U.S. patent application number 17/540121 was filed with the patent office on 2022-06-16 for raav-guanylate cyclase compositions and methods for treating leber's congenital amaurosis-1 (lca1).
This patent application is currently assigned to University of Florida Research Foundation, Incorporated. The applicant listed for this patent is University of Florida Research Foundation, Incorporated. Invention is credited to Sanford L. Boye, Shannon E. Boye, William W. Hauswirth.
Application Number | 20220186260 17/540121 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220186260 |
Kind Code |
A1 |
Boye; Shannon E. ; et
al. |
June 16, 2022 |
RAAV-GUANYLATE CYCLASE COMPOSITIONS AND METHODS FOR TREATING
LEBER'S CONGENITAL AMAUROSIS-1 (LCA1)
Abstract
Disclosed are viral vector compositions comprising
polynucleotide sequences that express one or more
biologically-active mammalian guanylate cyclase proteins. Also
disclosed are methods for their use in preventing, treating, and/or
ameliorating at least one or more symptoms of a disease, disorder,
abnormal condition, or dysfunction resulting at least in part from
a guanylate cyclase deficiency in vivo. In particular embodiments,
the use of recombinant adeno-associated viral (rAAV) vectors to
treat or ameliorate symptoms of Leber's congenital amaurosis, as
well as other conditions caused by an absence or reduction in the
expression of a functional retinal-specific guanylate cyclase 1
(retGC1).
Inventors: |
Boye; Shannon E.;
(Gainesville, FL) ; Hauswirth; William W.;
(Gainesville, FL) ; Boye; Sanford L.;
(Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Florida Research Foundation, Incorporated |
Gainesville |
FL |
US |
|
|
Assignee: |
University of Florida Research
Foundation, Incorporated
Gainesville
FL
|
Appl. No.: |
17/540121 |
Filed: |
December 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15728628 |
Oct 10, 2017 |
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17540121 |
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13643074 |
Feb 1, 2013 |
9816108 |
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PCT/US2011/033669 |
Apr 22, 2011 |
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15728628 |
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61327521 |
Apr 23, 2010 |
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International
Class: |
C12N 15/861 20060101
C12N015/861; A61K 48/00 20060101 A61K048/00; C12N 9/88 20060101
C12N009/88; C12N 15/86 20060101 C12N015/86 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Nos. EY011123 and EY008571 awarded by the National Institutes of
Health (NIH). The government has certain rights in the invention.
Claims
1-56. (canceled)
57. A recombinant adeno-associated viral (rAAV) vector comprising a
photoreceptor-specific human rhodopsin kinase promoter operably
linked to a nucleic acid encoding a mammalian guanylate cyclase
polypeptide.
58. The rAAV vector according to claim 57, wherein the mammalian
guanylate cyclase polypeptide comprises an amino acid sequence at
least about 90% identical to at least 80 contiguous amino acids of
a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
59. The rAAV vector according to claim 57, wherein the mammalian
guanylate cyclase polypeptide comprises an amino acid sequence at
least about 92% identical to at least 100 contiguous amino acids of
a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.
60. The rAAV vector according to claim 57, wherein the guanylate
cyclase polypeptide comprises an amino acid sequence at least about
95% identical to at least 120 contiguous amino acids of a sequence
as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
61. The rAAV vector according to claim 57, wherein the guanylate
cyclase polypeptide comprises an amino acid sequence at least about
98% identical to at least 140 contiguous amino acids of a sequence
as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
62. The rAAV vector according to claim 57, wherein the guanylate
cyclase polypeptide comprises an amino acid sequence at least about
98% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
63. The rAAV vector according to claim 57, wherein the rAAV vector
is a recombinant adeno-associated virus serotype 1 (rAAV1) vector,
a recombinant adeno-associated virus serotype 2 (rAAV2) vector,
recombinant adeno-associated virus serotype 3 (rAAV3) vector, a
recombinant adeno-associated virus serotype 4 (rAAV4) vector, a
recombinant adeno-associated virus serotype 5 (rAAV5) vector, a
recombinant adeno-associated virus serotype 6 (rAAV6) vector, a
recombinant adeno-associated virus serotype 7 (rAAV7) vector, a
recombinant adeno-associated virus serotype 8 (rAAV8) vector, or a
recombinant adeno-associated virus serotype 9 (rAAV) vector.
64. The rAAV vector according to claim 57, wherein the
photoreceptor-specific human rhodopsin kinase promoter is at least
90% identical to SEQ ID NO: 12.
65. The rAAV vector of claim 57, further comprising an enhancer
operably linked to the photoreceptor-specific human rhodopsin
kinase promoter.
66. The rAAV vector of claim 57, further comprising a mammalian
intron sequence operably linked to the photoreceptor-specific human
rhodopsin kinase promoter.
67. The rAAV vector according to claim 57, comprised within an
infectious adeno-associated viral particle, virion, or a plurality
of infectious AAV particles.
68. A virion or an infectious viral particle comprising the rAAV
vector of claim 57.
69. A plurality of infectious viral particles prepared from the
rAAV vector of claim 57.
70. A composition comprising: the rAAV vector of claim 57 and a
pharmaceutically-acceptable buffer, carrier, vehicle, or
diluent.
71. The composition according to claim 70, further comprising a
lipid, a liposome, a lipid complex, an ethosome, a niosome, a
nanoparticle, a microparticle, a liposphere, a nanocapsule, or any
combination thereof.
72. The composition according to claim 70, formulated
administration to the human eye.
73. The composition according to claim 70, for use in therapy or
prophylaxis of a human retinal dystrophy, disease, or disorder.
74. The composition according to claim 70, for use in the therapy
or prophylaxis of Leber congenital amaurosis-1 (LCA1).
75. The composition according to claim 70, for use in a subject
having a mutation in GUCY2D.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 15/728,628, entitled "rAAV-GUANYLATE CYCLASE
COMPOSITIONS AND METHODS FOR TREATING LEBER'S CONGENITAL
AMAUROSIS-1 (LCA1)" filed on Oct. 10, 2017, which is a continuation
of U.S. application Ser. No. 13/643,074, entitled "rAAV-GUANYLATE
CYCLASE COMPOSITIONS AND METHODS FOR TREATING LEBER'S CONGENITAL
AMAUROSIS-1 (LCA1)" filed on Feb. 1, 2013, which is a national
stage filing under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2011/033669, filed Apr. 22, 2011, which
claims priority to U.S. Provisional Patent Application No.
61/327,521, filed Apr. 23, 2010, the entire contents of each of
which are specifically incorporated herein in their entirety by
express reference thereto.
BACKGROUND OF THE INVENTION
Names of the Parties to a Joint Research Agreement
[0003] Not Applicable.
FIELD OF THE INVENTION
[0004] The present invention relates generally to the fields of
molecular biology and virology, and in particular, to methods for
using recombinant adeno-associated virus (rAAV) compositions that
express at least a first nucleic acid segment encoding at least a
first therapeutic gene product, and particularly those products
useful in the prevention, treatment, or amelioration of one or more
symptoms of diseases, disorders, trauma, injury, or dysfunction of
the mammalian eye. In particular embodiments, the invention
provides compositions including rAAV vectors that express a
biologically-functional guanylate cyclase peptide, polypeptide, or
protein for use in one or more investigative, diagnostic and/or
therapeutic regimens, including, for example, the treatment of one
or more disorders or diseases of the mammalian eye, and in
particular, for treating congenital retinal blindness including,
retinal dystrophy such as Leber's congenital amaurosis, type 1
(LCA1), in humans. Also provided are methods for preparing rAAV
vector-based guanylate cyclase medicaments for use in viral
vector-based gene therapies, including, for example rAAV-LCA1
vectors for treating or ameliorating one or more symptoms of
guanylate cyclase deficiency in humans.
DESCRIPTION OF RELATED ART
[0005] Leber's congenital amaurosis (LCA) (formerly "amaurosis
congenita of Leber"), first described as a congenital type of
retinitis pigmentosa (RP) by German ophthalmologist Dr. Theodor
Leber in 1869, is the earliest and most severe form of inherited
retinopathy, and accounts for about 6% of all inherited retinal
dystrophies. LCA is a group of degenerative diseases of the retina,
and is the most common cause of congenital blindness in children.
This autosomal recessive condition is usually recognized at birth
or during the first months of life in an infant with total
blindness or greatly impaired vision, normal fundus and
extinguished electroretinogram (ERG) (see e.g., Perrault et al.,
1996). Despite these functional deficits, LCA1 patients retain some
rod and cone photoreceptors in both their macular and peripheral
retina for years. Symptoms of the disease include retinal
dysfunction, wobbly eye movement (nystagmus), impaired vision, slow
pupil response, and ultimately, blindness.
[0006] Through genetic analyses, mutations in guanylate cyclase-1
(Gucy2d), assigned to the LCA1 locus, have been shown to account
for 20% of all reported cases of LCA (see e.g., Milam et al., 2003;
Perrault et al., 1996; Perrault et al., 2000). The number of
patients affected by LCA1 is approximately twice that of patients
affected by defects in the Retinal pigment epithelium-specific
65-kDa protein (RPE65) version of the disease (LCA2), which has
garnered much attention in the gene therapy community in recent
years.
[0007] It is estimated that 200,000 Americans have type 1 Leber's.
Gucy2d encodes guanylate cyclase (retGC1) which is expressed in
photoreceptor outer segment membranes (see e.g., Dizhoor et al.,
1994; Liu et al., 1994), and plays a role in the recovery phase of
phototransduction. Mutations which reduce or abolish activity of
this enzyme are thought to create the biochemical equivalent of
chronic light exposure in rod and cone photoreceptors. LCA is
usually regarded as the consequence of either impaired development
of photoreceptors or extremely early degeneration of cells that
have developed normally. The LCA1 locus (GUCY2D) has been mapped to
human chromosome 17p13.1 (LCA1) by homozygosity mapping.
DEFICIENCIES IN THE PRIOR ART
[0008] Presently there are no effective prophylactics or
therapeutics available to prevent or treat LCA1 in humans.
SUMMARY OF THE INVENTION
[0009] The present invention overcomes limitations inherent in the
prior art by providing new, non-obvious, and useful rAAV-based
genetic constructs that encode one or more therapeutic mammalian
polypeptides, and particularly those proteins, peptides,
polypeptides of the guanylate cyclase family, for the prophylaxis,
treatment and/or amelioration of one or more mammalian diseases,
disorders or dysfunctions, or one or more symptoms thereof, that
result from, or are exacerbated by, a deficit in, or a deficiency
of, biologically-active guanylate cyclase polypeptide activity. In
particular, the invention provides genetic constructs encoding one
or more mammalian retinal-specific guanylate cyclase (retGC1)
polypeptides, for use in the treatment of such conditions as LCA1,
and other conditions of the eye such as recessive and dominant
forms of cone-rod dystrophy that manifest from a deficiency or
absence of physiologically-normal levels of guanylate cyclase
polypeptide.
[0010] In one embodiment, the invention provides a recombinant
adeno-associated viral (rAAV) vector including at least a first
polynucleotide that comprises a promoter operably linked to at
least a first nucleic acid segment that encodes at least a first
mammalian guanylate cyclase protein, peptide, or polypeptide.
Preferably, the promoter is a photoreceptor-specific promoter (such
as, for example, a human rhodopsin kinase promoter), or a
ubiquitous promoter (such as, for example, a truncated chimeric
CMV-chicken .beta.-actin promoter). Preferably the first nucleic
acid segment encodes at least a first mammalian guanylate cyclase
protein, peptide, or polypeptide that comprises, consists
essentially of, or alternatively, consists of, at least a first
contiguous amino acid sequence region that is at least about 80%,
about 85%, or about 90% or greater in identity with at least a
first sequence region of at least about 60, about 70, about 80,
about 90, or about 100 or more contiguous amino acids of a sequence
as set forth in any one or more of the mammalian guanylate cyclase
proteins depicted in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9. SEQ ID NO:10, or SEQ ID NO:11.
[0011] In certain embodiments, the at least a first nucleic acid
segment preferably encodes at least a first mammalian guanylate
cyclase protein, peptide, or polypeptide that includes at least a
first contiguous amino acid sequence region that is at least about
91%, about 92%, about 93%, about 94%, or about 95% or greater in
primary amino acid sequence identity with at least a first sequence
region of at least about 100, about 110, about 120, about 130,
about 140, or about 150 or more contiguous amino acids of a
sequence as set forth in any one or more of the mammalian guanylate
cyclase protein sequences recited in SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
[0012] Preferably, the at least a first nucleic acid segment will
encode at least one or more mammalian guanylate cyclase proteins,
peptides, or polypeptides that each preferably include at least a
first contiguous primary amino acid sequence that is at least about
95%, at least about 96%, at least about 97%, at least about 98%, or
at least about 99% identical to at least a first sequence region
that includes at least about 90, about 110, about 130, about 150,
or about 170 or more contiguous amino acids of at least a first
guanylate cyclase protein as shown in one or more of SEQ ID NO:1.
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID
NO:11.
[0013] Preferably the rAAV vectors of the present invention include
at least a first nucleic acid segment encodes at least a first
mammalian guanylate cyclase protein, peptide, or polypeptide that
comprises, consists essentially of, or alternatively, consists of,
the amino acid sequence of any one or more of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8. SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, or
a polynucleotide sequence that is complementary to, or specifically
hybridizes to one or more such sequences under stringent, to
highly-stringent hybridization conditions. Preferably, the first
mammalian guanylate cyclase protein, peptide, or polypeptide will
possess guanylate cyclase activity in vitro and in vivo in
transformed mammalian cells, and preferably, in transformed human
host cells. In particular aspects, the guanylate cyclase protein,
peptide, or polypeptide will possess significant
biologically-active guanylate cyclase activity in vitro and in vivo
in transformed mammalian cells, and preferably, in transformed
human host cells when the nucleic acid segment encoding the
peptide, protein, or polypeptide is operably linked to at least a
first promoter capable of expressing the sequence in a mammalian,
and preferably, human, host cell.
[0014] While the rAAV vectors of the present invention are not
necessarily limited to a particular serotype, in certain
embodiments, the inventors contemplate beneficial results can be
achieved by utilizing an rAAV vector that is one or more of the
following known serotypes: recombinant adeno-associated virus
serotype 1 (rAAV1), recombinant adeno-associated virus serotype 2
(rAAV2), recombinant adeno-associated virus serotype 3 (rAAV3),
recombinant adeno-associated virus serotype 4 (rAAV4), recombinant
adeno-associated virus serotype 5 (rAAV5), recombinant
adeno-associated virus serotype 6 (rAAV6), recombinant
adeno-associated virus serotype 7 (rAAV7), recombinant
adeno-associated virus serotype 8 (rAAV8), or a recombinant
adeno-associated virus serotype 9 (rAAV) vector. In certain
applications, the rAAV vectors of the present invention may be a
self-complementary rAAV (scAAV) vector.
[0015] In embodiments in which a photoreceptor-specific promoter is
desired, the rAAV vectors disclosed herein may include at least a
first photoreceptor-specific rhodopsin kinase promoter. Exemplary
such promoters include the human rhodopsin kinase promoter, which
is illustrated in SEQ ID NO:12. In certain aspects of the
invention, the use of a promoter sequence that includes at least
about 20, at least about 25, at least about 30, at least about 35,
at least about 40, at least about 45, or at least about 50 or more
contiguous nucleotides from SEQ ID NO:12 is particularly preferred
when tissue-specific (and in particular, photoreceptor-specific)
expression of the therapeutic construct is desired.
[0016] Similarly, in embodiments in which a ubiquitous promoter is
desired, the rAAV vectors disclosed herein may include at least a
first truncated chimeric CMV-chicken .beta.-actin promoter.
Exemplary such promoters include the truncated chimeric CMV-chicken
.beta.-actin promoter, which is illustrated in SEQ ID NO:13. In
certain aspects of the invention, the use of a promoter sequence
that includes at least about 20, at least about 25, at least about
30, at least about 35, at least about 40, at least about 45, or at
least about 50 or more contiguous nucleotides from SEQ ID NO:13 is
particularly preferred when non-tissue specific expression of the
therapeutic gene is desired.
[0017] In some embodiments, the promoter sequence employed in the
disclosed therapeutic gene constructs may comprise, consist
essentially of, or alternately, consist of, a nucleic acid sequence
that includes at least 55, about 60, about 65, about 70, about 75,
about 80, about 85, about 90, about 95, about 100, about 105, about
110, about 115, or about 120 or more contiguous nucleotides from
the promoter sequences set forth in either SEQ ID NO:12 or SEQ ID
NO:13.
[0018] The gene therapy vectors disclosed herein may also further
optionally include one or more "upstream" or "downstream"
regulatory sequences, such as a first enhancer operably linked to
the at least a first nucleic segment, or a transcription regulatory
region such as the woodchuck hepatitis virus post-transcriptional
regulatory element. The constructs of the invention may also
further optionally include one or more intron sequences operably
linked to the at least a first nucleic segment encoding the
therapeutic agent.
[0019] The nucleic acid segments encoding the mammalian guanylate
cyclase proteins, peptides, and polypeptides of the invention may
be derived from natural, semi-synthetic, or fully synthetic
sequences, but will preferably be of mammalian origin. Exemplary
mammalian sources include, without limitation, human, non-human
primates, murines, felines, canines, porcines, ovines, bovine,
equines, epine, caprine, lupines, and the like.
[0020] The rAAV vectors disclosed herein may optionally be
comprised within an infectious adeno-associated viral particle, a
virion, or within one or more of a plurality of infectious AAV
particles. As such, the invention also encompasses virions, viral
particles, as well as isolated recombinant host cells that contain
one or more of the disclosed rAAV genetic constructs. Particularly
preferred host cells for the practice of the invention include,
without limitation, isolated mammalian host cells that include one
or more of: an rAAV vector, an AAV virion, or a plurality of
infectious viral particles.
[0021] In other aspects, the invention provides novel and useful
compositions that include one or more of (a) an rAAV vector, an
rAAV virion, an rAAV infectious viral particle, a plurality of such
virions or infectious particles, or an isolated mammalian host cell
that comprises the vector, the virion, the infectious particle, or
a plurality thereof. Preferably, such compositions will further
optionally include one or more pharmaceutically-acceptable buffers,
carriers, vehicles, diluents, and such like, and may further
optionally include one or more lipids, liposomes, lipid complexes,
ethosomes, niosomes, nanoparticles, microparticles, lipospheres,
nanocapsules, or any combination thereof. Preferably such
compositions are preferably formulated for administration to the
human eye, and may be used in therapy or prophylaxis, and in the
therapy or prophylaxis of a human retinal dystrophy, disease, or
disorder (such as LCA1), in particular.
[0022] As noted below, the invention also includes diagnostic,
therapeutic, and prophylactic kits that include one or more of the
rAAV vector constructs disclosed herein. Such kits may further
optionally include one or more protocols, dosing regimens, or
instructions for using the component in the diagnosis, prevention,
treatment, or amelioration of one or more symptoms of a retinal
dystrophy, disease, disorder, or abnormal condition in a human. In
certain aspects, therapeutic kits for the treatment of human
patients diagnosed with Leber congenital amaurosis-1 (LCA-1) are
particularly contemplated.
[0023] The present invention also encompasses the use of one or
more of the disclosed rAAV-based compositions in therapy, or in
prophylaxis of mammalian diseases or disorders. Likewise, the
invention include use of the disclosed compositions in the
manufacture of a medicament for diagnosing, preventing, treating or
ameliorating one or more symptoms of a disease, disorder,
dysfunction, or abnormal condition of a mammalian eye, and in
particular, for treating or ameliorating one or more symptoms of
Leber congenital amaurosis-1 (LCA-1) in a human.
[0024] The invention also provides a method for preventing,
treating or ameliorating one or more symptoms of a disease,
dysfunction, disorder, deficiency, or abnormal condition in a
mammal. Such method generally involves administering to a mammal in
need thereof, an effective amount of an rAAV composition disclosed
herein for a time sufficient to prevent, treat and/or ameliorate
the one or more symptoms of the disease, dysfunction, disorder,
deficiency, or abnormal condition in the mammal. Such a mammal
preferably has, is suspected of having, is at risk for developing,
or has been diagnosed with at least a first retinal disorder,
disease, or dystrophy, including, for example, Leber congenital
amaurosis-1 (LCA-1), or wherein the mammal is at risk for
developing, or has been diagnosed with one or more deficiencies,
defects, or absence of biologically-active, functional guanylate
cyclase protein, peptide, or polypeptide. The mammal may be of any
age, but will more preferably be a neonate, newborn, infant, or
juvenile that is at risk for developing or has been diagnosed with
a congenital retinal dystrophy such as Leber congenital amaurosis-1
(LCA-1).
[0025] The invention also further includes a method for providing a
mammal with a therapeutically-effective amount of a
biologically-active mammalian guanylate cyclase peptide,
polypeptide, or protein to a mammal in need thereof. Such a method
generally involves at least the step of introducing into suitable
cells of a mammal in need thereof, an effective amount of one or
more of the rAAV vectors disclosed herein, for a time sufficient to
produce a biologically-active guanylate cyclase peptide,
polypeptide or protein therefrom in at least a first population of
cells or at least a first tissue of the mamma into which the rAAV
vector has been introduced. In the practice of the method, mammal
in need thereof will preferably have one or more defects,
deficiencies, or a substantial or total absence of functional,
biologically-active retGC1 protein in one or more tissues within or
about the body of the mammal, when compared to the level of
biologically-active retGC1 protein in a normal mammal. In certain
applications of the method, a plurality of cells from the mammal is
provided with the rAAV vector ex vivo or in vitro, with the method
further including an additional step of subsequently introducing
the plurality of provided cells into at least a first tissue site
within or about the body of the mammal. For example, the plurality
of obtained cells may be introduced into at least a first site
within one or both eyes of the mammal, including for example, by
direct injection into the retina, the sub-retinal space, or to one
or more tissues surrounding the retina, or to the entire eye, or to
tissues surrounding the eye.
[0026] In particular aspects, the introduction of the rAAV-vectored
guanylate cyclase gene construct into the cell, and its subsequent
expression permits translation of functional guanylate cyclase
peptide, protein, or polypeptide, and as a result, cone
photoreceptors are preserved, and cone-mediated function is
restored. Importantly, such method provides for a return of normal
visual behavior in the eye of the mammal, and preferably, a return
of vision.
[0027] Administration of the rAAV vectors of the invention may be
part of a one-time therapy, or may be part of an ongoing therapy
regimen repeated two or more times during the lifetime of the
subject being treated. In certain aspects, a single administration
of the rAAV constructs produces sustained guanylate cyclase protein
formation, with preservation of the cone photoreceptors, and
restoration of cone-mediated function and visual behavior over a
period of at least one month, at least two months, at least three
months, or longer following administration. More preferably,
long-term therapy or prophylaxis is achieved using one or more
subsequent administrations of the therapeutic constructs to the
mammalian eye for periods of several months to several years.
Preferably, cone photoreceptors are preserved, and cone-mediated
function and visual behavior are restored in the mammal for a
period of at least four months, at least five months, at least six
months, or more following administration. In certain aspects,
preservation of photoreceptors, cone-mediated function, and visual
behavior are restored in the mammal for a period of at least one
year, at least two years, at least three years, or at least four
years or longer following completion of a treatment regimen that
includes the compositions disclosed herein. The invention further
provides a method for increasing the level of biologically-active
retGC1 protein in one or more retinal cells of a mammal that has,
is suspected of having, is diagnosed with, or is at risk for
developing, LCA1. Such a method generally involves introducing into
at least a first population of retinal cells of a mammal in need
thereof, one or more of the disclosed rAAV-guanylate cyclase viral
vector constructs, in an amount and for a time effective to
increase the level of biologically-active retGC1 protein in one or
more retinal cells of the mammal. Such method is particularly
contemplated for preventing, treating, or ameliorating one or more
symptoms of retinal dystrophy in a mammal, and may preferably
involve directly or indirectly administering to the retina,
sub-retinal space, or the eye of the mammal one or more of the
disclosed therapeutic constructs, in an amount and for a time
sufficient to treat or ameliorate the one or more symptoms of
retinal dystrophy in the mammal.
[0028] The invention also provides compositions and methods for
preventing, treating or ameliorating the symptoms of a guanylate
cyclase protein deficiency in a mammal, and particularly for
treating or reducing the severity or extent of deficiency in a
human manifesting one or more of the disorders linked to a
deficiency of biologically-active guanylate cyclase polypeptides.
In a general sense, the method involves administration of at least
a first rAAV-based genetic construct that encodes one or more
guanylate cyclase peptides, polypeptides, or proteins in a
pharmaceutically-acceptable vehicle to the animal in an amount and
for a period of time sufficient to treat or ameliorate the
deficiency in the animal suspected of suffering from such a
disorder, or one or more symptoms thereof. Exemplary guanylate
cyclase polypeptides useful in the practice of the invention
include, but are not limited to peptides, polypeptides and proteins
that have guanylate cyclase activity, and that are substantially
identical in primary amino acid sequence to any one of the
sequences disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, or SEQ ID NO:11, and to biologically-functional
equivalents, or derivatives thereof. Additional exemplary guanylate
cyclase peptides, proteins, and polypeptides useful in the practice
of the include, but are not limited to those the comprise, consist
essentially of, or consist of, an amino acid sequence encoding a
mammalian guanylate cyclase, and particularly those sequences as
disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, or SEQ ID NO:11, and to biologically-functional
equivalents, or derivatives thereof.
[0029] rAAV-Guanylate Cyclase Vector Compositions
[0030] In a first embodiment, the invention provides an rAAV vector
comprising a polypeptide that comprises at least a first nucleic
acid segment that encodes a guanylate cyclase protein, peptide or
polypeptide, and in particular, a mammalian guanylate cyclase
protein, peptide, or polypeptide (or a biologically-active fragment
or derivative thereof), operably linked to at least a first
promoter capable of expressing the nucleic acid segment in a
suitable host cell transformed with such a vector. In preferred
embodiments, the nucleic acid segment encodes a mammalian, and in
particular, a human, guanylate cyclase peptide, polypeptide or
protein, and in particular, a peptide, polypeptide, or protein that
comprises at least a first contiguous amino acid sequence that is
at least 90% homologous to at least a first 30 contiguous amino
acid sequence from one or more of the amino acid sequences
disclosed in SEQ ID NO:1. SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, or SEQ ID NO:11, or a biologically-active fragment or
variant thereof.
[0031] Preferably, the polypeptide comprises at least a first
contiguous amino acid sequence that is at least 90%, at least 95%,
or at least 98% homologous to an at least 30, an at least 40, an at
least 50, an at least 60, an at least 70, or an at least 80
contiguous amino acid sequence from SEQ ID NO:1, and more
preferably, the polypeptide comprises at least a first contiguous
amino acid sequence that is at least 99% homologous to an at least
90 contiguous amino acid sequence from SEQ ID NO:1.
[0032] Alternatively, the therapeutic constructs of the invention
may encompass nucleic acid segments that encode guanylate cyclase
polypeptides of any mammalian origin, such as for example nucleic
acids, peptides, and polypeptides of murine, primate, ovine,
porcine, bovine, equine, epine, caprine, canine, feline, and/or
lupine origin, or may encompass modified or site-specifically
mutagenized nucleic acid segments that were initially obtained from
one or more mammalian species, and genetically modified to be
expressed in human cells such that their guanylate cyclase activity
is retained.
[0033] In other preferred embodiments, the preferred nucleic acid
segments for use in the practice of the present invention, encodes
a mammalian, and in particular, a human guanylate cyclase
polypeptide or a biologically active fragment or variant
thereof.
[0034] The polynucleotides comprised in the vectors and viral
particles of the present invention preferably comprise at least a
first constitutive or inducible promoter operably linked to a
guanylate cyclase-encoding nucleic acid segment as described
herein. Such promoters may be homologous or heterologous promoters,
and may be operatively positioned upstream of the nucleic acid
segment encoding the guanylate cyclase polypeptide, such that the
expression of the guanylate cyclase-encoding segment is under the
control of the promoter. The construct may comprise a single
promoter, or alternatively, two or more promoters may be used to
facilitate expression of the guanylate cyclase-encoding DNA
sequence.
[0035] Exemplary promoters useful in the practice of the invention
include, but are in no way limited to, those promoter sequences
that are operable in mammalian, and in particular, human host
cells, tissues, and organs, such as for example, ubiquitous
promoters, such as a CMV promoter, promoter, a .beta.-actin
promoter, a hybrid CMV promoter, a hybrid CMV-.beta.-actin
promoter, a truncated CMV promoter, a truncated .beta.-actin
promoter, a truncated hybrid CMV-.beta.-actin promoter, an EF1
promoter, a U1a promoter, or a U1b promoter, or one or more cell-
or tissue-specific promoters (including, for example, a
photoreceptor-specific promoter such as a rhodopsin kinase promoter
[hGRK1]), or an inducible promoter such as a Tet-inducible promoter
or a VP16-LexA promoter.
[0036] In illustrative embodiments, a polynucleotide encoding a
therapeutic polypeptide was placed under the control of a
ubiquitous truncated hybrid chicken .beta.-actin (CBA) promoter, or
under the control of a photoreceptor cell-specific hGRK1) promoter,
and used to produce therapeutically-effective levels of the encoded
guanylate cyclase polypeptide when suitable host cells were
transformed with the genetic construct, and the DNA encoding the
guanylate cyclase polypeptide was expressed in such cells. An
example of a suitable hGRK1 promoter is shown in SEQ ID NO:12,
while a suitable ubiquitous promoter, such as the truncated hybrid
chicken .beta.-actin (CBA) promoter is shown in SEQ ID NO:13.
[0037] The polynucleotides comprised in the vectors and viral
particles of the present invention may also further optionally
comprise one or more native, synthetic, homologous, heterologous,
or hybrid enhancer or 5' regulatory elements, for example, a
natural enhancer, such as the CMV enhancer, or alternatively, a
synthetic enhancer. Cell- or tissue-specific enhancers, including
for example, those that increase expression of operably linked gene
sequences are also contemplated to be particularly useful in the
practice of the invention. Such enhancers may include, but are not
limited to, retinal-specific enhancers, rod-specific enhancers,
cone-specific enhancers, and such like.
[0038] The polynucleotides and nucleic acid segments comprised
within the vectors and viral particles of the present invention may
also further optionally comprise one or more intron sequences. In
such instances, the intron sequence(s) will preferably be mammalian
in origin, and more preferably, human in origin.
[0039] The DNA sequences, nucleic acid segments, and
polynucleotides comprised within a vector, virion, viral particle,
host cell, or composition of the present invention may also further
optionally comprise one or more native, synthetic, homologous,
heterologous, or hybrid post-transcriptional or 3' regulatory
elements operably positioned relative to the guanylate
cyclase-encoding nucleic acid segments disclosed herein to provide
greater expression, greater stability, and/or enhanced translation
of the encoded polypeptides. One such example is the woodchuck
hepatitis virus post-transcriptional regulatory element (WPRE),
operably positioned downstream of the guanylate cyclase gene. Use
of elements such as these in such circumstances is well-known to
those of skill in the molecular biological arts.
[0040] In illustrative embodiments, the invention concerns
administration of one or more biologically-active guanylate cyclase
proteins, peptides, or polypeptides that comprise an at least about
10, at least about 15, at least about 20, at least about 25, at
least about 30, at least about 35, at least about 40, at least
about 45, at least about 50, at least about 55, at least about 60,
at least about 65, at least about 70, at least about 75, at least
about 80, at least about 85, at least about 90, at least about 95,
or at about least 100, or more contiguous amino acid sequence from
the polypeptide and peptide sequences disclosed hereinbelow, and
particularly those polypeptides as recited in any one of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ
ID NO:11.
[0041] Likewise, in additional illustrative embodiments, the
invention concerns administration of one or more
biologically-active guanylate cyclase proteins, peptides or
polypeptides that are encoded by a nucleic acid segment that
comprises, consists essentially of, or consists of at least about
10, at least about 20, at least about 30, at least about 40, at
least about 50, at least about 60, at least about 70, at least
about 80, at least about 90, at least about 100, at least about
110, at least about 120, at least about 130, at least about 140, at
least about 150, at least about 160, at least about 170, at least
about 180, at least about 190, or at least about 200, about 250,
about 300, about 350, about 400, about 450, about 500, about 550,
about 600, about 650, about 700, about 750, or even about 800 or
more contiguous nucleic acid residues from the nucleic acid
segments disclosed hereinbelow, and particularly those DNA
sequences that encode any one or more mammalian guanylate cyclase
proteins, including for example, those that are recited in SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ
ID NO:11.
[0042] Exemplary adeno-associated viral vector constructs and
polynucleotides of the present invention include those that
comprise, consist essentially of, or consist of at least a first
nucleic acid segment that encodes a peptide or polypeptide that is
at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or at least about 99% identical to
the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, or SEQ ID NO:11, wherein the peptide or polypeptide
has guanylate cyclase activity when expressed in selected mammalian
cells and/or tissues.
[0043] In certain embodiments, the viral vector constructs and
polynucleotides of the present invention will preferably include
those vectors and polynucleotides that comprise, consist
essentially of, or consist of at least a first nucleic acid segment
that encodes a peptide or polypeptide that is at least about 82%,
at least about 84%, at least about 86%, at least about 88%, at
least about 92%, or at least about 94% identical to one or more of
the sequences disclosed in SEQ ID NO:1, SEQ ID NO:2. SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. Such constructs will
preferably encode one or more biologically-active peptides or
polypeptides that have guanylate cyclase activity when expressed in
selected mammalian cells and/or tissues and in human cells and/or
tissues in particular.
[0044] Exemplary polynucleotides of the present invention also
include those sequences that comprise, consist essentially of, or
consist of at least a first nucleic acid segment that is at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, or at least about 99% identical to a nucleic acid
sequence that encodes any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, wherein the
peptide or polypeptide encoded by the nucleic acid segment has
guanylate cyclase activity when expressed in selected mammalian
cells and/or tissues.
[0045] rAAV Viral Particles and Virions, and Host Cells Comprising
them
[0046] Other aspects of the invention concern rAAV particles and
virions that comprise the rAAV-guanylate cyclase vectors of the
present invention, pluralities of such particles and virions, as
well as pharmaceutical compositions and host cells that comprise
one or more of the rAAV-guanylate cyclase vectors disclosed herein,
such as for example pharmaceutical formulations of the
rAAV-guanylate cyclase vectors or virions intended for
administration to a mammal through suitable means, such as, by
intramuscular, intravenous, or direct injection to selected cells,
tissues, or organs of the mammal, for example, one or more regions
of the eye of the selected mammal. Typically, such compositions
will be formulated with pharmaceutically-acceptable excipients,
buffers, diluents, adjuvants, or carriers, as described
hereinbelow, and may further comprise one or more liposomes,
lipids, lipid complexes, microspheres, microparticles, nanospheres,
or nanoparticle formulations to facilitate administration to the
selected organs, tissues, and cells for which therapy is
desired.
[0047] Further aspects of the invention include mammalian host
cells, and pluralities thereof that comprise one or more of the
rAAV vectors, virions, or infectious viral particles as disclosed
herein. Particularly preferred cells are human host cells, and in
particular, human ocular tissues, including, for example, retinal
cells.
[0048] Therapeutic Kits and Pharmaceutical Compositions
[0049] Therapeutic kits for treating or ameliorating the symptoms
of a condition resulting from a guanylate cyclase deficiency in a
mammal are also part of the present invention. Exemplary kits are
those that preferably comprise one or more of the disclosed
AAV-guanylate cyclase vector constructs, virions, or pharmaceutical
compositions described herein, and instructions for using the kit.
The use of such kits in methods of treatment of guanylate cyclase
deficiency, and in particular, retinal-specific guanylate
cyclase-1, is preferable in the treatment of retGC1 defect or
deficiency and in the treatment of retinal dystrophies such as
LCA-1 in an affected mammal.
[0050] Another important aspect of the present invention concerns
use of the disclosed vectors, virions, compositions, and host cells
described herein in the preparation of medicaments for treating or
ameliorating the symptoms of guanylate cyclase deficiency in a
mammal, and in particular, a human. The use of such compositions in
the preparation of medicaments and in methods for the treatment of
neurological and/or central nervous system defects, including for
example, conditions resulting from a deficiency or defect in
retinal GC1, such as for example in retinal dystrophies such as
LCA-1, generally involve administration to a mammal, and
particularly to a human in need thereof, one or more of the
disclosed viral vectors, virions, host cells, or compositions
comprising one or more of them, in an amount and for a time
sufficient to treat or ameliorate the symptoms of such a deficiency
in the affected mammal. The methods may also encompass prophylactic
treatment of animals suspected of having such conditions, or
administration of such compositions to those animals at risk for
developing such conditions either following diagnosis, or prior to
the onset of symptoms.
[0051] Another aspect of the invention concerns compositions that
comprise one or more of the disclosed adeno-associated viral
vectors, virions, viral particles, and host cells as described
herein. Pharmaceutical compositions comprising such are
particularly contemplated to be useful in therapy, and particularly
in the preparation of medicaments for treating affected mammals,
and humans in particular.
[0052] Therapeutic Methods
[0053] The invention also provides methods for delivering
therapeutically-effective amounts of a guanylate cyclase
polypeptide to a mammal in need thereof. Such methods generally
comprise at least the step of providing or administering to such a
mammal, one or more of the guanylate cyclase compositions disclosed
herein. For example, the method may involve providing to such a
mammal, one or more of the rAAV vectors, virions, viral particles,
host cells, or pharmaceutical compositions as described herein.
Preferably such providing or such administration will be in an
amount and for a time effective to provide a
therapeutically-effective amount of one or more of the guanylate
cyclase polypeptides disclosed herein to selected cells, tissues,
or organs of the mammal, and in particular,
therapeutically-effective levels to the cells of the mammalian eye.
Such methods may include systemic injection(s) of the
therapeuticum, or may even involve direct or indirect
administration, injection, or introduction of the therapeutic
compositions to particular cells, tissues, or organs of the
mammal.
[0054] For example, the therapeutic composition may be provided to
mammal by direct injection to the tissues of the eye or to the
retina, or to the subretinal space, or to one or more particular
cell types within the mammalian eye.
[0055] The invention also provides methods of treating,
ameliorating the symptoms, and reducing the severity of guanylate
cyclase deficiency in an animal. These methods generally involve at
least the step of providing to an animal in need thereof, one or
more of the rAAV guanylate cyclase vector compositions disclosed
herein in an amount and for a time effective to treat retGC1
polypeptide defect or deficiency, or to treat a dysfunction
resulting from such accumulation, or resulting from an
underexpression or absence of sufficient biologically-active
guanylate cyclase polypeptide in the animal, including retinal
dystrophies such as LCA1 and the like. As described above, such
methods may involve systemic injection(s) of the therapeuticum, or
may even involve direct or indirect administration, injection, or
introduction of the therapeutic compositions to particular cells,
tissues, or organs of the animal.
[0056] The invention further concerns the use of the
adeno-associated viral vectors, virions, viral particles, host
cells, and/or the pharmaceutical compositions disclosed herein in
the manufacture of a medicament for treating guanylate cyclase
defect or deficiency, retinal dystrophy, or LCA1 or other
GC1-related ocular disease, disorder, or dysfunction in a mammal.
This use may involve systemic or localized injection, infection, or
administration to one or more cells, tissues, or organs of the
mammal. Such use is particularly contemplated in humans that have,
are suspected of having, or at risk for developing one or more
retinal dystrophies such as LCA-1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to the following description taken in
conjunction with the accompanying drawings, in which like reference
numerals identify like elements, and in which:
[0058] FIG. 1A and FIG. 1B shows representative cone--(left column)
and rod--(right column) mediated ERG traces from +/+ (upper
waveforms), untreated GC1KO (middle waveforms) and AAV-mGC1-treated
(bottom waveforms) mice. Black traces correspond to eyes injected
with hGRK1-mGC1 (bottom waveforms) and their un-injected
contralateral eyes (middle waveforms). Red traces correspond to
eyes injected with smCBA-mGC1 (bottom waveforms) and un-injected
contralateral eyes (middle waveforms). Cone responses in AAV-mGC1
treated eyes are restored to approximately 45% of normal;
[0059] FIG. 2A and FIG. 2B show average photopic b-wave maximum
amplitudes in GC1KO, isogenic +/+ controls, smCBA-mGC1-treated
(FIG. 2A) and hGRK1-mGC1-treated (FIG. 2B) GC1KO mice over time.
