication Nos.: WO
91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and
5,139,941; and European Patent No.: 0488528, all of which are
hereby incorporated by reference herein in their entireties). These
publications describe various AAV-derived constructs in which the
rep and/or cap genes are deleted and replaced by a gene of
interest, and the use of these constructs for transferring the gene
of interest in vitro (into cultured cells) or in vivo (directly
into an organism). The replication defective recombinant AAVs
according to the invention can be prepared by co-transfecting a
plasmid containing the nucleic acid sequence of interest flanked by
two AAV inverted terminal repeat (ITR) regions, and a plasmid
carrying the AAV encapsidation genes (rep and cap genes), into a
cell line that is infected with a human helper virus (for example,
an adenovirus). The AAV recombinants that are produced are then
purified by standard techniques.
[0088] In some embodiments, the vector(s) for use in the methods of
the invention are encapsidated into a virus particle (e.g. AAV
virus particle including, but not limited to, AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,
AAV14, AAV15, and AAV16). Accordingly, the invention includes a
recombinant virus particle (recombinant because it contains a
recombinant polynucleotide) comprising any of the vectors described
herein. Methods of producing such particles are known in the art
and are described in U.S. Pat. No. 6,596,535, the disclosure of
which is hereby incorporated by reference in its entirety.
[0089] Delivery of Light-Responsive Opsin Proteins and
Lanthanide-Doped Nanoparticles
[0090] In some aspects, polynucleotides encoding the
light-responsive opsin proteins disclosed herein (for example, an
AAV1 vector) can be delivered directly to neurons of the central or
peripheral nervous system with a needle, catheter, or related
device, using neurosurgical techniques known in the art, such as by
stereotactic injection (See, e.g., Stein et al., J. Virol., 1999,
73:34243429; Davidson et al., Proc. Nat. Acad. Sci. U.S.A., 2000,
97:3428-3432; Davidson et al., Nat. Genet., 1993, 3:219-223; and
Alisky & Davidson, Hum. Gene Ther., 2000, 11:2315-2329, the
contents of each of which are hereby incorporated by reference
herein in their entireties) or fluoroscopy. In some embodiments,
the polynucleotide encoding the light-responsive opsin proteins
disclosed herein (for example, an AAV1 vector) can be delivered to
neurons of the peripheral nervous system by injection into any one
of the spinal nerves (such as the cervical spinal nerves, the
thoracic spinal nerves, the lumbar spinal nerves, the sacral spinal
nerves, and/or the coccygeal spinal nerves).
[0091] Other methods to deliver the light-responsive opsin proteins
to the nerves of interest can also be used, such as, but not
limited to, transfection with ionic lipids or polymers,
electroporation, optical transfection, impalefection, or via gene
gun.
[0092] In another aspect, the polynucleotide encoding the
light-responsive opsin proteins disclosed herein (for example, an
AAV2 vector) can be delivered directly to muscles innervated by the
neurons of the peripheral nervous system. Because of the
limitations inherent in injecting viral vectors directly into the
specific cell bodies which innvervate particular muscles,
researchers have attempted to deliver transgenes to peripheral
neurons by injecting viral vectors directly into muscle. These
experiments have shown that some viral serotypes such as
adenovirus, AAV2, and Rabies glycoprotein-pseudotyped lentivirus
can be taken up by muscle cells and retrogradely transported to
motor neurons across the neuromuscular synapse (See, e.g., Azzouz
et al., 2009, Antioxid Redox Signal., 11(7):1523-34; Kaspar et al.,
2003, Science, 301(5634):839-842; Manabe et al., 2002, Apoptosis,
7(4):329-334, the disclosures of each of which are herein
incorporated by reference in their entireties).
[0093] Accordingly, in some embodiments, the vectors expressing the
light-responsive opsin proteins disclosed herein (for example, an
AAV2 vector) can be delivered to the neurons responsible for the
innervation of muscles by direct injection into the muscle of
interest.
[0094] The lanthanide-doped nanoparticles disclosed herein can be
delivered to neurons expressing one or more light-responsive opsin
proteins by any route, such as intravascularly, intracranially,
intracerebrally, intramuscularly, intradermally, intravenously,
intraocularly, orally, nasally, topically, or by open surgical
procedure, depending upon the anatomical site or sites to which the
nanoparticles are to be delivered. The nanoparticles can
additionally be delivered by the same route used for delivery of
the polynucleotide vectors expressing the light-responsive opsin
proteins, such as any of those described above. The nanoparticles
can also be administered in an open manner, as in the heart during
open heart surgery, or in the brain during stereotactic surgery, or
by intravascular interventional methods using catheters going to
the blood supply of specific organs, or by other interventional
methods.
[0095] Pharmaceutical compositions used for the delivery and/or
storage of polynucleotides encoding the light-responsive opsin
proteins disclosed herein and/or the lanthanide-doped nanoparticles
disclosed herein can be formulated according to known methods for
preparing pharmaceutically useful compositions. Formulations are
described in a number of sources which are well known and readily
available to those skilled in the art. For example, Remington's
Pharmaceutical Sciences (Martin E W, 1995, Easton Pa., Mack
Publishing Company, 19.sup.th ed.) describes formulations which can
be used in connection with the subject invention. Formulations
suitable for parenteral administration include, for example,
aqueous sterile injection solutions, which may contain
antioxidants, buffers, bacteriostats, and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampoules and vials, and may be stored in a freeze dried
(lyophilized) condition requiring only the condition of the sterile
liquid carrier, for example, water for injections, prior to
use.
[0096] The lanthanide-doped nanoparticles may also be administered
intravenously or intraperitoneally by infusion or injection.
Solutions of the nanoparticles and/or cells can be prepared in
water, optionally mixed with a nontoxic surfactant. Dispersions can
also be prepared in glycerol, liquid polyethylene glycols,
triacetin, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0097] The pharmaceutical dosage forms suitable for injection or
infusion of the lanthanide-doped nanoparticles described herein can
include sterile aqueous solutions or dispersions or sterile powders
comprising the active ingredient which are adapted for the
extemporaneous preparation of sterile injectable or infusible
solutions or dispersions. The liquid carrier or vehicle can be a
solvent or liquid dispersion medium comprising, for example, water,
ethanol, a polyol (for example, glycerol, propylene glycol, liquid
polyethylene glycols, and the like), vegetable oils, nontoxic
glyceryl esters, and suitable mixtures thereof. The prevention of
the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
[0098] Sources of Infrared or Near Infrared Electromagnetic
Radiation
[0099] Any device that is capable of producing a source of
electromagnetic radiation having a wavelength in the infrared (IR)
or near infrared (NIR) spectrum may be used to activate one or more
light-responsive proteins expressed on the surface of a neuron in
combination with the lanthanide-doped nanoparticles described
herein. The IR or NIR source can be configured to provide optical
stimulus to a specific target region of the brain. The IR or NIR
source can additionally provide continuous IR or NIR
electromagnetic radiation and/or pulsed IR or NIR electromagnetic
radiation, and may be programmable to provide IR or NIR
electromagnetic radiation in pre-determined pulse sequences.
