U.S. patent application number 15/676573 was filed with the patent office on 2018-08-02 for restoration of visual responses by in vivo delivery of rhodopsin nucleic acids.
The applicant listed for this patent is Salus University, Wayne State University. Invention is credited to Alexander M. DIZHOOR, Zhuo-Hua PAN.
Application Number | 20180214513 15/676573 |
Document ID | / |
Family ID | 38668588 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180214513 |
Kind Code |
A1 |
PAN; Zhuo-Hua ; et
al. |
August 2, 2018 |
Restoration of Visual Responses by In Vivo Delivery of Rhodopsin
Nucleic Acids
Abstract
Nucleic acid vectors encoding light-gated cation-selective
membrane channels, in particular channelrhodopsin-2 (Chop2),
converted inner retinal neurons to photosensitive cells in
photoreceptor-degenerated retina in an animal model. Such treatment
restored visual perception and various aspects of vision. A method
of restoring light sensitivity to a retina of a subject suffering
from vision loss due to photoreceptor degeneration, as in retinitis
pigmentosa or macular degeneration, is provided. The method
comprises delivering to the subject by intravitreal or subretinal
injection, the above nucleic acid vector which comprises an open
reading frame encoding a rhodopsin, to which is operatively linked
a promoter and transcriptional regulatory sequences, so that the
nucleic acid is expressed in inner retinal neurons. These cells,
normally light-insensitive, are converted to a light-sensitive
state and transmit visual information to the brain, compensating
for the loss, and leading to restoration of various visual
capabilities.
Inventors: |
PAN; Zhuo-Hua; (Troy,
MI) ; DIZHOOR; Alexander M.; (Dresher, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wayne State University
Salus University |
Detriot
Elkins Park |
MI
PA |
US
US |
|
|
Family ID: |
38668588 |
Appl. No.: |
15/676573 |
Filed: |
August 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13899198 |
May 21, 2013 |
9730981 |
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15676573 |
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12299574 |
Sep 15, 2009 |
8470790 |
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PCT/US2007/068263 |
May 4, 2007 |
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13899198 |
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60797357 |
May 4, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2830/002 20130101;
A61K 48/005 20130101; C12N 2750/14143 20130101; A61K 38/177
20130101; A61P 27/02 20180101; C07K 14/705 20130101; C12N 2830/008
20130101; A61K 38/00 20130101; C12N 15/86 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/705 20060101 C07K014/705; C12N 15/86 20060101
C12N015/86 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
EY004068, EY012180EY016097 and EY011522 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of restoring light sensitivity to a retina, comprising:
(a) delivering to retinal neurons a nucleic acid expression vector
that encodes a light-gated channel rhodopsin or a light-driven ion
pump rhodopsin expressible in said neurons, which vector comprises
an open reading frame encoding the rhodopsin, and operatively
linked thereto, a promoter sequence, and optionally,
transcriptional regulatory sequences; and (b) expressing said
vector in said neurons, thereby restoring light sensitivity.
2. The method of claim 1 wherein the rhodopsin is
channelrhodopsin-2 (Chop2) with the sequence SEQ ID NO:6, or a
biologically active fragment thereof, preferably SEQ ID NO:3, or a
conservative amino acid substitution variant thereof.
3. The method of claim 1 wherein the vector is a rAAV viral
vector.
4. The method of claim 1 wherein the promoter is a constitutive
promoter.
5. The method of claim 4 wherein the constitutive promoter is a
hybrid CMV enhancer/chicken 13-actin promoter (CAG)
6. The method of claim 2 wherein the promoter is a hybrid CAG.
7. The method of claim 4 wherein the constitutive promoter is a CMV
promoter.
8. The method of claim 1 wherein the promoter is an inducible
and/or a cell type-specific promoter.
9. The method of claim 8 wherein the cell type-specific promoter is
selected from the group consisting of a mGluR6 promoter, a Pcp2
(L7) promoter or a neurokinin-3 (NK-3) promoter.
10. The method of claim 9 wherein the promoter is the mGlu6
promoter and is part of a promoter sequence SEQ ID NO:7.
11. The method of claim 1 wherein the vector comprises hybrid CMV
enhancer/chicken (3-actin (CAG) promoter, a woodchuck
posttranscriptional regulatory element (WPRE), and a human or
bovine growth hormone polyadenylation sequence.
12. The method of claim 2 wherein the vector comprises a CAG
promoter, a woodchuck post-transcriptional regulatory element
(WPRE), and a human or bovine growth hormone polyadenylation
sequence.
13. The method of claim 1 wherein the retinal neurons are selected
from ON- and OFF-type retinal ganglion cells, retinal rod bipolar
cells, All amacrine cells and ON and OFF retinal cone bipolar
cells.
14. The method of claim 13 wherein the vector is targeted to and
expressed in ON type ganglion cells and/or ON type bipolar
cells.
15. The method of claim 14 wherein the vector comprises a mGluR6
promoter
16. The method of claim 15 wherein the mGluR6 promoter is part of a
promoter sequence SEQ ID NO:7.
17. The method of claim 9 wherein the promoter is an NK-3 promoter
and the vector is targeted to OFF cone bipolar cells.
18. A method of restoring photosensitivity to retinal neurons of a
subject suffering from vision loss or blindness in whom retinal
photoreceptor cells are degenerating or have degenerated and died,
which method comprises: (a) delivering to the retina of said
subject a nucleic acid vector that encodes a light-gated channel
rhodopsin or a light-driven ion pump rhodopsin expressible in said
neurons; which vector comprises an open reading frame encoding the
rhodopsin, and operatively linked thereto, a promoter sequence, and
optionally, transcriptional regulatory sequences; (b) expressing
said vector in said neurons, wherein the expression of the
rhodopsin renders said neurons photosensitive, thereby restoring of
photosensitivity to said retina.
19. The method of claim 18 wherein the rhodopsin is Chop2 or a
biologically active fragment or conservative amino acid
substitution variant thereof.
20. The method of claim 18 wherein the vector is a rAAV viral
vector.
21. The method of claim 18 wherein the promoter is a constitutive
promoter.
22. The method of claim 21 wherein the constitutive promoter is a
hybrid CAG promoter.
23. The method of claim 19 wherein the promoter is a hybrid CAG
promoter.
24. The method of claim 21 wherein the constitutive promoter is a
CMV promoter.
25. The method of claim 18 wherein the promoter is an inducible or
a cell type-specific promoter.
26. The method of claim 25 wherein the cell type-specific promoter
is selected from the group consisting of a mGluR6 promoter, a Pcp2
(L7) promoter or a neurokinin-3 (NK-3) promoter.
27. The method of claim 26 wherein the promoter is the mGlu6
promoter and is part of a promoter sequence SEQ ID NO:7.
28. The method of claim 18 wherein the retinal neurons are ON-type
retinal ganglion cells, OFF-type retinal ganglion cells, retinal
rod bipolar cells, All amacrine cells, ON-type retinal cone bipolar
cells or OFF-type retinal cone bipolar cells.
29. The method of claim 28 wherein the vector is targeted to and
expressed in ON type ganglion cells and/or ON type bipolar
cells.
30. The method of claim 29 wherein the vector comprises a mGluR6
promoter
31. The method of claim 30 wherein the mGluR6 promoter is part of a
promoter sequence SEQ ID NO:7.
32. The method of claim 26 wherein the promoter is the NK-3
promoter and the vector is targeted to OFF cone bipolar cells.
33. The method claim 18 wherein the restoration of photosensitivity
results in restoration of vision in said subject. 34 The method of
claim 33 wherein said vision is measured by one or more of the
following methods: (i) a light detection response by the subject
after exposure to a light stimulus (ii) a light projection response
by the subject after exposure to a light stimulus; (iii) light
resolution by the subject of a light versus a dark patterned visual
stimulus; (iv) electrical recording of a response in the visual
cortex to a light flash stimulus or a pattern visual stimulus
35. The method of claim 18 wherein said vision loss or blindness is
a result of a degenerative disease.
36. The method of claim 35 wherein said disease is retinitis
pigmentosa or age-related macular degeneration.
37. The method of claim 18 wherein the subject is also provided
with a visual prosthesis before, at the same time as, or after
delivery of said vector.
38. The method of claim 37, wherein the visual prosthesis is a
retinal implant, a cortical implant, a lateral geniculate nucleus
implant, or an optic nerve implant.
39. The method claim 18, wherein the subject's visual response is
subjected to training using one or more visual stimuli.
40. The method of claim 38 wherein the subject's visual response is
subjected to training using one or more visual stimuli.
41. The method of claim 39 wherein said training is achieved by one
of more of the following methods: (a) habituation training
characterized by training the subject to recognize (i) varying
levels of light and/or pattern stimulation, and/or (ii)
environmental stimulation from a common light source or object; and
(b) orientation and mobility training characterized by training the
subject to detect visually local objects and move among said
objects more effectively than without the training
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility
application Ser. No. 13/899,198, filed May 21, 2013, which is a
continuation of U.S. Utility application No. Ser. 12/299,574, filed
Sep. 15, 2009, now U.S. Pat. No. 8,470,790, which is a National
Stage Application, filed under 35 U.S.C. .sctn. 371, of
International Application No. PCT/US2007/068263, filed on May 4,
2007, which claims priority to, and the benefit of, U.S. Ser. No.
60/797,357, filed May 4, 2006, the contents of which are each
herein incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0003] The contents of the text file named
"RTRO-701/C02US_ST25.txt," which was created on Aug. 14, 2017, and
is 42.7 KB in size, are hereby incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] The present invention in the field of molecular biology and
medicine relates to the use of microbial-type rhodopsins, such as
the light-gated cation-selective membrane channel,
channelrhodopsin-2 (Chop2) to convert inner retinal neurons to
photosensitive cells in photoreceptor-degenerated retina, thereby
restoring visual perception and various aspects of vision.
Description of the Background Art
[0005] Vision normally begins when rods and cones, also called
photoreceptors, convert light signals to electrical signals that
are then relayed through second- and third-order retinal neurons
and the optic nerve to the lateral geniculate nucleus and, then to
the visual cortex where visual images are formed (Baylor, D, 1996,
Proc. Natl. Acad. Sci. USA 93:560-565; Wassle, H, 2004, Nat. Rev.
Neurosci. 5:747-57). For a patient who is vision-impaired due to
the loss of photoreceptors, visual perception may be induced by
providing electrical stimulation at one of these downstream
neuronal locations, depending on the nature of the particular
impairment.
[0006] The severe loss of photoreceptor cells can be caused by
congenital retinal degenerative diseases, such as retinitis
pigmentosa (RP) (Sung, C H et al., 1991, Proc. Natl. Acad Sci. USA
88 :6481-85; Humphries, P et al., 1992, Science 256:804-8; Weleber,
R G et al., in: S J Ryan, Ed, Retina, Mosby, St. Louis (1994), pp.
335-466), and can result in complete blindness. Age-related macular
degeneration (AMD) is also a result of the degeneration and death
of photoreceptor cells, which can cause severe visual impairment
within the centrally located best visual area of the visual
field.
[0007] Both rodents and humans go progressively blind because, as
rods and cones are lost, there is little or no signal sent to the
brain. Inherited retinal degenerations that cause partial or total
blindness affect one in 3000 people worldwide. Patients afflicted
with Usher's Syndrome develop progressive deafness in addition to
retinal degeneration. There are currently no effective treatments
or cures for these conditions.
[0008] Basic research on approaches for retinal degeneration has
long been classified into two approaches: (1) treatments to
preserve remaining photoreceptors in patients with retinal
degenerative disease, and (2) methods to replace photoreceptors
lost to retinal degeneration. Patients afflicted with retinal
disease often group themselves into those seeking ways to slow the
loss of their diminishing vision and those who are already legally
blind ("no light perception"), having lost their photoreceptors
because of an inherited eye disease or trauma.
[0009] For the first approach, neuroprotection with neurotrophic
factors (La Vail, M M et al., 1992, Proc. Natl. Acad. Sci. USA 89:
11249-53) and virus-vector-based delivery of wild-type genes for
recessive null mutations (Acland, G M et al., 2001, Nat. Genet.
28:92-95) have come the furthest to the point of a Phase I/II
clinical trial (Hauswirth, W W, 2005, Retina 25, S60; Jacobson, S,
Protocol #0410-677, World Wide Web URL:
webconferences.com/nihoba/16jun2005.html) gaining approval in the
U.S. for adeno-associated viral (AAV)-mediated gene replacement
therapy for Leber's Congenital Amaurosis (LCA), a specific form of
retinal degeneration. Unfortunately, for patients in advanced
stages of retinal degeneration, this approach is not applicable,
and the photoreceptor cells must be replaced.
[0010] For replacement, one approach involves transplantation
(replacement) of normal tissues or cells to the diseased retina.
Another involves electrical-stimulation of remaining non-visual
neurons via retinal implants in lieu of the lost photoreceptive
cells (prosthetic substitution). However, both methods face many
fundamental obstacles. For example, for successful transplantation,
the implanted tissue or cells must integrate functionally within
the host retina. The electrical-stimulation approaches are burdened
with mechanistic and technical difficulties as well as problems
related to lack of long-term biocompatibility of the implanted
bionic devices. In summary, there exist no effective
vision-restoring therapies for inherited blinding disease.
[0011] The present inventors' strategy as disclosed herein,
requires a suitable molecular "light-sensor." Previous studies
reported the heterologous expression of Drosophila rhodopsin
(Zemelman, B V et al., 2002, Neuron 33:15-22) and, more recently,
melanopsin, the putative photopigment of the intrinsic
photosensitive retinal ganglion cells (Mclyan, Z. et al., 2005,
Nature 433:741-5; Panda, S. et al., 2005, Science 307:600-604; Qiu,
X. et al., 2005, Nature 433:745-9). These photopigments, however,
are coupled to membrane channels via a G protein signaling cascade
and use cis-isoforms of retinaldehyde as their chromophore. As a
result, expression of multiple genes would be required to render
photosensitivity. In addition, their light response kinetics is
rather slow. Recent studies aimed to improve the temporal
resolution described the engineering of a light-sensitive K.sup.+
channel (Banghart et al., 2004, Nat. Neurosci. 7: 1381-6), though
this required introduction of an exogenous "molecular tether" and
use of UV light to unblock the channel. This engineered channel was
proposed to be potentially useful for restoring light sensitivity
in degenerate retinas, but its expression and function in retinal
neurons remain unknown.
[0012] The present invention makes use of microbial-type rhodopsins
similar to bacteriorhodopsin (Oesterhelt, D et al., 1973, Proc.
Natl. Acad. Sci. USA 70:2853-7), whose conformation change is
caused by reversible photoisomerization of their chromophore group,
the all-trans isoform of retinaldehyde, and is directly coupled to
ion movement through the membrane (Oesterhelt, D., 1998, Curr.
Opin. Struct. Biol. 8:489-500). Two microbial-type opsins,
channelopsin-1 and -2 (Chop1 and Chop2), have recently been cloned
from Chlamydomonas reinhardtii (Nagel, G. et al., 2002, Science
296:2395-8; Sineshchekov, O A et al., 2002, Proc. Natl. Acad Sci.
USA 99:8689-94; Nagel, G. et al., 2003, Proc. Natl. Acad Sci. USA
100, 13940-45) and shown to form directly light-gated membrane
channels when expressed in Xenopus laevis oocytes or HEK293 cells
in the presence of all-trans retinal. Chop2, a seven transmembrane
domain protein, becomes photo-switchable when bound to the
chromophore all-trans retinal. Chop2 is particularly attractive
because its functional light-sensitive channel, channelrhodopsin-2
(Chop2 retinalidene abbreviated ChR2) with the attached
chromnophore is penneable to physiological cations. Unlike animal
rhodopsins, which only bind the 11-cis conformation, Chop2 binds
all-trans retinal isomers, obviating the need for the all-trans to
11-cis isomerization reaction supplied by the vertebrate visual
cycle. However, the long-term compatibility of expressing ChR2 in
native neurons in vivo in general and the properties of
ChR2-mediated light responses in retinal neurons in particular
remained unknown until the present invention.
[0013] The present strategy is feasible because histological
studies, both in animal models of photoreceptor degeneration
(Chang, B. et al., 2002, Vision Res. 42:517-25; Olshevskaya, E V et
al., 2004, J. Neurosci. 24:6078-85) and in postmortem patient eyes
with almost complete photoreceptor loss due to RP (Santos, A H et
al., 1997, Arch. Ophthalmol. 115:511-15; Milam, A H et al., 1998,
Prag. Retin. Eye Res. 17: 175-205), reported the preservation of a
significant number of inner retinal neurons.
[0014] Retinal gene therapy has been considered a possible
therapeutic option for man. For example, U.S. Pat. No. 5,827,702
refers to methods for generating a genetically engineered ocular
cell by contacting the cell with an exogenous nucleic acid under
conditions in which the exogenous nucleic acid is taken up by the
cell for expression. The exogenous nucleic acid is described as a
retrovirus, an adenovirus, an adeno-associated virus or a plasmid.
See, also, WO 00/15822 (Mar. 23, 2000) and WO 98/48097 (Oct. 29,
1998)
[0015] Efforts in such gene therapy have focused mainly on slowing
down retinal degeneration in rodent models of primary photoreceptor
diseases. Normal genes and mutation-specific ribozymes delivered to
photoreceptors have prolonged the lifetime of these cells otherwise
doomed for apoptotic cell death (Bennett, J., et at 1996 Nat. Med
2, 649-54; Bennett, J., et at 1998, Gene Therapy 5, 1156-64;
Kumar-Singh, R et al., 1998 Hum. Mol. Genet. 7 1893-900; Lewin, A S
et at 1998, Nat. Med 4, 967-71; Ali, R et al 2000, Nat. Genet. 25,
306-10; Takahashi, M. et al., 1999, J Viral. 73, 7812-6; Lau, D.,
etal., 2000, Invest. Ophthalmol. Vis. Sci. 41, 3622-33; and LaVail,
M M, et al 2000, Proc Natl Acad Sci USA 97, 11488-93).
[0016] Retinal gene transfer of a reporter gene, green fluorescent
protein (GFP), using a recombinant adeno-associated virus (rAAV)
was demonstrated in normal primates (Bennett, J et at 1999 Proc.
Natl. Acad. Sci. USA 96, 9920-25). However, the restoration of
vision in a blinding disease of animals, particularly in humans and
other mammals, caused by genetic defects in retinal pigment
epithelium (RPE) and/or photoreceptor cells has not been achieved.
Jean Bennett and colleagues have described the rescue of
photoreceptors using gene therapy in a model of rapid degeneration
of photoreceptors using mutations of the RP65 gene and replacement
therapy with the normal gene to replace or supplant the mutant
gene. See, for example, US Patent Publication 2004/0022766 of
Acland, Bennett and colleagues. This therapy showed some success in
a naturally-occurring dog model of severe disease of retinal
degenerations--the RPE65 mutant dog, which is analogous to human
LCA.
[0017] Advantages of the present approach include the fact that it
does not require introducing exogenous cells and tissues or
physical devices, thus avoiding many obstacles encountered by
existing approaches; the present invention is applicable for the
reversal of vision loss or blindness caused by many retinal
degenerative diseases. By expressing photosensitive
membrane-channels or molecules in surviving retinal neurons of the
diseased retina by viral based gene therapy method, the present
invention can produce permanent treatment of the vision loss or
blindness with high spatial and temporal resolution for the
restored vision.
[0018] To the extent that any specific disclosure in the
aforementioned publications or other publications may be considered
to anticipate any generic aspect of the present invention, the
disclosure of the present invention should be understood to include
a proviso or provisos that exclude of disclaim any such species
that were previously disclosed. The aspects of the present
invention which are not anticipated by the disclosure of such
publications are also unobvious from the disclosure of these
publications, due at least in part to the unexpectedly superior
results disclosed or alleged herein.
SUMMARY OF THE INVENTION
[0019] The present invention is directed to the genetic conversion
of surviving light-insensitive inner retinal neurons in a retina in
which photoreceptors are degenerating or have already died, into
directly photosensitive neuronal cells, thereby imparting light
sensitivity to such retinas and restoring one or more aspects of
visual responses and functional vision to a subject suffering from
such degeneration. By restoring light sensitivity to a retina
lacking this capacity, due to disease, the invention provides a
mechanism for the most basic light-responses that are required for
vision. Said another way, the present invention introduces a "light
sensors" into retinal neurons that normally do not have them, to
compensate for loss of retinal photoreceptor cells.
[0020] The present inventors and colleagues investigated the
feasibility of using Chop2/ChR2 to restore light sensitivity to the
retinas that have undergone rod and cone degeneration. The results
presented herein show long-term expression of Chop2/ChR2 in rodent
inner retinal neurons in vivo. The results also show that these
inner retinal neurons can express a sufficient number of functional
ChR2 channels to produce robust membrane depolarization or action
potential firing without an exogenous supply of all-trans retinal.
Furthermore, the present inventors demonstrated that the expression
of ChR2 in a photoreceptor-deficient mouse model not only enables
retinal ganglion cells to encode light signals but also restores
visually evoked responses in the visual cortex.
[0021] The present invention is directed to the restoration of
vision loss to individuals that have lost vision or are blind as a
result of retinal photoreceptor degeneration. The invention enables
retinal neurons in such a diseased retina to respond to light by
expressing photosensitive membrane-channels or molecules in these
retinal neurons. Preferred the light-sensitive channels or
molecules are microbial type light-gate channel rhodopsins, such as
ChR2, ChR1, light-driven ion pump, such as bacteriorhodopsins
(Lanyi, J K, 2004, Annu Rev Physiol. 66:665-88), halorhodopsins
(Lanyi, J K, 1990, Physiol Rev. 70:319-30), and their
derivatives
[0022] As discovered by the present inventors, retinal neurons that
are normally not light sensitive (directly) in the retinas of blind
mice, such as retinal ganglion cells (RGCs) and bipolar cells, can
respond to light when a green algae protein called
channelrhodopsin-2 (ChR2), or a biologically active fragment or a
conservative amino acid substitution variant thereof, is inserted
into the neuronal cell membranes. The study was conducted with mice
that had been genetically bred to lose rods and cones, the
light-sensitive cells in the retina, a condition that models RP in
humans. In addition to RP, there are many forms of retinal
degenerative eye diseases that possibly could be treated by the
present approach.
[0023] As disclosed herein, visual function can be restored by
conveying light-sensitive properties to other surviving cells in
the retina after the rods and cones have died. Using a DNA transfer
approach, the present inventors introduced the light-absorbing
protein ChR2 into the mouse retinal neurons that survived after the
rods and cones had died. These cells became light sensitive and
sent signals via the optic nerve and higher order visual pathways
to the visual cortex where visual perception occurs. Using
electrophysiologic means, it was shown that the signals reached the
visual cortex in a majority of the ChR2-treated mice. The light
sensitivity persisted for at least six months, suggesting that the
subject might regain usable vision with additional maneuvers
disclosed herein, such as expressing ChR2 in other types of retinal
cells or modifying the light sensitivity and/or wavelength
selectivity of ChR2, or using similar microbial proteins, to
produce diverse light-sensitive channels to improve outcomes for
the restoration of normal vision.
[0024] As noted by persons of skill in this art, this strategy
represents a "paradigm shift in the field" referring to a "new
field of re-engineering retinal interneurons as genetically
modified `prosthetic` cells," The present invention "opened the
possibility of genetically modifying the surviving retinal
interneurons to function as a replacement light-sensing receptor,"
(Flannery, J and Greenberg, K., 2006, Neuron. 50 1-3; written as a
preview to a publication in the same issue of the present inventors
and colleagues, Bi J. et al., Neuron 50, 23-33, 2006).
[0025] The present inventors capitalized upon advancements in the
field by using viral vectors to transfer genes to retinal
photoreceptor cells (Flannery J G et al., 1997, Proc. Natl. Acad.
Sci. USA 94:6916-21). The conversion of light-insensitive retinal
interneurons into photosensitive cells introduces an entirely new
direction for treatments of blinding retinal degeneration.
[0026] In one embodiment of the present invention, retinal bipolar
cells, certain amacrine cells and ganglion cells are targeted for
transduction of the Chop2 DNA, to convert them functionally into
photosensitive cells that subsume the function of rods and cones.
The layering of cells in the retina is such that photoreceptor
cells excite bipolar cells which excite ganglion cells to transmit
signals to the visual cortex. It is preferred to express the
channel opsin of the present invention in bipolar ON-type cells.