Cone responses of both smCBA-mGC1 and hGRK1-mGC1-treated mice are
approximately 45% of normal for at least 3 months post
injection;
[0060] FIG. 3A, FIG. 3B, and FIG. 3C illustrate by optomotor
analysis that visually-elicited behavior was restored in GC1KO mice
treated with either smCBA-mGC1 or hGRK1-mGC1. M1 to M9 correspond
to the nine mice used for testing. Photopic acuities and contrast
sensitivities of +/+ control mice (M1, M2), naive GC1KO (M3, M4),
smCBA-mGC1 (M5, M6, M7) and hGRK1-mGC1-treated (M8, M9) mice reveal
that treated mice behave like normal-sighted mice (FIG. 3B and FIG.
3C). Averages of all +/+ eyes (n=4), GC1KO eyes (n=9) and
AAV-mGC1-treated eyes (n=5) are shown (FIG. 3C). Cone-mediated ERG
responses from each mouse (M1-M9) are shown for
electrophysiological comparison (FIG. 3A):
[0061] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F
show AAV5-hGRK1-mGC1 drives expression of GC1 in photoreceptor
outer segments of GC1KO mice (FIG. 4A). No GC1 expression is seen
in untreated contralateral control eye (FIG. 4B). AAV5-smCBA-mGC1
drives expression of GC1 in photoreceptor outer segments (FIG. 4C)
and occasionally in photoreceptor cell bodes (white arrows in FIG.
4F). No such GC1 expression is seen in the untreated contralateral
control eye (FIG. 4D). Levels of therapeutic transgene expression
in AAV5-mGC1-treated eyes are similar to that seen in isogenic +/+
control eyes (FIG. 4E). All retinas were taken from mice 3 months'
post treatment or age matched untreated controls. Scale bars in
FIG. 4A=100 .mu.m; in FIG. 4F=25 .mu.m. OS=outer segments, IS=inner
segments, ONL=outer nuclear layer;
[0062] FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D show cone arrestin
expression in cone photoreceptors of +/+, GC1KO,
AAV5-smCBA-mGC1-treated and AAV5-hGRK1-mGC1-treated mice. Untreated
GC1KO retinas contain characteristic disorganized, detached cone
outer segments (FIG. 5B), whereas cone outer segments were intact
and cone arrestin distribution appeared normal in treated GC1KO
(FIG. 5C and FIG. 5D) and +/+(FIG. 5A) retinal sections. All
retinas were taken from mice 3 months post treatment or age matched
untreated controls. Scale bars in FIG. 5D=100 .mu.m. OS=outer
segments, IS=inner segments, S=synaptic terminals;
[0063] FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D show that AAV-mGC1
treatment results in preservation of cone photoreceptors in treated
eyes for at least three months post treatment. Representative
retinal whole mounts from the hGRK1-mGC1 study (FIG. 6A: "no
TX"=untreated; FIG. 6C: "TX"=treated), and the smCBA-mGC1 study
(FIG. 6B: "no TX=untreated; FIG. 6D: "TX"=treated) and
contralateral un-injected eyes stained for cone arrestin reveal
that cone photoreceptors are preserved in GC1KO mice treated with
AAV-mGC1 for at least 3 months post treatment. Cone cell densities
were counted in central and inferior retinas of treated and
untreated mice. Significant differences were found in both areas
following treatment with either viral vector;
[0064] FIG. 7 illustrates the vertebrate phototransduction cascade.
Upon light stimulation, conformational changes in rhodopsin (R)
stimulate a cascade of events including activation of transducin
(T) and cGMP phosphodiesterase (PDE) eventually resulting in the
hydrolysis of cGMP. This lowering of intracellular cGMP causes a
closure of the cyclic nucleotide-gated channels (CNG) in
photoreceptor outer segment membranes. Closure of these channels
causes hyperpolarization of the cell and therefore a dramatic drop
in intracellular calcium. When calcium levels fall, unbound
guanylate cyclase activating protein (GCAP) is free to stimulate
guanylate cyclase (GC). GC plays a role in the recovery phase of
phototransduction in that its purpose is to produce cGMP. When
levels of cGMP are sufficiently increased by GC, cGMP-gated
channels re-open causing depolarization of the cell and a return to
the dark-adapted state;
[0065] FIG. 8 shows the predicted structure and topology of retGC1
shows homology to other guanylate cyclases with a single
transmembrane spanning region, an intracellular and extracellular
domain. The intracellular domain is further divided in a
"kinase-like" region and catalytic domain. The calcium and
GCAP1-dependent regulation of retGC1 is regulated through the
intracellular domains (KHD). When calcium concentration in the
photoreceptor cell is high (in the dark/depolarized state),
calcium-bound GCAP1 prevents activation of retGC1. Upon light
stimulation, calcium levels decrease. Calcium is unbound from
GCAP1, thereby allowing GCAP1 to activate retGC1. The role of
retGC1 is to produce cGMP;
[0066] FIG. 9A and FIG. 9B show cone photoreceptors in normal (WT)
vs. GC1KO mice. In WT cones, GC1 functions normally to produce cGMP
which can effectively reopen CNG gated channels and return the cell
to its dark-adapted/depolarized state. In cone photoreceptors of
the GC1KO mouse, GC1 fails to produce cGMP. This failure prevents
reopening of CNG-gated channels. These cells are in essence,
chronically hyperpolarized (light-adapted). They do not transduce
light for vision (as evidenced by a lack of ERG) and will
eventually degenerate;
[0067] FIGS. 10A-10B shows an amino acid sequence alignment of the
bovine GC1 (bov GC1) and mouse GC1 (mGC1) with consensus sequence
included. Variable region located in the N-terminal area is
highlighted by the red rectangle;
[0068] FIG. 11A and FIG. 11B show maps of the two illustrative
vectors. One contains the ubiquitous promoter smCBA, while the
other utilizes the photoreceptor-specific promoter, hGRK1;
[0069] FIG. 12 shows representative retinal section from a GC1KO
eye injected with AAV5-smCBA-mGC1 stained for GC1 (red) and PNA
lectin (green) reveals GC1 expression in cone outer segments
(yellow overlay) as well as in rod outer segments (red alone).
hGRK1-mGC1 injected eyes revealed the same pattern;
[0070] FIG. 13A, FIG. 13B, and FIG. 13C show AAV-mediated
restoration of retinal function in GC1KO mice. FIG. 13A:
Representative photopic (cone-mediated) traces recorded from eyes
of GC1KO mice treated at .about.P14 with AAV5-hGRK1-mGC1 (red),
AAV5-smCBA-mGC1 (green) or AAV8(Y733F)-hGRK1-mGC1 or age-matched,
isogenic GC1 +/+ controls. Traces were generated at 4 months
(left), 7 months (middle) and 9 months (right) post-injection. FIG.
13B: Average cone b-wave amplitudes generated monthly with a 12
cds/m2 stimulus in treated GC1KO mice, untreated GC1KO and
age-matched isogenic GC1 +/+ control mice. FIG. 13C: Scotopic
(rod-mediated) responses in treated vs. untreated GC1KO mice over
time. Values represent the ratio of rod b-wave amplitudes generated
at 5 cds/m2 in treated vs. untreated eyes. All three vectors confer
stable, long-term therapy to GC1KO mice, with
AAV8(Y733F)-hGRK1-mGC1 being the most efficient:
[0071] FIG. 14 shows GC1KO mice treated with AAV8(Y733F)-hGRK1-mGC1
were sacrificed at 7 months post injection. AAV5-smCBA-mGC1 and
AAV5-hGRK1-mGC1-treated mice were sacrificed at 9 months
post-injection. These eyes as well as that of an .about.11 month
old GC1 +/+ mice were sectioned and retinas stained with antibodies
raised against GC1 (green, top row) and cone arrestin (red, bottom
row). All three therapeutic vectors drove GC1 expression
exclusively in photoreceptors of GC1KO mice. Some retinal thinning
was observed in AAV5-hGRK1-mGC1 treated mice, a result likely due
to the high titer of this vector. GC1 expression and cone
density/morphology in AAV8(Y733F)- and AAV5-smCBA-treated mice
resembled that seen in age-matched GC1 +/+ controls. On the
contrary, retinas of an age-matched GC1KO mouse revealed an absence
of GC1 expression and a marked reduction in cone cell density;
[0072] FIG. 15 shows at 7.5 months post-injection with
AAV8(Y733F)-hGRK1-mGC1, one GC1KO mouse was sacrificed and its
retinas used for western blot. Antibodies directed against GC1 show
that the level of AAV-mediated GC1 expression in the treated GC1KO
eye are similar to that seen in the age-matched, isogenic GC1 +/+
control eye. Levels of guanylate cyclase activating protein-1
(GCAP1) expression (a biochemical partner of guanylate cyclase) was
also evaluated in treated and untreated GC1KO as well as GC1 +/+
control eyes. Consistent with previous reports, GCAP1 protein was
downregulated in the untreated GC1KO eyes. AAV-mediated GC1
expression results in increased GCAP1 expression, similar to levels
seen in the isogenic GC1 +/+ control;
[0073] FIG. 16A and FIG. 16B show results at 11 months post
injection with AAV5-smCBA-mGC1, one GC1KO mouse was sacrificed, its
retinas whole-mounted and stained with an antibody raised against
cone arrestin. The immunostain revealed that cones are absent in
the untreated GC1KO eye (FIG. 16A) except in the superior retina.
AAV-mediated GC1 expression preserves cone photoreceptors
throughout the retina of the treated eye (FIG. 16B) for at least 11
months (the latest time point studied);
[0074] FIG. 17 illustrates data in which GC1KO mice injected with
AAV8(733)-hGRK1-mGC1 were sacrificed at 4 months and 7 months post
injection. GC1KO mice injected with AAV5-smCBA-mGC1 and
AAV5-hGRK1-mGC1 were sacrificed at 7 months and 10 months post
injection. Age matched, naive GC1KO mice were used as controls.
Optic nerves from treated and untreated eyes, and portions of the
right and left brains containing visual pathways were isolated and
used for recovery of vector genomes. Note that
AAV8(Y733F)-hGRK1-mGC1 was injected into the LEFT eyes of GC1KO
mice whereas both AAV5 vectors were injected into RIGHT eyes of
GC1KO mice. Vector genomes were recovered only from the optic
nerves of treated eyes in all cases. By 10 months post-injection of
AAV5 vectors, no vector genomes were recovered from brain. The
highest number of vector genomes were recovered from GC1KO mice
injected with the strong, fast-acting AAV8(733) vector;
[0075] FIGS. 18A-18D illustrate data in which OCT and rod/cone ERGs
from a GCdko mouse TWO months post injection with
AAV8(Y733F)-hGRK1-mGC1;
[0076] FIG. 19A and FIG. 19B illustrate data in which
representative rod and cone ERGs from a GCdko mouse one month post
injection with AAV8(Y733F)-hGRK1-mGC1;
[0077] FIG. 20A and FIG. 20B shows real time RT-PCR standards.
Fluorescence (log units in Y-axis) is plotted against cycle
threshold C.tau.values (X-axis). Each panel represents the standard
curves (generated by a dilution series of total retina cDNA) for
GC1 and Gapdh transcript using retinal cDNA from either a wild
type, GC1 +/+ mouse (a) or a GC1KO mouse treated with
AAV8(Y733F)-hGRK1-mGC1. The standard curves generated by GC1 and
Gapdh primer sets were parallel using either template indicating
similar amplification kinetics. Cc cycle value increases with
decreasing amount of template;
[0078] FIG. 21A and FIG. 21B show GC1 and cone arrestin expression
in retinas of treated and untreated GC1KO mice and GC1 +/+
controls. FIG. 21A: Immunohistochemistry of frozen retinal
cross-sections was used to localize expression of GC1 (green, top
row) and cone arrestin (red, bottom row) in GC1KO mice treated with
AAV8(Y733F)-hGRK1-mGC1 (7 months post-injection), AAV5-smCBA-mGC1
(10 months post-injection) or AAV5-hGRK1-mGC1 (10 months
post-injection) vectors as well as retinas from 8 month old
untreated GC1KO and GC1 +/+ control mice. Nuclei were stained with
DAPI (blue). All sections were imaged at 20.times. magnification
and exposed at identical settings. FIG. 21B: Immunostaining of
retinal whole mounts from one GC1KO mouse 11 months post-treatment
with AAV5-smCBA-mGC1 (one eye only) with an antibody against cone
arrestin revealed marked preservation of cone photoreceptors in the
treated eye (bottom Tight) compared to the untreated contralateral
control eye (bottom left). Retinal whole mounts were oriented
similarly, with their temporal portions in the 12 o'clock position.
Portions of whole mounts were imaged at 10.times. magnification and
merged together for final presentation. OS=outer segments;
ONL=outer nuclear layer, INL=inner nuclear layer,
[0079] FIG. 22A and FIG. 22B show cone-mediated electroretinograms
(ERGs) of treated and untreated GC1KO and untreated GC1 +/+ control
eyes. FIG. 22A: Representative cone-mediated traces elicited by a
12 cds/m.sup.2 light stimulus from GC1KO eyes treated with
AAV5-hGRK1-mGC1 (red line), AAV5-smCBA-mGC1 (green line) or
AAV8(Y733F)-hGRK1-mGC1 (black line) or untreated age-matched GC1
+/+ control eyes. Representative traces generated between 4-months'
and 1-years' post-treatment are shown (top panel). Scale: y-axis=50
.mu.V, x-axis=20 ms. FIG. 22B: Maximum cone b-wave amplitudes
(those generated at 12 cds/m.sup.2) were calculated from each mouse
and averaged monthly in each treatment group as well as
age-matched, untreated GC1KO and GC1 +/+ controls. Comparisons were
made between groups of animals with an n>3. All AAV treatment
groups were statistically compared for 6-months' post-treatment.
AAV5 vector treated eyes were statistically compared for 9-months'
post-treatment;
[0080] FIGS. 23A-23C illustrate rod-mediated electroretinograms
(ERGs) of treated and untreated GC1KO and GC1 +/+ control eyes.
FIG. 23A: Rod b-wave amplitudes (top left) and a-wave amplitudes
(top right) elicited by a 1 cds/m.sup.2 stimulus under scotopic
conditions were determined in the treated and untreated eyes of
GC1KO mice treated with AAV8(Y733F)-hGRK1-mGC1 (black circles),
AAV5-hGRK1-mGC1 (red circles) or AAV5-smCBA-mGC1 (green triangles)
vector. Intra-mouse ratios of treated and untreated eyes were
generated by dividing the maximum a- or b-wave amplitude in treated
eyes by the maximum 27 amplitude in the untreated eye. These ratios
were averaged monthly in all treatment groups. Comparisons were
made between groups of animals with ann>3. All AAV treatment
groups were statistically compared for 6 months. AAV5 vectors were
also statistically compared for 9 months. Vector-mediated
improvement was defined by an average ratio>0.8. FIGS. 23B-23C:
Representative rod-mediated ERG traces from one GC1KO mouse reveal
that rod responses from the AAV8(Y733F)-hGRK1-mGC1-treated eye
(black line) were higher than those recorded from the untreated
contralateral control eye (green line). This treated rod response
was restored to .about.50% that of the normal GC1 +/+ rod response
(red line); and
[0081] FIG. 24A and FIG. 24B show protein and transcript levels in
treated and untreated GC1KO mice and GC1 +/+ controls. FIG. 24A:
Immunoblot of retinal lysates from one GC1KO mouse eye at 10 months
after treatment with AAV8(Y733F)-hGRK1-mGC1 and probed with
anti-GC1 and anti-GCAP1 antibodies. Anti-.beta.-actin antibody was
used as an internal loading control. FIG. 24B: Semiquantitative
real time RT-PCR of several transcripts (GC1, GCAP1, GNAT2, and
PDE6.alpha..quadrature. .quadrature.in one GC1KO retina treated
with AAV5-smCBA-mGC1, one GC1KO retina treated with
AAV8(Y733F)-hGRK1-mGC1 vector, and in individual untreated GC1KO or
GC1 +/+ control retinas. Samples were performed in triplicate using
Gapdh-specific primers as a standard. Data is presented as the
fold-change in mRNA levels relative to the GC1 +/+ control.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0082] Illustrative embodiments of the invention are described
below. In the interest of clarity, not all features of an actual
implementation are described in this specification. It will of
course be appreciated that in the development of any such actual
embodiment, numerous implementation-specific decisions must be made
to achieve the developers' specific goals, such as compliance with
system-related and business-related constraints, which will vary
from one implementation to another. Moreover, it will be
appreciated that such a development effort might be complex and
time-consuming, but would nevertheless be a routine undertaking for
those of ordinary skill in the art having the benefit of this
disclosure.
[0083] Adeno-Associated Virus
[0084] Adeno-associated virus-2 (AAV) is a human parvovirus that
can be propagated both as a lytic virus and as a provirus (Cukor et
al., 1984; Hoggan et al., 1972). The viral genome consists of
linear single-stranded DNA (Rose et al., 1969), 4679 bases long
(Srivastava et al., 1983), flanked by inverted terminal repeats of
145 bases (Lusby et al., 1982). For lytic growth AAV requires
co-infection with a helper virus. Either adenovirus (Atchinson et
al., 1965; Hoggan, 1965; Parks et al., 1967) or herpes simplex
(Buller et al., 1981) can supply helper function. Without helper,
there is no evidence of AAV-specific replication or gene expression
(Rose and Koczot, 1972; Carter et al., 1983). When no helper is
available, AAV can persist as an integrated provirus (Hoggan, 1965;
Berns et al., 1975; Handa et al., 1977; Cheung et al., 1980; Berns
et al., 1982).
[0085] Integration apparently involves recombination between AAV
termini and host sequences and most of the AAV sequences remain
intact in the provirus. The ability of AAV to integrate into host
DNA is apparently an inherent strategy for insuring the survival of
AAV sequences in the absence of the helper virus. When cells
carrying an AAV provirus are subsequently superinfected with a
helper, the integrated AAV genome is rescued and a productive lytic
cycle occurs (Hoggan, 1965).
[0086] AAV sequences cloned into prokaryotic plasmids are
infectious (Samulski et al., 1982). For example, when the wild type
AAV/pBR322 plasmid, pSM620, is transfected into human cells in the
presence of adenovirus, the AAV sequences are rescued from the
plasmid and a normal AAV lytic cycle ensues (Samulski et al.,
1982). This renders it possible to modify the AAV sequences in the
recombinant plasmid and, then, to grow a viral stock of the mutant
by transfecting the plasmid into human cells (Samulski et al.,
1983; Hermonat et al., 1984). AAV contains at least three
phenotypically distinct regions (Hermonat et al., 1984). The rep
region codes for one or more proteins that are required for DNA
replication and for rescue from the recombinant plasmid, while the
cap and lip regions appear to code for AAV capsid proteins and
mutants within these regions are capable of DNA replication
(Hermonat et al., 1984). It has been shown that the AAV termini are
required for DNA replication (Samulski et al., 1983).
[0087] Laughlin et al. (1983) have described the construction of
two E. coli hybrid plasmids, each of which contains the entire DNA
genome of AAV, and the transfection of the recombinant DNAs into
human cell lines in the presence of helper adenovirus to
successfully rescue and replicate the AAV genome (See also
Tratschin et al., 1984a; 1984b).
[0088] Adeno-associated virus (AAV) is particularly attractive for
gene transfer because it does not induce any pathogenic response
and can integrate into the host cellular chromosome (Kotin et al.,
1990). The AAV terminal repeats (TRs) are the only essential
cis-components for the chromosomal integration (Muzyczka and
McLaughin, 1988). These TRs are reported to have promoter activity
(Flotte et al., 1993). They may promote efficient gene transfer
from the cytoplasm to the nucleus or increase the stability of
plasmid DNA and enable longer-lasting gene expression (Bartlett and
Samulski, 1998). Studies using recombinant plasmid DNAs containing
AAV TRs have attracted considerable interest. AAV-based plasmids
have been shown to drive higher and longer transgene expression
than the identical plasmids lacking the TRs of AAV in most cell
types (Philip et al., 1994; Shafron et al., 1998; Wang et al.,
1998).
[0089] There are several factors that prompted researchers to study
the possibility of using rAAV as an expression vector. One is that
the requirements for delivering a gene to integrate into the host
chromosome are surprisingly few. It is necessary to have the 145-bp
ITRs, which are only 6% of the AAV genome. This leaves room in the
vector to assemble a 4.5-kb DNA insertion. While this carrying
capacity may prevent the AAV from delivering large genes, it is
amply suited for delivering the antisense constructs of the present
invention.
[0090] AAV is also a good choice of delivery vehicles due to its
safety. There is a relatively complicated rescue mechanism: not
only wild type adenovirus but also AAV genes are required to
mobilize rAAV. Likewise, AAV is not pathogenic and not associated
with any disease. The removal of viral coding sequences minimizes
immune reactions to viral gene expression, and therefore, rAAV does
not evoke an inflammatory response. AAV therefore, represents an
ideal candidate for delivery of the guanylate cyclase-encoding
polynucleotides of the present invention.
[0091] Production of rAAV Vectors
[0092] Traditional protocols to produce rAAV vectors have generally
been based on a three-component system. One component of this
system is a proviral plasmid encoding the recombinant DNA to be
packaged as rAAV. This recombinant DNA is located between 145 base
pair (bp) AAV-2 inverted terminal repeats (ITRs) that are the
minimal cis acting AAV-2 sequences that direct replication and
packaging of the vector. A second component of the system is a
plasmid encoding the AAV-2 genes, rep and cap. The AAV-2 rep gene
encodes four Rep proteins (Rep 78, 68, 52 and 40) that act in trans
to replicate the rAAV genome, resolve replicative intermediates,
and then package single-stranded rAAV genomes. The AAV-2 cap gene
encodes the three structural proteins (VP1, VP2, and VP3) that
comprise the virus capsid. Because AAV-2 does not proficiently
replicate on its own, the third component of a rAAV packaging
system is a set of helper functions from another DNA virus. These
helper functions create a cellular environment in which rAAV
replication and packaging can efficiently occur. The helper
functions provided by adenovirus (Ad) have almost exclusively been
used to produce rAAV and are encoded by the genes E1a, E1b, E2a,
E4orf6, and VA RNA. While the first two components of the system
are generally introduced into cells in which replication and
packaging is to occur by transfection, ad helper functions are
introduced by superinfection with wild type Ad virus.
[0093] The traditional rAAV production techniques are limited in
their ability to produce large quantities of vector because of
inherent inefficiencies in transfection. Serious difficulties are
also encountered when the scale of transfection is increased. The
requirement for wild type Ad may also reduce the amount of rAAV
produced since Ad may compete for cellular and viral substrates
that are required for viral replication but are present only in
limiting amounts. Another problem encountered in traditional
production protocols is that superinfection with Ad requires
development of effective procedures for purification of Ad from the
rAAV produced. While these purification processes are generally
successful at eliminating Ad contamination of rAAV preparations,
they also reduce rAAV titers. Stringent assays for Ad contamination
of rAAV are also necessary.
[0094] To produce rAAV, a double co-transfection procedure is used
to introduce a rAAV transfer vector plasmid together with pDG
(Grimm et al., 1998) AAV helper plasmid carrying the AAV rep and
cap genes, as well as Ad helper genes required for rAAV replication
and packaging at a 1:1 molar ratio. Plasmid DNA used in the
transfection is purified by a conventional alkaline lysis/CsCl
gradient protocol. The transfection is carried out as follows: 293
cells are split 1:2 the day prior to the experiment, so that, when
transfected, the cell confluence is about 75-80%. Ten 15-cm plates
are transfected as one batch. To make CaPO4 precipitate 0.7 mg of
pDG are mixed with 180 .mu.g of rAAV transfer vector plasmid in a
total volume of 12.5 mL of 0.25 M CaCl2). The old media is removed
from the cells and the formation of the CaPO4-precipitate is
initiated by adding 12.5 ml of 2.times.HBS (pH 7.05) that has been
pre-warmed to 37.degree. C. to the DNA-CaCl2 solution. The DNA is
incubated for 1 min; and transferring the mixture into 200 mL of
pre-warmed DMEM-10% FBS then stops the formation of the
precipitate. Twenty two mL of the medium is immediately dispensed
into each plate and cells are incubated at 37.degree. C. for 48 hr.
The CaPO4-precipitate is allowed to stay on the cells during the
whole incubation period without compromising cell viability.
Forty-eight hr post-transfection cells are harvested by
centrifugation at 1,140.times.g for 10 min. Cells are then lysed in
15 ml of 0.15 M MgCl, 50 mM Tris-HCl (pH 8.5) by 3 freeze/thaw
cycles in dry ice-ethanol and 37.degree. C. baths. Benzonase
(Nycomed Pharma A/S, pure grade) is added to the mixture (50 U/mL
final concentration) and the lysate is incubated for 30 min at
37.degree. C. The lysate is clarified by centrifugation at
3,700.times.g for 20 min and the virus-containing supernatant is
further purified using a discontinuous density gradient.
[0095] The typical discontinuous step gradient is formed by
underlayering and displacing the less dense cell lysate with
Iodixanol, 5,5''[(2-hydroxi-1-3-propanediyl)-bis(acetylamino)] bis
[N,N'bi(2,3dihydroxypropyl-2-4,6-triiodo-1,3-enzenecarboxamide],
prepared using a 60% (wt./vol.) sterile solution of OptiPrep
(Nycomed). Specifically, 15 mL of the clarified lysate are
transferred into Quick-Seal Ultra-Clear 25.times.89-mm centrifuge
tube (Beckman) using a syringe equipped with a 1/27.times.89 mm
spinal needle. Care is taken to avoid bubbles, which would
interfere with subsequent filling and sealing of the tube. A
variable speed peristaltic pump, Model EP-1 (Bio-Rad), is used to
underlay in order: 9 mL of 15% iodixanol and 1 M NaCl in PBS-MK
buffer containing Phenol Red (2.5 .mu.L of a 0.5% stock solution
per ml of the iodixanol solution); 5 mL of 40% iodixanol in PBS-MK
buffer; and finally, 5 mL of 60% iodixanol in PBS-MK buffer
containing Phenol Red (0.1 .mu.L/L). Tubes are sealed and
centrifuged in a Type 70 Ti rotor (Beckman) at 350,000.times.g for
1 hr at 18.degree. C. Four mL of the clear 40% step is aspirated
after puncturing the tube on the side with a syringe equipped with
an 18-gauge needle with the bevel uppermost. The iodixanol fraction
is further purified using conventional Heparin agarose affinity
chromatography.
[0096] For chromatography, typically, a pre-packed 2.5-mL Heparin
agarose type I column (Sigma) is equilibrated with 20 mL of PBS-MK
under gravity. The rAAV iodixanol fraction is then applied to the
pre-equilibrated column, and the column is washed with 10 mL of
PBS-MK. rAAV is eluted with the same buffer containing 1 M NaCl.
After applying the elution buffer, the first 2 ml of the eluant are
discarded, and the virus is collected in the subsequent 3.5 mL of
elution buffer.
[0097] Virus is then concentrated and desalted by centrifugation
through the BIOMAX.RTM. 100 K filter (Millipore, Bedford, Mass.,
USA) according to the manufacturer's instructions. The high salt
buffer is changed by repeatedly diluting the concentrated virus
with Lactated Ringer's solution, and repeating the titer both
genome containing particles and infectious rAAV particles. A
conventional dot-blot assay, quantitative competitive PCR (QC PCR)
assay, or more recently quantitative real-time PCR (aRT-PCR) are
used to determine physical particle titers (Zolotukhin et al.,
2002; Jacobson et al., 2006) Infectious titers are determined by
infectious center assay (ICA) and fluorescent cell assay (FCA),
which scores for expression of GFP (Zolotukhin et al., 2002).
[0098] QC PCR method is based on competitive co-amplified of a
specific target sequence with internal standard plasmid of known
concentration in on reaction tube. It provides precise and fast
quantitation of viral particles. The internal standard must hare
primer recognition sites with the specific template. Both the
specific template and the internal standard must be PCR-amplified
with the same efficiency and it must be possible to analyze the
PCR-amplified products separately. The easiest way to distinguish
between the template and the internal standard is to incorporate a
size difference in the two products. This can be achieved, for
example, by constructing standards having the same sequence as the
specific target but containing a deletion. Quantitation is then
performed by comparing the PCR signal of the specific template with
the PCR signal obtained with known concentrations of the competitor
(the internal standard). Quantitative real-time PCR (qRT-PCR) is a
standard method for evaluating DNA concentration of an unknown
sample by comparison of PCR product formation in real-time to a
known DNA standard.
[0099] The purified viral stock is first treated with DNAseI to
digest any contaminating unpackaged DNA. Ten .mu.L of a purified
virus stock is incubated with 10 U of DNAseI (Boehringer, Ingelheim
am Rhein, Germany) in a 100 .mu.L reaction mixture, containing 50
mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2 for 1 hr at 37.degree. C. At
the end of the reaction, 10 .mu.L of OX Proteinase K buffer (10 mM
Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS final concentration) was
added, followed by the addition of 1 .mu.L of Proteinase K (18.6
mg/mL, Boehringer). The mixture was incubated at 37.degree. C. for
1 hr. Viral DNA was purified by phenol/chloroform extraction
(twice), followed by chloroform extraction and ethanol
precipitation using 10 .mu.g of glycogen as a carrier. The DNA
pellet was dissolved in 100 .mu.L of water. QC PCR reaction
mixtures each contained 1 .mu.L of the diluted viral DNA and
two-fold serial dilutions of the internal standard plasmid DNA,
such as pdl-GFP. The most reliable range of standard DNA was found
to be between 1 and 100 .mu.g. An aliquot of each reaction was then
analyzed by 2% agarose gel electrophoresis, until two PCR products
were resolved. The analog image of the ethidium bromide stained gel
was digitized using and ImageStore 7500 system (UVP; Upland,
Calif., USA). The densities of the target and competitor bands in
each lane were measured using the ZERO-Dscan Image Analysis System,
version 1.0 (Scanalytics, Rockville, Md., USA) and their ratios are
plotted as a function of the standard DNA concentration. A ratio of
1.0, at which the number of viral DNA molecules equals the number
of competitor DNA molecules was used to determine the DNA
concentration of the virus stock.
[0100] A modification of the previously published protocol
(McLaughlin et al., 1988) was used to measure the ability of the
virus to infect C12 cells, unpackage, and replicate. Briefly, C2
cells containing integrated wtAAV rep and cap genes (Clark et al.,
1995) were plated in a 96-well dish at about 75% confluence, then
infected with Ad5 at a M.O.I of 20. One .mu.L of serially diluted
rAAV-sCNTF was visually scored using a fluorescence microscope.
High sensitivity CHROMA filter #41012 HighQ FITC LP (Chroma
Technology, Bellows Fall, Va., USA) was used to monitor the
fluorescence. To calculate the titer by hybridization, cells were
harvested and processed essentially as previously described
(McLaughlin et al., 1988).
[0101] Pharmaceutical Compositions
[0102] In certain embodiments, the present invention concerns
formulation of one or more of the rAAV-guanylate cyclase
compositions disclosed herein in pharmaceutically acceptable
solutions for administration to a cell or an animal, either alone,
or in combination with one or more other modalities of therapy.
[0103] It will also be understood that, if desired, the nucleic
acid segment, RNA, DNA or PNA compositions that express a
therapeutic gene product as disclosed herein may be administered in
combination with other agents as well, such as, e.g., proteins or
polypeptides or various pharmaceutically-active agents, including
one or more systemic or topical administrations of guanylate
cyclase polypeptides. In fact, there is virtually no limit to other
components that may also be included, given that the additional
agents do not cause a significant adverse effect upon contact with
the target cells or host tissues. The rAAV-vectored guanylate
cyclase compositions may thus be delivered along with various other
agents as required in the particular instance. Such compositions
may be purified from host cells or other biological sources, or
alternatively may be chemically synthesized as described herein.
Likewise, such compositions may further comprise substituted or
derivatized RNA, DNA, or PNA compositions.
[0104] Formulation of pharmaceutically-acceptable excipients and
carrier solutions is well-known to those of skill in the art, as is
the development of suitable dosing and treatment regimens for using
the particular compositions described herein in a variety of
treatment regimens, including e.g., oral, parenteral, intravenous,
intranasal, intramuscular, and direct administration to one or more
cells or tissue types within the animal, including for example,
ocular, retinal, and sub-retinal injection or such like.
[0105] Typically, these formulations may contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound(s) in each therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one of ordinary skill in the art of
preparing such pharmaceutical formulations, and as such, a variety
of dosages and treatment regimens may be desirable.
[0106] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally as
described (see e.g., U.S. Pat. Nos. 5,543,158; 5,641,515 and
5,399,363, each of which is specifically incorporated herein in its
entirety by express reference thereto). Solutions of the active
compounds as freebase or pharmacologically acceptable salts may be
prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0107] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions (see e.g., U.S. Pat. No. 5,466,468, specifically
incorporated herein in its entirety by express reference thereto).
In all cases the form must be sterile and must be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms, such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (e.g., glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and/or vegetable oils. Proper fluidity
may be maintained, for example, by the use of a coating, such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. The prevention of
the action of microorganisms can be brought about by various
antibacterial ad antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0108] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage may be dissolved in 1 ml of isotonic NaCl solution and
either added to 1000 mL of hypodermoclysis fluid or injected at the
proposed site of infusion, (see, e.g., Remington's Pharmaceutical
Sciences 15th Ed., pages 1035-1038 and 1570-1580). Some variation
in dosage will necessarily occur depending on the condition of the
subject being treated. The person responsible for administration
will, in any event, determine the appropriate dose for the
individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, and the general
safety and purity standards as required by the United States Food
and Drug Administration's (FDA) Office of Biologics Standards.
[0109] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various other ingredients as enumerated herein, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0110] The compositions disclosed herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts include the
acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug-release capsules,
and the like.
[0111] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0112] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0113] Sequence Comparison, Identity, and Homology
[0114] For sequence comparison and homology determination,
typically one sequence acts as a reference sequence to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison
algorithm can then be used to calculate the percent sequence
identity for the test sequence(s) relative to the reference
sequence, based on the designated program parameters.
[0115] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm (see e.g., Smith
and Waterman, 1981), by the homology alignment algorithm (see e.g.,
Needleman and Wunsch, 1970), by the search similarity comparison
method (see e.g., Pearson and Lipman, 1988), by computerized
implementations of algorithms such as GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, Madison, Wis., USA, or by visual inspection. One
example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm
(Altschul et al., 1990) and BLOSUM62 scoring matrix (see, e.g.,
Henikoff and Henikoff, 1989). Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (www.ncbi.nlm.nih.gov/).
[0116] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul,
1993). Another example of a useful sequence alignment algorithm is
the PILEUP program, which creates a multiple sequence alignment
from a group of related sequences using progressive, pairwise
alignments. It can also plot a tree showing the clustering
relationships used to create the alignment. PILEUP uses a
simplification of the progressive alignment comparison method (see
e.g., Feng and Doolittle, 1987), and employs a general alignment
matrix similar to that described by Higgins and Sharp (1989).
[0117] Therapeutic and Diagnostic Kits
[0118] The invention also encompasses one or more compositions
together with one or more pharmaceutically-acceptable excipients,
carriers, diluents, adjuvants, and/or other components, as may be
employed in the formulation of particular rAAV-guanylate cyclase
formulations, and in the preparation of therapeutic agents for
administration to a mammal, and in particularly, to a human, for
one or more of the guanylate cyclase-deficient conditions, such as
a retinal dystrophy like LCA1, as described herein. In particular,
such kits may comprise one or more rAAV-vectored guanylate cyclase
composition in combination with instructions for using the viral
vector in the treatment of such disorders in a mammal, and may
typically further include containers prepared for convenient
commercial packaging.
[0119] As such, preferred animals for administration of the
pharmaceutical compositions disclosed herein include mammals, and
particularly humans. Other preferred animals include non-human
primates, murines, epines, bovines, ovines, equines, hircines,
lupines, leporines, vulpines, porcines, canines, felines, and the
like. The composition may include partially or significantly
purified rAAV-guanylate cyclase compositions, either alone, or in
combination with one or more additional active ingredients, which
may be obtained from natural or recombinant sources, or which may
be obtainable naturally or either chemically synthesized, or
alternatively produced in vitro from recombinant host cells
expressing DNA segments encoding such additional active
ingredients.
[0120] Therapeutic kits may also be prepared that comprise at least
one of the compositions disclosed herein and instructions for using
the composition as a therapeutic agent. The container means for
such kits may typically comprise at least one vial, test tube,
flask, bottle, syringe or other container means, into which the
disclosed rAAV composition(s) may be placed, and preferably
suitably aliquoted. Where a second guanylate cyclase composition is
also provided, the kit may also contain a second distinct container
means into which this second composition may be placed.
Alternatively, the plurality of guanylate cyclase compositions may
be prepared in a single pharmaceutical composition, and may be
packaged in a single container means, such as a vial, flask,
syringe, bottle, or other suitable single container means. The kits
of the present invention will also typically include a means for
containing the vial(s) in close confinement for commercial sale,
such as, e.g., injection or blow-molded plastic containers into
which the desired vial(s) are retained.
[0121] Expression in Animal Cells
[0122] The inventors contemplate that a polynucleotide comprising a
contiguous nucleic acid sequence that encodes a therapeutic
guanylate cyclase polypeptide of the present invention may be
utilized to treat one or more cellular defects in a transformed
host cell. Such cells are preferably animal cells, including
mammalian cells such as those obtained from a human or a non-human
primate, or from one or more mammalian species including without
limitation, murines, canines, bovines, equines, epines, felines,
ovines, hircines, lupines, leporines, porcines, and the like. The
use of such constructs for the treatment and/or amelioration of one
or more symptoms of a retinal dystrophy such as LCA1, or of a
related retinal or ocular disease, disorder, condition, or
dysfunction in a human subject suspected of suffering from such a
disorder, or at risk for developing such a condition is
particularly contemplated by the present inventors.
[0123] The cells may be transformed with one or more rAAV vectors
comprising one or more therapeutic guanylate cyclase genes of
interest, such that the genetic construct introduced into and
expressed in the host cells of the animal is sufficient to alter,
reduce, ameliorate or prevent the deleterious or disease
condition(s) or one or more symptoms thereof, either ex vivo, in
vitro, ex situ, in situ, and/or in vivo.
[0124] Guanylate Cyclase
[0125] Guanylate cyclase (GC) (EC 4.6.1.2) is a lyase that
catalyzes the conversion of guanosine triphosphates (GTP) to
3',5'-cyclic guanosine monophosphate (cGMP) and pyrophosphate.
Referred to alternatively in the literature as "guanyl cyclase" or
"guanylyl cyclase," both membrane-bound (type 1) and soluble (type
2) forms of GC exist.
[0126] Leber Congenital Amaurosis
[0127] Leber congenital amaurosis (LCA) is an autosomal recessive
group of diseases that represent the earliest and most severe form
of all inherited retinal dystrophies. The first gene implicated in
the onset of this genetically and clinically heterogeneous disease,
and therefore assigned to the LCA1 locus was retinal-specific
Guanylate cyclase-1 (Gucy2d) (Perrault et al., 1996). Gucy2d
encodes for the retinal specific protein guanylate cyclase (retGC1)
which is expressed predominantly in photoreceptor outer segment
membranes and plays a role in the regulation of cGMP and Ca2+
levels within these cells. Following light stimulation, levels of
cGMP within photoreceptor outer segments rapidly fall due to
hydrolysis by cGMP phosphodiesterase (PDE). This reduction of cGMP
leads to a closure of cGMP-gated channels, reduced Ca2+ influx, and
hyperpolarization of the cell. This decrease in intracellular Ca2+
stimulates recovery of light-stimulated photoreceptors to the dark
state via its interaction with guanylate cyclase activating
proteins (GCAPs), a family of calcium binding proteins that
regulate the activity of GC. In the dark adapted photoreceptor,
Ca2+-bound GCAPs inhibit the activity of GC. Upon light
stimulation, however, Ca2+-free GCAPs stimulate GC activity which
produces an increase in cGMP levels, a reopening of the cGMP-gated
channels and a return of the cell to a depolarized state. Mutations
which reduce or abolish the ability of GC to replenish
intracellular cGMP and reopen cGMP-gated cation channels, as is the
case in LCA1, are thought to create the biochemical equivalent of
chronic light exposure in rod and cone photoreceptors.