[0100] In other aspects, the implantable IR or NIR source does not
require physical tethering to an external power source. In some
embodiments, the power source can be an internal battery for
powering the IR or NIR source. In another embodiment, the
implantable IR or NIR source can comprise an external antenna for
receiving wirelessly transmitted electromagnetic energy from an
external power source for powering the IR or NIR source. The
wirelessly transmitted electromagnetic energy can be a radio wave,
a microwave, or any other electromagnetic energy source that can be
transmitted from an external source to power the IR or
NIR-generating source. In one embodiment, the IR or NIR source is
controlled by an integrated circuit produced using semiconductor or
other processes known in the art.
[0101] In some aspects, the implantable IR or NIR electromagnetic
radiation source can be externally activated by an external
controller. The external controller can comprise a power generator
which can be mounted to a transmitting coil. In some embodiments of
the external controller, a battery can be connected to the power
generator, for providing power thereto. A switch can be connected
to the power generator, allowing an individual to manually activate
or deactivate the power generator. In some embodiments, upon
activation of the switch, the power generator can provide power to
the IR or NIR electromagnetic radiation source through
electromagnetic coupling between the transmitting coil on the
external controller and the external antenna of the implantable IR
or NIR source. When radio-frequency magnetic inductance coupling is
used, the operational frequency of the radio wave can be between
about 1 and 20 MHz, inclusive, including any values in between
these numbers (for example, about 1 MHz, about 2 MHz, about 3 MHz,
about 4 MHz, about 5 MHz, about 6 MHz, about 7 MHz, about 8 MHz,
about 9 MHz, about 10 MHz, about 11 MHz, about 12 MHz, about 13
MHz, about 14 MHz, about 15 MHz, about 16 MHz, about 17 MHz, about
18 MHz, about 19 MHz, or about 20 MHz). However, other coupling
techniques may be used, such as an optical receiver or a biomedical
telemetry system (See, e.g., Kiourti, "Biomedical Telemetry:
Communication between Implanted Devices and the External World,
Opticon 1826, (8): Spring, 2010).
[0102] In some aspects, the intensity of the IR or NIR
electromagnetic radiation reaching the neural cells (such as neural
cells expressing one or more light-responsive opsin proteins)
produced by the IR or NW electromagnetic radiation source has an
intensity of any of about 0.05 mW/mm.sup.2, 0.1 mW/mm.sup.2, 0.2
mW/mm.sup.2, 0.3 mW/mm.sup.2, 0.4 mW/mm.sup.2, 0.5 mW/mm.sup.2,
about 0.6 mW/mm.sup.2, about 0.7 mW/mm.sup.2, about 0.8
mW/mm.sup.2, about 0.9 mW/mm.sup.2, about 1.0 mW/mm.sup.2, about
1.1 mW/mm.sup.2, about 1.2 mW/mm.sup.2, about 1.3 mW/mm.sup.2,
about 1.4 mW/mm.sup.2, about 1.5 mW/mm.sup.2, about 1.6
mW/mm.sup.2, about 1.7 mW/mm.sup.2, about 1.8 mW/mm.sup.2, about
1.9 mW/mm.sup.2, about 2.0 mW/mm.sup.2, about 2.1 mW/mm.sup.2,
about 2.2 mW/mm.sup.2, about 2.3 mW/mm.sup.2, about 2.4
mW/mm.sup.2, about 2.5 mW/mm.sup.2, about 3 mW/mm.sup.2, about 3.5
mW/mm.sup.2, about 4 mW/mm.sup.2, about 4.5 mW/mm.sup.2, about 5
mW/mm.sup.2, about 5.5 mW/mm.sup.2, about 6 mW/mm.sup.2, about 7
mW/mm.sup.2, about 8 mW/mm.sup.2, about 9 mW/mm.sup.2, or about 10
mW/mm.sup.2, inclusive, including values in between these
numbers.
[0103] In other aspects, the IR or NIR electromagnetic radiation
produced by the IR or NW electromagnetic radiation source can have
a wavelength encompassing the entire infrared spectrum, such as
from about 740 nm to about 300,000 nm. In other embodiments, the IR
or NIR electromagnetic radiation produced by the IR or NIR
electromagnetic radiation source can have a wavelength
corresponding to the NIR spectrum, such as about 740 nm to about
1400 nm. In other embodiments, NIR electromagnetic radiation
produced has a wavelength between 700 nm and 1000 nm.
[0104] In some aspects, an IR or NIR electromagnetic radiation
source is used to hyperpolarize or depolarize the plasma membranes
of neural cells (such as neural cells expressing one or more
light-responsive opsin proteins) in the brain or central nervous
system of an individual when used in combination with the
lanthanide-doped nanoparticles disclosed herein. In some
embodiments, the skull of the individual is surgically thinned in
an area adjacent to the brain region of interest without puncturing
the bone. The IR or NW electromagnetic radiation source can then be
placed directly over the thinned-skull region. In other
embodiments, the IR or NIR electromagnetic radiation generator is
implanted under the skin of the individual directly adjacent to the
thinned skull region.
[0105] In some aspects, an IR or NIR electromagnetic radiation
source is used to hyperpolarize or depolarize the plasma membranes
of neural cells (such as neural cells expressing one or more
light-responsive opsin proteins) in the peripheral nervous system
of an individual when used in combination with the lanthanide-doped
nanoparticles disclosed herein. In some embodiments, the IR or NIR
electromagnetic radiation source is surgically implanted under the
skin of the individual directly adjacent to the peripheral neural
cell of interest. In other embodiments, the IR or NIR
electromagnetic radiation source is placed against the skin
directly adjacent to the peripheral neural cell of interest. In one
embodiment, the IR or NIR electromagnetic radiation source is held
against the skin in a bracelet or cuff configuration.
[0106] Examples of the IR or NIR electromagnetic radiation sources,
particularly those small enough to be implanted under the skin, can
be found in U.S. Patent Application Publication Nos.: 2009/0143842,
2011/0152969, 2011/0144749, and 2011/0054305, the disclosures of
each of which are incorporated by reference herein in their
entireties.