Intravitreal and/or subretinal injections arc used to deliver DNA
molecules and virus vectors to reach the cells being targeted.
[0027] In one embodiment, the promoter is from a mGluR6
promoter--region of the Grm6 gene (GenBank accession number
BC041684), a gene that controls expression of metabotropic
glutamate receptor 6 ((Ueda Y etal., 1997, J Neuroscl 7:3014-23).
The genomic sequence is shown in GenBank accession
number--AL627215. A preferred example of this promoter region
sequence from the above GenBank record is SEQ ID NO:9 consisting of
11023 nucleotides--as shown in FIG. 8. The original Umeda et al.,
study employed a 10 kb promoter, but the actual length of the
promoter and the sequence that comprises control elements of Grm6
can be adjusted by increasing or decreasing the fragment length. It
is a matter of routine testing to select and verify the action of
the optimally sized fragment from the Grm6 gene that drives
transgenic expression of a selected coding sequence, preferably
Chop2, in the desired target cells, preferably in bipolar cells
which are rich in glutamate receptors, particularly the "on" type
bipolar cells, which are the most bipolar cells in the retina
(Nakajima, Y., et al., 1993, J Biol Chem 268: 11868-73).
[0028] The present invention is directed to a method of restoring
light sensitivity to a retina, comprising: [0029] (a) delivering to
retinal neurons a nucleic acid expression vector that encodes a
light-gated channel rhodopsin or a light-driven ion pump rhodopsin
expressible in the neurons, which vector comprises an open reading
frame encoding the rhodopsin, and operatively linked thereto, a
promoter sequence, and optionally, transcriptional regulatory
sequences; and [0030] (b) expressing the vector in the neurons,
thereby restoring light sensitivity.
[0031] The rhodopsin is preferably channelrhodopsin-2 (Chop2) or a
biologically active fragment or conservative amino acid
substitution variant thereof.
[0032] The vector is preferably a rAAV viral vector.
[0033] The promoter may be a constitutive promoter such as a hybrid
CMV enhancer/chicken 13-actin promoter (CAG) (as indicated below as
part of SEQ ID NO: 1), or a CMV promoter. The promoter may also be
(i) an inducible or (ii) a cell type-specific promoter, preferred
examples of the latter being the mGluR6 promoter (e.g., part of a
promoter sequence SEQ ID NO:9), a Pcp2 (L7) promoter or a
neurokinin-3 (NK-3) promoter.
[0034] A preferred vector in the above method comprises the CAG
promoter, a woodchuck posttranscriptional regulatory element
(WPRE), and a bovine or human growth hormone polyadenylation
sequence.
[0035] In the present method, the retinal neurons are selected from
ON- and OFF-type retinal ganglion cells, retinal rod bipolar cells,
All amacrine cells and ON and OFF retinal cone bipolar cells.
Preferably, the vector is targeted to and expressed in ON type
ganglion cells and/or ON type bipolar cells If the vector comprises
the NK-3 promoter, the vector is preferably targeted to OH-cone
bipolar cells.
[0036] The invention is also directed to method of restoring
photosensitivity to retinal neurons of a subject suffering from
vision loss or blindness in whom retinal photoreceptor cells are
degenerating or have degenerated and died, which method comprises:
[0037] (a) delivering to the retina of the subject a nucleic acid
vector that encodes a light-gated channel rhodopsin or a
light-driven ion pump rhodopsin expressible in the neurons, which
vector comprises an open reading frame encoding the rhodopsin, and
operatively linked thereto, a promoter sequence, and optionally,
transcriptional regulatory sequences; [0038] (b) expressing the
vector in the neurons, wherein the expression of the rhodopsin
renders the neurons photosensitive, thereby restoring of
photosensitivity to the retina.
[0039] In this method the rhodopsin is preferably Chop2 or a
biologically active fragment or conservative amino acid
substitution variant thereof. The vector is preferably a rAAV viral
vector. Preferred promoters arc as described above for the
above-presented embodiment. Preferred target cells for the vector
are as described above.
[0040] The restoration of photosensitivity using the above method
preferably results in restoration of vision in the subject. The
vision is preferably measured by one or more of the following
methods: [0041] (i) a light detection response by the subject after
exposure to a light stimulus [0042] (ii) a light projection
response by the subject after exposure to a light stimulus; [0043]
(iii) light resolution by the subject of a light versus a dark
patterned visual stimulus; [0044] (iv) electrical recording of a
response in the visual cortex to a light flash stimulus or a
pattern visual stinmlus
[0045] In this foregoing method, the vision loss or blindness may
be a result of a degenerative disease, preferably, retinitis
pigmentosa or age-related macular degeneration.
[0046] In another embodiment, the subject is also provided with a
visual prosthesis before, at the same time as, or after delivery of
the vector. Preferred visual prostheses comprise retinal implants,
cortical implants, lateral geniculate nucleus implants, or optic
nerve implants.
[0047] When employing the foregoing method, the subject's visual
response may be subjected to training using one or more visual
stimuli. The training is preferably achieved by one or more of the
following methods: [0048] (a) habituation training characterized by
training the subject to recognize (i) varying levels of light
and/or pattern stimulation, and/or (ii) environmental stimulation
from a common light source or object; and [0049] (b) orientation
and mobility training characterized by training the subject to
detect visually local objects and move among the objects more
effectively than without the training.
BRIEF DESCRIPTION OF DRAWINGS
[0050] FIGS. 1A-1I. Expression of Chop2-GFP in Retinal Neurons In
vivo. FIG. 1A shows the rAAV-CAG-Chop2-GFP-WPRE expression
cassette. CAG: a hybrid CMV enhancer/chicken 13-actin promoter.
WPRE: woodchuck posttranscriptional regulatory element. BGHpA: a
bovine growth hormone polyadenylation sequence. (FIGS. 1B and 1C)
Chop2-GFP fluorescence viewed in low (FIG. 1B) and high (FIG. 1C)
magnifications from eyes two months after the viral vector
injection. (FIG. 1D) Confocal images of a ganglion cell, which show
a stacked image (left) and a single optical section image (right).
(FIG. 1E) Chop2-GFP fluorescence in a horizontal cell, which shows
GFP in soma, axon, and distal axon terminal. (FIGS. 1F and 1G)
Chop2-GFP fluorescence in amacrine cells (FIG. 1F) and a retinal
bipolar cell (FIGS. 1G). FIGS. 1H and 1I show fluorescence image
(FIG. 1H) and phase contrast image (FIG. 1I) taken from a retina 12
months after the injection of Chop2-GFP viral vectors. images in
(FIGS. 1B-1E) were taken from flat whole-mounts of rat retinas.
images in (FIGS. 1F-1I) were taken from vertical slice sections of
rat retinas. Scale bar: 200 .mu.min (FIG. 1B); 100 .mu.min (FIG.
1C); 15 .mu.min (FIG. 1D); 50 .mu.min (FIG. 1E), FIG. 1H), and
(FIG. 1I); 25 .mu.min (FIG. 1F) and (FIG. 1G). ONL: outer nuclear
layer; INL: inner nuclear layer; IPL: inner plexiform layer;
GCL:
[0051] FIGS. 2A-2H. Properties of Light-Evoked Currents of the
ChR2-expressing retinal neurons. (FIG. 2A) Phase contrast image
(left) and fluorescence image (right) of a GFP-positive retinal
neuron dissociated from the viral vector injected eye. Scale bar:
25 .mu.m (FIG. 2B) A recording of Chop2-GFP fluorescent retinal
cell to light stimuli of wavelengths ranging from 420 to 580 mn.
The light intensities were ranging from 1.0-1.6.times.10.sup.18
photons cm-.sup.2 s-.sup.1 (FIG. 2C) A representative recording of
the currents elicited by light stimuli at the wavelength of 460 nm
with light intensities ranging from 2.2.times.10.sup.15 to
1.8.times.10.sup.18 photons cm-.sup.2 s-.sup.1 (FIG. 2D) Current
traces after the onset of the light stimulation from FIG. 2C shown
in the expanded time scale. The line shows the fitting of one
current trace by an exponential function:
1.sub.(t)=a.sub.0+a.sub.1x(1-exp [-tfr i])+a.sub.2x(exp
[-tfr.sub.2]), in which .cndot..sub.1 and .sub.2 represent the
activation and inactivation time constant, respectively. (FIG. 2E)
Current traces after the termination of the light stimulation from
FIG. 2C shown in the expanded time scale. The line shows the
fitting of one current trace by a single exponential function:
1(.sub.t)=a.sub.0+a.sub.1x(exp[-tfr]), in which T represent the
deactivation time constant. (FIG. 2F) Light-intensity response
curve. The data points were fitted with a single logistic function
curve. (FIGS. 2F and H) The relationships of light-intensity and
activation time constant (FIG. 2G) and light-intensity and
inactivation time constant (FIG. 2H) obtained from the fitting
shown in FIG. 2D. All recordings were made at the holding potential
of -70 mV. The data points in FIG. 2F-2H are shown as mean.+-.SD
(n=7).
[0052] FIGS. 3A-3C. Properties of Light-Evoked Voltage Responses of
ChR2-Expressing Retinal Neurons. (FIG. 3A) A representative
recordings from GFP-positive nonspiking neurons. The voltage
responses were elicited by four incremental light stimuli at the
wavelength of 460 nm with intensities ranging from
2.2.times.10.sup.15 to 1.8.times.10.sup.18 photons cm-.sup.2
s-.sup.1 in current clamp. The dotted line indicates the saturated
potential level. (FIG. 3B) A representative recording from
GFP-positive nonspiking neurons to repeat light stimulations. The
light-evoked currents (top traces) and voltage responses (bottom
traces) from a same cells were shown. Left panel shows the
superimposition of the first (red) and second (black) traces in an
expanded time scale. The dotted line indicates the sustained
component of the currents (top) and plateau membrane potential
(bottom). (FIG. 3C) A representative recording of GFP-positive
spiking neurons to repeated light stimulations. The responses in
FIG. 3B and 3C were evoked by light at the wavelength of 460 nm
with the intensity of 1.8.times.10.sup.18 photons cm-.sup.2
s-.sup.1.
[0053] FIGS. 4A-4I. Expression and Light-Response Properties of
ChR2 in Retinal Neurons of rdl/rdl Mice. (FIG. 4A) Chop2-GFP
fluorescence viewed in flat retinal whole-mount of a 15 month old
mouse with the Chop2-GFP viral vector injection at 9 months of age.
(FIG. 4B) Chop2-GFP fluorescence viewed in vertical section from
the retina of a 6 month old mouse with the injection of Chop2-GFP
viral vectors at 3 months of age. (FIG. 4C) Light microscope image
of a semithin vertical retinal section from a 5 month old mouse
(Chop2-GFP viral vectors injected at postnatal day 1). Scale bar:
50 .mu.min (FIG. 4A) and 30 .mu.min (FIGS. 4B and 4C). (FIGS.
4D-4E) show representative recordings of transient spiking (FIG.
4D) and sustained spiking (FIG. 4E) GFP-positive neurons. The
responses were elicited by light of four incremental intensities at
the wavelength of 460 nm. The light intensity without neutral
density (Log I=0) was 3.6.times.10.sup.17 photons cm-.sup.2
s.sub.-1. The currents were recorded at the holding potential of
-70 mV. The superimposed second (solid black) and fourth (dashed or
red) current and voltage traces are shown in the right panel in the
expanded time scale. (FIGS. 4F-4I) show the relationships of the
amplitude of current (FIG. 4F), membrane depolarization (FIG. 4G),
the number of spikes (FIG. 4H), and the time to the first spike
peak (FIG. 4I) to light intensity. Recordings were made from
rdl/rdl mice at 4 months of age. The data points are the mean.+-.SE
(n=6 in FIG. 4F-4H and n=4 in FIG. 4I).
[0054] FIG. 5A-5D. Multielectrode Array Recordings of the
ChR2-Expressing Retinas of rdl/rdl Mice. (FIG. 5A) A sample
recording of light-evoked spike activities from the retinas of
rdl/rdl mice (ages 4 months). The recording was made in the present
of CNQX (25 .mu.M) and APS (25 .mu.M). Prominent light-evoked spike
activity was observed in 49 out of 58 electrodes (electrode 15 was
for grounding and electrode 34 was defective). (FIG. 58) Sample
light-evoked spikes recorded from a single electrode to three
incremental light intensities. (Fig. SC) The raster plots of 30
consecutive light-elicited spikes originated from a single neuron.
(Fig. SD) The averaged spike rate histograms. The light intensity
without neutral density filters (Log I=0) was 8.5.times.10.sup.17
photons cm-.sup.2 s-.sup.1. The responses shown in FIG. 5A were
elicited by a single light pulse without neutral density
filters.
[0055] FIG. 6A-6E. Central Projections of Chop2-GFP-Expressing
Retinal Ganglion Cells and Visual-Evoked Potentials in rdl/rdl
Mice. (FIG. 6A) GFP labeled terminal arbors of retinal ganglion
cells in ventral lateral geniculate nucleus and dorsal lateral
geniculate nucleus. (FIG. 6B) GFP-labeled terminal arbors of
retinal ganglion cells in superior colliculus. OT: optical track;
vLGN: ventral lateral geniculate nucleus; dLGN: dorsal lateral
geniculate nucleus; SC: superior colliculus. Scale bar: 200 .mu.min
FIG. 6A), 100 .mu.min FIG. 6B). (FIG. 6C) VEPs recorded from a
wild-type mouse. The responses were observed both to the
wavelengths of 460 and 580 nm. (FIG. 6D) VEPs recorded from an
rdl/rdl mouse injected with Chop2-GFP viral vectors. The responses
were elicited only by light at the wavelength of 460 nm but not at
the wavelength of 580 nm. (FIG. 6E) No detectable VEPs were
observed from rdl/rdl mice injected with viral vectors carrying GFP
alone. The light intensities measured at the corneal surface at the
wavelengths of 460 and 580 nm were 5.5.times.10.sup.16 and
5.2.times.10.sup.16 photons cm-.sup.2 s-.sup.1, respectively. (FIG.
6F) Plot of the amplitude of VEPs from rdl/rdl mice injected with
Chop2-GFP viral vectors to various light intensities at the
wavelengths of 420, 460, 500, 520, and 540 nm. For each eye, the
responses are normalized to the peak response obtained at 460 nm.
The data are the mean.+-.SD (n=3 eyes). Spectral sensitivity at
each wavelength was defined as the inverse of the interpolated
light intensity to produce 40% of the normalized peak response, as
indicated by the dot line. (FIG. 6G) The sensitivity data points
were fitted by a vitamin-Ai-based visual pigment template with a
peak wavelength of 461 nm.
[0056] FIG. 7 shows a map of the viral expression construct
rAAV2-CAG-Chop2-GFP-WPRE (SEQ ID NO: 1), which comprises a
Chop2-GFP fragment, an operatively linked a hybrid CMV
enhancer/chicken 13-actin promoter (CAG), a woodchuck
posttranscriptional regulatory element (WPRE), and a bovine growth
hormone (BGH) polyadenylation sequence.
[0057] FIG. 8 (sheets 1-3) presents the sequence (SEQ ID
NO:9)--11023 nt's --of the mGluR6 promoter region of the Grm6 gene
(GenBank No. BC041684). The genomic sequence is provided in GenBank
No. AL627215.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The invention provides a method for treating an ocular
disorder in a human, other mammalian or other animal subject. In
particular, the ocular disorder is one which involves a mutated or
absent gene in a retinal pigment epithelial cell or a photoreceptor
cell. The method of this invention comprises the step of
administering to the subject by intravitreal or subretinal
injection of an effective amount of a recombinant virus carrying a
nucleic acid sequence encoding an ocular cell-specific normal gene
operably linked to, or under the control of, a promoter sequence
which directs the expression of the product of the gene in the
ocular cells and replaces the lack of expression or incorrect
expression of the mutated or absent gene.
[0059] Ocular Disorders
[0060] The ocular disorders for which the present methods are
intended and may be used to improve one or more parameters of
vision include, but are not limited to, developmental abnormalities
that affect both anterior and posterior segments of the eye.
Anterior segment disorders include glaucoma, cataracts, corneal
dystrophy, keratoconus. Posterior segment disorders include
blinding disorders caused by photoreceptor malfunction and/or death
caused by retinal dystrophies and degenerations. Retinal disorders
include congenital stationary night blindness, age-related macular
degeneration, congenital cone dystrophies, and a large group of
retinitis-pigmentosa (RP)-related disorders. These disorders
include genetically pre-disposed death of photoreceptor cells, rods
and cones in the retina, occurring at various ages. Among those are
severe retinopathies, such as subtypes of RP itself that progresses
with age and causes blindness in childhood and early adulthood and
RP-associated diseases, such as genetic subtypes of LCA, which
frequently results in loss of vision during childhood, as early as
the first year of life. The latter disorders are generally
characterized by severe reduction, and of ten complete loss of
photoreceptor cells, rods and cones. (Trabulsi, E I, ed., Genetic
Diseases of the Eye, Oxford University Press, NY, 1998).
[0061] In particular, this method is useful for the treatment
and/or restoration of at least partial vision to subjects that have
lost vision due to ocular disorders, such as RPE-associated
retinopathies, which are characterized by a long-term preservation
of ocular tissue structure despite loss of function and by the
association between function loss and the defect or absence of a
normal gene in the ocular cells of the subject. A variety of such
ocular disorders are known, such as childhood onset blinding
diseases, retinitis pigmentosa, macular degeneration, and diabetic
retinopathy, as well as ocular blinding diseases known in the art.
It is anticipated that these other disorders, as well as blinding
disorders of presently unknown causation which later are
characterized by the same description as above, may also be
successfully treated by this method. Thus, the particular ocular
disorder treated by this method may include the above-mentioned
disorders and a number of diseases which have yet to be so
characterized.
[0062] Visual information is processed through the retina through
two pathways: an ON pathway which signals the light ON, and an OFF
pathway which signals the light OFF (Wassle, supra). It is
generally believed that the existence of the ON and OFF pathway is
important for the enhancement of contrast sensitivity. The visual
signal in the ON pathway is relay front ON-cone bipolar cells to ON
ganglion cells. Both ON-cone bipolar cells and ON-ganglion cells
are depolarized in response to light. On the other hand, the visual
signal in the OFF pathway is carried from OFF-cone bipolar cells to
OFF ganglion cells. Both OFF-cone bipolar cells and OFF-ganglion
cells are hypopolarized in response to light. Rod bipolar cells,
which are responsible for the ability to see in dim light (scotopic
vision), are ON bipolar cells (depolarized in response to light).
Rod bipolar cells relay the vision signal through All amacrine
cells (an ON type retinal cells) to ON an OFF cone bipolar
cells.
[0063] The present Examples show functional consequence of
expressing ubiquitously expressing light sensitive channels, namely
ChR2, in retinal ganglion cells by CAG promoter, and suggest that
this sufficient for restoring useful vision. However, targeting of
depolarizing membrane channels, such as ChR2, to the ON-type
retinal neurons might result in better useful vision. In addition,
expression of light sensors in more distal retinal neurons, such as
bipolar cells, would utilize the remaining signal processing
functions of the degenerate retina. Furthermore, by expressing a
depolarizing light sensor, such as ChR2, in ON type retinal neurons
(ON type ganglion cells and/or ON type bipolar cells) and
expressing a hypopolarizing light sensor, such as halorhodopsin (a
chloride pump) (Han, X et al., 2007, PLoS ONE, March 21; 2:e299;
Zhang, F etal., 2007; Nature 446:633-9; present inventors' results)
in OFF type retinal neurons (OFF type ganglion cells and/or OFF
type bipolar cells) could create ON and OFF pathways in
photoreceptor degenerated retinas.
[0064] An alternative approach to restore ON and OFF pathways in
the retina is achieved by, expressing a depolarizing light sensor,
such as ChR2, to rod bipolar cells or All amacrine. This is because
the depolarization of rod bipolar cells or All amacrine cells can
lead to the ON and OFF responses at the levels of cone bipolar
cells and the downstream retinal ganglion cells and, thus, the ON
and OFF pathways that are inherent in the retina could be
maintained (Wassle, 2004).
[0065] According to the present invention, the followings
approaches are used to restore the light sensitivity of inner
retinal neurons:
[0066] (1) Ubiquitously expressing light sensitive channels, such
as ChR2, are employed to produced membrane depolarization in all
types of ganglion cells (both ON and OFF ganglion cells), or all
types of bipolar cells (rod bipolar cells, and ON and OFF cone
bipolar cells). The AAV vector with CAG promoter has already
partially achieved this approach in rodent retinas, as exemplified
herein.
[0067] (2) A depolarizing light sensor, such as ChR2, is targeted
to ON type retinal neurons such as ON type ganglion cells or ON
type bipolar cells. A study from Dr. J. G. Flannery's group has
identified the fragments of a human gap junctional protein
(connexin-36) promoter to target GFP in ON-type retinal ganglion
cells by using AAV-2 virus vector (Greenberg K P et al., 2007, In
vivo Transgene Expression in ON-Type Retinal Ganglion Cells:
Applications to Retinal Disease. ARVO abstract, 2007). A readily
packable shorter version of mGluR6 promoter of (<2.5 kb) would
allow targeting of ChR2 to ON type bipolar cells (both rod bipolar
cells and ON type cone bipolar cells).
[0068] (3) Cell specific promoters are used to target the specific
types of retinal neurons. A promoter that could target rod bipolar
cells is Pcp2 (L7) promoter (Tomomura, M et al., 2001, Eur J
Neurosci. 14:57-63). The length of the active promoter is
preferably less that 2.5 Kb so it can be packaged into the AAV
viral cassette.
[0069] (4) A depolarizing light sensor, such as ChR2, is targeted
to ON type ganglion cells or
[0070] ON type cone bipolar cells and a hypopolarizing light
sensor, such as halorhodopsin, to 014 type ganglion cells or 014
type cone bipolar cells to create ON and OFF pathways. As described
above, an adequately short (packable) version of mGluR6 promoter
(<2.5 kb) would allow targeting of ChR2 to ON type bipolar
cells. The Neurokinin-3 (NK-3) promoter would be used to target
halorhodopsin to OFF cone bipolar cells (Haverkamp, S et al., 2002,
J Comparative Neurology, 455:463-76.
[0071] Vectors
[0072] According to the various embodiments of the present
invention, a variety of known nucleic acid vectors may be used in
these methods, e.g., recombinant viruses, such as recombinant
adeno-associated virus (rAAV), recombinant adenoviruses,
recombinant retroviruses, recombinant poxviruses, and other known
viruses in the art, as well as plasmids, cosmids and phages, etc.
Many publications well-known in the art discuss the use of a
variety of such vectors for delivery of genes. See, e.g., Ausubel
et al., Current Protocols in Molecular Biology, John Wiley &
Sons, New York, latest edition; Kay, M A. et al, 2001, Nat. Med,
7:33-40; and Walther W e t al., 2000, Drugs 60:249-71).
[0073] Methods for assembly of the recombinant vectors are
well-known. See, for example, WO 00/15822 and other references
cited therein, all of which are incorporated by reference.
[0074] There are advantages and disadvantages to the various viral
vector systems. The limits of how much DNA can be packaged is one
determinant in choosing which system to employ. rAAV tend to be
limited to about 4.5 kb of DNA, whereas lentivirus (e.g.,
retrovirus) system can accommodate 4-5 kb.
[0075] AAVVectors
[0076] Adeno-associated viruses are small, single-stranded DNA
viruses which require a helper virus for efficient replication
(Berns, K I, Parvoviridae: the viruses and their replication, p.
1007-1041 (vol. 2), in Fields, B N et al., Fundamental Virology,
3rd Ed., (Lippincott-Raven Publishers, Philadelphia (1995)). The
4.7 kb genome of AAV has two inverted terminal repeats (ITR) and
two open reading frames (ORFs) which encode the Rep proteins and
Cap proteins, respectively. The Rep reading frame encodes four
proteins of molecular weights 78, 68, 52 and 40 kDa. These proteins
primarily function in regulating AAV replication and rescue and
integration of the AAV into the host cell chromosomes. The Cap
reading frame encodes three structural proteins of molecular
weights 85 (VP 1), 72 (VP2) and 61 (VP3) kDa which form the virion
capsid (Berns, supra). VP3 comprises >80% of total AAV virion
proteins.