[0128] Mutations in Gucy2d account for .about.15% of all cases of
LCA making it one of the leading causes of this disease. The number
of patients affected by LCA1 is approximately double that affected
by the well known RPE65 version of the disease (LCA2), a form for
which successful AAV-mediated gene therapy trials have recently
garnered worldwide attention. Diagnosis of LCA1 is typically made
within the first few months of life in an infant with total
blindness or severely impaired vision, extinguished
electroretinogram (ERG) and pendular nystagmus (Perrault et al.,
1999; Chung and Traboulsi, 2009). Despite these functional
deficits, LCA1 patients present with normal fundus (Perrault et
al., 1999) and retain some rod and cone photoreceptors in both
their macular and peripheral retina for years (Milam et al., 2003;
Simonelli et al., 2007; Pasadhika et al., 2009). Using
spectral-domain optical coherence tomography (SDOCT) to scan the
central macular and perifoveal areas, a recent study revealed that
LCA1 patients (age range, 20-53 years) retained all 6 retinal
layers with visible photoreceptor inner/outer segment juncture.
Maintenance of retinal structure in LCA1 is unlike other forms of
the disease which exhibit marked retinal thinning that generally
worsens with age (Pasadhika et al., 2009). While the preservation
of retinal structure does not parallel better visual acuity in LCA1
patients, it does suggest that they are better suited for future
therapeutic strategies.
[0129] Animal Models
[0130] Two animal models carrying null mutations in the retGC1 gene
have been used to evaluate gene replacement therapy, the naturally
occurring GUCY1*B chicken and the guanylate-cyclase-1 (GC1)
knockout mouse (see e.g., Williams et al., 2006; Haire et al.,
2006). The GUCY1*B chicken is blind at hatch, exhibits extinguished
scotopic (rod-mediated) and photopic (cone-mediated) ERG and
retinal degeneration (see e.g., Ulshafer et al., 1984; Huang et
al., 1998; Semple-Rowland et al., 1998). Lentiviral-mediated
transfer of Gucy2d to the GUCY1*B retina restored vision to these
animals as evidenced by behavioral testing and ERG (see e.g.,
Williams et al., 2006). Despite the short term therapeutic success,
this therapy fell short of preserving retinal structure or function
in the long term. The transient nature of this result, obtained in
a non-mammalian species with an integrating viral vector delivered
in ovo suggested the need for more appropriate translational
studies towards the development of clinical application.
[0131] A mammalian model of LCA1, the GC1KO mouse, exhibits cone
photoreceptor degeneration (see e.g., Yang et al., 1999; Coleman et
al., 2004). Like LCA1 patients, loss of cone function in this mouse
model precedes cone degeneration (Yang et al., 1999). In addition,
light-induced translocation of cone arrestin is disrupted. Rod
photoreceptors in this model do not degenerate and continue to
generate electrical responses to light (Yang et al., 1999), a
result likely owed to the presence of GC2, a close relative of GC1
in these cells (see e.g., Lowe et al., 1995; Yang et al., 1995;
Yang and Garbers, 1997; Karan et al., 2010). AAV-mediated transfer
of Gucy2d to the post-natal GC1KO retina restored light-driven
translocation of cone arrestin in transduced cells, but failed to
restore cone ERG responses or prevent cone degeneration (Haire et
al., 2006). In both the chicken and mouse studies, which were
conducted by the same investigators, the therapeutic cDNA was of
bovine origin which is the protein species historically used in
biochemical assays evaluating GC1 functionality (Otto-Bruc et al.,
1997; Williams et al., 2006).
[0132] Exemplary Definitions
[0133] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and compositions similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and compositions are
described herein. For purposes of the present invention, the
following terms are defined below:
[0134] In accordance with long standing patent law convention, the
words "a" and "an" when used in this application, including the
claims, denotes "one or more."
[0135] As used herein, the term "about" should generally be
understood to refer to both numbers in a range of numerals. For
example, "about 1 to 10" should be understood as "about 1 to about
10." Moreover, all numerical ranges herein should be understood to
include each whole integer within the range, as well as each tenth.
The term "about," as used herein, should generally be understood to
mean "approximately", and typically refers to numbers approximately
equal to a given number recited within a range of numerals.
Moreover, all numerical ranges herein should be understood to
include each whole integer within the range.
[0136] In accordance with the present invention, polynucleotides,
nucleic acid segments, nucleic acid sequences, and the like,
include, but are not limited to, DNAs (including and not limited to
genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs)
RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs),
nucleosides, and suitable nucleic acid segments either obtained
from natural sources, chemically synthesized, modified, or
otherwise prepared or synthesized in whole or in part by the hand
of man.
[0137] As used herein, the term "nucleic acid" includes one or more
types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
polyribonucleotides (containing D-ribose), and any other type of
polynucleotide that is an N-glycoside of a purine or pyrimidine
base, or modified purine or pyrimidine bases (including abasic
sites). The term "nucleic acid," as used herein, also includes
polymers of ribonucleosides or deoxyribonucleosides that are
covalently bonded, typically by phosphodiester linkages between
subunits, but in some cases by phosphorothioates,
methylphosphonates, and the like. "Nucleic acids" include single-
and double-stranded DNA, as well as single- and double-stranded
RNA. Exemplary nucleic acids include, without limitation, gDNA;
hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA
(siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA),
and small temporal RNA (stRNA), and the like, and any combination
thereof.
[0138] As used herein, the term "DNA segment" refers to a DNA
molecule that has been isolated free of total genomic DNA of a
particular species. Therefore, a DNA segment obtained from a
biological sample using one of the compositions disclosed herein
refers to one or more DNA segments that have been isolated away
from, or purified free from, total genomic DNA of the particular
species from which they are obtained. Included within the term "DNA
segment," are DNA segments and smaller fragments of such segments,
as well as recombinant vectors, including, for example, plasmids,
cosmids, phage, viruses, and the like.
[0139] Similarly, the term "RNA segment" refers to an RNA molecule
that has been isolated free of total cellular RNA of a particular
species. Therefore, RNA segments can refer to one or more RNA
segments (either of native or synthetic origin) that have been
isolated away from, or purified free from, other RNAs. Included
within the term "RNA segment," are RNA segments and smaller
fragments of such segments.
[0140] In the context of the invention the term "expression" is
intended to include the combination of intracellular processes,
including transcription and translation undergone by a
polynucleotide such as a structural gene to synthesize the encoded
peptide or polypeptide.
[0141] The term "e.g.," as used herein, is used merely by way of
example, without limitation intended, and should not be construed
as referring only those items explicitly enumerated in the
specification.
[0142] As used herein, the term "promoter" is intended to generally
describe the region or regions of a nucleic acid sequence that
regulates transcription.
[0143] As used herein, the term "regulatory element" is intended to
generally describe the region or regions of a nucleic acid sequence
that regulates transcription. Exemplary regulatory elements
include, but are not limited to, enhancers, post-transcriptional
elements, transcriptional control sequences, and such like.
[0144] As used herein, the term "structural gene" is intended to
generally describe a polynucleotide, such as a gene, that is
expressed to produce an encoded peptide, polypeptide, protein,
ribozyme, catalytic RNA molecule, or antisense molecule.
[0145] As used herein, the term "transformation" is intended to
generally describe a process of introducing an exogenous
polynucleotide sequence (e.g., a viral vector, a plasmid, or a
recombinant DNA or RNA molecule) into a host cell or protoplast in
which the exogenous polynucleotide is incorporated into at least a
first chromosome or is capable of autonomous replication within the
transformed host cell. Transfection, electroporation, and "naked"
nucleic acid uptake all represent examples of techniques used to
transform a host cell with one or more polynucleotides.
[0146] As used herein, the term "transformed cell" is intended to
mean a host cell whose nucleic acid complement has been altered by
the introduction of one or more exogenous polynucleotides into that
cell.
[0147] As used herein, the term "transgenic cell" is generally
intended to mean any cell that is derived or regenerated from a
transformed cell or derived from another transgenic cell, or from
the progeny or offspring of any generation of such a transformed or
transgenic host cell.
[0148] As used herein, the term "vector" is generally intended to
mean a nucleic acid molecule (typically comprised of DNA) capable
of replication in a host cell and/or to which another nucleic acid
segment can be operatively linked so as to bring about replication
of the attached segment. A plasmid, cosmid, or a virus is an
exemplary vector.
[0149] The terms "substantially corresponds to," "substantially
homologous," or "substantial identity" as used herein denotes a
characteristic of a nucleic acid or an amino acid sequence, wherein
a selected nucleic acid or amino acid sequence has at least about
70 or about 75 percent sequence identity as compared to a selected
reference nucleic acid or amino acid sequence. More typically, the
selected sequence and the reference sequence will have at least
about 76, about 77, about 78, about 79, about 80, about 81, about
82, about 83, about 84 or even about 85 percent sequence identity,
and more preferably at least about 86% sequence identity, at least
about 87% sequence identity, at least about 88% sequence identity,
at least about 89% sequence identity, at least about 90% sequence
identity, at least about 91% sequence identity, at least about 92%
sequence identity, at least about 93% sequence identity, at least
about 94% sequence identity, or at least about 95% percent or
greater sequence identity. More preferably still, highly homologous
sequences often share greater than at least about 96% sequence
identity, at least about 97% sequence identity, at least about 98%
sequence identity, or at least about 99% sequence identity between
the selected sequence and the reference sequence to which it was
compared. The percentage of sequence identity may be calculated
over the entire length of the sequences to be compared, or may be
calculated by excluding small deletions or additions which total
less than about 25 percent or so of the chosen reference sequence.
The reference sequence may be a subset of a larger sequence, such
as a portion of a gene or flanking sequence, or a repetitive
portion of a chromosome.
[0150] However, in the case of sequence homology of two or more
polynucleotide sequences, the reference sequence and the target
sequence will typically comprise at least about 18 to about 25
contiguous identical nucleotides, more typically at least about 26
to about 35 contiguous nucleotides that are identical, and even
more typically at least about 40, about 50, about 60, about 70,
about 80, about 90, or even about 100 or so contiguous nucleotides
that are identical. Desirably, which highly homologous fragments
are desired, the extent of overall percent sequence identity
between two given sequences will be at least about 80% identical
preferably at least about 85% identical, and more preferably about
90% identical, about 91% identical, about 92% identical, about 93%
identical, about 94% identical, or even about 95% or greater
identical, as readily determined by one or more of the sequence
comparison algorithms well-known to those of skill in the art, such
as, e.g., the FASTA program analysis described by Pearson and
Lipman (1988).
[0151] The terms "identical" or percent "identity," in the context
of two or more nucleic acid or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the sequence comparison algorithms described
below (or other algorithms available to persons of ordinary skill)
or by visual inspection.
[0152] The phrase "substantially identical," in the context of two
nucleic acids refers to two or more sequences or subsequences that
have at least about 90%, preferably 91%, most preferably about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 98.5%, about 99%, about 99.1%, about 99.2%, about 99.3%,
about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or
about 99.9% or more nucleotide residue identity, when compared and
aligned for maximum correspondence, as measured using a sequence
comparison algorithm or by visual inspection. Such "substantially
identical" sequences are typically considered "homologous," without
reference to actual ancestry.
[0153] The term "naturally occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by the hand of man in a laboratory is naturally-occurring.
As used herein, laboratory strains of rodents that may have been
selectively bred according to classical genetics are considered
naturally occurring animals.
[0154] As used herein, a "heterologous" is defined in relation to a
predetermined referenced gene sequence. For example, with respect
to a structural gene sequence, a heterologous promoter is defined
as a promoter which does not naturally occur adjacent to the
referenced structural gene, but which is positioned by laboratory
manipulation. Likewise, a heterologous gene or nucleic acid segment
is defined as a gene or segment that does not naturally occur
adjacent to the referenced promoter and/or enhancer elements.
[0155] As used herein, the term "homology" refers to a degree of
complementarity between two or more polynucleotide or polypeptide
sequences. The word "identity" may substitute for the word
"homology" when a first nucleic acid or amino acid sequence has the
exact same primary sequence as a second nucleic acid or amino acid
sequence. Sequence homology and sequence identity can be determined
by analyzing two or more sequences using algorithms and computer
programs known in the art. Such methods may be used to assess
whether a given sequence is identical or homologous to another
selected sequence.
[0156] As used herein, "homologous" means, when referring to
polynucleotides, sequences that have the same essential nucleotide
sequence, despite arising from different origins. Typically,
homologous nucleic acid sequences are derived from closely related
genes or organisms possessing one or more substantially similar
genomic sequences. By contrast, an "analogous" polynucleotide is
one that shams the same function with a polynucleotide from a
different species or organism, but may have a significantly
different primary nucleotide sequence that encodes one or more
proteins or polypeptides that accomplish similar functions or
possess similar biological activity. Analogous polynucleotides may
often be derived from two or more organisms that are not closely
related (e.g., either genetically or phylogenetically).
[0157] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and includes any chain or chains of two or more
amino acids. Thus, as used herein, terms including, but not limited
to "peptide," "dipeptide." "tripeptide," "protein," "enzyme,"
"amino acid chain," and "contiguous amino acid sequence" are all
encompassed within the definition of a "polypeptide," and the term
"polypeptide" can be used instead of, or interchangeably with, any
of these terms. The term further includes polypeptides that have
undergone one or more post-translational modification(s), including
for example, but not limited to, glycosylation, acetylation,
phosphorylation, amidation, derivatization, proteolytic cleavage,
post-translation processing, or modification by inclusion of one or
more non-naturally occurring amino acids. Conventional nomenclature
exists in the art for polynucleotide and polypeptide structures.
For example, one-letter and three-letter abbreviations are widely
employed to describe amino acids: Alanine (A; Ala), Arginine (R;
Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C;
Cys), Glutamine (Q; Gln), Glutamic Acid (E; Glu), Glycine (G; Gly),
Histidine (H; His), Isoleucine (I; Ile), Leucine (L; Leu),
Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro),
Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine
(Y; Tyr), Valine (V; Val), and Lysine (K: Lys). Amino acid residues
described herein are preferred to be in the "L" isomeric form.
However, residues in the "D" isomeric form may be substituted for
any L-amino acid residue provided the desired properties of the
polypeptide are retained.
[0158] "Protein" is used herein interchangeably with "peptide" and
"polypeptide," and includes both peptides and polypeptides produced
synthetically, recombinantly, or in vitro and peptides and
polypeptides expressed in vivo after nucleic acid sequences are
administered into a host animal or human subject. The term
"polypeptide" is preferably intended to refer to all amino acid
chain lengths, including those of short peptides of about 2 to
about 20 amino acid residues in length, oligopeptides of about 10
to about 100 amino acid residues in length, and polypeptides of
about 100 to about 5,000 or more amino acid residues in length. The
term "sequence," when referring to amino acids, relates to all or a
portion of the linear N-terminal to C-terminal order of amino acids
within a given amino acid chain, e.g., polypeptide or protein;
"subsequence" means any consecutive stretch of amino acids within a
sequence, e.g., at least 3 consecutive amino acids within a given
protein or polypeptide sequence. With reference to nucleotide and
polynucleotide chains, "sequence" and "subsequence" have similar
meanings relating to the 5' to 3' order of nucleotides.
[0159] As used herein, the term "substantially homologous"
encompasses two or more biomolecular sequences that are
significantly similar to each other at the primary nucleotide
sequence level. For example, in the context of two or more nucleic
acid sequences, "substantially homologous" can refer to at least
about 75%, preferably at least about 80%, and more preferably at
least about 85%, or at least about 90% identity, and even more
preferably at least about 95%, more preferably at least about 97%
identical, more preferably at least about 98% identical, more
preferably at least about 99% identical, and even more preferably
still, entirely identical (i.e., 100% or "invariant").
[0160] Likewise, as used herein, the term "substantially identical"
encompasses two or more biomolecular sequences (and in particular
polynucleotide sequences) that exhibit a high degree of identity to
each other at the nucleotide level. For example, in the context of
two or more nucleic acid sequences, "substantially identical" can
refer to sequences that at least about 80%, and more preferably at
least about 85% or at least about 90% identical to each other, and
even more preferably at least about 95%, more preferably at least
about 97% identical, more preferably at least about 98% identical,
more preferably at least about 99% identical, and even more
preferably still, entirely identical (i.e., 100% identical or
"non-degenerate").
[0161] The term "recombinant" indicates that the material (e.g., a
polynucleotide or a polypeptide) has been artificially or
synthetically (non-naturally) altered by human intervention. The
alteration can be performed on the material within or removed from,
its natural environment or state. Specifically, e.g., a promoter
sequence is "recombinant" when it is produced by the expression of
a nucleic acid segment engineered by the hand of man. For example,
a "recombinant nucleic acid" is one that is made by recombining
nucleic acids, e.g., during cloning, DNA shuffling or other
procedures, or by chemical or other mutagenesis; a "recombinant
polypeptide" or "recombinant protein" is a polypeptide or protein
which is produced by expression of a recombinant nucleic acid; and
a "recombinant virus," e.g., a recombinant AAV virus, is produced
by the expression of a recombinant nucleic acid.
[0162] As used herein, the term "operably linked" refers to a
linkage of two or more polynucleotides or two or more nucleic acid
sequences in a functional relationship. A nucleic acid is "operably
linked" when it is placed into a functional relationship with
another nucleic acid sequence. For instance, a promoter or enhancer
is operably linked to a coding sequence if it affects the
transcription of the coding sequence. "Operably linked" means that
the nucleic acid sequences being linked are typically contiguous,
or substantially contiguous, and, where necessary to join two
protein coding regions, contiguous and in reading frame. Since
enhancers generally function when separated from the promoter by
several kilobases and intronic sequences may be of variable
lengths; however, some polynucleotide elements may be operably
linked but not contiguous.
[0163] "Transcriptional regulatory element" refers to a
polynucleotide sequence that activates transcription alone or in
combination with one or more other nucleic acid sequences. A
transcriptional regulatory element can, for example, comprise one
or more promoters, one or more response elements, one or more
negative regulatory elements, and/or one or more enhancers.
[0164] As used herein, a "transcription factor recognition site"
and a "transcription factor binding site" refer to a polynucleotide
sequence(s) or sequence motif(s) which are identified as being
sites for the sequence-specific interaction of one or more
transcription factors, frequently taking the form of direct
protein-DNA binding. Typically, transcription factor binding sites
can be identified by DNA footprinting, gel mobility shift assays,
and the like, and/or can be predicted on the basis of known
consensus sequence motifs, or by other methods known to those of
skill in the art.
[0165] "Transcriptional unit" refers to a polynucleotide sequence
that comprises at least a first structural gene operably linked to
at least a first cis-acting promoter sequence and optionally linked
operably to one or more other cis-acting nucleic acid sequences
necessary for efficient transcription of the structural gene
sequences, and at least a first distal regulatory element as may be
required for the appropriate tissue-specific and developmental
transcription of the structural gene sequence operably positioned
under the control of the promoter and/or enhancer elements, as well
as any additional cis sequences that are necessary for efficient
transcription and translation (e.g., polyadenylation site(s), mRNA
stability controlling sequence(s), etc.
[0166] The term "substantially complementary," when used to define
either amino acid or nucleic acid sequences, means that a
particular subject sequence, for example, an oligonucleotide
sequence, is substantially complementary to all or a portion of the
selected sequence, and thus will specifically bind to a portion of
an mRNA encoding the selected sequence. As such, typically the
sequences will be highly complementary to the mRNA "target"
sequence, and will have no more than about 1, about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, or about 10
or so base mismatches throughout the complementary portion of the
sequence. In many instances, it may be desirable for the sequences
to be exact matches, i.e., be completely complementary to the
sequence to which the oligonucleotide specifically binds, and
therefore have zero mismatches along the complementary stretch. As
such, highly complementary sequences will typically bind quite
specifically to the target sequence region of the mRNA and will
therefore be highly efficient in reducing, and/or even inhibiting
the translation of the target mRNA sequence into polypeptide
product.
[0167] Substantially complementary nucleic acid sequences will be
greater than about 80 percent complementary (or "% exact-match") to
a corresponding nucleic acid target sequence to which the nucleic
acid specifically binds, and will, more preferably be greater than
about 85 percent complementary to the corresponding target sequence
to which the nucleic acid specifically binds. In certain aspects,
as described above, it will be desirable to have even more
substantially complementary nucleic acid sequences for use in the
practice of the invention, and in such instances, the nucleic acid
sequences will be greater than about 90 percent complementary to
the corresponding target sequence to which the nucleic acid
specifically binds, and may in certain embodiments be greater than
about 95 percent complementary to the corresponding target sequence
to which the nucleic acid specifically binds, and even up to and
including about 96%, about 97%, about 98%, about 99%, and even
about 100% exact match complementary to all or a portion of the
target sequence to which the designed nucleic acid specifically
binds.
[0168] Percent similarity or percent complementary of any of the
disclosed nucleic acid sequences may be determined, for example, by
comparing sequence information using the GAP computer program,
version 6.0, available from the University of Wisconsin Genetics
Computer Group (UWGCG). The GAP program utilizes the alignment
method of Needleman and Wunsch (1970). Briefly, the GAP program
defines similarity as the number of aligned symbols (i.e.,
nucleotides or amino acids) that are similar, divided by the total
number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a
unary comparison matrix (containing a value of 1 for identities and
0 for non-identities) for nucleotides, and the weighted comparison
matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps.
[0169] As used herein, the terms "protein," "polypeptide," and
"peptide" are used interchangeably, and include molecules that
include at least one amide bond linking two or more amino acid
residues together. Although used interchangeably, in general, a
peptide is a relatively short (e.g., from 2 to about 100 amino acid
residues in length) molecule, while a protein or a polypeptide is a
relatively longer polymer (e.g., 100 or more residues in length).
However, unless specifically defined by a chain length, the terms
peptide, polypeptide, and protein are used interchangeably.
[0170] As used herein, the term "patient" (also interchangeably
referred to as "host" or "subject") refers to any host that can
serve as a recipient for one or more of the rAAV-based guanylate
cyclase compositions as discussed herein. In certain aspects, the
recipient will be a vertebrate animal, which is intended to denote
any animal species (and preferably, a mammalian species such as a
human being). In certain embodiments, a "patient" refers to any
animal host, including but not limited to, human and non-human
primates, bovines, canines, caprines, cavines, corvines, epines,
equines, felines, hircines, lapines, leporines, lupines, murines,
ovines, porcines, racines, vulpines, and the like, including,
without limitation, domesticated livestock, herding or migratory
animals, exotics or zoological specimens, as well as companion
animals, pets, and any animal under the care of a veterinary
practitioner.
[0171] As used herein, the term "carrier" is intended to include
any solvent(s), dispersion medium, coating(s), diluent(s),
buffer(s), isotonic agent(s), solution(s), suspension(s),
colloid(s), inert(s) or such like, or a combination thereof that is
pharmaceutically acceptable for administration to the relevant
animal or acceptable for a diagnostic purpose, as applicable. The
use of one or more delivery vehicles for gene therapy constructs,
viral particles, vectors, and the like, is well known to those of
ordinary skill in the pharmaceutical and molecular arts. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the prophylactic, and/or therapeutic
compositions is contemplated. One or more supplementary active
ingredient(s) may also be incorporated into, or administered in
association with, one or more of the disclosed compositions.
[0172] As used herein, "an effective amount" would be understood by
those of ordinary skill in the art to provide a therapeutic,
prophylactic, or otherwise beneficial effect to a recipient
patient.
[0173] The phrases "isolated" or "biologically pure" refer to
material that is substantially, or essentially, free from
components that normally accompany the material as it is found in
its native state. Thus, isolated polynucleotides in accordance with
the invention preferably do not contain materials normally
associated with those polynucleotides in their natural, or in situ,
environment.
[0174] "Link" or "join" refers to any method known in the art for
functionally connecting one or more proteins, peptides, nucleic
acids, or polynucleotides, including, without limitation,
recombinant fusion, covalent bonding, disulfide bonding, ionic
bonding, hydrogen bonding, electrostatic bonding, and the like.
[0175] As used herein, the term "plasmid" or "vector" refers to a
genetic construct that is composed of genetic material (i.e.,
nucleic acids). Typically, a plasmid or a vector contains an origin
of replication that is functional in bacterial host cells, e.g.,
Escherichia coli, and selectable markers for detecting bacterial
host cells including the plasmid. Plasmids and vectors of the
present invention may include one or more genetic elements as
described herein arranged such that an inserted coding sequence can
be transcribed and translated in a suitable expression cells. In
addition, the plasmid or vector may include one or more nucleic
acid segments, genes, promoters, enhancers, activators, multiple
cloning regions, or any combination thereof, including segments
that are obtained from or derived from one or more natural and/or
artificial sources.
[0176] The term "a sequence essentially as set forth in SEQ ID
NO:X" means that the sequence substantially corresponds to a
portion of SEQ ID NO:X and has relatively few nucleotides (or amino
acids in the case of polypeptide sequences) that are not identical
to, or a biologically functional equivalent of, the nucleotides (or
amino acids) of SEQ ID NO:X. The term "biologically functional
equivalent" is well understood in the art, and is further defined
in detail herein. Accordingly, sequences that have about 85% to
about 90%; or more preferably, about 91% to about 95%; or even more
preferably, about 96% to about 99%; of nucleotides that are
identical or functionally equivalent to one or more of the
nucleotide sequences provided herein are particularly contemplated
to be useful in the practice of the invention.
[0177] Suitable standard hybridization conditions for the present
invention include, for example, hybridization in 50% formamide,
5.times.Denhardts' solution, 5.times.SSC, 25 mM sodium phosphate,
0.1% SDS and 100 .mu.g/ml of denatured salmon sperm DNA at
42.degree. C. for 16 h followed by 1 hr sequential washes with
0.1.times.SSC, 0.1% SDS solution at 60.degree. C. to remove the
desired amount of background signal. Lower stringency hybridization
conditions for the present invention include, for example,
hybridization in 35% formamide, 5.times.Denhardts' solution,
5.times.SSC, 25 mM sodium phosphate, 0.1% SDS and 100 .mu.g/ml
denatured salmon sperm DNA or E. coli DNA at 42.degree. C. for 16 h
followed by sequential washes with 0.8.times.SSC, 0.1% SDS at
55.degree. C. Those of skill in the art will recognize that
conditions can be readily adjusted to obtain the desired level of
stringency.
[0178] Naturally, the present invention also encompasses nucleic
acid segments that are complementary, essentially complementary,
and/or substantially complementary to at least one or more of the
specific nucleotide sequences specifically set forth herein.
Nucleic acid sequences that are "complementary" are those that are
capable of base-pairing according to the standard Watson-Crick
complementarity rules. As used herein, the term "complementary
sequences" means nucleic acid sequences that are substantially
complementary, as may be assessed by the same nucleotide comparison
set forth above, or as defined as being capable of hybridizing to
one or more of the specific nucleic acid segments disclosed herein
under relatively stringent conditions such as those described
immediately above.
[0179] As described above, the probes and primers of the present
invention may be of any length. By assigning numeric values to a
sequence, for example, the first residue is 1, the second residue
is 2, etc., an algorithm defining all probes or primers contained
within a given sequence can be proposed:
[0180] n to n+y, where n is an integer from 1 to the last number of
the sequence and y is the length of the probe or primer minus one,
where n+y does not exceed the last number of the sequence. Thus,
for a 25-basepair probe or primer (i.e., a "25-mer"), the
collection of probes or primers correspond to bases 1 to 25, bases
2 to 26, bases 3 to 27, bases 4 to 28, and so on over the entire
length of the sequence. Similarly, for a 35-basepair probe or
primer (i.e., a "35-mer), exemplary primer or probe sequence
include, without limitation, sequences corresponding to bases 1 to
35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the
entire length of the sequence. Likewise, for 40-mers, such probes
or primers may correspond to the nucleotides from the first
basepair to bp 40, from the second bp of the sequence to bp 41,
from the third bp to bp 42, and so forth, while for 50-mers, such
probes or primers may correspond to a nucleotide sequence extending
from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp
4 to bp 53, and so forth.
[0181] In certain embodiments, it will be advantageous to employ
one or more nucleic acid segments of the present invention in
combination with an appropriate detectable marker (i.e., a
"label,"), such as in the case of employing labeled polynucleotide
probes in determining the presence of a given target sequence in a
hybridization assay. A wide variety of appropriate indicator
compounds and compositions are known in the art for labeling
oligonucleotide probes, including, without limitation, fluorescent,
radioactive, enzymatic or other ligands, such as avidin/biotin,
etc., which are capable of being detected in a suitable assay. In
particular embodiments, one may also employ one or more fluorescent
labels or an enzyme tag such as urease, alkaline phosphatase or
peroxidase, instead of radioactive or other environmentally
less-desirable reagents. In the case of enzyme tags, colorimetric,
chromogenic, or fluorogenic indicator substrates are known that can
be employed to provide a method for detecting the sample that is
visible to the human eye, or by analytical methods such as
scintigraphy, fluorimetry, spectrophotometry, and the like, to
identify specific hybridization with samples containing one or more
complementary or substantially complementary nucleic acid
sequences. In the case of so-called "multiplexing" assays, where
two or more labeled probes are detected either simultaneously or
sequentially, it may be desirable to label a first oligonucleotide
probe with a first label having a first detection property or
parameter (for example, an emission and/or excitation spectral
maximum), which also labeled a second oligonucleotide probe with a
second label having a second detection property or parameter that
is different (i.e., discreet or discernable from the first label.
The use of multiplexing assays, particularly in the context of
genetic amplification/detection protocols are well-known to those
of ordinary skill in the molecular genetic arts.
EXAMPLES
[0182] The following examples are included to demonstate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--AAV-Mediated Gene Therapy Restores Visual Function and
Behavior to a Mouse Model of LCA1
[0183] In this example, the inventors evaluated whether delivery of
a species-specific version of retGC1 (i.e., murine) to cone cells
of the postnatal GC1KO mouse could restore function to these cells.
Serotype 5 AAV vectors were used to deliver mGC1 to photoreceptors
of postnatal day 14 (P14) GC1KO mice. Electroretinogram (ERG) and
behavioral testing were used to assess visual function and
immunocytochemistry was used to examine therapeutic transgene
expression, cone arrestin localization and cone photoreceptor
densities in treated and untreated eyes.
[0184] This example demonstrates that an AAV vector subretinally
delivered to one eye of P14 GC1 KO mice facilitated expression of
wild type retGC1, restoration of visual function and behavior, and
preservation of cone photoreceptors. Four weeks following
injection, visual function (ERG) was analyzed in treated and
untreated eyes. ERG was performed every two weeks thereafter until
3 months post injection (the latest time point evaluated). Mice
with positive ERG responses as well as isogenic wild type and
un-injected control mice were evaluated for restoration of visual
behavior using optokinetic reflex testing. At 3 months post
injection, all animals were sacrificed and their treated and
untreated retinas were evaluated for expression of GC1 and
localization of cone arrestin.
[0185] The results also confirm that cone-mediated function was
restored to treated eyes of GC1KO mice (ERG amplitudes were
.about.60% of normal). Moreover, the treatment effect was stable
for at least 3 months post-administration. Behavior testing
revealed robust improvements in cone-mediated visual behavior, with
responses of treated mice being similar or identical to that of
wild type mice. Histology revealed AAV-mediated GC1 expression in
photoreceptors and a restoration of cone arrestin translocation in
treated mice. In addition, cone cell densities were higher in
treated eyes than untreated contralateral controls. This result
suggests that treatment is capable of preserving cone
photoreceptors for at least three months post treatment. This is
the first demonstration that postnatal gene therapy is capable of
restoring visual function and behavior to, and preserving retinal
structure in, a mammalian model of LCA1. Importantly, results were
obtained using a well characterized, clinically relevant AAV
vector, the in vivo animal model data thus obtained provide the
foundation for an AAV-based gene therapy vector for treatment of
children affected with LCA1.
[0186] Materials and Methods:
[0187] Experimental Animals:
[0188] GC1 +/- heterozygote embryos were removed from a
cryopreserved stock at The Jackson Laboratory (Bar Harbor, Me.,
USA). Heterozygotes were mated at the inventors' facilities to
produce GC1 KO (-/-) and isogenic +/+ control offspring. All mice
were bred and maintained in a centralized facility at the
inventors' institution under a 12 hr/12 hr light/dark cycle. Food
and water were available ad libitum. All animal studies were
approved by the local Institutional Animal Care and Use Committee
and conducted in accordance with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and NIH regulations.
[0189] Construction of AAV Vectors:
[0190] Serotype 5 Adeno-associated virus (AAV5) vectors were used
to deliver murine GC1 (mGC1) as they have been shown to exhibit
robust transduction efficiency and a faster onset of expression in
retinal photoreceptors than other AAV serotypes (Yang et al.,
2002). Both a cell-specific and a ubiquitous promoter were selected
to drive expression of mGC1. The cell-specific, G protein-coupled
receptor kinase 1 (GRK1), also known as rhodopsin kinase promoter
was chosen for its ability to specifically target robust transgene
expression in rod and cone photoreceptors when used in conjunction
with AAV (Khani et al., 2007). The ubiquitous smCBA promoter which
exhibits a similar expression pattern to full-length CBA in retina
was chosen for its ability to efficiently target the neural retina
(Haire et al., 2006). Polymerase chain reaction utilizing the
following forward primer:
TABLE-US-00001 (SEQ ID NO: 14)
5'-AAAAGCGGCCGCATGAGCGCTTGGCTCCTGCCAGCC-3'
and the following reverse primer:
TABLE-US-00002 (SEQ ID NO: 15)
5'-AAAAGCGGCCGCTCACTTCCCAGTAAACTGGCCTGG-3'
was used to amplify mGC1 from a plasmid containing a mGC1-eGFP
fusion (Bhowmick et al., 2009). The resulting fragment was cloned
into pCRblunt plasmid (Invitrogen, Carlsbad, Calif., USA) and
sequence verified. AAV vector plasmid containing smCBA driving
expression of mGC1 (pTR-smCBA-mGC1) was created by replacing
full-length CBA with smCBA in plasmid pTR-CB.sup.SB-hRPE65
(Jacobson et al., 2006) via EcoRI digestion and subsequent
ligation. Subsequently, hRPE65 was replaced with mGC1 via NotI
digestion and ligation, resulting in the creation of pTR-smCBA-mGC1
(FIG. 11A and FIG. 11B). An AAV vector plasmid containing human
GRK1 promoter driving expression of mGC1, pTR-GRK1-mGC1 was created
by removing hGFP from pTR-hGRK1-hGFP (Beltran et al., 2010) and
replacing it with mGC1 via NotI digest and ligation (FIG. 11A and
FIG. 11B). AAV vectors were packaged according to previously
published methods (Haire et al., 2006). Viral particles were
resuspended in Balanced Salt Solution (Alcon, Fort Worth, Tex.,
USA) and titered by quantitative real-time PCR (Jacobson et al.,
2006). Resulting titers were 4.69.times.10.sup.12 viral genomes per
mL (vg/mL) and 4.12.times.10.sup.1 vg/mL for AAV5-smCBA-mGC1 and
AAV5-hGRK1-mGC1, respectively.
[0191] Subretinal Injections:
[0192] One .mu.L of AAV5-GRK1-mGC1 (4.12.times.10.sup.10 delivered
vector genomes) or AAV5-smCBA-mGC1 (4.69.times.10.sup.9 delivered
vector genomes) was delivered subretinally at postnatal day 14
(P14) to the right eye of each GC1KO mouse, leaving the left eye as
a contralateral control. Subretinal injections were performed as
previously described (Timmers et al., 2001; Pang et al., 2006).
Further analysis was carried out only on animals which received
comparable, successful injections (>60% retinal detachment and
minimal complications). It is well established that the area of
retinal detachment corresponds to the area of viral transduction
(Cideciyan et al., 2008; Timmers et al., 2001).
[0193] Electroreinographic Analysis:
[0194] Electroretinograms (ERGs) of treated GC1 KO (n=14) and
isogenic +/+ controls (n=2) were recorded using a PC-based control
and recording unit (Toennies Multiliner Vision; Jaeger/Toennies,
Hochberg, Germany) according to methods previously described with
minor modifications (Haire et al., 2006). Initial ERG measurements
were recorded at 4 weeks' post-injection, and each subsequent 2
weeks thereafter, until 3 months' post-injection (the latest time
point evaluated in the study). Age matched +/+ isogenic controls
were recorded alongside treated animals at every time point. Mice
were dark-adapted overnight (more than 12 hours) and anesthetized
with a mixture of 100 mg/kg ketamine, 20 mg/kg xylazine and saline
in a 1:1:5 ratio, respectively. Pupils were dilated with 1%
tropicamide and 2.5% phenylephrine hydrochloride. A heated
circulating water bath was used to maintain the body temperature at
38.degree. C. Hydroxypropyl methylcellulose 2.5% was applied to
each eye to prevent corneal dehydration. Full-field ERGs were
recorded using custom, gold wire loop corneal electrodes. Reference
and ground electrodes were placed subcutaneously between the eyes
and in the tail, respectively. Scotopic rod recordings were
elicited with a series of white flashes of seven increasing
intensities (0.01 mcds/m.sup.2 to 5 cds/m.sup.2). Interstimulus
intervals for low intensity stimuli were 1.1 second. At the three
highest intensities (100 mcds/m.sup.2, 1 cds/m.sup.2 and 5
cds/m.sup.2), interstimulus intervals were 2.5, 5.0 and 20.0
seconds, respectively. Ten responses were recorded and averaged at
each intensity. Mice were then light adapted to a 100 cds/m.sup.2
white background for 2 min. Photopic cone responses were elicited
with a series of five increasing light intensities (100
mcds/m.sup.2 to 12 cds/m.sup.2). Fifty responses were recorded and
averaged at each intensity. All stimuli were presented in the
presence of the 100 cds/m.sup.2 background. B-wave amplitudes were
defined as the difference between the a-wave troughs to the
positive peaks of each waveform.
[0195] Photopic b-wave maximum amplitudes (those generated at 12
cds/m.sup.2) of all smCBA-mGC1-treated (n=6) and hGRK1-mGC1-treated
(n=8) GC1KO (both treated and untreated eyes) and isogenic +/+
control mice were averaged and used to generate standard errors.
These calculations were made at every time point (4 weeks'-13
weeks' post-injection). This data was imported into Sigma Plot for
final graphical presentation. The paired t-test was used to
calculate P-values between treated and untreated eyes within each
promoter group (smCBA or hGRK1) and between each promoter group
over time (4 weeks post-injection vs. 3 months' post-injection).
The standard t-test was used to calculate P-values between
smCBA-mGC1 vs. hGRK1-mGC1 treated eyes. Significant difference was
defined as a P-value<0.05. Because some of the mice from each
treated group were temporarily removed from the study for
behavioral analyses, the total number of mice averaged and
presented at each time point in FIG. 2A and FIG. 2B differs. Three
mice from the smCBA-mGC1-treated group were sent for optomotor
testing, leaving an "n" of 3 mice used for ERG analysis during the
8, 10 and 12 week measurements (FIG. 2A). Two mice from the
hGRK1-mGC1-treated group were sent for optomotor testing, leaving
an "n" of 6 used for ERG analysis during the 6, 8, 10 and 12 week
measurements (FIG. 2B). All mice sent for behavioral analysis were
measured at 13 weeks' post-injection upon their return to the
inventors' laboratories (smCBA-mGC1: n=3, hGRK1-mGC1: n=2)
following completion of behavioral analyses.
[0196] Optomotor Testing:
[0197] Photopic visual acuities and contrast sensitivities of
treated and untreated GC1KO mouse eyes were measured using a
two-alternative forced choice paradigm as described previously (see
e g., Umino et al., 2008; Alexander et al., 2007). To test the
sensitivity of individual eyes from the same animal we took
advantage of the fact that mouse vision has minimal binocular
overlap and that the left eye is more sensitive to clockwise
rotation and the right to counter-clockwise rotation (Douglas et
al., 2005). Thus in the inventors' "randomize-separate" optomotor
protocol, each eye's acuity and contrast sensitivity threshold was
determined separately and simultaneously via stepwise functions for
correct responses in both the clockwise and counter-clockwise
directions. Correct detection of patterns rotating in the clockwise
direction was driven primarily by visual signals originating from
the left eye and correct responses in the counterclockwise
direction were derived from visual signals originating from the
right eye. Acuity was defined the highest spatial frequency (100%
contrast) yielding a threshold response, and contrast sensitivity
was defined as 100 divided by the lowest percent contrast yielding
a threshold response. For photopic acuity, the initial stimulus was
a 0200 cycles/degree sinusoidal pattern with a fixed 100% contrast.