[0107] In still other aspects, the lanthanide-doped nanoparticles
disclosed herein can be exposed to higher wavelength light in the
visible spectrum (such as red light) to upconvert the higher
wavelength visible light into lower wavelength visible light (such
as blue or green light). As described above, light passes through
biological tissue poorly. However, when visible light does
penetrate into tissues, it typically does so in higher wavelengths
which correspond to red light (for example, between about 620 nm to
740 nm). Accordingly, the lanthanide-doped nanoparticles disclosed
herein can additionally be used in combination with optical sources
of visible light to upshift wavelengths corresponding to red light
into wavelengths corresponding to green or blue light (for example,
between about 440 nm and 570 nm). Examples of light stimulation
devices, including light sources, can be found in International
Patent Application Nos.: PCT/US08/50628 and PCT/US09/49936 and in
Llewellyn et al., 2010, Nat. Med., 16(10):161-165, the disclosures
of each of which are hereby incorporated herein in their
entireties.
Methods of the Invention
[0108] Depolarization of Neural Cells
[0109] Provided herein are methods to depolarize the plasma
membrane of a neural cell in an individual comprising placing a
plurality of lanthanide-doped nanoparticles in proximity to the
neural cell; and exposing the plurality of nanoparticles to
electromagnetic radiation in the infrared (IR) or near infrared
(NM) spectrum, wherein the electromagnetic radiation in the IR or
NIR spectrum is upconverted into light in the visible spectrum by
the nanoparticles, and wherein a light-responsive opsin is
expressed on the plasma membrane of the neural cells and activation
of the opsin by the light in the visible spectrum induces the
depolarization of the plasma membrane.
[0110] Also provided herein is a method to depolarize the plasma
membrane of a neural cell in an individual comprising administering
a polynucleotide encoding a light-responsive opsin to a neural cell
in the brain of an individual, wherein the light-responsive protein
is expressed on the plasma membrane of the neural cell and the
opsin is capable of inducing membrane depolarization of the neural
cell when illuminated with light administering a plurality of
lanthanide-doped nanoparticles in proximity to the neural cell; and
exposing the plurality of nanoparticles to electromagnetic
radiation in the infrared (IR) or near (IR) spectrum, wherein the
electromagnetic radiation in the IR or near IR spectrum is
upconverted into light in the visible spectrum and the activation
of the opsin by the light in the visible spectrum induces the
depolarization of the plasma membrane.
[0111] In some embodiments, the light-responsive opsin protein is
ChR2, VChR1, or C1V1. In other embodiments, the light-responsive
opsin protein is selected from the group consisting of SFO, SSFO,
C1V1-E122, C1V1-E162, and C1V1-E122/E162.
[0112] The lanthanide metal can be ions or atoms from any of the
lanthanide series of elements, such as Lanthanum, Cerium,
Praseodymium, Neodymium, Promethium, Samarium, Europium,
Gadolinium, Terbium, Dysprosium, Holmium, Erbium, Thulium,
Ytterbium, or Lutetium. In other embodiments, the nanoparticles
comprise NaYF4:Yb/X/Gd, wherein X is Er, Tm, or Er/Tm.
[0113] The electromagnetic radiation in the IR or near IR spectrum
can be upconverted into light having a wavelength of about 450 nm
to about 550 nm. The light can have wavelengths corresponding to
red, yellow, amber, orange, green, or blue light. In some
embodiments, the individual is a human or a non-human animal. In
other embodiments, the neural cell is in the peripheral nervous
system. In another embodiment, the neural cell is in the central
nervous system.
[0114] Hyperpolarization of Neural Cells
[0115] Provided herein are methods to hyperpolarize the plasma
membrane of a neural cell in an individual comprising placing a
plurality of lanthanide-doped nanoparticles in proximity to the
neural cell; and exposing the plurality of nanoparticles to
electromagnetic radiation in the infrared (IR) or near infrared
(NIR) spectrum, wherein the electromagnetic radiation in the IR or
NIR spectrum is upconverted into light in the visible spectrum by
the nanoparticles, and wherein a light-responsive opsin is
expressed on the plasma membrane of the neural cells and activation
of the opsin by the light in the visible spectrum induces the
hyperpolarization of the plasma membrane.
[0116] Also provided herein is a method to hyperpolarize the plasma
membrane of a neural cell in an individual comprising administering
a polynucleotide encoding a light-responsive opsin to a neural cell
in the brain of an individual, wherein the light-responsive protein
is expressed on the plasma membrane of the neural cell and the
opsin is capable of inducing membrane depolarization of the neural
cell when illuminated with light administering a plurality of
lanthanide-doped nanoparticles in proximity to the neural cell; and
exposing the plurality of nanoparticles to electromagnetic
radiation in the infrared (IR) or near (IR) spectrum, wherein the
electromagnetic radiation in the IR or near IR spectrum is
upconverted into light in the visible spectrum and the activation
of the opsin by the light in the visible spectrum induces the
hyperpolarization of the plasma membrane.
[0117] In some embodiments, the light-responsive opsin protein is
an NpHR or a GtR3.
[0118] The lanthanide metal can be ions or atoms from any of the
lanthanide series of elements, such as Lanthanum, Cerium,
Praseodymium, Neodymium, Promethium, Samarium, Europium,
Gadolinium, Terbium, Dysprosium, Holmium, Erbium, Thulium,
Ytterbium, or Lutetium. In other embodiments, the nanoparticles
comprise NaYF4:Yb/X/Gd, wherein X is Er, Tm, or Er/Tm.
[0119] The electromagnetic radiation in the IR or near IR spectrum
can be upconverted into light having a wavelength of about 450 nm
to about 550 nm. The light can have wavelengths corresponding to
red, yellow, amber, orange, green, or blue light. In some
embodiments, the individual is a human or a non-human animal. In
other embodiments, the neural cell is in the peripheral nervous
system. In another embodiment, the neural cell is in the central
nervous system.
[0120] Kits
[0121] Also provided herein are kits comprising polynucleotides
encoding a light-responsive opsin protein (such as any of the
light-responsive opsin proteins described herein) and
lanthanide-doped nanoparticles for use in any of the methods
disclosed herein to alter the membrane polarization state of one or
more neurons of the central and/or peripheral nervous system. In
some embodiments, the kits further comprise an infrared or near
infrared electromagnetic radiation source. In other embodiments,
the kits further comprise instructions for using the
polynucleotides and lanthanide-doped nanoparticles described
herein. In still other embodiments, the lanthanide-doped
nanoparticles described herein are embedded and/or trapped in a
biocompatible material (such as any of the biocompatible materials
described above).
EXEMPLARY EMBODIMENTS
[0122] Aspects of the present disclosure may be more completely
understood in consideration of the detailed description of various
embodiments of the present disclosure that follows in connection
with the accompanying drawings. This description and the various
embodiments are presented as follows:
[0123] The embodiments and specific applications discussed herein
may be implemented in connection with one or more of the
above-described aspects, embodiments and implementations, as well
as with those shown in the figures and described below. Reference
may also be made to Wang et al., 2010, Nature, 463(7284):1061-5,
which is fully incorporated herein by reference. For further
details on light responsive molecules and/or opsins, including
methodology, devices and substances, reference may also be made to
the following background publications: U.S. Patent Publication No.