[0077] Flanking the rep and cap ORFs at the 5' and 3' ends are 145
bp TTRs, the first 125 bp's of which can form Y- or T-shaped duplex
structures. The two ITRs are the only cis elements essential for
AAV replication, rescue, packaging and integration of the genome.
Two conformations of AAV ITRs called "flip" and "flop" exist
(Snyder, R O et at, 1993, J Viral, 67:6096-6104; Berns, K I, 1990
Microbial Rev, 54:316-29). The entire rep and cap domains can be
excised and replaced with a transgene such as a reporter or
therapeutic transgene (Carter, B J, in Handbook of Parvoviruses, P.
Tijsser, ed., CRC Press, pp. 155-168 (1990)).
[0078] AAVs have been found in many animal species, including
primates, canine, fowl and human (Murphy, F A et al, The
Classification and Nomenclature of Viruses: Sixth Rept of the Int'l
Comme on Taxonomy of Viruses, Arch Viral, Springer-Verlag, 1995).
Six primate serotypes are known (AAV1, AAV2, AAV3, AAV4, AAV5 and
AAV6).
[0079] The AAV ITR sequences and other AAV sequences employed in
generating the minigenes, vectors, and capsids, and other
constructs used in the present invention may be obtained from a
variety of sources. For example, the sequences may be provided by
any of the above 6 AAV serotypes or other AAV serotypes or other
densoviruses, including both presently known human AAV and yet to
yet-to-be-identified serotypes. Similarly, AAVs known to infect
other animal species may be the source of ITRs used in the present
molecules and constructs. Capsids from a variety of serotypes of
AAV may be combined in various mixtures with the other vector
components (e.g., WOO1/83692 (Nov. 8, 2001) incorporated by
reference). Many of these viral strains or serotypes are available
from the American Type Culture Collection (ATCC), Manassas, Va., or
are available from a variety of other sources (academic or
commercial).
[0080] It may be desirable to synthesize sequences used in
preparing the vectors and viruses of the invention using known
techniques, based on published AAV sequences, e.g., available from
a variety of databases. The source of the sequences utilized to
prepare the present constructs is not considered to be limiting.
Similarly, the selection of the AAV serotype and species (of
origin) is within the skill of the art and is not considered
limiting
[0081] The Minigene
[0082] As used herein, the AAV sequences are typically in the form
of a rAAV construct (e.g., a minigene or cassette) which is
packaged into a rAAV virion. At minimum, the rAAV minigene is
formed by AAV ITRs and a heterologous nucleic acid molecule for
delivery to a host cell. Most suitably, the minigene comprises ITRs
located 5' and 3' to the heterologous sequence. However, minigene
comprising 5' ITR and 3' ITR sequences arranged in tandem, e.g., 5'
to 3' or a head-to-tail, or in another configuration may also be
desirable. Other embodiments include a minigene with multiple
copies of the TTRs, or one in which 5' TIRs (or conversely, 3'
TTRs) are located both 5' and 3' to the heterologous sequence. The
ITRs sequences may be located immediately upstream and/or
downstream of the heterologous sequence; intervening sequences may
be present. The ITRs may be from AAV5, or from any other AAV
serotype. A minigene may include 5' ITRs from one serotype and 3'
ITRs from another.
[0083] The AAV sequences used are preferably the 145 bp cis-acting
5' and 3' ITR sequences (e.g., Carter, B J, supra). Preferably, the
entire ITR sequence is used, although minor modifications are
permissible. Methods for modifying these ITR sequences are
well-known (e.g., Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, 3.sup.rd Edition, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., 2001; Brent, R et al., eds., Current Protocols
in Molecular Biology, John Wiley & Sons, Inc., 2003; Ausubel, F
M et al., eds., Short Protocols in Molecular Biology, 5.sup.th
edition, Current Protocols, 2002; Carter et al., supra; and Fisher,
K et al., 1996 J Viral 70:520-32). It is conventional to engineer
the rAAV vims using known methods (e.g., Bennett, J et al 1999,
supra). An example of such a molecule employed in the present
invention is a "cis-acting" plasmid containing the heterologous
sequence, preferably the Chop2 sequence, flanked by the 5' and 3'
AAV ITR sequences.
[0084] The heterologous sequence encodes a protein or polypeptide
which is desired to be delivered to and expressed in a cell. The
present invention is directed to Chop2 sequences under the control
of a selected promoter and other conventional vector regulatory
components.
[0085] The Transgene Being Targeted and Expressed
[0086] In a most preferred embodiment, the heterologous sequence is
a nucleic acid molecule that functions as a transgene. The term
"transgene" as used herein refers to a nucleic acid sequence
heterologous to the AAV sequence, and encoding a desired product,
preferably Chop2 and the regulatory sequences which direct or
modulate transcription and/or translation of this nucleic acid in a
host cell, enabling expression in such cells of the encoded
product. Preferred polypeptide products are those that can be
delivered to the eye, particularly to retinal neurons.
[0087] The transgene is delivered and expressed in order to treat
or otherwise improve the vision status of a subject with an ocular
disorder that may result from any of a number of causes, including
mutations in a normal photoreceptor-specific gene. The targeted
ocular cells may be photoreceptor cells (if not totally
degenerated) or, more preferably, other retinal neurons, namely,
bipolar cells and retinal ganglion cells.
[0088] Using an mGluR6 promoter operatively linked to a Chop2 opsin
coding sequence and a reporter gene, e.g., GFP or another
fluorescent protein, an insert of about 4.5 kb is preferred -1 kb
for the opsin, 0.7 kb for the reporter, 10 kb-for the mGluR6
promoter region and about 0.4 kb for conventional transcriptional
regulatory factors.
[0089] Use of different opsin genes allows selection of desired
wavelengths as the absorption maxima of different channel proteins
differ. In one embodiment, the reported gene is moved to the red
part of the visual spectrum.
[0090] Similarly, based on the studies reported herein, the
brightness of the light needed to stimulate evoked potential in
transduced mouse retinas, indicates that a channel opsin with
increased light sensitivity may be more desirable. This can be
achieved by selection of a suitable naturally occurring opsin, for
example other microbial-type rhodopsins, or by modifying the light
sensitivity of Chop2 as well as its other properties, such as ion
selectivity and spectral sensitivity, to produce diversified
light-sensitive channels to better fit the need for vision
restoration.
[0091] Different transgenes may be used to encode separate subunits
of a protein being delivered, or to encode different polypeptides
the co-expression of which is desired. If a single transgene
includes DNA encoding each of several subunits, the DNA encoding
each subunit may be separated by an internal ribozyme entry site
(IRES), which is preferred for short subunit-encoding DNA sequences
(e.g., total DNA, including IRES i s<5kB). Other methods which
do not employ an IRES may be used for co-expression, e.g., the use
of a second internal promoter, an alternative splice signal, a
co-or post-translational proteolytic cleavage strategy, etc., all
of which are known in the art.
[0092] The coding sequence or non-coding sequence of the nucleic
acids useful herein preferably are codon-optimized for the species
in which they are to be expressed. Such codon-optimization is
routine in the art.
[0093] While a preferred transgene encodes a full length
polypeptide, preferably Chop2 (SEQ ID NO:6, the present invention
is also directed to vectors that encode a biologically active
fragment or a conservative amino acid substitution variant of Chop2
(or of any other polypeptide of the invention to be expressed in
retinal neurons). Non-limiting examples of useful fragments are the
polypeptide with the sequence SEQ ID NO:3 and SEQ ID NO:8. The
fragment or variant is expressed by the targets cells being
transformed and is able to endow such cells with light sensitivity
that is functionally equivalent to that of the full length or
substantially full length polypeptide having a native, rather than
variant, amino acid sequence. A biologically active fragment or
variant is a "functional equivalent" --a term that is well
understood in the art and is further defined in detail herein. The
requisite biological activity of the fragment or variant, using any
method disclosed herein or known in the art to establish activity
of a channel opsin, has the following activity relative to the
wild-type native polypeptide: about 50%, about 55%, about 60 %,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, about 99%, and any range derivable therein, such as, for
example, from about 70% to about 80%, and more preferably from
about 81% to about 90% or even more preferably, from about 91%to
about 99%.
[0094] It should be appreciated that any variations in the coding
sequences of the present nucleic acids and vectors that, as a
result of the degeneracy of the genetic code, express a polypeptide
of the same sequence, are included within the scope of this
invention.
[0095] The amino acid sequence identity of the variants of the
present invention are determined using standard methods, typically
based on certain mathematical algorithms. In a preferred
embodiment, the percent identity between two amino acid sequences
is determined using the Needleman and Wunsch (J. Mal. Biol.
48:444-453 (1970) algorithm which has been incorporated into the
GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
Meyers and Miller (CABIOS, 4: 11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The nucleotide and amino acid sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases, for example, to identify other
family members or related sequences. Such searches can be performed
using the NBLAST and)(BLAST programs (Altschul et al. (1990) J.
Mal. Biol. 215:403-10). BLAST nucleotide searches can be performed
with the NBLAST program, score=100, wordlength=12 to obtain
nucleotide sequences homologous to, e.g., DAN encoding Chop2 of C.
reinhardtii. BLAST protein searches can be performed with the
XBLAST program, score 50, wordlength=3 to obtain amino acid
sequences homologous to the appropriate reference protein such as
Chop2. To obtain gapped alignments for comparison purposes, Gapped
BLAST can be utilized (Altschul et at (1997) Nucleic Acids Res.
25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See World Wide Web URL ncbi.nlm.nih.gov.
[0096] The preferred amino acid sequence variant has the following
degrees of sequence identity with the native, full length channel
opsin polypeptide, preferably Chop2 from C. reinhardtii (SEQ ID
NO:6) or with a fragment thereof (e.g., SEQ ID NO: 3 or 8): about
50%, about 55%, abou 60%, about 65%, about 70%, about 71%, about
72%, about 73%, about 74%, about 75%, about 76%, about 77%, about
78%, about 79%, about 80%, about 81%, about 82%, about 83%, about
84%, about 85%, about 86%, about 87%, about 88%, about 89%, about
90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, or about 99%, and any range derivable
therein, such as, for example, from about 70% to about 80%, and
more preferably from about 81% to about 90%; or even more
preferably, from about 91% to about 99% identity. A preferred
biologically active fragment comprises or consists of SEQ ID NO: 3,
which corresponds to residues 1-315 of SEQ ID NO:6, or comprises or
consists of SEQ ID NO:8.
[0097] Any of a number of known recombinant methods are used to
produce a DNA molecule encoding the fragment or variant. For
production of a variant, it is routine to introduce mutations into
the coding sequence to generate desired amino acid sequence
variants of the invention. Site-directed mutagenesis is a
well-known technique for which protocols and reagents are
commercially available (e.g., Zoller, M J et al, 1982, Nucl Acids
Res 10:6487-6500; Adelman, J P et al., 1983, DNA 2: 183-93). These
mutations include simple deletions or insertions, systematic
deletions, insertions or substitutions of clusters of bases or
substitutions of single bases.
[0098] In terms of functional equivalents, it is well understood by
those skilled in the art that, inherent in the definition of a
"biologically functional equivalent" protein, polypeptide, gene or
nucleic acid, is the concept that there is a limit to the number of
changes that may be made within a defined portion of the molecule
and still result in a molecule with an acceptable level of
equivalent biological activity. Biologically functional equivalent
peptides are thus defined herein as those peptides in which
certain, not most or all, of the amino acids may be
substituted.
[0099] In particular, the shorter the length of the polypeptide,
the fewer amino acids changes should be made. Longer fragments may
have an intermediate number of changes. The full length polypeptide
protein will have the most tolerance for a larger number of
changes. It is also well understood that where certain residues are
shown to be particularly important to the biological or structural
properties of a polypeptide residues in a binding regions or an
active site, such residues may not generally be exchanged. In this
manner, functional equivalents are defined herein as those poly
peptides which maintain a substantial amount of their native
biological activity.
[0100] For a detailed description of protein chemistry and
structure, see Schulz, G E et al., Principles of Ppotein Structure,
Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins:
Structure and Molecular Properties, W. H. Freeman & Co., San
Francisco, 1983, which are hereby incorporated by reference. The
types of substitutions that may be made in the protein molecule may
be based on analysis of the frequencies of amino acid changes
between a homologous protein of different species, such as those
presented in Table 1-2 of Schulz et al (supra) and FIG. 3-9 of
Creighton (supra). Based on such an analysis, conservative
substitutions are defined herein as exchanges within one of the
following five groups:
TABLE-US-00001 1 Small aliphatic, nonpolar or Ala, Ser, Thr (Pro,
Gly); slightly polar residues 2 Polar, negatively charged residues
Asp, Asn, Glu, Gln; and their amides 3 Polar, positively charged
residues His, Arg, Lys; 4 Large aliphatic, nonpolar residues Met,
Leu, Ile, Val (Cys) 5 Large aromatic residues Phe, Tyr, Trp.
[0101] The three amino acid residues in parentheses above have
special roles in protein architecture. Gly is the only residue
lacking a side chain and thus imparts flexibility to the chain.
Pro, because of its unusual geometry, tightly constrains the chain.
Cys can participate in disulfide bond formation, which is important
in protein folding.
[0102] The hydropathy index of amino acids may also be considered
in selecting variants. Each amino acid has been assigned a
hydropathy index on the basis of their hydrophobicity and charge
characteristics, these are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe
(+2.8); Cys (+2.5); Met (+1.9); Ala(+1.8); Glycine (-0.4); Thr
(-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2);
Glu (-3.5); Gln (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg
(-4.5). The importance of the hydropathy index in conferring
interactive biological function on a proteinaceous molecule is
generally understood in the art (Kyte and Doolittle, 1982, J. Mol.
Biol. 157:105-32). It is known that certain amino acids may be
substituted for other amino acids having a similar hydropathy index
or score and still retain a similar biological activity. In making
changes based upon the hydropathy index, the substitution of amino
acids whose hydropathy indices are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. It is also understood
in the art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity, particularly where the
biological functional equivalent polypeptide thereby created is
intended for use in certain of the present embodiments. U.S. Pat.
No. 4,554,101, discloses that the greatest local average
hydrophilicity of a proteinaceous molecule, as governed by the
hydrophilicity of its adjacent amino acids, correlates with a
biological property of the molecule. See U.S. Pat. No. 4,554,101
for a hydrophilicity values. In making changes based upon similar
hydrophilicity values, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those which
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0103] Regulatory Sequences
[0104] The minigene or transgene of the present invention includes
appropriate sequences operably linked to the coding sequence or ORF
to promote its expression in a targeted host cell. "Operably
linked" sequences include both expression control sequences such
as. promoters that are contiguous with the coding sequences and
expression control sequences that act in trans or distally to
control the expression of the polypeptide product.
[0105] Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (e.g., Kozak
consensus sequence); sequences that enhance nucleic acid or protein
stability; and when desired, sequences that enhance protein
processing and/or secretion. Many varied expression control
sequences, including native and non-native, constitutive, inducible
and/or tissue-specific, are known in the art and may be utilized
herein. depending upon the type of expression desired.
[0106] Expression control sequences for eukaryotic cells typically
include a promoter, an enhancer, such as one derived from an
immunoglobulin gene, SV40, CMV, etc., and a polyadenylation
sequence which may include splice donor and acceptor sites. The
polyadenylation sequence generally is inserted 3' to the coding
sequence and 5' to the 3' ITR sequence. PolyA from bovine growth
hormone is a suitable sequence.
[0107] The regulatory sequences useful herein may also contain an
intron, such as one located between the promoter/enhancer sequence
and the coding sequence. One useful intron sequence is derived from
SV40, and is referred to as the SV40 T intron sequence. Another
includes the woodchuck hepatitis virus post-transcriptional
element. (See, for example, Wang L and Verma, I, 1999, Proc Nat'l
Acad Sci USA, 96:3906-10).
[0108] An IRES sequence, or other suitable system as discussed
above, may be used to produce more than one polypeptide from a
single transcript. In exemplary IRES is the poliovirus IRES which
supports transgene expression in photoreceptors, RPE and ganglion
cells. Preferably, the IRES is located 3' to the coding sequence in
the rAAV vector.
[0109] The promoter may be selected from a number of constitutive
or inducible promoters that can drive expression of the selected
transgene in an ocular setting, preferably in retinal neurons. A
preferred promoter is "cell-specific", meaning that it is selected
to direct expression of the selected transgene in a particular
ocular cell type, such as photoreceptor cells. p Examples of useful
constitutive promoters include the exemplified??? CMV immediate
early enhancer/chicken-actin (C A) promoter-exon 1-intron 1
element, the RSV LTR promoter/enhancer, the SV40 promoter, the CMV
promoter, the dihydrofolate reductase (DHFR) promoter, and the
phosphoglycerol kinase (PGK) promoter.
[0110] Additional useful promoters are disclosed in W. W. Hauswirth
et al., 1998, W098/48027 and A. M. Timmers et al., 2000,
W000/15822. Promoters that were found to drive RPE cell-specific
gene expression in vivo include (1) a 528-bp promoter region (bases
1-528 of a murine 11-cis retinol dehydrogenase (RDH) gene
(Driessen, C A et at, 1995, Invest. Ophthalmol. Vis Sci. 36:
1988-96; Simon, A. et at, 1995, J. Biol. Chem 270: 1107-12, 1995;
Simon, A. et at, 1996, Genomics 36:424-3) Genbank Accession Number
X97752); (2) a 2274-bp promoter region) from a human cellular
retinaldehyde-binding protein (CRALBP) gene (Intres, R et al, 1994,
J. Biol. Chem. 269:25411-18; Kennedy, B N et al., 1998,1.. Bio!.
Chem. 273:5591-8, 1998), Genbank Accession Number L34219); and (3)
a 1485-bp promoter region from human RPE65 (Nicoletti, A et al.,
1998, Invest. Ophthalmol. Vis Sci. 39:637-44, Genbank Accession
Number U20510). These three promoters (labeled with the following
SEQ ID numbers in W000/15822'' 2. 3 and 3) promoted RPE-cell
specific expression of GFP. It is envisioned that minor sequence
variations in the various promoters and promoter regions discussed
herein--whether additions, deletions or mutations, whether
naturally occurring or introduced in vitro, will not affect their
ability to drive expression in the cellular targets of the present
invention. Furthermore, the use of other promoters, even if not yet
discovered, that are characterized by abundant and/or specific
expression in retinal cells, particularly in bipolar or ganglion
cells, is specifically included within the scope of this
invention.
[0111] An inducible promoter is used to control the amount and
timing of production of the transgene product in an ocular cell.
Such promoters can be useful if the gene product has some
undesired, e.g., toxic, effects in the cell if it accumulates
excessively. Inducible promoters include those known in the art,
such as the Zn-inducible sheep metallothionine (MT) promoter, the
dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV)
promoter; the T7 promoter; the ecdysone insect promoter; the
tetracycline-repressible system; the tetracycline-inducible system;
the RU486-inducible system; and the rapamycin-inducible system. Any
inducible promoter the action of which is tightly regulated and is
specific for the particular target ocular cell type, may be used.
Other useful types of inducible promoters are ones regulated by a
specific physiological state, e.g., temperature, acute phase, a
cell's replicating or differentiation state.
[0112] Selection of the various vector and regulatory elements for
use herein are conventional, well-described, and readily available.
See, e.g., Sambrook et al., supra; and Ausubel et al., supra. It
will be readily appreciated that not all vectors and expression
control sequences will function equally well to express the present
transgene, preferably Chop2. Clearly, the skilled artisan may apply
routine selection among the known expression control sequences
without departing from the scope of this invention and based upon
general knowledge as well as the guidance provided herein. One
skilled in the art can select one or more expression control
sequences, operably link them to the coding sequence being
expressed to make a minigene, insert the mini gene or vector into
an AAV vector, and cause packaging of the vector into infectious
particles or virions following one of the known packaging methods
for rAAV.
[0113] Production of the rAAV
[0114] The rAAV used in the present invention may be constructed
and produced using the materials and methods described herein and
those well-known in the art. The methods that are preferred for
producing any construct of this invention are conventional and
include genetic engineering, recombinant engineering, and synthetic
techniques, such as those set forth in reference cited above.
[0115] Briefly, to package an rAAV construct into an rAAV virion, a
sequences necessary to express AAV rep and AAV cap or functional
fragments thereof as well as helper genes essential for AAV
production must be present in the host cells. See, for example U.S.
Patent Pub. 2007/0015238, which describes production of pseudotyped
rAAV virion vectors encoding AAV Rep and Cap proteins of different
serotypes and AdV transcription products that provide helper
functions For example, AAV rep and cap sequences may be introduced
into the host cell in any known manner including, without
limitation, transfection, electroporation, liposome delivery,
membrane fusion, biolistic deliver of DNA-coated pellets, viral
infection and protoplast fusion. Devices specifically adapted for
delivering DNA to specific regions within and around the eye for
the purpose of gene therapy have been described recently (for
example, U.S. Patent Pub. 2005/0277868, incorporated by reference)
are used within the scope of this invention. Such devices utilize
electroporation and electromigration, providing, e.g., two
electrodes on a flexible support that can be placed behind the
retina. A third electrode is part of a hollow support, which can
also be used to inject the molecule to the desired area. The
electrodes can be positioned around the eye, including behind the
retina or within the vitreous.
[0116] These sequences may exist stably in the cell as an episome
or be stably integrated into the cell's genome. They may also be
expressed more transiently in the host cell. As an example, a
useful nucleic acid molecule comprises, from 5' to 3', a promoter,
an optional spacer between the promoter and the start site of the
rep sequence, an AAV rep sequence, and an AAV cap sequence.
[0117] The rep and cap sequences, along with their expression
control sequences, are preferably provided in a single vector,
though they may be provided separately in individual vectors. The
promoter may be any suitable constitutive, inducible or native
promoter. The delivery molecule that provides the Rep and Cap
proteins may be in any form, preferably a plasmid which may contain
other non-viral sequences, such as those to be employed as markers.
This molecule typically excludes the AAV ITRs and packaging
sequences. To avoid the occurrence of homologous recombination,
other viral sequences, particularly adcnoviral sequences, arc
avoided. This plasmid is preferably one that is stably
expressed.
[0118] Conventional genetic engineering or recombinant DNA
techniques described in the cited references are used. The rAAV may
be produced using a triple transfection method with either the
calcium phosphate (Clontech) or Effectene.TM. reagent (Qiagen)
according to manufacturer's instructions. See, also, Herzog et al.,
1999, Nat. Med. 5:56-63.
[0119] The rAAV virions are produced by culturing host cells
comprising a rAAV as described herein which includes a rAAV
construct to be packaged into a rAAV virion, an AAV rep sequence
and an AAV cap sequence, all under control of regulatory sequences
directing expression.
[0120] Suitable viral helper genes, such as adenovirus E2A, E40rf6
and VA, may be added to the culture preferably on separate
plasmids. Thereafter, the rAAV virion which directs expression of
the transgene is isolated in the absence of contaminating helper
virus or wildtype AAV.
[0121] It is conventional to assess whether a particular expression
control sequence is suitable for a given transgene, and choose the
one most appropriate for expressing the transgene. For example, a
target cell may be infected in vitro, and the number of copies of
the transgene in the cell monitored by Southern blots or
quantitative PCR. The level of RNA expression may be monitored by
Northern blots quantitative RT-PCR. The level of protein expression
may be monitored by Western blot, immunohistochemistry, immunoassay
including enzyme immunoassay (EIA) such as enzyme-linked
immunosorbent assays (ELISA), radioimmunoassays (RIA) or by other
methods. Specific embodiments are described in the Examples
below.
Pharmaceutical Compositions and Methods of the Invention
[0122] The rAAV that comprises the Chop2 transgene and
cell-specific promoter for use in the target ocular cell as
described above should be assessed for contamination using
conventional methods and formulated into a sterile or aseptic
pharmaceutical composition for administration by, for example,
subretinal injection.
[0123] Such formulations comprise a pharmaceutically and/or
physiologically acceptable vehicle, diluent, carrier or excipient,
such as buffered saline or other buffers, e.g., HEPES, to maintain
physiologic pH. For a discussion of such components and their
formulation, see, generally, Gennaro, A E., Remington: The Science
and Practice of Pharmacy, Lippincott Williams & Wilkins
Publishers; 2003 or latest edition). See also, W000/15822. If the
preparation is to be stored for long periods, it may be frozen, for
example, in the presence of glycerol.
[0124] The pharmaceutical composition described above is
administered b a subject having a visual or blinding disease by any
appropriate route, preferably by intravitreal or subretinal
injection, depending on the retinal layer being targeted.
