U.S. patent application number 12/993092 was filed with the patent office on 2012-04-19 for vectors for delivery of light sensitive proteins and methods of use.
Invention is credited to Edward S. Boyden, III, William Hauswirth, Alan Horsager, Jianwen Liu, Benjamin Matteo.
Application Number | 20120093772 12/993092 |
Document ID | / |
Family ID | 41570800 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120093772 |
Kind Code |
A1 |
Horsager; Alan ; et
al. |
April 19, 2012 |
VECTORS FOR DELIVERY OF LIGHT SENSITIVE PROTEINS AND METHODS OF
USE
Abstract
Provided herein are compositions and methods for gene and
etiology-nonspecific and circuit-specific treatment of diseases,
utilizing vectors for delivery of light-sensitive proteins to
diseased and normal cells and tissues of interest.
Inventors: |
Horsager; Alan; (Los
Angeles, CA) ; Hauswirth; William; (Gainesville,
FL) ; Liu; Jianwen; (Gainesville, FL) ;
Matteo; Benjamin; (San Francisco, CA) ; Boyden, III;
Edward S.; (Chestnut Hill, MA) |
Family ID: |
41570800 |
Appl. No.: |
12/993092 |
Filed: |
May 20, 2009 |
PCT Filed: |
May 20, 2009 |
PCT NO: |
PCT/US09/44753 |
371 Date: |
June 10, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61054571 |
May 20, 2008 |
|
|
|
61199241 |
Nov 14, 2008 |
|
|
|
61200430 |
Nov 26, 2008 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
435/320.1; 435/455; 435/456; 604/20 |
Current CPC
Class: |
C07K 14/70571 20130101;
C12N 2710/10043 20130101; A61K 45/06 20130101; C12N 2830/008
20130101; A61K 48/005 20130101; C07K 14/723 20130101; C12N 15/86
20130101; A61P 27/00 20180101; C12N 2799/025 20130101; A61P 27/02
20180101; A61K 48/0058 20130101 |
Class at
Publication: |
424/93.2 ;
435/320.1; 435/455; 435/456; 604/20 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/867 20060101 C12N015/867; A61M 37/00 20060101
A61M037/00; C12N 15/86 20060101 C12N015/86; A61P 27/02 20060101
A61P027/02; C12N 15/85 20060101 C12N015/85; C12N 15/863 20060101
C12N015/863 |
Claims
1. A recombinant nucleic acid comprising a nucleic acid encoding a
light-sensitive protein operatively linked to a metabotropic
glutamate receptor 6 (mGluR6) regulatory sequence or fragment
thereof.
2. The nucleic acid of claim 1 wherein the light-sensitive protein
is selected from the group consisting of ChR1, ChR2, VChR1, ChR2
C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD,
ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants
thereof.
3. The nucleic acid of claim 1 wherein the light-sensitive protein
is ChR2 or a light-sensitive protein that is at least about 70%
identical to ChR2.
4. The nucleic acid of claim 1 wherein the mGluR6 regulatory
sequence fragment comprises less than about 1000 base pairs.
5. The nucleic acid of claim 1 wherein the mGluR6 regulatory
sequence or fragment thereof is an mGluR6 promoter or enhancer.
6. The nucleic acid of claim 1 further comprising a green
fluorescent protein.
7. The nucleic acid of claim 1 wherein the nucleic acid is
encapsidated within a recombinant adeno-associated virus (AAV).
8. The nucleic acid of claim 7 wherein the recombinant
adeno-associated virus is of a serotype selected from the group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAV12, and hybrids thereof.
9. The nucleic acid of claim 7 wherein the recombinant
adeno-associated virus is of a serotype selected from the group
consisting of AAV2, AAV5, AAV7, AAV8, and hybrids thereof.
10. The nucleic acid of claim 1 wherein the nucleic acid is
encapsidated within a recombinant virus selected from the group
consisting of recombinant adeno-associated virus (AAV), recombinant
retrovirus, recombinant lentivirus, and recombinant poxvirus.
11. A vector comprising a nucleic acid encoding a light-sensitive
protein, said nucleic acid operatively linked to a metabotropic
glutamate receptor 6 (mGluR6) regulatory sequence or fragment
thereof.
12-22. (canceled)
23. A method of treating a subject suffering from a disease or
disorder of the eye comprising introducing into an affected eye a
recombinant adeno-associated virus (AAV) comprising a
light-sensitive protein operatively linked to a metabotropic
glutamate receptor 6 regulatory sequence (mGluR6 regulatory
sequence) or fragment thereof.
24. The method of claim 23 wherein the disease or disorder of the
eye is caused by photoreceptor cell degeneration.
25. (canceled)
26. The method of claim 23 wherein the light-sensitive protein is
ChR2 or a light-sensitive protein that is at least about 70%
identical to ChR2.
27. The method of claim 23 wherein the mGluR6 regulatory sequence
fragment is less than about 1000 base pairs.
28. The method of claim 27 wherein the mGluR6 regulatory sequence
fragment is represented by the sequence in FIG. 6.
29. The method of claim 23 wherein the AAV is of a serotype
selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids
thereof.
30. The method of claim 29 wherein the AAV comprises a mutated
capsid protein.
31. (canceled)
32. The method of claim 31 wherein the mutated tyrosine residue is
selected from the group consisting of Y252F, Y272F, Y444F, Y500F,
Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and
Y720F.
33. (canceled)
34. The method of claim 23 wherein the AAV is introduced using
intravitreal injection, subretinal injection and/or ILM peel.
35. The method of claim 23 wherein the AAV is introduced into a
retinal bipolar cell.
36. The method of claim 23 wherein the method further comprises
using a light-generating device external to the eye.
37. A method of expressing an exogenous nucleic acid in a retinal
bipolar cell comprising introducing into a retina a vector
comprising the exogenous nucleic operatively linked to a retinal
bipolar cell-specific regulatory sequence wherein the method
results in at least about a 10% transduction efficiency.
38. (canceled)
39. (canceled)
40. The method of claim 37 wherein the exogenous nucleic acid
comprises a light-sensitive protein.
41. (canceled)
42. The nucleic acid of claim 40 wherein the light-sensitive
protein is ChR2 or a light-sensitive protein that is at least about
70% identical to ChR2.
43. The method of claim 37 wherein the regulatory sequence
comprises a metabotropic glutamate receptor 6 regulatory sequence
(mGluR6) or a fragment thereof.
44. The method of claim 43 wherein the mGluR6 regulatory sequence
fragment is less than about 1000 base pairs.
45. (canceled)
46. The method of claim 36 wherein the exogenous nucleic acid is
introduced using a recombinant adeno-associated viral vector
(AAV).
47. (canceled)
48. The method of claim 47 wherein the capsid protein comprises a
mutated tyrosine residue.
49. The method of claim 48 wherein the mutated tyrosine residue is
selected from the group consisting of Y252F, Y272F, Y444F, Y500F,
Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and
Y720F.
50. (canceled)
51. The method of claim 46 wherein the exogenous nucleic acid is
introduced using intravitreal injection, subretinal injection,
and/or ILM peel.
52. The method of claim 46 wherein the AAV is of a serotype is
selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids
thereof.
53. A method of introducing an exogenous nucleic acid into the
nucleus of a retinal cell comprising introducing a vector
comprising an exogenous nucleic acid operatively linked to a
retinal cell-specific regulatory sequence into a retinal cell,
wherein the vector is specifically designed to avoid
ubiquitin-mediated protein degradation.
54. (canceled)
55. The method of claim 53 wherein the exogenous nucleic acid
comprises a light-sensitive protein.
56. (canceled)
57. The nucleic acid of claim 55 wherein the light-sensitive
protein is ChR2 or a light-sensitive protein that is at least about
70% identical to ChR2.
58. The method of claim 53 wherein the retinal cell is a retinal
bipolar cell.
59. The method of claim 58 wherein the regulatory sequence
comprises a metabotropic glutamate receptor 6 regulatory sequence
(mGluR6) or fragment thereof.
60. The method of claim 59 wherein the mGluR6 fragment is less than
1000 base pairs.
61. The method of claim 59 wherein the mGluR6 regulatory sequence
fragment is represented by the sequence in FIG. 6.
62. (canceled)
63. The method of claim 53 the vector is a recombinant
adeno-associated viral vector (AAV).
64. The method of claim 63 wherein the AAV is of a serotype
selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids
thereof.
65. The method of claim 63 wherein the AAV comprises a mutated
capsid protein.
66. The method of claim 65 wherein the capsid protein comprises a
mutated tyrosine residue.
67. The method of claim 66 wherein the mutated tyrosine residue is
selected from the group consisting of Y252F, Y272F, Y444F, Y500F,
Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and
Y720F.
68. (canceled)
69. The method of claim 53 wherein the vector is introduced using
intravitreal injection, subretinal injection, and/or ILM peel.
70. A method of transducing a retinal bipolar cell comprising
introducing into a retina a vector comprising an exogenous nucleic
acid operatively linked to a regulatory sequence.
71-83. (canceled)
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 61/054,571 filed May 20, 2008, 61/199,241 filed
Nov. 14, 2008, and 61/200,430 filed Nov. 26, 2008, which
applications are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] A gene-delivery therapy to treat a disease or disorder
independent of treating an underlying mutation could have potential
value. Methods capable of controlling, regulating, and/or driving
specific neural circuits so as to mediate naturalistic neural
responses and high resolution perception and control could also be
of enormous potential therapeutic value. Neurons are an example of
a type of cell that uses the electrical currents created by
depolarization to generate communication signals (e.g., nerve
impulses). Other electrically excitable cells include skeletal
muscle, cardiac muscle, and endocrine cells. Neurons use rapid
depolarization to transmit signals throughout the body and for
various purposes, such as motor control (e.g., muscle
contractions), sensory responses (e.g., touch, hearing, and other
senses) and computational functions (e.g., brain functions). By
facilitating or inhibiting the flow of positive or negative ions
through cell membranes, the cell can be briefly depolarized,
depolarized and maintained in that state, or hyperpolarized. Thus,
the control of the depolarization of cells can be beneficial for a
number of different purposes, including visual, muscular and
sensory control. Light-sensitive protein channels, pumps, and
receptors can permit millisecond-precision optical control of
cells. Although light-sensitive proteins in combination with light
can be used to control the flow of ions through cell membranes,
targeting and delivery remain to be addressed for specific
diseases, disorders, and circuits.
SUMMARY OF THE INVENTION
[0003] In one aspect, the invention provides a recombinant nucleic
acid comprising a nucleic acid encoding a light-sensitive protein
operatively linked to a metabotropic glutamate receptor 6 (mGluR6)
regulatory sequence or fragment thereof. In one embodiment the
light-sensitive protein can be selected from the group consisting
of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof. In another embodiment the light-sensitive
protein is ChR2 or a light-sensitive protein that is at least about
70%, at least about 80%, at least about 90% or at least about 95%
identical to ChR2. In another embodiment the mGluR6 regulatory
sequence fragment comprises less than about 1000, less than about
750, less than about 500, less than about 250, or less than about
100 base pairs. In a related embodiment the mGluR6 regulatory
sequence or fragment thereof is a mGluR6 promoter or enhancer. In a
specific related embodiment the nucleic acid further comprises a
green fluorescent protein. In another embodiment the nucleic acid
is encapsidated within a recombinant adeno-associated virus (AAV).
In certain embodiments, the recombinant AAV is of a combinatorial
hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more
serotypes or mutants thereof. In certain embodiments, the
recombinant adeno-associated virus is of a serotype selected from
the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In a related
embodiment, the nucleic acid is encapsidated within a recombinant
virus selected from the group consisting of recombinant
adeno-associated virus (AAV), recombinant retrovirus, recombinant
lentivirus, and recombinant poxvirus.
[0004] In another aspect the invention provides a vector comprising
a nucleic acid encoding a light-sensitive protein, said nucleic
acid operatively linked to a metabotropic glutamate receptor 6
(mGluR6) regulatory sequence or fragment thereof. In one embodiment
the light-sensitive protein is selected from the group consisting
of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof. In a relate embodiment the light-sensitive
protein is ChR2 or a light-sensitive protein that is at least about
70%, at least about 80%, at least about 90% or at least about 95%
identical to ChR2. In another embodiment the mGluR6 regulatory
sequence fragment is less than about 1000, less than about 750,
less than about 500, less than about 250, or less than about 100
base pairs. In a related embodiment the mGluR6 regulatory sequence
fragment is represented by the sequence in FIG. 6. In another
embodiment the vector comprises a recombinant adeno-associated
virus (AAV). In a related embodiment the vector comprises a
recombinant virus selected from the group consisting of recombinant
adeno-associated virus (AAV), recombinant retrovirus, recombinant
lentivirus, and recombinant poxvirus. In a specific embodiment the
AAV is of a serotype selected from the group consisting of AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,
AAV12, and hybrids thereof. In other specific embodiments, the
recombinant AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutants thereof.
In a related embodiment the AAV comprises mutated capsid protein.
In one specific embodiment the capsid protein comprises a mutated
tyrosine residue. The mutated tyrosine residue can be selected from
the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F,
Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. In a
specific embodiment the mutated capsid protein comprises a tyrosine
residue mutated to a phenylalanine.
[0005] In another aspect the present invention provides a method of
treating a subject suffering from a disease or disorder of the eye
comprising introducing into an affected eye a recombinant
adeno-associated virus (AAV) comprising a light-sensitive protein
operatively linked to a metabotropic glutamate receptor 6
regulatory sequence (mGluR6) or fragment thereof. In one embodiment
the disease or disorder of the eye is caused by photoreceptor cell
degeneration. In another embodiment the light-sensitive protein is
selected from the group consisting of ChR1, ChR2, VChR1, ChR2
C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD,
ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. In
a related embodiment the light-sensitive protein is ChR2 or a
light-sensitive protein that is at least about 70%, at least about
80%, at least about 90% or at least about 95% identical to ChR2. In
another embodiment the mGluR6 promoter fragment is less than about
1000, less than about 750, less than about 500, less than about
250, or less than about 100 base pairs. In a related embodiment the
mGluR6 promoter fragment is represented by the sequence in FIG. 6.
In another embodiment the AAV is of a serotype selected from the
group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAV12, and hybrids thereof. In other
embodiments, the recombinant AAV is of a combinatorial hybrid of 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or
mutants thereof. In a related embodiment the AAV comprises a
mutated capsid protein. In another related embodiment the capsid
protein comprises a mutated tyrosine residue. In a specific
embodiment the mutated tyrosine residue is selected from the group
consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F,
Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. In a related
embodiment the mutated capsid protein comprises a tyrosine residue
mutated to a phenylalanine. In another embodiment the AAV is
introduced using intravitreal injection, subretinal injection
and/or ILM peel. In a specific embodiment the AAV is introduced
into a retinal bipolar cell (e.g. ON or OFF retinal bipolar cells;
rod and cone bipolar cells). In another embodiment the method
further comprises using a light-generating device external to the
eye.
[0006] In another aspect the present invention provides a method of
expressing an exogenous nucleic acid in a retinal bipolar cell
(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells)
comprising introducing into a retina a vector comprising the
exogenous nucleic operatively linked to a retinal bipolar (e.g. ON
or OFF retinal bipolar cells; rod and cone bipolar cells)
cell-specific regulatory sequence wherein the method results in at
least about a 25-30% transduction efficiency. In other embodiment,
such method results in at least about a 10% transduction
efficiency. In one embodiment the method results in at least about
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% transduction efficiency.