2010/0190229, entitled "System for Optical Stimulation of Target
Cells" to Zhang et al.; U.S. Patent Publication No. 2007/0261127,
entitled "System for Optical Stimulation of Target Cells" to Boyden
et al. These applications form part of the provisional patent
document and are fully-incorporated herein by reference. Consistent
with these publications, numerous opsins can be used in mammalian
cells in vivo and in vitro to provide optical stimulation and
control of target cells. For example, when ChR2 is introduced into
an electrically-excitable cell, such as a neuron, light activation
of the ChR2 channel rhodopsin can result in excitation and/or
firing of the cell. In instances when NpHR is introduced into an
electrically-excitable cell, such as a neuron, light activation of
the NpHR opsin can result in inhibition of firing of the cell.
These and other aspects of the disclosures of the above-referenced
patent applications may be useful in implementing various aspects
of the present disclosure.
[0124] In various embodiments of the present disclosure, minimally
invasive delivery of light, for example as can be useful for
manipulation of neural circuits with optogenetics, using near
infrared up-conversion nanocrystals, is achieved. This is used to
avoid the implantation of light sources within living tissues,
including, for example, a subject's brain. Mammalian tissue has a
transparency window in near infrared part of the spectrum (700-1000
nm). Accordingly, aspects of the present disclosure relate to the
use of nanoparticles for the purpose of using (near) infrared light
to deliver energy into the depth of a brain by converting the
infrared light into visible wavelengths at a site of interest.
[0125] In certain embodiments, delivering visible wavelengths at a
site of interest within the brain is achieved through a process of
optical upconversion in Lanthanide-doped nanocrystals. During
upconversion 3-4 photons are absorbed by the material which then
emits one photon with the energy .about.1.5-2 times the energy of
absorbed photons. For example NaYF4:Yb/X/Gd nanocrystals can absorb
980 nm light and emit light with spectra centered between 450-550
nm depending on the nature and relative content of dopants
(X.dbd.Er, Tm, Er/Tm). For more information regarding modifying the
light emitted from the nanoparticles, see Wang et al., Nature,
2010, 463(7284):1061-5, the disclosure of which is incorporated by
reference herein in its entirety.
[0126] In certain embodiments a single step surgery is performed to
modify a target cell population and provide nanoparticles to
convert near infrared light to visible light that stimulates the
modified target cell population. During the surgery, the surgeon
injects both an adeno-associated virus carrying an opsin gene and a
nanoparticle solution to a site of interest.
[0127] The virus is optimized to only infect the target cell
population. Similarly, the nanoparticles are functionalized with
antibodies so that the nanoparticles anchor to the target cell
population as well. In certain more specific embodiments the target
cell population is a particular neuron type. After surgery is
completed, a LED that emits near infrared light is placed on a
thinned portion of the patient's skull, underneath the skin. A
battery can also be implanted underneath the skin to power the LED.
In certain embodiments the battery has characteristics similar to
those of a pacemaker battery. A microcontroller can be used to
control the battery to deliver energy to the LED at specified
intervals, resulting in LED light pulses at specified
intervals.
[0128] Certain aspects of the present disclosure are directed to
the use of optogenetics in vivo. Optogenetics, applied in vivo,
relies on light delivery to specific neuron populations that can be
located deep within the brain. Mammalian tissue is highly
absorptive and scatters light in the visible spectrum. However,
near infrared light is able to penetrate to deep levels of the
brain without excessive absorption or scattering.
[0129] Certain aspects of the present disclosure are directed to
imbedding nanoparticles in the brain near target neurons. The
nanoparticles can be lanthanide doped-nanoparticle. Nanoparticles
doped with Lanthanides or with other dopants can be optimized with
respect to a particular opsin's activation spectra. As discussed in
more detail in Wang et al., Nature, 2010, 463(7284):1061-5, the
disclosure of which is incorporated by reference herein in its
entirety, the spectra of the light emitted from lanthanide-doped
nanocrystals can be manipulated based on which dopants are used,
and how much. Similarly, the light emitted from nanoparticles doped
with other molecules can be manipulated based on the concentration
of dopants.
[0130] The ability to provide different output spectra depending on
the doping of nanoparticles allows for a non-invasive approach to
acute neural manipulation. A light source, such as a LED can be
mounted onto a thinned skull under the skin. Depending on the
composition of nanoparticles, and the opsin delivered to the target
neurons, aspects of the present disclosure can be used for neural
excitation or silencing. Similarly, multiple neural populations may
be controlled simultaneously through the use of various dopants and
opsins in combination.
[0131] Turning to FIG. 1, a patient's head 100 is shown. A target
(neural) cell population 114 includes light responsive molecules.
These light responsive molecules can include, but are not
necessarily limited to, opsins derived from Channel rhodopsins
(e.g. ChR1 or ChR2) or Halorhodopins (NpHR). The specific molecule
can be tailored/selected based upon the desired effect on the
target cell population and/or the wavelength at which the molecules
respond to light.
[0132] Nanocrystals 110 are introduced near or at the target cell
populate. Various embodiments of the present disclosure are
directed toward methods and devices for positioning and maintaining
positioning of the nanocrystals near the target cell population.
Certain embodiments are directed toward anchoring the nanocrystals
to cells of (or near) the target cell population using
antibodies.
[0133] According to other example embodiments, a structure can be
introduced that includes the nanocrystals. For instance, a mesh
structure can be coated with the nanocrystals. The synthetic mesh
can be constructed so as to allow the dendrites and axons to pass
through the mess without allowing the entire neuron (e.g., the cell
body) to pass. One example of such a mesh has pores that are on the
order of 3-7 microns in diameter and is made from polyethylene
terephthalate. This mesh structure can be constructed with
light-responsive cells/neurons contained therein and/or be placed
near the target cell population, which includes the
light-responsive cells. Consistent with another embodiment, one or
more transparent capsules, each containing a solution of
nanocrystals, can be positioned near the target cell
populations.
[0134] Embodiments of the present disclosure are also directed
toward various optical sources of stimulation. These sources can
include, but are not limited to, external laser sources and
light-emitting didoes (LEDs). Particular aspects of the present
disclosure are directed toward the relatively low absorption and/or
scattering/diffusion caused by intervening material when the light
is at certain wavelengths (e.g., (near) infrared). Accordingly, the
light source can be externally located because of the ability to
penetrate the tissue with little loss of optical intensity or
power. Moreover, reduced diffusion can be particularly useful for
providing a relatively-high spatial-precision for the delivery of
the light. Thus, embodiments of the present disclosure are directed
toward multiple target cell populations with respective
nanocrystals that can be individually controlled using
spatially-precise optical stimulus. For instance, the nanocrystals
can be implanted in several locations within the brain. The light
source can then be aimed at a respective and particular location.