[0125] Disclosures from Bennett and colleagues (cited herein)
concern targeting of retinal pigment epithelium--the most distal
layer from the vitreal space. According to the present invention,
the DNA construct is targeted to either retinal ganglion cells or
bipolar cells. The ganglion cells are reasonably well-accessible to
intravitreal injection as disclosed herein. Intravitreal and/or
subretinal injection can provide the necessary access b the bipolar
cells, especially in circumstances in which the photoreceptor cell
layer is absent due to degeneration--which is the case in certain
forms of degeneration that the present invention is intended to
overcome.
[0126] To test for the vector's ability to express the transgene,
specifically in mammalian retinal neurons, by AAV-mediated
delivery, a combination of a preferred promoter sequence linked to
a reporter gene such as Lacz or GFP linked to a SV40 poly A
sequence can be inserted into a plasmid and packaged into rAAV
virus particles, concentrated, tested for contaminating adenovirus
and titered for rAAV using an infectious center assay. The right
eyes of a number of test subjects, preferably inbred mice, are
injected sub-retinally with about 1 .mu.l of the rAAV preparation
(e.g., greater than about 10.sup.10 infectious units ml). Two weeks
later, the right (test) and left (control) eyes of half the animals
are removed, fixed and stained with an appropriate substrate or
antibody or other substance to reveal the presence of the reporter
gene. A majority of the test retinas in injected eyes will
exhibited a focal stained region, e.g., blue for LacZ/Xgal, or
green for GFP consistent with a subretinal bleb of the injected
virus creating a localized retinal detachment. All control eyes are
negative for the reporter gene product. Reporter gene expression
examined in mice sacrificed at later periods is detected for at
least 10 weeks post-injection, which suggests persistent expression
of the reporter transgene.
[0127] An effective amount of rAAV virions carrying a nucleic acid
sequence encoding the Chop2 DNA under the control of the promoter
of choice, preferably a constitutive CMV promoter or a
cell-specific promoter such as mGluR6, is preferably in the range
of between about 10.sup.1.sup.0 to about 10.sup.1.sup.3 rAAV
infectious units in a volume of between about 150 and about 800
.mu.l per injection. The rAAV infectious units can be measured
according to McLaughlin, S K et al., 1988, J Viral 62: 1963. More
preferably, the effective amount is between about 10.sup.1.sup.0
and about 10.sup.12 rAAV infectious units and the injection volume
is preferably between about 250 and about 500 .mu.l. Other dosages
and volumes, preferably within these ranges but possibly outside
them, may be selected by the treating professional, taking into
account the physical state of the subject (preferably a human), who
is being treated, including, age, weight, general health, and the
nature and severity of the particular ocular disorder.
[0128] It may also be desirable to administer additional doses
("boosters") of the present nucleic acid or rAAV compositions. For
example, depending upon the duration of the transgene expression
within the ocular target cell, a second treatment may be
administered after 6 months or yearly, and may be similarly
repeated. Neutralizing antibodies to AAV are not expected to be
generated in view of the routes and doses used, thereby permitting
repeat treatment rounds.
[0129] The need for such additional doses can be monitored by the
treating professional using, for example, well-known
electrophysiological and other retinal and visual function tests
and visual behavior tests. The treating professional will be able
to select the appropriate tests applying routine skill in the art.
It may be desirable to inject larger volumes of the composition in
either single or multiple doses to further improve the relevant
outcome parameters.
Restoration or Improvement of Light Sensitivity and Vision
[0130] Both in vitro and in vivo studies to assess the various
parameters of the present invention may be used, including
recognized animal models of blinding human ocular disorders. Large
animal models of human retinopathy, e.g., childhood blindness, are
useful. The examples provided herein allow one of skill in the art
to readily anticipate that this method may be similarly used in
treating a range of retinal diseases.
[0131] While earlier studies by others have demonstrated that
retinal degeneration can be retarded by gene therapy techniques,
the present invention demonstrates a definite physiological
recovery of function, which is expected to generate or improve
various parameters of vision, including behavioral parameters.
[0132] Behavioral measures can be obtained using known animal
models and tests, for example performance in a water maze, wherein
a subject in whom vision has been preserved or restored to varying
extents will swim toward light (Hayes, J M et al., 1993, Behav
Genet 23:395-403).
[0133] In models in which blindness is induced during adult life or
congenital blindness develops slowly enough that the individual
experiences vision before losing it, training of the subject in
various tests may be done. In this way, when these tests are
re-administered after visual loss to test the efficacy of the
present compositions and methods for their vision-restorative
effects, animals do not have to learn the tasks de nova while in a
blind state. Other behavioral tests do not require learning and
rely on the instinctiveness of certain behaviors. An example is the
optokinetic nystagmus test (Balkema G W et al., 1984, Invest
Ophthalmol Vis Sci 25:795-800; Mitchiner J C et al., 1976,
VisionRes.16:1169-71).
[0134] As is exemplified herein, the transfection of retinal
neurons with DNA encoding Chop2 provides residual retinal neurons,
principally bipolar cells and ganglion cells, with photosensitive
membrane channels. Thus, it was possible to measure, with a strong
light stimulus, the transmission of a visual stimulus to the
animal's visual cortex, the area of the brain responsible for
processing visual signals; this therefore constitutes a form of
vision, as intended herein. Such vision may differ from forms of
normal human vision and may be referred to as a sensation of light,
also termed "light detection" or "light perception."
[0135] Thus, the term "vision" as used herein is defined as the
ability of an organism to usefully detect light as a stimulus for
differentiation or action. Vision is intended to encompass the
following: [0136] 1. Light detection or perception--the ability to
discern whether or not light is present [0137] 2. Light
projection--the ability to discern the direction from which a light
stimulus is coming; [0138] 3. Resolution--the ability to detect
differing brightness levels (i.e., contrast) in a grating or letter
target; and [0139] 4. Recognition--the ability to recognize the
shape of a visual target by reference to the differing contrast
levels within the target.
[0140] Thus, "vision" includes the ability to simply detect the
presence of light. This opens the possibility to train an affected
subject who has been treated according to this invention to detect
light, enabling the individual to respond remotely to his
environment however crude that interaction might be. In one
example, a signal array is produced to which a low vision person
can respond to that would enhance the person's ability to
communicate by electronic means remotely or to perform everyday
tasks. In addition such a person's mobility would be dramatically
enhanced if trained to use such a renewed sense of light resulting
from "light detection." The complete absence of light perception
leaves a person with no means (aside from hearing and smell) to
discern anything about objects remote to himself.
[0141] The methods of the present invention that result in light
perception, even without full normal vision, also improve or permit
normally regulated circadian rhythms which control many
physiological processes including sleep-wake cycles and associated
hormones. Although some blind individuals with residual retinal
ganglion cells (RGCs) can mediate their rhythms using RGC
melanopsin, it is rare for them to do so. Thus, most blind persons
have free-running circadian rhythms. Even when such individuals do
utilize the melanopsin pathway, the effect is very weak effect. The
methods of the present invention are thus expected to improve
health status of blind individuals by enabling absent light
entrainment or improving weakened (melanopsin-mediated) light
entrainment of their circadian rhythms. This leads to better health
and well-being of these subjects.
[0142] In addition to circadian rhythms, the present invention
provides a basis to improve deficits in other light-induced
physiological phenomena. Photoreceptor degeneration may result in
varying degrees of negative masking, or suppression, of locomotor
activity during the intervals in the circadian cycle in which the
individual should be sleeping. Another result is suppression of
pineal melatonin. Both of these contribute to the entrainment
process. Thus, improvement in these responses or activities in a
subject in whom photoreceptors are degenerating or have degenerated
contributes, independently of vision per se, to appropriate
sleep/wake cycles that correspond with the subject's environment in
the real world.
[0143] Yet another benefit of the present invention is
normalization of pupillary light reflexes because regulation of
pupil size helps modulate the effectivenees of light stimuli in a
natural feed back loop. Thus, the present invention promotes
re-establishment of this natural feedback loop, making vision more
effective in subject treated as described herein.
[0144] In certain embodiments, the present methods include the
measurement of vision before, and preferably after, administering a
vector comprising, for example, DNA encoding Chop2. Vision is
measured using any of a number of methods well-known in the art or
ones not yet establshed. Most preferred herein are the following
visual responses: [0145] (1) A light detection response by the
subject after exposure to a light stimulus--in which evdence is
sought for a reliable response of an indication or movement in the
general direction of the light by the subject individual when the
light it is turned on is. [0146] (2) a light projection response by
the subject after exposure to a light stimulus in which evidence is
sought for a reliable response of indication or movement in the
specific direction of the light by the individual when the light is
turned on. [0147] (3) light resolution by the subject of a light
vs. dark patterned visual stimulus, which measures the subject's
capability of resolving light vs dark patterned visual stimuli as
evidenced by: [0148] (a) the presence of demonstrable reliable
optokinetically produced mystagmoid eye movements and/or related
head or body movements that demonstrate tracking of the target (see
above) and/or [0149] (b). the presence of a reliable ability to
discriminate a pattern visual stimulus and to indicate such
discrimination by verbal or non-verbal means, including, for
example pointing, or pressing a bar or a button; or [0150] (4)
electrical recording of a visual cortex response to a light flash
stimulus or a pattern visual stimulus, which is an endpoint of
electrical transmission from a restored retina to the visual
cortex. Measurement may be by electrical recording on the scalp
surface at the region of the visual cortex, on the cortical
surface, and/or recording within cells of the visual cortex.
[0151] It is known in the art that it is often difficult to make
children who have only light perception appreciate that they have
this vision. Training is required to get such children to react to
their visual sensations. Such a situation is mimicked in the animal
studies exemplified below. Promoting or enhancing light perception,
which the compositions and methods of the present invention will
accomplish, is valuable because patients with light perception not
only are trainable to see light, but they can usually be trained to
detect the visual direction of the light, thus enabling them to be
trained in mobility in their environment. In addition, even basic
light perception can be used by visually impaired individuals,
including those whose vision is improved using the present
compositions and methods, along with specially engineered
electronic and mechanical devices to enable these individuals to
accomplish specific daily tasks. Beyond this and depending on their
condition, they may even be able to be trained in resolution tasks
such as character recognition and even reading i f their impairment
permits. Thus it is expected that the present invention enhances
the vision of impaired subjects to such a level that by applying
additional training methods, these individuals will achieve the
above objectives.
[0152] Low sensitivity vision may emulate the condition of a person
with a night blinding disorder, an example of which is Retinitis
Pigmentosa (RP), who has difficulty adapting to light levels in his
environment and who might use light amplification devices such as
supplemental lighting and/or night vision devices.
[0153] Thus, the visual recovery that has been described in the
animal studies described below would, in human terms, place the
person on the low end of vision function. Nevertheless, placement
at such a level would be a significant benefit because these
individuals could be trained in mobility and potentially in low
order resolution tasks which would provide them with a greatly
improved level of visual independence compared to total
blindness.
[0154] The mice studied in the present Examples were rendered
completely devoid of photoreceptors; this is quite rare, even in
the worst human diseases. The most similar human state is RP. In
most cases of RP, central vision is retained till the very end. In
contrast, in the studied mouse model, the mouse becomes completely
blind shortly after birth.
[0155] Common disorders encountered in low vision are described by
J. Tasca and E. A. Deglin in Chap. 6 of Essentials of Low Vision
Practice, R. L. Brilliant, ed, Butterworth Heinemann Publ., 1999,
which is incorporated by reference in its entirety. There is
reference to similar degenerative conditions, but these references
show.form vision that is measurable as visual acuity. Ganglion cell
layers are not retained in all forms of RP, so the present approach
will not work for such a disorder.
[0156] When applying the present methods to humans with severe
cases of RP, it is expected that central vision would be maintained
for a time at some low level while the peripheral retina
degenerated first. It is this degenerating retina that is the
target for re-activation using the present invention. In essence,
these individuals would be able to retain mobility vision as they
approached blindness gradually.
[0157] Subjects with macular degeneration, characterized by
photoreceptor loss within the central "sweet spot" of vision
(Macula Lutea), are expected to benefit by treatment in accordance
with the present invention, in which case the resolution capability
of the recovered vision would be expected to be higher due to the
much higher neuronal density within the human macula.
[0158] While it is expected that bright illumination of daylight
and artificial lighting that may be used by a visually impaired
individual will suffice for many visual activities that are
performed with vision that has recovered as a result of the present
treatments. It is also possible that light amplification devices
may be used, as needed, to further enhance the affected person's
visual sensitivity. The human vision system can operate over a 10
log unit range of luminance. On the other hand, microbial type
rhodopsins, such as ChR2, operate over up to a 3 log unit range of
luminance. In addition, the light conditions the patient encounters
could fall outside of the operating range of the light sensor. To
compensate for the various light conditions, a light
pre-amplification or attenuation device could be used to expand the
operation range of the light conditions. Such device would contain
a camera, imaging processing system, and microdisplays, which can
be assembled from currently available technologies, such as night
vision goggles and/or 3D adventure and entertainment system. (See,
for example the following URL on the Worldwide
web--emagin.com/.)
[0159] The present invention may be used in combination with other
forms of vision therapy known in the art. Chief among these is the
use of visual prostheses, which include retinal implants, cortical
implants, lateral geniculate nucleus implants, or optic nerve
implants. Thus, in addition to genetic modification of surviving
retinal neurons using the present methods, the subject being
treated may be provided with a visual prosthesis before, at the
same time as, or after the molecular method is employed.
[0160] The effectiveness of visual prosthetics can be improved with
training of the individual, thus enhancing the potential impact of
the Chop2 transformation of patient cells as contemplated herein.
An example of an approach to training is found in US 2004/0236389
(Fink et al.), incorporated by reference. The training method may
include providing a non-visual reference stimulus to a patient
having a visual prosthesis based on a reference image. The
non-visual reference stimulus is intended to provide the patient
with an expectation of the visual image that the prosthesis will
induce. Examples of non-visual reference stimuli are a pinboard,
Braille text, or a verbal communication. The visual prosthesis
stimulates the patient's nerve cells, including those cells whose
responsiveness has been improved by expressing Chop2 as disclosed
herein, with a series of stimulus patterns attempting to induce a
visual perception that matches the patient's expected perception
derived from the non-visual reference stimulus. The patient
provides feedback to indicate which of the series of stimulus
patterns induces a perception that most closely resembles the
expected perception. The patient feedback is used as a "fitness
function" (also referred to as a cost function or an energy
function). Subsequent stimuli provided to the patient through the
visual prosthesis are based, at least in part, on the previous
feedback of the patient as to which stimulus pattern(s) induce the
perception that best matches the expected perception. The
subsequent stimulus patterns may also be based, at least in part,
on a fitness function optimization algorithm, such as a simulated
annealing algorithm or a genetic algorithm.
[0161] Thus, in certain embodiments of this invention, the method
of improving or restoring vision in a subject further comprises
training of that subject, as discussed above. Preferred examples of
training methods are: [0162] (a) habituation training characterized
by training the subject to recognize (i) varying levels of light
and/or pattern stimulation, and/or (ii) environmental stimulation
from a common light source or object as would be understood by one
skilled in the art; and [0163] (b) orientation and mobility
training characterized by training the subject to detect visually
local objects and move among said objects more effectively than
without the training.
[0164] In fact, any visual stimulation techniques that are
typically used in the field of low vision rehabilitation are
applicable here.
[0165] The remodeling of inner retinal neurons triggered by
photoreceptor degeneration has raised a concerns about
retinal-based rescue strategics after the death of photoreceptors
(Strettoi and Pignatelli 2000, Proc Natl Acad Sci USA. 97: 11020-5;
Jones, B W et al., 2003, J Comp Neural 464:1-16; Jones, B W and
Marc, R E, 2005, Exp Eye Res. 81:123-37; Jones, B W et al., 2005,
Clin Exp Optom. 88:282-91). Retinal remodeling is believed to
result from deafferentation, the loss of afferent inputs from
photoreceptors--in other words, the loss of light induced
activities So after death of rods and coned, there is m light
evoked input to retinal bipolar cells and ganglion cells, and
through them to higher visual centers. In response to the loss of
such input, the retina and higher visual network are triggered to
undergo remodeling, in a way seeking other forms of inputs. Said
otherwise, the retina needs to be used to sense light in order to
maintain its normal network, and with the loss of light sensing,
the network will deteriorate via a remodeling process. This process
is not an immediate consequence of photoreceptor death; rather it
is a slow process, providing a reasonably long window for
intervention.
[0166] Thus, an additional utility of restoring light sensitivity
to inner retinal neurons in accordance with the present invention
is the prevention or delay in the remodeling processes in the
retina, and, possibly, in the higher centers. Such retinal
remodeling may have undesired consequences such as corruption of
inner retinal network, primarily the connection between bipolar and
retinal ganglion cells. By introducing the light-evoked activities
in bipolar cells or ganglion cells, the present methods would
prevent or diminish the remodeling due to the lack of input; the
present methods introduce this missing input (either starting from
bipolar cells or ganglion cells), and thereby stabilize the retinal
and higher visual center network. Thus, independently of its direct
effects on vision, the present invention would benefit other
therapeutic approaches such as photoreceptor transplantation or
device implants.
[0167] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
SYNOPSIS OF EXAMPLES
[0168] (references cited in the following sections may appear in a
list at the end)
Methods
[0169] A Chop2-GFP chimera was made by linking a nucleic acid
encoding green fluorescent protein (GFP) (part of SEQ ID NO: 1 as
shown below) to a nucleic acid (SEQ ID NO:2) encoding an active
fragment (SEQ ID NO:3) of channelopsin-2 (Chop2) such that an
expressed protein has the GFP linked to the C-terminus of the Chop2
region. Both these sequences constitute the "transgene" as
discussed above. The Chop2-GFP DNA was transfected into HEK293
cells under control of a CMV promoter.
[0170] A viral construct (SEQ ID NO: 1) was made by subcloning the
Chop2-GFP into an AAV-2 viral cassette containing a CAG promoter. A
map of this construct is shown in FIG. 7. The viral vectors were
injected into the eye of newborn rats. The expression of Chop2-GFP
was examined by GFP fluorescence in retinal whole-mounts or slice
sections. The function of the Chop2-GFP was assessed by whole-cell
patch clamp recordings.
Results
[0171] Bright GFP fluorescence was detected within 18-24 hrs in HEK
cells after the transfection. The fluorescence was localized
predominantly to the plasma membrane. The preserve of the function
of the Chop2-GFP chimera was confirmed by patch-clamp recordings.
Substantial light-gated currents were also observed in the
Chop2-GFP-expressing HEK cells without adding the exogenous
all-trans retinal, indicating that a significant number of
functional Chop2-GFP channels were formed in HEK cells using only
endogenous precursor for the chromophore group. Three to four weeks
after the injection, GFP fluorescence was observed in the retinal
neurons of the injected eyes. Bright GFP-fluorescence was observed
in many ganglion cells and horizontal cells, some amacrine cells,
and, occasionally, bipolar cells for at least 10 weeks following
injection. The Chop2-GFP-expressing retinal neurons exhibited
robust membrane depolarization in response to light stimulation and
did not require an exogenous source of all-trans retinal.
[0172] Thus, the inventors demonstrated that the selected AAV
vector construct efficiently targeted retinal ganglion cells and
effectively delivered the Chop2-GFP cDNA and expressed protein at
high levels after intravitreal injection in both normal and
diseased retinas. When endogenous retinal was bound to the Chop2,
it could be photoswitched, and neural activity could be evoked in
retinas and at cortical levels. This was shown by several
techniques-initially by in vitro patch-clamp recordings of
individual dissociated RGCs, followed by multielectrode array
recordings of whole-mount retina preparations representative of a
large population of RGCs. Finally, in vivo cortical recordings from
live blind mice demonstrated that critical connections were
functionally maintained to higher visual centers.
Conclusion
[0173] The progressive in vitro and in vivo results show that
ectopic expression of Chop2 is a therapeutic strategy for restoring
light sensitivity to a "blind" retina. Functional expression of a
directly light-gated membrane channel, Chop2, was demonstrated in
rat retinal neurons in vivo. Thus, expression of light-gated
membrane channels in second- or third-order retinal neurons is a
useful strategy for restoration of light perception after
photoreceptor degeneration.
Example I
Materials and Methods
DNA and Viral Vector Constructions
[0174] The DNA fragment encoding the N-terminal fragment
(Met.sup.1-Lys.sup.315) of Chop2 (Nagel et al., 2003) was cloned
into pBluescript vector (Stratagene) containing the last exon of a
mouse protamine 1 gene containing polyadenylation signal (mP 1) and
GFP cDNA inserted in frame at the 3' end of the Chop2 coding
fragment to produce a Chop2-GFP fusion protein. The function of
Chop2-GFP chimera was verified in transfected HEK293 cells.
[0175] The viral expression construct rAAV2-CAG-Chop2-GFP-WPRE was
made by subcloning the Chop2-GFP fragment into an adeno-associated
(serotype-2) viral expression cassette. The viral cassette
comprised a hybrid CMV enhancer/chicken-actin promoter (CAG), a
woodchuck posttranscriptional regulatory element (WPRE), and a
bovine growth hormone (BGH) polyadenylation sequence. Viral vectors
were packaged and affinity purified (GeneDetect).
[0176] The vector map is shown in FIG. 7.
[0177] The nucleic acid sequence of this vector (SEQ ID NO:1) is
shown below in annotated form (with the annotations as
described):
[0178] ITR's (at both ends) (UPPER CASE underscore)
[0179] CAG promoter sequence (Lower case, bold italic)
[0180] Kozak sequence (lower case double underscore)
[0181] Chop2 coding sequence (lower case, bold)
[0182] Green fluorescent protein coding sequence (lower case, bold
underscored)
[0183] WPRE (regulatory element): (UPPER CASE)
[0184] The BGH Poly A sequence is not marked.