In a related embodiment the transduction efficiency is measured by
quantifying the total number of retinal bipolar cells (e.g. ON or
OFF retinal bipolar cells; rod and cone bipolar cells) infected. In
another embodiment the exogenous nucleic acid comprises a
light-sensitive protein. In a related embodiment the
light-sensitive protein is selected from the group consisting of
ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof. In a specific embodiment the light-sensitive
protein is ChR2 or a light-sensitive protein that is at least about
70%, at least about 80%, at least about 90% or at least about 95%
identical to ChR2. In another embodiment the regulatory sequence
comprises a metabotropic glutamate receptor 6 regulatory sequence
(mGluR6) or a fragment thereof. In a related embodiment the mGluR6
regulatory sequence fragment is less than about 1000, less than
about 750, less than about 500, less than about 250, or less than
about 100 base pairs. In a specific embodiment the mGluR6
regulatory sequence fragment is represented by the sequence in FIG.
6. In another embodiment the exogenous nucleic acid is introduced
using a recombinant adeno-associated viral vector (AAV). In a
related embodiment the AAV comprises a mutated capsid protein. In a
specific embodiment the capsid protein comprises a mutated tyrosine
residue. In a related embodiment the mutated tyrosine residue is
selected from the group consisting of Y252F, Y272F, Y444F, Y500F,
Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and
Y720F. In another embodiment the mutated capsid protein comprises a
tyrosine residue mutated to a phenylalanine. In another embodiment
the exogenous nucleic acid is introduced using intravitreal
injection, subretinal injection, and/or ILM peel. In another
embodiment the AAV is of a serotype is selected from the group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAV12, and hybrids thereof. In yet another
embodiments, the recombinant AAV is of a combinatorial hybrid of 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or
mutants thereof.
[0007] In yet another aspect, the present invention provides a
method of introducing an exogenous nucleic acid into the nucleus of
a retinal cell comprising introducing a vector comprising an
exogenous nucleic acid operatively linked to a retinal
cell-specific regulatory sequence into a retinal cell, wherein the
vector is specifically designed to avoid ubiquitin-mediated protein
degradation. In one embodiment the degradation is
proteasome-mediated. In another embodiment the exogenous nucleic
acid comprises a light-sensitive protein. In a related embodiment
the light-sensitive protein is selected from the group consisting
of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof. In another related embodiment the
light-sensitive protein is ChR2 or a light-sensitive protein that
is at least about 70%, at least about 80%, at least about 90% or at
least about 95% identical to ChR2. In another embodiment the
retinal cell is a retinal bipolar cell (e.g. ON or OFF retinal
bipolar cells; rod and cone bipolar cells). In a related embodiment
the regulatory sequence comprises a metabotropic glutamate receptor
6 promoter (mGluR6 promoter) or fragment thereof. In another
embodiment the mGluR6 fragment is less than 1000, 750, 500, 250, or
100 base pairs. In another embodiment the mGluR6 promoter fragment
is represented by the sequence in FIG. 6. In another embodiment the
vector is selected from the group consisting of recombinant
adeno-associated virus (AAV), recombinant retrovirus, recombinant
lentivirus, and recombinant poxvirus. In a related embodiment the
vector is a recombinant adeno-associated viral vector (AAV). In
another embodiment the AAV is of a serotype selected from the group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAV12, and hybrids thereof. In other embodiments, the
recombinant AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutants thereof.
In another embodiment the AAV comprises a mutated capsid protein.
In another embodiment the capsid protein comprises a mutated
tyrosine residue. In another embodiment the mutated tyrosine
residue is selected from the group consisting of Y252F, Y272F,
Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F,
Y612G, Y673F and Y720F. In another embodiment the mutated capsid
protein comprises a tyrosine residue mutated to a phenylalanine. In
another embodiment the vector is introduced using intravitreal
injection, subretinal injection, and/or ILM peel.
[0008] In another aspect the present invention provides a method of
transducing a retinal bipolar cell (e.g. ON or OFF retinal bipolar
cells; rod and cone bipolar cells) comprising introducing into a
retina a vector comprising an exogenous nucleic acid operatively
linked to a regulatory sequence. In one embodiment the regulatory
sequence is a non-cell type specific promoter. In another
embodiment the regulatory sequence is a guanine nucleotide binding
protein alpha activating activity polypeptide 0 (GNAO1) promoter or
a fusion of the cytomegalovirus (CMV) immediate early enhancer and
the bovine .beta.-actin promoter plus intron1-exon1 junction (CBA,
smCBA). In another embodiment the exogenous nucleic acid comprises
a light-sensitive protein. In a related embodiment the
light-sensitive protein is selected from the group consisting of
ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof. In another related embodiment the
light-sensitive protein is ChR2 or a light-sensitive protein that
is at least about 70%, at least about 80%, at least about 90% or at
least about 95% identical to ChR2. In another embodiment the vector
is selected from the group consisting of recombinant
adeno-associated virus (AAV), recombinant retrovirus, recombinant
lentivirus, and recombinant poxvirus. In a related embodiment the
vector is a recombinant adeno-associated viral vector (AAV). In
another embodiment the AAV is of a serotype selected from the group
consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAV12, and hybrids thereof. In certain embodiments,
the recombinant AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutants
thereof. In a related embodiment the AAV comprises a mutated capsid
protein. In another embodiment the capsid protein comprises a
mutated tyrosine residue. In another embodiment the mutated
tyrosine residue is selected from the group consisting of Y252F,
Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F,
Y576F, Y612G, Y673F and Y720F. In another embodiment the mutated
capsid protein comprises a tyrosine residue mutated to a
phenylalanine. In another embodiment the vector is introduced using
intravitreal injection, subretinal injection, and/or ILM peel.
INCORPORATION BY REFERENCE
[0009] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0011] FIG. 1 depicts the ChR1 nucleic acid sequence.
[0012] FIG. 2 depicts the ChR2 nucleic acid sequence.
[0013] FIG. 3 depicts the NpHR nucleic acid sequence.
[0014] FIG. 4 depicts the melanopsin nucleic acid sequence.
[0015] FIG. 5 depicts the ChR2 nucleic acid sequence that is
mammalian codon-optimized and that encodes a ChR2 fused with Green
Fluorescent Protein (GFP).
[0016] FIG. 6 depicts a fragment of the GRM6 (metabotropic
glutamate receptor 6) regulatory nucleic acid sequence capable of
regulating expression in a bipolar cell specific manner.
[0017] FIG. 7 depicts a smCBA regulatory nucleic acid sequence.
[0018] FIG. 8 depicts: neurons expressing ChR2 and firing. (A)
Neurons expressing ChR2 with light stimulation; (B) Neurons firing
in response to fast trains of blue light pulses.
[0019] FIG. 9 depicts: AAV Delivery to retinal bipolar cells.
Column 1 shows GFP expression in retinal bipolar cells after a
subretinal injection with the AAV7-CBA-GFP vector after 8 weeks of
age. Column 2 shows PKC.alpha. staining (an antibody that is
specific to bipolar cells), column 3 shows DAPI staining for cell
nuclei, and column 4 shows merged images of GFP expression,
PKC.alpha. and DAPI stains. The first row is 20.times.
magnification and the second row is 40.times. magnification.
[0020] FIG. 10 depicts: Expression of the ChR2-GFP fused protein in
rd1, rho -/-, and rd16 in retinal bipolar cells. In each image, the
retinal pigment epithelium (RPE), bipolar cells or inner nuclear
layer (INL), inner plexiform layer (IPL), and ganglion cell layer
(GCL) are noted. The brighter white areas show GFP expression.
There are ringlets of expression in the bipolar cells of the INL
(except for the AAV5 intravitreal injection).
[0021] FIG. 11 depicts: analysis of EGFP expression in frozen
retinal sections by immunohistochemistry at 1 month following
subretinal injections with the Tyrosine mutant AAV vectors. Example
sections depicting spread and intensity of EGFP fluorescence
throughout the retina after transduction with serotype 2 Y444 (a)
or serotype 8 Y733 (b). The images are oriented with the vitreous
toward the bottom and the photoreceptor layer toward the top. EGFP
fluorescence in photoreceptors, RPE and ganglion cells from mouse
eyes injected subretinally with serotype 2 Y444 (c) EGFP
fluorescence in photoreceptors, RPE and Muller cells after serotype
8 Y733 delivery (d) Detection of Muller cells processes (red) by
immunostaining with a glutamine-synthetase (GS) antibody (e) Merged
image showing colocalization of EGFP fluorescence (green) and GS
staining (red) in retinal sections from eyes treated with serotype
8 Y733 (f) Calibration bar 100 .mu.M. gcl, ganglion cell layer;
ipl, inner plexiform layer; inl, inner nuclear layer; onl, outer
nuclear; os, outer segment; rpe, retinal pigment epithelium.
[0022] FIG. 12 depicts: Training mice on a water maze task. (A) A
schematic of the water maze used to measure scotopic threshold
(Hayes and Balkema, 1993). (B) Time it took each mouse group
(retinal degenerated--treated, retinal degenerated--untreated, and
wild type) to find the target (black platform+LED array) as a
function of training sessions. (C) Time it took each mouse group
(treated rd1, treated rd16, treated rho -/-, untreated retinal
degenerated, and wild type) to find the target (black platform+LED
array) as a function of different light intensities.
[0023] FIG. 13 depicts: goggle-like device with an associated light
generation/production element (LED array/laser system) that can
trigger expression of light-sensitive proteins.
DETAILED DESCRIPTION OF THE INVENTION
Light-Sensitive Proteins
[0024] The present invention provides recombinant nucleic acids
encoding light-sensitive proteins, viral and non-viral vectors for
the delivery of recombinant nucleic acids encoding light-sensitive
proteins, and methods for delivery of light-sensitive proteins.
[0025] Light-sensitive proteins are proteins that belong to the
opsin family and include vertebrate (animal) and invertebrate
rhodopsins. The animal opsins, rhodopsins, are G-protein coupled
receptors (GPCRs) with 7-transmembrane helices which can regulate
the activity of ion channels. Invertebrate rhodopsins are usually
not GPCRs, but are light-sensitive or light-activated ion pumps or
ion channels.
[0026] An algal opsin such as channelrhodopsin (ChR2) from
Chlamydomonas reinhardtii allows blue light-induced action
potentials to be triggered with millisecond-precision in cells due
to depolarizing cation flux through a light-gated pore. An archael
opsin such as halorhodopsin Natronomonas pharaonis allows for
light-activated chloride pumping; the pump can be hyperpolarized
and inhibited from firing action potentials when exposed to yellow
light. Use of such light-sensitive opsins allows for temporal and
spatial regulation of neuronal firing activity.
[0027] As referred to herein, a "light-sensitive" protein includes
channelrhodopsins (ChR1, ChR2), halorhodopsins (NpHR), melanopsins,
pineal opsins, bacteriorhodopsin, and variants thereof. A
light-sensitive protein of this invention can occur naturally in
plant, animal, archaebacterial, algal, or bacterial cells, or can
alternatively be created through laboratory techniques.
[0028] Channelrhodopsins ChR1 (GenBank accession number
AB058890/AF385748; FIG. 1) and ChR2 (GenBank accession number
AB058891/AF461397; FIG. 2) are two rhodopsins from the green alga
Chlamydomonas reinhardtii (Nagel, 2002; Nagel, 2003). Both are
light-sensitive channels that, when expressed and activated in
neural tissue, allow for a cell to be depolarized when stimulated
with light (Boyden, 2005).
[0029] In some embodiments hybrid or chimeric channelrhodopsins can
be created and used by combining different portions of the ChR1 and
ChR2 proteins.
[0030] In one embodiment a hybrid or chimeric channelrhodopsin can
be created and used by replacing the N-terminal segments of ChR2
with the homologous counterparts of ChR1 (and vice-versa). In some
embodiments the hybrid channelrhodopsins result in a shift of
sensitivity into a different wavelength spectrum (for example into
the red wavelength spectrum) with negligible desensitization and
slowed turning-on and turning-off kinetics.
[0031] In another embodiment a ChR1 (amino acids 1-345) and ChR2
(amino acids 1-315) hybrid/chimera can be created and used.
[0032] In yet another embodiment ChR1-ChR2 hybrids/chimeras
retaining the N-terminal portion of ChR1 and replacing the
C-terminal portion with the corresponding ChR2 segment can be
created and used. In specific embodiments hybrids/chimeras of ChR1
and ChR2 can be constructed and utilized including mutant residues
around the retinal binding pockets of the chimeras. In exemplary
embodiments the following chimeras can be created and used: [0033]
a. ChD: a hybrid/chimera of a ChR1 N-terminal portion and a ChR2
C-terminal portion where the crossover site is at a point of
homology at helixD of the two channelrhodopsins [0034] b. ChEF: a
hybrid/chimera of a ChR1 N-terminal portion and a ChR2 C-terminal
portion where the crossover site is at the loop between helices E
and F of the two channelrhodopsins [0035] c. ChIEF: a variant of
the ChEF chimera with isoleucine 170 mutated to valine [0036] d.
ChF: a hybrid/chimera of a ChR1 N-terminal portion and a ChR2
C-terminal portion where the crossover site is at the end of helix
F of the two channelrhodopsins.
[0037] In some embodiments the chimeras retain the reduced
inactivation of ChR1 in the presence of persistent light, but can
allow the permeation of sodium and potassium ions in addition to
protons. In other embodiments the chimeras can improve the kinetics
of the channel by enhancing the rate of the channel closure after
stimulation.
[0038] In some embodiments other ChR1 and ChR2 variants can be
engineered. In specific embodiments single or multiple point
mutations to the ChR2 protein can result in ChR2 variants. In
exemplary embodiments, mutations at the C128 location of ChR2 can
result in altered channel properties. In related embodiments,
C128A, C128S, and C128T ChR2 mutations can result in greater
overall mean open times (Berndt, 2009). In other related
embodiments, ChR2 variants can result in altered kinetics.
[0039] In another embodiment, a VChR1 can be used (GenBank
accession number EU622855).
[0040] In specific embodiments a mammalian codon optimized version
of ChR2 is utilized (FIG. 5).
[0041] NpHR (Halorhodopsin) (GenBank accession number EF474018;
FIG. 3) is from the haloalkaliphilic archaeon Natronomonas
pharaonis. In certain embodiments variants of NpHR can be created.
In specific embodiments single or multiple point mutations to the
NpHR protein can result in NpHR variants. In specific embodiments a
mammalian codon optimized version of NpHR can be utilized.
[0042] In one embodiment NpHR variants are utilized. In one
specific embodiment eNpHR (enhanced NpHR) is utilized. Addition of
the amino acids FCYENEV to the NpHR C-terminus along with the
signal peptide from the .beta. subunit of the nicotinic
acetylcholine receptor to the NpHR N-terminus results in the
construction of eNpHR.
[0043] Melanopsin (GenBank accession number 6693702; FIG. 4) is a
photopigment found in specialized photosensitive ganglion cells of
the retina that are involved in the regulation of circadian
rhythms, pupillary light reflex, and other non-visual responses to
light. In structure, melanopsin is an opsin, a retinylidene protein
variety of G-protein-coupled receptor. Melanopsin resembles
invertebrate opsins in many respects, including its amino acid
sequence and downstream signaling cascade. Like invertebrate
opsins, melanopsin appears to be a bistable photopigment, with
intrinsic photoisomerase activity. In certain embodiments variants
of melanopsin can be created. In specific embodiments single or
multiple point mutations to the melanopsin protein can result in
melanopsin variants.
[0044] Light-sensitive proteins may also include proteins that are
at least about 10%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, or at least about 99% identical to the
light-sensitive proteins ChR1, ChR2, NpHR and melanopsin. For
example, the ChR2 protein may include proteins that are at least
about 60%, at least about 70%, at least about 80%, at least about
90% or at least about 95% identical to ChR2. In addition, these
proteins may include ChR2 that is photosensitive and can be
activated by specific wavelengths of high intensity light.