Multiple light sources can also be used for simultaneous
stimulation of a plurality of locations.
[0135] Consistent with a particular embodiment of the present
disclosure, the skull 102 has a thinned portion 106. An LED 104 is
located above the thinned portion of the skull and emits near
infrared light 108. When the IR hits nanocrystal 110, it is
absorbed. The nanocrystal emits visible light 112 in response to
absorbing the IR light 108. The visible light 112 is absorbed by
modified cell 114.
[0136] The system shown in FIG. 1 allows for delivery of light to a
target cell deep within a patient's brain tissue. The light
responsive molecule can be specifically targeted to a neural cell
type of interest. Similarly, the nanocrystals 112 are anchored to
the neural cell with antibodies chosen based on the type of neural
cell 114 being targeted.
[0137] Turning to FIG. 2, a group of neurons is illuminated with
infrared light 208 between 700-1000 nm. Target neurons 214 express
an opsin gene, allowing the neurons to be activated or inhibited,
depending on which opsin, and what wavelength of light is absorbed
by the neurons 214. The target neurons 214 can be interspersed
between other neurons 216. As shown in inset 202, target neurons
214 are coated with upconverting nanoparticles 210 that are
anchored to the neural membrane via antibodies. The nanoparticles
210 absorb IR photons and emit visible photons that are then
absorbed by opsins triggering neural activation.
[0138] The system of FIG. 2 can be used with a variety of target
neurons 214. The opsin gene 215 expressed in the target neurons 214
is modified based on the target neuron. Similarly, the antibodies
used to anchor the nanoparticles 210 to the target neuron membranes
are modified to attach to a specific membrane type. As shown in
inset 202, the nanoparticles 210 are closely linked to the target
neurons so that visible light photons emitted by the nanoparticles
210 are absorbed by the target neurons 214.
[0139] FIG. 3 depicts a system that uses multiple light sources,
consistent with an embodiment of the present disclosure. A patient
has nanoparticles located at target locations 308-312. The system
includes light sources 302-306, which can be configured to generate
light at a frequency that is upconverted by the nanoparticles
located at target locations 308-312. Although three light sources
are depicted, there can be any number of light sources. These light
sources can be external to the patient (e.g., a targeting system
that directs several light sources using mechanical positioning),
using embedded lights sources (e.g., LEDs implanted on the skull)
or combinations thereof. The target locations 308-312 include cells
that have optically-responsive membrane molecules. These
optically-responsive membrane molecules react to light at the
upconverted frequency.
[0140] Nanoparticles located at the intersection 314 of the light
from the different light sources 302-306 receive increased
intensity of optical stimulus relative to other locations,
including those locations within the path of light from a single
light source. In this manner, the light intensity of each of the
light sources can be set below a threshold level. When multiple
light sources are directed at the same location, the threshold
intensity level can be exceeded at the location. This allows for
spatial control in three-dimensions and also allows for reduced
inadvertent effects on non-targeted tissue. Consistent with one
embodiment, the threshold level can be set according to an amount
of light necessary to cause the desired effect (e.g., excitation or
inhibition) on the target cells. Consistent with other embodiments,
the threshold level can be set to avoid adverse effects on
non-targeted tissue (e.g., heating).
[0141] The use of multiple light sources can also bring about a
step-wise increase in light intensity. For instance, a disease
model could be tested by monitoring the effects of additional
stimulation caused by the increase in light intensity. The use of
independent light sources allows for relatively simple control over
temporal and spatial increases or decreases. Consistent with other
embodiments of the present disclosure, the spatial precision of the
light sources can be varied between the different light sources.
For example, a first light source can provide light that
illuminates the entire target cell location. This allows for all
cells within the population to be illuminated. A second light
source can provide light having a focal point that illuminates less
than all of the entire target cell location. The combination of the
first and second (or more) light sources can be used to provide
different levels of stimulation within the same cell
population.
[0142] Embodiments of the present disclosure relate to the use of
one or more light sources operating in a scanning mode. The light
source(s) are aimed at specific locations within a target cell
population. The effects of the stimulation can be monitored as the
light source is used to scan or otherwise move within the target
cell population. This can be particularly useful in connection with
the three-dimensional control provided by the use of multiple light
sources.
[0143] Various embodiments of the present disclosure are directed
toward the use of nanocrystals that emit light at different
wavelengths. This can be particularly useful when using multiple
opsins having different light-absorption spectrums. The
nanocrystals can be targeted toward different opsins and/or placed
in the corresponding locations. While the present disclosure is
amenable to various modifications and alternative forms, specifics
thereof have been shown by way of example in the drawings and will
be described in further detail. It should be understood that the
intention is not to limit the disclosure to the particular
embodiments and/or applications described. On the contrary, the
intention is to cover all modifications, equivalents, and
alternatives falling within the spirit and scope of the present
disclosure.
EXAMPLES
Example 1: Use of Lanthanide-Doped Nanoparticles in the Use of
Optogenetics to Hyperpolarize the Cholinergic Interneurons of the
Nucleus Accumbens
[0144] The nucleus accumbens (NAc) is a collection of neurons that
forms the main part of the ventral striatum. The NAc is thought to
play an important role in the complex mammalian behaviors
associated with reward, pleasure, laughter, addiction, aggression,
fear, and the placebo effect. Cholinergic interneurons within the
NAc constitute less than 1% of the local neural population, yet
they project throughout the NAc and provide its only known
cholinergic input. In this Example, an optogenetic approach using a
light-responsive chloride pump protein in combination with
lanthanide-doped nanoparticles is used to block action potential
firing in these cells, with both high temporal resolution and high
cell-type specificity. To express microbial opsin genes
specifically in cholinergic interneurons, a transgenic mouse line
expressing Cre recombinase is employed under the choline
acetyltransferase (ChAT) promoter. A Cre-inducible adeno-associated
virus (AAV) vector carrying a yellow-light gated third-generation
chloride pump halorhodopsin (eNpHR3.0) gene fused in-frame with
coding sequence for enhanced yellow fluorescent protein (eYFP) is
stereotactically injected.
[0145] Specifically, mice are anesthetized and then placed in a
stereotactic head apparatus. Surgeries are performed on 4-6 week
old mice and ophthalmic ointment is applied throughout to prevent
the eyes from drying. A midline scalp incision is made followed by
a craniotomy, and then AAV vector is injected with a 10 .mu.l
syringe and a 34 gauge metal needle. The injection volume and flow
rate (1 .mu.l at 0.15 .mu.l/min) are controlled by an injection
pump. Each NAc receives two injections (injection 1: AP 1.15 mm, ML
0.8 mm, DV -4.8 mm; injection 2: AP 1.15 mm, ML 0.8 mm, DV -4.2
mm). The virus injection and fiber position are chosen so that
virtually the entire shell is stimulated.