[0185] The remaining sequence (all lower case), including between
Chop2 and GFP, is vector sequence
TABLE-US-00002 --------ITR ---------- CCTGCAGGCA GCTGCGCGCT
CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC 60 GGGCGACCTT
TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA 120
-----ITR-----------7 --------CAG Promoter---------- ##STR00001##
180 ##STR00002## 240 ##STR00003## 300 ##STR00004## 360 ##STR00005##
420 ##STR00006## 480 ##STR00007## 540 ##STR00008## 600 ##STR00009##
660 ##STR00010## 720 ##STR00011## 780 ##STR00012## 840 ##STR00013##
900 ##STR00014## 960 --------------------CAG
Promoter--------------------> ##STR00015## 1020 gctctgactg
accgcgttac tcccacaggt gagcgggcgg gacggccctt ctccttcggg 1080
ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
1140 <Kozak> ---------chop2----- tgaggggctc cgggagggcc
cgagctcgcg atccgcagcc atggattatg gaggcgccct 1200 gagtgccgtt
gggcgcgagc tgctatttgt aacgaaccca gtagtcgtca atggctctgt 1260
acttgtgcct gaggaccagt gttactgcgc gggctggatt gagtcgcgtg gcacaaacgg
1320 tgcccaaacg gcgtcgaacg tgctgcaatg gcttgctgct ggcttctcca
tcctactgct 1380 tatgttttac gcctaccaaa catggaagtc aacctgcggc
tgggaggaga tctatgtgtg 1440 cgctatcgag atggtcaagg tgattcttga
gttcttcttc gagtttaaga acccgtccat 1500 gctgtatcta gccacaggcc
accgcgtcca gtggttgcgt tacgccgagt ggcttctcac 1560 ctgcccggtc
attctcattc acctgtcaaa cctgacgggc ttgtccaacg actacagcag 1620
gcgcactatg ggtctgcttg tgtctgatat tggcacaatt gtgtggggcg ccacttccgc
1680 tatggccacc ggatacgtca aggtcatctt cttctgcctg ggtctgtgtt
atggtgctaa 1740 cacgttcttt cacgctgcca aggcctacat cgagggttac
cataccgtgc cgaagggccg 1800 gtgtcgccag gtggtgactg gcatggcttg
gctcttcttc gtatcatggg gtatgttccc 1860 catcctgttc atcctcggcc
ccgagggctt cggcgtcctg agcgtgtacg gctccaccgt 1920 cggccacacc
atcattgacc tgatgtcgaa gaactgctgg ggtctgctcg gccactacct 1980
gcgcgtgctg atccacgagc atatcctcat ccacggcgac attcgcaaga ccaccaaatt
2040 gaacattggt ggcactgaga ttgaggtcga gacgctggtg gaggacgagg
ccgaggctgg 2100 ---------chop2-----------> ----- GFP----
cgcggtcaac aagggcaccg gcaaggaatt cggaggcgga ggtggagcta gcaaaggaga
2160 agaactcttc actggagttg tcccaattct tgttgaatta gatggtgatg
ttaacggcca 2220 caagttctct gtcagtagag aaggtgaaag tgatgcaaca
tacagaaaac ttaccctgaa 2280 gttcatctgc actactggca aactgcctgt
tccatggcca acactagtca ctactctgtg 2340 ctatggtgtt caatgctttt
caagataccc ggatcatatg aaacagcatg actttttcaa 2400 gagtgccatg
cccgaaggtt atgtacagga aaggaccatc ttcatcaaag atgacggcaa 2460
ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt
2520 aaaaagtatt gacttcaagg aagatagcaa cattctaaga cacaaattag
aatacaacta 2590 taactcacac aatgtataca tcatggcaga caaacaaaag
aatggaatca aagtgaactt 2640 caagacccgc cacaacattg aagatagaag
cgttcaacta gcagaccatt atcaacaaaa 2700 tactccaatt ggcgatggcc
ctgtcctttt accagacaac cattacctgt ccacacaatc 2760 tgccctttcg
aaagatccca acgaaaagag aaaccacata gtccttcttg aatttgtaac 2820
-----------------GFP------------------7 agctgctggg attacacatg
gcatggatga actgtacaac atcgattgac taagcttgcc 2880
-------------------WPRE----------------- tcgagaattc acgcgtggta
cCGATAATCA ACCTCTGGAT TACAAAATTT GTGAAAGATT 2940 GACTGGTATT
CTTAACTATG TTGCTCCTTT TACGCTATGT GGATACGCTG CTTTAATGCC 3000
TTIGTATCAT GCTATTGCTT CCCGTATGGC TTTCATTTTC TCCTCCTTGT ATAAATCCTG
3060 GTTGCTGTCT CTTTATGAGG AGTTGTGGCC CGTTGTCAGG CAACGTGGCG
TGGTGTGCAC 3120 TGTGTTTGCT GACGCAACCC CCACTGGTTG GGGCATTGCC
ACCACCTGTC AGCTCCTTTC 3180 CGGGACTTTC GCTTTCCCCC TCCCTATTGC
CACGGCGGAA CTCATCGCCG CCTGCCTTGC 3240 CCGCTGCTGG ACAGGGGCTC
GGCTGTTGGG CACTGACAAT TCCGTGGTGT TGTCGGGGAA 3300 GCTGACGTCC
TTTCCATGGC TGCTCGCCTG TGTTGCCACC TGGATTCTGC GCGGGACGTC 3360
CTTCTGCTAC GTCCCTTCGG CCCTCAATCC AGCGGACCTT CCTTCCCGCG GCCTGCTGCC
3420 GGCTCTGCGG CCTCTTCCGC GTCTTCGCCT TCGCCCTCAG ACGAGTCGGA
TCTCCCTTTG 3480 --------WPRE-------7 GGCCGCCTCC CCGCCTGATC
cggccgcggg gatccagaca tgataagata cattgatgag 3540 tttggacaaa
ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat 3600
gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
3660 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttcgga
tcctctagag 3720 <-----------------------bGH
PolyA-------------------- tcgagagatc tacggg.sub.--rgg.sub.--c
arcccrgrga ccccrcccca grgccrcrcc rgg.sub.--cccrgg.sub.--a 3780
agrrgccacr ccagrgccca ccagccrrgr ccraaraaaa rraagrrgca rcarrrrgrc
3840 rgacragg.sub.--rg rccrrcrara
ararrarggg.sub.--grgg.sub.--aggggg.sub.-- grgg.sub.--rargg.sub.--a
gcaagggg.sub.--ca 3900 agrrggg.sub.--aag acaaccrgra
ggg.sub.--ccrgcgg.sub.-- gg.sub.--rcrarrgg.sub.-- gaaccaagcr
gg.sub.-- agrgcagr 3960 .gg.sub.--cacaarcr rgg.sub.--crcacrg
caarcrccgc cfccrggg.sub.--rr caagcgarrc rccrgccrca 4020 .gccrcccgag
rrgrrggg.sub.--ar rccagg.sub.-- carg cargaccagg.sub.-- crcagcraar
rrrrgrrrrr 4080 rrgg.sub.--ragaga cgggg.sub.--rrrca
ccararrgg.sub.--c cagg.sub.--crgg.sub.--rc rccaacrccr
aarcrcagg.sub.--r 4140 .garcraccca ccrrgg.sub.--ccrc ccaaarrgcr
ggg.sub.--arracag gcgrgaacca crgcrcccrr 4200 -bGH Po1yA7 ---ITR---
cccrgrccrr ctgattttgt aggtaaccac gtgcggaccg agcggccgcA GGAACCCCTA
4260 GTGATGGAGT TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC
CGGGCGACCA 4320 AAGGTCGCCC GACGCCCGGG CTTTGCCCGG GCGGCCTCAG
TGAGCGAGCG AGCGCGCAGC 4380 ---ITR---7 TGCCTGCAGG ggcgcctgat
gcggtatttt ctccttacgc atctgtgcgg tatttcacac 4440 cgcatacgtc
aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg 4500
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
4560 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
ctaaatcggg 4620 ggctcccttt agggttccga tttagtgctt tacggcacct
cgaccccaaa aaacttgatt 4680 tgggtgatgg ttcacgtagt gggccatcgc
cctgatagac ggtttttcgc cctttgacgt 4740 tggagtccac gttctttaat
agtggactct tgttccaaac tggaacaaca ctcaacccta 4800 tctcgggcta
ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 4860
atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
4920 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
ccccgacacc 4980 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
cccggcatcc gcttacagac 5040 aagctgtgac cgtctccggg agctgcatgt
gtcagaggtt ttcaccgtca tcaccgaaac 5100 gcgcgagacg aaagggcctc
gtgatacgcc tatttttata ggttaatgtc atgataataa 5160 tggtttctta
gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 5220
tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
5280 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
gcccttattc 5340 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
agaaacgctg gtgaaagtaa 5400 aagatgctga agatcagttg ggtgcacgag
tgggttacat cgaactggat ctcaacagcg 5460 gtaagatcct tgagagtttt
cgccccgaag aacgttttcc aatgatgagc acttttaaag 5520 ttctgctatg
tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 5580
gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
5640 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg 5700 cggccaactt acttctgaca acgatcggag gaccgaagga
gctaaccgct tttttgcaca 5760 acatggggga tcatgtaact cgccttgatc
gttgggaacc ggagctgaat gaagccatac 5820 caaacgacga gcgtgacacc
acgatgcctg tagcaatggc aacaacgttg cgcaaactat 5880 taactggcga
actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 5940
ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
6000 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
ccagatggta 6060 agccctcccg tatcgtagtt atctacacga cggggagtca
ggcaactatg gatgaacgaa 6120 atagacagat cgctgagata ggtgcctcac
tgattaagca ttggtaactg tcagaccaag 6180 tttactcata tatactttag
attgatttaa aacttcattt ttaatttaaa aggatctagg 6240 tgaagatcct
ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 6300
gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
6360 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
ttgccggatc 6420 aagagctacc aactcttttt ccgaaggtaa ctggcttcag
cagagcgcag ataccaaata 6480 ctgtccttct agtgtagccg tagttaggcc
accacttcaa gaactctgta gcaccgccta 6540 catacctcgc tctgctaatc
ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 6600 ttaccgggtt
ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 6660
ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
6720 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg 6780 taagcggcag ggtcggaaca ggagagcgca cgagggagct
tccaggggga aacgcctggt 6840 atctttatag tcctgtcggg tttcgccacc
tctgacttga gcgtcgattt ttgtgatgct 6900 cgtcaggggg gcggagccta
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 6960 ccttttgctg
gccttttgct cacatgt 6987
[0186] The Chop2 coding sequence from the above vector is shown
below as SEQ ID NO:2. Numbering indicates both nucleotide number
and codon number. The encoded polypeptide (SEQ ID NO:3) is also
shown. Again, this is the N-terminal 315 residues of Chop2
polypeptide (SEQ ID NO:6).
TABLE-US-00003 atg gat tat gga gge gee etg agt gee gtt ggg ege gag
etg eta ttt 48 M D Y G G A L S A V G R E L L F 16 gta acg aac cca
gta gtc gtc aat ggc tct gta ctt gtg cct gag gac 96 V T N P V V V N
G S V L V P E D 32 cag tgt tac tgc gcg ggc tgg att gag tcg cgt ggc
aca aac ggt gee 144 Q C Y C A G W I E S R G T N G A 48 caa acg gcg
tcg aac gtg ctg caa tgg ctt get get ggc ttc tee ate 192 Q T A S N V
L Q W L A A G F S I 64 eta ctg ctt atg ttt tac gee tac caa aca tgg
aag tea ace tgc ggc 240 L L L M F Y A Y Q T W K S T C G 80 tgg gag
gag ate tat gtg tgc get ate gag atg gtc aag gtg att ctt 288 W E E I
y V C A I E M V K V I L 96 gag ttc ttc ttc gag ttt aag aac ccg tee
atg ctg tat eta gee aca 336 E F F F E F K N p S M L y L A T 112 ggc
cac cgc gtc cag tgg ttg cgt tac gee gag tgg ctt etc ace tgc 384 G H
R V Q W L R y A E W L L T C 128 ccg gtc att etc att cac ctg tea aac
ctg acg ggc ttg tee aac gac 432 p V I L I H L S N L T G L S N D 144
tac age agg cgc act atg ggt ctg ctt gtg tct gat att ggc aca att 480
y S R R T M G L L V S D I G T I 160 gtg tgg ggc gee act tee get atg
gee ace gga tac gtc aag gtc ate 528 V W G A T S A M A T G y V K V I
176 ttc ttc tgc ctg ggt ctg tgt tat ggt get aac acg ttc ttt cac get
576 F F C L G L C y G A N T F F H A 192 gee aag gee tac ate gag ggt
tac cat ace gtg ccg aag ggc egg tgt 624 A K A y I E G y H T V p K G
R C 208 cgc cag gtg gtg act ggc atg get tgg etc ttc ttc gta tea tgg
ggt 672 R Q V V T G M A W L F F V S W G 224 atg ttc ccc ate ctg ttc
ate etc ggc ccc gag ggc ttc ggc gtc ctg 720 M F p I L F I L G p E G
F G V L 240 age gtg tac ggc tee ace gtc ggc cac ace ate att gac ctg
atg tcg 768 S V y G S T V G H T I I D L M S 256 aag aac tgc tgg ggt
ctg etc ggc cac tac ctg cgc gtg ctg ate cac 816 K N C W G L L G H y
L R V L I H 272 gag cat ate etc ate cac ggc gac att cgc aag ace ace
aaa ttg aac 864 E H I L I H G D I R K T T K L N 288 att ggt ggc act
gag att gag gtc gag acg ctg gtg gag gac gag gee 912 I G G T E I E V
E T L V E D E A 304 gag get ggc gcg gtc aac aag ggc ace ggc aag 945
E A G A V N K G T G K 315
[0187] A native nucleic acid sequence that encodes the full length
Chop2 protein of C. reinhardtii (GenBank Accession #AF461397) has
the following nucleotide sequence (SEQ TD NO:4). Note that the
coding sequence begins at the ATG codon beginning at nt 28.
TABLE-US-00004 1 gcatctgtcg ccaagcaagc attaaacATG gattatggag
gcgccctgag tgccgttggg 61 cgcgagctgc tatttgtaac gaacccagta
gtcgtcaatg gctctgtact tgtgcctgag 121 gaccagtgtt actgcgcggg
ctggattgag tcgcgtggca caaacggtgc ccaaacggcg 181 tcgaacgtgc
tgcaatggct tgctgctggc ttctccatcc tactgcttat gttttacgcc 241
taccaaacat ggaagtcaac ctgcggctgg gaggagatct atgtgtgcgc tatcgagatg
301 gtcaaggtga ttctcgagtt cttcttcgag tttaagaacc cgtccatgct
gtatctagcc 361 acaggccacc gcgtccagtg gttgcgttac gccgagtggc
ttctcacctg cccggtcatt 421 ctcattcacc tgtcaaacct gacgggcttg
tccaacgact acagcaggcg caccatgggt 481 ctgcttgtgt ctgatattgg
cacaattgtg tggggcgcca cttccgccat ggccaccgga 541 tacgtcaagg
tcatcttctt ctgcctgggt ctgtgttatg gtgctaacac gttctttcac 601
gctgccaagg cctacatcga gggttaccac accgtgccga agggccggtg tcgccaggtg
661 gtgactggca tggcttggct cttcttcgta tcatggggta tgttccccat
cctgttcatc 721 ctcggccccg agggcttcgg cgtcctgagc gtgtacggct
ccaccgtcgg ccacaccatc 781 attgacctga tgtcgaagaa ctgctggggt
ctgctcggcc actacctgcg cgtgctgatc 841 cacgagcata tcctcatcca
cggcgacatt cgcaagacca ccaaattgaa cattggtggc 901 actgagattg
aggtcgagac gctggtggag gacgaggccg aggctggcgc ggtcaacaag 961
ggcaccggca agtacgcctc ccgcgagtcc ttcctggtca tgcgcgacaa gatgaaggag
1021 aagggcattg acgtgcgcgc ctctctggac aacagcaagg aggtggagca
ggagcaggcc 1081 gccagggctg ccatgatgat gatgaacggc aatggcatgg
gtatgggaat gggaatgaac 1141 ggcatgaacg gaatgggcgg tatgaacggg
atggctggcg gcgccaagcc cggcctggag 1201 ctcactccgc agctacagcc
cggccgcgtc atcctggcgg tgccggacat cagcatggtt 1261 gacttcttcc
gcgagcagtt tgctcagcta tcggtgacgt acgagctggt gccggccctg 1321
ggcgctgaca acacactggc gctggttacg caggcgcaga acctgggcgg cgtggacttt
1381 gtgttgattc accccgagtt cctgcgcgac cgctctagca ccagcatcct
gagccgcctg 1441 cgcggcgcgg gccagcgtgt ggctgcgttc ggctgggcgc
agctggggcc catgcgtgac 1501 ctgatcgagt ccgcaaacct ggacggctgg
ctggagggcc cctcgttcgg acagggcatc 1561 ctgccggccc acatcgttgc
cctggtggcc aagatgcagc agatgcgcaa gatgcagcag 1621 atgcagcaga
ttggcatgat gaccggcggc atgaacggca tgggcggcgg tatgggcggc 1681
ggcatgaacg gcatgggcgg cggcaacggc atgaacaaca tgggcaacgg catgggcggc
1741 ggcatgggca acggcatggg cggcaatggc atgaacggaa tgggtggcgg
caacggcatg 1801 aacaacatgg gcggcaacgg aatggccggc aacggaatgg
gcggcggcat gggcggcaac 1861 ggtatgggtg gctccatgaa cggcatgagc
tccggcgtgg tggccaacgt gacgccctcc 1921 gccgccggcg gcatgggcgg
catgatgaac ggcggcatgg ctgcgcccca gtcgcccggc 1981 atgaacggcg
gccgcctggg taccaacccg ctcttcaacg ccgcgccctc accgctcagc 2041
tcgcagctcg gtgccgaggc aggcatgggc agcatgggag gcatgggcgg aatgagagga
2101 atgggaggca tgggtggaat ggggggcatg ggcggcgccg gcgccgccac
gacgcaggct 2161 gcgggcggca acgcggaggc ggagatgctg cagaatctca
tgaacgagat caatcgcctg 2221 aagcgcgagc ttggcgag
[0188] The coding portion of SEQ ID NO:4 is shown below as SEQ ID
NO:5, organized as 737 triplet codons (plus a stop codon) that
encode a 737 amino acid polypeptide. The ATG start codon and the
TAA stop codon are highlighted.
TABLE-US-00005 ATG gat tat gga ggc gee ctg agt gee gtt ggg cgc gag
ctg eta ttt gta acg aac cca gta gtc gtc aat ggc tct gta ctt gtg cct
gag gac cag tgt tac tgc gcg ggc tgg att gag tcg cgt ggc aca aac ggt
gee caa acg gcg tcg aac gtg ctg caa tgg ctt get get ggc ttc tee ate
eta ctg ctt atg ttt tac gee tac caa aca tgg aag tea ace tgc ggc tgg
gag gag ate tat gtg tgc get ate gag atg gtc aag gtg att etc gag ttc
ttc ttc gag ttt aag aac ccg tee atg ctg tat eta gee aca ggc cac cgc
gtc cag tgg ttg cgt tac gee gag tgg ctt etc ace tgc ccg gtc att etc
att cac ctg tea aac ctg acg ggc ttg tee aac gac tac age agg cgc ace
atg ggt ctg ctt gtg tct gat att ggc aca att gtg tgg ggc gee act tee
gee atg gee ace gga tac gtc aag gtc ate ttc ttc tgc ctg ggt ctg tgt
tat ggt get aac acg ttc ttt cac get gee aag gee tac ate gag ggt tac
cac ace gtg ccg aag ggc egg tgt cgc cag gtg gtg act ggc atg get tgg
etc ttc ttc gta tea tgg ggt atg ttc ccc ate ctg ttc ate etc ggc ccc
gag ggc ttc ggc gtc ctg age gtg tac ggc tee ace gtc ggc cac ace ate
att gac ctg atg tcg aag aac tgc tgg ggt ctg etc ggc cac tac ctg cgc
gtg ctg ate cac gag cat ate etc ate cac ggc gac att cgc aag ace ace
aaa ttg aac att ggt ggc act gag att gag gtc gag acg ctg gtg gag gac
gag gee gag get ggc gcg gtc aac aag ggc ace ggc aag tac gee tee cgc
gag tee ttc ctg gtc atg cgc gac aag atg aag gag aag ggc att gac gtg
cgc gee tct ctg gac aac age aag gag gtg gag cag gag cag gee gee agg
get gee atg atg atg atg aac ggc aat ggc atg ggt atg gga atg gga atg
aac ggc atg aac gga atg ggc ggt atg aac ggg atg get ggc ggc gee aag
ccc ggc ctg gag etc act ccg cag eta cag ccc ggc cgc gtc ate ctg gcg
gtg ccg gac ate age atg gtt gac ttc ttc cgc gag cag ttt get cag eta
tcg gtg acg tac gag ctg gtg ccg gee ctg ggc get gac aac aca ctg gcg
ctg gtt acg cag gcg cag aac ctg ggc ggc gtg gac ttt gtg ttg att cac
ccc gag ttc ctg cgc gac cgc tct age ace age ate ctg age cgc ctg cgc
ggc gcg ggc cag cgt gtg get gcg ttc ggc tgg gcg cag ctg ggg ccc atg
cgt gac ctg ate gag tee gca aac ctg gac ggc tgg ctg gag ggc ccc tcg
ttc gga cag ggc ate ctg ccg gee cac ate gtt gee ctg gtg gee aag atg
cag cag atg cgc aag atg cag cag atg cag cag att ggc atg atg ace ggc
ggc atg aac ggc atg ggc ggc ggt atg ggc ggc ggc atg aac ggc atg ggc
ggc ggc aac ggc atg aac aac atg ggc aac ggc atg ggc ggc ggc atg ggc
aac ggc atg ggc ggc aat ggc atg aac gga atg ggt ggc ggc aac ggc atg
aac aac atg ggc ggc aac gga atg gee ggc aac gga atg ggc ggc ggc atg
ggc ggc aac ggt atg ggt ggc tee atg aac ggc atg age tee ggc gtg gtg
gee aac gtg acg ccc tee gee gee ggc ggc atg ggc ggc atg atg aac ggc
ggc atg get gcg ccc cag tcg ccc ggc atg aac ggc ggc cgc ctg ggt ace
aac ccg etc ttc aac gee gcg ccc tea ccg etc age tcg cag etc ggt gee
gag gca ggc atg ggc age atg gga ggc atg ggc gga atg age gga atg gga
ggc atg ggt gga atg ggg ggc atg ggc ggc gee ggc gee gee acg acg cag
get gcg ggc ggc aac gcg gag gcg gag atg ctg cag aat etc atg aac gag
ate aat cgc ctg aag cgc gag ctt ggc gag taa 2214 nt's
[0189] The full length Chop2 protein of C. reinhardtii (GenBank:
Accession #AF461397) encoded by SEQ ID NO's 3 and 4, has the
following amino acid sequence, SEQ ID NO:6:
TABLE-US-00006 MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQT
SO ASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFF 100
EFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTM 150
GLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGY 200
HTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHT 250
IIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV 300
EDEAEAGAVNKGTGKYASRESFLVMRDKMKEKGIDVRASLDNSKEVEQEQ 350
AARAAMMMMNGNGMGMGMGMNGMNGMGGMNGMAGGAKPGLELTPQLQPGR 400
VILAVPDISMVDFFREQFAQLSVTYELVPALGADNTLALVTQAQNLGGVD 450
FVLIHPEFLRDRSSTSILSRLRGAGQRVAAFGWAQLGPMRDLIESANLDG 500
WLEGPSFGQGILPAHIVALVAKMQQMRKMQQMQQIGMMTGGMNGMGGGMG 550
GGMNGMGGGNGMNNMGNGMGGGMGNGMGGNGMNGMGGGNGMNNMGGNGMA 600
GNGMGGGMGGNGMGGSMNGMSSGVVANVTPSAAGGMGGMMNGGMAAPQSP 650
GMNGGRLGTNPLFNAAPSPLSSQLGAEAGMGSMGGMGGMSGMGGMGGMGG 700
MGGAGAATTQAAGGNAEAEMLQNLMNEINRLKRELGE 737
[0190] Another useful Chop2 sequence useful for the present
invention is a nucleic acid of 933 nt's (including the stop codon)
encoding a 310 aa polypeptide (a biologically active fragment of
the full length native Chop2) is a synthetic construct derived from
Chlamydomonas reinhardtii" (See EF474017 and Zhang et al, 2007,
Nature in press). This sequence is codon-optimized for human
expression. The nt sequence shown below is SEQ ID NO:7, and the
encoded a.a. sequence shown is
[0191] SEQ ID NO:8. The polypeptide with the a.a. sequence SEQ ID
NO:8 is a fragment of SEQ ID NO:6 truncated at the C-terminus and
with Pro replacing Asn at 310.
TABLE-US-00007 atg gac tat ggc ggc get ttg tct gee gtc gga cgc gaa
ctt ttg ttc 48 M D y G G A L S A G R E L L F 16 gtt act aat cct gtg
gtg gtg aac ggg tee gtc ctg gtc cct gag gat 96 T N p N G S L p E D
32 caa tgt tac tgt gee gga tgg att gaa tct cgc ggc acg aac ggc get
144 Q C y C A G W I E S R G T N G A 48 cag ace gcg tea aat gtc ctg
cag tgg ctt gca gca gga ttc age att 192 Q T A S N L Q W L A A G F S
I 64 ttg ctg ctg atg ttc tat gee tac caa ace tgg aaa tct aca tgc
ggc 240 L L L M F y A y Q T W K S T C G 80 tgg gag gag ate tat gtg
tgc gee att gaa atg gtt aag gtg att etc 288 W E E I y C A I E M K I
L 96 gag ttc ttt ttt gag ttt aag aat ccc tct atg etc tac ctt gee
aca 336 E F F F E F K N p S M L y L A T 112 gga cac egg gtg cag tgg
ctg cgc tat gca gag tgg ctg etc act tgt 384 G H R Q W L R y A E W L
L T C 128 cct gtc ate ctt ate cac ctg age aac etc ace ggc ctg age
aac gac 432 p I L I H L S N L T G L S N D 144 tac age agg aga ace
atg gga etc ctt gtc tea gac ate ggg act ate 480 y S R R T M G L L S
D I G T I 160 gtg tgg ggg get ace age gee atg gca ace ggc tat gtt
aaa gtc ate 528 W G A T S A M A T G y K I 176 ttc ttt tgt ctt gga
ttg tgc tat ggc gcg aac aca ttt ttt cac gee 576 F F C L G L C y G A
N T F F H A 192 gee aaa gca tat ate gag ggt tat cat act gtg cca aag
ggt egg tgc 624 A K A y I E G y H T p K G R C 208 cgc cag gtc gtg
ace ggc atg gca tgg ctg ttt ttc gtg age tgg ggt 672 R Q V V T G M A
W L F F V S W G 224 atg ttc cca att etc ttc att ttg ggg ccc gaa ggt
ttt ggc gtc ctg 720 M F p I L F I L G p E G F G L 240 age gtc tat
ggc tee ace gta ggt cac acg att att gat ctg atg agt 768 S y G S T G
H T I I D L M S 256 aaa aat tgt tgg ggg ttg ttg gga cac tac ctg cgc
gtc ctg ate cac 816 E H I L I H G D I R K T T K L N 272 gag cac ata
ttg att cac gga gat ate cgc aaa ace ace aaa ctg aac 864 I G G T E I
E E T L E D E A 288 ate ggc gga acg gag ate gag gtc gag act etc gtc
gaa gac gaa gee 912 I G G T E I E E T L E D E A 304 gag gee gga gee
gtg cca taa 933 E A G A V p 310
AAV Vector Injection
[0192] All of the animal experiments were at the institutional
level and were in accord with the NIH Guide for the Care and Use of
Laboratory Animals.