For photopic contrast sensitivity measurements, the initial pattern
was presented at 100% contrast, with a fixed spatial frequency of
0.128 cycles/degree. Photopic vision was measured at a mean
luminance of 70 cd/m.sup.2. Visual acuities and contrast
sensitivities were measured for both eyes of each mouse four to six
times over a period of 1 week. Age matched, isogenic +/+ control
animals (M1, M2) and naive GC1KO mice (M3, M4) are presented along
with the smCBA-mGC1-treated (M5, M6, M7) and hGRK1-mGC1-treated
mice (M8, M9) in FIG. 3. Cone-mediated ERG amplitudes generated
from a 12 cds/m.sup.2 stimulus of all mice (M1-M9) are presented
alongside the behavior results. Unpaired t-tests were carried out
on acuity and percent contrast values to determine significance of
results.
[0198] Tissue Preparation:
[0199] Three months' post-injection, P14-treated GC1KO mice and age
matched isogenic +/+ controls were dark adapted for 2 hr.
Immediately following dark adaptation, mice were sacrificed under
dim red light (>650 nm). The limbus of injected and un-injected
eyes was marked with a hot needle at the 12:00 position,
facilitating orientation. Enucleation was performed under dim red
light and eyes were placed immediately in 4% paraformaldehyde. Eyes
that were to be used for cryosectioning were prepared according to
previously described methods (Haire et al., 2006). Briefly, corneas
were removed from each eye, leaving the lens inside the remaining
eye cup. A small "V" shaped cut was made into the sclera adjacent
to the burned limbus to maintain orientation. After overnight
fixation, the lens and vitreous were removed. The remaining
retina/RPE-containing eyecup was placed in 30% sucrose in PBS for
at least 1 hr at 4.degree. C. Eyecups were then placed in cryostat
compound (Tissue Tek OCT 4583; Sakura Finetek, Inc., Torrance,
Calif., USA) and snap-frozen in a bath of dry ice/ethanol. Eyes
were serially sectioned at 10 .mu.m with a cryostat (Microtome
HM550; Walldorf, Germany). Eyes that were to be used for whole
mount analysis were prepared according to previously described
methods (Pang et al., 2010). Orientation was achieved as previously
mentioned. After overnight fixation, cornea, lens, vitreous and
retinal pigment epithelia were removed from each eye without
disturbing the retina. A cut was made in the superior (dorsal)
portion of the retina adjacent to the original limbus burn to
maintain orientation.
[0200] Immunohistochemistry and Microscopy:
[0201] Retinal cryosections and whole mounts were washed 3.times.
in 1.times.PBS. Following these washes, samples were incubated in
0.5% Triton X-100.RTM. for 1 hr in the dark at room temperature.
Next, samples were blocked in a solution of 1% bovine serum albumin
(BSA) in PBS for 1 hr at room temperature. Retinal sections were
incubated overnight at 37.degree. C. with a rabbit polyclonal GC1
antibody (1:200, sc-50512, Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif., USA) or rabbit polyclonal cone arrestin antibody
("Lumij" 1:1000; provided by Dr. Cheryl Craft, University of
Southern California, Los Angeles, Calif., USA) diluted in 0.3%
Triton X-100.RTM./1% BSA. Retinal whole mounts were incubated
overnight at room temperature with the same cone arrestin antibody,
diluted 1:1000 in 0.3% Triton X-100.RTM./1% BSA. Following primary
incubation, retinal sections and whole mounts were washed 3.times.
with 1.times.PBS.
[0202] Retinal sections were incubated for 1 hr at room temperature
with IgG secondary antibodies tagged with either Alexa-594 or
Alexa-488 fluorophore (Molecular Probes, Eugene, Oreg., USA)
diluted 1:500 in 1.times.PBS. Following incubation with secondary
antibodies, sections and whole mounts were washed with 1.times.PBS.
Retinal sections were counterstained with
4',6'-diamino-2-phenylindole (DAPI) for 5 min at room temperature.
After a final rinse with 1.times.PBS and water, sections were
mounted in an aqueous-based medium (DAKO) and cover-slipped.
Retinal whole mounts were oriented on slides with the superior
(dorsal) portion of the retina positioned at the 12:00 position.
Samples were mounted in DAKO and cover-slipped.
[0203] Retinal sections were analyzed with confocal microscopy
(Leica TCS SP2 AOBS Spectral Confocal Microscope equipped with LCS
Version 2.61, Build 1537 software, (Bannockburn, Ill., USA). All
images were taken with identical exposure settings at either
20.times. or 63.times. magnification. Excitation wavelengths used
for DAPI, GC1 and cone arrestin stains were 405 nm, 488 nm, and 594
nm, respectively. Emission spectra were 440-470 nm, 500-535 nm and
605-660 nm, respectively. Retinal whole mounts were analyzed with a
widefield fluorescent microscope (Axioplan 2) (Zeiss, Thornwood,
N.Y., USA) equipped with a QImaging Retiga 4000R Camera and
QImaging QCapture Pro software (QImaging, Inc., Surrey, BC,
Canada). Quadrants of each whole mount were imaged at 5.times.
under identical exposure settings and then merged together in
Photoshop.RTM. (Version 7.0) (Adobe, San Jose, Calif., USA)
[0204] Image Analysis:
[0205] Cone photoreceptor densities were analyzed in retinal whole
mounts by counting cells labelled with secondary fluorophore
directed against cone arrestin antibody in the central and inferior
retina using ImageJ.RTM. software (National Institutes of Health,
Bethesda, Md., USA). These values were obtained by zooming in on
the 5.times.TIFF files shown in FIG. 6. Five squares (500
.mu.m.sup.2) were placed over identical areas in central and
inferior retina of both treated and untreated GC1KO eyes. For
central retina, squares were placed at an equal eccentricity around
the optic nerve head in all eyes (125 .mu.m). Cone photoreceptors
were counted in each respective retinal area, values were averaged
and standard deviations calculated. The standard t-test was used to
calculate P-values between desired samples. Significant difference
was defined as a P-value<0.05.
[0206] Results
[0207] Photoreceptor Function (ERG) was Restored in AAV-Treated
GC1KO Mice:
[0208] It was previously reported that cone responses in the GC1KO
mouse are barely detectable by 1 month of age. Here the inventors
have shown that P14-treatment of this mouse with an AAV vector
carrying the mouse GC1 gene under the control of either a
photoreceptor-specific (hGRK1) or ubiquitous (smCBA) promoter led
to substantial restoration of cone photoreceptor function as
measured by ERG. Representative cone traces (FIG. 1A and FIG. 1B)
(as well as the average photopic b-wave amplitudes (FIG. 2A and
FIG. 2B) from hGRK1-mGC1-treated, smCBA-mGC1-treated, GC1KO and
isogenic +/+ controls) showed that cone function in treated eyes
was restored to approximately 45% of normal at four weeks'
post-injection. Similar to previous reports, cone responses in
contralateral, untreated eyes were ablated by this time point. At 4
weeks' post-injection, the average cone-mediated b-wave amplitude
in smCBA-mGC1-treated eyes (65.1 .mu.V) was significantly higher
(P=0.006) than that in the untreated eyes (3.9 .mu.V). The average
cone mediated b-wave amplitude in hGRK1-mGC1-treated eyes (59.1
.mu.V) was significantly higher (P<0.001) than that in untreated
eyes (3.2 .mu.V). The level of restoration achieved four weeks'
post-delivery of the photoreceptor-specific hGRK1-mGC1 vector was
not significantly different from that achieved with the ubiquitous
promoter-containing smCBA-mGC1 vector (P=0.604). At 3 months'
post-injection, the average cone-mediated b-wave amplitude in
smCBA-mGC1-treated eyes (53.3 .mu.V) was significantly higher
(P<0.001) than that in the untreated eyes (2.8 .mu.V). The
average cone mediated b-wave amplitude in hGRK1-mGC1-treated eyes
(45.3 .mu.V) was significantly higher (P<0.001) than that in
untreated eyes (3.4 .mu.V). The level of restoration achieved 3
months following delivery of the photoreceptor-specific GRK1-mGC1
vector was not significantly different from that achieved with the
ubiquitous promoter-containing smCBA-mGC1 vector (P=0.331). Both
promoters conferred similar levels of functional restoration to
cones in treated eyes of the GC1KO mouse in the short term.
Importantly, restoration of cone photoreceptor function remained
stable for 3 months (the latest time point evaluated in this study
(see FIG. 1A, FIG. 1B, FIG. 2A and FIG. 2B). There was no
significant difference in photopic b-wave amplitudes of
smCBA-mGC1-treated or hGRK1-mGC1-treated eyes between 4 weeks and 3
months post treatment (P=0.174 and 0.125, respectively).
[0209] ERG implicit times which are an important feature in the
diagnosis of various retinal disorders including other forms of LCA
(Sun et al., 2010) were also determined. While no such measurement
can be obtained from a GC1KO eye (there are no ERG responses in
these eyes), it was possible to compare cone b-wave implicit times
in AAV-mGC1 treated and isogenic +/+ control mice. At 4 weeks post
injection, there was no significant difference between cone b-wave
implicit times in treated and +/+ control eyes (P=0.884); average
values in AAV-mGC1-treated and +/+ eyes at this time point were
50.8 ms and 50.4 ms, respectively. At 3 months post injection,
there was also no significant difference between the two groups
(P=0.697); averages of all cone b-wave implicit times in treated
and +/+ control eyes were 59.7 ms and 58.3 ms, respectively. The
response kinetics of cones in the treated GC1KO retina (as
determined by implicit time measurements) appeared to be normal and
stable in the short term.
[0210] It was previously reported that rod ERGs in the GC1 KO mouse
show alterations by 1 month of age, with the rod a-wave and b-wave
both markedly reduced (Yang et al., 1999). This reduction plateaus
at 5 months of age with responses approximately 50-70% that of a
wild-type (WT) mouse. While some instances of AAV-mGC1-mediated
improvements were observed in treated eyes of GC1KO mice relative
to untreated controls (example seen in FIG. 1A and FIG. 1B), this
result was not as consistent as that seen in the cone-mediated
responses.
[0211] Visual Behavior was Restored in AAV-Treated GC1KO Mice:
[0212] Optomotor analysis revealed that eyes of GC1KO mice treated
with either smCBA-mGC1 (M5, M6, M7) or hGRK1-mGC1 (M8, M9)
responded significantly better than untreated eyes under all
photopic, cone-mediated conditions. Untreated GC1KO eyes perform
poorly with a visual acuity of 0.163 t 0.040 cycles per degree
(FIG. 3B and FIG. 3C, bar, mean.+-.s.d., n=9 eyes). Isogenic
GC1.sup.+/+ control eyes (M1, M2) respond significantly better,
showing an average acuity of 0.418.+-.0.046 cycles per degree (n=4
eyes). AAV-mGC1-treated eyes (M5-M9) have an average acuity of
0.392.+-.0.077 cycles per degree (n=5 eyes), a level essentially
identical to control +/+ eyes and significantly better than
untreated GC1KO eyes (P<0.0001). Photopic contrast sensitivities
(FIG. 3B and FIG. 3C) paralleled the photopic acuity results, with
AAV-mGC1-treated eyes (contrast sensitivity of 11.9.+-.7.37, n=5
eyes) showing contrast thresholds nearly identical to +/+ mice
(11.94 t 3.03, n=4 eyes). Again, GC1KO eyes treated at P14 with
AAV-mGC1 performed significantly better than untreated eyes, which
showed an average contrast sensitivity of 1.27.+-.0.31 (n=9,
P<0.0001). In all photopic tests, untreated GC21 KO eyes perform
extremely poorly, essentially equivalent to no cone-mediated
function. Statistical comparisons of these measurements are shown
in Table 1. Cone-mediated ERG traces of all GC1 +(M1, M2), GC1KO
(M3, M4), smCBA-mGC1-treated (M5, M5, M7) and hGRK1-mGC1-treated
(M8. M9) mice used in behavior analysis are shown in FIG. 3A to
relate visual function (optmotor behavior) to retinal function
(electrophysiology).
[0213] Rod retinal function (ERG) is partially preserved in the
GC1KO mouse. Studies have shown that even very small ERG amplitudes
translate into robust visual behavior (Williams et al., 2006). In
fact, LCA2 patients who received AAV-RPE65 therapy were found to
exhibit behavioral restoration despite a complete lack of ERG
response (Maguire et al., 2008). Optomotor testing revealed that
scotopic, rod-mediated visual acuities and contrast sensitivities
of GC1KO eyes are very similar to +/+ controls. For this reason, it
was impossible to compare visual restoration of treated vs.
untreated eyes on a behavioral level. Statistical comparisons of
these measurements are shown in Table 1:
TABLE-US-00003 TABLE 1 STATISTICAL COMPARISON OF THE PHOTOPIC
VISUAL FUNCTIONS OF WT, AAV-MGC1-TREATED AND UNTREATED GC1KO EYES
AS MEASURED BY OPTOMOTOR BEHAVIOR Photopic Acuity Wild Type(WT)
Treated Untreated Number of Values 4 5 9 Mean 0.4483 0.3919 0.163
Standard Deviation 0.0456 0.07731 0.03954 P-value WT vs. Treated
0.5671 Not significant WT vs, Untreated <0.0001 * Treated vs.
Untreated <9.9001 * Photopic Contrast Sensitivity WT Treated
Untreated Number of Values 4 5 9 Mean 1.1.94 11.16 1.27 Standard
Deviation 3.03 7.37 0.31 P-value WT vs, Treated 0.4186 Not
significant WT is. Untreated <9.9001 * Treated vs. Untreated
<0.0001 * * = P < 0.0001
[0214] Photoreceptor-Specific and Ubiquitous Promoters Both Drive
mGC1 Transgene Expression in Rods and Cones of GC1KO Mice:
[0215] GC1-deficiency affects both rod and cone photoreceptors in
LCA1 patients. The photoreceptor-specific human RK promoter and the
ubiquitous smCBA promoter were therefore chosen for this study as a
means of targeting both cell types. The human RK promoter was
chosen for its small size and ability to efficiently drive
transgene expression specifically in photoreceptor cells.
Immunostaining of GC1KO retinas 3 months' post-treatment with
AAV-hGRK1-mGC1 revealed that this promoter drove robust GC1
expression in photoreceptor outer segments. A representative image
of a retinal cross section from an eye injected with this
therapeutic vector (FIG. 4A) shows intense GC1 staining in the OS
layer whereas the contralateral, untreated eye from the same mouse
lacks any GC1 expression (FIG. 4B). The smCBA promoter also
efficiently drove GC1 expression in photoreceptor cells.
Photoreceptor OS exhibited robust smCBA-mediated GC1 expression in
treated eyes (FIG. 4C), relative to the contralateral, untreated
eye (FIG. 4D). Levels of hGRK1 and smCBA-mediated GC1 expression
approached that seen in isogenic, +/+ control eyes (FIG. 4E). GC1
expression in hGRK1-mGC1-treated eyes was restricted to OS. In
smCBA-mGC1-treated eyes, GC1 expression was occasionally found in
photoreceptor cell bodies of the outer nuclear layer (see e.g.,
arrows FIG. 4F). Notably however, neither promoter construct drove
therapeutic GC1 expression outside the photoreceptor cells. This
lack of off-target expression is relevant to the development of
future clinical applications.
[0216] Cone Arrestin Translocation was Restored in AAV-mGC1-Treated
GC1KO Mice:
[0217] AAV-mGC1 treatment restored light-induced cone arrestin
translocation to cone photoreceptors in the treated GC1KO retina.
Representative treated, untreated and +/+ retinal cross sections
immunostained with an antibody generated against cone arrestin
showed that cone arrestin was localized to the outer segments,
inner segments, axons and synaptic termini of +/+,
smCBA-mGC1-treated and hGRK1-mGC1-treated cone photoreceptors (FIG.
5A, FIG. 5C, and FIG. 5D, respectively). On the contrary, cone
arrestin remained localized mostly to the outer segments of cones
in untreated GC1KO retinas (FIG. 5B). This result was consistent
with the notion that cones in the GC1KO mouse retina are
chronically hyperpolarized. Not only was a restoration of cone
arrestin localization in dark-adapted, treated retinas observed,
but an apparent up-regulation of the protein was seen in treated
eyes relative to untreated controls. Significantly, cone cell
densities also appeared higher in treated eyes relative to
untreated controls (see e.g., FIG. 5A. FIG. 5B, and FIG. 5C).
[0218] Cone Photoreceptors were Preserved in AAV-mGC1-Treated GC1KO
Mice:
[0219] Analysis of smCBA-mGC1 and hGRK-mGC1 treated and
un-injected, contralateral retinal whole mounts 3 months'
post-injection with therapeutic vector that were stained with an
antibody directed against cone arrestin revealed that cone
photoreceptors were preserved as a result of treatment with the
therapeutic vector (FIG. 6). Counts of cone photoreceptors in
inferior and central retinas of both treated and untreated retinal
whole mounts revealed that there was a statistically significant
difference in the cone cell densities of treated vs. untreated
eyes. This result was consistent with the observation that robust
electrophysiological and behavioral restoration was clearly
evident. P14-treatment of GC1KO mice with either therapeutic
construct was capable of preserving cone photoreceptor structure
for at least three months.
Example 2--Animal Model Containing a GC1/GC2 Double Knockout
[0220] It is important to note that while only cone photoreceptors
are affected in the GC1KO mouse (rods only lose partial function
and they do not degenerate), LCA1 patients exhibit rod function
loss and rod degeneration. The reason for this difference is
speculated to be a species-specific difference in the dependence on
GC2, a close relative of GC1 that is expressed in rod
photoreceptors. Mouse rods are able to function in the absence of
GC1 presumably because GC2 is capable of reconstituting activity;
however in humans this is not the case. GC1 is required for rod
function, hence the rod degeneration. A GC1/GC2 double knockout
mouse model was generated and shown to have rod function loss (in
addition to cone function loss as seen in the GC1 K/O) (Baehr et
al., 2007). It was proven through biochemical studies with this
model that GC2 is what provides rod function in the absence of GC1.
Having said that, it is the GC1/GC2 double knockout mouse that more
reliably mimics the human condition (both cones and rods affected)
(Karan et al., 2010). To test both the rAAV-smCBA-mGC1 and
rAAV-hGRK1-mGC1 vectors in the GC1/GC2 double knock-out mouse, rAAV
vectors are delivered in precisely the same manner and time
(post-natal day 14) as with the aforementioned GC knock-out study.
Analysis of restoration of vision, both physiologically and
behaviorally, is also performed in the same manner as described
above for the GC1 knock-out study. Particular emphasis is paid to
scotopic (i.e., rod) responses, as a measurable recovery of rod
function is expected in GC1/GC2 double knock-out mice treated with
a GC1 vector construct.
Example 3-`Humanized` Murine Animal Model of LCA1
[0221] This example describes the creation of a "humanized" murine
animal model of LCA1. In one embodiment, the mouse model contains a
GC1/GC2/GCAP1 knockout. GCAP1 is the protein that activates GC1. To
create an in-vivo system in which human GC1 expressed from a
clinical grade rAAV vector designed for use in humans can be
evaluated for function, a GC1/GC2/GCAP1 triple knockout hGCAP1
transgenic mouse is utilized. In this mouse, visual function is
resorted by rAAV-mediated hGC1 interacting with hGCAP1 only (i.e.,
no endogenous murine GCAP1 is present). From this study, it is
possible to determine whether the human CrCAP1 protein is required
to stimulate human GC1 activity in the mouse model, and whether
function can be restored to cones and rods when the two human
polypeptides are reconstituted and expressed in the non-human
(i.e., murine) model of the disease. To generate the GC1/GC2/GCAP1
triple knockout hGCAP1 transgenic mouse the GC1/GC2 double knockout
mouse (Baehr et al., 2007) is crossed with the GCAP1 knock-out
mouse (Mendez et al., 2001). Human GCAP1 is then
transgenically-expressed in the animal model to generate a
GC1/GC2/GCAP1 triple knockout hGCAP1 transgenic mouse. Studies in
which rAAV-vectored hGC1 is provided to these animals are conducted
in a manner substantially identical to the methods used in the GC1
knock-out study. Analysis of restoration of vision, both
physiologically and behaviorally, is then performed in the same
manner as was carried in the GC1 knock-out study.
Example 4--Exemplary Vector Constructs Useful in the Practice of
the Invention
[0222] Maps of the two illustrative vectors are shown in FIG. 11A
and FIG. 11B. One contains the nonspecific promoter smCBA and the
other has the rod/cone limited promoter GRK1. Both have been
packaged into serotype 5 AAV. All vector doses tested to date are
safe in the mouse retina. Cohorts of GC1 -/- mice were then
sub-retinally injected at postnatal day 14 (P14) and then analyzed
periodically by ERG and by photopic optokinetic (cone mediated)
behavior. Since the GC1.sup.1 mouse maintains a rod mediated ERG,
monitoring functional rescue focused primarily on restoration of
cone function. For the smCBA vector ERGs were assessed at 4 weeks
post-treatment and every 2 weeks thereafter until 12-13 weeks
post-treatment. All 9 eyes treated in 9 mice responded to
treatment. The results, shown below demonstrate a significant
restoration of photopic ERG amplitudes from essentially
unrecordable in control untreated eyes to approximately 50% of
normal in partner vector treated eyes.
[0223] Four GC1.sup.-/- mice were then analyzed by scotopic
optokinetic behavior for differences mediated by treated vs.
untreated partner eyes (shown below). All four treated eyes (289,
290, 294, 295, red bars) showed significant improvement in visual
acuity over their control eyes, and three of the four showed
significantly improved contrast sensitivity. Mice 297 and 298 were
wild type controls, and mouse 299 was an untreated GC1.sup.-/-
mouse. The results demonstrate that the vector achieved functional
and behavioral restoration of cone mediated vision in the animal
model of LCA1.
[0224] For the GRK1 vector that limits expression to rods and
cones, ERGs were assessed in 14 GC1.sup.-/- mice treated in one eye
at 4 weeks post-treatment and every 2 weeks thereafter until 12-13
weeks post-treatment. Twelve of the 14 treated eyes in 12 animals
responded. The results (shown below) revealed a significant
restoration of photopic ERG amplitudes from essentially
unrecordable in control untreated eyes to approximately 40% of
normal in partner vector treated eyes.
[0225] One GC1.sup.-/- mouse (#293) was then analyzed by scotopic
optokinetic behavior for differences in treated vs. untreated
partner eyes (shown above). This mouse showed significant
improvement in both visual acuity and contrast sensitivity in the
vector treated right eye (red bar) relative to its control left eye
(blue bar). Responses were nearly equivalent to control wild type
mice (297 and 298) and significantly improved over an untreated
GC1-/- mouse (299). It was therefore concluded that the GRK1 vector
also achieves functional and behavioral restoration of cone
mediated vision in this model of LCA1.
Example 5--Specific Cone Targeting of wtGC1 Improves Rescue
[0226] The data presented above clearly demonstrate that cone
function and cone-mediated behavior can be rescued with the
rod/cone limited GRK1 promoter. Since human LCA1 shows both rod and
cone deficits (unlike the GC1.sup.-/- mouse that shows primarily
cone deficits), expression need not be further limited to gain pure
cone specificity. However, there is one final cone phenotype in the
mouse model which is important to study; in dark adapted
conditions, cone arrestin does not move normally from cone outer
segments into inner segments, axons and synaptic termini as it does
in the wild type retina. Studies were therefore performed to assess
whether this cell biological phenotype was also corrected in
vector-treated GC1.sup.-/- eyes. In the results shown, a
GC1.sup.-/- mouse was treated in one eye with the GRK1 vector, then
at 7 weeks post-injection the mouse was dark adapted. Treated
(bottom panel) and control (top panel) retinas were then analyzed
for cone arrestin localization by immunohistochemistry. In the
untreated GC1.sup.-/- retina (top panel), cone arrestin remained
largely in cone outer segments (OS) and the synaptic layer (SL). In
contrast, in the contralateral treated retina (bottom panel) a
substantial fraction (.about.50%) has translocated into the inner
segments and synaptic termini. It was concluded, therefore, that
vector treatment also restored correct translocation of cone
arrestin.
Example 6--Long-Term Therapy of LCA1 Using rAAV-Vectored Genetic
Constructs
[0227] The previous examples have demonstrated that subretinal
injection of rAAV vectors containing the murine GC1 cDNA (driven by
either the photoreceptor-specific human rhodopsin kinase [hGRK1] or
the ubiquitous [smCBA] promoter) were capable of restoring
cone-mediated function and visual behavior and preserving cone
photoreceptors in the GC1KO mouse for at least three months.
[0228] In the present Example, the inventors evaluated whether
long-term therapy was also achievable in the rodent model of LCA1.
Additionally, the inventors examined whether delivery of GC to
photoreceptors of the GC1/GC2 double knockout mouse (GCdko), a
model which exhibits loss of both rod and cone structure and
function and phenotypically resembles human LCA1, would confer
therapy to these cells.
[0229] Methods
[0230] Subretinal injections of AAV5-hGRK1-mGC1, AAV5-smCBA-mGC1 or
the highly efficient capsid tyrosine mutant AAV8(Y733F)-hGRK1-mGC1
were performed in one eye of GC1KO or GCdko mice between postnatal
day 14 (P14) and P25. Rod and cone photoreceptor function were
assayed electroretinographically. Localization of therapeutic GC1
expression and extent of cone photoreceptor preservation were
determined by immunohistochemistry. Biodistribution studies were
used to evaluate the presence of vector genomes in optic nerves and
brains of treated animals.
[0231] Results
[0232] Cone photoreceptor function was restored in GC1KO mice
treated with all vectors, with AAV8(733) being the most efficient.
Responses were stable for at least 10 months post-treatment.
Therapeutic GC1 was found in photoreceptor outer segments. By 10
months post-injection, AAV5 and AAV8(733) vector genomes were
detected only in the optic nerves of treated eyes of GC KO mice.
AAV8(733)-vectored mGC1 restored function to both rods and cones in
treated GCdko mice.
CONCLUSION
[0233] Long-term therapy is achievable in a mammalian model of GC1
deficiency, the GC1KO mouse, using the rAAV vector constructs
disclosed herein. Importantly, therapy was also achievable in the
GCdko mouse which mimics the LCA1 rod/cone phenotype. These results
provide evidence for the use of rAAV-based gene therapy vectors for
treatment of retinal dystrophies, and LCA1 in particular.
Example 7--Long Term Preservation of Cone Photoreceptors and
Restoration of Cone Function by Gene Therapy in the GC1KO Mouse
[0234] In previous examples, it was shown that subretinal AAV5
vectors containing murine GC1 cDNA driven by either the
photoreceptor-specific (hGRK1) or the ubiquitous (smCBA) promoter
were capable of restoring cone-mediated function and visual
behavior and preserving cone photoreceptors in the GC1KO mouse for
three months. In the present example, long term therapy is
evaluated using the same murine model. AAV5-hGRK1-mGC1,
AAV5-smCBA-mGC1 or the highly efficient capsid tyrosine mutant
AAV8(Y733F)-hGRK1-mGC1 were delivered subretinally to GC1KO mice
between postnatal day 14 (P14) and postnatal day (P25). Retinal
function was assayed by electroretinograms (ERGs). Localization of
AAV-mediated GC1 expression and cone survival were assayed with
immunohistochemistry and the spread of vector genomes beyond the
retina was quantified by PCR of optic nerve and brain tissue. Cone
function was restored with all vectors tested, with AAV8(Y733F)
being the most efficient. AAV-mediated expression of GC1 was found
exclusively in photoreceptors. By 10 months post-injection, AAV
genomes were detected only in optic nerve of treated eyes. These
results demonstrate for the first time that long-term therapy is
achievable in a mammalian model of GC1 deficiency.
[0235] Retinal guanylate cyclase-1 (GC1) encoded by GUCY2D serves a
key function in vertebrate phototransduction (Pugh et al., 1997).
Following light stimulus, second messenger cyclic GMP (cGMP) is
rapidly hydrolyzed by phosphodiesterase (PDE6) within photoreceptor
cells leading to a closure of cGMP-gated cation channels and
hyperpolarization of the cell. When cytoplasmic [Ca.sup.2+] drops
below 50 nM, GC1 is activated by small Ca.sup.2+-binding proteins,
GCAPs (guanylate cyclase activating proteins). GC1 synthesizes cGMP
which binds and reopens cGMP-gated channels, returning the
photoreceptor to the "dark", depolarized state (Pugh et al., 1997;
Polans et al., 1996; Wensel, 2008; Lamb and Pugh, 2006; Arshavsky
et al, 2002). Thus, GC1 plays a vital role in the light-dark and
recovery cycles, anchoring, via cGMP, the feedback loop linking
intracellular calcium levels and the polarization state of
photoreceptors.
[0236] GC1 is expressed in the outer segments of rod and cone
photoreceptors of human, monkey and mouse retinas (Dizhoor et al.,
1994; Liu et al., 1994; Haire et al., 2006). Like other membrane
guanylate cyclases, it contains an N'-terminal signal sequence, an
extracellular domain (ECD), a single transmembrane domain, a
kinase-like homology domain (KHD), a dimerization domain (DD) and a
C'-terminal catalytic domain (CCD), and is present likely as
homomeric dimers (Yang and Garbers, 1997). Mutations in GUCY2D are
associated with recessive Leber congenital amaurosis-1 (LCA1) as
well as dominant and recessive forms of cone-rod dystrophy, CORD6
and CORD, respectively (Perrault et al., 1996; Perrault et al.,
2000; Kelsell et al., 1998; Perrault et al., 1998; Gregory-Evans et
al., 2000; Weigell-Weber et al., 2000; Ugur et al., 2010). LCA1 is
a severe, early onset, autosomal recessive blinding disorder
characterized by extinguished electroretinogram (ERG) which
precedes photoreceptor degeneration (Perrault et al., 1999; Chung
and Traboulsi, 2009). CORD6 is a dominant disorder characterized by
progressive degeneration of photoreceptors beginning with cones
causing early loss of visual acuity and color vision followed by
degeneration of rods leading to progressive night blindness and
peripheral visual field loss (Kelsell et al., 1998; Perrault et
al., 1998). CORD6 mutations are restricted to the dimerization
domain (DD) and generally cause an increase in GCAP-mediated
activation of GC1 (Payne et al., 2001; Downes et al., 2001; Wilkie
et al., 2000). A recently found recessive CORD-causing mutation is
located in the catalytic domain (CD) of GC1 and is thought to
reduce overall enzyme function (Ugur et al., 2010). LCA1-causing
mutations are distributed throughout the ECD, KHD, DD and CCD
domains of GC1 (Karan et al., 2010). These mutations alter enzyme
structure and stability, may impact retrograde transport of other
peripheral membrane associated proteins and are frequently
null.
[0237] The GC1KO mouse carries a null mutation in Gucy2e, the
murine homologue of GUCY2D. Like LCA1 patients, loss of cone
function in this model precedes cone degeneration (Timmers et al.,
2001). Rods retain 30-50% of their function and do not degenerate
due to the presence of GC2, another functional guanylate cyclase in
murine photoreceptors (Yang and Garbers, 1997; Jacobson et al.,
2006; Timmers et al., 2001; Cideciyan et al., 2008; Song et al.,
2002). In the earlier examples, it was shown that subretinal
injection of serotype 5 adeno-associated viral (AAV) vectors
containing the murine GC1 cDNA driven by either the
photoreceptor-specific human rhodopsin kinase (hGRK1) or the
ubiquitous (smCBA) promoter were capable of restoring cone-mediated
function and visual behavior and preserving cone photoreceptors in
the GC1KO mouse for three months. In the present study,
AAV-mediated gene replacement therapy was evaluated for its ability
to provide therapy to the GC1KO mouse over the long term.
AAV5-hGRK1-mGC1 and AAV5-smCBA-mGC1 and the highly efficient capsid
tyrosine mutant vector AAV8(Y733F)-hGRK1-mGC1 were delivered
subretinally to GC1KO mice between postnatal day 14 (P14) and
postnatal day 25 (P25). These findings demonstrate for the first
time that long-term therapy is achievable in a mammalian model of
GC1 deficiency. Vector genome biodistribution was also evaluated
for AAV5- and AAV8(733)-based vectors. These findings have direct
bearing on the development of an AAV-based gene therapy clinical
trial for LCA1 (and possibly cone-rod dystrophies), and help to
develop a standardized vector design for a wide range of recessive
retinal degenerations mediated by defects in
photoreceptor-associated genes.
[0238] Materials and Methods
[0239] Experimental Animals:
[0240] GC1KO and congenic +/+ controls derived from heterozygous
matings of GC1 +/- mice provided by The Jackson Laboratory (Bar
Harbor, Me., USA) were bred and maintained in the inventors'
institutional animal care facility under a 12 hr/12 hr light/dark
cycle. Food and water were available ad libitum. All studies were
conducted in accordance with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and NIH regulations.
[0241] Construction of AAV Vectors:
[0242] Serotype 5 Adeno-associated virus (AAV5) vector plasmids
containing either the ubiquitous (smCBA) or photoreceptor-specific
human rhodopsin kinase (hGRK1) promoter driving murine GC1 (mGC1)
cDNA were generated according to previously described methods (Boye
et al., 2010). Site-directed mutagenesis of surface-exposed
tyrosine residues on the AAV2 capsid have been reported (Zhong et
al., 2008). Similar methods were used to generate the AAV8(Y733F)
capsid mutant described here. All vectors were packaged, purified
and titered according to previously described methods (Zolotukhin
et al., 2002; Jacobson et al., 2006). Resulting titers for
AAV5-smCBA-mGC1, AAV5-hGRK1-mGC1 and AAV8(Y733F)-hGRK1-mGC1 were
4.69.times.10.sup.12 vector genomes per ml (vg/mL),
4.12.times.10.sup.13 vg/mL and 1.08.times.10.sup.13 vg/mL,
respectively.
[0243] Subretinal Injections:
[0244] One .mu.L of AAV5-smCBA-mGC1 (4.69.times.10.sup.9 vector
genomes). AAV5-hGRK1-mGC1 (4.12.times.10.sup.10 vector genomes) or
AAV8(Y733F)-hGRK1-mGC1 (1.08.times.10.sup.10 vector genomes) were
injected subretinally into one eye of GC1KO mice between postnatal
day 14 (P14) and postnatal day 25 (P25). The contralateral control
eye remained uninjected. Subretinal injections were performed as
previously described (Timmers et al., 2001). Further analysis was
carried out only on animals which received comparable, successful
injections (>60% retinal detachment with minimal complications).
Approximately 75% of all cohorts received "successful" injections.
It is well established that the area of vector transduction
corresponds to at least the area of retinal detachment (Timmers et
al., 2001; Cideciyan et al., 2008).
[0245] Electroretinographic Analysis:
[0246] ERGs of treated GC1KO and age-matched, congenic (+/+)
controls were recorded using a PC-based control and recording unit
(Toennies Multiliner Vision; Jaeger/Toennies, Hochberg, Germany)
according to methods previously described with minor modifications
(Haire et al., 2006; Boye et al., 2010). Recordings of
AAV5-smCBA-mGC1-treated GC1KO mice (n=10), AAV5-hGRK1-mGC1-treated
GC1KO mice (n=6), AAV8(Y733F)-treated GC1KO mice (n=6) and congenic
(+/+) controls (n=8) commenced on different dates and therefore
each subset of mice was monitored for slightly different lengths of
time. ERGs of treated GC1KO mice were recorded 4-weeks'
post-injection and every month thereafter until 1 year
post-injection (AAV5-treated mice) or 9 months post-injection
(AAV8[Y733F]-treated mice). Age-matched, congenic (+/+) control
mice were followed for 8 months. Mice were removed from the study
at different time points throughout the experiment for various
postmortem studies (biodistribution studies, retinal
immunohistochemical analysis, real time RT-PCR of retinal tissue)
or unexpected sickness/death. ERG data was presented only for
groups of animals with ann>3. Therefore, this study compares
findings out to 9 months post-injection for AAV5-treated mice and 6
months post-injection for AAV8(Y733F)-treated mice. Treated mice
continued to exhibit ERG responses beyond these time points,
however sample sizes were sufficiently reduced such that
statistical analysis was no longer practical. Representative
cone-mediated traces from individual mice 1 year post-treatment
with AAV5 vectors and 9 months post-treatment with AAV8(Y733F) are
presented to support this contention. Scotopic (rod-mediated) and
photopic (cone-mediated) recordings were elicited using recording
parameters previously described (Boye et al., 2010). B-wave
amplitudes were defined as the difference between the a-wave
troughs and the subsequent positive peak of each waveform.
Rod-mediated ERG responses in untreated GC1KO mice are variable
from animal to animal (Yang et al., 1999), hence, large standard
deviations were observed when averaging scotopic a- and b-wave
amplitudes from different animals. Rod ERG data is presented in
ratio form (the average of intra-individual, treated versus
untreated rod a- and b-wave amplitudes). As such, any value above 1
indicates AAV-mGC1 treatment improved the rod response. Ratios were
calculated using amplitudes generated with a 1 cds/m.sup.2
stimulus. Photopic, cone-mediated b-wave maximum amplitudes in
injected and uninjected eyes of all treated GC1KO mice and congenic
(+/+) control mice generated at 12 cds/m.sup.2 were averaged at
each time point and used to generate standard errors. All data was
imported into Sigma Plot for final graphical presentation. The
standard t-test was used to calculate P-values between data sets.
Significant difference was defined as a P-value<0.05.
[0247] Biodistribution:
[0248] The spread of vector DNA in tissues of the treated GC1KO
mice was determined in samples collected at sacrifice according to
previously described methods with minor modifications (Jacobson et
al., 2006). Vector-treated mice were sacrificed at the following
time points; AAV8(Y733F)-hGRK1-mGC1-treated mice (4-months'
post-injection: n=1; 7-months' post-injection: n=1),
AAV5-smCBA-mGC1 (7-months' post-injection: n=1; 10-months'
post-injection: n=5), AAV5-hGRK1-mGC1 (7-months' post injection:
n=1; 10-months' post-injection: n=1). Control tissues from GC1KO
mice age-matched to the 7-month-post injection or 10-month-post
injection time points were also evaluated alongside experimental
animals. Following sacrifice, different new forceps were used to
enucleate treated and untreated eyes which retained approximately
0.5 cm of proximal optic nerve. Different, new dissection scissors
were then used to cut the optic nerves away from the eyeballs after
which they were snap frozen in liquid nitrogen and transferred to
-80.degree. C. where they remained until the time of DNA
extraction. Eyeballs were immersed in 4% paraformaldehyde (PAF) and
processed for immunohistochemistry (see below).
[0249] Brains were removed and a stainless steel mouse coronal
brain matrix (Harvard Apparatus, Holliston, Mass., USA) was used to
isolate visual-specific regions. Right and left lateral geniculate
nuclei were collected from one mouse per treatment group (at the
latest time point), formalin fixed and saved in the event that
vector genomes were recovered from brain and immunohistochemistry
was necessary. Separate portions of right and left brain containing
visual pathways were collected, snap frozen in liquid nitrogen and
transferred to -80.degree. C. where they remained until the time of
DNA extraction. Precautions were taken to avoid cross-contamination
while harvesting tissues. Genomic DNA was extracted from tissues
according to the manufacturer's protocol (Qiagen DNeasy tissue
kit). Resulting DNA concentrations were determined using an
Eppendorf Biophotomoter (Model 6131; Eppendorf, Hamburg, Germany).
Quantitative PCRs were performed according to previously described
methods with minor modifications (Jacobson et al., 2006; Song et
al., 2002; Poirier et al., 2004).
[0250] Primer pairs were designed to the SV40 poly-adenylation
signal (SV40 polyA) region in each vector genome and standard
curves established using known concentrations of plasmid DNA
containing the same SV40 polyA target sequence. DNA samples were
assayed in triplicate. In order to rule out false negatives due to
inhibition of PCR, the third replicate was `spiked` with plasmid
DNA containing target (SV40 polyA) at a ratio of 100 copies/.mu.g
of genomic DNA. If >40 copies of the spike-in DNA were detected,
the sample was considered acceptable for reporting vector genome
copies. In some cases samples failing `spike in` were reanalyzed
using less than 1 .mu.g of genomic DNA in PCR reactions, thereby
diluting out PCR inhibitors copurifying with DNA in the extracted
tissue. Spike-in copy number was reduced proportionally to maintain
the 100 copies/.mu.g DNA ratio. Criteria for reporting vector
genome copies were established according to previously described
methods (Jacobson et al., 2006). Briefly, greater than 100 genome
copies/.mu.g was considered positive and the measured copy
number/.mu.g reported. Fewer than 100 copies/.mu.g was considered
negative.