[0146] Next, before withdrawing the needle, NaYF.sub.4:Yb/Er/Gd,
nanoparticles are injected into the Nac. Concentrations of 3.4,
8.5, or 17 nmoles of NaYF4:Yb/Er/Gd, nanoparticles are used. After
injection of both the AAV vector and the lanthanide-doped
nanoparticles is complete, the needle is left in place for 5
additional minutes and then very slowly withdrawn.
[0147] Following a recovery period, the mice are again
anesthetized, the skulls of the mice are thinned and an NIR source
of electromagnetic radiation is placed adjacent to the thinned
skull-region. Simultaneous NW stimulation and extracellular
electrical recording are performed based on methods described
previously using optical stimulation (Gradinaru et al., J.
Neurosci., 27, 14231-14238 (2007)). The electrode consists of a
tungsten electrode (1 M.OMEGA.; 0.005 in; parylene insulation) with
the tip of the electrode projecting beyond the fiber by 300-500
.mu.m. The electrode is lowered through the NAc in approximately
100 .mu.m increments, and NIR-upconverted optical responses are
recorded at each increment. Signals are amplified and band-pass
filtered (300 Hz low cut-off, 10 kHz high cut-off) before
digitizing and recording to disk. At each site, 5 stimulation
repetitions are presented and saved.
[0148] The examples, which are intended to be purely exemplary of
the invention and should therefore not be considered to limit the
invention in any way, also describe and detail aspects and
embodiments of the invention discussed above. The foregoing
examples and detailed description are offered by way of
illustration and not by way of limitation. All publications, patent
applications, and patents cited in this specification are herein
incorporated by reference as if each individual publication, patent
application, or patent were specifically and individually indicated
to be incorporated by reference. In particular, all publications
cited herein are expressly incorporated herein by reference for the
purpose of describing and disclosing compositions and methodologies
which might be used in connection with the invention. Although the
foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
151273PRTNatronomonas pharaonis 1Val Thr Gln Arg Glu Leu Phe Glu
Phe Val Leu Asn Asp Pro Leu Leu1 5 10 15Ala Ser Ser Leu Tyr Ile Asn
Ile Ala Leu Ala Gly Leu Ser Ile Leu 20 25 30Leu Phe Val Phe Met Thr
Arg Gly Leu Asp Asp Pro Arg Ala Lys Leu 35 40 45Ile Ala Val Ser Thr
Ile Leu Val Pro Val Val Ser Ile Ala Ser Tyr 50 55 60Thr Gly Leu Ala
Ser Gly Leu Thr Ile Ser Val Leu Glu Met Pro Ala65 70 75 80Gly His
Phe Ala Glu Gly Ser Ser Val Met Leu Gly Gly Glu Glu Val 85 90 95Asp
Gly Val Val Thr Met Trp Gly Arg Tyr Leu Thr Trp Ala Leu Ser 100 105
110Thr Pro Met Ile Leu Leu Ala Leu Gly Leu Leu Ala Gly Ser Asn Ala
115 120 125Thr Lys Leu Phe Thr Ala Ile Thr Phe Asp Ile Ala Met Cys
Val Thr 130 135 140Gly Leu Ala Ala Ala Leu Thr Thr Ser Ser His Leu
Met Arg Trp Phe145 150 155 160Trp Tyr Ala Ile Ser Cys Ala Cys Phe
Leu Val Val Leu Tyr Ile Leu 165 170 175Leu Val Glu Trp Ala Gln Asp
Ala Lys Ala Ala Gly Thr Ala Asp Met 180 185 190Phe Asn Thr Leu Lys
Leu Leu Thr Val Val Met Trp Leu Gly Tyr Pro 195 200 205Ile Val Trp
Ala Leu Gly Val Glu Gly Ile Ala Val Leu Pro Val Gly 210 215 220Val
Thr Ser Trp Gly Tyr Ser Phe Leu Asp Ile Val Ala Lys Tyr Ile225 230
235 240Phe Ala Phe Leu Leu Leu Asn Tyr Leu Thr Ser Asn Glu Ser Val
Val 245 250 255Ser Gly Ser Ile Leu Asp Val Pro Ser Ala Ser Gly Thr
Pro Ala Asp 260 265 270Asp2559PRTArtificial SequenceSynthetic
polypeptide 2Met Thr Glu Thr Leu Pro Pro Val Thr Glu Ser Ala Val
Ala Leu Gln1 5 10 15Ala Glu Val Thr Gln Arg Glu Leu Phe Glu Phe Val
Leu Asn Asp Pro 20 25 30Leu Leu Ala Ser Ser Leu Tyr Ile Asn Ile Ala
Leu Ala Gly Leu Ser 35 40 45Ile Leu Leu Phe Val Phe Met Thr Arg Gly
Leu Asp Asp Pro Arg Ala 50 55 60Lys Leu Ile Ala Val Ser Thr Ile Leu
Val Pro Val Val Ser Ile Ala65 70 75 80Ser Tyr Thr Gly Leu Ala Ser
Gly Leu Thr Ile Ser Val Leu Glu Met 85 90 95Pro Ala Gly His Phe Ala
Glu Gly Ser Ser Val Met Leu Gly Gly Glu 100 105 110Glu Val Asp Gly
Val Val Thr Met Trp Gly Arg Tyr Leu Thr Trp Ala 115 120 125Leu Ser
Thr Pro Met Ile Leu Leu Ala Leu Gly Leu Leu Ala Gly Ser 130 135
140Asn Ala Thr Lys Leu Phe Thr Ala Ile Thr Phe Asp Ile Ala Met
Cys145 150 155 160Val Thr Gly Leu Ala Ala Ala Leu Thr Thr Ser Ser
His Leu Met Arg 165 170 175Trp Phe Trp Tyr Ala Ile Ser Cys Ala Cys
Phe Leu Val Val Leu Tyr 180 185 190Ile Leu Leu Val Glu Trp Ala Gln
Asp Ala Lys Ala Ala Gly Thr Ala 195 200 205Asp Met Phe Asn Thr Leu
Lys Leu Leu Thr Val Val Met Trp Leu Gly 210 215 220Tyr Pro Ile Val
Trp Ala Leu Gly Val Glu Gly Ile Ala Val Leu Pro225 230 235 240Val
Gly Val Thr Ser Trp Gly Tyr Ser Phe Leu Asp Ile Val Ala Lys 245 250
255Tyr Ile Phe Ala Phe Leu Leu Leu Asn Tyr Leu Thr Ser Asn Glu Ser
260 265 270Val Val Ser Gly Ser Ile Leu Asp Val Pro Ser Ala Ser Gly
Thr Pro 275 280 285Ala Asp Asp Ala Ala Ala Lys Ser Arg Ile Thr Ser
Glu Gly Glu Tyr 290 295 300Ile Pro Leu Asp Gln Ile Asp Ile Asn Val
Val Ser Lys Gly Glu Glu305 310 315 320Leu Phe Thr Gly Val Val Pro
Ile Leu Val Glu Leu Asp Gly Asp Val 325 330 335Asn Gly His Lys Phe
Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr 340 345 350Tyr Gly Lys
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro 355 360 365Val
Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Leu Gln Cys 370 375
380Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
Ser385 390 395 400Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
Phe Phe Lys Asp 405 410 415Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
Lys Phe Glu Gly Asp Thr 420 425 430Leu Val Asn Arg Ile Glu Leu Lys
Gly Ile Asp Phe Lys Glu Asp Gly 435 440 445Asn Ile Leu Gly His Lys
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val 450 455 460Tyr Ile Met Ala
Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys465 470 475 480Ile
Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr 485 490
495Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
500 505 510His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn
Glu Lys 515 520 525Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala
Ala Gly Ile Thr 530 535 540Leu Gly Met Asp Glu Leu Tyr Lys Phe Cys
Tyr Glu Asn Glu Val545 550 5553542PRTArtificial SequenceSynthetic
polypeptide 3Met Val Thr Gln Arg Glu Leu Phe Glu Phe Val Leu Asn
Asp Pro Leu1 5 10 15Leu Ala Ser Ser Leu Tyr Ile Asn Ile Ala Leu Ala
Gly Leu Ser Ile 20 25 30Leu Leu Phe Val Phe Met Thr Arg Gly Leu Asp
Asp Pro Arg Ala Lys 35 40 45Leu Ile Ala Val Ser Thr Ile Leu Val Pro
Val Val Ser Ile Ala Ser 50 55 60Tyr Thr Gly Leu Ala Ser Gly Leu Thr