[0193] Newborn (PI) rat pups (Sprague-Dawley and Long-Evans) and
mouse pups (C57BL/6J and C3H/HcJ or rdl/rdl) were anesthetized by
chilling on ice. Adult mice (rdl/rdl) were anesthetized by IP
injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Under a
dissecting microscope, an incision was made by scissors through the
eyelid to expose the sclera. A small perforation was made in the
sclera region posterior to the lens with a needle and viral vector
suspension of 0.8-1.5 .mu.l at the concentration of approximately
10.sup.11 genomic particles/ml was injected into intravitreal space
through the hole with a Hamilton syringe with a 32-gauge
blunt-ended needle. For each animal, usually only one eye was
injected with viral vectors carrying Chop2-GFP and the other eye
was uninjected or injected with control viral vectors carrying GFP
alone. After the injection, animals were kept on a 12/12 hr
light/dark cycle. The light illumination of the room housing the
animals measured at the wavelength of 500 nm was
6.0.times.10.sup.14 photons cm-.sup.2 s-.sup.1.
Histology
[0194] Animals were sacrificed at various time points after the
vector injection. The expression of Chop2-GFP fluorescence was
examined in flat whole-mount retinas, vertical retinal, and coronal
brain sections. The dissected retinas and brains were fixed with 4%
paraformaldehyde in PBS for 0.5-2 hr at room temperature and 24 hr
at 4.degree. C., respectively. The fixed retinas (embedded in 3%
agarose) and brains were cut by using a vibrato me. The retinal and
brain sections or the retinal whole mounts were mounted on slides
and covered with Vectashield medium (Vector Laboratories). GFP
fluorescence was visualized under a fluorescence microscope
equipped with exciter, dichroic, and emission filters of 465-495
nm, 505 nm, and 515-555 nm, respectively, and most images were
obtained with a digital camera (Axiocam, Zeiss). Some images were
obtained with a confocal microscope (TCS SP2, Leica). For light
microscopy of semithin vertical retinal section, eyes were
enucleated, rinsed in PBS, and fixed in 1% osmium tetroxide, 2.5%
glutaraldehyde, and 0.2 M Sorenson' s phosphate buffer (pH 7.4) at
4.degree. C. for 3 hr. The eyes were then dehydrated in graded
ethanols and embedded in plastic and cut into 1 .mu.m sections and
stained with a methylene blue/azure mixture.
Patch-Clamp Recordings
[0195] Dissociated retinal cells and retinal slice were prepared as
previously described (Pan, 2000 and Cui et al., 2003). Recordings
with patch electrodes in the whole-cell configuration were made by
an EPC-9 amplifier and PULSE software (Heka Electronik, Lambrecht,
Germany). Recordings were made in Hanks' solution containing (in
mM): NaCl, 138; NaHC0.sub.3, 1; Na.sub.2HP0.sub.4, 0.3; KCl, 5;
KH2P0.sub.4, 0.3; CaCh, 1.25; MgS0.sub.4, 0.5; MgOH, 0.5;
HEPES-NaOH, 5; glucose, 22.2; with phenol red, 0.001% v/v; adjusted
to pH 7.2 with 0.3 N NaOH.
[0196] The electrode solution contained (in mM): K-gluconate, 133;
KCl, 7; MgCh, 4; EGTA, 0.1; HEPES, 10, Na-GTP, 0.5; and Na-ATP, 2;
pH adjusted with KOH to 7.4. The resistance of the electrode was 13
to 15 MO. The recordings were performed at room temperature
(-22.degree. C.).
Multielectrode Array Recordings
[0197] The multielectrode array recordings were based on the
procedures reported by Tian and Copenhagen (2003). Briefly, the
retina was dissected and placed photoreceptor side down on a
nitrocellulose filter paper strip Millipore Corp., Bedford, Mass.).
The mounted retina was placed in the MEA-60 multi electrode array
recording chamber of 30 .mu.m diameter electrodes spaced 200 .mu.m
apart (Multi Channel System MCS GmbH, Reutlingen, Germany), with
the ganglion cell layer facing the recording electrodes. The retina
was continuously perfused in oxygenated extracellular solution at
34.degree. C. during all experiments. The extracellular solution
contained (in mM): NaCl, 124; KCl, 2.5; CaCh, 2; MgCh, 2; NaH2P04,
1.25; NaHC01, 26; and glucose, 22 (pH 7.35 with 95% 02 and 5%
CO.sub.2). Recordings were usually started 60 min after the retina
was positioned in the recording chamber. The interval between
onsets of each light stimulus was 10-15 s. The signals were
filtered between 200 Hz (low cut off) and 20 kHz (high cut off).
The responses from individual neurons were analyzed using Offline
Sorter software (Plexon, Inc., Dallas, Tex.).
Visual-Evoked Potential Recordings
[0198] Visual-evoked potential recordings were carried out in
wild-type mice of the C57BL/6 and 129/Sv strains aged 4-6 months
and in the rdl/rdl mice aged 6-11 months. Recordings were performed
2-6 months after viral vector injection.
[0199] After general anesthesia (i.p. injection of ketamine (100
mg/kg) and acepromazine (0.8 mg/kg), animals were mounted in a
stereotaxic apparatus. Body temperature was either unregulated or
maintained at 34.degree. C. with a heating pad and a rectal probe.
Pupils were dilated with 1% atropine and 2.5% accu-phenylephrine. A
small portion of the skull (1.5.times.1.5 mm) centered about 2.5 mm
from the midline and 1 mm rostral to the lambdoid suture was
drilled and removed. Recordings were made from visual cortex (area
V1) by a glass micropipette (resistance 0.5 M after filling with 4
M NaCl) advanced 0.4 mm beneath the surface of the cortex at the
contralateral side of the stimulated eye. The stimuli were 20 ms
pluses at 0.5 Hz. Responses were amplified (1,000 b 10,000),
band-pass filtered (0.3-100 Hz), digitized (1 kHz), and averaged
over 30-250 trials.
Light Stimulation
[0200] For dissociated cell and retinal slice recordings, light
stimuli were generated by a 150 W xenon lamp-based scanning
monochromator with bandwidth of 10 nm (TILL Photonics, Germany) and
coupled to the microscope with an optical fiber. For multielectrode
array recordings, light responses were evoked by the monochromator
or a 175 W xenon lamp-based illuminator (Lambda L S, Sutter
Instrument) with a band-pass filter of 400-580 nm and projected b
the bottom of the recording chamber through a liquid light guider.
For visual evoked potential, light stimuli were generated by the
monochromator and projected to the eyes through the optical fiber.
The light intensity was attenuated by neutral density filters. The
light energy was measured by a thin-type sensor (TQ82017) and an
optical power meter (Model: TQ8210) (Advantest, Tokyo, Japan).
Example 2
Expression of Chop2 in Retinal Neurons In Vivo
[0201] To directly visualize the expression and localization of
Chop2 proteins, the C-terminal portion of the Chop2 channel was
replaced with GFP, to make a Chop2-GFP chimera. The
adeno-associated virus (AAV) vectors was selected to target the
expression of Chop2-GFP fusion protein into retinal neurons because
the capability of AAV vectors to deliver transgenes into
nondividing cells, including inner retinal neurons (Harvey et al.,
2002 and Martin et al., 2003), and to integrate the transgenes into
the host genome (Flotte, 2004).
[0202] A viral expression cassette, rAAV2-CAG-Chop2-GFP-WPRE, was
made by subcloning the Chop2-GFP chimera into an AAV scrotype-2
expression cassette containing a hybrid CMV enhancer/chicken
13-actin (CAG) promoter (FIG. 1A). To establish the expression and
function of Chop2 channels in retinal neurons in general, we first
examined the expression of Chop2 in nondystrophic retinas. The
viral vector was injected into the intravitreal space in the eyes
of postnatal day 1 rats and mice. Three to four weeks after the
injection, bright GFP fluorescence was observed in retinal neurons
of all injected eyes (FIGS. 1B-1H), confirming that Chop2-GFP was
expressed. The expression was usually confluent throughout the
retina (FIG. 1B).
[0203] The Chop2-GFP-fluorescence was predominantly observed in
retinal ganglion cells (Figures IC and ID; also see Figure IH). The
fluorescence signal was observed throughout the inner plexiform
layer (IPL) (FIG. 1H), indicating that the viral vector targeted
the expression of Chop2-GFP both in ON and OFF ganglion cells. The
expressing of Chop2-GFP was also frequently observed in horizontal
cells (FIG. 1E), amacrine cells (FIG. 1F), and, occasionally, in
bipolar cells (FIG. 1G).
[0204] The GFP signal was predominantly localized to the plasma
membrane (Figure ID), consistent with the GFP tag being anchored to
the membrane by a seven-transmembrane portion of the Chop2 channel.
Once expressed in a cell, the GFP signal was extended over the
entire cell including distal processes and axon terminals (see
FIGS. 1C and 1E). Bright GFP fluorescence was found to be stable
for 12 months or more after the injection (FIG. 1H), whereas no
gross changes in retinal nl.orphology were noticed (Figure II).
These results indicated that long-term stable expression of
Chop2-GFP was achieved in inner retinal neurons in vivo.
Example3
Properties of Light-Evoked Currents of ChR2-Expressing Inner
Retinal Neurons
[0205] Functional properties of the Chop2 channels were examined in
inner retinal neurons by using whole-cell patch-clamp recordings.
The recordings were performed in acutely dissociated cells so that
photoreceptor-mediated light responses were confidently excluded.
Chop2-GFP-positive cells were identified by their GFP fluorescence
(FIG. 2A). The precursor for the Chop2 chromophore group, all-trans
retinal, was not added because it might be ubiquitously present in
cells (Kim et al., 1992 and Thompson and Gal, 2003). Light-evoked
responses were observed in all recorded GFP fluorescent cells
(n=34), indicating that functional ChR2 (Chop2 with the chromophore
attached) can be formed in retinal neurons with the retinal
chromophore groups already present in the cells. Consistently, the
expression of functional ChR2 channels has also been recently
reported in cultured hippocampal neurons without the supply of
exogenous retinal chromophore groups (Boyden et al., 2005; but see
Li et al., 2005).
[0206] The properties of the ChR2-mediated light responses were
first examined in voltage clamp. Light-evoked currents were
observed in Chop2-GFP-expressing inner retinal neurons by light
stimuli up to the wavelength of 580 nm with the most sensitive
wavelength around 460 nm (FIG. 2B), consistent with the reported
peak spectrum sensitivity of ChR2 (Nagel et al., 2003). The
amplitude and the kinetics of the currents were dependent on the
light intensity (FIG. 2C). FIGS. 2D and 2E show in the expanded
time scale the current traces right after the onset and the
termination of the light stimulation, respectively. Detectable
currents were observed in most recorded cells at a light intensity
of 2.2.times.10.sup.15 photons cm-.sup.2 s-.sup.1 In some cells,
currents were observed at a light intensity of 2.times.10.sup.14
photons cm.sup.2 s.sup.1 (not shown). At higher light intensities,
the currents displayed both transient and sustained components,
similar to the properties of the nonfusion ChR2 (Nagel et al.,
2003). The relationship between the light intensity and peak
current is shown in FIG. 2F (n=7). The activation and inactivation
kinetics of the currents were also dependent on the light intensity
(FIG. 2D). The initial phase of the current could be well fitted by
an exponential function with a single activation and inactivation
constant, as illustrated in FIG. 2D (red trace). The activation and
inactivation time constants versus light intensity are plotted in
FIGS. 2G and 2H, respectively. On the other hand, the deactivation
kinetics of the currents after the light off was not
light-intensity dependent. The current decay trace could be well
fitted by a single exponential function as shown in FIG. 2E (red
trace). The time constant was 17.1.+-.6.5 ms (mean.+-.SD, n=7).
[0207] The next experiment examined whether the ChR2-mediated
currents were sufficient to drive membrane depolarization. FIG. 3A
shows the representative responses from a nonspiking neuron in
response to four incremental light intensities at the wavelength of
460 nm. Detectable responses were observed in most recorded cells
at a light intensity of 2.2.times.10.sup.15 photons cm-.sup.2 s-1.
At higher light intensities, the membrane depolarization approached
a saturated level. The ChR2-mediated light responses to repeated
light stimulations were further examined. The transient component
of the currents diminished to repeated stimulations whereas the
sustained component of the currents was stable (top traces in FIG.
3B). This was clearly seen in the expanded time scale in the right
panel of FIG. 3B by comparing the superimposed first (red trace)
and the second (black trace) light-evoked currents. For the same
cell, in current clamp, the stimulations evoked robust membrane
depolarizations (bottom traces in FIG. 3B). The membrane
depolarizations reached an almost identical level, except for the
initial portion of the response. This was also shown in the
expanded time scale (right panel), which superimposed the first
(red trace) and the second (black trace) light-evoked responses.
FIG. 3C shows a representative recording of spiking neurons to
repeated light stimulations. Again, the stimulations elicited
almost identical membrane depolarizations accompanied by multiple
spikes. Taken together, these results demonstrated that the
ChR2-mediated currents in second- and third-order retinal neurons
arc sufficient to drive membrane depolarization and/or spike
firing.
Example4
Expression of Chop2 in Photoreceptor-Deficient rdl/rdl Mice
[0208] Having established the expression and function of ChR2 in
wild-type retinas, we went on to address whether the expression of
ChR2 could restore light responses in retinas after photoreceptor
degeneration. To this end, the experiments were carried out in
homozygous rdl (rdl/rdl) mice (Bowes et al., 1990), a photoreceptor
degeneration model with a null mutation in a cyclic GMP
phosphodiesterase, PDE6, similar to some forms of retinitis
pigmentosa in humans (McLaughlin et al., 1993). The Chop2-GFP viral
vector was injected intravitreally into the eyes of newborn (P1) or
adult mice at 2-12 months of age. Similar to the results observed
in wild-type animals, bright GFP signal was observed in
Chop2-GFP-injected retinas, predominately in retinal ganglion cells
(FIGS. 4A and 4B). At the time of the recording experiments
(::C::4months of age unless otherwise indicated), photoreceptor
cells were absent (FIG. 4C). The expression of Chop2-GFP was
observed in the rd]/rd] mice up to 16 months of age (3-6 months
after the viral injection) as the case shown in FIG. 4A from a 15
month old rdl/rdl mouse. These results indicate that inner retinal
neurons in this photoreceptor-deficient model not only survive long
after the complete death of photoreceptors but also retain the
capability of stable expression of Chop2-GFP.
Examples
Light-Evoked Responses of ChR2-Expressing Surviving Inner Retinal
Neurons of rdl/rdl Mice
[0209] The light response properties of the ChR2-expressing retinal
neurons in rdl/rdl mice were examined by whole-cell patch-clamp
recording in retinal slices. The recordings were made from the
GFP-positive cells located in the ganglion cell layer. Light-evoked
currents were observed in GFP-positive cells. The magnitude of the
current was again dependent on the light intensity (top traces in
FIGS. 4D and 4E; also see light intensity and current relationships
shown in FIG. 4F). Two groups of ChR2-expressing retinal neurons
were observed based on their response properties: a group of
transient spiking neurons (FIG. 4D) and a group of sustained
spiking neurons (FIG. 4E). The membrane depolarization and/or spike
rates were also dependent on the light intensity (bottom traces in
FIGS. 4D and 4E). Furthermore, light at higher intensities markedly
accelerated the kinetics of the voltage responses as illustrated in
the right panels of FIGS. 4D and 4E by superimposing the second
traces (black) and the fourth traces (red) in an expanded time
scale. The relationships of light intensity to the membrane
depolarization, the spike firing rate, and the time to the first
spike peak are shown in FIGS. 4G, 4H, and 4I, respectively. These
results demonstrate that the surviving retinal third-order neurons
with the expression of ChR2 are capable of encoding light intensity
with membrane depolarization and/or action potential firing and
response kinetics.
Example6
Multielectrode Array Recordings of ChR2-Mediated Retinal
Activities
[0210] The spike coding capability of the photoreceptor-deficient
retina of rd]/rd] mice were examined after the expression of ChR2
by use of multielectrode array recordings from whole-mount retinas.
As shown from a sample recording in Figure SA, spike firings with
fast kinetics in response to light on and off were observed in
Chop2-GFP-expressing retinas (n=11 retinas). The light-evoked spike
firings were not affected by the application of CNQX (25-50 .mu.M)
plus APV (25-50 .mu.M) (n=3), indicating that the responses are
originated from the ChR2 of the recorded cells. No such
light-evoked spike firings were observed in retinas that were
either injected with viral vectors carrying GFP alone (n=2 retinas)
or left uninjected (n=3). The latter confirmed the absence of
photoreceptor-originated light responses. The light-evoked spike
firings were not affected by suramine (100 .mu.M) (n=2), which has
been reported to be able to block melanopsin receptor-mediated
photocurrent (Melyan et al., 2005 and Qiu et al., 2005).
[0211] In addition, the response kinetics to both light on and off
(see FIG. 58) were much faster than those generated by the
intrinsically photosensitive retinal ganglion cells (Tu et at,
2005). These results indicated that a significant contribution to
the observed light responses from the intrinsically photosensitive
ganglion cells under our recording conditions is unlikely. The
light-evoked responses were often found to be picked up by the
majority of the electrodes (see FIG. 5A), consistent with the
observation that Chop2-GFP was extensively expressed in the
retinas. The vast majority of the responses were sustained during
light stimulation. FIG. 58 illustrates the raw traces recorded by a
single electrode in response to three incremental light stimuli.
The raster plots of the spike activity sorted from a single neuron
of the recording were shown in FIG. 5C. The firing frequency was
remarkably stable during the course of the recording. The averaged
spike rate histograms are shown in FIG. 5D. Again, the spike
frequency was increased to the higher light intensity. The light
responses could be recorded for up to 5 hr. These results
demonstrate further that the ChR2-expressing retinal ganglion cells
can reliably encode light intensity with spike firing rate.
Example 7
Visual-Evoked Potentials
[0212] A study was conducted to test whether the ChR2-mediated
light responses in the retinas of rdl/rdl mice were transmitted to
the visual cortex. The expression of transgenes, such as GFP, in
retinal ganglion cells as achieved by AAV infection was reported to
be able to extend to their terminations in higher visual centers in
the brain (Harvey et al, 2002). Therefore the anatomical
projections of the axon terminals of Chop2-GFP-expressing retinal
ganglion cells were first examined. Consistently, Chop2-GFP labeled
axon terminals of retinal ganglion cells were observed in several
regions of the brain, including ventral lateral geniculate nucleus
and dorsal lateral geniculate nucleus (FIG. 6A), as well as
superior colliculus (FIG. 68). These results indicate that the
central projections of retinal ganglion cells in the degenerate
retinas are maintained.
[0213] Visual evoked potentials (VEPs) from visual cortex were then
examined. First, as illustrated in FIG. 6C, VEPs were observed in
all tested wild-type mice (4-6 months of age) in response to light
stimuli at the wavelengths of both 460 and 580 nm (n=6 eyes). When
tested in Chop2-GFP-injected eyes of rdl/rdl mice (6-11 months of
age), VEPs were observed in the majority of the eyes (nine out of
13) in response to light stimulus at the wavelength of 460 nm but
not to light stimulus at the wavelength of 580 nm (FIG. 6D),
consistent with the light sensitivity of ChR2 channels (see FIG.
28). The average amplitude of the VEPs in the Chop2-GFP-injected
eyes in response to the light stimulus at the wavelength of 460 nm
was 110.+-.34 tV (mean.+-.SE; n=10), which is smaller than that
observed in wild-type mice (274.+-.113 .mu.V; n=6), although these
two values are not significantly different (one-way ANOVA test,
p<0.1). The lower amplitudes of the VEPs in the
Chop2-transfected mice compared to the wild-type mice are not
surprising because the expression of ChR2 was probably only
achieved in a small portion of the retinal ganglion cells. The
average latency to the peak of the VEPs in the Chop2-GFP-injected
eyes was 45.+-.1.7 ms (n=10), which is shorter than that observed
in wild-type mice (62.+-.2.8 ms; n=6). These two values were
significantly different (p<0.01). The latter would be predicted
because the light response mediated by ChR2 in retinal ganglion
cells originates two synapses downstream of the photoreceptors. As
a control, no detectable VEPs were observed to light stimulus at
the wavelength of 460 nm in the eyes of the age-matched rdl/rdl
mice that were injected with viral vectors carrying GFP alone (n=5)
(FIG. 6E). In addition, no detectable VEPs were observed in
uninjected rdl/rdl mice (n=3; 5 months of age) to the wavelengths
ranging from 420 to 620 nm (not shown), confirming that rd]/rd]
mice at 5 months of age are completely blind based on VEPs.
[0214] To further ensure that the VEPs in the blind rdl/rdl mice
originate from ChR2 expressed in their retinas, the action spectrum
of the VEP were measured by plotting their normalized amplitudes in
response to varying light wavelengths and intensities to obtain the
relative sensitivity of the response (FIG. 6F) (n=3). The data
points were well fitted by a vitamin-Ai-based visual pigment
template (Partridge and De Grip, 1991) with a peak wavelength at
461 nm (FIG. 6G), a good match to the reported peak action spectrum
of ChR2 at -A60 nm (Nagel et al., 2003). Taken together, these
results demonstrated that expression of ChR2 in the
photoreceptor-deficient retinas can restore visually evoked
responses in the brain.
Examples
Discussion of Examples 1-7
[0215] The results presented herein demonstrated that the strategy
of restoration of light responses in photoreceptor-deficient rodent
retinas based on the expression of ChR2 is mechanistically and
technically feasible. Most importantly, the results showed that
ChR2 satisfies several major criteria for its use as a light sensor
in retinal neurons. First, by delivery of an AAV vector carrying
fused Chop2-GFP, the inventors showed the ability of retinal
neurons to tolerate the prolonged expression of Chop2. To date, the
expression of Chop2-GFP proteins had been achieved in nondystrophic
rat retinal neurons for 12 months and in photoreceptor deficient
rdl/rdl mice for 6 months in vivo after the viral injection. The
present results therefore indicate that the expression of ChR2 in
retinal neurons is biocompatible under normal light cycle
conditions.
[0216] Second, these results showed that a sufficient number of
ChR2 can be formed in retinal neurons, with only endogenous
chromophore groups as supplied by regular diet, to produce robust
membrane depolarizations and/or action potential firings in the
retina and VEPs in visual cortex. It is worth emphasizing here
that, unlike animal visual pigments that rapidly lose their
chromophore after its photoisomerization from 11-cis to all-trans
retinal (Wald, 1968), for microbial-type rhodopsins,
photoisomerization from all-trans to 11-cis retinal is reversible
and both isomers remain attached to the protein (Oesterhelt, 1998).
Once the ChR2 complex is formed, the light-sensitive channel can
sustain multiple cycles of photoisomerization with the same
chromophore moiety. Although the efficacy of the de nova ChR2
formation might be expected to depend on the availability of the
chromophore group, the need for constant resupply of the
chromophore to form new ChR2 does not appear to impose a limitation
on overall ChR2 function. As observed in the multielectrode array
recordings, ChR2 respond repeatedly to light stimulation for
several hours in vitro without loss of activity. These results thus
indicate that the tum-over rate for ChR2 is fairly slow, an
additional advantage for use as an artificially produced light
sensor.