[0251] Tissue Preparation, Immunohistochemistry and Microscopy:
[0252] At sacrifice, concomitant with biodistribution studies
performed at 7 months post-[AAV8(Y733F)-hGRK1-mGC1] and 10 months
post-(AAV5-smCBA-mGC1 and AAV5-hGRK1-mGC1) injection, the limbus of
treated GC1KO mice, age-matched, untreated GC1KO mice as well as
age-matched congenic GC1 +/+ mice were marked with a hot needle at
the 12 o'clock position, facilitating orientation. Untreated GC1KO
and GC1 +/+ controls were age-matched to the AAV8(Y733F)-treated
mice (8 months of age at the time of sacrifice). Eyes designated
for cryosectioning were processed and immunostained according to
previously described methods (Haire et al., 2006). Briefly, 10
.mu.m retinal sections were incubated with antibodies directed
against GC1 (rabbit polyclonal 1:200, sc-50512 Santa Cruz
Biotechnology, USA) or mouse cone arrestin (rabbit polyclonal
"LUMIj", 1:1000, provided by Dr. Cheryl Craft. University of
Southern California, Los Angeles, Calif., USA). Following primary
incubation, IgG secondary antibodies Alexa-488 or Alexa-594,
respectively, were applied for 1 hour at room temperature (1:500 in
IX PBS). Sections were counterstained with
4',6'-diamino-2-phenyl-indole (DAPI) for 5 min at room temperature.
At 11-months' post-injection, one GC1KO mouse that received
treatment with AAV5-smCBA-mGC1 in one eye only was sacrificed and
retinal whole mounts from treated and untreated eyes processed
according to previously described methods (Pang et al., 2010).
Briefly, whole mounts were stained with LUMIj (1:1000) followed by
IgG secondary Alexa-594 (1:500 in 1.times.PBS) and positioned on
slides with the superior (dorsal) portion of the retina oriented at
12-o'clock. Retinal sections were analyzed by confocal microscopy
(Leica TCS SP2 AOBS Spectral Confocal Microscope equipped with LCS
Version 2.61, Build 1537 software). Images were taken at identical
exposure settings at 20.times. magnification. Retinal whole mounts
were analyzed with a wide-field fluorescent microscope (Zeiss
Axioplan 2) equipped with QImaging Retiga 4000R Camera and QImaging
QCapture Pro software. Quadrants of each whole mount were imaged at
10.times. under identical exposure settings and then merged
together in Adobe Photoshop.
[0253] Immunoblotting:
[0254] At 7 months post-injection, one mouse injected with
AAV8(Y733F)-hGRK1-mGC1 and an age-matched, congenic GC1
+/+(control) mouse were sacrificed, their eyes enucleated and
placed in IX PBS. Retinas were immediately dissected and processed
as follows. Individual retinas were solubilized in PBS (137 mM
NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4)
with 1% Triton X-100 and complete protease inhibitor (Roche) for 1
hour at 4.degree. C., followed by centrifugation at 14000 rpm. The
protein concentration of the supernatant was determined by BCA
(Pierce) and 15 .mu.g of each sample was separated on a 12%
polyacrylamide gel (Bio-Rad) and transferred onto Immobilon-FL
membranes for 1 hour in transfer buffer (25 mM Tris, 192 mM
glycine) containing 15% methanol. Blots were treated with blocking
buffer (Li-Cor) and labeled for 1 hour with a mouse monoclonal
antibody recognizing GC1 (IS4, 1:3000, provided by Dr. Kris
Palcweski, Case Western University, USA.) and rabbit polyclonal
antibodies raised against GCAP1 (pAb UW14, 1:25,000, provided by
Dr. Wolfgang Baehr, University of Utah) and .beta.-actin (1:5000,
Abcam). Secondary antibodies (goat anti-mouse Ig conjugated to
CW800 and goat anti-rabbit conjugated with IR680) were applied for
1 hour and blots imaged with an Odyssey Infrared Imaging System
(Licor, Lincoln, Nebr., USA).
[0255] mRNA Quantification by rtPCR, Retinal Genome Recovery and
Optic Nerve IHC
[0256] Individual treated eyes with optic nerve attached were
harvested from GC1KO mice 1 year post-treatment with either
AAV8(Y733F)-hGRK1-mGC1 or AAV5-smCBA-mGC1 and an age-matched,
untreated GC1 +/+ mouse. Retinas were dissected from the eye
immediately and snap frozen in liquid nitrogen. Optic nerves were
dissociated from the eyes, fixed in 4% paraformaldehyde overnight
at 4.degree. C., immersed in 30% sucrose for 2 hours at 4.degree.
C., and then quick frozen in cryostat compound (Tissue Tek.RTM. OCT
4583; Sakura Finetek USA, Inc., Torrance, Calif., USA) in a bath of
dry ice/ethanol. Optic nerves were sectioned at 10 .mu.m and
stained according to previously described methods (Boye et al.,
2010). Retinas were homogenized in 350 mL of Buffer RLT
(RNeasy.RTM. Protect Mini Kit, Qiagen, Inc., Valencia, Calif., USA)
plus BME for 45 sec. Samples were centrifuged and the lysate was
split in half (one half designated for genome recovery and the
other half for RNA extraction) (Traint and Whitehead, 2009). Genome
recovery was performed as described above. RNA extraction was
performed with an RNeasy.RTM. Protect Mini Kit (Qiagen, Inc.). RNA
was reverse transcribed (iScript.RTM. cDNA synthesis kit. Biorad
Laboratories, Hercules, Calif., USA) and used in real-time PCR (iQ
SYBR.RTM. Green Supermix and MyiQ real-time PCR detection system
interfaced with iCycler.RTM. thermal cycler, Biorad Laboratories)
to measure the following retinal specific mRNAs: guanylate
cyclase-1 (GC1), guanylate cyclase activating protein-1 (GCAP1),
cone transducin a (GNAT2), rod cGMP-specific 3',5' cyclic
phosphodiesterase subunit alpha (PDE6a) and the housekeeping gene,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
[0257] Primer pairs for GCAP1, GNAT2, PDE.alpha..quadrature. and
GAPDH were identical to those used by Baehr et al. (2007). Primers
for murine GC1 (forward primer: 5'-GACCCTTCCTGCTGGTTCGATCCA-3' [SEQ
ID NO:16], reverse primer: 5''-CTGCATGTGTAGCAGCCTGTGCCTC-3' [SEQ ID
NO:17]) were designed to flank exon 5, the site of gene disruption
in the GC1KO mouse (Yang et al., 1999) and generate an amplicon of
151 bp. PCR produced appropriately sized amplicons in GC1 +/+ and
AAV-mGC1-treated GC1KO retina samples, but not in untreated GC1KO
retina as expected. Amplicon identity was verified by restriction
digest with StuI (NEB) which cleaves within the target sequence to
yield fragments of 56 bps and 95 bps. rtPCR with GC1 and GAPDH
primers on dilution series of reverse transcribed DNA (from both
GC1 +/+ and AAV-mGC1-treated GC1KO retina samples) resulted in
similar slopes, indicating suitability of GC1 primers for
quantifying both endogenous and vector mediated GC1 message (FIG.
20A and FIG. 20B).
[0258] Results are the average of 3 replicate reactions and were
calculated using the 2.sup.-.DELTA..DELTA.C.sup..tau. method (Livak
and Schmittgen, 2001) with GAPDH signal used to normalize samples
and the GC1 +/+ sample serving as the calibrator. Standard
deviations were calculated from the 3 replicate reactions done for
each sample. Data is presented as the fold change in mRNA levels
relative to the GC1 +/+ sample.
[0259] Results
[0260] Long-Term, Photoreceptor-Specific GC1 Expression:
[0261] Immunostaining with an antibody directed against GC1
revealed that AAV-vectored therapeutic protein expression persisted
exclusively in photoreceptors of treated GC1KO mice for a
significant fraction of the animal's lifetime;
AAV8(Y733F)-hGRK1-mGC1 for at least 7 months, AAV5-smCBA-mGC1 for
at least 10 months, and AAV5-hGRK1-mGC1 for at least 10 months
(FIG. 21A and FIG. 21B). GC1 expression was limited to the outer
segments of rods and cones treated with AAV8(Y733F)-hGRK1-mGC1
vector whereas it was found in both outer segments and more rarely
in photoreceptor cell bodies of eyes treated with AAV5-smCBA-mGC1,
a result consistent with the strength of this ubiquitous promoter
relative to photoreceptor-specific, hGRK1 (Beltran et al., 2010).
Two examples of retinal thinning were observed. The first was a
GC1KO retina treated with AAV5-smCBA-mGC1 (4.69.times.10.sup.9
total vector genomes delivered). The outer nuclear layer (ONL) was
slightly thinned relative to that seen in naive GC1 KO or GC1 +/+
control retinas (both 8 months of age). This may be a result of
over-expression of GC1 mediated by the smCBA promoter (Beltran et
al., 2010).
[0262] The second involved a GC1KO retina treated with the more
concentrated AAV5-hGRK1-mGC1 and as before showed
photoreceptor-specific GC1 expression but with profound thinning of
the outer nuclear layer. It should be noted that this vector was
the most concentrated of the three evaluated in this study
(4.12.times.10.sup.10 vector genomes delivered versus
4.69.times.10.sup.9 and 1.08.times.10.sup.10, for the
AAV5-smCBA-mGC1 prep and AAV8(Y733F)-hGRK1-mGC1, respectively), and
again highlights that over expression of GC may be the cause of the
observed thinning. At a minimum, these results suggest that a dose
limiting toxicity may be observable in the mouse. GC1 expression
was absent from the untreated GC1KO retina (FIG. 21A and FIG.
21B).
[0263] Long Term Cone Photoreceptor Survival is Achieved by
AAV-Vectored GC1
[0264] Cone photoreceptors in treated and untreated GC1KO mice as
well as GC11+/+ controls were identified by staining for mouse cone
arrestin. Retinal cross sections from mice sacrificed for the final
biodistribution study and retinal whole mounts from a GC1KO mouse
11 months post-treatment with AAV5-smCBA-mGC1 (right eye only) were
analyzed. Here it was shown that cone photoreceptor densities were
markedly reduced in untreated GC1KO retinas by 10 months of age and
confirm previous reports that cones are lost in a topographically
specific manner in this mouse model (Coleman et al., 2004) (FIG.
21A and FIG. 21B). Whole mount analysis revealed the 11 month old,
untreated retina exhibited a sparse cone density, with residual
cones found exclusively in superior retinal regions whereas the
partner, P14-treated retina retained much higher cone density
throughout, with the exception of a small patch of temporal retina
which likely was not exposed to vector during the subretinal
injection and therefore did not contain transgene product. Compared
to that seen in AAV5-treated retinas, cone densities and structure
in retinal cross sections of AAV8(Y733F)-treated mice appeared
qualitatively most similar to that seen in the normal, GC1 +/+
retina (FIG. 21A and FIG. 21B). While their densities were
increased relative to untreated controls, cones in AAV5-treated
retinas appeared slightly disorganized, a result likely due to the
slight overall disorder/thinning of the outer nuclear layers in
these mice.
[0265] Long-Term Restoration of Photoreceptor Function (ERG) in
AAV-Treated GC1KO Mice
[0266] In the previous examples, cone-mediated function could be
restored to GC1KO mice for 3 months following P14 delivery of
AAV5-smCBA-mGC1 or AAV5-hGRK1-mGC1 (Boye et al., 2010). Average
photopic b-wave amplitudes in treated mice were partially restored
at 4 weeks post-injection and remained stable throughout that
study. In the present example, cone-mediated responses out to 9
months post-treatment were compared in GC1KO mice injected between
P14 and P25 with identical vectors used in the previous study. All
remaining mice treated with AAV5-mGC1 vector continued to exhibit
measurable cone-mediated function out to at least 1 year
post-treatment. Representative traces elicited at 12 cds/m.sup.2
from an individual mouse treated with AAV5-hGRK1-mGC1 are shown in
FIG. 22A and FIG. 22B. Cone responses were stable over time and
were significantly higher than responses generated from untreated,
contralateral controls (p<0.001), suggesting that restoration of
cone function is possible over the lifetime of the animal (FIG.
22A). Consistent with the previous example, the level of
restoration achieved following delivery of the
photoreceptor-specific promoter (hGRK1)-containing vector was not
significantly different from that achieved with the ubiquitous
promoter (smCBA)-containing vector at any post-treatment time
point. Representative traces reveal that the kinetics of the
restored cone ERG appeared normal throughout the course of the
study (FIG. 22B). In addition, it was shown in this example that
cone photoreceptor function was stably restored for at least 6
months following injection with AAV8(Y733F)-hGRK1-mGC1.
[0267] Cone b-wave amplitudes in GC1KO mice injected with this
strong, fast-acting AAV8 tyrosine capsid mutant were higher than
those seen in GC1KO mice injected with either AAV5 vector at every
time point evaluated. At 6 months post-treatment, the latest time
point in which all vectors could be compared in parallel, there was
a significant difference between cone b-wave amplitudes in
AAV8(Y733)-hGRK1-mGC1 vs. AAV5-hGRK1-mGC1-treated mice (p=0.033)
and AAV(Y733F)-hGRK1-mGC1 vs. AAV5-smCBA-mGC1-treated mice
(p=0.025). A representative trace recorded 9 months post-injection
with AAV8(Y733F)-hGRK1-mGC1 (n=1) was noticeably smaller than that
recorded at 6-months' post-injection.
[0268] Due to the inter-mouse variability in untreated GC1KO rod
responses (50-70% of WT by 5 months of age (23), statistical
comparison of average rod responses of treated vs. untreated eyes
is problematic. However, within an animal, rod ERG amplitudes are
nearly equal between partner eyes, therefore we calculated the
average intra-mouse rod a- and b-wave amplitude ratios for treated
versus untreated eyes and then plotted these ratios over time (FIG.
23A and FIG. 23B). AAV-mediated restoration of rod function is
indicated by ratios with a value>1.0. FIG. 23A and FIG. 23B show
that, with the exception of one time point (4 months
post-treatment), the average ratios of rod b-wave amplitudes in
AAV8(Y733F)-hGRK1-mGC1-treated vs. untreated eyes were all>1.0.
Ratios of AAV5-treated vs. untreated eyes were only
occasionally>1.0. Similarly, rod a-wave ratios were consistently
higher in AAV8(Y733F)-hGRK1-mGC1-treated mice, whereas they often
declined following treatment with either AAV5 vector (FIG. 23B).
These results suggest that while the therapeutic effects on rods
were subtle, AAV8(Y733F) conferred the most robust rod-mediated
functional improvement to the GC1KO mouse (FIG. 23B).
Representative rod-mediated scotopic ERG traces elicited by a 1
cds/m.sup.2 stimulus were demonstrated in an
AAV8(Y733F)-hGRK1-mGC1-treated GC1KO mouse (6 months
post-treatment), the untreated contralateral control eye and an
age-matched GC1 +/+ control. AAV8(Y733F)-mediated improvements in
rod ERG amplitudes are clear in this example and indicate that
aside from the sub-wild type amplitudes, treated eye response
kinetics resemble that seen in the GC1 +/+ control.
[0269] Vector Biodistribution:
[0270] Biodistribution studies were performed in GC1KO mice treated
with each vector to establish whether AAV5 or AAV8(Y733F)-delivered
vector genomes could be detected in the optic nerves and/or brains
of treated mice after a period of months. Mice injected with AAV5
vectors were evaluated at 7 (n=2) and 10 (n=5) months
post-treatment and mice injected with AAV8(Y733F)-hGRK1-mGC1 were
evaluated at 4 (n=1) and 7 (n=1) months post-treatment. The optic
nerves from injected and uninjected eyes were examined as well as
portions of left and right brain that contained visual pathways.
AAV5 vectors were injected in the right eyes of GC1KO mice.
Accordingly, vector genomes were detected in the right optic nerve
of AAV5-treated mice at both 7 and 10 months post-injection. At 7
months post-injection, vector genomes were also detected in the
left brain of one mouse injected with AAV5-hGRK1-mGC1. No vector
genomes were detected from the right brain of that animal. The
observation that right (injected) optic nerve and left brain were
positive is anatomically consistent since the left hemisphere is
predominantly "wired" to the right eye.
[0271] By 10 months post-injection. AAV5 delivered vector genomes
were still detected in right (injected) optic nerve but were absent
from both brain hemispheres. AAV8(Y733) vector was injected into
the left eyes of GC1KO mice. Accordingly, AAV8(Y733F)-delivered
vector genomes were detected in the left optic nerves at both 4 and
7 months post-injection. At no time point were vector genomes in
the AAV8(733)-treated mouse detected in either brain hemisphere. A
higher average number of vector genomes were detected in optic
nerves of eyes injected with AAV5-GRK1-mGC1 compared to
AAV5-smCBA-mGC1. This result is likely due to the higher titer of
the former (4.12.times.10.sup.13 vg/mL) compared to the latter
(4.69.times.10.sup.12 vg/mL).
[0272] In addition, only AAV5-hGRK1-mGC1-delivered genomes were
detected in brain tissue over the course of this study, another
observation likely due to the relatively high titer of this vector.
Despite the fact that the titer of AAV8(Y733F)-hGRK1-mGC1 vector
used (1.08.times.10.sup.13 vg/mL) was less than that of the
AAV5-hGRK1-mGC1 vector, a higher average number of vector genomes
was detected in optic nerves of AAV8(Y733F)-treated eyes. While
AAV5 is known to be ineffective for transducing ganglion cells of
the mouse retina (Stieger et al., 2008), it was shown that AAV8
does transduce this cell type (Jacobson et al., 2006). Some
exposure of vector to retinal ganglion cells is expected as the
syringe transverses the inner retina during subretinal injection
and because the ratio of injection volume to total eye size is high
in mouse. The higher number of vector genomes detected in optic
nerves of AAV8(Y733F)-treated eyes therefore could be due to the
increased affinity of AAV8(Y733F), relative to AAV5, for retinal
ganglion cells. As expected, no AAV vector genomes were recovered
from any tissue of naive GC1KO control mice.
[0273] AAV-mGC1 Treatment Restores Wild-type Levels of GC1 and
GCAP1 to Treated GC1 KO Retina
[0274] At 7 months post-injection with AAV8(Y733F)-hGRK1-mGC1,
treated and untreated retinas from one GC1KO mouse as well as one
age-matched GC1 +/+ control mouse were used to assay levels of GC1
and GCAP1 protein expression. The goal of this experiment was not
to compare GC1 levels across treatment groups but rather to compare
levels of vector-mediated GC expression to levels of GC1 in a wild
type animal. Similarly we evaluated the effects of AAV-delivered
GC1 on GCAP1 expression. As expected, GC1 protein was absent from
the untreated eye of the GC1KO mouse. In contrast, levels of GC1 in
the AAV8(Y733F)-treated eye approached that seen in the normal, GC1
+/+ control (FIG. 4). Consistent with previous reports that GCAP1
is post-translationally downregulated in the GC1KO mouse, we show
that GCAP1 was downregulated in untreated GC1KO retina relative to
the GC1 +/+ control (39). However, AAV8(Y733F)-mediated delivery of
GC1 leads to an upregulation in GCAP1 expression in the treated
GC1KO mouse retina. Levels of GCAP1 expression were also comparable
to that seen in GC1 +/+ controls.
[0275] In treated GC1KO mice, GC1 mRNA is present and GNAT2 mRNA
levels are increased relative to untreated GC1KO mice. Using a GC1
primer pair that flanks the neomycin gene disruption located within
Exon 5 of the GC1KO mouse (Timmers et al., 2001) it was possible to
measure GC1 mRNA in both GC1 +/+ and vector-treated GC1KO mice.
Interestingly a second GC1 primer pair targeted to exon 18 and 19
of GC1, well downstream of the gene disruption, produced a PCR
product in the untreated GC1KO mouse sample and therefore these
primers were not used. At one-year post-treatment, levels of GC1
mRNA in treated retinas were approximately seven-fold
(AAV5-treated) and 14-fold [AAV8(YY733F)-treated] higher than that
seen in the age-matched GC1 +/+ control mouse (FIG. 24A and FIG.
24B). By using a nucleic acid recovery technique that enabled
homogeneous partitioning of the sample into 2 equal halves, one for
RNA extraction and the other for DNA (Pang et al., 2011), albeit
was possible to measure mRNA levels and determine the number of
vector genomes within the same sample. It was found that high
levels of GC1 mRNA in treated retinas corresponded to recovery of
many vector genomes; 1.57.times.10.sup.7 vector genomes/.mu.g of
DNA for AAV8(Y733F) and 4.7.times.10.sup.6 vector genomes/.mu.g for
AAV5. Despite the high levels of GC1 mRNA in treated retinas, no
GC1 expression was detected in optic nerves of treated eyes. This
result further supports the notion that vectors evaluated in this
study did not result in off-target transgene expression. Consistent
with previous reports that the reduction of GCAP1 in GC1KO mice is
post-translational (i.e., mRNA levels are unchanged), we found no
substantial changes in the levels of GCAP1 mRNA across samples
(FIG. 24A and FIG. 24B).
[0276] As an initial estimate of treatment on other cone specific
RNAs, several other transcripts were also evaluated in these
samples. To establish a baseline for levels of cone transducin a
(GNAT2), GNAT2 RNA was evaluated in untreated GC1KO samples and
found to be reduced relative to GC1 +/+ controls, a result likely
due to the loss of cone photoreceptors in these retinas (FIG. 24A
and FIG. 24B). In contrast, there were appreciable increases GNAT2
mRNA levels in eyes treated with either AAV5 or AAV8(Y733F)
vectors, a result which further supports the notion that cone
photoreceptors are preserved in AAV-mGC1-treated GC1KO mice. Levels
of rod PDE6a were relatively unchanged across samples likely
because rod photoreceptors do not degenerate in the GC1KO mouse
(FIG. 24A and FIG. 24B).
[0277] In conclusion, these studies demonstrate that persistent
AAV-mediated GC1 expression is capable of restoring long term
retinal function and preserving cone photoreceptors in the GC1KO
mouse. Cohorts of AAV5- and AAV8(Y733F)-treated GC1KO mice were
evaluated for ERG recovery for 9 months and 6 months
post-injection, respectively. While the statistical comparison of
cone ERG amplitudes did not continue beyond these time points due
to dwindling sample sizes, all treated mice continued to exhibit
functional (ERG) rescue. A variety of assays performed on subsets
of these remaining mice all show clear indications of continuing
therapy. This therapeutic longevity was validated on a number of
different levels: 1) the existence of GC1 protein in treated eyes
at 10 months post-treatment, 2) the restoration of cone function as
measured by ERG at 12 months post-treatment, 3) the increased cone
survival in treated eyes at 11 months post-treatment and 4) the
recovery of vector genomes and GC1 mRNA in retinas at 12 months
post-treatment. When viewed as individual, discrete analyses, the
sample sizes used in these assays were often small. However when
all are considered as correlates of therapeutic efficacy in mice
exhibiting clear signs of functional rescue, the sample size is
effectively much larger. Within this context, therefore, it appears
that therapy persists beyond the period statistically evaluated for
ERG rescue. This is the first demonstration of long-term therapy in
an animal model of GC1 deficiency.
[0278] Restored cone ERGs were observed in AAV5 and
AAV8(Y733)-treated GC1KO mice for at least 9 months and 6 months
post-treatment, respectively. Responses were stable and
significantly higher than untreated GC1KO cone responses throughout
the course of the study. Recovery was most pronounced in mice
treated with AAV8(Y733F) vector. Average cone b-wave amplitudes in
AAV8(Y733F)-treated mice were consistently .about.20 .mu.V higher
than those recorded from GC1KO mice treated with standard AAV5
vectors (.about.55 .mu.V vs. .about.35 .mu.V, respectively). At 6
months post-treatment, the latest time point that all vectors were
statistically compared, this difference remained significant. This
result confirms that an AAV8(Y733F) vector stably restored retinal
structure and function to the rd10 mouse, a model refractory to
treatment with standard AAV vectors.
[0279] Quantifying differences in rod amplitudes between treated
and untreated eyes in the GC1KO mouse is complicated by the fact
that rod function in this model is partially subserved by guanylate
cyclase-2 (GC2) (Sun et al., 2010). Rod ERG responses are therefore
variable from animal to animal (30-50% of normal). Therefore,
unlike comparisons of treated and untreated cone responses, treated
rod responses cannot be compared to a zero baseline. Nevertheless,
paired GC1KO eyes have comparable rod ERG amplitudes, and the
intra-animal ratio of rod ERGs in partner eyes, one treated and the
other untreated, provides a valid metric for evaluating treatment
effects on rod function. Improvements in rod-mediated responses in
AAV8(Y733F)-treated GC1KO mice were observed more consistently than
those recorded from AAV5-treated mice as indicated by comparing the
intra-individual ratio of rod a- and b-wave amplitudes from the
treated and untreated eye. This suggests that aggressive expression
of GC1 in the GC1KO eye can supplement the partial effect of GC2 on
murine rod function.
[0280] Long-term cone photoreceptor survival (11 months
post-injection) was demonstrated by immunostaining treated and
untreated retinal whole mounts from one mouse treated with
AAV5-smCBA-mGC1 with an antibody directed against cone arrestin.
Cones were identified throughout the treated GC1KO retina.
AAV5-smCBA-mGC1-treated retina also clearly contained more cones
than the untreated eye which, consistent with previous reports,
retained only a small fraction of cones in its superior hemisphere
(Provost et al., 2005). While the preserved cones in treated GC1KO
retina were not examined on an ultrastructural level (e.g.,
electron microscopy), the observation that cones remained
functional over time by ERG analysis suggests that their structure
was intact. Long term preservation of cone photoreceptors mediated
by therapeutic AAV-GC1 has obvious clinical relevance because it
suggests the potential to preserve macular cones and restore usable
daytime/color vision to patients with GC1 deficiency.
[0281] AAV-mediated GC1 expression persisted for at least 10 months
post-treatment (the latest time point evaluated by IHC), and was
located exclusively in photoreceptors, regardless of the serotype
used or whether a photoreceptor-specific (hGRK1) or ubiquitous
(smCBA) promoter drove its expression. While transgene expression
was limited to the target cell type, the hGRK1 promoter was more
specific in that it resulted in expression exclusively within the
proper compartment of the target cell (photoreceptor outer
segments). This result, along with other successful
proof-of-concept studies utilizing this promoter suggests that the
hGRK1 promoter should be considered in the design of a clinical AAV
vector targeting photoreceptors.
[0282] Immunostaining of transverse GC1KO retinal sections at 10
months post-treatment with AAV5-smcBA-mGC1 revealed moderate
thinning of the ONL relative to the wild type and untreated GC1KO
controls. Additionally, in this retina GC1 was occasionally found
in cell bodies of photoreceptors. It is possible that the strong,
ubiquitous smCBA promoter drove expression of GC1 at levels that
overwhelmed the trafficking machinery of some photoreceptors and
that the accumulation of transgene product in photoreceptor cell
bodies constituted a stress-initiated apoptosis in these cells.
More dramatic ONL thinning was observed in one mouse injected with
AAV5-hGRK1-mGC1. With an n of 1, it cannot be definitively conclude
that retinal thinning was present in all mice treated with this
vector. Nevertheless, consistent with the notion of overexpression
toxicity, the titer of the AAV5-hGRK1-mGC1 vector was the highest
of the three vectors evaluated in this study. However, it should
also be noted that there was no accumulation of GC1 in
photoreceptor cell bodies with the high titer AAV5-hGRK1-mGC1
vector.
[0283] Despite the photoreceptor-exclusive nature of AAV-mediated
GC1 expression, the inventors were interested in evaluating the
spread of vector genomes to tissues outside the subretinal space.
Importantly, these data were collected from `diseased` animals.
This is relevant based on evidence that the pattern of vector
transduction is different in diseased vs. healthy retina (Kolstad
et al., 2010). This would suggest that biodistribution patterns may
also be different. For this reason, it was important to evaluate
the spread of genomes within the rescued animal model itself (i.e.,
within subjects that exhibited clear ERG recovery). Although the
sample size was limited, useful information was collected about the
distribution of AAV5- and AAV8(Y733F)-delivered genomes in optic
nerve and brain.
[0284] This is the first evaluation of biodistribution for an AAV
vector containing a capsid surface exposed tyrosine mutation. AAV5-
and AAV8(Y733F)-delivered vector genomes were detected in the optic
nerves of injected eyes at all time points assessed. At only one
time point (7 months post-injection) were AAV5 vector genomes
detected in the brain of a treated GC1KO mouse. Genomes were
recovered only in the hemisphere opposite the injected eye. This
result contrasts the finding by Provost et al., 2005 who reported a
lack of AAV5-delivered sequence in brains of subretinally-injected
rats and dogs. By 10 months post-injection, no vector genomes were
recovered from brains of AAV5-treated GC1KO mice nor from brains of
mice treated with AAV8(Y733F) at any time point. However, due to
the relatively small number of mice analyzed, it cannot
unequivocally be excluded that AAV5-delivered genomes were present
in brains at 10 months post-injection or that AAV8(Y733F) delivered
genomes are never present in brains of treated GC1KO mice at any
time.
[0285] Despite recovering vector genomes from optic nerves of
treated eyes, immunostaining revealed a lack of GC1 expression in
optic nerves of eyes treated with either AAV5-smCBA-mGC1 or
AAV8(Y733F)-hGRK1-mGC1 vectors. A previous study by Stieger, et
al., (2005) detected transgene expression in optic nerves and
brains of rats and dogs at 2 months and 4 weeks post-subretinal
injection with AAV8 containing green fluorescent protein (GFP).
Taking into account that the AAV8(Y733F) vector contained the
photoreceptor-specific hGRK1 promoter and the previous finding that
GC1 expression was limited to photoreceptors even when under the
control of a ubiquitous promoter like smCBA, a lack of GC1
expression in optic nerves is not unexpected. Stieger et al.,
(2005) incorporated the strong, ubiquitous CMV promoter into their
vector to drive GFP, a protein which is capable of being stably
expressed in a wide variety of tissues when delivered via viral
vectors.
[0286] While both AAV5 and AAV8(Y733F) vectors were capable of
providing long term therapy to the GC1KO mouse, there are apparent
advantages associated with using AAV8(Y733F). First and foremost,
AAV8(Y733F) with a photoreceptor-specific promoter conferred
significantly higher cone ERG responses to treated mice than either
AAV5 vector. The reason for this may be due to the ability of AAV8
vectors to transduce areas outside of the injection bleb in rodent
retina whereas the area of retina transduced by AAV5 remains
largely confined to the bleb (47). Thus, AAV8(733F) may simply
transduce on average a larger area of retina relative to AAV5
vectors and in turn result in more cone transduction and a robust
full-field cone ERG response, through either or both an increased
overall cone survival and/or an increased level of light response
in each transduced cone.