[0045] In some embodiments, light-sensitive proteins can modulate
signaling within neural circuits and bidirectionally control
behavior of ionic conductance at the level of a single neuron. In
some embodiments the neuron is a retinal neuron, a retinal bipolar
cell (e.g. ON or OFF retinal bipolar cells; rod and cone bipolar
cells), a retinal ganglion cell, a photoreceptor cell, or a retinal
amacrine cell.
Adeno-Associated Viral Vectors
[0046] The present invention provides viral vectors comprising
nucleic acids encoding a light-sensitive proteins and methods of
use, as described herein.
[0047] Adeno-associated virus (AAV) is a small (25-nm),
nonenveloped virus that packages a linear single-stranded DNA
genome of 4.7 Kb. The small size of the AAV genome and concerns
about potential effects of Rep on the expression of cellular genes
led to the construction of AAV vectors that do not encode Rep and
that lack the cis-active IEE, which is required for frequent
site-specific integration. The ITRs are kept because they are the
cis signals required for packaging. Thus, current recombinant AAV
(rAAV) vectors persist primarily as extrachromosomal elements.
[0048] A variety of recombinant adeno-associated viral vectors
(rAAV) may be used to deliver genes of interest to a cell and to
effect the expression of a gene of interest, e.g., a gene encoding
a light-sensitive protein. For example, rAAV can be used to express
light-sensitive proteins, e.g., ChR1, ChR2, VChR1, ChR2 C128A, ChR2
C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF,
ChIEF, NpHR, eNpHR, melanopsin, and variants thereof or any
light-sensitive protein described herein, in a target cell. At
times herein, "transgene" is used to refer to a polynucleotide
encoding a polypeptide of interest, wherein the polynucleotide is
encapsidated in a viral vector (e.g., rAAV).
[0049] Adeno-associated viruses are small, single-stranded DNA
viruses which require helper virus to facilitate efficient
replication. The 4.7 kb genome of AAV is characterized by two
inverted terminal repeats (ITR) and two open reading frames which
encode the Rep proteins and Cap proteins, respectively. The Rep
reading frame encodes four proteins of molecular weight 78 kD, 68
kD, 52 kD and 40 kD. These proteins function mainly in regulating
AAV replication and rescue and integration of the AAV into a host
cell's chromosomes. The Cap reading frame encodes three structural
proteins of molecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD
(VP3) (Berns, cited above) which form the virion capsid. More than
80% of total proteins in AAV virion comprise VP3.
[0050] The genome of rAAV is generally comprised of: (1) a
5'adeno-associated virus ITR, (2) a coding sequence (e.g.,
transgene) for the desired gene product (e.g., a light-sensitive
protein) operatively linked to a sequence which regulates its
expression in a cell (e.g., a promoter sequence such as a mGluR6 or
fragment thereof), and (3) a 3'adeno-associated virus inverted
terminal repeat. In addition, the rAAV vector may preferably
contain a polyadenylation sequence.
[0051] Generally, rAAV vectors have one copy of the AAV ITR at each
end of the transgene or gene of interest, in order to allow
replication, packaging, and efficient integration into cell
chromosomes. The ITR consists of nucleotides 1 to 145 at the 5'end
of the AAV DNA genome, and nucleotides 4681 to 4536 (i.e., the same
sequence) at the 3'end of the AAV DNA genome. The rAAV vector may
also include at least 10 nucleotides following the end of the ITR
(i.e., a portion of the "D region").
[0052] The transgene sequence (e.g., the polynucleotide encoding a
light-sensitive protein) can be of about 2 to 5 kb in length (or
alternatively, the transgene may additionally contain a "stuffer"
or "filler" sequence to bring the total size of the nucleic acid
sequence between the two ITRs to between 2 and 5 kb).
Alternatively, the transgene may be composed of repeated copies of
the same or similar heterologous sequence several times (e.g., two
nucleic acid molecules which encode one or more light-sensitive
proteins separated by a ribosome readthrough, or alternatively, by
an Internal Ribosome Entry Site or "IRES"), or several different
heterologous sequences (e.g., ChR2 and NpHR separated by a ribosome
readthrough or an IRES; or any two ore more of the light-sensitive
proteins described herein including but not limited to ChR1, ChR2,
VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof).
[0053] Recombinant AAV vectors of the present invention may be
generated from a variety of adeno-associated viruses, including for
example, any of serotypes 1 through 12, as described herein. For
example, ITRs from any AAV serotype are expected to have similar
structures and functions with regard to replication, integration,
excision and transcriptional mechanisms.
[0054] In some embodiments, a cell-type specific promoter (or other
regulatory sequence such as an enhancer) is employed to drive
expression of a gene of interest, e.g., a light-sensitive protein,
ChR2, etc., in one or more specific cell types. In other cases,
Within certain embodiments of the invention, expression of the
light-sensitive transgene may be accomplished by a separate
promoter (e.g., a viral, eukaryotic, or other promoter that
facilitates expression of an operatively linked sequence in an
eukaryotic cell, particularly a mammalian cell). Representative
examples of suitable promoters in this regard include a mGluR6
promoter, a GNA01 promoter, a CBA/smCBA (fusion of the CMV
immediate early enhancer and bovine beta actin promoter plus
intro1-exon1 junction) promoter, CBA promoter (chicken beta actin),
CMV promoter, RSV promoter, SV40 promoter, MoMLV promoter, or
derivatives, mutants and/or fragments thereof. Promoters and other
regulatory sequences are further described herein.
[0055] Other promoters that may similarly be utilized within the
context of the present invention include cell or tissue specific
promoters (e. g, a rod, cone, or ganglia derived promoter), or
inducible promoters. Representative examples of suitable inducible
promoters include inducible promoters sensitive to an antibiotic,
e.g., tetracycline-responsive promoters such as "tet-on" and/or
"tet-off" promoters. Inducible promoters may also include promoters
sensitive to chemicals other than antibiotics.
[0056] The rAAV vector may also contain additional sequences, for
example from an adenovirus, which assist in effecting a desired
function for the vector. Such sequences include, for example, those
which assist in packaging the rAAV vector into virus particles.
[0057] Packaging cell lines suitable for producing adeno-associated
viral vectors may be accomplished given available techniques (see
e.g., U.S. Pat. No. 5,872,005). Methods for constructing and
packaging rAA7I vectors are described in, for example, WO
00/54813.
[0058] Flanking the rep and cap open reading frames at the 5' and
3' ends are 145 bp inverted terminal repeats (ITRs), the first 125
bp of which are capable of forming Y- or T-shaped duplex
structures. The two ITRs are the only cis elements essential for
AAV replication, rescue, packaging and integration of the AAV
genome. There are two conformations of AAV ITRs called "flip" and
"flop". These differences in conformation originated from the
replication model of adeno-associated virus which uses the ITR to
initiate and reinitiate the replication (R. O. Snyder et al, 1993,
J. Virol., 67:6096-6104 (1993); K. I. Berns, 1990 Microbiological
Reviews, 54:316-329). The entire rep and cap domains can be excised
and replaced with a therapeutic or reporter transgene.
[0059] In some embodiments self-complementary AAV vectors are used.
Self-complementary vectors have been developed to circumvent
rate-limiting second-strand synthesis in single-stranded AAV vector
genomes and to facilitate robust transgene expression at a minimal
dose. In specific embodiments a self-complementary AAV of any
serotype or hybrid serotype or mutant serotype, or mutant hybrid
serotype increases expression of a light-sensitive protein such as
ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof by at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 100%, at least 125%, at least 150%, at least
175%, at least 200%, or more than 200%, when compared to a non-self
complementary rAAV of the same serotype.
Adeno-Associated Viral Serotypes
[0060] In one embodiment the vector comprises a recombinant AAV of
a particular serotype, either naturally occurring or
engineered.
[0061] AAVs have been found in many animal species, including
primates, canine, fowl and human.
[0062] Viral serotypes are strains of microorganisms having a set
of recognizable antigens in common. There are several known
serotypes of AAV, and the efficacy of transfection or transduction
within the retina may vary as a function of the specific serotype
and the nature of the target cells. rAAV, or a specific serotype of
rAAV or AAV, may provide tissue-specific or cell-type specific
tropism for gene delivery to retinal bipolar cells (e.g. ON or OFF
retinal bipolar cells; rod and cone bipolar cells). While rAAV
and/or AAV is likely a relatively safe method to deliver a
transgene to a target tissue, the efficacy of delivery, and
possibly the safety of delivery, may depend on the coat proteins of
the AAV. The protein coat, or capsid, determines which cells can
take up the viral payload. Different AAV serotypes, i.e., viruses
that differ in their proteins coats or capsids, may differ in their
tissue tropism and ability to transduce targeted cells. Transgenes
can be packaged within AAV particles with many functionally
different coat proteins, or capsids. These different capsids are
what define the serotype and may contribute (entirely or in part)
to its ability to transduce particular cell types. The entry of the
viral vector begins with the interaction of the capsid and the
target cell surface proteins. Without wishing to be bound by
theory, it is at this point in the transduction pathway that
different serotypes may significantly influence the efficiency of
transgene delivery.
[0063] In certain embodiments the AAV vector is of a serotype or
variant/mutant thereof including but not limited to: AAV1 (GenBank
accession number AY724675), AAV2 (GenBank accession number
AF043303), AAV3, AAV4, AAV5 (GenBank accession number M61166),
AAV6, AAV7 (GenBank accession number AF513851), AAV8 (GenBank
accession number AF513852), AAV9 (GenBank accession number
AX753250), AAV10, AAV11 (GenBank accession number AY631966), or
AAV12 (GenBank accession number DQ813647), or mutants, hybrids, or
fragments thereof. In certain embodiments the AAV vector comprises
one or more, two or more, three or more, four or more, or five or
more of the following serotypes: AAV1 (GenBank accession number
AY724675), AAV2 (GenBank accession number AF043303), AAV3, AAV4,
AAV5 (GenBank accession number M61166), AAV6, AAV7 (GenBank
accession number AF513851), AAV8 (GenBank accession number
AF513852), AAV9 (GenBank accession number AX753250), AAV10, AAV11
(GenBank accession number AY631966), or AAV12 (GenBank accession
number DQ813647), or mutants, hybrids, or fragments thereof. In
other embodiments, the AAV vector is of a natural serotype or
variant/mutant thereof, heretofore yet undiscovered and
uncharacterized.
[0064] In certain embodiments, the recombinant AAV is of a
combinatorial hybrid of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or more serotypes or mutants thereof.
[0065] In some embodiments, the AAV vector may be used to
specifically transduce a specific cell type, e.g., retinal cells or
retinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rod
and cone bipolar cells). In some cases, a specific serotype, e.g.,
AAV2, AAV5, AAV7 or AAV8, may be better than other serotypes at
transducing a particular cell type (e.g., retinal bipolar cells
(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells),
neurons) or tissue. For example, a specific AAV serotype such as
AAV2, AAV5, AAV7 or AAV8 may transduce a specific cell type, e.g.,
retinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rod
and cone bipolar cells), with an increased transduction efficiency
of at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
100%, at least 125%, at least 150%, at least 175%, at least 200%,
or more than 200%, when compared to a different AAV serotype. In
some cases, a specific serotype e.g., AAV2, AAV5, AAV7 or AAV8, may
permit transduction of at least 2%, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, or 90% of cells of a particular cell type, e.g.,
retinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rod
and cone bipolar cells), or of cells within a particular tissue,
e.g., retinal tissue.
[0066] There is a need in the art for AAV serotypes that can
effectively transduce retinal bipolar cells (e.g. ON or OFF retinal
bipolar cells; rod and cone bipolar cells), particularly when such
transduction enables the delivery and expression of a
light-sensitive protein, e.g., ChR2. An important therapy to treat
eye disorders or diseases (e.g., visual impairment, blindness), may
involve using a particular AAV serotype to express light-sensitive
proteins such as ChR2 in retinal bipolar cells (e.g. ON or OFF
retinal bipolar cells; rod and cone bipolar cells). For example, in
a preferred embodiment, AAV5 or AAV7 serotypes are used to target
expression of a gene of interest (e.g., a light-sensitive protein,
ChR2, etc.) in retinal cells, e.g., retinal bipolar cells (e.g. ON
or OFF retinal bipolar cells; rod and cone bipolar cells). In some
cases, AAV5 and/or AAV7 may more efficiently transduce retinal
cells, e.g., retinal bipolar cells (e.g. ON or OFF retinal bipolar
cells; rod and cone bipolar cells), than other AAV serotypes. For
example, in some embodiments, the AAV5 and/or AAV7 serotypes, but
not AAV1 serotype, are used to transduce retinal bipolar cells
(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells).
In other cases AAV2 and/or AAV8 serotypes are used to transduce
retinal bipolar cells (e.g. ON or OFF retinal bipolar cells; rod
and cone bipolar cells). In some cases, a specific serotype, e.g.,
AAV2, AAV5, AAV7 or AAV8, may be generally applied to a tissue,
e.g., retinal tissue, but then preferentially transduces a specific
cell-type over another cell type.
[0067] In some cases, an AAV e.g., AAV2, AAV5, AAV7 or AAV8, that
is introduced to the retina may preferentially transduce retinal
bipolar cells (e.g. ON or OFF retinal bipolar cells; rod and cone
bipolar cells) so that the transgene is expressed more highly in
retinal bipolar cells compared to other retinal cells. In some
cases, the particular serotype e.g., AAV2, AAV5, AAV7 or AAV8, of
the AAV may be the cause or contribute to the cause of such
preferential transduction. In some cases, only a small subset of
the bipolar cells are transduced.
[0068] In some embodiments, a specific serotype of AAV, e.g., AAV5
and/or AAV7 (or any other AAV serotype or mutant described herein)
comprising a non-cell-type-specific promoter is used to drive
expression of a light-sensitive protein in a particular cell type.
In some cases, a specific serotype of AAV that has been
demonstrated to preferentially transduce a particular cell type is
used along with a cell-type specific promoter to drive expression
of a protein of interest, e.g., a light-sensitive protein, in a
specific cell-type.
[0069] The AAV ITR sequences and other AAV sequences employed in
generating the minigenes, vectors, and capsids, and other
constructs used in certain embodiments may be obtained from a
variety of sources. For example, the sequences may be provided by
presently identified human AAV types and AAV serotypes yet to be
identified Similarly, AAVs known to infect other animals may also
provide these ITRs employed in the molecules or constructs of this
invention. Similarly, the capsids from a variety of serotypes of
AAV may be "mixed and matched" with the other vector components.
See, e.g., International Patent Publication No. WO01/83692,
published Nov. 8, 2001, and incorporated herein by reference. A
variety of these viral serotypes and strains are available from the
American Type Culture Collection, Manassas, Va., or are available
from a variety of academic or commercial sources. Alternatively, it
may be desirable to synthesize sequences used in preparing the
vectors and viruses of the invention using known techniques, which
may utilize AAV sequences which are published and/or available from
a variety of databases.