Ile Ser Val Leu Glu Met Pro65 70 75 80Ala Gly His Phe Ala Glu Gly
Ser Ser Val Met Leu Gly Gly Glu Glu 85 90 95Val Asp Gly Val Val Thr
Met Trp Gly Arg Tyr Leu Thr Trp Ala Leu 100 105 110Ser Thr Pro Met
Ile Leu Leu Ala Leu Gly Leu Leu Ala Gly Ser Asn 115 120 125Ala Thr
Lys Leu Phe Thr Ala Ile Thr Phe Asp Ile Ala Met Cys Val 130 135
140Thr Gly Leu Ala Ala Ala Leu Thr Thr Ser Ser His Leu Met Arg
Trp145 150 155 160Phe Trp Tyr Ala Ile Ser Cys Ala Cys Phe Leu Val
Val Leu Tyr Ile 165 170 175Leu Leu Val Glu Trp Ala Gln Asp Ala Lys
Ala Ala Gly Thr Ala Asp 180 185 190Met Phe Asn Thr Leu Lys Leu Leu
Thr Val Val Met Trp Leu Gly Tyr 195 200 205Pro Ile Val Trp Ala Leu
Gly Val Glu Gly Ile Ala Val Leu Pro Val 210 215 220Gly Val Thr Ser
Trp Gly Tyr Ser Phe Leu Asp Ile Val Ala Lys Tyr225 230 235 240Ile
Phe Ala Phe Leu Leu Leu Asn Tyr Leu Thr Ser Asn Glu Ser Val 245 250
255Val Ser Gly Ser Ile Leu Asp Val Pro Ser Ala Ser Gly Thr Pro Ala
260 265 270Asp Asp Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu
Tyr Ile 275 280 285Pro Leu Asp Gln Ile Asp Ile Asn Val Val Ser Lys
Gly Glu Glu Leu 290 295 300Phe Thr Gly Val Val Pro Ile Leu Val Glu
Leu Asp Gly Asp Val Asn305 310 315 320Gly His Lys Phe Ser Val Ser
Gly Glu Gly Glu Gly Asp Ala Thr Tyr 325 330 335Gly Lys Leu Thr Leu
Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 340 345 350Pro Trp Pro
Thr Leu Val Thr Thr Phe Gly Tyr Gly Leu Gln Cys Phe 355 360 365Ala
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 370 375
380Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
Asp385 390 395 400Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
Gly Asp Thr Leu 405 410 415Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
Phe Lys Glu Asp Gly Asn 420 425 430Ile Leu Gly His Lys Leu Glu Tyr
Asn Tyr Asn Ser His Asn Val Tyr 435 440 445Ile Met Ala Asp Lys Gln
Lys Asn Gly Ile Lys Val Asn Phe Lys Ile 450 455 460Arg His Asn Ile
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln465 470 475 480Gln
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 485 490
495Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg
500 505 510Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
Thr Leu 515 520 525Gly Met Asp Glu Leu Tyr Lys Phe Cys Tyr Glu Asn
Glu Val 530 535 5404223PRTGuillardia theta 4Ala Ser Ser Phe Gly Lys
Ala Leu Leu Glu Phe Val Phe Ile Val Phe1 5 10 15Ala Cys Ile Thr Leu
Leu Leu Gly Ile Asn Ala Ala Lys Ser Lys Ala 20 25 30Ala Ser Arg Val
Leu Phe Pro Ala Thr Phe Val Thr Gly Ile Ala Ser 35 40 45Ile Ala Tyr
Phe Ser Met Ala Ser Gly Gly Gly Trp Val Ile Ala Pro 50 55 60Asp Cys
Arg Gln Leu Phe Val Ala Arg Tyr Leu Asp Trp Leu Ile Thr65 70 75
80Thr Pro Leu Leu Leu Ile Asp Leu Gly Leu Val Ala Gly Val Ser Arg
85 90 95Trp Asp Ile Met Ala Leu Cys Leu Ser Asp Val Leu Met Ile Ala
Thr 100 105 110Gly Ala Phe Gly Ser Leu Thr Val Gly Asn Val Lys Trp
Val Trp Trp 115 120 125Phe Phe Gly Met Cys Trp Phe Leu His Ile Ile
Phe Ala Leu Gly Lys 130 135 140Ser Trp Ala Glu Ala Ala Lys Ala Lys
Gly Gly Asp Ser Ala Ser Val145 150 155 160Tyr Ser Lys Ile Ala Gly
Ile Thr Val Ile Thr Trp Phe Cys Tyr Pro 165 170 175Val Val Trp Val
Phe Ala Glu Gly Phe Gly Asn Phe Ser Val Thr Phe 180 185 190Glu Val
Leu Ile Tyr Gly Val Leu Asp Val Ile Ser Lys Ala Val Phe 195 200
205Gly Leu Ile Leu Met Ser Gly Ala Ala Thr Gly Tyr Glu Ser Ile 210
215 2205310PRTChlamydomonas reinhardtii 5Met Asp Tyr Gly Gly Ala
Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro Val
Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr Cys
Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr Ala
Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu Leu
Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75
80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu
85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala
Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu
Leu Thr Cys 115 120 125Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr
Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu
Val Ser Asp Ile Gly Thr Ile145 150 155 160Val Trp Gly Ala Thr Ser
Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu
Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys
Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200
205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly
210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly
Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile
Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His
Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile His Gly
Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu
Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly
Ala Val Pro305 3106310PRTArtificial SequenceSynthetic polypeptide
6Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5
10 15Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu
Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn
Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly
Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys
Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu
Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro
Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu
Arg Tyr Ala Glu Trp Leu Leu Thr Ser 115 120 125Pro Val Ile Leu Ile
His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg
Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155
160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile
165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe
His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro
Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu
Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu
Gly Pro Glu Gly Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser
Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys
Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu
His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280
285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala
290 295 300Glu Ala Gly Ala Val Pro305 3107310PRTArtificial
SequenceSynthetic polypeptide 7Met Asp Tyr Gly Gly Ala Leu Ser Ala
Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro Val Val Val Asn
Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp
Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr Ala Ser Asn Val
Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu Leu Leu Met Phe
Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75 80Trp Glu Glu
Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu 85 90 95Glu Phe
Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala Thr 100 105
110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu Leu Thr Ser