[0217] Furthermore, as reported originally in cell expression
systems (Nagel et al., 2003), later in hippocampal neurons (Boyden
et al., 2005, Ishizuka et al., 2006 and Li et al., 2005), and now
shown in retinal neurons, a number of properties of the ChR2
channel are highly advantageous for its use as a light sensor.
[0218] First, the ChR2 channel is permeable to the cations that
underlie neuronal membrane excitability. Thus, activation of ChR2
channels by light can directly produce membrane depolarizations to
mimic the ON-responses of inner retinal neurons. Indeed, as shown
herein, the light-evoked responses mediated by ChR2 in nonspiking
and spiking retinal neurons remarkably resemble the light responses
of ON-bipolar cells and sustained ON-ganglion cells (Werblin and
Dowling, 1%9 and Kaneko, 1970).
[0219] Second, the activation kinetics of the current in response
to light are extremely fast, whereas the sustained components of
the currents do not show apparent inactivation to continuous or
repeated light illuminations. Thus, the ChR2-expressing neurons can
signal with rapid kinetics but without pigment inactivation.
Consistently, the expression of ChR2 has been shown to allow
optical control of neural excitability with high temporal
resolution (Boyden et al., 2005, Ishizuka et al., 2006 and Li et
al., 2005). Furthermore, it is shown here that the magnitude and
activation kinetics of the light-evoked current depend upon light
irradiance over a 3-log-unit range. As demonstrated in the
whole-cell and multielectrode array recordings, this would allow
the encoding of various light intensities with graded membrane
depolarizations and/or spike rates.
[0220] Also of importance for the feasibility of the strategy of
restoring light sensitivity in retinas after photoreceptor
degeneration, results of this study show that many inner retinal
neurons survive in aged rdl/rdl mice (up to 16 months of age) and
are capable of expressing ChR2 long after the death of all
photoreceptors. This is consistent with histological studies
showing that many inner retinal neurons survive, despite some
remodeling, in this mouse model (Jimenez et al., 1996, Strettoi and
Pignatelli, 2000 and Chang et al., 2002). Moreover, the present
studies using ChR2 showed that the surviving inner retinal neurons
retained their physiological capability to encode light signals
with membrane depolarizations and/or action potential firings and
to transmit visual signals to the visual cortex. Thus, the strategy
based on the expression of ChR2 is suitable at least for certain
retinal degenerative diseases at certain stages.
[0221] The remodeling of inner retinal neurons triggered by
photoreceptor degeneration raised some concerns for the
retinal-based rescue strategy after the death of photoreceptors
(Strettoi and Pignatelli, 2000, Jones et al., 2003 and Jones and
Marc, 2005). However, retinal degenerative diseases are
heterogeneous as to the time course of the degeneration, survival
and functional state of different cell types (Chang et al., 2002).
The use of ChR2 is a powerful tool for undertaking such
studies.
[0222] Retinal remodeling is believed to be caused by
deafferentation (Jones and Marc, 2005). Therefore, the restoration
of the light sensitivity in inner retinal neurons may be able to
prevent or delay the remodeling processes.
[0223] Finally, according to the present invention, viral-based
gene delivery systems, such as AAV vectors (Flannery et al., 1997,
Bennett et al., 1999, Ali et al., 2000 and Acland et al., 2001),
are tools for introducing Chop2 into retinal neurons as
demonstrated herein.
[0224] The present results showed that that viral construct with
AAV serotype-2 and CAG promoter achieved robust expression of Chop2
in ganglion cells. However, because the expression of Chop2 with
this construct appears to target both ON- and OFF-type ganglion
cells, it remains to be determined how the conversion of both ON-
and OFF-ganglion cells into ON-type affects the visual
perception.
[0225] Behavior studies in primates reported that pharmacological
blockade of the ON channel in the retina did not severely impair
such vision functions as the detection of light decrement and the
perception of shape (Schiller et al., 1986). Therefore, targeting
of ChR2 to the ON channel, for example to ON-type ganglion cells,
is expected to result in useful vision.
[0226] It is also contemplated herein to express ChR2 in the more
distal retinal neurons, such as bipolar cells; this approach would
utilize the remaining signal processing functions of the degenerate
retina. Targeting ChR2 to rod bipolar cells is particularly
attractive because the depolarization of rod bipolar cells can lead
to the ON and OFF responses at the levels of cone bipolar cells and
retinal ganglion cells (Wassle, 2004), thereby maintaining the ON
and OFF channels that are inherent in the retina.
[0227] The threshold light intensity required for producing
responses in ChR2-expressing retinas appeared to be near
10.sup.14-10.sup.15 photons cm-.sup.2 s-.sup.1. For comparison, the
thresholds for normal rod and cone photoreceptors are about
10.sup.6 and 10.sup.10 photons cm-z s-.sub.1, respectively (Dacey
et al., 2005). Therefore, the ChR2-expressing retinas would operate
in substantially higher photonic range. The relatively low light
sensitivity of the ChR2-expressing retinas compared to the normal
retinas could be due to a number of factors. First, there may be a
low cross-sectional density of ChR2 molecules in the transfected
retinal neurons compared with the visual pigments in rods and
cones. Second, the ChR2-expressing inner retinal neurons lack the
unique multilayer photoreceptor membrane organization, typical for
the outer segments of rods and cones, which developed to achieve
higher pigment density and thus increase the probability of
catching photons (Steinberg, et al., 1980). Third, unlike visual
pigments that propagate their signal through amplification cascade
(Stryer, 1991), the directly light-gated ChR2 channels lack such
amplification capabilities. Finally, in normal retinas,
amplification of visual signals occurs as the signals converge from
multiple photoreceptors to ganglion cells (Barlow et al., 1971).
This process was not yet achieved in the ChR2-transfected retinas.
It is not yet evident which of these factors contributes the most
to the decreased light sensitivity of the ChR2-expressing retinas
remains. Interestingly, ChR2 mediated phototaxis to low-intensity
light in green algae (Sineshchekov et al., 2002; but see Katcriya
et al. [2004]). Therefore, the light sensitivity of ChR2 in retinal
neurons may have been altered by modifications introduced in the
Chop2 molecule for the heterologous expression. Such a difference
may also reflect different structural and functional organization
of algae and mammalian cells.
[0228] Nevertheless, for clinical usage, light intensifying devices
can be used to expand the light operation range.
[0229] At present, no treatment is available for restoring vision
once the photoreceptor cells have been lost. As noted above,
transplantation of normal photoreceptor cells or progenitor cells
(Bok, 1993 and Lund et al., 2001) or direct electrical stimulation
of the surviving second- and third-order retinal neurons via
retinal implants (Zrenner, 2002) have been proposed as possible
strategies for restoration of light responses in the retina after
rod and cone degeneration. An important advantage of the present
invention is that it does not involve the introduction of tissues
or devices into the retina and, therefore, may largely avoid the
complications of immune reactions and bioincompatibilities. In
addition, the present approach is expected to achieve high spatial
resolution for the restored "vision" because the approach targets
the cellular level. Thus, the expression of microbial-type channel
rhodopsins, such as ChR2, in surviving retinal neurons is a
strategy for the treatment of complete blindness caused by rod and
cone degeneration.
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[0287] All references cited herein, including journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued U.S. or foreign patents, or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures, and text presented in the
cited references. Additionally, the entire contents of the
references cited within the references cited herein are also
entirely incorporated by references.
[0288] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not in any way an
admission that any aspect, description or embodiment of the present
invention is disclosed, taught or suggested in the relevant
art.
[0289] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various applications such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning and range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by those skilled in
the art in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the art.
Sequence CWU 1
1
916987DNAArtificial SequenceExpression vector
rAAV2-CAG-Chop2-GFP-WPRE 1cctgcaggca gctgcgcgct cgctcgctca
ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga
gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc
tgcggccgca acgcgttacg tatcggatcc agaattcgtg 180atatctgaat
tcgtcgacaa gcttctcgag cctaggctag ctctagacca cacgtgtggg
240ggccggccgt aatgagacgc acaaactaat atcacaaact ggaaatgtct
atcaatatat 300agttgctcta gttattaata gtaatcaatt acggggtcat
tagttcatag cccatatatg 360gagttccgcg ttacataact tacggtaaat
ggcccgcctg gctgaccgcc caacgacccc 420cgcccattga cgtcaataat
gacgtatgtt cccatagtaa cgccaatagg gactttccat 480tgacgtcaat
gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
540catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
ctggcattat 600gcccagtaca tgaccttatg ggactttcct acttggcagt
acatctacgt attagtcatc 660gctattacca tgcatggtcg aggtgagccc
cacgttctgc ttcactctcc ccatctcccc 720cccctcccca cccccaattt
tgtatttatt tattttttaa ttattttgtg cagcgatggg 780ggcggggggg
gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg
840ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
agtttccttt 900tatggcgagg cggcggcggc ggcggcccta taaaaagcga
agcgcgcggc gggcgggagt 960cgctgcgcgc tgccttcgcc ccgtgccccg
ctccgccgcc gcctcgcgcc gcccgccccg 1020gctctgactg accgcgttac
tcccacaggt gagcgggcgg gacggccctt ctccttcggg 1080ctgtaattag
cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
1140tgaggggctc cgggagggcc cgagctcgcg atccgcagcc atggattatg
gaggcgccct 1200gagtgccgtt gggcgcgagc tgctatttgt aacgaaccca
gtagtcgtca atggctctgt 1260acttgtgcct gaggaccagt gttactgcgc
gggctggatt gagtcgcgtg gcacaaacgg 1320tgcccaaacg gcgtcgaacg
tgctgcaatg gcttgctgct ggcttctcca tcctactgct 1380tatgttttac
gcctaccaaa catggaagtc aacctgcggc tgggaggaga tctatgtgtg
1440cgctatcgag atggtcaagg tgattcttga gttcttcttc gagtttaaga
acccgtccat 1500gctgtatcta gccacaggcc accgcgtcca gtggttgcgt
tacgccgagt ggcttctcac 1560ctgcccggtc attctcattc acctgtcaaa
cctgacgggc ttgtccaacg actacagcag 1620gcgcactatg ggtctgcttg
tgtctgatat tggcacaatt gtgtggggcg ccacttccgc 1680tatggccacc
ggatacgtca aggtcatctt cttctgcctg ggtctgtgtt atggtgctaa
1740cacgttcttt cacgctgcca aggcctacat cgagggttac cataccgtgc
cgaagggccg 1800gtgtcgccag gtggtgactg gcatggcttg gctcttcttc
gtatcatggg gtatgttccc 1860catcctgttc atcctcggcc ccgagggctt
cggcgtcctg agcgtgtacg gctccaccgt 1920cggccacacc atcattgacc
tgatgtcgaa gaactgctgg ggtctgctcg gccactacct 1980gcgcgtgctg
atccacgagc atatcctcat ccacggcgac attcgcaaga ccaccaaatt
2040gaacattggt ggcactgaga ttgaggtcga gacgctggtg gaggacgagg
ccgaggctgg 2100cgcggtcaac aagggcaccg gcaaggaatt cggaggcgga
ggtggagcta gcaaaggaga 2160agaactcttc actggagttg tcccaattct
tgttgaatta gatggtgatg ttaacggcca 2220caagttctct gtcagtggag
agggtgaagg tgatgcaaca tacggaaaac ttaccctgaa 2280gttcatctgc
actactggca aactgcctgt tccatggcca acactagtca ctactctgtg
2340ctatggtgtt caatgctttt caagataccc ggatcatatg aaacggcatg
actttttcaa 2400gagtgccatg cccgaaggtt atgtacagga aaggaccatc
ttcatcaaag atgacggcaa 2460ctacaagaca cgtgctgaag tcaagtttga
aggtgatacc cttgttaata gaatcgagtt 2520aaaaggtatt gacttcaagg
aagatggcaa cattctggga cacaaattgg aatacaacta 2580taactcacac
aatgtataca tcatggcaga caaacaaaag aatggaatca aagtgaactt
2640caagacccgc cacaacattg aagatggaag cgttcaacta gcagaccatt
atcaacaaaa 2700tactccaatt ggcgatggcc ctgtcctttt accagacaac
cattacctgt ccacacaatc 2760tgccctttcg aaagatccca acgaaaagag
agaccacatg gtccttcttg agtttgtaac 2820agctgctggg attacacatg
gcatggatga actgtacaac atcgattgac taagcttgcc 2880tcgagaattc
acgcgtggta ccgataatca acctctggat tacaaaattt gtgaaagatt
2940gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
ctttaatgcc 3000tttgtatcat gctattgctt cccgtatggc tttcattttc
tcctccttgt ataaatcctg 3060gttgctgtct ctttatgagg agttgtggcc
cgttgtcagg caacgtggcg tggtgtgcac 3120tgtgtttgct gacgcaaccc
ccactggttg gggcattgcc accacctgtc agctcctttc 3180cgggactttc
gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
3240ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
tgtcggggaa 3300gctgacgtcc tttccatggc tgctcgcctg tgttgccacc
tggattctgc gcgggacgtc 3360cttctgctac gtcccttcgg ccctcaatcc
agcggacctt ccttcccgcg gcctgctgcc 3420ggctctgcgg cctcttccgc
gtcttcgcct tcgccctcag acgagtcgga tctccctttg 3480ggccgcctcc
ccgcctgatc cggccgcggg gatccagaca tgataagata cattgatgag
3540tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
aatttgtgat 3600gctattgctt tatttgtaac cattataagc tgcaataaac
aagttaacaa caacaattgc 3660attcatttta tgtttcaggt tcagggggag
gtgtgggagg ttttttcgga tcctctagag 3720tcgagagatc tacgggtggc
atccctgtga cccctcccca gtgcctctcc tggccctgga 3780agttgccact
ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc
3840tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
gcaaggggca 3900agttgggaag acaacctgta gggcctgcgg ggtctattgg
gaaccaagct ggagtgcagt 3960ggcacaatct tggctcactg caatctccgc
ctcctgggtt caagcgattc tcctgcctca 4020gcctcccgag ttgttgggat
tccaggcatg catgaccagg ctcagctaat ttttgttttt 4080ttggtagaga
cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt
4140gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
ctgctccctt 4200ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg
agcggccgca ggaaccccta 4260gtgatggagt tggccactcc ctctctgcgc
gctcgctcgc tcactgaggc cgggcgacca 4320aaggtcgccc gacgcccggg
ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 4380tgcctgcagg
ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
4440cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
gcggcgggtg 4500tggtggttac gcgcagcgtg accgctacac ttgccagcgc
cctagcgccc gctcctttcg 4560ctttcttccc ttcctttctc gccacgttcg
ccggctttcc ccgtcaagct ctaaatcggg 4620ggctcccttt agggttccga
tttagtgctt tacggcacct cgaccccaaa aaacttgatt 4680tgggtgatgg
ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
4740tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
ctcaacccta 4800tctcgggcta ttcttttgat ttataaggga ttttgccgat
ttcggcctat tggttaaaaa 4860atgagctgat ttaacaaaaa tttaacgcga
attttaacaa aatattaacg tttacaattt 4920tatggtgcac tctcagtaca
atctgctctg atgccgcata gttaagccag ccccgacacc 4980cgccaacacc
cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
5040aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
tcaccgaaac 5100gcgcgagacg aaagggcctc gtgatacgcc tatttttata
ggttaatgtc atgataataa 5160tggtttctta gacgtcaggt ggcacttttc
ggggaaatgt gcgcggaacc cctatttgtt 5220tatttttcta aatacattca
aatatgtatc cgctcatgag acaataaccc tgataaatgc 5280ttcaataata
ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
5340ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa 5400aagatgctga agatcagttg ggtgcacgag tgggttacat
cgaactggat ctcaacagcg 5460gtaagatcct tgagagtttt cgccccgaag
aacgttttcc aatgatgagc acttttaaag 5520ttctgctatg tggcgcggta
ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 5580gcatacacta
ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
5640cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg 5700cggccaactt acttctgaca acgatcggag gaccgaagga
gctaaccgct tttttgcaca 5760acatggggga tcatgtaact cgccttgatc
gttgggaacc ggagctgaat gaagccatac 5820caaacgacga gcgtgacacc
acgatgcctg tagcaatggc aacaacgttg cgcaaactat 5880taactggcga
actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
5940ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
attgctgata 6000aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
agcactgggg ccagatggta 6060agccctcccg tatcgtagtt atctacacga
cggggagtca ggcaactatg gatgaacgaa 6120atagacagat cgctgagata
ggtgcctcac tgattaagca ttggtaactg tcagaccaag 6180tttactcata
tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
6240tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
tcgttccact 6300gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
agatcctttt tttctgcgcg 6360taatctgctg cttgcaaaca aaaaaaccac
cgctaccagc ggtggtttgt ttgccggatc 6420aagagctacc aactcttttt
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 6480ctgtccttct
agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
6540catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc 6600ttaccgggtt ggactcaaga cgatagttac cggataaggc
gcagcggtcg ggctgaacgg 6660ggggttcgtg cacacagccc agcttggagc
gaacgaccta caccgaactg agatacctac 6720agcgtgagct atgagaaagc
gccacgcttc ccgaagggag aaaggcggac aggtatccgg 6780taagcggcag
ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
6840atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
ttgtgatgct 6900cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
ggccttttta cggttcctgg 6960ccttttgctg gccttttgct cacatgt
69872945DNAArtificial sequenceChop2 sequence from
rAAV2-CAG-Chop2-GFP-WPRE vector 2atggattatg gaggcgccct gagtgccgtt
gggcgcgagc tgctatttgt aacgaaccca 60gtagtcgtca atggctctgt acttgtgcct
gaggaccagt gttactgcgc gggctggatt 120gagtcgcgtg gcacaaacgg
tgcccaaacg gcgtcgaacg tgctgcaatg gcttgctgct 180ggcttctcca
tcctactgct tatgttttac gcctaccaaa catggaagtc aacctgcggc
240tgggaggaga tctatgtgtg cgctatcgag atggtcaagg tgattcttga
gttcttcttc 300gagtttaaga acccgtccat gctgtatcta gccacaggcc
accgcgtcca gtggttgcgt 360tacgccgagt ggcttctcac ctgcccggtc
attctcattc acctgtcaaa cctgacgggc 420ttgtccaacg actacagcag
gcgcactatg ggtctgcttg tgtctgatat tggcacaatt 480gtgtggggcg
ccacttccgc tatggccacc ggatacgtca aggtcatctt cttctgcctg
540ggtctgtgtt atggtgctaa cacgttcttt cacgctgcca aggcctacat
cgagggttac 600cataccgtgc cgaagggccg gtgtcgccag gtggtgactg
gcatggcttg gctcttcttc 660gtatcatggg gtatgttccc catcctgttc
atcctcggcc ccgagggctt cggcgtcctg 720agcgtgtacg gctccaccgt
cggccacacc atcattgacc tgatgtcgaa gaactgctgg 780ggtctgctcg
gccactacct gcgcgtgctg atccacgagc atatcctcat ccacggcgac
840attcgcaaga ccaccaaatt gaacattggt ggcactgaga ttgaggtcga
gacgctggtg 900gaggacgagg ccgaggctgg cgcggtcaac aagggcaccg gcaag
9453315PRTArtificial sequenceChop2 sequence from
rAAV2-CAG-Chop2-GFP-WPRE vector 3Met Asp Tyr Gly Gly Ala Leu Ser
Ala Val Gly Arg Glu Leu Leu Phe 1 5 10 15 Val Thr Asn Pro Val Val
Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30 Gln Cys Tyr Cys
Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45 Gln Thr
Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60
Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly 65
70 75 80 Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val
Ile Leu 85 90 95 Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu
Tyr Leu Ala Thr 100 105 110 Gly His Arg Val Gln Trp Leu Arg Tyr Ala
Glu Trp Leu Leu Thr Cys 115 120 125 Pro Val Ile Leu Ile His Leu Ser
Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140 Tyr Ser Arg Arg Thr Met
Gly Leu Leu Val Ser Asp Ile Gly Thr Ile 145 150 155 160 Val Trp Gly
Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175 Phe
Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185
190 Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys
195 200 205 Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser
Trp Gly 210 215 220 Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly
Phe Gly Val Leu 225 230 235 240 Ser Val Tyr Gly Ser Thr Val Gly His
Thr Ile Ile Asp Leu Met Ser 245 250 255 Lys Asn Cys Trp Gly Leu Leu
Gly His Tyr Leu Arg Val Leu Ile His 260 265 270 Glu His Ile Leu Ile
His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285 Ile Gly Gly
Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300 Glu