Adeno-Associated Viruses and Mutations of Surface-Exposed
Residues
[0070] Recombinant adeno-associated virus vectors are in use in
several clinical trials, but relatively large vector doses are
needed to achieve therapeutic benefits. Large vector doses may also
trigger an immune response as a significant fraction of the vectors
may fail to traffic efficiently to the nucleus and may be targeted
for degradation by the host cell proteasome machinery. It has been
reported that epidermal growth factor receptor protein tyrosine
kinase (EGFR-PTK) signaling negatively affects transduction by AAV
Serotype 2 vectors by impairing nuclear transport of the vectors
(Zhong 2007). Tyrosine-phosphorylated AAV2 vectors enter but do not
transduce effectively, in part because of the ubiquitination of AAV
capsids followed by proteasome-mediated degradation. Point
mutations in tyrosines in AAV2 may lead to high-efficiency
transduction at lower virus titers (Zhong 2008). In one embodiment,
tyrosine-mutated AAVs e.g., AAV2 or AAV8, are used in order to
improve the efficiency of transduction of retinal cells, e.g.,
retinal bipolar cells (FIGS. 9-12). In one embodiment, mutations of
the surface-exposed tyrosine residues of rAAV capsid allow the
vectors to evade phosphorylation and subsequent ubiquitination and,
thus, prevent proteasome-mediated degradation, leading to greater
transduction and subsequent gene expression of light-sensitive
proteins.
[0071] In a related embodiment any one or more surface exposed
residues other than tyrosine may be mutated to improve the
transduction efficiency, tissue/cell-type tropism, expression
characteristics, and titers needed for effective infection.
[0072] As described herein, modification and changes to the
structure of the polynucleotides and polypeptides of wild-type rAAV
vectors may result in improved rAAV virions possessing desirable
characteristics. For example, mutated rAAV vectors may improve
delivery of light-sensitive gene constructs to selected mammalian
cell, tissues, and organs for the treatment, prevention, and
prophylaxis of various diseases and disorders. Such approach may
also provide a means for the amelioration of symptoms of such
diseases, and to facilitate the expression of exogenous therapeutic
and/or prophylactic polypeptides of interest via rAAV
vector-mediated gene therapy. The mutated rAAV vectors may encode
one or more proteins, e.g., the light-sensitive proteins, e.g.,
ChR2, described herein. The creation (or insertion) of one or more
mutations into specific polynucleotide sequences that encode one or
more of the light-sensitive proteins encoded by the disclosed rAAV
constructs are provided herein. In certain circumstances, the
resulting light-sensitive polypeptide sequence is altered by these
mutations, or in other cases, the sequence of the polypeptide is
unchanged by one or more mutations in the encoding polynucleotide
to produce modified vectors with improved properties for effecting
gene therapy in mammalian systems. As described herein,
codon-optimization of the polynucleotide encoding the
light-sensitive protein may also improve transduction
efficiency.
[0073] The ubiquitin-proteasome pathway plays a role in
AAV-intracellular trafficking. Substitution of surface exposed
tyrosine residues on, for example, AAV2 or AAV8 capsids permits the
vectors to either have limited ubiquitination or to escape
ubiquitination altogether. The reduction in, or absence of,
ubiquitination may help prevent the capsid from undergoing
proteasome-mediated degradation. AAV or rAAV capsids can be
phosphorylated at tyrosine residues by EGFR-PTK in an in vitro
phosphorylation assay, and the phosphorylated AAV capsids retain
their structural integrity. Although phosphorylated AAV vectors may
enter cells as efficiently as their unphosphorylated counterparts,
their transduction efficiency may be significantly impaired.
[0074] In some cases, a recombinant adeno-associated viral (rAAV)
vector comprises a capsid protein with a mutated tyrosine residue
which enables to the vector to have improved transduction
efficiency of a target cell, e.g., a retinal bipolar cell (e.g. ON
or OFF retinal bipolar cells; rod and cone bipolar cells). In some
cases, the rAAV further comprises a promoter (e.g., mGluR6, or
fragment thereof) capable of driving the expression of a protein of
interest in the target cell.
[0075] In some cases, expression in a specific cell type is further
achieved by including a cell-type specific promoter described
herein within the rAAV vector.
[0076] In one embodiment, a recombinant adeno-associated viral
(rAAV) vector comprises at least a first capsid protein comprising
at least a first phosphorylated tyrosine amino acid residue, and
wherein said vector further comprises at least a first nucleic acid
segment that encodes a light-sensitive protein operably linked to a
promoter capable of expressing said segment in a host cell.
[0077] In one embodiment, a mutation may be made in any one or more
of tyrosine residues of the capsid protein of AAV 1-12 or hybrid
AAVs. In specific embodiments these are surface exposed tyrosine
residues. In a related embodiment the tyrosine residues are part of
the VP1, VP2, or VP3 capsid protein. In exemplary embodiments, the
mutation may be made at one or more of the following amino acid
residues of an AAV-VP3 capsid protein: Tyr252, Tyr272, Tyr444,
Tyr500, Tyr700, Tyr704, Tyr730; Tyr275, Tyr281, Tyr508, Tyr576,
Tyr612, Tyr673 or Tyr720. Exemplary mutations are
tyrosine-to-phenylalanine mutations including, but not limited to,
Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F,
Y508F, Y576F, Y612G, Y673F and Y720F. In a specific embodiment
these mutations are made in the AAV2 serotype. In some cases, an
AAV2 serotype comprises a Y444F mutation and/or an AAV8 serotype
comprises a Y733F mutation, wherein 444 and 733 indicate the
location of a point tyrosine mutation of the viral capsid. In
further embodiments, such mutated AAV2 and AAV8 serotypes encode a
light-sensitive protein, e.g., ChR2, and may also comprise a
regulatory sequence (e.g., mGluR6) to drive expression of such
light-sensitive protein.
[0078] In a related embodiment, 1, 2, 3, 4, 5, 6, or 7 mutations
are made to the tyrosine residue on an AAV 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, or hybrid serotype. In one exemplary embodiment, 3
tyrosines are mutated to create an AAV serotype with a triple
mutation consisting of: Y444F, Y500F, and Y730F.
[0079] The rAAV vectors of the present invention may be comprised
within an adeno-associated viral particle or infectious rAAV
virion, including for example, virions selected from the group
consisting of an AAV serotype 1, an AAV serotype 2, an AAV serotype
3, an AAV serotype 4, an AAV serotype 5 and an AAV serotype 6, an
AAV serotype 7, an AAV serotype 8, an AAV serotype 9, an AAV
serotype 10, an AAV serotype 11, an AAV serotype 12, or a hybrid
AAV serotype.
[0080] The rAAV vectors of the present invention may also be
comprised within an isolated mammalian host cell, including for
example, human, primate, murine, feline, canine, porcine, ovine,
bovine, equine, epine, caprine and lupine host cells. The rAAV
vectors may be comprised within an isolated mammalian host cell
such as a human endothelial, epithelial, vascular, liver, lung,
heart, pancreas, intestinal, kidney, muscle, bone, neural, blood,
or brain cell.
[0081] In certain embodiments the transduction efficiency of an AAV
comprising a mutated capsid protein (e.g., a mutation of a tyrosine
residue described herein) expressing a light-sensitive protein such
as ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof is increased by at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 100%, at least 125%, at least
150%, at least 175%, at least 200%, or more than 200%, when
compared to a wild-type AAV expressing a light-sensitive protein.
This disclosure also provides mutated rAAV vectors (e.g., the AAV2
Y444F vector or the AAV8Y733F vector) capable of transducing at
least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of
(e.g. ON or OFF retinal bipolar cells; rod and cone bipolar cells)
bipolar cells. The improvement in transduction created by the
mutated capsid may permit transduction of bipolar cells by
intravitreal injection. For example, in some embodiments, a mutated
rAAV vector or a rAAV combinatorial serotype hybrid vector or a
mutated combinatorial serotype hybrid rAAV vector may be capable of
transducing at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90% of retinal bipolar cells (e.g. ON or OFF retinal
bipolar cells; rod and cone bipolar cells) is introduced to the
retina by intravitreal injection. In a specific embodiment, only a
subset of the retinal bipolar cells is transduced. In another
specific embodiment only the highly sensitive bipolar cells are
transduced.
[0082] In certain embodiments the ubiquitin or proteasome-mediated
degradation of an AAV comprising a capsid protein with a mutation
expressing a light-sensitive protein, such as ChR1, ChR2, VChR1,
ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras,
ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants
thereof, is decreased by at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, or at least
90%, when compared to a wild-type AAV expressing a light-sensitive
protein.
Other Gene Delivery Vectors
[0083] Any of a variety of other vectors adapted for expression of
ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof or any light-sensitive protein in a cell of
the eye, particularly within a retinal cell, more particularly
within a non-photoreceptor cell (e.g. amacrine cells, retinal
ganglion cells, retinal bipolar cells, (ON or OFF retinal bipolar
cells; rod and cone bipolar cells)), are within the scope of the
present invention. Gene delivery vectors can be viral (e.g.,
derived from or containing sequences of viral DNA or RNA,
preferably packaged within a viral particle), or non-viral (e.g.,
not packaged within a viral particle, including "naked"
polynucleotides, nucleic acid associated with a carrier particle
such as a liposome or targeting molecule, and the like).
[0084] Other exemplary gene delivery vectors are described
below.
[0085] Recombinant Adenoviral Vectors (Ad): in other embodiments,
the gene delivery vector is a recombinant adenoviral vector. U.S.
Pat. No. 6,245,330 describes recombinant adenoviruses which may be
suitable for use in the invention. Ad vectors do not integrate into
the host cell genome, particularly preferred when short term gene
is required, typically about 14 days. Thus, use of Ad vectors can
require repeated intraocular injections to treat a retinal disease
which continues over decades in the average patient.
[0086] The viral tropism of Ad and AAV in the retina is can be
different. The subset of cells that are transduced by the vector is
usually a receptor-mediated event. Ad vectors have been shown to
primarily transduce retinal Muller cells and Retinal pigment
epithelial cells following injection. AAV vectors are very
efficient at transferring their genetic payload to retinal
photoreceptor and non-photoreceptor cells when injected into the
eye.
[0087] Retroviral gene delivery vectors: the gene delivery vectors
of the invention can be a retroviral gene delivery vector adapted
to express a selected gene (s) or sequence (s) of interest (e.g.,
ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof). Retroviral gene delivery vectors of the
present invention may be readily constructed from a wide variety of
retroviruses, including for example, B, C, and D type retroviruses
as well as spumaviruses and lentiviruses. For example, in some
cases, a retrovirus, e.g., a lentivirus, is pseudotyped with an
envelope protein or other viral protein to facilitate entry into
target cells. In some cases, a lentivirus is pseudotyped with
vesicular-stomatitis virus g protein. (see RNA Tumor Viruses,
Second Edition, Cold Spring Harbor Laboratory, 1985). Such
retroviruses may be readily obtained from depositories or
collections such as the American Type Culture Collection ("ATCC";
Rockville, Md.), or isolated from known provided herein, and
standard recombinant techniques (e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual 2d ed., Cold Spring Harbor Laboratory
Press, 1989; Kunkel, PNAS 52: 488, 1985).
[0088] In addition, within certain embodiments of the invention,
portions of the retroviral gene delivery vectors may be derived
from different retroviruses. For example, within one embodiment of
the invention, retrovirus LTRs may be derived from a Murine Sarcoma
Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging
signal from a Murine Leukemia Virus, and an origin of second strand
synthesis from an Avian Leukosis Virus.
[0089] Within one aspect of the present invention, retroviral
vector constructs are provided comprising a 5'LTR, a tRNA binding
site, a packaging signal, one or more heterologous sequences, an
origin of second strand DNA synthesis and a 3'LTR, wherein the
vector construct lacks gag, pol or env coding sequences.
[0090] Other retroviral gene delivery vectors may likewise be
utilized within the context of the present invention, and are well
known in the art.
[0091] Packaging cell lines suitable for use with the above
described retroviral vector constructs can be readily prepared
according to methods well known in the art, and utilized to create
producer cell lines for the production of recombinant vector
particles.
[0092] Alphavirus delivery vectors: gene delivery vectors suitable
for use in the invention can also be based upon alphavirus vectors.
For example, the Sindbis virus is the prototype member of the
alphavirus genus of the togavirus family. The unsegmented genomic
RNA (49S RNA) of Sindbis virus is approximately 11,703 nucleotides
in length, contains a 5'cap and a 3' poly-adenylated tail, and
displays positive polarity. Infectious enveloped Sindbis virus is
produced by assembly of the viral nucleocapsid proteins onto the
viral genomic RNA in the cytoplasm and budding through the cell
membrane embedded with viral encoded glycoproteins. Entry of virus
into cells is by endocytosis through clatharin coated pits, fusion
of the viral membrane with the endosome, release of the
nucleocapsid, and uncoating of the viral genome. During viral
replication the genomic 49S RNA serves as template for synthesis of
the complementary negative strand. This negative strand in turn
serves as template for genomic RNA and an internally initiated 26S
subgenomic RNA.
[0093] The Sindbis viral nonstructural proteins are translated from
the genomic RNA while structural proteins are translated from the
subgenomic 26S RNA. All viral genes are expressed as a polyprotein
and processed into individual proteins by post translational
proteolytic cleavage. The packaging sequence resides within the
nonstructural coding region, therefore only the genomic 49S RNA is
packaged into virions.
[0094] Several different Sindbis vector systems may be constructed
and utilized within the present invention. Representative examples
of such systems include those described within U.S. Pat. Nos.
5,091,309 and 5,217,879, and PCT Publication No. WO 95/07994.
[0095] Other viral gene delivery vectors In addition to retroviral
vectors and alphavirus vectors, numerous other viral vectors
systems may also be utilized as a gene delivery vector.
Representative examples of such gene delivery vectors include
viruses such as pox viruses, such as canary pox virus or vaccinia
virus
[0096] Non-viral gene delivery vectors: in addition to the above
viral-based vectors, numerous non-viral gene delivery vectors may
likewise be utilized within the context of the present invention.
Representative examples of such gene delivery vectors include
direct delivery of nucleic acid expression vectors, naked DNA
(e.g., DNA not contained in a viral vector) (WO 90/11092),
polycation condensed DNA linked or unlined to killed adenovirus
(Curiel et al., Hum. Gene Ther. 3: 147-154, 1992), DNA ligand
linked to a ligand with or without one of the high affinity pairs
described above (Wu et al., R of Biol. Chem (264: 16985-16987,
1989), nucleic acid containing liposomes (e.g., WO 95/24929 and WO
95/12387) and certain eukaryotic cells.
Regulatory Sequences
[0097] In some embodiments, regulatory sequences or elements are
utilized to allow for cell-type or tissue-type specific targeting
of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof. In related embodiments regulatory elements
are used to specifically target retinal neurons, or retinal bipolar
cells (e.g. ON or OFF retinal bipolar cells; rod and cone bipolar
cells), or retinal ganglion cells, or photoreceptor cells, or
amacrine cells. Examples of regulatory sequences or elements
include, but are not limited to promoter, silencer, enhancer, and
insulator sequences.
[0098] In some embodiments regulatory sequences such as promoters
suitable for use in the present invention include constitutive
promoters, strong promoters (e.g., CMV promoters), inducible
promoters, and tissue-specific or cell-specific promoters (e.g.,
promoters that preferentially facilitate expression in a limited
number of tissues or cell types (e.g., eye tissues, retina, retinal
cells, photoreceptor cells, and the like).
[0099] Any of a variety of regulatory sequences can be used in the
gene delivery vectors of the invention to provide for a suitable
level or pattern of expression of the light-sensitive protein of
interest. The regulatory sequences are generally derived from
eukaryotic regulatory sequences.