115 120 125Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr Gly Leu Ser
Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu Val Ser Ala
Ile Gly Thr Ile145 150 155 160Val Trp Gly Ala Thr Ser Ala Met Ala
Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu Gly Leu Cys
Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys Ala Tyr Ile
Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200 205Arg Gln Val
Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly 210 215 220Met
Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly
Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His
Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu
Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile
His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly
Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu
Ala Gly Ala Val Pro305 3108344PRTArtificial SequenceSynthetic
polypeptide 8Met Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala
Val Ala Leu1 5 10 15Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp
Ala Thr Val Pro 20 25 30Val Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe
His Arg Ala His Glu 35 40 45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu
Glu Asn Asn Gly Ser Val 50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys
Phe Cys Leu Ala Trp Leu Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu
Lys Leu Ala Ala Asn Ile Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser
Ala Leu Cys Leu Met Phe Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser
Thr Cys Gly Trp Glu Glu Ile Tyr Val Ala Thr Ile 115 120 125Glu Met
Ile Lys Phe Ile Ile Glu Tyr Phe His Glu Phe Asp Glu Pro 130 135
140Ala Val Ile Tyr Ser Ser Asn Gly Asn Lys Thr Val Trp Leu Arg
Tyr145 150 155 160Ala Glu Trp Leu Leu Thr Cys Pro Val Leu Leu Ile
His Leu Ser Asn 165 170 175Leu Thr Gly Leu Lys Asp Asp Tyr Ser Lys
Arg Thr Met Gly Leu Leu 180 185 190Val Ser Asp Val Gly Cys Ile Val
Trp Gly Ala Thr Ser Ala Met Cys 195 200 205Thr Gly Trp Thr Lys Ile
Leu Phe Phe Leu Ile Ser Leu Ser Tyr Gly 210 215 220Met Tyr Thr Tyr
Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe His225 230 235 240Thr
Val Pro Lys Gly Ile Cys Arg Glu Leu Val Arg Val Met Ala Trp 245 250
255Thr Phe Phe Val Ala Trp Gly Met Phe Pro Val Leu Phe Leu Leu Gly
260 265 270Thr Glu Gly Phe Gly His Ile Ser Pro Tyr Gly Ser Ala Ile
Gly His 275 280 285Ser Ile Leu Asp Leu Ile Ala Lys Asn Met Trp Gly
Val Leu Gly Asn 290 295 300Tyr Leu Arg Val Lys Ile His Glu His Ile
Leu Leu Tyr Gly Asp Ile305 310 315 320Arg Lys Lys Gln Lys Ile Thr
Ile Ala Gly Gln Glu Met Glu Val Glu 325 330 335Thr Leu Val Ala Glu
Glu Glu Asp 3409344PRTArtificial SequenceSynthetic polypeptide 9Met
Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10
15Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro
20 25 30Val Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His
Glu 35 40 45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly
Ser Val 50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala
Trp Leu Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala
Asn Ile Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu
Met Phe Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp
Glu Thr Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile
Ile Glu Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr
Ser Ser Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala
Glu Trp Leu Leu Thr Cys Pro Val Leu Leu Ile His Leu Ser Asn 165 170
175Leu Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu
180 185 190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala
Met Cys 195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser
Leu Ser Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val
Tyr Ile Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys
Arg Glu Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala
Trp Gly Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly
Phe Gly His Ile Ser Pro Tyr Gly Ser Ala Ile Gly His 275 280 285Ser
Ile Leu Asp Leu Ile Ala Lys Asn Met Trp Gly Val Leu Gly Asn 290 295
300Tyr Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp
Ile305 310 315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu
Met Glu Val Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp
34010344PRTArtificial SequenceSynthetic polypeptide 10Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu
Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala Thr Trp
Leu Leu Thr Cys Pro Val Leu Leu Ile His Leu Ser Asn 165 170 175Leu
Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys
195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser
Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile
Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys Arg Glu
Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala Trp Gly
Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly Phe Gly
His Ile Ser Pro Tyr Gly Ser Ala Ile Gly His 275 280 285Ser Ile Leu
Asp Leu Ile Ala Lys Asn Met Trp Gly Val Leu Gly Asn 290 295 300Tyr
Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp Ile305 310
315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val
Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp
34011344PRTArtificial SequenceSynthetic polypeptide 11Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Thr
Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala Thr Trp
Leu Leu Thr Cys Pro Val Leu Leu Ile His Leu Ser Asn 165 170 175Leu
Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys
195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser
Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile
Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys Arg Glu
Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala Trp Gly
Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly Phe Gly
His Ile Ser Pro Tyr Gly Ser Ala Ile Gly His 275 280 285Ser Ile Leu
Asp Leu Ile Ala Lys Asn Met Trp Gly Val Leu Gly Asn 290 295 300Tyr
Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp Ile305 310
315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val
Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp 3401220PRTArtificial
SequenceSynthetic peptide 12Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr
Ile Pro Leu Asp Gln Ile1 5 10 15Asp Ile Asn Val 20136PRTArtificial
SequenceSynthetic peptideVARIANT(2)...(2)x = any amino acid 13Phe
Xaa Tyr Glu Asn Glu1 5147PRTArtificial SequenceSynthetic peptide
14Phe Cys Tyr Glu Asn Glu Val1 51518PRTArtificial SequenceSynthetic
peptide 15Met Thr Glu Thr Leu Pro Pro Val Thr Glu Ser Ala Val Ala
Leu Gln1 5 10 15Ala Glu
* * * * *