Ala Gly Ala Val Asn Lys Gly Thr Gly Lys 305 310 315
42241DNAChlamydomonas reinhardtii 4gcatctgtcg 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
224152214DNAChlamydomonas reinhardtii 5atggattatg gaggcgccct
gagtgccgtt gggcgcgagc tgctatttgt aacgaaccca 60gtagtcgtca atggctctgt
acttgtgcct gaggaccagt gttactgcgc gggctggatt 120gagtcgcgtg
gcacaaacgg tgcccaaacg gcgtcgaacg tgctgcaatg gcttgctgct
180ggcttctcca tcctactgct tatgttttac gcctaccaaa catggaagtc
aacctgcggc 240tgggaggaga tctatgtgtg cgctatcgag atggtcaagg
tgattctcga gttcttcttc 300gagtttaaga acccgtccat gctgtatcta
gccacaggcc accgcgtcca gtggttgcgt 360tacgccgagt ggcttctcac
ctgcccggtc attctcattc acctgtcaaa cctgacgggc 420ttgtccaacg
actacagcag gcgcaccatg ggtctgcttg tgtctgatat tggcacaatt
480gtgtggggcg ccacttccgc catggccacc ggatacgtca aggtcatctt
cttctgcctg 540ggtctgtgtt atggtgctaa cacgttcttt cacgctgcca
aggcctacat cgagggttac 600cacaccgtgc cgaagggccg gtgtcgccag
gtggtgactg gcatggcttg gctcttcttc 660gtatcatggg gtatgttccc
catcctgttc atcctcggcc ccgagggctt cggcgtcctg 720agcgtgtacg
gctccaccgt cggccacacc atcattgacc tgatgtcgaa gaactgctgg
780ggtctgctcg gccactacct gcgcgtgctg atccacgagc atatcctcat
ccacggcgac 840attcgcaaga ccaccaaatt gaacattggt ggcactgaga
ttgaggtcga gacgctggtg 900gaggacgagg ccgaggctgg cgcggtcaac
aagggcaccg gcaagtacgc ctcccgcgag 960tccttcctgg tcatgcgcga
caagatgaag gagaagggca ttgacgtgcg cgcctctctg 1020gacaacagca
aggaggtgga gcaggagcag gccgccaggg ctgccatgat gatgatgaac
1080ggcaatggca tgggtatggg aatgggaatg aacggcatga acggaatggg
cggtatgaac 1140gggatggctg gcggcgccaa gcccggcctg gagctcactc
cgcagctaca gcccggccgc 1200gtcatcctgg cggtgccgga catcagcatg
gttgacttct tccgcgagca gtttgctcag 1260ctatcggtga cgtacgagct
ggtgccggcc ctgggcgctg acaacacact ggcgctggtt 1320acgcaggcgc
agaacctggg cggcgtggac tttgtgttga ttcaccccga gttcctgcgc
1380gaccgctcta gcaccagcat cctgagccgc ctgcgcggcg cgggccagcg
tgtggctgcg 1440ttcggctggg cgcagctggg gcccatgcgt gacctgatcg
agtccgcaaa cctggacggc 1500tggctggagg gcccctcgtt cggacagggc
atcctgccgg cccacatcgt tgccctggtg 1560gccaagatgc agcagatgcg
caagatgcag cagatgcagc agattggcat gatgaccggc 1620ggcatgaacg
gcatgggcgg cggtatgggc ggcggcatga acggcatggg cggcggcaac
1680ggcatgaaca acatgggcaa cggcatgggc ggcggcatgg gcaacggcat
gggcggcaat 1740ggcatgaacg gaatgggtgg cggcaacggc atgaacaaca
tgggcggcaa cggaatggcc 1800ggcaacggaa tgggcggcgg catgggcggc
aacggtatgg gtggctccat gaacggcatg 1860agctccggcg tggtggccaa
cgtgacgccc tccgccgccg gcggcatggg cggcatgatg 1920aacggcggca
tggctgcgcc ccagtcgccc ggcatgaacg gcggccgcct gggtaccaac
1980ccgctcttca acgccgcgcc ctcaccgctc agctcgcagc tcggtgccga
ggcaggcatg 2040ggcagcatgg gaggcatggg cggaatgagc ggaatgggag
gcatgggtgg aatggggggc 2100atgggcggcg ccggcgccgc cacgacgcag
gctgcgggcg gcaacgcgga ggcggagatg 2160ctgcagaatc tcatgaacga
gatcaatcgc ctgaagcgcg agcttggcga gtaa 22146737PRTChlamydomonas
reinhardtii 6Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu
Leu Leu Phe 1 5 10 15 Val Thr Asn Pro Val Val Val Asn Gly Ser Val
Leu Val Pro Glu Asp 20 25 30
Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35
40 45 Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser
Ile 50 55 60 Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser
Thr Cys Gly 65 70 75 80 Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met
Val Lys Val Ile Leu 85 90 95 Glu Phe Phe Phe Glu Phe Lys Asn Pro
Ser Met Leu Tyr Leu Ala Thr 100 105 110 Gly His Arg Val Gln Trp Leu
Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125 Pro Val Ile Leu Ile
His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140 Tyr Ser Arg
Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile 145 150 155 160
Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165
170 175 Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His
Ala 180 185 190 Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys
Gly Arg Cys 195 200 205 Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe
Phe Val Ser Trp Gly 210 215 220 Met Phe Pro Ile Leu Phe Ile Leu Gly
Pro Glu Gly Phe Gly Val Leu 225 230 235 240 Ser Val Tyr Gly Ser Thr
Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255 Lys Asn Cys Trp
Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270 Glu His
Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285
Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290
295 300 Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys Tyr Ala Ser Arg
Glu 305 310 315 320 Ser Phe Leu Val Met Arg Asp Lys Met Lys Glu Lys
Gly Ile Asp Val 325 330 335 Arg Ala Ser Leu Asp Asn Ser Lys Glu Val
Glu Gln Glu Gln Ala Ala 340 345 350 Arg Ala Ala Met Met Met Met Asn
Gly Asn Gly Met Gly Met Gly Met 355 360 365 Gly Met Asn Gly Met Asn
Gly Met Gly Gly Met Asn Gly Met Ala Gly 370 375 380 Gly Ala Lys Pro
Gly Leu Glu Leu Thr Pro Gln Leu Gln Pro Gly Arg 385 390 395 400 Val
Ile Leu Ala Val Pro Asp Ile Ser Met Val Asp Phe Phe Arg Glu 405 410
415 Gln Phe Ala Gln Leu Ser Val Thr Tyr Glu Leu Val Pro Ala Leu Gly
420 425 430 Ala Asp Asn Thr Leu Ala Leu Val Thr Gln Ala Gln Asn Leu
Gly Gly 435 440 445 Val Asp Phe Val Leu Ile His Pro Glu Phe Leu Arg
Asp Arg Ser Ser 450 455 460 Thr Ser Ile Leu Ser Arg Leu Arg Gly Ala
Gly Gln Arg Val Ala Ala 465 470 475 480 Phe Gly Trp Ala Gln Leu Gly
Pro Met Arg Asp Leu Ile Glu Ser Ala 485 490 495 Asn Leu Asp Gly Trp
Leu Glu Gly Pro Ser Phe Gly Gln Gly Ile Leu 500 505 510 Pro Ala His
Ile Val Ala Leu Val Ala Lys Met Gln Gln Met Arg Lys 515 520 525 Met
Gln Gln Met Gln Gln Ile Gly Met Met Thr Gly Gly Met Asn Gly 530 535
540 Met Gly Gly Gly Met Gly Gly Gly Met Asn Gly Met Gly Gly Gly Asn
545 550 555 560 Gly Met Asn Asn Met Gly Asn Gly Met Gly Gly Gly Met
Gly Asn Gly 565 570 575 Met Gly Gly Asn Gly Met Asn Gly Met Gly Gly
Gly Asn Gly Met Asn 580 585 590 Asn Met Gly Gly Asn Gly Met Ala Gly
Asn Gly Met Gly Gly Gly Met 595 600 605 Gly Gly Asn Gly Met Gly Gly
Ser Met Asn Gly Met Ser Ser Gly Val 610 615 620 Val Ala Asn Val Thr
Pro Ser Ala Ala Gly Gly Met Gly Gly Met Met 625 630 635 640 Asn Gly
Gly Met Ala Ala Pro Gln Ser Pro Gly Met Asn Gly Gly Arg 645 650 655
Leu Gly Thr Asn Pro Leu Phe Asn Ala Ala Pro Ser Pro Leu Ser Ser 660
665 670 Gln Leu Gly Ala Glu Ala Gly Met Gly Ser Met Gly Gly Met Gly
Gly 675 680 685 Met Ser Gly Met Gly Gly Met Gly Gly Met Gly Gly Met
Gly Gly Ala 690 695 700 Gly Ala Ala Thr Thr Gln Ala Ala Gly Gly Asn
Ala Glu Ala Glu Met 705 710 715 720 Leu Gln Asn Leu Met Asn Glu Ile
Asn Arg Leu Lys Arg Glu Leu Gly 725 730 735 Glu 7933DNAArtificial
Sequencehuman codon-optimized Chop2 7atggactatg 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 taa
9338310PRTArtificial Sequencehuman codon-optimized Chop2 8Met Asp
Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe 1 5 10 15
Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20
25 30 Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly
Ala 35 40 45 Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly
Phe Ser Ile 50 55 60 Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp
Lys Ser Thr Cys Gly 65 70 75 80 Trp Glu Glu Ile Tyr Val Cys Ala Ile
Glu Met Val Lys Val Ile Leu 85 90 95 Glu Phe Phe Phe Glu Phe Lys
Asn Pro Ser Met Leu Tyr Leu Ala Thr 100 105 110 Gly His Arg Val Gln
Trp Leu Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125 Pro Val Ile
Leu Ile His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140 Tyr
Ser Arg Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile 145 150
155 160 Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val
Ile 165 170 175 Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe
Phe His Ala 180 185 190 Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val
Pro Lys Gly Arg Cys 195 200 205 Arg Gln Val Val Thr Gly Met Ala Trp
Leu Phe Phe Val Ser Trp Gly 210 215 220 Met Phe Pro Ile Leu Phe Ile
Leu Gly Pro Glu Gly Phe Gly Val Leu 225 230 235 240 Ser Val Tyr Gly
Ser Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255 Glu His
Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 260 265 270
Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 275
280 285 Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu
Ala 290 295 300 Glu Ala Gly Ala Val Pro 305 310 910123DNAArtificial
SequencemGluR6 promoter region 9tagaagttag tccactcttc ctcatgggcc
ttcgcctctg gtccaaagta ttaccaaagt 60cacttaaatt aacaagaaca gacacacaca
cacccaagct agaactagca gcactagcca 120gaactcaatt tacattttag
agaaaaaggg ggtggaggac agctcctgta gagggaatga 180tattaacacg
ttctgggctc cgtgcccagc atcgttctgc tcctttccaa cagtaaaacc
240ttagagcaaa ggcacaagtg gaaaaaatgg actgtggaat tcagttaaga
tactgtccag 300caccgaagac tgacagaaac taagtttcac ctccaggatt
gaaagcctac aggcgatctg 360ctcaaggccg acttgactag ctaacctgaa
gccggaggct tctttgaccg ctgttcgggc 420agcagaacct ggagtcaggg
cccgaggccc tcaccagcag ctgaggcctc tgcgtgcttc 480cgccaggctc
tcagccctgg cccgcaggtt cccggccgtt ccagctctgc cagaaaaccc
540agaaagctca atgcccagag cgggtaagac taggctcaac tgcgcgtgcg
cgcgagccac 600ctggtttcca ctgtggacta catttcccag aaggcactgt
gacactccta cccaccctgt 660atggtgcaga gtgggacaca ggcgcctaaa
gactgagaat caacttttca gttgccacca 720gctttcaggt ttctgtgcag
gcttcattca taattacaat ggtaatacta ctaaagagga 780aaaagtgagt
gtgcattaaa atgttgaaga ataaggctct gactgcttag tttcagatag
840cgaaaggact gtcctctttc attttttaat agaaaattat gctttttcta
ggctacaaaa 900gatacataac atacacaatt tttcattgct ggctcatact
ttgtattaag caaaaaactg 960ccatattagt cattactgtc atggacaact
cagattttca ggggaagcaa acaggtagaa 1020ataatttatt cattacttaa
gttggaaatg tctgtttttt acaaaaattt tttcctgtct 1080ttgtccactg
taaaagttct gaagaatgat tattcggtct caacaagata caaattatgt
1140tctctaggta gcaattaaca caaggaacgc cttgaggtat gggaggggtg
aggaagctca 1200caagatagac cctggtgcct ggaaggaaga cagccaacta
aaggtcatat cacagtgtcc 1260cgggaaccaa cttgaagggc ttctgctgta
caaatgtggg agaatttcat cgtcagaagg 1320ctctgcaaag gtctgaaagt
caccgaactc tgtaagattc tatcctgctt ctattcctgt 1380caaaatatac
cagaaggaat ggaactaccc cctccaaaaa ataaataaac aaacaaacca
1440ccaaaccacg cacagacaaa gcattcaata cacatgctaa aacataccac
tttagtttaa 1500ggactatagt gattccacac taggtaaggt gctttctgta
ggcttttagt taatagtttt 1560gtcaagctaa agaagatctc cagatggcta
aacttttaaa tcatgaatga agtagatatt 1620accaaattgc tttttcagca
tccatttaga taatcatgtt ttttgccttt aatctgttaa 1680tgtagtgaat
tacagaaata catttcctaa atcattacat cccccaaatc gttaatctgc
1740taaagtacat ctctggctca aacaagactg gttgtgacag gtttgtctct
gtcagtttgt 1800gactgttggg ctggctcttc ctacccctct gcttcttggt
ttggcctgaa cattaatttt 1860attttatttt tttaatttta cctacaatca
atttcacaat gtgtgttgtc attttctcct 1920attgtgtgat attttgtgaa
cagagaaatt cctttgcaac ataactgagt atcatgggtt 1980agttttttct
tcagtagaag gcttcacatg ggtcttttct gctctgagtg agagcagctc
2040aatgctgtga gctgacacag cagactgcaa tacaacctgt tgtgttttat
aaaaagataa 2100ggaggaatga aatctgtttg gtggatgtgt ggtcaggtgt
ggggaaaggg ggtgcctcca 2160cgggcccatg ctgaggcttt ccttccccgt
gaaggaccag cctcaggaca gtatgttata 2220gaatagagtt tattcagggc
atgaggaggg gagttgagag aaaggcagag agagagagag 2280agagagagag
agagagagag agagaatata tagaggagta gaggctgacc atgagcacag
2340ggagagaggg ggagagggga atggggaggg agaataagga acaggagcaa
gagagcaaga 2400acaagagaga caagagaagg caagctgccc ctgttatagt
gagtcaggca tacctggcta 2460ttgccaggta actgtggggc ggatcccaga
ctaaatgcca acacaaccag aggaagggga 2520gatgtgtttg gtgttccttc
gtctccctca gcacactgtg tgtgcctgtt ctctgaaaaa 2580tgctctggcc
atttcttttt aactcctccg tgctgaactg gaacccagtt gtgcaagcga
2640ggcaggcagt ctaccgtagc gctagatttt tacttttaaa ccgggatctc
gctttgcatt 2700aatgccctgc ttccacatct gcttacagct tagtgtgttg
ttttgctttt atccccctca 2760cactctcagt ttttcctgtg gagtttcaca
cacaaatttt cagcagggac accctttctg 2820gttccttgat attactgctg
ttgtcatttt gacattgttc ttcgtctggg ctccagctac 2880tgttctttct
acctcccaga caccaacatt gttcttcact caggtttctg cccatgcatc
2940atctaccttg ctgtgtattc aactggatat ccatatgcaa atggttgaat
ttggacccaa 3000catcatatta cactcaaaaa ttccctcaac atggatcaac
gatctaaatg ttagcgctag 3060aatcacaaaa cactaaaaat aaaacacggg
agtgtttagt gatgtcttag ttatggtttc 3120tattgctgtg ataaaacact
gtgattaaaa gcagcagcag tggggtgagg cagggtaggc 3180aggaaaggtt
caatctcagc ttagaactct ctctctctgg tcatgctcca tcagtgaatg
3240gagtaagagc aggaacttga ggcaggagct gatgcagagg cccagaagga
aggaacctgc 3300ttactggctt gctccttgtg gcttgctcaa cctactttct
tgttgactcc aggaccacct 3360gccaagggct ggcacctccc ctaggggact
ggaccctccc acttcaatca ttaatcaaga 3420aaatgcccca cagggggcat
tttcaattgt gactctctct tctcagagga ctcttgtttg 3480ttaacaaaaa
actaaccagg gcaggtataa atcttcatga ctttggaatt gcctgtggga
3540tctcagatgt gctatccaaa cacaaacaat aaaagaaaaa tgcaatttga
atcttaacaa 3600atgttttgaa tcttaaaatg ttatgtatta tgaagaaagt
aaaccgataa ctcacagaca 3660ggaacaaaaa tctttgcaag tcaaaagttt
aataagtcca ggctttacac cttaacaaga 3720agactgagtc tgtggctaca
taccgtggca catattacta ctagagcatg ggatgcccct 3780ggtaacggca
acttctgggg accacgtgga tgtccgggga ctgtgcataa cttgtcccac
3840ccctcactgg atgcggcact ctagagagct ggccccatct ctcacctatg
gcggcactct 3900ggagagtggg ccgggcagca cagtggagct gctcctggct
tcgagggtag agatgagcca 3960gctccaagga tgtgagtgtg ggagagctga
ccctgccact tgtctgccat gggtagcaca 4020ggtgcagatg tgatacacac
acacacacac acacacacac acacacacac acacactgcc 4080ccgcactact
cctgaagtca ggagctagtc ccacccctac cagctacagc tctcagaaca
4140gtcccgggac cttgtctgga gagcacagca gaactaaccc tggtgttgag
ggtgcaggaa 4200acccagcccc aagagtgaaa gctcggaaaa gctggctcca
tcattcatct gctgtgaggt 4260gccatgggtg tgcaggtgat gctctcccca
cctcttcact ccctgccacc taaggcagta 4320gggacagctg gtccccaggg
acatcagagt gggagagctg gctctgctcc tcactggctg 4380tagaactcag
aatgggccct gcatcttgtc tgggcagcac aatagggctg gccttgttgg
4440agggagagag gatggggcag cccagagggt agagtgtagg agaggtggcc
ccgacacttg 4500tctggtgtga ggtggtgtgg atgtaggggc aatgccctcc
ctgggcccct cactgcctga 4560ggcagtcagg agaactgacc ccagggtcat
gagagcaggt gagctggccc tgctcccact 4620ggctgcactt cagagagcag
gccctgaatc tcctctgggc agcatagtcg cactagcact 4680ggtagagggg
gcacgggtga actccacccc ccatccccca ccaggttaag agcatgggag
4740agctggcctt gccacttgtc ttgtgtgagg tgggtgcggg gattatgtcc
tcttccccca 4800cctgcagtgg ctgggagagt tgaccctgga ccatgagagg
gagagagagc taatggcccc 4860tcattggcta tagcacttgg gtgagtgggc
ctgcacctca cctgggcaat acagtggagc 4920tggccctgat ggtgaaagta
tgggtgagtt agtccagagg gtatgagagt gtgagaaatg 4980gcccagcctc
tcacaggctg cagcacctgg gagagcgcac cctgaacctt gaatggatag
5040ctaggtggag cggctctgga ggaatgggtg caaatgaccc atcctgaggg
caagagagca 5100ggagtgctga ccttgcctcc tgccaatgga gggaggtatt
gactggccta gctggaagag 5160tgctggagag tttactctag tggtgtgggt
aagggagagc tggcaagctg accagctcag 5220ctactaccca gggcaagatc
ctgggctctg agccagccca ccccaaaatc gatatcatct 5280gtgaacagtt
gacatgcatg aaaggggcat ccttgctatt ctaaaactgc aggctctcca
5340tgacacagag caacaacagg ataacccaga ggggtctcaa tgaagatcca
atattgatgg 5400tatcacagaa gctaaagact tcaaaccaga ccgttgactc
attataatga acaccttacc 5460ttcaagtgaa gatgtgtgga cagagggaaa
tactgtagga cacactgtga cacactacag 5520cttgcatggt gagatgtttt
ctatgctttg ttttgttgtt gttgttggtg gtggtggtgg 5580tggtggtggt
ggtgtggtgg tggtagtggg gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
5640gtgtattatt ttggggggag gttgcgaggg tgaagaatag atatgaggag
atggggagat 5700gagcagaact ggggtgcatg atgtgaaact cacaaagaat
caataaagtt tttaaaaact 5760cagaaactaa gccgggcggt agtggcgcac
gcctttaatc ccagcacttg ggaggcagag 5820gcaggtggat ttctgagttc
aaggccagcc tggtctacaa agtgagttcc aggacagcca 5880gggatacaca
gagaaacctt gtcttaaaaa acaaacaaac aaacaaacaa acaagaaaca
5940aaaacaaata aaacaaaaaa ccctcagaaa ctagttttaa agcttatcaa
agcagactct 6000actcgctgtt ttactgaatt tcatcaagct aagtacttta
ggggagagag aatctcctcc 6060tcagcctgca gtttctatac tactggactg
taaaattccc gagagtaaga tatgaatcct 6120gggcctgtaa attatattta
aactaatata tattcaaaac agtgaattat agggaaaaaa 6180agaaaactcc
gtttatatgg tgcttcattc acccttagtg agctatttcc ctggttgcca
6240ccaggccacc ctgtggtggc agcactgagt actcctagct gccaagtcag
tctttgcaca 6300gcacattcac atggcgatcg aaccaaagag cgtgtttaat
ggtgcagagc tatattgaag 6360gaagcttgca tagctgggtg tcaacaagtg
ctgatggctg attgttttaa taccccatcc 6420tgctacattg aaaggtctgc
agttgccttg ggcttggcag aggagcctag cggaaagaca 6480ggctgtcaaa
gcagcagtgg gatgagggat gaggtgatag ttagtcctcc ctgtcaactt
6540actagtttcg aatcacctgg gagacacatt gctgagtgta actgtgaggg
gctctctagg 6600gaggttaaac tgagggggaa acacccaccc tgaatgtgcg
tggcactata actgaatgca 6660aacgggaaag aaagaaacaa gctggctggg
gagcaccaga attcatctct cctttcttcc 6720tgactgtgga caccatgtga
ccagctgcct cacactcttt ccagcaaacc ttgctgtcat 6780ggaagactgt
gttccctcca actgtgagcc agaatagtcc ttctcttgta tcacttgtct
6840ttgtcaggta ttttgtcata gcaatgagaa acataaccca gtatggagtt
acttagttac 6900acttgctcca tactggtcca cgcaggaccc taaggcctct
gtggacattc tcagttgcag 6960acatcatgct tttcaacacc ttgtgctaga
gatggtgaag aatgctccaa ctctcctgcc 7020tacatgttct ctaaaagtga
gaaggtggac agcactctta gcactcctag tcagagggca 7080gaggtttgac
ccatacattg aaccctcaaa ggtatagtct taagtctatt tgtgtgcaca
7140tgcttgcaca cacacacaca cacacacaca cacacacaca cacacacaca
cacgtgcacg 7200ctcccacaca gaagctctgc ttggatagtc tcctgcagtg
tcacccactc tggtcaagcc 7260ccactaagct ggcttccatc acaggaatga
actgctctgg gtgtgacaag agaaatcgga 7320ggatagtggt tatatttctg
ctgcttgctc tctccaccag tcatgtccag atctctgctg 7380ccaccctaat
ccaccctgac taatgcactg aagagcccat taatccctgg aggctggggc
7440tcagcaactg tctccaagat gcctttgctg tccagcatca gagagctcaa
tcctgtcctc 7500tgttgacaat gatgggaaaa tatctttggg ttgaacatct
tcacggtgta aatcagttcc 7560agagagctag gaaactcaga aatgatgtgg
ggagacaact gagggctcct gacccacatg 7620ggagcttcag ggtgaactaa
ccccatcttc ccccctccaa gccagtgggt aggctggtgt 7680ttcacaccac
tctgaaatgc aaatctagtt gctgacaaag gccagctgca gagccttagg
7740gccatagggc agccagtcat ttcctgaggt gtctatttgt ctgtctgcag
atggagagat 7800tctctgcaag gctttggtgt gtttgctgct gctgaaggtc
tgttcagcat tgtttccagc 7860cttaccaagg cttcttgcat ctgtccttca
gattcactgt gctggcacac cctggctggc 7920tcagctccta tcatctgcca
cttacgggtt tgcttcagag aaagttgggg tggcttttat 7980gcagctgcat
taaaaagaac tactaaactc tgataagatg gctcagctgg taaaagtgcc
8040tgctgccaag actcacaacc tgtgttcagt cctcaggacc aacatggtga
aaggtgatag 8100gttatttctc tgccgctagt gaaatgagcc aagttgggat
atgttaaagg caggtttatt 8160gggaagctgc tcttaggtga gttcacagac
ccggaggatt gagggcaggg cagttgccat 8220ggggggaaga ggggaggtga
gggagaatag aaagcgagaa agggggcaca gatgtcccga 8280cccgcaggac
ccagttattc agggggtctg agggagacct tgcctgaaag agaaacgggc
8340gggaaataag agacagacca agtagatcca tcaaggtctg tttattgaga
gtaaggttac 8400agaatataag cggcaaggag gaaggaagta aagagggaga
aacttgcccg tgcctcagcc 8460cgcaggcagg ggtggttctg cacaactgcc
cgggaaggtg ctatctactc ttagctcagg 8520ggacattctg tgttttttca
cagaaagttt gcagatacta ttatctgccc ttgatgttgt 8580gtcagctgtc
atttcaaaag gtcggaagtc tctcctccag gagggagcgg aactttggct
8640tatgactcag tgtcagtccc caacatctct caaaaggtcc gaagtttctc
acgaaggaag 8700gggagctttg gcttatggct caatgccggt ctccaacaca
gagagagaag agaaggaaca 8760gaaggaagag agaaagagac caaaatgtct
ggatcacata gggaagaacc tctgggaaaa 8820aggcagccca gcccctgggc
tggaaagttc agggtgggag gcagggtatg tcaggtaggg 8880actgggggat
gctgggagat ccctgaagtc aggtctgctt tgatatgcaa actatgcacc
8940ttgtcccggt cccaaaccaa aagggagaga actaactctg gcgtgagagg
gcatgtgccg 9000catatcacac acacacacac acacacacac acacacacac
acacacacac acacacacac 9060aaaaccatgc acgctcgcac acgatagata
atacatacac caatatctga aaagagaaaa 9120ggttctagtg gtcaggacag
agaatgaaaa cggcaggaag gcaagaaagt ttgagaacgt 9180agggggtggg
gtagggagac actacgagtg gaataagcca cgtttggaga acgtctaggc
9240agatacagaa atgcagaaca cagagagacc gagaccagag cagcgtcaga
ccggctgcaa 9300ggctcttgtt aggggcttta gaaacacctg tgtgctctcc
cggaagcctg gtgcagtcag 9360agaggaagct tgcttcccag acagagatga
cacagtttca caacctgtca gaccaccttg 9420caggagagac tgaaccccag
caaccagaac cacttggcta tgcatgtcct tttctgttta 9480aacctaagtc
tctgaagacc gaccagggga gtccctggac ttctttgttc ctcttctcgg
9540ggtggcggga ctgattgtgt aaatctctta tctccaactt tcactcttat
ctgtctcttt 9600aatcggcata ttgaggatga gtggccaagc ttattggtgt
tgctgggtca gacaatttaa 9660aggcagtcta ggggagaagc agacccaggg
agtcagagag gcagagagag aagagagccc 9720ttcctccact ctcaagctct
ggagggggtc tctgccctca ccctcatccc tccccagaat 9780ccttaaatcc
tctagactgt agctctgatt ttacagctgt cacagactcg tcctactagc
9840cagaggttgg ctcaggtaag caccactggg gaggtagcct agggtgcgct
ggggtgggtc 9900cagaggaaga gctgcccaga actgtggggg aaggagcggg
accgaccatc aacaggggga 9960cttttcaggg agaatgagag caatcctctg
gaggcctggg agaggctgct gagttgctgg 10020tgcgcgagtc accaactttt
cctgcgctct cggtgtccgg ccagaatccc gaagtggcag 10080ctgagcacgg
ggtggcagct tcgtccgcca gcggccggga tcc 10123
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References