[0100] In some embodiments non-cell specific regulatory elements
are used. In one embodiment, the promoter comprises (from 5' to 3')
a viral enhancer (a CMV immediate early enhancer), and a beta-actin
promoter (a bovine or chicken beta-actin promoter-exon 1-intron 1
element). In a specific embodiment, the promoter comprises (from 5'
to 3') CMV immediate early enhancer (381 bp)/bovine or chicken
beta-actin (CBA) promoter-exon 1-intron 1 (1352 bp) element, which
together are termed herein the "CBA promoter" (FIG. 7). In some
embodiments a nucleic acid encoding a light-sensitive protein is
delivered to a cell using a viral vector such as AAV carrying a
selected light-sensitive transgene-encoding DNA regulated by a non
cell-specific promoter and/or other regulatory sequences that
expresses the product of the DNA. In some related embodiments the
non cell-specific promoter is general promoter such as a
ubiquitin-based promoter, for e.g. a ubiquitin C promoter.
[0101] In other embodiments, a light-sensitive protein is delivered
to a cell type or tissue type of interest using a viral vector such
as AAV carrying a selected light-sensitive transgene-encoding DNA
regulated by a promoter and/or other regulatory sequences that
expresses the product of the DNA in selected retinal cells of a
subject. In specific embodiments, expression is targeted to
particular types of cells within the retina through the use of a
specific promoter nucleotide sequence and/or other regulatory
regions such as silencer, enhancer, or insulator sequences which
are engineered into the vector. In some embodiments, different
regulatory sequences are used to drive expression of different
engineered genes in different populations of cells.
[0102] In other embodiments retinal bipolar cell-specific
regulatory sequences such as promoter, enhancer, silencer, and
insulator sequences are used. In specific embodiments the ON
bipolar cells are targeted. In other embodiments the OFF bipolar
cells are targeted. In other embodiments the rode bipolar cells are
targeted. In other embodiments the cone bipolar cells are
targeted.
[0103] In one embodiment specific expression of a light sensitive
proteins in ON bipolar cells is targeted using a light-sensitive
protein such as ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2
C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR,
eNpHR, melanopsin, and variants thereof operatively linked to a
GRM6 (metabotropic glutamate receptor 6, mGluR6) regulatory
sequence or a fragment thereof. In one embodiment the full length
mGluR6 regulatory sequence is utilized.
[0104] In another embodiment specific expression of a light
sensitive proteins in ON bipolar cells is targeted using a
light-sensitive protein such as ChR1, ChR2, VChR1, ChR2 C128A, ChR2
C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF,
ChIEF, NpHR, eNpHR, melanopsin, and variants thereof operatively
linked to a mGluR6 regulatory sequence fragment. In a specific
embodiment, the mGluR6 regulatory sequence fragment is the sequence
presented in FIG. 6. In a related embodiment the mGluR6 regulatory
sequence fragment is substantially the same as the sequence
presented in FIG. 6, or is about 60% identical, or is about 70%
identical, or is about 80% identical, or is about 90% identical, or
is about 95% identical to the sequence presented in FIG. 6.
[0105] Many known promoters are too large to fit into the genome of
the AAV. Indeed, the original cell-specific regulatory sequence for
mGluR6 (Dhingra, 2008) was far too large (approximately 10.5 Kb) to
be used in AAV. In a preferred embodiment of the current invention,
a mGluR6 regulatory sequence fragment is used, one that is small
enough to be used in AAV-mediated delivery. In related embodiments
the mGluR6 regulatory sequence fragment is less than about 2000
base pairs, less than about 1000 base pairs, less than about 750
base pairs, less than about 500 base pairs, less than about 250
base pairs, or less than about 100 base pairs in length. In another
related embodiment, the mGluR6 regulatory sequence fragment is a
variant of the sequence presented in FIG. 6.
[0106] In another embodiment, specific expression of a light
sensitive proteins in ON bipolar cells is targeted using a
light-sensitive protein such as ChR1, ChR2, VChR1, ChR2 C128A, ChR2
C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF,
ChIEF, NpHR, eNpHR, melanopsin, and variants thereof operatively
linked to a GNA01 (guanine nucleotide binding protein (G protein),
alpha activating activity polypeptide O) regulatory sequence. In a
related embodiment the regulatory sequence is substantially the
same as GNA01 sequence, or is about 60% identical, or is about 70%
identical, or is about 80% identical, or is about 90% identical, or
is about 95% identical to the GNA01 sequence.
Retinal Bipolar Cells and Targeting
[0107] Although only a fraction of the human visual system, the
retina is a complex system that filters, amplifies, and modulates
the visual signal before it is sent to the rest of the visual
system (Wassle 2004). The vast majority of this processing happens
within the inner plexiform layer (IPL) where a system of bipolar
and amacrine cells refine the visual signal into its primary
components (e.g., motion, contrast, resolution) (Mills 1999; Roska
2001). Some groups are currently targeting ChR2 to the ganglion
cell layer, which bypasses the processing power of the IPL and
system of amacrine cells (Bi 2006; Greenberg 2007; Tomita 2007).
These groups have reported very few behavioral changes.
[0108] The majority of retinal cells are either ON-center
(increased firing rate as a result of a step increase in contrast
within the center of the receptive field) or OFF center type
(increased firing rate as a result of a step decrease in contrast
in the center of the receptive field), working in a push-pull
inhibitory fashion (Wassle 2004). In order to maintain this
relationship between the two pathways, these two pathways can be
driven independently. ON and OFF channels of information traveling
from bipolar to ganglion cells are partially modulated through a
network of inhibitory amacrine cells within the inner nuclear layer
(Roska 2001). This bipolar-amacrine network produces
temporally-distinct parallel channels of information:
sustained-activity neurons, for example, maintain activity
throughout the light step, whereas transient-activity neurons have
activity only at the onset or offset. These distinct patterns of
response code for visual information luminance, shape, edges, and
motion (Wassle 2004). In some embodiments, cells that are
pre-synaptic to the retinal ganglion cells are genetically targeted
to maintain the naturalism of these pathways and elicit
naturalistic ganglion cell spiking.
[0109] In some embodiments, retinal bipolar cells (e.g. ON or OFF
retinal bipolar cells; rod and cone bipolar cells) are genetically
targeted. Targeting retinal bipolar cells may allow the retina to
respond to external light and, more importantly, convey meaningful
image information to the brain even in the absence of natural
photoreceptors.
Conditions Amenable to Treatment
[0110] In some embodiments, the present invention provides methods
of treating a subject suffering from a disease or disorder. The
compositions and methods described herein can be utilized to treat
central and peripheral nervous system diseases and disorders.
[0111] In one aspect, the compositions and methods of this
invention are utilized to treat photoreceptor diseases.
Photoreceptor diseases such as retinitis pigmentosa (RP) and
age-related macular degeneration (ARMD) cause blindness (Congdon
2004) in 15 million people worldwide (Chader 2002), a number that
is increasing with the age of the population. There have been
attempts to restore basic visual function through gene replacement
therapy or cellular transplantation (Acland 2001, Acland 2005,
Batten 2005, Pawlyk 2005, Aguirre 2007, MacLaren 2006). However,
current approaches are fundamentally limited in scope and extent of
potential impact, as they attempt to correct mechanistically
distinct genetic pathways on a one-at-a-time basis (Punzo 2007).
Photoreceptor diseases are genetically diverse, with over 160
different mutations leading to degeneration (Punzo 2007). There
have also been efforts in utilizing electrical stimulation with
implanted acute, semi-acute, and long-term retinal prostheses in
human subjects (de Balthasar, 2008; Horsager, 2009). They have
shown elementary progress but are gene-nonspecific; electrical
stimulation offers only gross specificity and indiscriminately
drives visual information channels mediated by unique cell types.
Activating retinal neurons requires large disc electrodes (at least
20 times the diameter of a retinal ganglion cell), leading to
stimulation of broad areas of retina in a nonselective fashion,
greatly limiting the achievable visual resolution (Winter 2007). In
this aspect, the compositions and methods of this invention consist
of introducing a gene encoding a light-sensitive protein (e.g.,
ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof) to induce light sensitivity in 2.sup.nd order
neurons (e.g., bipolar cells) delivered using a viral vector such
as an AAV8 with a single tyrosine to phenylalanine mutation, under
the control of a regulatory element (e.g., GRM6). The activation of
these light-sensitive proteins could be controlled by ambient light
or through a light-delivery device such as the goggles described in
FIG. 13.
[0112] The methods of the invention can be used to treat (e.g.,
prior to or after the onset of symptoms) in a susceptible subject
or subject diagnosed with a variety of eye diseases. The eye
disease may be a results of environmental (e.g., chemical insult,
thermal insult, and the like), mechanical insult (e.g., injury due
to accident or surgery), or genetic factors. The subject having the
condition may have one or both eyes affected, and therapy may be
administered according to the invention to the affected eye or to
an eye at risk of photoreceptor degeneration due to the presence of
such a condition in the subject's other, affected eye.
[0113] The present invention provides methods which generally
comprise the step of intraocularly administering (e.g., by
subretinal injection or by intravitreal injection) a gene delivery
vector which directs the expression of a light-sensitive protein to
the eye to treat, prevent, or inhibit the onset or progression of
an eye disease. As utilized herein, it should be understood that
the terms "treated, prevented, or, inhibited" refers to the
alteration of a disease onset, course, or progress in a
statistically significant manner.
[0114] Another condition amenable to treatment according to the
invention is Age-related Macular Degeneration (AMD). The macula is
a structure near the center of the retina that contains the fovea.
This specialized portion of the retina is responsible for the
high-resolution vision that permits activities such as reading. The
loss of central vision in AMD is devastating. Degenerative changes
to the macula (maculopathy) can occur at almost any time in life
but are much more prevalent with advancing age. Conventional
treatments are short-lived, due to recurrent choroidal
neovascularization. AMD has two primary pathologic processes,
choroidal neovascularization (CNV) and macular photoreceptor cell
death.
[0115] Exemplary conditions of particular interest which are
amenable to treatment according to the methods of the invention
include, but are not necessarily limited to, retinitis pigmentosa
(RP), diabetic retinopathy, and glaucoma, including open-angle
glaucoma (e.g., primary open-angle glaucoma), angle-closure
glaucoma, and secondary glaucomas (e.g., pigmentary glaucoma,
pseudoexfoliative glaucoma, and glaucomas resulting from trauma and
inflammatory diseases).
[0116] Further exemplary conditions amenable to treatment according
to the invention include, but are not necessarily limited to,
retinal detachment, age-related or other maculopathies, photic
retinopathies, surgery-induced retinopathies, toxic retinopathies,
retinopathy of prematurity, retinopathies due to trauma or
penetrating lesions of the eye, inherited retinal degenerations,
surgery-induced retinopathies, toxic retinopathies, retinopathies
due to trauma or penetrating lesions of the eye.
[0117] Specific exemplary inherited conditions of interest for
treatment according to the invention include, but are not
necessarily limited to, Bardet-Biedl syndrome (autosomal
recessive); Congenital amaurosis (autosomal recessive); Cone or
cone-rod dystrophy (autosomal dominant and X-linked forms);
Congenital stationary night blindness (autosomal dominant,
autosomal recessive and X-linked forms); Macular degeneration
(autosomal dominant and autosomal recessive forms); Optic atrophy,
autosomal dominant and X-linked forms); Retinitis pigmentosa
(autosomal dominant, autosomal recessive and X-linked forms);
Syndromic or systemic retinopathy (autosomal dominant, autosomal
recessive and X-linked forms); and Usher syndrome (autosomal
recessive).
[0118] In another aspect, the compositions and methods of this
invention are utilized to treat peripheral injury, nociception, or
chronic pain. Nociception (pain) for prolonged periods of time can
give rise to chronic pain and may arise from injury or disease to
visceral, somatic and neural structures in the body. Although the
range of pharmacological treatments for neuropathic pain has
improved over the past decade, many patients do not get effective
analgesia, and even effective medications often produce undesirable
side effects. Substance P (SP) is involved in nociception,
transmitting information about tissue damage from peripheral
receptors to the central nervous system to be converted to the
sensation of pain. It has been theorized that it plays a part in
fibromyalgia A role of substance P in nociception is suggested by
the reduction in response thresholds to noxious stimuli by central
administration of NK1 and NK2 agonists. Pain behaviors induced by
mechanical, thermal and chemical stimulation of somatic and
visceral tissues were reduced in the mutant mice lacking SP/NKA. In
one embodiment light-sensitive proteins can silence the activity of
over-active neurons (i.e., substance P expressing peripheral
neurons) due to peripheral injury or chronic pain using NpHR or
eNpHR. NpHR/eNpHR can be genetically targeted to substance P
expressing cells using the substance P promoter sequence. In
another embodiment light-sensitive proteins enhance the activity of
neurons that are inactive due to peripheral injury or chronic
pain.
[0119] In another aspect, the compositions and methods of this
invention are utilized to treat spinal cord injury and/or motor
neuron diseases. Spinal cord injury can cause myelopathy or damage
to white matter and myelinated fiber tracts that carry sensation
and motor signals to and from the brain. It can also damage gray
matter in the central part of the spine, causing segmental losses
of interneurons and motor neurons. Spinal cord injury can occur
from many causes, including but not limited to trauma, tumors,
ischemia, abnormal development, neurodevelopmental,
neurodegenerative disorders or vascular malformations. In one
embodiment light-sensitive proteins activate damaged neural
circuits to restore motor or sensory function. In one specific
embodiment the elements act to allow control of autonomic and
visceral functions. In other embodiments the elements act to allow
control of somatic skeletal function. The neural control of storage
and voiding of urine is complex and dysfunction can be difficult to
treat. One treatment for people with refractory symptoms is
continuous electrical nerve stimulation of the sacral nerve roots
using implanted electrodes and an implanted pulse generator.
However, stimulation of this nerve root can result in a number of
different complications or side effects. Being able to directly
control the sacral nerve through genetically-targeted tools would
be highly beneficial. In one embodiment, both ChR2 and NpHR could
be expressed in this nerve to control storage and voiding of the
bladder.
[0120] In another aspect, the compositions and methods of this
invention are utilized to treat Parkinson's disease. Parkinson's
disease belongs to a group of conditions called movement disorders.
They are characterized by muscle rigidity, tremor, a slowing of
physical movement (bradykinesia) and, in extreme cases, a loss of
physical movement (akinesia). The primary symptoms are the results
of decreased stimulation of the motor cortex by the basal ganglia,
normally caused by the insufficient formation and action of
dopamine, which is produced in the dopaminergic neurons of the
brain. Parkinson's disease is both chronic and progressive. Deep
brain stimulation (DBS) is an effective surgical treatment for
advanced Parkinson's disease (PD), with significant advantages in
morbidity-mortality and quality of life when compared to lesion
techniques such as thalamotomy and/or pallidotomy. The procedure is
indicated in patients with severe resting tremor, unresponsive to
conventional medical treatment or with motor complications. The
most commonly reported complications in the intra- and
post-surgical period are aborted procedure, misplaced leads,
intracranial hemorrhage, seizures and hardware complications,
whereas in the long-term period, symptoms may include high level
cognitive dysfunction, psychiatric, and subtle language problems.
Indeed, this method of therapy would be improved by being able to
target specific cell types within a given region to avoid these
side effects. In one embodiment light-sensitive proteins
specifically activate dopaminergic circuits.
[0121] In another specific aspect, the compositions and methods of
this invention are utilized to treat epilepsy and seizures.
Epilepsy is a neurological disorder that is often characterized by
seizures. These seizures are transient signs and/or symptoms due to
abnormal, excessive or asynchronous neuronal activity in the brain.
Over 30% of people with epilepsy do not have seizure control even
with the best available medications. Epilepsy is not a single
disorder, but rather as a group of syndromes with vastly divergent
symptoms but all involving episodic abnormal electrical activity in
the brain. Acute deep brain stimulation (DBS) in various thalamic
nuclei and medial temporal lobe structures has recently been shown
to be efficacious in small pilot studies. There is little
evidence-based information on rational targets and stimulation
parameters. Amygdalohippocampal DBS has yielded a significant
decrease of seizure counts and interictal EEG abnormalities during
long-term follow-up. Data from pilot studies suggest that chronic
DBS for epilepsy may be a feasible, effective, and safe procedure.