Example 8--Exemplary Mammalian GC1 Polypeptide Sequences
[0287] Exemplary amino acid sequences useful in the practice of the
present invention include, without limitation, one or more amino
acid sequences that encode a biologically-active mammalian
guanylate cyclase protein. Such sequences include, without
limitation, those of human, non-human primate, murine, bovine, and
canine origin, such as those guanylate cyclase proteins set forth
in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID
NO:11, hereinbelow:
TABLE-US-00004 Homo sapiens (human; GenPept Accession Number: NP
000171) (SEQ ID NO: 1)
MTACARRAGGLPDPGLCGPAWWAPSLPRLPRALPRLPLLLLLLLLQPPALSAVFTVGVLGPWACDP
IFSRARPDLAARLAAARLNRDPGLAGGPRFEVALLPEPCRTPGSLGAVSSALARVSGLVGPVNPAA
CRPAELLAEEAGIALVPWGCPWTQAEGTTAPAVTPAADALYALLRAFGWARVALVTAPQDLWVEAG
RSLSTALRARGLPVASVTSMEPLDLSGAREALRKVRDGPRVTAVIMVMHSVLLGGEEQRYLLEAAE
ELGLTDGSLVFLPFDTIHYALSPGFEALAALANSSQLRRAHDAVLTLTRHCPSEGSVLDSLRRAQE
RRELPSDLNLQQVSPLFGTIYDAVFLLARGVAEARAAAGGRWVSGAAVARHIRDAQVPGFCGDLGG
DEEPPFVLLDTDAAGDRLFATYMLDPARGSFLSAGTRMHFPRGGSAPGPDPSCWFDPNNICGGGLE
PGLVFLGFLLVVGMGLAGAFLAHYVRHRLLHMQMVSGPNKIILTVDDITFLHPHGGTSRKVAQGSR
SSLGARSMSDIRSGPSQHLDSPNIGVYEGDRVWLKKFPGDQHIAIRPATKTAFSKLQELRHENVAL
YLGLFLARGAEGPAALWEGNLAVVSEHCTRGSLQDLLAQREIKLDWMFKSSLLLDLIKGIRYLHHR
GVAHGRLKSRNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPRAEDQLWTAPELLRDPALERRGTLA
GDVFSLAIIMQEVVCRSAPYAMLELTPEEVVQRVRSPPPLCRPLVSMDQAPVECILLMKQCWAEQP
ELRPSMDHTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLLTQMLP
PSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIEVVDLLNDLYTLFDAIIGSHDVYKV
ETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAVGTFRMRHMPEVPVRIRIGLHSGPCVAGVVG
LTMPRYCLFGDTVNTASRMESTGLPYRIHVNLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWL
VGRRGFNKPIPKPPDLQPGSSNHGISLQEIPPERRRKLEKARPGQFS Mus musculus
(mouse: GenPept Accession Number: NP 032218) (SEQ ID NO: 2)
MSAWLLPAGGLPGAGFCVPARQSPSSFSRVLRWPRPGLPGLLLLLLLPSPSALSAVFKVGVLGPWA
CDPIFARARPDLAARLAANRLNRDFALDGGPRFEVALLPEPCLTPGSLGAVSSALSRVSGLVGPVN
PAACRPAELLAQEAGVALVPWGCPGTRAAGTTAPAVTPAADALYVLLRAFRWARVALITAPQDLWV
EAGRALSTALRARGLPWALVISMETSDRSGAREALGRIRDGPRVRVVIMVMHSVLLGGEEQRYLLE
AAEELALIDGSLVFLPFDTLHYALSPGPEALAAFVNSSQLRRAHDAVLTLTRRCPPGGSVQDSLRR
AQEHQELPLDLNLKQVSPLFGTIYDAVFLLAGGVKRARTAVGGGWVSGASVARQVREAQVSGFCGV
LGRTEEPSFVLLDTDASGEQLFATHLLDPVLGSLRSAGTPMHEPRGGPAPGPDPSCWFDPDVICNG
GVEPGLVFVGFLLVIGMGLTGAFLAHYLRHRLLHMQMASGPNKIILTLEDVTFLHPPGGSSRFVVQ
GSRSSLATRSASDIRSVPSQPQESTNVGLYEGDWVWLKKFPGEHHMAIRPATKTAFSKLRELRHEN
VALYLGLFLAGTADSPATPGEGILAVVSEHCARGSLHDLLAQREIKLDWMFKSSLLLDLIKGMRYL
HHRGVAHGRLKSRNCVVDGRFVLKVTDHGHGRLLEAQRVLPEPPSAEDQLMTAPELLRDPSLERRG
TLAGDVFSLAIIMQEVVCRSTPYAMLELTPEEVIQRVRSPPPLCRPLVSMDQAPMECIQLMTQCWA
EHPELRPSMDLTFDLFKSINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELEQEKQKTDRLLTQ
MLPPSVAEALKMGTSVEPEYFEEVTLYESDIVGFTTISAMSEPIEVVDLLNDLYTLFDAIIGAHDV
YKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAVGSFRMRHMPEVTWRIRIGLHSGPCVAG
VVGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNMSTVRILRALDQGFQMECRGRTELKGKGIEDT
YWLVGRLGFNKPIPKPPDLQPGASNHGISLQEIPPERRKKLEKARPGQFTGK Rattus
norvegicus (Norway rat; GenPept Accession Number: NP 077356) (SEQ
ID NO: 3)
MSAWLLPAGGFPGAGFCIPAWQSRSSLSRVLRWPGPGLPGLLLLLLLPSPSAFSAVEKVGVLGPWA
CDPIFARARPDLAARLATDRLNRDLALDGGPWFEVTLLPEPCLTPGSLGAVSSALTRVSGLVGPVN
PAACRPAELLAQEAGVALVPWGCPGTRAAGTTAPAVTPAADALYVLLKAFRWARVALITAPQDLWV
EAGRALSTALRARGLPVALVTSMVPSDLSGAREALRRIRDGPRVRVVIMVMHSVLLGGEEQRYLLE
AAEELGLTDGSLVFLPFDTLHYALSPGPEALAAFVNSSKLRRAHDAVLTLTRRCPPGGSVQDSLRR
AQEHQELPLDLDLKQVSPLFGTIYDAVFLLAGGVTRARAAVGGGWVSGASVARQMREAQVFGFCGI
LGRTEEPSFVLLDTDAAGERLFTTHLLDPVLGSLRSAGTPVHFPRGAPAPGPDPSCWFDPDVICNG
GVEPGLVFVGFLLVIVVGLTGAFLAHYLRHRLLHMQMVSGPNKIILTLEDVTFLHPQGGSSRKVAQ
GSRSSLATRSTSDIRSVPSQFQESTNIGLYEGDWVWLKKFPGEHHMAIRPATKMAFSKLRELRHEN
VALYLGLFLAGTADSPATPGEGILAVVSEHCARGSLHDLLAQRDIKLDWMFKSSLLLDLIKGMRYL
HHRGVAHGRLKSRNCVVDGRFVLKVTDHGHGRLLEAQRVLPEPPSAEDQLWTAPELLRDPALERRG
TLAGDVFSLAIIMQEVVCRSTPYAMLELTPEEVIQRVRSPPPLCRPLVSMDQAPMECIQLMTQCWA
EHPELRPSMDLTFDLFKSINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELEQEKQKTDRLLTQ
MLPPSVAEALKMGTSVEPEYFEEVTLYFSDIVGFTTISAMSEPIEVVDLLNDLYTLFDAIIGAHDV
YKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAVGSFRMRHMPEVPVRIRIGLHSGPCVAG
VVGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNMSTVRILRALDQGFQMECRGRTELKGKGIEDT
YWLVGRLGFNKPIPKPPDLQPGASNHGISLQEIPPERRKKLEKARPGQFTGK Bos taurus GC1
(bovine; GenPept Accession Number: NP 776973) (SEQ ID NQ: 4)
MTACTFLAGGLRDPGLCAPTRWSPSPPGLPPIPPRPRLRLRPPLLLLLLLPRSVLSAVFTVGVLGP
WACDPIFARARPDLAARLAASRLNHAAALEGGPRFEVALLPEPCRTPGSLGAVSSALTRVSGLVGP
VNPAACRPAELLAQEAGVALVPWGCPGTRAAGTTAPVVTPAADALYALLRAFRWAHVALVTAPQDL
WVEAGHALSTALRARGLPVALVTSMEPSDLSGAREALRRVQDGPRVRAVIMVMHSVLLGGEEQRCL
LEAAEELGLADGSLVFLPFDTLHYALSPGPDALAVLANSSQLRKAHDAVLTLTRHCPLGGSVRDSL
RRAQEHRELPLDLNLQQVSPLEGTIYDSVFLLAGGVARARVAAGGGWVSGAAVARHIRDARVPGFC
GALGGAEEPSFVLLDTDATGDQLFATYVLDPTQGFFHSAGTPVHFPKGGRGPGPDPSCWFDPDTIC
NGGVEPSVVFIGFLLVVGMGLAGAFLAHYCRHRLLHIQMVSGPNKIILTLDDITFLHPHGGNSRKV
AQGSRTSLAARSISDVRSIHSQLPDYTNIGLYEGDWVWLNKFPGDRHIAIRPATKMAFSKIRELRH
ENVALYLGLFLAGGAGGPAAPGEGVLAVVSEHCARGSLQDLLAQRDIKLDWMFKSSLLLDLIKGIR
YLHHRGVARGRLKSRNCVVDGRFVLKVTDHGHGRLLEAQRVLPEPPSAEDQLWTAPELLRDPVLER
RGTLAGDVFSLGIIMQEVVCRSAPYAMLELTPEEVVKRVQSPPPLCRPSVSIDQAPMECIQLMKQC
WAEQPELRPSMDRTFELFKSINKGRKMNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLL
TQMLPPSVAEALKMGTPVEPEYFEEVTLYFSDIVGFTTISAMSEPIEVVDLLNDLYTLFDAIIGSH
DVYKVETIGDAYMVASGLPQRNGHRHAAEIANMALDILSAVGTFRMRHMPEVPVRIRIGLHSGPCV
AGVVGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNRSTVQILSALNEGFLTEVRGRTELKGKGAE
ETYWLVGRRGFNKPIPKPPDLQPGASNHGISLHEIPPDRRQKLEKARPGQFSGK Canis 1upus
familiaris (canine; GenPept Accession Number: NP 001003207) (SEQ ID
NO: 5)
MSACALLAGGLPDPRLCAPARWARSPPGVPGAPPWPQPRLRLLLLLLLLPPSALSAVFTVGVLGPW
ACDPIFARARPDLAARLAAARLNRDAALEDGPRFEVTLLPEPCRTPGSLGAVSSALGRVSGLVGPV
NPAACRPAELLAQEAGVALVPWSCPGTRAGGTTAPAGTPAADALYALLRAFRWARVALITAPQDLW
VEAGRALSAALRARGLPVALVTTMEPSDLSGAREALRRVQDGPRVRAVIMVMHSVLLGGEEQRCLL
QAAEELGLADGSLVFLPFDTLHYALSPGPEALAVLANSSQLRRAHDAVLILTRHCPPGGSVMDNLR
RAQEHQELPSDLDLQQVSPFFGTIYDAVLLLAGGVARARAAAGGGWVSGATVAHHIPDAQVPGFCG
TLGGAQEPPFVLLLTDAAGDRLFATYMLDPTRGSLLSAGTPVHFPRGGGTPGSDPSCWFEPGVICN
GGVEPGLVFLGFLLVVGMGLTGAFLAHYLRHRLLHIQMVSGPNKIILTLDDYTFLHPHGGSTRKVV
QGSRSSLAARSTSDIRSVPSQPLDNSNIGLFEGDWVWLKKFPGDQHIAIRPATKTAFSKLRELRHE
NVVLYLGLFLGSGGAGGSAAGEGVLAVVSEHCARGSLHDLLAQRDIKLDWMFKSSLLLDLIKGMRY
LHHRGVAHGRLKSPNCVVDGRFVLKVTDHGHARLMEAQRVLLEPPSAEDQLWTAPELLRDPALERR
GTLPGDYFSLGIIMQEVVCRSAPYAMLELTPEEVVERVRSPPPLCRPSVSMDQAPVECIQLMKQCW
AEHPDLRPSLGHIFDQFKSINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLLT
QMLPPSVAEALKMGTPVEPEYFEEVTLYFSDIVGFITISAMSEPIEVVDLLNDLYTLFDAIIGSHD
VYKVETIGDAYMYASGLPQRNGQRHAAEIANMALDILSAVGSFRMRHMPEVPVRIRIGLHSGPCVA
GVVGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNMSTVRILHALDEGFQTEVRGRTELKGKGAED
TYWLVGRRGFNKPIPKPPDLQPGASNHGISLQEIPLDRRWKLEKARPGQFSGK Macaca
mulatta (Rhesus macaque; predicted sequence from XP 001111670) (SEQ
ID NO: 6)
MTACARRAGGLPDPRLCGPARWAPALPRLPRALPRLPLLLLLLLLQPPALSAVFTVGVLGPWACDP
IFSRARADLAARLAAARLNRDPDLAGGPRFEVALLPEPCRTPGSLGAVSSALTRVSGLVGPVNPAA
CRPAELLAEEAGIALVPWGCPGTQAAGTTAPALTPAADALYALLRAFGWARVALVTAPQDLWVEAG
HSLSTALRARGLPVASVTSMEPLDLSGAREALRKVPDGPRVTAVIMVMHSVLLGGEEQRYLLEAAE
ELGLTDGSLVFLPFDTVHYALSPGPEALAALANSSQLRRAHDAVLTLTRHCPSEGSVLDSLRRAQE
RRELPSDLNLQOVSPLFGTIYDAVFLLVRGVAEARAAAGGRWVSGAAVARHVWDAQVPGFCGDLGG
DEEPPFVLLDTDAVGDRLFATYMLDPTRGSLLSAGTPMHFPRGGSAPGPDPSCWFDPNNICGGGLE
PGLVFLGFLLVVGMGLAGAFLAHYVRHQLLHIQMVSGPNKIILTVDDITFLHPHGGTSRKVAQGSR
SSLAARSMSDVRSGPSQPTDSPNVGVYEGDRVWLKKFPGDQHIAIRPATKTAFSKLQELRHENVAL
YLGLFLAQGAEGPAALWEGNLAVVSEHCTRGSLQDLLAQREIKLDWMFKSSLLLDLIKGIRYLHHR
GVAHGRLKSPNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPRAEDQLWTAPELLRDPALERRGTLA
GDVFSLAIIMQEVVCRSAPYAMLELTPEEVVQRVRSPPPLCRPLVSMDQAPVECIHLMKQCWAEQP
ELRPSMDHTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLLTQMLP
PSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIEVVDLLNDLYTLFDAIIGSHDVYKV
ETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAVGTFRMRHMPEVPVRIRIGLHSGPCVAGVVG
LTMPRYCLFGDTVNTASRMESTGLPYRIHVNLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWL
VGRRGFNKPIPKPPDLQPGSSNHGISLQEIPPERRRKLEKARPGQFS. Pongo abelii
(Sumatran Orangutan; predicted sequence from XP_002827037) (SEQ ID
NO: 7) MTACARRAGGLPDPGLCGPARWAPSLPRLPRALPRLPLLLLLLLLQPPALSAVFTVGVLG
PWACDPIFSRAPPDLAARLAAARLNRDPGLAGGPRFEVALLPEPCRTPGSLGAVSSALAR
VSGLVGPVNPAACRPAELLADNPGIALVPWGCPWTQAEGTTAPCVTPAADALYALLRAFG
WARVALVTAPQDLWVEAGRSLSTALRARGLPVASVTSMEPLDLSGAREALRKVRDGFRVT
AWIMVMHSVLLGGEEQRYLLEAAEELGLTDGSLVFLPFDTIHYALSPGPEALAALANSSQ
LRRAHDAVLTLTRHCPSEGSVLDSLRRAQERRELPSDLNLQOVSPLFGTIYDAVFLLARG
VAEAWAAAGGRWVSGAAVARHIRDAQVPGFCGDLGGDGEPPFVLLDTDAAGDRLFATYML
DPARGSFLSAGTRMHFPRGGSAPGPDPSCWFDPNNICGGGLEPGLVFLGFLLVVGMGLAG
AFLAHYVPHRLLHIQMVSGPNKIILTVNDITFLHPHGGTSRKVAQGSRSSLAARSMSDIR
SGPSQPLDSPNVGVYEGDRVWLKKFPGDQHIAIRPATKTAFSKLQELRHENVALYLGLFL
ARGAEGPAALWEGNIAVVSEHCTRGSLQDLLSQREIKLDWMFKSSLLLDLIKGIRYLHHR
GVAHGRLKSRNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPRAEDQLWTAPELLPDPALE
RRGTLAGDVFSLAIIMQEVVCRSAPYAMLELTPEEVVQRVRSPPPLCRPLVSMDQAPVEC
IHLMKQCWAEQPELRPSMDHTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTE
ELELEKQKTDRLLTQMLPPSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIE
VVDLLNDLYTLFDAIIGSHDVYKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAV
GTFRMRHMPEVPVRIRIGLHSGPCVAGVVGLTMPRYCLFGDTVNTASRMESTGLPYRIHV
NLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWLVGRRGFNKPIPKPPDLQPGSSNHG
ISLQEIPPERRRKLEKARPGQFS Callithrix jacchus (white tufted-ear
marmoset; predicted sequence from XP_002747985) (SEQ ID NO: 8)
MTACARRAGGLPDPGLCGPARWAPALSRLPRALPRLPLLLLLLLLQPPALSAQFTVGVLG
PWACDPIFSRARPDLAARLAAARLNRDPSLAGGPRFEVALLPEPCRTPGSLGAVSSALAR
VSGLVGPVNPAACRPAELLAEEAGIALVPWGCPGTQAAGTTAPVVTPAADALYALLRAFG
WARVALVTAPQDLWVEAGLSLSTALRARGLPVVSVTSMEPLDLSGAREALRKVHNGPRVT
AVIMVMHSVLLGGEEQRYLLEAAEELGLTDGSLVFLPFDTIHYALSPGREALAALVNSSQ
LRRAHDAVLTLTRHCSSEGSVLDSLRKAQQRRELPSDLNLEQVSPLEGTIYDAVVLLARG
VADARAAVGGRWVSGAAVARHVWDAQASGFCGDLGRDEEPSFVLLDTDAAGDQLFATYML
DPARGSLLSAGTPMHFPRGGPAPGPDPSCWFDPNNICDGGLEPGFIFLGFLLVVGMGLAG
ALLAHYVRHQLLHIQMVSGPNKIILTVDDITFLHPHGGASRKVAQGSRSSLAAHSTSDIR
SGPSQPSDSPNIGVYEGDRVWLKKFPGEQHIAIRPATKTAFSKLQELRHENVALYLGLFL
AQGAEGPAALWEGNLAVVSEHCTRGSLQDLLAQREIKLDWMFKSSLLLDLIKGIRYLHHR
GVAHGRLKSRNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPKAEDQLWTAPELLRDPALE
RRGTLAGDVFSLGIIMQEVVCRSAPYAMLELTPDEVVQRVRSPPPLCRPFVSMDQAPVEC
IHLMKQCWAEQPELRPSMDLTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTE
ELELEKQKTDRLLTQMLPPSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIE
VVDLLNDLYTLFDAIIGSHDVYKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAV
GTFRMRHMPEVPVRIRIGLHSGPCVAGVVGLTMPRYCLFGDTVNTASRMESTGLPYRIHV
NLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWLVGRRGFNKPIPKPPDLQPGASNHG
ISLQEIPPERRRKLEKARPGQFS Ailutropoda melanoleuca (giant panda;
predicted sequence from XP_002921218) (SEQ ID NO: 9)
MRACALLAGGLPYPRLCAPTRWAPARPGVSRALPWPRPRLRLLLLLLLRPPSVLSAVFTV
GVLGPWACDPIFARARPDLXXXXXXXXXDALYVLLRAFRWARVALVTAPQDLWVEAGRAL
SAALRARGLPVALVTTMEPSDLSGAREALRRVQHGPRVSAVIMVMHSVLLGGEEQRCLLQ
AAEELGLADGSLVFLPFDTLHYALSPGPEALAALANSSQLRRAHDAVLILTRHCPPGGSV
MDSLRRAQERQELPSDLNLEQVSPLFGTIYDAVFLLAGGVARARAAAADSRVPGFCGALG
GAEEPPFVLLDTDAAGDRFFATYVLDPTRGSLHSAGTPVHFPRGGGAPGPDPSCWFEPDS
ICNGGVEPGLVFTGELLVVGMGLMGAFLAHYVRHRLLHIQMVSGPNKIILTLDDITFLHP
QGGSARKVVQGSRSSLAARSTSDYRSVPSQPSDGGNIGLYEGDWVWLKKFPGSQHIAIRP
ATKTAFSKLRELRHENVALYLGLFLGGGEGGSAAAGGGMLAVVSEHCTRGSLHDLLAQRD
IKLDWMFKSSLLLDLIKGMRYLHHRGVAHGRLKSRNCVVDGRFVLKVTDHGHGRLLEAQK
VLAEPPSAEDQLWTAPELLRDPALERRGTLAGDVFSLGIIMQEVVCRSSPYAMLELSARE
VVQRVRSPPPLCRPSVSVDQAPAECIQLMKQCWAEQPELRPSLDRTFDQFKSINKGRKTN
IIDSMLRMLEQYSSNLEGLIRERTEELELEKRKTDRLRAASLPSSVAEALKMGTPVEPEY
FEEVTLYFSDIVGFTTISAMSEPIEVVDLLNDLYTLFDAIIGSHDVYKVETIGDAYMVAS
GLPQRNGQRHAAEIANMALDILSAVGSFRMRHMPEVPVRIRIGLHSGPCVAGVVGLTMPR
YCLFGDTVNTASRMESTGLPYRIHVNMSTVRILRALDEGFQTEVRGRTELKGKGAEDTYW
LVGXXXXXXXXPIPKPPDLQPGASNHGISLQEIPLDRRQKLEKARPGQSGK Monodelphis
domestica (gray short-tailed opossum; predicted sequence from
XP_001369029) (SEQ ID NO: 10)
MLVPSINGLFHHPPWCFPPLPLPLEFLFLLLLLPVPVLPATFTIGVLGPWSCDPIFSRAR
PDLAARLAATRMNHDQALEGGPWFEVILLPEPCRTSGSLGALSPSLARVSGLVGPVNPAA
CHPAELLAQEAGVPLVPWGCPQGKARTTAPALPLALDALYALLRAFHWAKVALITAPQDL
WVEAGQALAGGLRSRGLPVAMVTSLETTDLESAKNALKRVRDGPKVKVLIMVMHSVLLGG
EEQRLLLEAAEELGLVEGTMVFLPFDTLHYALPPGPEALRPITNSSRLRKAHDAVLTLTR
YCPKGSVSASLRQAQEHRELPLDLKPQQVSPLFGTIYDAIYLLAGAVAGAQVAGGGGWVS
GAAVARHIPNTLVSGFCGDLGGTKEPPFVLLDTDGMRDQLLPTYTLDPAQGVLHHAGNPI
HFPHGGQGPGPDPPCWFDPNVICSGGIEPRFILLVILIIIGGGLVVATLAYYVRRQLLHA
QMVSGPNKMILTLEDITFFPRQGSSSRKATEGSRSSLIAHSASDMRSIPSQPPDNSNIGM
YEGDWVWLKKFPGEHYTEIRPATKMAFSKLRELRHENVAVQMGLFLAGSMEGAAAGGLGG
GILAVVSEYCSRGSLQDLLIQRDIKLDWMFKSSLLLDLIKGLRYLHHRGVAHGRLKSRNC
VVDGRFVLKITDHAHGRLLEAQRVSLEPPQAEDRLWTAPELLRNEALERQGTLQGDVFSV
GIIMQEVVCRCEPYAMLELTPEEIIQKVQSPPPMCRPSVSVDQAPMECIQLMKQCWAEQP
DLRPNMDTTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDKL
DAIIGSHDVYKVETIGDAYMVASGLPKRNGQRHAAEIANMSLDILSSVGSFRMRHMPEVP
VRIRIGLHSGPCVAGVVGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNLSTVKILQGLN
EGFQIEIRGRTELKGKGVEDTYWLVGRKGFDKPIPIPPDLLPGASNHGISLQEIPEDRRK
KLEKARPGQPLGK Equus caballus (horse; predicted sequence from
XP_001918412) (SEQ ID NO: 11)
MVMHSVLLGGEEQRCLLEAAEELGLADGSLVFLPFDTLHYALSPGPEALAVLANNSQLRR
AHDAVLTLTRHCPLGGSVLDSLRRAQEHQELPSDLNLQQVSPLFGTIYDAVYLLAGGVAR
ARAAAGGSWVSGAAVAHHVRDAQVPGFCGALGGAEEPQFVLLDTDAAGDRLFATYMLDPT
RGSLWSAGTPVHFPRGGRGPGPDPWCWFDPDDICNGGVEPRLVFIGFLLAVGMGLAGVFL
AHYVRHRLLHIQMASGPNKIILTLDDITFLHPQGGSSRKVIQGSRSSLAARSVSDIRSVP
SQPMDSSNIGLYEGDWVWLKKFPGDQHIAIRPATKTAFSKLRELNHENVALYLGLFLAGG
SSGAAAPREGMLAVVSEHCARGSLHDLLAQRDIKLDWMFKSSLLLDLIKGMRYLHHRGVA
HGRLKSRNCVVDGRFVLKVTDHGHGRLLEAQKVLPEPPSAEDQLWTAPELLRDPALERQG
TLAGDVFSLGIIIQEVVCRSTPYAMLELTPEEVVQRLQSPPPLCRPSVSMDQAPMECIQL
MKQCWAEQPDLRPSMDRTFDLFKSINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELE
LEKQKTDRLLTQMLPPSVAEALKMGTPVEPEYFEEVTLYFSDIVGFTTISAMSEPIEVVD
LLNDLYTLFDAIIGSHDVYKVETIGDAYMVASGLPQRNGQRHAAEIANMALDILSAVGSF
RMRNMPEVPVRIRIGLHSGPCVAGVVGLTMPRYCLFGDTVNTASRMESTGLPYRINVNMS
TVRILRALDEGFQVEVRGRTELKGKGVEDTYWLVGRRGFNKPIPKPPDLQPGASNHGISL
QEIPPERRQKLEKARPGQFSGK
Example 9--Sequence Analysis of Known Mammalian GC1
Polypeptides
[0288] All GC1 alignment data generated using amino acid sequence
for the following species: Bos taurus (bovine; 1110 residues),
Canis lupus familiaris (canine; 1109 residues), Mus musculus
(murine; 1108 residues), and Homo sapiens (human; 1103 residues).
Positions of consensus and variable regions are based on numerical
residues corresponding to Bos taurus as this is the longest GC1
protein, 1110 residues, and has no gaps in the alignment.
[0289] Similarity graph of alignment of GC1 proteins from Bos
taurus, Canis lupus familiaris, Mus musculus, and Homo sapiens.
[0290] GC1 Consensus Regions: [0291] Amino acid positions: 44-49,
55-90, 98-155, 164-321, 464-549, 561-604, 620-761, 813-1026,
1045-1054, and 1060-1110.
[0292] Variable Regions: [0293] Amino acid positions: 4-43, 50-54,
91-97, 156-163, 322-463, 550-560, 605-619, 762-812, 1027-1044, and
1055-1059.
[0294] Other Notable Regions of the GC1 Consensus Alignment
Include: [0295] (1) Kinase homology domain: amino acid positions
531 to 541 of the consensus sequence (known to be essential for
activity in photoreceptors--see, e.g., Bereta et al., 2010). [0296]
(2) Phosphorylated serine residues within the kinase homology
domain of murine GC1 protein (consensus/bovine position shown in
parenthesis): 530 (532), 532 (534), 533 (535) and 538(540).
Example 10--Nucleotide Sequence of the smCBA Promoter
[0297] The nucleic acid sequence of an illustrative human GRK1
(hGRK1) promoter which was used in the studies described above is
shown below:
TABLE-US-00005 (SEQ ID NO: 12)
GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGG
CGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAG
CAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGT
CCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGTC
TCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCTCGACAGGGCCCGGTCT
CTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCCAAGGGC
[0298] The nucleic acid sequence of an illustrative smCBA promoter
which was used in the studies described above is shown below:
TABLE-US-00006 (SEQ ID NO: 13)
AATTCGGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTC
ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCG
CCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA
TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG
ACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG
CCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA
TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCT
ACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGC
TTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATT
TATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGC
GCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG
AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTT
TATGGCCAGGCGGCGCCGGCGGCCGCCCTATAAAAACCGAAGCGCCCGGC
GGGCGGGACTCGCTGCGACGCTGCCTTCGCCCCGTGCCGCGCTCCGCCGC
CGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG
TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT
TAATGACGGCTTGTTTCTTTTCTCTGGCTGCGTGAAAGCCTTGAGGGGCT
CCGGGAGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCT
ACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCA AAG
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[0469] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of ordinary skill in the
art that variations may be applied to the compositions and methods
and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and
scope of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those of ordinary skill
in the art are deemed to be within the spirit, scope and concept of
the invention as defined by the appended claims. [0470] Pang J J,
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D. G.; Harrison, W. R.; Elder, F. F. B.; Heckenlively, J. R.;
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gene transfer," Vis. Res., 48:353-359, 2007. [0500] Tan M H, Smith
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pigmentosa and Leber congenital amaurosis caused by defects in
AIPL1: effective rescue of mouse models of partial and complete
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Genet., 18:2099-114, 2009. [0501] Timmers A M, Zhang H. Squitieri
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[0524] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of ordinary skill in the
art that variations may be applied to the compositions and methods
and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and
scope of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those of ordinary skill
in the art are deemed to be within the spirit, scope and concept of
the invention as defined by the appended claims.
Sequence CWU 1
1
1811103PRTHomo sapiens 1Met Thr Ala Cys Ala Arg Arg Ala Gly Gly Leu
Pro Asp Pro Gly Leu1 5 10 15Cys Gly Pro Ala Trp Trp Ala Pro Ser Leu
Pro Arg Leu Pro Arg Ala 20 25 30Leu Pro Arg Leu Pro Leu Leu Leu Leu
Leu Leu Leu Leu Gln Pro Pro 35 40 45Ala Leu Ser Ala Val Phe Thr Val
Gly Val Leu Gly Pro Trp Ala Cys 50 55 60Asp Pro Ile Phe Ser Arg Ala
Arg Pro Asp Leu Ala Ala Arg Leu Ala65 70 75 80Ala Ala Arg Leu Asn
Arg Asp Pro Gly Leu Ala Gly Gly Pro Arg Phe 85 90 95Glu Val Ala Leu
Leu Pro Glu Pro Cys Arg Thr Pro Gly Ser Leu Gly 100 105 110Ala Val
Ser Ser Ala Leu Ala Arg Val Ser Gly Leu Val Gly Pro Val 115 120
125Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu Leu Ala Glu Glu Ala Gly
130 135 140Ile Ala Leu Val Pro Trp Gly Cys Pro Trp Thr Gln Ala Glu
Gly Thr145 150 155 160Thr Ala Pro Ala Val Thr Pro Ala Ala Asp Ala
Leu Tyr Ala Leu Leu 165 170 175Arg Ala Phe Gly Trp Ala Arg Val Ala
Leu Val Thr Ala Pro Gln Asp 180 185 190Leu Trp Val Glu Ala Gly Arg
Ser Leu Ser Thr Ala Leu Arg Ala Arg 195 200 205Gly Leu Pro Val Ala
Ser Val Thr Ser Met Glu Pro Leu Asp Leu Ser 210 215 220Gly Ala Arg
Glu Ala Leu Arg Lys Val Arg Asp Gly Pro Arg Val Thr225 230 235
240Ala Val Ile Met Val Met His Ser Val Leu Leu Gly Gly Glu Glu Gln
245 250 255Arg Tyr Leu Leu Glu Ala Ala Glu Glu Leu Gly Leu Thr Asp
Gly Ser 260 265 270Leu Val Phe Leu Pro Phe Asp Thr Ile His Tyr Ala
Leu Ser Pro Gly 275 280 285Pro Glu Ala Leu Ala Ala Leu Ala Asn Ser
Ser Gln Leu Arg Arg Ala 290 295 300His Asp Ala Val Leu Thr Leu Thr
Arg His Cys Pro Ser Glu Gly Ser305 310 315 320Val Leu Asp Ser Leu
Arg Arg Ala Gln Glu Arg Arg Glu Leu Pro Ser 325 330 335Asp Leu Asn
Leu Gln Gln Val Ser Pro Leu Phe Gly Thr Ile Tyr Asp 340 345 350Ala
Val Phe Leu Leu Ala Arg Gly Val Ala Glu Ala Arg Ala Ala Ala 355 360
365Gly Gly Arg Trp Val Ser Gly Ala Ala Val Ala Arg His Ile Arg Asp
370 375 380Ala Gln Val Pro Gly Phe Cys Gly Asp Leu Gly Gly Asp Glu
Glu Pro385 390 395 400Pro Phe Val Leu Leu Asp Thr Asp Ala Ala Gly
Asp Arg Leu Phe Ala 405 410 415Thr Tyr Met Leu Asp Pro Ala Arg Gly
Ser Phe Leu Ser Ala Gly Thr 420 425 430Arg Met His Phe Pro Arg Gly
Gly Ser Ala Pro Gly Pro Asp Pro Ser 435 440 445Cys Trp Phe Asp Pro
Asn Asn Ile Cys Gly Gly Gly Leu Glu Pro Gly 450 455 460Leu Val Phe
Leu Gly Phe Leu Leu Val Val Gly Met Gly Leu Ala Gly465 470 475
480Ala Phe Leu Ala His Tyr Val Arg His Arg Leu Leu His Met Gln Met
485 490 495Val Ser Gly Pro Asn Lys Ile Ile Leu Thr Val Asp Asp Ile
Thr Phe 500 505 510Leu His Pro His Gly Gly Thr Ser Arg Lys Val Ala
Gln Gly Ser Arg 515 520 525Ser Ser Leu Gly Ala Arg Ser Met Ser Asp
Ile Arg Ser Gly Pro Ser 530 535 540Gln His Leu Asp Ser Pro Asn Ile
Gly Val Tyr Glu Gly Asp Arg Val545 550 555 560Trp Leu Lys Lys Phe
Pro Gly Asp Gln His Ile Ala Ile Arg Pro Ala 565 570 575Thr Lys Thr
Ala Phe Ser Lys Leu Gln Glu Leu Arg His Glu Asn Val 580 585 590Ala
Leu Tyr Leu Gly Leu Phe Leu Ala Arg Gly Ala Glu Gly Pro Ala 595 600
605Ala Leu Trp Glu Gly Asn Leu Ala Val Val Ser Glu His Cys Thr Arg
610 615 620Gly Ser Leu Gln Asp Leu Leu Ala Gln Arg Glu Ile Lys Leu
Asp Trp625 630 635 640Met Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile
Lys Gly Ile Arg Tyr 645 650 655Leu His His Arg Gly Val Ala His Gly
Arg Leu Lys Ser Arg Asn Cys 660 665 670Ile Val Asp Gly Arg Phe Val
Leu Lys Ile Thr Asp His Gly His Gly 675 680 685Arg Leu Leu Glu Ala
Gln Lys Val Leu Pro Glu Pro Pro Arg Ala Glu 690 695 700Asp Gln Leu
Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro Ala Leu Glu705 710 715
720Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser Leu Ala Ile Ile Met
725 730 735Gln Glu Val Val Cys Arg Ser Ala Pro Tyr Ala Met Leu Glu
Leu Thr 740 745 750Pro Glu Glu Val Val Gln Arg Val Arg Ser Pro Pro
Pro Leu Cys Arg 755 760 765Pro Leu Val Ser Met Asp Gln Ala Pro Val
Glu Cys Ile Leu Leu Met 770 775 780Lys Gln Cys Trp Ala Glu Gln Pro
Glu Leu Arg Pro Ser Met Asp His785 790 795 800Thr Phe Asp Leu Phe
Lys Asn Ile Asn Lys Gly Arg Lys Thr Asn Ile 805 810 815Ile Asp Ser
Met Leu Arg Met Leu Glu Gln Tyr Ser Ser Asn Leu Glu 820 825 830Asp
Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Leu Glu Lys Gln Lys 835 840
845Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro Ser Val Ala Glu Ala
850 855 860Leu Lys Thr Gly Thr Pro Val Glu Pro Glu Tyr Phe Glu Gln
Val Thr865 870 875 880Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr
Ile Ser Ala Met Ser 885 890 895Glu Pro Ile Glu Val Val Asp Leu Leu
Asn Asp Leu Tyr Thr Leu Phe 900 905 910Asp Ala Ile Ile Gly Ser His
Asp Val Tyr Lys Val Glu Thr Ile Gly 915 920 925Asp Ala Tyr Met Val
Ala Ser Gly Leu Pro Gln Arg Asn Gly Gln Arg 930 935 940His Ala Ala
Glu Ile Ala Asn Met Ser Leu Asp Ile Leu Ser Ala Val945 950 955
960Gly Thr Phe Arg Met Arg His Met Pro Glu Val Pro Val Arg Ile Arg
965 970 975Ile Gly Leu His Ser Gly Pro Cys Val Ala Gly Val Val Gly
Leu Thr 980 985 990Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn
Thr Ala Ser Arg 995 1000 1005Met Glu Ser Thr Gly Leu Pro Tyr Arg
Ile His Val Asn Leu Ser 1010 1015 1020Thr Val Gly Ile Leu Arg Ala
Leu Asp Ser Gly Tyr Gln Val Glu 1025 1030 1035Leu Arg Gly Arg Thr
Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr 1040 1045 1050Phe Trp Leu
Val Gly Arg Arg Gly Phe Asn Lys Pro Ile Pro Lys 1055 1060 1065Pro
Pro Asp Leu Gln Pro Gly Ser Ser Asn His Gly Ile Ser Leu 1070 1075
1080Gln Glu Ile Pro Pro Glu Arg Arg Arg Lys Leu Glu Lys Ala Arg
1085 1090 1095Pro Gly Gln Phe Ser 110021108PRTMus musculus 2Met Ser
Ala Trp Leu Leu Pro Ala Gly Gly Leu Pro Gly Ala Gly Phe1 5 10 15Cys
Val Pro Ala Arg Gln Ser Pro Ser Ser Phe Ser Arg Val Leu Arg 20 25
30Trp Pro Arg Pro Gly Leu Pro Gly Leu Leu Leu Leu Leu Leu Leu Pro
35 40 45Ser Pro Ser Ala Leu Ser Ala Val Phe Lys Val Gly Val Leu Gly
Pro 50 55 60Trp Ala Cys Asp Pro Ile Phe Ala Arg Ala Arg Pro Asp Leu
Ala Ala65 70 75 80Arg Leu Ala Ala Asn Arg Leu Asn Arg Asp Phe Ala
Leu Asp Gly Gly 85 90 95Pro Arg Phe Glu Val Ala Leu Leu Pro Glu Pro
Cys Leu Thr Pro Gly 100 105 110Ser Leu Gly Ala Val Ser Ser Ala Leu
Ser Arg Val Ser Gly Leu Val 115 120 125Gly Pro Val Asn Pro Ala Ala
Cys Arg Pro Ala Glu Leu Leu Ala Gln 130 135 140Glu Ala Gly Val Ala
Leu Val Pro Trp Gly Cys Pro Gly Thr Arg Ala145 150 155 160Ala Gly
Thr Thr Ala Pro Ala Val Thr Pro Ala Ala Asp Ala Leu Tyr 165 170
175Val Leu Leu Arg Ala Phe Arg Trp Ala Arg Val Ala Leu Ile Thr Ala
180 185 190Pro Gln Asp Leu Trp Val Glu Ala Gly Arg Ala Leu Ser Thr
Ala Leu 195 200 205Arg Ala Arg Gly Leu Pro Val Ala Leu Val Thr Ser
Met Glu Thr Ser 210 215 220Asp Arg Ser Gly Ala Arg Glu Ala Leu Gly
Arg Ile Arg Asp Gly Pro225 230 235 240Arg Val Arg Val Val Ile Met
Val Met His Ser Val Leu Leu Gly Gly 245 250 255Glu Glu Gln Arg Tyr
Leu Leu Glu Ala Ala Glu Glu Leu Ala Leu Thr 260 265 270Asp Gly Ser
Leu Val Phe Leu Pro Phe Asp Thr Leu His Tyr Ala Leu 275 280 285Ser
Pro Gly Pro Glu Ala Leu Ala Ala Phe Val Asn Ser Ser Gln Leu 290 295
300Arg Arg Ala His Asp Ala Val Leu Thr Leu Thr Arg Arg Cys Pro
Pro305 310 315 320Gly Gly Ser Val Gln Asp Ser Leu Arg Arg Ala Gln
Glu His Gln Glu 325 330 335Leu Pro Leu Asp Leu Asn Leu Lys Gln Val
Ser Pro Leu Phe Gly Thr 340 345 350Ile Tyr Asp Ala Val Phe Leu Leu
Ala Gly Gly Val Lys Arg Ala Arg 355 360 365Thr Ala Val Gly Gly Gly
Trp Val Ser Gly Ala Ser Val Ala Arg Gln 370 375 380Val Arg Glu Ala
Gln Val Ser Gly Phe Cys Gly Val Leu Gly Arg Thr385 390 395 400Glu
Glu Pro Ser Phe Val Leu Leu Asp Thr Asp Ala Ser Gly Glu Gln 405 410
415Leu Phe Ala Thr His Leu Leu Asp Pro Val Leu Gly Ser Leu Arg Ser
420 425 430Ala Gly Thr Pro Met His Phe Pro Arg Gly Gly Pro Ala Pro
Gly Pro 435 440 445Asp Pro Ser Cys Trp Phe Asp Pro Asp Val Ile Cys
Asn Gly Gly Val 450 455 460Glu Pro Gly Leu Val Phe Val Gly Phe Leu
Leu Val Ile Gly Met Gly465 470 475 480Leu Thr Gly Ala Phe Leu Ala
His Tyr Leu Arg His Arg Leu Leu His 485 490 495Met Gln Met Ala Ser
Gly Pro Asn Lys Ile Ile Leu Thr Leu Glu Asp 500 505 510Val Thr Phe
Leu His Pro Pro Gly Gly Ser Ser Arg Lys Val Val Gln 515 520 525Gly
Ser Arg Ser Ser Leu Ala Thr Arg Ser Ala Ser Asp Ile Arg Ser 530 535
540Val Pro Ser Gln Pro Gln Glu Ser Thr Asn Val Gly Leu Tyr Glu
Gly545 550 555 560Asp Trp Val Trp Leu Lys Lys Phe Pro Gly Glu His
His Met Ala Ile 565 570 575Arg Pro Ala Thr Lys Thr Ala Phe Ser Lys
Leu Arg Glu Leu Arg His 580 585 590Glu Asn Val Ala Leu Tyr Leu Gly
Leu Phe Leu Ala Gly Thr Ala Asp 595 600 605Ser Pro Ala Thr Pro Gly
Glu Gly Ile Leu Ala Val Val Ser Glu His 610 615 620Cys Ala Arg Gly
Ser Leu His Asp Leu Leu Ala Gln Arg Glu Ile Lys625 630 635 640Leu
Asp Trp Met Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile Lys Gly 645 650
655Met Arg Tyr Leu His His Arg Gly Val Ala His Gly Arg Leu Lys Ser
660 665 670Arg Asn Cys Val Val Asp Gly Arg Phe Val Leu Lys Val Thr
Asp His 675 680 685Gly His Gly Arg Leu Leu Glu Ala Gln Arg Val Leu