Again, being able to genetically-target activation to specific
subsets of cells would improve the quality of the therapy as well
as minimize overall side effects. In specific embodiments, the
light-sensitive proteins are utilized to alter the asynchronous
electrical activity leading to seizures in these deep brain
areas.
[0122] In another aspect the compositions and methods of this
invention are utilized to effect the light-stimulated release of
implanted drug or vaccine stores for the prevention, treatment, and
amelioration of diseases.
[0123] In another aspect the compositions and methods of this
invention are utilized to treat neurodegenerative disease selected
from but not limited to alcoholism, Alexander's disease, Alper's
disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia
telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform
encephalopathy (BSE), chronic pain, Canavan disease, Cockayne
syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,
Huntington's disease, HIV-associated dementia, Kennedy's disease,
Krabbe's disease, Lewy body dementia, Machado-Joseph disease
(Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple
System Atrophy, Narcolepsy, Neuroborreliosis, Parkinson's disease,
Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral
sclerosis, Prion diseases, Refsum's disease, Sandhoffs disease,
Schilder's disease, Subacute combined degeneration of spinal cord
secondary to Pernicious Anaemia, Schizophrenia,
Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten
disease), Spinocerebellar ataxia (multiple types with varying
characteristics), Spinal muscular atrophy,
Steele-Richardson-Olszewski disease, and Tables dorsalis.
[0124] In another aspect the compositions and methods of this
invention are utilized to treat a neurodevelopmental disease
selected from but not limited to attention deficit hyperactivity
disorder (ADHD), attention deficit disorder (ADD), schizophrenia,
obsessive-compulsive disorder (OCD), mental retardation, autistic
spectrum disorders (ASD), cerebral palsy, Fragile-X Syndrome, Downs
Syndrome, Rett's Syndrome, Asperger's syndrome, Williams-Beuren
Syndrome, childhood disintegrative disorder, articulation disorder,
learning disabilities (i.e., reading or arithmetic), dyslexia,
expressive language disorder and mixed receptive-expressive
language disorder, verbal or performance aptitude. Diseases that
can result from aberrant neurodevelopmental processes can also
include, but are not limited to bi-polar disorders, anorexia,
general depression, seizures, obsessive compulsive disorder (OCD),
anxiety, bruixism, Angleman's syndrome, aggression, explosive
outburst, self injury, post traumatic stress, conduct disorders,
Tourette's disorder, stereotypic movement disorder, mood disorder,
sleep apnea, restless legs syndrome, dysomnias, paranoid
personality disorder, schizoid personality disorder, schizotypal
personality disorder, antisocial personality disorder, borderline
personality disorder, histrionic personality disorder, narcissistic
personality disorder, avoidant personality disorder, dependent
personality disorder, reactive attachment disorder; separation
anxiety disorder; oppositional defiant disorder; dyspareunia,
pyromania, kleptomania, trichotillomania, gambling, pica, neurotic
disorders, alcohol-related disorders, amphetamine-related
disorders, cocaine-related disorders, marijuana abuse,
opioid-related disorders, phencyclidine abuse, tobacco use
disorder, bulimia nervosa, delusional disorder, sexual disorders,
phobias, somatization disorder, enuresis, encopresis, disorder of
written expression, expressive language disorder, mental
retardation, mathematics disorder, transient tic disorder,
stuttering, selective mutism, Crohn's disease, ulcerative colitis,
bacterial overgrowth syndrome, carbohydrate intolerance, celiac
sprue, infection and infestation, intestinal lymphangiectasia,
short bowel syndrome, tropical sprue, Whipple's disease,
Alzheimer's disease, Parkinson's Disease, ALS, spinal muscular
atrophies, and Huntington's Disease. Further examples, discussion,
and information on neurodevelopmental disorders can be found, for
example, through the Neurodevelopmental Disorders Branch of the
National Institute of Mental Health (worldwide website address at
nihm.nih.gov/dptr/b2-nd.cfm). Additional information on
neurodevelopmental disorders can also be found, for example, in
Developmental Disabilities in Infancy and Childhood:
Neurodevelopmental Diagnosis and Treatment, Capute and Accardo,
eds. 1996, Paul H Brookes Pub Co.; Hagerman, Neurodevelopmental
Disorders: Diagnosis and Treatment, 1999, Oxford Univ Press;
Handbook of Neurodevelopmental and Genetic Disorders in Children,
Goldstein and Reynolds, eds., 1999, Guilford Press; Handbook of
Neurodevelopmental and Genetic Disorders in Adults, Reynolds and
Goldstein, eds., 2005, Guilford Press; and Neurodevelopmental
Disorders, Tager-Flusberg, ed., 1999, MIT Press.
Assessment of Therapy
[0125] The effects of therapy according to the invention as
described herein can be assessed in a variety of ways, using
methods known in the art. For example, the subject's vision can be
tested according to conventional methods. Such conventional methods
include, but are not necessarily limited to, electroretinogram
(ERG), focal ERG, tests for visual fields, tests for visual acuity,
ocular coherence tomography (OCT), Fundus photography, Visual
Evoked Potentials (VEP) and Pupillometry. In other embodiments, the
subject can be assessed behaviorally. In general, the invention
provides for maintenance of a subject's vision (e.g., prevention or
inhibition of vision loss of further vision loss due to
photoreceptor degeneration), slows onset or progression of vision
loss, or in some embodiments, provides for improved vision relative
to the subject's vision prior to therapy.
Methods of Administration
[0126] The gene delivery vectors of the present invention can be
delivered to the eye through a variety of routes. They may be
delivered intraocularly, by topical application to the eye or by
intraocular injection into, for example the vitreous (intravitreal
injection) or subretinal (subretinal injection) inter-photoreceptor
space. Alternatively, they may be delivered locally by insertion or
injection into the tissue surrounding the eye. They may be
delivered systemically through an oral route or by subcutaneous,
intravenous or intramuscular injection. Alternatively, they may be
delivered by means of a catheter or by means of an implant, wherein
such an implant is made of a porous, non-porous or gelatinous
material, including membranes such as silastic membranes or fibers,
biodegradable polymers, or proteinaceous material. The gene
delivery vector can be administered prior to the onset of the
condition, to prevent its occurrence, for example, during surgery
on the eye, or immediately after the onset of the pathological
condition or during the occurrence of an acute or protracted
condition.
[0127] In another embodiment the inner limiting membrane (ILM) is
broken down to effect delivery. The ILM is the boundary between the
retina and the vitreous body, formed by astrocytes and the end feet
of Muller cells. In both nonhuman primates and humans, the ILM is
thick and provides a substantial barrier to the retina. Indeed,
using intravitreal injections, most viral particles are incapable
of transducing retinal cells. In one embodiment, to improve
transduction efficiency, an ILM peel is conducted comprising
carrying out a surgical procedure that comprises peeling off a
small part of the ILM. In another embodiment, to improve
transduction efficiency, the ILM barrier can be partially or wholly
broken down comprising using enzymatic techniques and one or more
enzymes.
[0128] In one embodiment the ILM is maintained to limit the
therapeutic effect of the light-sensitive protein to the macula. In
another embodiment the ILM peel procedure and/or the ILM enzymatic
digestion procedure, both described herein is used to achieve a
broader distribution of the light-sensitive protein.
[0129] The gene delivery vector can be modified to enhance
penetration of the blood-retinal barrier. Such modifications may
include increasing the lipophilicity of the pharmaceutical
formulation in which the gene delivery vector is provided.
[0130] The gene delivery vector can be delivered alone or in
combination, and may be delivered along with a pharmaceutically
acceptable vehicle. Ideally, such a vehicle would enhance the
stability and/or delivery properties. The invention also provides
for pharmaceutical compositions containing the active factor or
fragment or derivative thereof, which can be administered using a
suitable vehicle such as liposomes, microparticles or
microcapsules. In various embodiments of the invention, it may be
useful to use such compositions to achieve sustained release of the
active component.
[0131] The amount of gene delivery vector (e.g., the number of
viral particles), and the amount of light-sensitive protein
expressed, effective in the treatment of a particular disorder or
condition may depend of the nature of the disorder or condition and
a variety of patient-specific factors, and can be determined by
standard clinical techniques.
[0132] In one embodiment, the gene delivery vectors are
administered to the eye, intraocularly to a variety of locations
within the eye depending on the type of disease to be treated,
prevented, or, inhibited, and the extent of disease. Examples of
suitable locations include the retina (e.g., for retinal diseases),
the vitreous, or other locations in or adjacent the retina or in or
adjacent the eye.
[0133] The human retina is organized in a fairly exact mosaic. In
the fovea, the mosaic is a hexagonal packing of cones. Outside the
fovea, the rods break up the close hexagonal packing of the cones
but still allow an organized architecture with cones rather evenly
spaced surrounded by rings of rods. Thus in terms of densities of
the different photoreceptor populations in the human retina, it is
clear that the cone density is highest in the foveal pit and falls
rapidly outside the fovea to a fairly even density into the
peripheral retina (see Osterberg, G. (1935) Topography of the layer
of rods and cones in the human retina. Acta Ophthal. (suppl.) 6,
1-103; see also Curcio, C. A., Sloan, K. R., Packer, O.,
Hendrickson, A. E. and Kalina, R. E. (1987) Distribution of cones
in human and monkey retina: individual variability and radial
asymmetry. Science 236, 579-582).
[0134] Access to desired portions of the retina, or to other parts
of the eye may be readily accomplished by one of skill in the art
(see, generally Medical and Surgical Retina: Advances,
Controversies, and Management, Hilel Lewis, Stephen J. Ryan, Eds.,
medical-"illustrator, Timothy C. Hengst. St. Louis: Mosby, c1994.
xix, 534; see also Retina, Stephen J. Ryan, editor in chief, 2nd
ed., St. Louis, Mo.: Mosby, c1994. 3 v. (xxix, 2559).
[0135] In one embodiment, the amount of the specific viral vector
applied to the retina is uniformly quite small as the eye is a
relatively contained structure and the agent is injected directly
into it. The amount of vector that needs to be injected is
determined by the intraocular location of the chosen cells targeted
for treatment. The cell type to be transduced may be determined by
the particular disease entity that is to be treated.
[0136] For example, a single 20-microliter volume (e.g., containing
about 10.sup.13 physical particle titer/ml rAAV) may be used in a
subretinal injection to treat the macula and fovea of a human eye.
A larger injection of 50 to 100 microliters may be used to deliver
the rAAV to a substantial fraction of the retinal area, perhaps to
the entire retina depending upon the extent of lateral spread of
the particles.
[0137] A 100 microliter injection may provide several million
active rAAV particles into the subretinal space. This calculation
is based upon a titer of 10.sup.13 physical particles per
milliliter. Of this titer, it is estimated that 1/1000 to 1/10,000
of the AAV particles are infectious. The retinal anatomy constrains
the injection volume possible in the subretinal space (SRS).
Assuming an injection maximum of 100 microliters, this could
provide an infectious titer of 10.sup.5 to 10.sup.9 rAAV in the
SRS. This would have the potential of infecting all of the
approximately 150.times.10.sup.6 photoreceptors in the entire human
retina with a single injection.
[0138] Smaller injection volumes focally applied to the fovea or
macula may adequately transfect the entire region affected by the
disease in the case of macular degeneration or other regional
retinopathies.
[0139] Depending on the application at least 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or more particles can
be delivered into the tissue of interest.
[0140] Gene delivery vectors can alternately be delivered to the
eye by intraocular injection into the vitreous, e.g., to treat
glaucomatous loss of retinal ganglion cells through apoptosis. In
the treatment of glaucoma, the primary target cells to be
transduced are the retinal ganglion cells, the retinal cells
primarily affected. In such an embodiment, the injection volume of
the gene delivery vector could be substantially larger, as the
volume is not constrained by the anatomy of the subretinal space.
Acceptable dosages in this instance can range from about 25
microliters to 1000 microliters.
Pharmaceutical Compositions
[0141] Gene delivery vectors can be prepared as a pharmaceutically
acceptable composition suitable for administration. In general,
such pharmaceutical compositions comprise an amount of a gene
delivery vector suitable for delivery of light-sensitive
protein-encoding polynucleotide to a cell of the eye for expression
of a therapeutically effective amount of the light-sensitive
protein, combined with a pharmaceutically acceptable carrier or
excipient. Preferably, the pharmaceutically acceptable carrier is
suitable for intraocular administration. Exemplary pharmaceutically
acceptable carriers include, but are not necessarily limited to,
saline or a buffered saline solution (e.g., phosphate-buffered
saline).
[0142] Various pharmaceutically acceptable excipients are well
known in the art. As used herein, "pharmaceutically acceptable
excipient" includes any material which, when combined with an
active ingredient of a composition, allows the ingredient to retain
biological activity, preferably without causing disruptive
reactions with the subject's immune system or adversely affecting
the tissues surrounding the site of administration (e.g., within
the eye).
[0143] Exemplary pharmaceutically carriers include sterile aqueous
of non-aqueous solutions, suspensions, and emulsions. Examples
include, but are not limited to, any of the standard pharmaceutical
excipients such as a saline, buffered saline (e.g., phosphate
buffered saline), water, emulsions such as oil/water emulsion, and
various types of wetting agents.
[0144] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, hyaluronic acid, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate.
[0145] Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's or fixed
oils. Intravenous vehicles can include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like.
[0146] A composition of gene delivery vector of the invention may
also be lyophilized using means well known in the art, for
subsequent reconstitution and use according to the invention. Where
the vector is to be delivered without being encapsulated in a viral
particle (e.g., as "naked" polynucleotide), formulations for
liposomal delivery, and formulations comprising microencapsulated
polynucleotides, may also be of interest.
[0147] Compositions comprising excipients are formulated by well
known conventional methods (see, for example, Remington's
Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Col,
Easton Pa. 18042, USA).
[0148] In general, the pharmaceutical compositions can be prepared
in various forms, preferably a form compatible with intraocular
administration. Stabilizing agents, wetting and emulsifying agents,
salts for varying the osmotic pressure or buffers for securing an
adequate pH value may also optionally be present in the
pharmaceutical composition.
[0149] The amount of gene delivery vector in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.1%,
usually at or at least about 2% to as much as 20% to 50% or more by
weight, and may be selected primarily by fluid volumes,
viscosities, etc., in accordance with the particular mode of
administration selected.
[0150] The pharmaceutical composition can comprise other agents
suitable for administration, which agents may have similar to
additional pharmacological activities to the light-sensitive
protein to be delivered (e.g., ChR1, ChR2, VChR1, ChR2 C128A, ChR2
C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF,
ChIEF, NpHR, eNpHR, melanopsin, and variants thereof).
Kits
[0151] The invention also provides kits comprising various
materials for carrying out the methods of the invention. In one
embodiment, the kit comprises a vector encoding a light-sensitive
protein polypeptide (e.g. ChR1, ChR2, VChR1, ChR2 C128A, ChR2
C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF,
ChIEF, NpHR, eNpHR, melanopsin, and variants thereof), which vector
is adapted for delivery to a subject, particularly an eye of the
subject, and adapted to provide for expression of the
light-sensitive polypeptide in a cell of an eye, particularly a
mammalian cell. The kit can comprise the vector in a sterile vial,
which may be labeled for use. The vector can be provided in a
pharmaceutical composition. In one embodiment, the vector is
packaged in a virus. The kit can further comprise a needle and/or
syringe suitable for use with the vial or, alternatively,
containing the vector, which needle and/or syringe are preferably
sterile. In another embodiment, the kit comprises a catheter
suitable for delivery of a vector to the eye, which catheter may be
optionally attached to a syringe for delivery of the vector. The
kits can further comprise instructions for use, e.g., instructions
regarding route of administration, dose, dosage regimen, site of
administration, and the like.