Pro Glu Pro Pro 690 695 700Ser Ala Glu Asp Gln Leu Trp Thr Ala Pro
Glu Leu Leu Arg Asp Pro705 710 715 720Ser Leu Glu Arg Arg Gly Thr
Leu Ala Gly Asp Val Phe Ser Leu Ala 725 730 735Ile Ile Met Gln Glu
Val Val Cys Arg Ser Thr Pro Tyr Ala Met Leu 740 745 750Glu Leu Thr
Pro Glu Glu Val Ile Gln Arg Val Arg Ser Pro Pro Pro 755 760 765Leu
Cys Arg Pro Leu Val Ser Met Asp Gln Ala Pro Met Glu Cys Ile 770 775
780Gln Leu Met Thr Gln Cys Trp Ala Glu His Pro Glu Leu Arg Pro
Ser785 790 795 800Met Asp Leu Thr Phe Asp Leu Phe Lys Ser Ile Asn
Lys Gly Arg Lys 805 810 815Thr Asn Ile Ile Asp Ser Met Leu Arg Met
Leu Glu Gln Tyr Ser Ser 820 825 830Asn Leu Glu Asp Leu Ile Arg Glu
Arg Thr Glu Glu Leu Glu Gln Glu 835 840 845Lys Gln Lys Thr Asp Arg
Leu Leu Thr Gln Met Leu Pro Pro Ser Val 850 855 860Ala Glu Ala Leu
Lys Met Gly Thr Ser Val Glu Pro Glu Tyr Phe Glu865 870 875 880Glu
Val Thr Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr Ile Ser 885 890
895Ala Met Ser Glu Pro Ile Glu Val Val Asp Leu Leu Asn Asp Leu Tyr
900 905 910Thr Leu Phe Asp Ala Ile Ile Gly Ala His Asp Val Tyr Lys
Val Glu 915 920 925Thr Ile Gly Asp Ala Tyr Met Val Ala Ser Gly Leu
Pro Gln Arg Asn 930 935 940Gly Gln Arg His Ala Ala Glu Ile Ala Asn
Met Ser Leu Asp Ile Leu945 950 955 960Ser Ala Val Gly Ser Phe Arg
Met Arg His Met Pro Glu Val Pro Val 965 970 975Arg Ile Arg Ile Gly
Leu His Ser Gly Pro Cys Val Ala Gly Val Val 980 985 990Gly Leu Thr
Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Thr 995 1000
1005Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile His Val
1010 1015 1020Asn Met Ser Thr Val Arg Ile Leu Arg Ala Leu Asp Gln
Gly Phe 1025 1030 1035Gln Met Glu Cys Arg Gly Arg Thr Glu Leu Lys
Gly Lys Gly Ile 1040 1045 1050Glu Asp Thr Tyr Trp Leu Val Gly Arg
Leu Gly Phe Asn Lys Pro 1055 1060 1065Ile Pro Lys Pro Pro Asp Leu
Gln Pro Gly Ala Ser Asn His Gly 1070 1075 1080Ile Ser Leu Gln Glu
Ile Pro Pro Glu Arg Arg Lys Lys Leu Glu 1085 1090 1095Lys Ala Arg
Pro Gly Gln Phe Thr Gly Lys 1100 110531108PRTRattus norvegicus 3Met
Ser Ala Trp Leu Leu Pro Ala Gly Gly Phe Pro Gly Ala Gly Phe1 5 10
15Cys Ile Pro Ala Trp Gln Ser Arg Ser Ser Leu Ser Arg Val Leu Arg
20 25 30Trp Pro Gly Pro Gly Leu Pro Gly Leu Leu Leu Leu Leu Leu Leu
Pro 35 40 45Ser Pro Ser Ala Phe Ser Ala Val Phe Lys Val Gly Val Leu
Gly Pro 50 55 60Trp Ala Cys Asp Pro Ile Phe Ala Arg Ala Arg Pro Asp
Leu Ala Ala65 70 75 80Arg Leu Ala Thr Asp Arg Leu Asn Arg Asp Leu
Ala Leu Asp Gly Gly 85 90 95Pro Trp Phe Glu Val Thr Leu Leu Pro Glu
Pro Cys Leu Thr Pro Gly 100 105 110Ser Leu Gly Ala Val Ser Ser Ala
Leu Thr Arg Val Ser Gly Leu Val 115 120 125Gly Pro Val Asn Pro Ala
Ala Cys Arg Pro Ala Glu Leu Leu Ala Gln 130 135 140Glu Ala Gly Val
Ala Leu Val Pro Trp Gly Cys Pro Gly Thr Arg Ala145 150 155 160Ala
Gly Thr Thr Ala Pro Ala Val Thr Pro Ala Ala Asp Ala Leu Tyr 165 170
175Val Leu Leu Lys Ala Phe Arg Trp Ala Arg Val Ala Leu Ile Thr Ala
180 185 190Pro Gln Asp Leu Trp Val Glu Ala Gly Arg Ala Leu Ser Thr
Ala Leu 195 200 205Arg Ala Arg Gly Leu Pro Val Ala Leu Val Thr Ser
Met Val Pro Ser 210 215 220Asp Leu Ser Gly Ala Arg Glu Ala Leu Arg
Arg Ile Arg Asp Gly Pro225 230 235 240Arg Val Arg Val Val Ile Met
Val Met His Ser Val Leu Leu Gly Gly 245
250 255Glu Glu Gln Arg Tyr Leu Leu Glu Ala Ala Glu Glu Leu Gly Leu
Thr 260 265 270Asp Gly Ser Leu Val Phe Leu Pro Phe Asp Thr Leu His
Tyr Ala Leu 275 280 285Ser Pro Gly Pro Glu Ala Leu Ala Ala Phe Val
Asn Ser Ser Lys Leu 290 295 300Arg Arg Ala His Asp Ala Val Leu Thr
Leu Thr Arg Arg Cys Pro Pro305 310 315 320Gly Gly Ser Val Gln Asp
Ser Leu Arg Arg Ala Gln Glu His Gln Glu 325 330 335Leu Pro Leu Asp
Leu Asp Leu Lys Gln Val Ser Pro Leu Phe Gly Thr 340 345 350Ile Tyr
Asp Ala Val Phe Leu Leu Ala Gly Gly Val Thr Arg Ala Arg 355 360
365Ala Ala Val Gly Gly Gly Trp Val Ser Gly Ala Ser Val Ala Arg Gln
370 375 380Met Arg Glu Ala Gln Val Phe Gly Phe Cys Gly Ile Leu Gly
Arg Thr385 390 395 400Glu Glu Pro Ser Phe Val Leu Leu Asp Thr Asp
Ala Ala Gly Glu Arg 405 410 415Leu Phe Thr Thr His Leu Leu Asp Pro
Val Leu Gly Ser Leu Arg Ser 420 425 430Ala Gly Thr Pro Val His Phe
Pro Arg Gly Ala Pro Ala Pro Gly Pro 435 440 445Asp Pro Ser Cys Trp
Phe Asp Pro Asp Val Ile Cys Asn Gly Gly Val 450 455 460Glu Pro Gly
Leu Val Phe Val Gly Phe Leu Leu Val Ile Val Val Gly465 470 475
480Leu Thr Gly Ala Phe Leu Ala His Tyr Leu Arg His Arg Leu Leu His
485 490 495Met Gln Met Val Ser Gly Pro Asn Lys Ile Ile Leu Thr Leu
Glu Asp 500 505 510Val Thr Phe Leu His Pro Gln Gly Gly Ser Ser Arg
Lys Val Ala Gln 515 520 525Gly Ser Arg Ser Ser Leu Ala Thr Arg Ser
Thr Ser Asp Ile Arg Ser 530 535 540Val Pro Ser Gln Pro Gln Glu Ser
Thr Asn Ile Gly Leu Tyr Glu Gly545 550 555 560Asp Trp Val Trp Leu
Lys Lys Phe Pro Gly Glu His His Met Ala Ile 565 570 575Arg Pro Ala
Thr Lys Met Ala Phe Ser Lys Leu Arg Glu Leu Arg His 580 585 590Glu
Asn Val Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gly Thr Ala Asp 595 600
605Ser Pro Ala Thr Pro Gly Glu Gly Ile Leu Ala Val Val Ser Glu His
610 615 620Cys Ala Arg Gly Ser Leu His Asp Leu Leu Ala Gln Arg Asp
Ile Lys625 630 635 640Leu Asp Trp Met Phe Lys Ser Ser Leu Leu Leu
Asp Leu Ile Lys Gly 645 650 655Met Arg Tyr Leu His His Arg Gly Val
Ala His Gly Arg Leu Lys Ser 660 665 670Arg Asn Cys Val Val Asp Gly
Arg Phe Val Leu Lys Val Thr Asp His 675 680 685Gly His Gly Arg Leu
Leu Glu Ala Gln Arg Val Leu Pro Glu Pro Pro 690 695 700Ser Ala Glu
Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro705 710 715
720Ala Leu Glu Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser Leu Gly
725 730 735Ile Ile Met Gln Glu Val Val Cys Arg Ser Thr Pro Tyr Ala
Met Leu 740 745 750Glu Leu Thr Pro Glu Glu Val Ile Gln Arg Val Arg
Ser Pro Pro Pro 755 760 765Leu Cys Arg Pro Leu Val Ser Met Asp Gln
Ala Pro Met Glu Cys Ile 770 775 780Gln Leu Met Ala Gln Cys Trp Ala
Glu His Pro Glu Leu Arg Pro Ser785 790 795 800Met Asp Leu Thr Phe
Asp Leu Phe Lys Gly Ile Asn Lys Gly Arg Lys 805 810 815Thr Asn Ile
Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr Ser Ser 820 825 830Asn
Leu Glu Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Gln Glu 835 840
845Lys Gln Lys Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro Ser Val
850 855 860Ala Glu Ala Leu Lys Met Gly Thr Ser Val Glu Pro Glu Tyr
Phe Glu865 870 875 880Glu Val Thr Leu Tyr Phe Ser Asp Ile Val Gly
Phe Thr Thr Ile Ser 885 890 895Ala Met Ser Glu Pro Ile Glu Val Val
Asp Leu Leu Asn Asp Leu Tyr 900 905 910Thr Leu Phe Asp Ala Ile Ile
Gly Ser His Asp Val Tyr Lys Val Glu 915 920 925Thr Ile Gly Asp Ala
Tyr Met Val Ala Ser Gly Leu Pro Gln Arg Asn 930 935 940Gly Gln Arg
His Ala Ala Glu Ile Ala Asn Met Ser Leu Asp Ile Leu945 950 955
960Ser Ala Val Gly Ser Phe Arg Met Arg His Met Pro Glu Val Pro Val
965 970 975Arg Ile Arg Ile Gly Leu His Ser Gly Pro Cys Val Ala Gly
Val Val 980 985 990Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp
Thr Val Asn Thr 995 1000 1005Ala Ser Arg Met Glu Ser Thr Gly Leu
Pro Tyr Arg Ile His Val 1010 1015 1020Asn Met Ser Thr Val Arg Ile
Leu Arg Ala Leu Asp Gln Gly Phe 1025 1030 1035Gln Met Glu Cys Arg
Gly Arg Thr Glu Leu Lys Gly Lys Gly Val 1040 1045 1050Glu Asp Thr
Tyr Trp Leu Val Gly Arg Val Gly Phe Asn Lys Pro 1055 1060 1065Ile
Pro Lys Pro Pro Asp Leu Gln Pro Gly Ala Ser Asn His Gly 1070 1075
1080Ile Ser Leu Gln Glu Ile Pro Pro Glu Arg Arg Lys Lys Leu Glu
1085 1090 1095Lys Ala Arg Pro Gly Gln Phe Thr Gly Lys 1100
110541110PRTBos taurus 4Met Thr Ala Cys Thr Phe Leu Ala Gly Gly Leu
Arg Asp Pro Gly Leu1 5 10 15Cys Ala Pro Thr Arg Trp Ser Pro Ser Pro
Pro Gly Leu Pro Pro Ile 20 25 30Pro Pro Arg Pro Arg Leu Arg Leu Arg
Pro Pro Leu Leu Leu Leu Leu 35 40 45Leu Leu Pro Arg Ser Val Leu Ser
Ala Val Phe Thr Val Gly Val Leu 50 55 60Gly Pro Trp Ala Cys Asp Pro
Ile Phe Ala Arg Ala Arg Pro Asp Leu65 70 75 80Ala Ala Arg Leu Ala
Ala Ser Arg Leu Asn His Ala Ala Ala Leu Glu 85 90 95Gly Gly Pro Arg
Phe Glu Val Ala Leu Leu Pro Glu Pro Cys Arg Thr 100 105 110Pro Gly
Ser Leu Gly Ala Val Ser Ser Ala Leu Thr Arg Val Ser Gly 115 120
125Leu Val Gly Pro Val Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu Leu
130 135 140Ala Gln Glu Ala Gly Val Ala Leu Val Pro Trp Gly Cys Pro
Gly Thr145 150 155 160Arg Ala Ala Gly Thr Thr Ala Pro Val Val Thr
Pro Ala Ala Asp Ala 165 170 175Leu Tyr Ala Leu Leu Arg Ala Phe Arg
Trp Ala His Val Ala Leu Val 180 185 190Thr Ala Pro Gln Asp Leu Trp
Val Glu Ala Gly His Ala Leu Ser Thr 195 200 205Ala Leu Arg Ala Arg
Gly Leu Pro Val Ala Leu Val Thr Ser Met Glu 210 215 220Pro Ser Asp
Leu Ser Gly Ala Arg Glu Ala Leu Arg Arg Val Gln Asp225 230 235
240Gly Pro Arg Val Arg Ala Val Ile Met Val Met His Ser Val Leu Leu
245 250 255Gly Gly Glu Glu Gln Arg Cys Leu Leu Glu Ala Ala Glu Glu
Leu Gly 260 265 270Leu Ala Asp Gly Ser Leu Val Phe Leu Pro Phe Asp
Thr Leu His Tyr 275 280 285Ala Leu Ser Pro Gly Pro Asp Ala Leu Ala
Val Leu Ala Asn Ser Ser 290 295 300Gln Leu Arg Lys Ala His Asp Ala
Val Leu Thr Leu Thr Arg His Cys305 310 315 320Pro Leu Gly Gly Ser
Val Arg Asp Ser Leu Arg Arg Ala Gln Glu His 325 330 335Arg Glu Leu
Pro Leu Asp Leu Asn Leu Gln Gln Val Ser Pro Leu Phe 340 345 350Gly
Thr Ile Tyr Asp Ser Val Phe Leu Leu Ala Gly Gly Val Ala Arg 355 360
365Ala Arg Val Ala Ala Gly Gly Gly Trp Val Ser Gly Ala Ala Val Ala
370 375 380Arg His Ile Arg Asp Ala Arg Val Pro Gly Phe Cys Gly Ala
Leu Gly385 390 395 400Gly Ala Glu Glu Pro Ser Phe Val Leu Leu Asp
Thr Asp Ala Thr Gly 405 410 415Asp Gln Leu Phe Ala Thr Tyr Val Leu
Asp Pro Thr Gln Gly Phe Phe 420 425 430His Ser Ala Gly Thr Pro Val
His Phe Pro Lys Gly Gly Arg Gly Pro 435 440 445Gly Pro Asp Pro Ser
Cys Trp Phe Asp Pro Asp Thr Ile Cys Asn Gly 450 455 460Gly Val Glu
Pro Ser Val Val Phe Ile Gly Phe Leu Leu Val Val Gly465 470 475
480Met Gly Leu Ala Gly Ala Phe Leu Ala His Tyr Cys Arg His Arg Leu
485 490 495Leu His Ile Gln Met Val Ser Gly Pro Asn Lys Ile Ile Leu
Thr Leu 500 505 510Asp Asp Ile Thr Phe Leu His Pro His Gly Gly Asn
Ser Arg Lys Val 515 520 525Ala Gln Gly Ser Arg Thr Ser Leu Ala Ala
Arg Ser Ile Ser Asp Val 530 535 540Arg Ser Ile His Ser Gln Leu Pro
Asp Tyr Thr Asn Ile Gly Leu Tyr545 550 555 560Glu Gly Asp Trp Val
Trp Leu Lys Lys Phe Pro Gly Asp Arg His Ile 565 570 575Ala Ile Arg
Pro Ala Thr Lys Met Ala Phe Ser Lys Ile Arg Glu Leu 580 585 590Arg
His Glu Asn Val Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gly Gly 595 600
605Ala Gly Gly Pro Ala Ala Pro Gly Glu Gly Val Leu Ala Val Val Ser
610 615 620Glu His Cys Ala Arg Gly Ser Leu Gln Asp Leu Leu Ala Gln
Arg Asp625 630 635 640Ile Lys Leu Asp Trp Met Phe Lys Ser Ser Leu
Leu Leu Asp Leu Ile 645 650 655Lys Gly Ile Arg Tyr Leu His His Arg
Gly Val Ala His Gly Arg Leu 660 665 670Lys Ser Arg Asn Cys Val Val
Asp Gly Arg Phe Val Leu Lys Val Thr 675 680 685Asp His Gly His Gly
Arg Leu Leu Glu Ala Gln Arg Val Leu Pro Glu 690 695 700Pro Pro Ser
Ala Glu Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg705 710 715
720Asp Pro Val Leu Glu Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser
725 730 735Leu Gly Ile Ile Met Gln Glu Val Val Cys Arg Ser Ala Pro
Tyr Ala 740 745 750Met Leu Glu Leu Thr Pro Glu Glu Val Val Lys Arg
Val Gln Ser Pro 755 760 765Pro Pro Leu Cys Arg Pro Ser Val Ser Ile
Asp Gln Ala Pro Met Glu 770 775 780Cys Ile Gln Leu Met Lys Gln Cys
Trp Ala Glu Gln Pro Glu Leu Arg785 790 795 800Pro Ser Met Asp Arg
Thr Phe Glu Leu Phe Lys Ser Ile Asn Lys Gly 805 810 815Arg Lys Met
Asn Ile Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr 820 825 830Ser
Ser Asn Leu Glu Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu 835 840
845Leu Glu Lys Gln Lys Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro
850 855 860Ser Val Ala Glu Ala Leu Lys Met Gly Thr Pro Val Glu Pro
Glu Tyr865 870 875 880Phe Glu Glu Val Thr Leu Tyr Phe Ser Asp Ile
Val Gly Phe Thr Thr 885 890 895Ile Ser Ala Met Ser Glu Pro Ile Glu
Val Val Asp Leu Leu Asn Asp 900 905 910Leu Tyr Thr Leu Phe Asp Ala
Ile Ile Gly Ser His Asp Val Tyr Lys 915 920 925Val Glu Thr Ile Gly
Asp Ala Tyr Met Val Ala Ser Gly Leu Pro Gln 930 935 940Arg Asn Gly
His Arg His Ala Ala Glu Ile Ala Asn Met Ala Leu Asp945 950 955
960Ile Leu Ser Ala Val Gly Thr Phe Arg Met Arg His Met Pro Glu Val
965 970 975Pro Val Arg Ile Arg Ile Gly Leu His Ser Gly Pro Cys Val
Ala Gly 980 985 990Val Val Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe
Gly Asp Thr Val 995 1000 1005Asn Thr Ala Ser Arg Met Glu Ser Thr
Gly Leu Pro Tyr Arg Ile 1010 1015 1020His Val Asn Arg Ser Thr Val
Gln Ile Leu Ser Ala Leu Asn Glu 1025 1030 1035Gly Phe Leu Thr Glu
Val Arg Gly Arg Thr Glu Leu Lys Gly Lys 1040 1045 1050Gly Ala Glu
Glu Thr Tyr Trp Leu Val Gly Arg Arg Gly Phe Asn 1055 1060 1065Lys
Pro Ile Pro Lys Pro Pro Asp Leu Gln Pro Gly Ala Ser Asn 1070 1075
1080His Gly Ile Ser Leu His Glu Ile Pro Pro Asp Arg Arg Gln Lys
1085 1090 1095Leu Glu Lys Ala Arg Pro Gly Gln Phe Ser Gly Lys 1100
1105 111051109PRTCanis familiaris 5Met Ser Ala Cys Ala Leu Leu Ala
Gly Gly Leu Pro Asp Pro Arg Leu1 5 10 15Cys Ala Pro Ala Arg Trp Ala
Arg Ser Pro Pro Gly Val Pro Gly Ala 20 25 30Pro Pro Trp Pro Gln Pro
Arg Leu Arg Leu Leu Leu Leu Leu Leu Leu 35 40 45Leu Pro Pro Ser Ala
Leu Ser Ala Val Phe Thr Val Gly Val Leu Gly 50 55 60Pro Trp Ala Cys
Asp Pro Ile Phe Ala Arg Ala Arg Pro Asp Leu Ala65 70 75 80Ala Arg
Leu Ala Ala Ala Arg Leu Asn Arg Asp Ala Ala Leu Glu Asp 85 90 95Gly
Pro Arg Phe Glu Val Thr Leu Leu Pro Glu Pro Cys Arg Thr Pro 100 105
110Gly Ser Leu Gly Ala Val Ser Ser Ala Leu Gly Arg Val Ser Gly Leu
115 120 125Val Gly Pro Val Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu
Leu Ala 130 135 140Gln Glu Ala Gly Val Ala Leu Val Pro Trp Ser Cys
Pro Gly Thr Arg145 150 155 160Ala Gly Gly Thr Thr Ala Pro Ala Gly
Thr Pro Ala Ala Asp Ala Leu 165 170 175Tyr Ala Leu Leu Arg Ala Phe
Arg Trp Ala Arg Val Ala Leu Ile Thr 180 185 190Ala Pro Gln Asp Leu
Trp Val Glu Ala Gly Arg Ala Leu Ser Ala Ala 195 200 205Leu Arg Ala
Arg Gly Leu Pro Val Ala Leu Val Thr Thr Met Glu Pro 210 215 220Ser
Asp Leu Ser Gly Ala Arg Glu Ala Leu Arg Arg Val Gln Asp Gly225 230
235 240Pro Arg Val Arg Ala Val Ile Met Val Met His Ser Val Leu Leu
Gly 245 250 255Gly Glu Glu Gln Arg Cys Leu Leu Gln Ala Ala Glu Glu
Leu Gly Leu 260 265 270Ala Asp Gly Ser Leu Val Phe Leu Pro Phe Asp
Thr Leu His Tyr Ala 275 280 285Leu Ser Pro Gly Pro Glu Ala Leu Ala
Val Leu Ala Asn Ser Ser Gln 290 295 300Leu Arg Arg Ala His Asp Ala
Val Leu Ile Leu Thr Arg His Cys Pro305 310 315 320Pro Gly Gly Ser
Val Met Asp Asn Leu Arg Arg Ala Gln Glu His Gln 325 330 335Glu Leu
Pro Ser Asp Leu Asp Leu Gln Gln Val Ser Pro Phe Phe Gly 340 345
350Thr Ile Tyr Asp Ala Val Leu Leu Leu Ala Gly Gly Val Ala Arg Ala
355 360 365Arg Ala Ala Ala Gly Gly Gly Trp Val Ser Gly Ala Thr Val
Ala His 370 375 380His Ile Pro Asp Ala Gln Val Pro Gly Phe Cys Gly
Thr Leu Gly Gly385 390 395 400Ala Gln Glu Pro Pro Phe Val Leu Leu
Asp Thr Asp Ala Ala Gly Asp 405 410 415Arg Leu Phe Ala Thr Tyr Met
Leu Asp Pro Thr Arg Gly Ser Leu Leu 420 425 430Ser Ala Gly Thr Pro
Val His Phe Pro Arg Gly Gly Gly Thr Pro Gly 435 440 445Ser Asp Pro
Ser Cys Trp Phe Glu Pro Gly Val Ile Cys Asn Gly Gly 450 455 460Val
Glu Pro Gly Leu Val Phe Leu Gly Phe Leu Leu Val Val Gly Met465 470
475 480Gly Leu Thr Gly Ala Phe Leu Ala His Tyr Leu Arg His Arg Leu
Leu 485 490 495His Ile Gln
Met Val Ser Gly Pro Asn Lys Ile Ile Leu Thr Leu Asp 500 505 510Asp
Val Thr Phe Leu His Pro His Gly Gly Ser Thr Arg Lys Val Val 515 520
525Gln Gly Ser Arg Ser Ser Leu Ala Ala Arg Ser Thr Ser Asp Ile Arg
530 535 540Ser Val Pro Ser Gln Pro Leu Asp Asn Ser Asn Ile Gly Leu
Phe Glu545 550 555 560Gly Asp Trp Val Trp Leu Lys Lys Phe Pro Gly
Asp Gln His Ile Ala 565 570 575Ile Arg Pro Ala Thr Lys Thr Ala Phe
Ser Lys Leu Arg Glu Leu Arg 580 585 590His Glu Asn Val Val Leu Tyr
Leu Gly Leu Phe Leu Gly Ser Gly Gly 595 600 605Ala Gly Gly Ser Ala
Ala Gly Glu Gly Val Leu Ala Val Val Ser Glu 610 615 620His Cys Ala
Arg Gly Ser Leu His Asp Leu Leu Ala Gln Arg Asp Ile625 630 635
640Lys Leu Asp Trp Met Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile Lys
645 650 655Gly Met Arg Tyr Leu His His Arg Gly Val Ala His Gly Arg
Leu Lys 660 665 670Ser Arg Asn Cys Val Val Asp Gly Arg Phe Val Leu
Lys Val Thr Asp 675 680 685His Gly His Ala Arg Leu Met Glu Ala Gln
Arg Val Leu Leu Glu Pro 690 695 700Pro Ser Ala Glu Asp Gln Leu Trp
Thr Ala Pro Glu Leu Leu Arg Asp705 710 715 720Pro Ala Leu Glu Arg
Arg Gly Thr Leu Pro Gly Asp Val Phe Ser Leu 725 730 735Gly Ile Ile
Met Gln Glu Val Val Cys Arg Ser Ala Pro Tyr Ala Met 740 745 750Leu
Glu Leu Thr Pro Glu Glu Val Val Glu Arg Val Arg Ser Pro Pro 755 760
765Pro Leu Cys Arg Pro Ser Val Ser Met Asp Gln Ala Pro Val Glu Cys
770 775 780Ile Gln Leu Met Lys Gln Cys Trp Ala Glu His Pro Asp Leu
Arg Pro785 790 795 800Ser Leu Gly His Ile Phe Asp Gln Phe Lys Ser
Ile Asn Lys Gly Arg 805 810 815Lys Thr Asn Ile Ile Asp Ser Met Leu
Arg Met Leu Glu Gln Tyr Ser 820 825 830Ser Asn Leu Glu Asp Leu Ile
Arg Glu Arg Thr Glu Glu Leu Glu Leu 835 840 845Glu Lys Gln Lys Thr
Asp Arg Leu Leu Thr Gln Met Leu Pro Pro Ser 850 855 860Val Ala Glu
Ala Leu Lys Met Gly Thr Pro Val Glu Pro Glu Tyr Phe865 870 875
880Glu Glu Val Thr Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr Ile
885 890 895Ser Ala Met Ser Glu Pro Ile Glu Val Val Asp Leu Leu Asn
Asp Leu 900 905 910Tyr Thr Leu Phe Asp Ala Ile Ile Gly Ser His Asp
Val Tyr Lys Val 915 920 925Glu Thr Ile Gly Asp Ala Tyr Met Val Ala
Ser Gly Leu Pro Gln Arg 930 935 940Asn Gly Gln Arg His Ala Ala Glu
Ile Ala Asn Met Ala Leu Asp Ile945 950 955 960Leu Ser Ala Val Gly
Ser Phe Arg Met Arg His Met Pro Glu Val Pro 965 970 975Val Arg Ile
Arg Ile Gly Leu His Ser Gly Pro Cys Val Ala Gly Val 980 985 990Val
Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn 995
1000 1005Thr Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile
His 1010 1015 1020Val Asn Met Ser Thr Val Arg Ile Leu His Ala Leu
Asp Glu Gly 1025 1030 1035Phe Gln Thr Glu Val Arg Gly Arg Thr Glu
Leu Lys Gly Lys Gly 1040 1045 1050Ala Glu Asp Thr Tyr Trp Leu Val
Gly Arg Arg Gly Phe Asn Lys 1055 1060 1065Pro Ile Pro Lys Pro Pro
Asp Leu Gln Pro Gly Ala Ser Asn His 1070 1075 1080Gly Ile Ser Leu
Gln Glu Ile Pro Leu Asp Arg Arg Trp Lys Leu 1085 1090 1095Glu Lys
Ala Arg Pro Gly Gln Phe Ser Gly Lys 1100 110561103PRTMacaca mulatta
6Met Thr Ala Cys Ala Arg Arg Ala Gly Gly Leu Pro Asp Pro Arg Leu1 5
10 15Cys Gly Pro Ala Arg Trp Ala Pro Ala Leu Pro Arg Leu Pro Arg
Ala 20 25 30Leu Pro Arg Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Gln
Pro Pro 35 40 45Ala Leu Ser Ala Val Phe Thr Val Gly Val Leu Gly Pro
Trp Ala Cys 50 55 60Asp Pro Ile Phe Ser Arg Ala Arg Ala Asp Leu Ala
Ala Arg Leu Ala65 70 75 80Ala Ala Arg Leu Asn Arg Asp Pro Asp Leu
Ala Gly Gly Pro Arg Phe 85 90 95Glu Val Ala Leu Leu Pro Glu Pro Cys
Arg Thr Pro Gly Ser Leu Gly 100 105 110Ala Val Ser Ser Ala Leu Thr
Arg Val Ser Gly Leu Val Gly Pro Val 115 120 125Asn Pro Ala Ala Cys
Arg Pro Ala Glu Leu Leu Ala Glu Glu Ala Gly 130 135 140Ile Ala Leu
Val Pro Trp Gly Cys Pro Gly Thr Gln Ala Ala Gly Thr145 150 155
160Thr Ala Pro Ala Leu Thr Pro Ala Ala Asp Ala Leu Tyr Ala Leu Leu
165 170 175Arg Ala Phe Gly Trp Ala Arg Val Ala Leu Val Thr Ala Pro
Gln Asp 180 185 190Leu Trp Val Glu Ala Gly His Ser Leu Ser Thr Ala
Leu Arg Ala Arg 195 200 205Gly Leu Pro Val Ala Ser Val Thr Ser Met
Glu Pro Leu Asp Leu Ser 210 215 220Gly Ala Arg Glu Ala Leu Arg Lys
Val Arg Asp Gly Pro Arg Val Thr225 230 235 240Ala Val Ile Met Val
Met His Ser Val Leu Leu Gly Gly Glu Glu Gln 245 250 255Arg Tyr Leu
Leu Glu Ala Ala Glu Glu Leu Gly Leu Thr Asp Gly Ser 260 265 270Leu
Val Phe Leu Pro Phe Asp Thr Val His Tyr Ala Leu Ser Pro Gly 275 280
285Pro Glu Ala Leu Ala Ala Leu Ala Asn Ser Ser Gln Leu Arg Arg Ala
290 295 300His Asp Ala Val Leu Thr Leu Thr Arg His Cys Pro Ser Glu
Gly Ser305 310 315 320Val Leu Asp Ser Leu Arg Arg Ala Gln Glu Arg
Arg Glu Leu Pro Ser 325 330 335Asp Leu Asn Leu Gln Gln Val Ser Pro
Leu Phe Gly Thr Ile Tyr Asp 340 345 350Ala Val Phe Leu Leu Val Arg
Gly Val Ala Glu Ala Arg Ala Ala Ala 355 360 365Gly Gly Arg Trp Val
Ser Gly Ala Ala Val Ala Arg His Val Trp Asp 370 375 380Ala Gln Val
Pro Gly Phe Cys Gly Asp Leu Gly Gly Asp Glu Glu Pro385 390 395
400Pro Phe Val Leu Leu Asp Thr Asp Ala Val Gly Asp Arg Leu Phe Ala
405 410 415Thr Tyr Met Leu Asp Pro Thr Arg Gly Ser Leu Leu Ser Ala
Gly Thr 420 425 430Pro Met His Phe Pro Arg Gly Gly Ser Ala Pro Gly
Pro Asp Pro Ser 435 440 445Cys Trp Phe Asp Pro Asn Asn Ile Cys Gly
Gly Gly Leu Glu Pro Gly 450 455 460Leu Val Phe Leu Gly Phe Leu Leu
Val Val Gly Met Gly Leu Ala Gly465 470 475 480Ala Phe Leu Ala His
Tyr Val Arg His Gln Leu Leu His Ile Gln Met 485 490 495Val Ser Gly
Pro Asn Lys Ile Ile Leu Thr Val Asp Asp Ile Thr Phe 500 505 510Leu
His Pro His Gly Gly Thr Ser Arg Lys Val Ala Gln Gly Ser Arg 515 520
525Ser Ser Leu Ala Ala Arg Ser Met Ser Asp Val Arg Ser Gly Pro Ser
530 535 540Gln Pro Thr Asp Ser Pro Asn Val Gly Val Tyr Glu Gly Asp
Arg Val545 550 555 560Trp Leu Lys Lys Phe Pro Gly Asp Gln His Ile
Ala Ile Arg Pro Ala 565 570 575Thr Lys Thr Ala Phe Ser Lys Leu Gln
Glu Leu Arg His Glu Asn Val 580 585 590Ala Leu Tyr Leu Gly Leu Phe
Leu Ala Gln Gly Ala Glu Gly Pro Ala 595 600 605Ala Leu Trp Glu Gly
Asn Leu Ala Val Val Ser Glu His Cys Thr Arg 610 615 620Gly Ser Leu
Gln Asp Leu Leu Ala Gln Arg Glu Ile Lys Leu Asp Trp625 630 635
640Met Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile Lys Gly Ile Arg Tyr
645 650 655Leu His His Arg Gly Val Ala His Gly Arg Leu Lys Ser Arg
Asn Cys 660 665 670Ile Val Asp Gly Arg Phe Val Leu Lys Ile Thr Asp
His Gly His Gly 675 680 685Arg Leu Leu Glu Ala Gln Lys Val Leu Pro
Glu Pro Pro Arg Ala Glu 690 695 700Asp Gln Leu Trp Thr Ala Pro Glu
Leu Leu Arg Asp Pro Ala Leu Glu705 710 715 720Arg Arg Gly Thr Leu
Ala Gly Asp Val Phe Ser Leu Ala Ile Ile Met 725 730 735Gln Glu Val
Val Cys Arg Ser Ala Pro Tyr Ala Met Leu Glu Leu Thr 740 745 750Pro
Glu Glu Val Val Gln Arg Val Arg Ser Pro Pro Pro Leu Cys Arg 755 760
765Pro Leu Val Ser Met Asp Gln Ala Pro Val Glu Cys Ile His Leu Met
770 775 780Lys Gln Cys Trp Ala Glu Gln Pro Glu Leu Arg Pro Ser Met
Asp His785 790 795 800Thr Phe Asp Leu Phe Lys Asn Ile Asn Lys Gly
Arg Lys Thr Asn Ile 805 810 815Ile Asp Ser Met Leu Arg Met Leu Glu
Gln Tyr Ser Ser Asn Leu Glu 820 825 830Asp Leu Ile Arg Glu Arg Thr
Glu Glu Leu Glu Leu Glu Lys Gln Lys 835 840 845Thr Asp Arg Leu Leu
Thr Gln Met Leu Pro Pro Ser Val Ala Glu Ala 850 855 860Leu Lys Thr
Gly Thr Pro Val Glu Pro Glu Tyr Phe Glu Gln Val Thr865 870 875
880Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr Ile Ser Ala Met Ser
885 890 895Glu Pro Ile Glu Val Val Asp Leu Leu Asn Asp Leu Tyr Thr
Leu Phe 900 905 910Asp Ala Ile Ile Gly Ser His Asp Val Tyr Lys Val
Glu Thr Ile Gly 915 920 925Asp Ala Tyr Met Val Ala Ser Gly Leu Pro
Gln Arg Asn Gly Gln Arg 930 935 940His Ala Ala Glu Ile Ala Asn Met
Ser Leu Asp Ile Leu Ser Ala Val945 950 955 960Gly Thr Phe Arg Met
Arg His Met Pro Glu Val Pro Val Arg Ile Arg 965 970 975Ile Gly Leu
His Ser Gly Pro Cys Val Ala Gly Val Val Gly Leu Thr 980 985 990Met
Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Thr Ala Ser Arg 995
1000 1005Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile His Val Asn Leu
Ser 1010 1015 1020Thr Val Gly Ile Leu Arg Ala Leu Asp Ser Gly Tyr
Gln Val Glu 1025 1030 1035Leu Arg Gly Arg Thr Glu Leu Lys Gly Lys
Gly Ala Glu Asp Thr 1040 1045 1050Phe Trp Leu Val Gly Arg Arg Gly
Phe Asn Lys Pro Ile Pro Lys 1055 1060 1065Pro Pro Asp Leu Gln Pro
Gly Ser Ser Asn His Gly Ile Ser Leu 1070 1075 1080Gln Glu Ile Pro
Pro Glu Arg Arg Arg Lys Leu Glu Lys Ala Arg 1085 1090 1095Pro Gly
Gln Phe Ser 110071103PRTPongo pygmaeus 7Met Thr Ala Cys Ala Arg Arg
Ala Gly Gly Leu Pro Asp Pro Gly Leu1 5 10 15Cys Gly Pro Ala Arg Trp
Ala Pro Ser Leu Pro Arg Leu Pro Arg Ala 20 25 30Leu Pro Arg Leu Pro
Leu Leu Leu Leu Leu Leu Leu Leu Gln Pro Pro 35 40 45Ala Leu Ser Ala
Val Phe Thr Val Gly Val Leu Gly Pro Trp Ala Cys 50 55 60Asp Pro Ile
Phe Ser Arg Ala Arg Pro Asp Leu Ala Ala Arg Leu Ala65 70 75 80Ala
Ala Arg Leu Asn Arg Asp Pro Gly Leu Ala Gly Gly Pro Arg Phe 85 90
95Glu Val Ala Leu Leu Pro Glu Pro Cys Arg Thr Pro Gly Ser Leu Gly
100 105 110Ala Val Ser Ser Ala Leu Ala Arg Val Ser Gly Leu Val Gly
Pro Val 115 120 125Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu Leu Ala
Asp Asn Pro Gly 130 135 140Ile Ala Leu Val Pro Trp Gly Cys Pro Trp
Thr Gln Ala Glu Gly Thr145 150 155 160Thr Ala Pro Cys Val Thr Pro
Ala Ala Asp Ala Leu Tyr Ala Leu Leu 165 170 175Arg Ala Phe Gly Trp
Ala Arg Val Ala Leu Val Thr Ala Pro Gln Asp 180 185 190Leu Trp Val
Glu Ala Gly Arg Ser Leu Ser Thr Ala Leu Arg Ala Arg 195 200 205Gly
Leu Pro Val Ala Ser Val Thr Ser Met Glu Pro Leu Asp Leu Ser 210 215
220Gly Ala Arg Glu Ala Leu Arg Lys Val Arg Asp Gly Pro Arg Val
Thr225 230 235 240Ala Val Ile Met Val Met His Ser Val Leu Leu Gly
Gly Glu Glu Gln 245 250 255Arg Tyr Leu Leu Glu Ala Ala Glu Glu Leu
Gly Leu Thr Asp Gly Ser 260 265 270Leu Val Phe Leu Pro Phe Asp Thr
Ile His Tyr Ala Leu Ser Pro Gly 275 280 285Pro Glu Ala Leu Ala Ala
Leu Ala Asn Ser Ser Gln Leu Arg Arg Ala 290 295 300His Asp Ala Val
Leu Thr Leu Thr Arg His Cys Pro Ser Glu Gly Ser305 310 315 320Val
Leu Asp Ser Leu Arg Arg Ala Gln Glu Arg Arg Glu Leu Pro Ser 325 330
335Asp Leu Asn Leu Gln Gln Val Ser Pro Leu Phe Gly Thr Ile Tyr Asp
340 345 350Ala Val Phe Leu Leu Ala Arg Gly Val Ala Glu Ala Trp Ala
Ala Ala 355 360 365Gly Gly Arg Trp Val Ser Gly Ala Ala Val Ala Arg
His Ile Arg Asp 370 375 380Ala Gln Val Pro Gly Phe Cys Gly Asp Leu
Gly Gly Asp Gly Glu Pro385 390 395 400Pro Phe Val Leu Leu Asp Thr
Asp Ala Ala Gly Asp Arg Leu Phe Ala 405 410 415Thr Tyr Met Leu Asp
Pro Ala Arg Gly Ser Phe Leu Ser Ala Gly Thr 420 425 430Arg Met His
Phe Pro Arg Gly Gly Ser Ala Pro Gly Pro Asp Pro Ser 435 440 445Cys
Trp Phe Asp Pro Asn Asn Ile Cys Gly Gly Gly Leu Glu Pro Gly 450 455
460Leu Val Phe Leu Gly Phe Leu Leu Val Val Gly Met Gly Leu Ala
Gly465 470 475 480Ala Phe Leu Ala His Tyr Val Arg His Arg Leu Leu
His Ile Gln Met 485 490 495Val Ser Gly Pro Asn Lys Ile Ile Leu Thr
Val Asn Asp Ile Thr Phe 500 505 510Leu His Pro His Gly Gly Thr Ser
Arg Lys Val Ala Gln Gly Ser Arg 515 520 525Ser Ser Leu Ala Ala Arg
Ser Met Ser Asp Ile Arg Ser Gly Pro Ser 530 535 540Gln Pro Leu Asp
Ser Pro Asn Val Gly Val Tyr Glu Gly Asp Arg Val545 550 555 560Trp
Leu Lys Lys Phe Pro Gly Asp Gln His Ile Ala Ile Arg Pro Ala 565 570
575Thr Lys Thr Ala Phe Ser Lys Leu Gln Glu Leu Arg His Glu Asn Val
580 585 590Ala Leu Tyr Leu Gly Leu Phe Leu Ala Arg Gly Ala Glu Gly
Pro Ala 595 600 605Ala Leu Trp Glu Gly Asn Leu Ala Val Val Ser Glu
His Cys Thr Arg 610 615 620Gly Ser Leu Gln Asp Leu Leu Ser Gln Arg
Glu Ile Lys Leu Asp Trp625 630 635 640Met Phe Lys Ser Ser Leu Leu
Leu Asp Leu Ile Lys Gly Ile Arg Tyr 645 650 655Leu His His Arg Gly
Val Ala His Gly Arg Leu Lys Ser Arg Asn Cys 660 665 670Ile Val Asp
Gly Arg Phe Val Leu Lys Ile Thr Asp His Gly His Gly 675 680 685Arg
Leu Leu Glu Ala Gln Lys Val Leu Pro Glu Pro Pro Arg Ala Glu 690 695
700Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro Ala Leu
Glu705 710 715 720Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser Leu
Ala Ile Ile Met 725 730 735Gln Glu Val Val Cys Arg Ser Ala Pro Tyr
Ala Met Leu Glu Leu Thr 740 745
750Pro Glu Glu Val Val Gln Arg Val Arg Ser Pro Pro Pro Leu Cys Arg
755 760 765Pro Leu Val Ser Met Asp Gln Ala Pro Val Glu Cys Ile His
Leu Met 770 775 780Lys Gln Cys Trp Ala Glu Gln Pro Glu Leu Arg Pro
Ser Met Asp His785 790 795 800Thr Phe Asp Leu Phe Lys Asn Ile Asn
Lys Gly Arg Lys Thr Asn Ile 805 810 815Ile Asp Ser Met Leu Arg Met
Leu Glu Gln Tyr Ser Ser Asn Leu Glu 820 825 830Asp Leu Ile Arg Glu
Arg Thr Glu Glu Leu Glu Leu Glu Lys Gln Lys 835 840 845Thr Asp Arg
Leu Leu Thr Gln Met Leu Pro Pro Ser Val Ala Glu Ala 850 855 860Leu