Devices
[0152] The data in FIG. 12C demonstrate that the delivery of
light-sensitive proteins can work in the range of normal vision. In
some embodiments, for greater efficacy, an internal or external
device may be used. In one embodiment an external device, such as a
goggle, can be used for generation and/or amplification of light.
In embodiments where a subject having partial vision is being
treated, i.e. a treatment of a subject whose photoreceptors are
only partially damaged and a light-sensitive protein such as ChR1,
ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2
hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin,
and variants thereof is being delivered, the stimulation may be
adjusted so that the surviving and/or healthy photoreceptors are
not overdriven by the light generation/amplification device. In
various embodiments, limiting overdriving by the light
generation/amplification device can be achieved by i) stimulating
evenly, but shielding the surviving or healthy photoreceptor cells
from bright light through an implanted or external contact-lens
type partial sunglass (tinting over photoreceptors, clear over
light-sensitive protein transduction area); ii) adjustment of the
stimulation intensity to match the cell types being stimulated; or
iii) adjustment of the stimulation to the be near the top of the
visual dynamic range.
[0153] In one embodiment, an internal light-generating device is
implanted.
[0154] In another embodiment, a protective optic, or a contact
lens-type barrier is implanted either in conjunction with or
independent of the device. In a specific embodiment such an optic
or contact lens protects photoreceptors from light stimulation. In
a specific embodiment the lens comprises tinting over
photoreceptors, and clear over light-sensitive protein transduction
area.
[0155] In some embodiments a head-mounted, external device or
eyewear is utilized. In certain embodiments where the
light-sensitive element is not triggered to the extent desired by
natural or ambient light, an additional light production or
generation source such as a LED array/laser system is provided. In
certain embodiments the external eyewear can additionally include a
camera and an image processing unit for the filtering, enhancement,
processing, and resolution of the presented images. FIG. 13 depicts
a goggle-like device with an associated light production element
(LED array/laser system) that may trigger expression of
light-sensitive proteins.
[0156] In one embodiment, an exemplary camera system would comprise
at least three main components: 1) A small camera built into the
glasses, 2) an imaging processing unit, and 3) a light delivery
system that includes either or both LEDs or a laser system. The
camera could either be a single lens camera or a dual camera system
that could potentially provide binocular imaging and depth
information. The camera could capture either visual light or
infrared light. The camera could either be adaptive to various
lighting conditions or could be fixed. The image processing unit
(IPU) could provide any number of signal transformations including
amplification, increased or decreased contrast, structure from
motion, edge enhancement, or temporal filtering (i.e.,
integration). Additionally, saliency algorithms could be employed
such that only certain objects within the field of view are
enhanced (e.g., moving cars, doorways) and less important objects
(e.g., clouds), are filtered out. The LED and/or laser lighting
array system could contain a high-density LED array or a scanning
laser system that consists of either one (1) or more lasers. The
position of the lights could be either fixed or could move. For
example, the orientation of the lights relative to the eye could
move as a function of eye movements, using an eye movement tracking
device as an input. This is depicted in FIG. 13.
[0157] In another related exemplary embodiment, an image
intensifying device, such as those provided by Telesensory
(http://www.telesensory.com), may be combined with a retinal
scanning device (RSD) as developed by Microvision
(http://www.microvision.com/milprod.html), to provide a head-worn
apparatus capable of delivering a bright, intensified image
directly to the retina of a patient with impaired vision. Briefly,
a RSD projects images onto the retina such that an individual can
view a large, full-motion image without the need for additional
screens or monitors. Thus, by projecting an intensified image
directly onto the retina of an individual with impaired vision, it
may be possible to improve vision in those considered to be blind
or near-blind.
[0158] While some embodiments of the present invention have been
shown and described herein, it will be obvious to those skilled in
the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to
those skilled in the art without departing from the invention. It
should be understood that various alternatives to the embodiments
of the invention described herein may be employed in practicing the
invention.
EXAMPLES
Example 1
Injection Methods
[0159] All procedures in animals were handled according to the
statement for the use of animals in Ophthalmic and Vision Research
of the Association of Research in Vision and Ophthalmology and the
guidelines of the Institutional Animal Care and Use Committee at
the University of Florida.
[0160] For intravitreal injections, mice were anesthetized with
ketamine (72 mg/kg)/xylazine (4 mg/kg) by intraperitoneal
injection. Following anesthetization, a Hamilton syringe fitted
with a 33-gauge beveled needle was used. The needle was passed
through the sclera, at the equator, next to the limbus, into the
vitreous cavity. Injection occurred with direct observation of the
needle in the center of the vitreous cavity. The total volume
delivered was 1.5 containing different concentrations of the AAV
vectors tested.
[0161] For subretinal injections, one hour before the anesthesia,
eyes of mice were dilated with eye drops of 1% atropine, followed
by topical administration of 2.5% phenylephrine. Mice were then
anesthetized with ketamine (72 mg/kg)/xylazine (4 mg/kg) by
intraperitoneal injection An aperture within the pupil was made
through the cornea with a 301/2-gauge disposable needle and a
33-gauge unbeveled blunt needle in a Hamilton syringe was
introduced through the corneal opening into the subretinal space
and 1.5 .mu.l of AAV was delivered.
[0162] Typical titers of the AAV vectors were between
1.3.times.10.sup.12 and 3.0.times.10.sup.13.
Example 2
Screening for AAV Serotypes 1, 2, 5, 7, 8, and 9 for Transduction
of Retinal Bipolar Cells
[0163] Screening of known and characterized viral vectors for
optimal transduction of retina bipolar cells was carried out. AAV
serotypes 1, 2, 5, 7, 8 and 9 carrying green fluorescent protein
(GFP) were individually subretinally injected in 4 week old rd1
mice. Rd1 homozygous mice carry a rd1 mutation and rod
photoreceptor degeneration in these mice begins around postnatal
day (P)10 and is almost completed by P21. GFP was placed under
control of the strong, non-cell type specific promoter CBA (fusion
of the CMV immediate early enhancer and the bovine .beta.-actin
promoter plus intron1-exon1 junction). 1 month later, mice were
tested for expression of GFP. Double labeling with the PKC.alpha.
antibody of mice injected with AAV7 demonstrated that the
transduced cells were most likely residual (no outer segment)
photoreceptors rather than bipolar cells. Subretinal injections
with AAV7 were then performed in 8 week old mice, where there is
less of a chance of residual photoreceptors. It was found that AAV7
was highly effective at transducing retinal bipolar cells, leading
to GFP expression in at least 75% of all bipolar cells after a
single injection (FIG. 9). These images were obtained 16 weeks
after injection, which additionally show that GFP expression using
an AAV7 delivery mechanism is stable for at least 4 months. AA7 is
a serotype that can be utilized to transduce bipolar cells in an
effective and stable manner.
Example 3
Transduction of Retinal Bipolar Cells with Serotypes AAV5, AAV2
Y444F Mutant, and AAV8 Y733F Mutant
[0164] As depicted in FIG. 10, mice were subretinally (right eye)
and intravitreally (left eye) injected with 1.5 .mu.l of
adeno-associated viruses (AAV) of different serotypes. The
serotypes tested included AAV2, AAV5, and AAV8, all of which are
traditional wild type serotypes. Additionally, single tyrosine to
phenylalanine mutated serotypes AAV2 Y444F mutant and AAV8 Y733F
mutant, where 444 and 733 indicate the location of the point
tyrosine mutation of the viral capsid, respectively. The virus
contained the self-complementary DNA construct GRM6-ChR2-GFP, where
GRM6 is the metabotropic glutamate receptor 6 regulatory sequence
driving cell-specific expression in the ON bipolar cells (including
rod bipolar), ChR2 is the therapeutic, light-sensitive protein
gene, and GFP is the reporter gene.
[0165] The images in FIG. 10 show the overall expression of GFP
(the reporter gene). This expression is shown as white in the black
and white images. This is indicative of the overall expression of
ChR2 as the ChR2-GFP complex is a fused protein. Note the ringlets
of GFP expression in the INL, showing expression of the ChR2-GFP
protein complex is membrane bound. These data show that delivery of
the construct with an adeno-associated virus leads to robust
expression of ChR2-GFP in all 3 mouse models of blindness (rd1,
rd16, and rho -/-). This is conducted with 3 different serotypes
using a subretinal injection (column 1). When using a tyrosine to
phenylalanine mutant serotype, it is possible to get good
expression in bipolar cells (INL) using either a subretinal or
intravitreal injection. However, wild type serotypes require a
subretinal injection to get reasonable transduction of bipolar
cells; intravitreal injections using wild type serotypes do not
effectively transduce bipolar cells (column 2).
Example 4
Creation of AAV7-GRM6-ChR2 to Establish Light Sensitivity in
Retinal On-Bipolar Cells
[0166] mGluR6 is a G-protein coupled metabotropic glutamate
receptor that is, in the retina, specifically expressed in ON
bipolar cells (Tian 2006). The adeno-associated virus, serotype 7
(AAV7), an mGluR6 regulatory sequence fragment gene sequence
(presented in FIG. 6), GRM6, and channelrhodopsin-2, ChR2 was
constructed to form the AAV7-GRM6-ChR2 construct. The cDNA encoding
ChR2 with eGFP was cloned downstream (i.e., 3') of the mGluR6
regulatory sequence fragment-SV40 minimal promoter. No IRES was
used; ChR2 and GFP were fused. This viral vector construct once
delivered using a viral delivery mechanism, and expressed, can
establish photosensitivity in retinal ON bipolar cells with
high-spatial and temporal resolution. This method can restore
retinal responsiveness to optical information, using the ChR2 class
of light-activated molecules to directly sensitize spared retinal
neurons to light.
Example 5
AAV8 Mutant Y733F-GRM6-ChR2 and AAV8 Mutant Y446F-CBA-ChR2 to
Establish Light Sensitivity in Retinal ON-Bipolar Cells
[0167] Using a tyrosine-mutated version of AAV8 (at the 733
location), under the control of bipolar cell specific promoter
GRM6, in the self-complementary configuration, it was possible to
restore visual behavioral efficacy in rd1 mice, as depicted in
FIGS. 11 and 12. FIG. 11 depicts the analysis of EGFP expression in
frozen retinal sections by immunohistochemistry at 1 month
following subretinal injections with the Tyrosine mutant AAV
vectors. Example sections depicting spread and intensity of EGFP
fluorescence throughout the retina after transduction with serotype
2 Y444 (a) or serotype 8 Y733 (b). The images are oriented with the
vitreous toward the bottom and the photoreceptor layer toward the
top. EGFP fluorescence in photoreceptors, RPE and ganglion cells
from mouse eyes injected subretinally with serotype 2 Y444 (c) EGFP
fluorescence in photoreceptors, RPE and Muller cells after serotype
8 Y733 delivery (d) Detection of Muller cells processes (red) by
immunostaining with a glutamine-synthetase (GS) antibody (e) Merged
image showing colocalization of EGFP fluorescence (green) and GS
staining (red) in retinal sections from eyes treated with serotype
8 Y733 (f) Calibration bar 100 .mu.M. gcl, ganglion cell layer;
ipl, inner plexiform layer; inl, inner nuclear layer; onl, outer
nuclear; os, outer segment; rpe, retinal pigment epithelium.
[0168] Using a tyrosine-mutated version of AAV8 (at the 446
location), under the control of the non-cell specific promoter CBA
(fusion of the CMV immediate early enhancer and the bovine
.beta.-actin promoter plus intron1-exon1 junction, and ChR2), in
the self-complementary configuration, most or all bipolar cells can
be targeted and visual function is restored as depicted in FIG.
12.
Example 6
AAV5-CBA-ChR2 to Establish Light Sensitivity in Retinal On Bipolar
Cells
[0169] Using the AAV5, non-cell type specific promoter CBA (fusion
of the CMV immediate early enhancer and the bovine .beta.-actin
promoter plus intron1-exon1 junction, and ChR2, in the
self-complementary configuration, all bipolar cells can be targeted
and visual function and behavior is restored (FIG. 12). Treated
mice were subretinally (right eye) and intravitreally (left eye)
injected with 1.5 .mu.l of adeno-associated viruses (AAV) of
different serotypes. The serotypes tested included AAV2, AAV5, and
AAV7, all of which are traditional wild type serotypes.
Additionally, the single tyrosine to phenylalanine mutated
serotypes AAV2 Y444F mutant and AAV8 Y733F mutant, where 444 and
733 indicate the location of the point tyrosine mutation of the
viral capsid, respectively. The virus contained the
self-complementary DNA construct GRM6-ChR2-GFP, where GRM6 is the
regulatory sequence driving cell-specific expression in the ON
bipolar cells (including rod bipolar), ChR2 is the therapeutic,
light-sensitive protein gene, and GFP is the reporter gene.
[0170] These mice were then trained on a water maze task FIG. 12A
for 14 days (7 days for the wild type mice) and the time to find
the target (a black platform with a 4.times.6 LED light source) was
recorded. FIG. 12B shows the average time it took for the treated,
untreated, and wild type mice to find the target, as a function of
the training session. Both the untreated and treated groups
contained samples from the rd1, rd16, and rho -/- (different mouse
models of blindness that have different types of gene mutations
that lead to photoreceptor disease) groups. These data demonstrate
that mice treated with ChR2 are able to learn a behavior task by
using visual information, suggesting that a light sensitive protein
such as ChR2 has the ability to restore at least some visual
function.
[0171] The animals' performance on the task was then evaluated at
different light levels. FIG. 12C shows the average time it took for
the rd1, rd16, and rho -/- treated, sham injected (sham injected
mice represent an average of rd1, rd16, and rho -/- untreated), and
wild type mice to find the target, as a function of the light
intensity. These data show that the treated mice can perform the
task at multiple light levels and their performance is dependent on
the amount of light presented.