Lys Thr Gly Thr Pro Val Glu Pro Glu Tyr Phe Glu Gln Val Thr865 870
875 880Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr Ile Ser Ala Met
Ser 885 890 895Glu Pro Ile Glu Val Val Asp Leu Leu Asn Asp Leu Tyr
Thr Leu Phe 900 905 910Asp Ala Ile Ile Gly Ser His Asp Val Tyr Lys
Val Glu Thr Ile Gly 915 920 925Asp Ala Tyr Met Val Ala Ser Gly Leu
Pro Gln Arg Asn Gly Gln Arg 930 935 940His Ala Ala Glu Ile Ala Asn
Met Ser Leu Asp Ile Leu Ser Ala Val945 950 955 960Gly Thr Phe Arg
Met Arg His Met Pro Glu Val Pro Val Arg Ile Arg 965 970 975Ile Gly
Leu His Ser Gly Pro Cys Val Ala Gly Val Val Gly Leu Thr 980 985
990Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Thr Ala Ser Arg
995 1000 1005Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile His Val Asn
Leu Ser 1010 1015 1020Thr Val Gly Ile Leu Arg Ala Leu Asp Ser Gly
Tyr Gln Val Glu 1025 1030 1035Leu Arg Gly Arg Thr Glu Leu Lys Gly
Lys Gly Ala Glu Asp Thr 1040 1045 1050Phe Trp Leu Val Gly Arg Arg
Gly Phe Asn Lys Pro Ile Pro Lys 1055 1060 1065Pro Pro Asp Leu Gln
Pro Gly Ser Ser Asn His Gly Ile Ser Leu 1070 1075 1080Gln Glu Ile
Pro Pro Glu Arg Arg Arg Lys Leu Glu Lys Ala Arg 1085 1090 1095Pro
Gly Gln Phe Ser 110081103PRTCallithrix jacchus 8Met Thr Ala Cys Ala
Arg Arg Ala Gly Gly Leu Pro Asp Pro Gly Leu1 5 10 15Cys Gly Pro Ala
Arg Trp Ala Pro Ala Leu Ser Arg Leu Pro Arg Ala 20 25 30Leu Pro Arg
Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Gln Pro Pro 35 40 45Ala Leu
Ser Ala Gln Phe Thr Val Gly Val Leu Gly Pro Trp Ala Cys 50 55 60Asp
Pro Ile Phe Ser Arg Ala Arg Pro Asp Leu Ala Ala Arg Leu Ala65 70 75
80Ala Ala Arg Leu Asn Arg Asp Pro Ser Leu Ala Gly Gly Pro Arg Phe
85 90 95Glu Val Ala Leu Leu Pro Glu Pro Cys Arg Thr Pro Gly Ser Leu
Gly 100 105 110Ala Val Ser Ser Ala Leu Ala Arg Val Ser Gly Leu Val
Gly Pro Val 115 120 125Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu Leu
Ala Glu Glu Ala Gly 130 135 140Ile Ala Leu Val Pro Trp Gly Cys Pro
Gly Thr Gln Ala Ala Gly Thr145 150 155 160Thr Ala Pro Val Val Thr
Pro Ala Ala Asp Ala Leu Tyr Ala Leu Leu 165 170 175Arg Ala Phe Gly
Trp Ala Arg Val Ala Leu Val Thr Ala Pro Gln Asp 180 185 190Leu Trp
Val Glu Ala Gly Leu Ser Leu Ser Thr Ala Leu Arg Ala Arg 195 200
205Gly Leu Pro Val Val Ser Val Thr Ser Met Glu Pro Leu Asp Leu Ser
210 215 220Gly Ala Arg Glu Ala Leu Arg Lys Val Arg Asn Gly Pro Arg
Val Thr225 230 235 240Ala Val Ile Met Val Met His Ser Val Leu Leu
Gly Gly Glu Glu Gln 245 250 255Arg Tyr Leu Leu Glu Ala Ala Glu Glu
Leu Gly Leu Thr Asp Gly Ser 260 265 270Leu Val Phe Leu Pro Phe Asp
Thr Ile His Tyr Ala Leu Ser Pro Gly 275 280 285Arg Glu Ala Leu Ala
Ala Leu Val Asn Ser Ser Gln Leu Arg Arg Ala 290 295 300His Asp Ala
Val Leu Thr Leu Thr Arg His Cys Ser Ser Glu Gly Ser305 310 315
320Val Leu Asp Ser Leu Arg Lys Ala Gln Gln Arg Arg Glu Leu Pro Ser
325 330 335Asp Leu Asn Leu Glu Gln Val Ser Pro Leu Phe Gly Thr Ile
Tyr Asp 340 345 350Ala Val Val Leu Leu Ala Arg Gly Val Ala Asp Ala
Arg Ala Ala Val 355 360 365Gly Gly Arg Trp Val Ser Gly Ala Ala Val
Ala Arg His Val Trp Asp 370 375 380Ala Gln Ala Ser Gly Phe Cys Gly
Asp Leu Gly Arg Asp Glu Glu Pro385 390 395 400Ser Phe Val Leu Leu
Asp Thr Asp Ala Ala Gly Asp Gln Leu Phe Ala 405 410 415Thr Tyr Met
Leu Asp Pro Ala Arg Gly Ser Leu Leu Ser Ala Gly Thr 420 425 430Pro
Met His Phe Pro Arg Gly Gly Pro Ala Pro Gly Pro Asp Pro Ser 435 440
445Cys Trp Phe Asp Pro Asn Asn Ile Cys Asp Gly Gly Leu Glu Pro Gly
450 455 460Phe Ile Phe Leu Gly Phe Leu Leu Val Val Gly Met Gly Leu
Ala Gly465 470 475 480Ala Leu Leu Ala His Tyr Val Arg His Gln Leu
Leu His Ile Gln Met 485 490 495Val Ser Gly Pro Asn Lys Ile Ile Leu
Thr Val Asp Asp Ile Thr Phe 500 505 510Leu His Pro His Gly Gly Ala
Ser Arg Lys Val Ala Gln Gly Ser Arg 515 520 525Ser Ser Leu Ala Ala
His Ser Thr Ser Asp Ile Arg Ser Gly Pro Ser 530 535 540Gln Pro Ser
Asp Ser Pro Asn Ile Gly Val Tyr Glu Gly Asp Arg Val545 550 555
560Trp Leu Lys Lys Phe Pro Gly Glu Gln His Ile Ala Ile Arg Pro Ala
565 570 575Thr Lys Thr Ala Phe Ser Lys Leu Gln Glu Leu Arg His Glu
Asn Val 580 585 590Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gln Gly Ala
Glu Gly Pro Ala 595 600 605Ala Leu Trp Glu Gly Asn Leu Ala Val Val
Ser Glu His Cys Thr Arg 610 615 620Gly Ser Leu Gln Asp Leu Leu Ala
Gln Arg Glu Ile Lys Leu Asp Trp625 630 635 640Met Phe Lys Ser Ser
Leu Leu Leu Asp Leu Ile Lys Gly Ile Arg Tyr 645 650 655Leu His His
Arg Gly Val Ala His Gly Arg Leu Lys Ser Arg Asn Cys 660 665 670Ile
Val Asp Gly Arg Phe Val Leu Lys Ile Thr Asp His Gly His Gly 675 680
685Arg Leu Leu Glu Ala Gln Lys Val Leu Pro Glu Pro Pro Lys Ala Glu
690 695 700Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro Ala
Leu Glu705 710 715 720Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser
Leu Gly Ile Ile Met 725 730 735Gln Glu Val Val Cys Arg Ser Ala Pro
Tyr Ala Met Leu Glu Leu Thr 740 745 750Pro Asp Glu Val Val Gln Arg
Val Arg Ser Pro Pro Pro Leu Cys Arg 755 760 765Pro Phe Val Ser Met
Asp Gln Ala Pro Val Glu Cys Ile His Leu Met 770 775 780Lys Gln Cys
Trp Ala Glu Gln Pro Glu Leu Arg Pro Ser Met Asp Leu785 790 795
800Thr Phe Asp Leu Phe Lys Asn Ile Asn Lys Gly Arg Lys Thr Asn Ile
805 810 815Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr Ser Ser Asn
Leu Glu 820 825 830Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Leu
Glu Lys Gln Lys 835 840 845Thr Asp Arg Leu Leu Thr Gln Met Leu Pro
Pro Ser Val Ala Glu Ala 850 855 860Leu Lys Thr Gly Thr Pro Val Glu
Pro Glu Tyr Phe Glu Gln Val Thr865 870 875 880Leu Tyr Phe Ser Asp
Ile Val Gly Phe Thr Thr Ile Ser Ala Met Ser 885 890 895Glu Pro Ile
Glu Val Val Asp Leu Leu Asn Asp Leu Tyr Thr Leu Phe 900 905 910Asp
Ala Ile Ile Gly Ser His Asp Val Tyr Lys Val Glu Thr Ile Gly 915 920
925Asp Ala Tyr Met Val Ala Ser Gly Leu Pro Gln Arg Asn Gly Gln Arg
930 935 940His Ala Ala Glu Ile Ala Asn Met Ser Leu Asp Ile Leu Ser
Ala Val945 950 955 960Gly Thr Phe Arg Met Arg His Met Pro Glu Val
Pro Val Arg Ile Arg 965 970 975Ile Gly Leu His Ser Gly Pro Cys Val
Ala Gly Val Val Gly Leu Thr 980 985 990Met Pro Arg Tyr Cys Leu Phe
Gly Asp Thr Val Asn Thr Ala Ser Arg 995 1000 1005Met Glu Ser Thr
Gly Leu Pro Tyr Arg Ile His Val Asn Leu Ser 1010 1015 1020Thr Val
Gly Ile Leu Arg Ala Leu Asp Ser Gly Tyr Gln Val Glu 1025 1030
1035Leu Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr
1040 1045 1050Phe Trp Leu Val Gly Arg Arg Gly Phe Asn Lys Pro Ile
Pro Lys 1055 1060 1065Pro Pro Asp Leu Gln Pro Gly Ala Ser Asn His
Gly Ile Ser Leu 1070 1075 1080Gln Glu Ile Pro Pro Glu Arg Arg Arg
Lys Leu Glu Lys Ala Arg 1085 1090 1095Pro Gly Gln Phe Ser
110091012PRTAiluropoda melanoleucamisc_feature(80)..(88)Xaa can be
any naturally occurring amino acidmisc_feature(964)..(971)Xaa can
be any naturally occurring amino acid 9Met Arg Ala Cys Ala Leu Leu
Ala Gly Gly Leu Pro Tyr Pro Arg Leu1 5 10 15Cys Ala Pro Thr Arg Trp
Ala Pro Ala Arg Pro Gly Val Ser Arg Ala 20 25 30Leu Pro Trp Pro Arg
Pro Arg Leu Arg Leu Leu Leu Leu Leu Leu Leu 35 40 45Arg Pro Pro Ser
Val Leu Ser Ala Val Phe Thr Val Gly Val Leu Gly 50 55 60Pro Trp Ala
Cys Asp Pro Ile Phe Ala Arg Ala Arg Pro Asp Leu Xaa65 70 75 80Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ala Leu Tyr Val Leu Leu Arg 85 90
95Ala Phe Arg Trp Ala Arg Val Ala Leu Val Thr Ala Pro Gln Asp Leu
100 105 110Trp Val Glu Ala Gly Arg Ala Leu Ser Ala Ala Leu Arg Ala
Arg Gly 115 120 125Leu Pro Val Ala Leu Val Thr Thr Met Glu Pro Ser
Asp Leu Ser Gly 130 135 140Ala Arg Glu Ala Leu Arg Arg Val Gln His
Gly Pro Arg Val Ser Ala145 150 155 160Val Ile Met Val Met His Ser
Val Leu Leu Gly Gly Glu Glu Gln Arg 165 170 175Cys Leu Leu Gln Ala
Ala Glu Glu Leu Gly Leu Ala Asp Gly Ser Leu 180 185 190Val Phe Leu
Pro Phe Asp Thr Leu His Tyr Ala Leu Ser Pro Gly Pro 195 200 205Glu
Ala Leu Ala Ala Leu Ala Asn Ser Ser Gln Leu Arg Arg Ala His 210 215
220Asp Ala Val Leu Thr Leu Thr Arg His Cys Pro Pro Gly Gly Ser
Val225 230 235 240Met Asp Ser Leu Arg Arg Ala Gln Glu Arg Gln Glu
Leu Pro Ser Asp 245 250 255Leu Asn Leu Glu Gln Val Ser Pro Leu Phe
Gly Thr Ile Tyr Asp Ala 260 265 270Val Phe Leu Leu Ala Gly Gly Val
Ala Arg Ala Arg Ala Ala Ala Ala 275 280 285Asp Ser Arg Val Pro Gly
Phe Cys Gly Ala Leu Gly Gly Ala Glu Glu 290 295 300Pro Pro Phe Val
Leu Leu Asp Thr Asp Ala Ala Gly Asp Arg Phe Phe305 310 315 320Ala
Thr Tyr Val Leu Asp Pro Thr Arg Gly Ser Leu His Ser Ala Gly 325 330
335Thr Pro Val His Phe Pro Arg Gly Gly Gly Ala Pro Gly Pro Asp Pro
340 345 350Ser Cys Trp Phe Glu Pro Asp Ser Ile Cys Asn Gly Gly Val
Glu Pro 355 360 365Gly Leu Val Phe Thr Gly Phe Leu Leu Val Val Gly
Met Gly Leu Met 370 375 380Gly Ala Phe Leu Ala His Tyr Val Arg His
Arg Leu Leu His Ile Gln385 390 395 400Met Val Ser Gly Pro Asn Lys
Ile Ile Leu Thr Leu Asp Asp Ile Thr 405 410 415Phe Leu His Pro Gln
Gly Gly Ser Ala Arg Lys Val Val Gln Gly Ser 420 425 430Arg Ser Ser
Leu Ala Ala Arg Ser Thr Ser Asp Val Arg Ser Val Pro 435 440 445Ser
Gln Pro Ser Asp Gly Gly Asn Ile Gly Leu Tyr Glu Gly Asp Trp 450 455
460Val Trp Leu Lys Lys Phe Pro Gly Ser Gln His Ile Ala Ile Arg
Pro465 470 475 480Ala Thr Lys Thr Ala Phe Ser Lys Leu Arg Glu Leu
Arg His Glu Asn 485 490 495Val Ala Leu Tyr Leu Gly Leu Phe Leu Gly
Gly Gly Glu Gly Gly Ser 500 505 510Ala Ala Ala Gly Gly Gly Met Leu
Ala Val Val Ser Glu His Cys Thr 515 520 525Arg Gly Ser Leu His Asp
Leu Leu Ala Gln Arg Asp Ile Lys Leu Asp 530 535 540Trp Met Phe Lys
Ser Ser Leu Leu Leu Asp Leu Ile Lys Gly Met Arg545 550 555 560Tyr
Leu His His Arg Gly Val Ala His Gly Arg Leu Lys Ser Arg Asn 565 570
575Cys Val Val Asp Gly Arg Phe Val Leu Lys Val Thr Asp His Gly His
580 585 590Gly Arg Leu Leu Glu Ala Gln Lys Val Leu Ala Glu Pro Pro
Ser Ala 595 600 605Glu Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg
Asp Pro Ala Leu 610 615 620Glu Arg Arg Gly Thr Leu Ala Gly Asp Val
Phe Ser Leu Gly Ile Ile625 630 635 640Met Gln Glu Val Val Cys Arg
Ser Ser Pro Tyr Ala Met Leu Glu Leu 645 650 655Ser Ala Arg Glu Val
Val Gln Arg Val Arg Ser Pro Pro Pro Leu Cys 660 665 670Arg Pro Ser
Val Ser Val Asp Gln Ala Pro Ala Glu Cys Ile Gln Leu 675 680 685Met
Lys Gln Cys Trp Ala Glu Gln Pro Glu Leu Arg Pro Ser Leu Asp 690 695
700Arg Thr Phe Asp Gln Phe Lys Ser Ile Asn Lys Gly Arg Lys Thr
Asn705 710 715 720Ile Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr
Ser Ser Asn Leu 725 730 735Glu Gly Leu Ile Arg Glu Arg Thr Glu Glu
Leu Glu Leu Glu Lys Arg 740 745 750Lys Thr Asp Arg Leu Arg Ala Ala
Ser Leu Pro Ser Ser Val Ala Glu 755 760 765Ala Leu Lys Met Gly Thr
Pro Val Glu Pro Glu Tyr Phe Glu Glu Val 770 775 780Thr Leu Tyr Phe
Ser Asp Ile Val Gly Phe Thr Thr Ile Ser Ala Met785 790 795 800Ser
Glu Pro Ile Glu Val Val Asp Leu Leu Asn Asp Leu Tyr Thr Leu 805 810
815Phe Asp Ala Ile Ile Gly Ser His Asp Val Tyr Lys Val Glu Thr Ile
820 825 830Gly Asp Ala Tyr Met Val Ala Ser Gly Leu Pro Gln Arg Asn
Gly Gln 835 840 845Arg His Ala Ala Glu Ile Ala Asn Met Ala Leu Asp
Ile Leu Ser Ala 850 855 860Val Gly Ser Phe Arg Met Arg His Met Pro
Glu Val Pro Val Arg Ile865 870 875 880Arg Ile Gly Leu His Ser Gly
Pro Cys Val Ala Gly Val Val Gly Leu 885 890 895Thr Met Pro Arg Tyr
Cys Leu Phe Gly Asp Thr Val Asn Thr Ala Ser 900 905 910Arg Met Glu
Ser Thr Gly Leu Pro Tyr Arg Ile His Val Asn Met Ser 915 920 925Thr
Val Arg Ile Leu Arg Ala Leu Asp Glu Gly Phe Gln Thr Glu Val 930 935
940Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr Tyr
Trp945 950 955 960Leu Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Ile Pro Lys Pro 965 970 975Pro Asp Leu Gln Pro Gly Ala Ser Asn His
Gly Ile Ser Leu Gln Glu 980 985
990Ile Pro Leu Asp Arg Arg Gln Lys Leu Glu Lys Ala Arg Pro Gly Gln
995 1000 1005Phe Ser Gly Lys 1010101093PRTMonodelphis domestica
10Met Leu Val Pro Ser Ile Asn Gly Leu Phe His His Pro Pro Trp Cys1
5 10 15Phe Pro Pro Leu Pro Leu Pro Leu Phe Phe Leu Phe Leu Leu Leu
Leu 20 25 30Leu Pro Val Pro Val Leu Pro Ala Thr Phe Thr Ile Gly Val
Leu Gly 35 40 45Pro Trp Ser Cys Asp Pro Ile Phe Ser Arg Ala Arg Pro
Asp Leu Ala 50 55 60Ala Arg Leu Ala Ala Thr Arg Met Asn His Asp Gln
Ala Leu Glu Gly65 70 75 80Gly Pro Trp Phe Glu Val Ile Leu Leu Pro
Glu Pro Cys Arg Thr Ser 85 90 95Gly Ser Leu Gly Ala Leu Ser Pro Ser
Leu Ala Arg Val Ser Gly Leu 100 105 110Val Gly Pro Val Asn Pro Ala
Ala Cys His Pro Ala Glu Leu Leu Ala 115 120 125Gln Glu Ala Gly Val
Pro Leu Val Pro Trp Gly Cys Pro Gln Gly Lys 130 135 140Ala Arg Thr
Thr Ala Pro Ala Leu Pro Leu Ala Leu Asp Ala Leu Tyr145 150 155
160Ala Leu Leu Arg Ala Phe His Trp Ala Lys Val Ala Leu Ile Thr Ala
165 170 175Pro Gln Asp Leu Trp Val Glu Ala Gly Gln Ala Leu Ala Gly
Gly Leu 180 185 190Arg Ser Arg Gly Leu Pro Val Ala Met Val Thr Ser
Leu Glu Thr Thr 195 200 205Asp Leu Glu Ser Ala Lys Asn Ala Leu Lys
Arg Val Arg Asp Gly Pro 210 215 220Lys Val Lys Val Leu Ile Met Val
Met His Ser Val Leu Leu Gly Gly225 230 235 240Glu Glu Gln Arg Leu
Leu Leu Glu Ala Ala Glu Glu Leu Gly Leu Val 245 250 255Glu Gly Thr
Met Val Phe Leu Pro Phe Asp Thr Leu His Tyr Ala Leu 260 265 270Pro
Pro Gly Pro Glu Ala Leu Arg Pro Ile Thr Asn Ser Ser Arg Leu 275 280
285Arg Lys Ala His Asp Ala Val Leu Thr Leu Thr Arg Tyr Cys Pro Lys
290 295 300Gly Ser Val Ser Ala Ser Leu Arg Gln Ala Gln Glu His Arg
Glu Leu305 310 315 320Pro Leu Asp Leu Lys Pro Gln Gln Val Ser Pro
Leu Phe Gly Thr Ile 325 330 335Tyr Asp Ala Ile Tyr Leu Leu Ala Gly
Ala Val Ala Gly Ala Gln Val 340 345 350Ala Gly Gly Gly Gly Trp Val
Ser Gly Ala Ala Val Ala Arg His Ile 355 360 365Pro Asn Thr Leu Val
Ser Gly Phe Cys Gly Asp Leu Gly Gly Thr Lys 370 375 380Glu Pro Pro
Phe Val Leu Leu Asp Thr Asp Gly Met Arg Asp Gln Leu385 390 395
400Leu Pro Thr Tyr Thr Leu Asp Pro Ala Gln Gly Val Leu His His Ala
405 410 415Gly Asn Pro Ile His Phe Pro His Gly Gly Gln Gly Pro Gly
Pro Asp 420 425 430Pro Pro Cys Trp Phe Asp Pro Asn Val Ile Cys Ser
Gly Gly Ile Glu 435 440 445Pro Arg Phe Ile Leu Leu Val Ile Leu Ile
Ile Ile Gly Gly Gly Leu 450 455 460Val Val Ala Thr Leu Ala Tyr Tyr
Val Arg Arg Gln Leu Leu His Ala465 470 475 480Gln Met Val Ser Gly
Pro Asn Lys Met Ile Leu Thr Leu Glu Asp Ile 485 490 495Thr Phe Phe
Pro Arg Gln Gly Ser Ser Ser Arg Lys Ala Thr Glu Gly 500 505 510Ser
Arg Ser Ser Leu Ile Ala His Ser Ala Ser Asp Met Arg Ser Ile 515 520
525Pro Ser Gln Pro Pro Asp Asn Ser Asn Ile Gly Met Tyr Glu Gly Asp
530 535 540Trp Val Trp Leu Lys Lys Phe Pro Gly Glu His Tyr Thr Glu
Ile Arg545 550 555 560Pro Ala Thr Lys Met Ala Phe Ser Lys Leu Arg
Glu Leu Arg His Glu 565 570 575Asn Val Ala Val Gln Met Gly Leu Phe
Leu Ala Gly Ser Met Glu Gly 580 585 590Ala Ala Ala Gly Gly Leu Gly
Gly Gly Ile Leu Ala Val Val Ser Glu 595 600 605Tyr Cys Ser Arg Gly
Ser Leu Gln Asp Leu Leu Ile Gln Arg Asp Ile 610 615 620Lys Leu Asp
Trp Met Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile Lys625 630 635
640Gly Leu Arg Tyr Leu His His Arg Gly Val Ala His Gly Arg Leu Lys
645 650 655Ser Arg Asn Cys Val Val Asp Gly Arg Phe Val Leu Lys Ile
Thr Asp 660 665 670His Ala His Gly Arg Leu Leu Glu Ala Gln Arg Val
Ser Leu Glu Pro 675 680 685Pro Gln Ala Glu Asp Arg Leu Trp Thr Ala
Pro Glu Leu Leu Arg Asn 690 695 700Glu Ala Leu Glu Arg Gln Gly Thr
Leu Gln Gly Asp Val Phe Ser Val705 710 715 720Gly Ile Ile Met Gln
Glu Val Val Cys Arg Cys Glu Pro Tyr Ala Met 725 730 735Leu Glu Leu
Thr Pro Glu Glu Ile Ile Gln Lys Val Gln Ser Pro Pro 740 745 750Pro
Met Cys Arg Pro Ser Val Ser Val Asp Gln Ala Pro Met Glu Cys 755 760
765Ile Gln Leu Met Lys Gln Cys Trp Ala Glu Gln Pro Asp Leu Arg Pro
770 775 780Asn Met Asp Thr Thr Phe Asp Leu Phe Lys Asn Ile Asn Lys
Gly Arg785 790 795 800Lys Thr Asn Ile Ile Asp Ser Met Leu Arg Met
Leu Glu Gln Tyr Ser 805 810 815Ser Asn Leu Glu Asp Leu Ile Arg Glu
Arg Thr Glu Glu Leu Glu Leu 820 825 830Glu Lys Gln Lys Thr Asp Lys
Leu Leu Thr Gln Met Leu Pro Pro Ser 835 840 845Val Ala Glu Ala Leu
Lys Leu Gly Ile Pro Val Glu Pro Glu Tyr Phe 850 855 860Glu Glu Val
Thr Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr Ile865 870 875
880Ser Ala Met Ser Glu Pro Ile Glu Val Val Asp Leu Leu Asn Asp Leu
885 890 895Tyr Thr Leu Phe Asp Ala Ile Ile Gly Ser His Asp Val Tyr
Lys Val 900 905 910Glu Thr Ile Gly Asp Ala Tyr Met Val Ala Ser Gly
Leu Pro Lys Arg 915 920 925Asn Gly Gln Arg His Ala Ala Glu Ile Ala
Asn Met Ser Leu Asp Ile 930 935 940Leu Ser Ser Val Gly Ser Phe Arg
Met Arg His Met Pro Glu Val Pro945 950 955 960Val Arg Ile Arg Ile
Gly Leu His Ser Gly Pro Cys Val Ala Gly Val 965 970 975Val Gly Leu
Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn 980 985 990Thr
Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile His Val 995
1000 1005Asn Leu Ser Thr Val Lys Ile Leu Gln Gly Leu Asn Glu Gly
Phe 1010 1015 1020Gln Ile Glu Ile Arg Gly Arg Thr Glu Leu Lys Gly
Lys Gly Val 1025 1030 1035Glu Asp Thr Tyr Trp Leu Val Gly Arg Lys
Gly Phe Asp Lys Pro 1040 1045 1050Ile Pro Ile Pro Pro Asp Leu Leu
Pro Gly Ala Ser Asn His Gly 1055 1060 1065Ile Ser Leu Gln Glu Ile
Pro Glu Asp Arg Arg Lys Lys Leu Glu 1070 1075 1080Lys Ala Arg Pro
Gly Gln Pro Leu Gly Lys 1085 109011862PRTEquus caballus 11Met Val
Met His Ser Val Leu Leu Gly Gly Glu Glu Gln Arg Cys Leu1 5 10 15Leu
Glu Ala Ala Glu Glu Leu Gly Leu Ala Asp Gly Ser Leu Val Phe 20 25
30Leu Pro Phe Asp Thr Leu His Tyr Ala Leu Ser Pro Gly Pro Glu Ala
35 40 45Leu Ala Val Leu Ala Asn Asn Ser Gln Leu Arg Arg Ala His Asp
Ala 50 55 60Val Leu Thr Leu Thr Arg His Cys Pro Leu Gly Gly Ser Val
Leu Asp65 70 75 80Ser Leu Arg Arg Ala Gln Glu His Gln Glu Leu Pro
Ser Asp Leu Asn 85 90 95Leu Gln Gln Val Ser Pro Leu Phe Gly Thr Ile
Tyr Asp Ala Val Tyr 100 105 110Leu Leu Ala Gly Gly Val Ala Arg Ala
Arg Ala Ala Ala Gly Gly Ser 115 120 125Trp Val Ser Gly Ala Ala Val
Ala His His Val Arg Asp Ala Gln Val 130 135 140Pro Gly Phe Cys Gly
Ala Leu Gly Gly Ala Glu Glu Pro Gln Phe Val145 150 155 160Leu Leu
Asp Thr Asp Ala Ala Gly Asp Arg Leu Phe Ala Thr Tyr Met 165 170
175Leu Asp Pro Thr Arg Gly Ser Leu Trp Ser Ala Gly Thr Pro Val His
180 185 190Phe Pro Arg Gly Gly Arg Gly Pro Gly Pro Asp Pro Trp Cys
Trp Phe 195 200 205Asp Pro Asp Asp Ile Cys Asn Gly Gly Val Glu Pro
Arg Leu Val Phe 210 215 220Ile Gly Phe Leu Leu Ala Val Gly Met Gly
Leu Ala Gly Val Phe Leu225 230 235 240Ala His Tyr Val Arg His Arg
Leu Leu His Ile Gln Met Ala Ser Gly 245 250 255Pro Asn Lys Ile Ile
Leu Thr Leu Asp Asp Ile Thr Phe Leu His Pro 260 265 270Gln Gly Gly
Ser Ser Arg Lys Val Ile Gln Gly Ser Arg Ser Ser Leu 275 280 285Ala
Ala Arg Ser Val Ser Asp Ile Arg Ser Val Pro Ser Gln Pro Met 290 295
300Asp Ser Ser Asn Ile Gly Leu Tyr Glu Gly Asp Trp Val Trp Leu
Lys305 310 315 320Lys Phe Pro Gly Asp Gln His Ile Ala Ile Arg Pro
Ala Thr Lys Thr 325 330 335Ala Phe Ser Lys Leu Arg Glu Leu Arg His
Glu Asn Val Ala Leu Tyr 340 345 350Leu Gly Leu Phe Leu Ala Gly Gly
Ser Ser Gly Ala Ala Ala Pro Arg 355 360 365Glu Gly Met Leu Ala Val
Val Ser Glu His Cys Ala Arg Gly Ser Leu 370 375 380His Asp Leu Leu
Ala Gln Arg Asp Ile Lys Leu Asp Trp Met Phe Lys385 390 395 400Ser
Ser Leu Leu Leu Asp Leu Ile Lys Gly Met Arg Tyr Leu His His 405 410
415Arg Gly Val Ala His Gly Arg Leu Lys Ser Arg Asn Cys Val Val Asp
420 425 430Gly Arg Phe Val Leu Lys Val Thr Asp His Gly His Gly Arg
Leu Leu 435 440 445Glu Ala Gln Lys Val Leu Pro Glu Pro Pro Ser Ala
Glu Asp Gln Leu 450 455 460Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro
Ala Leu Glu Arg Gln Gly465 470 475 480Thr Leu Ala Gly Asp Val Phe
Ser Leu Gly Ile Ile Ile Gln Glu Val 485 490 495Val Cys Arg Ser Thr
Pro Tyr Ala Met Leu Glu Leu Thr Pro Glu Glu 500 505 510Val Val Gln
Arg Leu Gln Ser Pro Pro Pro Leu Cys Arg Pro Ser Val 515 520 525Ser
Met Asp Gln Ala Pro Met Glu Cys Ile Gln Leu Met Lys Gln Cys 530 535
540Trp Ala Glu Gln Pro Asp Leu Arg Pro Ser Met Asp Arg Thr Phe
Asp545 550 555 560Leu Phe Lys Ser Ile Asn Lys Gly Arg Lys Thr Asn
Ile Ile Asp Ser 565 570 575Met Leu Arg Met Leu Glu Gln Tyr Ser Ser
Asn Leu Glu Asp Leu Ile 580 585 590Arg Glu Arg Thr Glu Glu Leu Glu
Leu Glu Lys Gln Lys Thr Asp Arg 595 600 605Leu Leu Thr Gln Met Leu
Pro Pro Ser Val Ala Glu Ala Leu Lys Met 610 615 620Gly Thr Pro Val
Glu Pro Glu Tyr Phe Glu Glu Val Thr Leu Tyr Phe625 630 635 640Ser
Asp Ile Val Gly Phe Thr Thr Ile Ser Ala Met Ser Glu Pro Ile 645 650
655Glu Val Val Asp Leu Leu Asn Asp Leu Tyr Thr Leu Phe Asp Ala Ile
660 665 670Ile Gly Ser His Asp Val Tyr Lys Val Glu Thr Ile Gly Asp
Ala Tyr 675 680 685Met Val Ala Ser Gly Leu Pro Gln Arg Asn Gly Gln
Arg His Ala Ala 690 695 700Glu Ile Ala Asn Met Ala Leu Asp Ile Leu
Ser Ala Val Gly Ser Phe705 710 715 720Arg Met Arg His Met Pro Glu
Val Pro Val Arg Ile Arg Ile Gly Leu 725 730 735His Ser Gly Pro Cys
Val Ala Gly Val Val Gly Leu Thr Met Pro Arg 740 745 750Tyr Cys Leu
Phe Gly Asp Thr Val Asn Thr Ala Ser Arg Met Glu Ser 755 760 765Thr
Gly Leu Pro Tyr Arg Ile His Val Asn Met Ser Thr Val Arg Ile 770 775
780Leu Arg Ala Leu Asp Glu Gly Phe Gln Val Glu Val Arg Gly Arg
Thr785 790 795 800Glu Leu Lys Gly Lys Gly Val Glu Asp Thr Tyr Trp
Leu Val Gly Arg 805 810 815Arg Gly Phe Asn Lys Pro Ile Pro Lys Pro
Pro Asp Leu Gln Pro Gly 820 825 830Ala Ser Asn His Gly Ile Ser Leu
Gln Glu Ile Pro Pro Glu Arg Arg 835 840 845Gln Lys Leu Glu Lys Ala
Arg Pro Gly Gln Phe Ser Gly Lys 850 855 86012292DNAHomo sapiens
12gggccccaga agcctggtgg ttgtttgtcc ttctcagggg aaaagtgagg cggccccttg
60gaggaagggg ccgggcagaa tgatctaatc ggattccaag cagctcaggg gattgtcttt
120ttctagcacc ttcttgccac tcctaagcgt cctccgtgac cccggctggg
atttagcctg 180gtgctgtgtc agccccggtc tcccaggggc ttcccagtgg
tccccaggaa ccctcgacag 240ggcccggtct ctctcgtcca gcaagggcag
ggacgggcca caggccaagg gc 29213953DNAArtificial Sequencehybrid
CBA/CMV promoter 13aattcggtac cctagttatt aatagtaatc aattacgggg
tcattagttc atagcccata 60tatggagttc cgcgttacat aacttacggt aaatggcccg
cctggctgac cgcccaacga 120cccccgccca ttgacgtcaa taatgacgta
tgttcccata gtaacgccaa tagggacttt 180ccattgacgt caatgggtgg
actatttacg gtaaactgcc cacttggcag tacatcaagt 240gtatcatatg
ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
300ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
acgtattagt 360catcgctatt accatggtcg aggtgagccc cacgttctgc
ttcactctcc ccatctcccc 420cccctcccca cccccaattt tgtatttatt
tattttttaa ttattttgtg cagcgatggg 480ggcggggggg gggggggggc
gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 540ggcgaggcgg
agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
600tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
gggcgggagt 660cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc
cgcctcgcgc cgcccgcccc 720ggctctgact gaccgcgtta ctcccacagg
tgagcgggcg ggacggccct tctcctccgg 780gctgtaatta gcgcttggtt
taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 840ttgaggggct
ccgggagcta gagcctctgc taaccatgtt catgccttct tctttttcct
900acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aag
9531436DNAArtificial SequenceForward Primer 14aaaagcggcc gcatgagcgc
ttggctcctg ccagcc 361536DNAArtificial SequenceReverse Primer
15aaaagcggcc gctcacttcc cagtaaactg gcctgg 361624DNAArtificial
SequenceForward Primer 16gacccttcct gctggttcga tcca
241725DNAArtificial SequenceReverse Primer 17ctgcatgtgt agcagcctgt
gcctc 2518992PRTArtificial SequenceConsensus Sequence 18Met Ser Ala
Ala Gly Gly Leu Gly Cys Pro Arg Ala Pro Ser Ile Pro1 5 10 15Arg Leu
Leu Leu Leu Leu Leu Leu Ser Leu Ser Ala Val Phe Val Gly 20 25 30Val
Leu Gly Pro Trp Ala Cys Asp Pro Ile Phe Ala Arg Ala Arg Pro 35 40
45Asp Ile Ala Ala Arg Leu Ala Ala Arg Leu Asn Ala Leu Asp Gly Gly
50 55 60Pro Arg Phe Glu Val Ala Leu Leu Pro Glu Pro Cys Thr Pro Gly
Ser65 70 75 80Leu Gly Ala Val Ser Ser Ala Ser Arg Val Ser Gly Leu
Val Gly Pro 85 90 95Val Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu Leu
Ala Gln Glu Ala 100 105 110Gly Val Ala Leu Val Pro Trp Gly Val Pro
Gly Thr Arg Ala Ala Gly 115 120 125Thr Thr Ala Pro Val Thr Pro Ala
Ala Asp Ala Leu Tyr Leu Leu Arg 130 135 140Ala Phe Arg Trp Ala Val
Ala Leu Ile Thr Ala Pro Gln Asp Leu Trp145 150 155 160Val Glu Ala
Gly Ala Leu Ser Thr Ala Leu Arg Ala Arg Gly Leu Pro 165 170 175Val
Ala Leu Val Thr Ser Met Glu Val Arg Val Ile Met Val Met Gly 180 185
190Ser Val Leu Leu Gly Gly Glu Glu Gln Arg Leu Leu Glu Ala
Ala Glu 195 200 205Glu Leu Ala Leu Asp Gly Ser Leu Val Phe Leu Pro
Phe Asp Thr Leu 210 215 220His Trp Ala Leu Ser Pro Gly Pro Asp Ala
Ile Ala Asn Ser Ser Gln225 230 235 240Leu Arg Lys Ala His Asp Ala
Val Leu Thr Leu Thr Arg Cys Pro Gly 245 250 255Gly Ser Val Asp Ser
Leu Arg Arg Ala Gln Glu His Glu Leu Pro Leu 260 265 270Asp Leu Asn
Leu Gln Val Ser Pro Leu Phe Gly Thr Ile Tyr Asp Ala 275 280 285Val
Phe Leu Leu Ala Gly Gly Thr Ala Thr Ala Gly Gly Gly Trp Val 290 295
300Ser Gly Ala Ala Val Ala Arg Ile Arg Asp Ala Val Gly Phe Cys
Gly305 310 315 320Leu Gly Glu Glu Pro Ser Phe Val Leu Ile Asp Thr
Asp Ala Ser Gly 325 330 335Asp Gln Leu Phe Ala Thr His Leu Leu Asp
Pro Gly Ser Ala Gly Thr 340 345 350Pro Met His Phe Pro Lys Gly Gly
Ala Pro Gly Pro Asp Pro Ser Cys 355 360 365Trp Phe Asp Pro Asp Ile
Cys Asn Gly Gly Val Glu Pro Leu Val Phe 370 375 380Ile Gly Phe Leu
Leu Val Ile Gly Met Gly Leu Gly Ala Phe Leu Ala385 390 395 400Phe
Leu Ala His Tyr Arg His Arg Leu Leu His Ile Gln Met Ser Gly 405 410
415Pro Asn Lys Ile Ile Leu Thr Leu Asp Asp Ile Thr Phe Leu His Pro
420 425 430Gly Gly Ser Arg Lys Val Gln Gly Ser Arg Ser Ser Leu Ala
Arg Ser 435 440 445Ser Asp Ile Arg Ser Ile Ser Gln Asp Thr Asn Ile
Gly Leu Tyr Glu 450 455 460Gly Asp Trp Val Trp Leu Lys Lys Phe Pro
Gly Asp His Ile Ala Ile465 470 475 480Arg Pro Ala Thr Lys Ala Phe
Ser Lys Ile Arg Glu Leu Arg His Glu 485 490 495Asn Val Ala Leu Tyr
Leu Gly Leu Phe Leu Ala Gly Ala Gly Ala Pro 500 505 510Ala Pro Gly
Glu Gly Ile Leu Ala Val Val Ser Glu His Cys Ala Arg 515 520 525Gly
Ser Leu Asp Leu Leu Ala Gln Arg Asp Ile Lys Leu Asp Trp Met 530 535
540Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile Lys Gly Ile Arg Tyr
Leu545 550 555 560His His Arg Gly Val Ala His Gly Arg Leu Lys Ser
Arg Asn Cys Val 565 570 575Val Asp Gly Arg Phe Val Leu Lys Val Thr
Asp His Gly His Gly Arg 580 585 590Leu Leu Glu Ala Gln Arg Val Leu
Pro Glu Pro Pro Ser Ala Glu Asp 595 600 605Gln Leu Trp Thr Ala Pro
Glu Leu Leu Arg Asp Pro Leu Glu Arg Arg 610 615 620Arg Gly Thr Leu
Ala Gly Asp Val Phe Ser Leu Ala Ile Ile Met Gln625 630 635 640Glu
Val Val Cys Arg Ser Pro Tyr Ala Met Leu Glu Leu Thr Pro Glu 645 650
655Glu Val Ile Arg Val Ser Pro Pro Pro Leu Cys Arg Pro Val Ser Ile
660 665 670Asp Gln Ala Pro Met Glu Cys Ile Gln Leu Met Gln Val Trp
Ala Glu 675 680 685Pro Glu Leu Arg Pro Ser Met Asp Thr Phe Asp Leu
Phe Lys Ser Ile 690 695 700Asn Lys Gly Arg Lys Asn Ile Ile Asp Ser
Met Leu Arg Met Leu Glu705 710 715 720Gln Tyr Ser Ser Asn Leu Glu
Asp Leu Ile Arg Glu Arg Thr Glu Glu 725 730 735Leu Glu Glu Glu Lys
Gln Lys Thr Asp Arg Leu Leu Thr Gln Met Leu 740 745 750Pro Pro Ser
Val Ala Glu Ala Leu Lys Met Gly Thr Val Glu Pro Glu 755 760 765Tyr
Phe Glu Glu Val Thr Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr 770 775
780Thr Ile Ser Ala Met Ser Glu Pro Ile Glu Val Val Asp Leu Leu
Asn785 790 795 800Asp Leu Tyr Thr Leu Phe Asp Ala Ile Ile Gly Ala
His Asp Val Tyr 805 810 815Lys Val Glu Thr Ile Gly Asp Ala Tyr Met
Val Ala Ser Gly Leu Pro 820 825 830Gln Arg Asn Gly Arg His Ala Ala
Glu Ile Ala Asn Met Ala Leu Asp 835 840 845Ile Leu Ser Ala Val Gly
Ser Phe Arg Phe Arg Met Arg His Met Pro 850 855 860Glu Val Pro Val
Arg Ile Arg Ile Gly Leu His Ser Gly Pro Cys Val865 870 875 880Ala
Gly Val Val Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp 885 890
895Thr Val Asn Thr Ala Ser Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile
900 905 910His Val Asn Ser Thr Val Ile Leu Ala Leu Gly Phe Glu Arg
Gly Arg 915 920 925Thr Glu Leu Lys Gly Lys Gly Glu Asp Thr Tyr Trp
Leu Val Gly Arg 930 935 940Gly Phe Asn Lys Pro Ile Pro Lys Pro Pro
Asp Leu Gln Pro Gly Ala945 950 955 960Ser Asn His Gly Ile Ser Leu
His Gly Ile Ser Leu Glu Ile Pro Pro 965 970 975Asp Arg Arg Lys Leu
Glu Lys Ala Arg Pro Gly Gln Phe Ser Gly Lys 980 985 990
* * * * *