Sequence CWU 1
1
817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Phe Cys Tyr Glu Asn Glu Val1
522236DNAChlamydomonas reinhardtii 2gcgttgcttg actacgcttc
gctgtaataa tagcagcgcc acaagtagtg tcgccaaaca 60actctcactt tgagcttgag
cacaccgctg agccccgatg tcgcggaggc catggcttct 120tgccctagcg
ctggcagtgg cgctggcggc cggcagcgca ggagcctcga ctggcagtga
180cgcgacggtg ccggtcgcga ctcaggatgg ccccgactac gttttccacc
gtgcccacga 240gcgcatgctc ttccaaacct catacactct tgagaacaat
ggttctgtta tttgcatccc 300gaacaacggc cagtgcttct gcttggcttg
gcttaaatcc aacggaacaa atgccgagaa 360gttggctgcc aacattctgc
agtggattac ttttgcgctt tcagcgctct gcctgatgtt 420ctacggctac
cagacctgga agtctacttg cggctgggag gagatttacg tggccacgat
480cgagatgatc aagttcatca tcgagtattt ccatgagttt gacgaacctg
cggtgatcta 540ctcatccaac ggcaacaaga ccgtgtggct tcgttacgcg
gagtggctgc tgacctgccc 600tgtcattctt atccatctga gcaaccttac
gggtctggcg aacgactata acaagcgtac 660catgggtctg ctggtgtcag
atatcggcac gatcgtgtgg ggcaccacgg ccgcgctgtc 720caagggatac
gtccgtgtca ttttcttcct gatgggcctg tgctacggca tctacacatt
780cttcaacgca gccaaggtct acattgaggc gtaccacacc gtgcccaagg
gcatttgccg 840cgacctggtc cgctaccttg cctggctcta cttctgttca
tgggctatgt tcccggtgct 900gttcctgctg ggccccgagg gctttggcca
catcaaccaa ttcaactctg ccatcgccca 960cgccatcctg gaccttgcct
ccaagaacgc ttggagtatg atgggtcact ttctgcgtgt 1020caagatccac
gagcacatcc tgctgtacgg cgacatccgc aagaagcaga aggtcaacgt
1080ggctggccag gagatggagg tggagaccat ggtgcacgag gaggacgacg
agacgcagaa 1140ggtgcccacg gcaaagtacg ccaaccgcga ctcgttcatc
atcatgcgcg accgcctcaa 1200ggagaagggc ttcgagaccc gcgcctcgct
ggacggcgac ccgaacggcg acgccgaggc 1260caacgctgca gccggcggca
agcccggaat ggagatgggc aagatgaccg gcatgggcat 1320gggcatgggt
gccggcatgg gcatggcgac catcgattcg ggccgcgtca tcctcgccgt
1380gccggacatc tccatggtgg actttttccg cgagcagttc gcgcggctgc
ccgtgcccta 1440cgaactggtg cccgcgctgg gcgcggagaa caccctccag
ctggtgcagc aggcgcagtc 1500actgggaggc tgcgacttcg tcctcatgca
ccccgagttc ctgcgcgacc gcagtcccac 1560gggtctgctg ccccgcctca
agatgggcgg gcagcgcgcc gcggccttcg gctgggcggc 1620aatcggcccc
atgcgggact tgatcgaggg ttcgggcgtt gacggctggc tggagggccc
1680cagctttggc gccggcatca accagcaggc gctggtggcg ctgatcaacc
gcatgcagca 1740ggccaagaag atgggcatga tgggcggtat gggtatgggc
atgggcggcg gcatgggtat 1800gggcatgggt atgggcatgg gcatggcccc
cagcatgaac gccggcatga ctggcggcat 1860gggcggcgcc tccatgggcg
gtgccgtgat gggcatgggc atgggcatgc agcccatgca 1920gcaggctatg
ccggccatgt cgcccatgat gactcagcag cccagcatga tgagtcagcc
1980ctccgccatg agcgccggcg gcgccatgca ggccatgggt ggcgtcatgc
ccagccccgc 2040ccccggcggc cgcgtgggca ccaacccgct gtttggctct
gcgccctctc cgctgagctc 2100gcagcccggc atcagccctg gcatggcgac
gccgcccgcc gccaccgccg cacccgccgc 2160tggcggcagc gaggccgaga
tgctgcagca gctgatgagc gagatcaacc gcctgaagaa 2220cgagctgggc gagtaa
223632241DNAChlamydomonas reinhardtii 3gcatctgtcg ccaagcaagc
attaaacatg gattatggag gcgccctgag tgccgttggg 60cgcgagctgc tatttgtaac
gaacccagta gtcgtcaatg gctctgtact tgtgcctgag 120gaccagtgtt
actgcgcggg ctggattgag tcgcgtggca caaacggtgc ccaaacggcg
180tcgaacgtgc tgcaatggct tgctgctggc ttctccatcc tactgcttat
gttttacgcc 240taccaaacat ggaagtcaac ctgcggctgg gaggagatct
atgtgtgcgc tatcgagatg 300gtcaaggtga ttctcgagtt cttcttcgag
tttaagaacc cgtccatgct gtatctagcc 360acaggccacc gcgtccagtg
gttgcgttac gccgagtggc ttctcacctg cccggtcatt 420ctcattcacc
tgtcaaacct gacgggcttg tccaacgact acagcaggcg caccatgggt
480ctgcttgtgt ctgatattgg cacaattgtg tggggcgcca cttccgccat
ggccaccgga 540tacgtcaagg tcatcttctt ctgcctgggt ctgtgttatg
gtgctaacac gttctttcac 600gctgccaagg cctacatcga gggttaccac
accgtgccga agggccggtg tcgccaggtg 660gtgactggca tggcttggct
cttcttcgta tcatggggta tgttccccat cctgttcatc 720ctcggccccg
agggcttcgg cgtcctgagc gtgtacggct ccaccgtcgg ccacaccatc
780attgacctga tgtcgaagaa ctgctggggt ctgctcggcc actacctgcg
cgtgctgatc 840cacgagcata tcctcatcca cggcgacatt cgcaagacca
ccaaattgaa cattggtggc 900actgagattg aggtcgagac gctggtggag
gacgaggccg aggctggcgc ggtcaacaag 960ggcaccggca agtacgcctc
ccgcgagtcc ttcctggtca tgcgcgacaa gatgaaggag 1020aagggcattg
acgtgcgcgc ctctctggac aacagcaagg aggtggagca ggagcaggcc
1080gccagggctg ccatgatgat gatgaacggc aatggcatgg gtatgggaat
gggaatgaac 1140ggcatgaacg gaatgggcgg tatgaacggg atggctggcg
gcgccaagcc cggcctggag 1200ctcactccgc agctacagcc cggccgcgtc
atcctggcgg tgccggacat cagcatggtt 1260gacttcttcc gcgagcagtt
tgctcagcta tcggtgacgt acgagctggt gccggccctg 1320ggcgctgaca
acacactggc gctggttacg caggcgcaga acctgggcgg cgtggacttt
1380gtgttgattc accccgagtt cctgcgcgac cgctctagca ccagcatcct
gagccgcctg 1440cgcggcgcgg gccagcgtgt ggctgcgttc ggctgggcgc
agctggggcc catgcgtgac 1500ctgatcgagt ccgcaaacct ggacggctgg
ctggagggcc cctcgttcgg acagggcatc 1560ctgccggccc acatcgttgc
cctggtggcc aagatgcagc agatgcgcaa gatgcagcag 1620atgcagcaga
ttggcatgat gaccggcggc atgaacggca tgggcggcgg tatgggcggc
1680ggcatgaacg gcatgggcgg cggcaacggc atgaacaaca tgggcaacgg
catgggcggc 1740ggcatgggca acggcatggg cggcaatggc atgaacggaa
tgggtggcgg caacggcatg 1800aacaacatgg gcggcaacgg aatggccggc
aacggaatgg gcggcggcat gggcggcaac 1860ggtatgggtg gctccatgaa
cggcatgagc tccggcgtgg tggccaacgt gacgccctcc 1920gccgccggcg
gcatgggcgg catgatgaac ggcggcatgg ctgcgcccca gtcgcccggc
1980atgaacggcg gccgcctggg taccaacccg ctcttcaacg ccgcgccctc
accgctcagc 2040tcgcagctcg gtgccgaggc aggcatgggc agcatgggag
gcatgggcgg aatgagcgga 2100atgggaggca tgggtggaat ggggggcatg
ggcggcgccg gcgccgccac gacgcaggct 2160gcgggcggca acgcggaggc
ggagatgctg cagaatctca tgaacgagat caatcgcctg 2220aagcgcgagc
ttggcgagta a 22414869DNANatronomonas pharaonis 4atgactgaga
ccctcccacc cgtgactgaa agcgccgtcg ctctgcaagc agaggttacc 60cagcgggagc
tgttcgagtt cgtcctcaac gaccccctcc tggcttctag cctctacatc
120aacatgctct ggcaggcctg tctatactgc tgttcgtctt catgaccagg
ggactcgatg 180accctagggc taaactgatt gcagtgagca caattctggt
tcccgtggtc tctatcgctt 240cctacactgg ctggcatctg gtctcacaat
cagtgtcctg gaaatgccag ctggccactt 300tgccgaaggg agttctgtca
tgctgggagg cgaagaggtc gatggggttg tcacaatgtg 360gggtcgctac
ccacctgggc tctcagtacc cccatgatcc tgctggcact cggactcctg
420gccggaagta acgccaccaa actcttcact gctattacat tcgatatcgc
catgtgcgtg 480accgggctcg cagctccctc accaccagca gccatctgat
gagatggttt tggtatgcca 540tctcttgtgc ctgctttctg gtggtgctgt
atatcctgct ggtggagtgg gctcaggatg 600ccaaggctgc agggacagcg
acatgtttaa tacactgaag ctgctcactg tggtgatgtg 660gctgggttac
cctatcgttt gggcactcgg cgtggaggga atcgcagttc tgcctgttgg
720tgtgacaagc tggggctact cctcctggac attgtggcca agtatatttt
tgcctttctg 780ctgctgaatt atctgacttc caatgagtcc gtggtgtccg
gctccatact ggacgtgcca 840tccgccagcg gcacacctgc cgatgctga
86951495DNAMus musculus 5gaggatccgc caccatgaac cctccttcgg
gccctagagt cctgcccagc ccaacccaag 60agcccagctg catggccacc ccagcaccac
ccagctggtg ggacagctcc cagagcagca 120tctccagcct gggccggctt
ccatccatca gtcccacagc acctgggact tgggctgctg 180cctgggtccc
cctccccacg gttgatgttc cagaccatgc ccactatacc ctgggcacag
240tgatcttgct ggtgggactc acgggcatgc ttggcaacct gacggtcatc
tataccttct 300gcaggagcag aagcctccgg acacctgcca acatgttcat
tatcaacctc gcggtcagcg 360acttcctcat gagtttcacc caggcccctg
tcttcttcac cagtagcctc tataagcagt 420ggctctttgg ggagacaggc
tgcgagttct atgccttctg tggagctctc tttggcattt 480cctccatgat
caccctgacg gccatcgccc tggaccgcta cctggtaatc acacgcccgc
540tggccacctt tggtgtggcg tccaagaggc gtgcggcatt tgtcctgctg
ggcgtttggc 600tctatgcgct agcttggagt ctgccaccct tcttcggctg
gagcgcctac gtgcccgagg 660ggttgctgac atcctgctcc tgggactaca
tgagcttcac gccggccgtg cgtgcctaca 720ccatgcttct ctgctgcttc
gtgttcttcc tccctttatt aattatcatc tactgctaca 780tcttcatctt
cagggccatc cgggagacag gacgggctct ccagaccttc ggggcctgca
840agggcaatgg cgagtccctg tggcagcggc agcggctgca gagcgagtgc
aagatggcca 900agatcatgct gctggtcatc ctcctcttcg tgctctcctg
ggctccctat tccgctgtgg 960ccctggtggc ctttgctggg tacgcacacg
tcctgacacc ctacatgagc tcggtgccag 1020ccgtcatcgc caaggcctct
gcaatccaca accccatcat ttacgccatc acccacccca 1080agtacagggt
ggccattgcc cagcacctgc cctgcctagg tgtgctgctg ggtgtatcac
1140gccggcacag tcgcccctac cccagctacc gctccaccca ccgctccacg
ctgaccagcc 1200acacctccaa cctcagctgg atctccatac ggaggcgcca
ggagtccctg ggctcggaga 1260gtgaggtggg ctggacacac atggaggcag
cagctgtgtg gggagctgcc cagcaagcaa 1320atgggcggtc cctctacggt
cagggtctgg aggacttgga agccaaggca ccccccagac 1380cccagggaca
cgaagcagag actccaggga agaccaaggg gctgatcccc agccaggacc
1440cgcggatggg cggcggcgac tacaaggacg atgatgacaa gtaataagaa ttcag
149561665DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 6atggactatg gcggcgcttt gtctgccgtc
ggacgcgaac ttttgttcgt tactaatcct 60gtggtggtga acgggtccgt cctggtccct
gaggatcaat gttactgtgc cggatggatt 120gaatctcgcg gcacgaacgg
cgctcagacc gcgtcaaatg tcctgcagtg gcttgcagca 180ggattcagca
ttttgctgct gatgttctat gcctaccaaa cctggaaatc tacatgcggc
240tgggaggaga tctatgtgtg cgccattgaa atggttaagg tgattctcga
gttctttttt 300gagtttaaga atccctctat gctctacctt gccacaggac
accgggtgca gtggctgcgc 360tatgcagagt ggctgctcac ttgtcctgtc
atccttatcc acctgagcaa cctcaccggc 420ctgagcaacg actacagcag
gagaaccatg ggactccttg tctcagacat cgggactatc 480gtgtgggggg
ctaccagcgc catggcaacc ggctatgtta aagtcatctt cttttgtctt
540ggattgtgct atggcgcgaa cacatttttt cacgccgcca aagcatatat
cgagggttat 600catactgtgc caaagggtcg gtgccgccag gtcgtgaccg
gcatggcatg gctgtttttc 660gtgagctggg gtatgttccc aattctcttc
attttggggc ccgaaggttt tggcgtcctg 720agcgtctatg gctccaccgt
aggtcacacg attattgatc tgatgagtaa aaattgttgg 780gggttgttgg
gacactacct gcgcgtcctg atccacgagc acatattgat tcacggagat
840atccgcaaaa ccaccaaact gaacatcggc ggaacggaga tcgaggtcga
gactctcgtc 900gaagacgaag ccgaggccgg agccgtgcca gcggcaccgg
tagtagcagt gagcaagggc 960gaggagctgt tcaccggggt ggtgcccatc
ctggtcgagc tggacggcga cgtaaacggc 1020cacaagttca gcgtgtccgg
cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 1080aagttcattt
gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
1140acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
cgacttcttc 1200aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
tcttcttcaa ggacgacggc 1260aactacaaga cccgcgccga ggtgaagttc
gagggcgaca ccctggtgaa ccgcatcgag 1320ctgaagggca tcgacttcaa
ggaggacggc aacatcctgg ggcacaagct ggagtacaac 1380tacaacagcc
acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
1440ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
ctaccagcag 1500aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
accactacct gagcacccag 1560tccgccctga gcaaagaccc caacgagaag
cgcgatcaca tggtcctgct ggagttcgtg 1620accgccgccg ggatcactct
cggcatggac gagctgtaca agtaa 16657199DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
7atctccagat ggctaaactt ttaaatcatg aatgaagtag atattaccaa attgcttttt
60cagcatccat ttagataatc atgttttttg cctttaatct gttaatgtag tgaattacag
120aaatacattt cctaaatcat tacatccccc aaatcgttaa tctgctaaag
tacatctctg 180gctcaaacaa gactggttg 1998952DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
8aattcggtac cctagttatt aatagtaatc aattacgggg tcattagttc atagcccata
60tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
120cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
tagggacttt 180ccattgacgt caatgggtgg actatttacg gtaaactgcc
cacttggcag tacatcaagt 240gtatcatatg ccaagtacgc cccctattga
cgtcaatgac ggtaaatggc ccgcctggca 300ttatgcccag tacatgacct
tatgggactt tcctacttgg cagtacatct acgtattagt 360catcgctatt
accatggtcg aggtgagccc cacgtttgct tcactctccc catctccccc
420ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
agcgatgggg 480gcgggggggg ggggggggcg cgcgccaggc ggggcggggc
ggggcgaggg gcggggcggg 540gcgaggcgga gaggtgcggc ggcagccaat
cagagcggcg cgctccgaaa gtttcctttt 600atggcgaggc ggcggcggcg
gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc 660gctgcgacgc
tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
720gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
ctcctccggg 780ctgtaattag cgcttggttt aatgacggct tgtttctttt
ctgtggctgc gtgaaagcct 840tgaggggctc cgggagctag agcctctgct
aaccatgttc atgccttctt ctttttccta 900cagctcctgg gcaacgtgct
ggttattgtg ctgtctcatc attttggcaa ag 952
* * * * *
References