U.S. patent application number 17/308785 was filed with the patent office on 2021-12-16 for compositions and methods for intravitreal delivery of polynucleotides to retinal cones.
The applicant listed for this patent is Adverum Biotechnologies, Inc., University of Washington. Invention is credited to Thomas W. CHALBERG, JR., Jay NEITZ, Maureen NEITZ.
Application Number | 20210388030 17/308785 |
Document ID | / |
Family ID | 1000005798617 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210388030 |
Kind Code |
A1 |
CHALBERG, JR.; Thomas W. ;
et al. |
December 16, 2021 |
COMPOSITIONS AND METHODS FOR INTRAVITREAL DELIVERY OF
POLYNUCLEOTIDES TO RETINAL CONES
Abstract
Methods and compositions are provided for intravitreally
delivering a polynucleotide to cone photoreceptors. Aspects of the
methods include injecting a recombinant adeno-associated virus
comprising a polynucleotide of interest into the vitreous of the
eye. These methods and compositions find particular use in treating
ocular disorders associated with cone dysfunction and/or death.
Inventors: |
CHALBERG, JR.; Thomas W.;
(Redwood City, CA) ; NEITZ; Jay; (Seattle, WA)
; NEITZ; Maureen; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Adverum Biotechnologies, Inc.
University of Washington |
Redwood City
Seattle |
CA
WA |
US
US |
|
|
Family ID: |
1000005798617 |
Appl. No.: |
17/308785 |
Filed: |
May 5, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15554664 |
Aug 30, 2017 |
11021519 |
|
|
PCT/US2016/020482 |
Mar 2, 2016 |
|
|
|
17308785 |
|
|
|
|
62127194 |
Mar 2, 2015 |
|
|
|
62134466 |
Mar 17, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/861 20130101;
C12N 7/00 20130101; A61K 48/0066 20130101; C12N 2750/14122
20130101; C12N 2750/14143 20130101; A61K 48/0075 20130101; C07K
14/005 20130101; C12N 15/86 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; A61K 48/00 20060101 A61K048/00; C12N 15/86 20060101
C12N015/86; C12N 15/861 20060101 C12N015/861 |
Claims
1-29. (canceled)
30. A method for delivering a polynucleotide of interest to a cone
photoreceptor in a subject, the method comprising: delivering into
the vitreous of the eye an effective amount of recombinant
adeno-associated virus (rAAV) variant comprising the polynucleotide
of interest, wherein: a) the rAAV variant comprises a variant AAV2
VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ
ID NO: 11) inserted into the GH loop between amino acids 587 and
588 of the parental AAV2 VP1 capsid protein; and b) the therapeutic
polynucleotide comprises a regulatory cassette operably linked to a
polynucleotide encoding a therapeutic protein, wherein the
regulatory cassette comprises a human L/M opsin Locus Control
Region ("LCR") enhancer and a truncated M-opsin promoter consisting
of about 140 nucleotides upstream of the transcription start
site.
31. The method according to claim 30, wherein the variant AAV2 VP1
capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID
NO: 13) inserted into the GH loop between amino acids 587 and 588
of the parental AAV2 VP1 capsid protein.
32. The method according to claim 30, wherein the rAAV variant
comprises a VP1 protein having a sequence identity of at least 80%
to the polypeptide of SEQ ID NO: 19.
33. The method according to claim 32, wherein the VP1 protein has a
sequence identity of at least 95% to the polypeptide of SEQ ID NO:
19.
34. The method according to claim 33, wherein the VP1 protein has a
sequence identity of at least 99% to the polypeptide of SEQ ID NO:
19.
35. The method according to claim 34, wherein the VP1 protein has a
sequence identity of 100% to the polypeptide of SEQ ID NO: 19.
36. The method according to claim 30, wherein the cone
photoreceptor is a foveal cone.
37. The method according to claim 30, wherein the subject is a
primate.
38. A method for expressing a gene product in a cone photoreceptor
in a subject, the method comprising: delivering into the vitreous
of the eye an effective amount of recombinant adeno-associated
virus (rAAV) variant comprising a polynucleotide that encodes the
gene product, wherein: a) the rAAV variant comprises a variant AAV2
VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ
ID NO: 11) inserted into the GH loop between amino acids 587 and
588 of the parental AAV2 VP1 capsid protein; and b) the therapeutic
polynucleotide comprises a regulatory cassette operably linked to a
polynucleotide encoding a therapeutic protein, wherein the
regulatory cassette comprises a human L/M opsin Locus Control
Region ("LCR") enhancer and a truncated M-opsin promoter consisting
of about 140 nucleotides upstream of the transcription start
site.
39. The method according to claim 38, wherein the variant AAV2 VP1
capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID
NO: 13) inserted into the GH loop between amino acids 587 and 588
of the parental AAV2 VP1 capsid protein.
40. The method according to claim 38, wherein the rAAV variant
comprises a VP1 protein having a sequence identity of at least 80%
to the polypeptide of SEQ ID NO: 19.
41. The method according to claim 40, wherein the VP1 protein has a
sequence identity of at least 95% to the polypeptide of SEQ ID NO:
19.
42. The method according to claim 41, wherein the VP1 protein has a
sequence identity of at least 99% to the polypeptide of SEQ ID NO:
19.
43. The method according to claim 42, wherein VP1 protein has a
sequence identity of 100% to the polypeptide of SEQ ID NO: 19.
44. The method according to claim 38, wherein the method further
comprises detecting the expression of the polynucleotide in the
cone photoreceptor.
45. The method according to claim 38, wherein the cone
photoreceptor is a foveal cone.
46. The method according to claim 38, wherein the subject is a
primate.
47. A method for treating or preventing a cone-associated retinal
disorder in a subject having or at risk for developing a
cone-associated retinal disorder, the method comprising:
administering intravitreally a recombinant adeno-associated virus
(rAAV) variant comprising a therapeutic polynucleotide in an amount
effective to treat or prevent the cone-associated retinal disorder,
wherein: a) the rAAV variant comprises a variant AAV2 VP1 capsid
protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11)
inserted into the GH loop between amino acids 587 and 588 of the
parental AAV2 VP1 capsid protein; and b) the therapeutic
polynucleotide comprises a regulatory cassette operably linked to a
polynucleotide encoding a therapeutic protein, wherein the
regulatory cassette comprises a human L/M opsin Locus Control
Region ("LCR") enhancer and a truncated M-opsin promoter consisting
of about 140 nucleotides upstream of the transcription start
site.
48. The method according to claim 47, wherein the variant AAV2 VP1
capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID
NO: 13) inserted into the GH loop between amino acids 587 and 588
of the parental AAV2 VP1 capsid protein.
49. The method according to claim 47, wherein the VP1 protein has a
sequence identity of at least 80% to the polypeptide of SEQ ID NO:
19.
50. The method according to claim 49, wherein the VP1 protein has a
sequence identity of at least 95% to the polypeptide of SEQ ID NO:
19.
51. The method according to claim 50, wherein the VP1 protein has a
sequence identity of at least 99% to the polypeptide of SEQ ID NO:
19.
52. The method according to claim 51, wherein the VP1 protein has a
sequence identity of 100% to the polypeptide of SEQ ID NO: 19.
53. The method according to claim 47, wherein the retinal disorder
is a cone-associated disorder.
54. The method according to claim 53, wherein the cone-associated
disorder is selected from the group consisting of rod-cone
dystrophy; cone-rod dystrophy; progressive cone dystrophy;
retinitis pigmentosa (RP); Stargardt Disease; macular
telangiectasia, Leber hereditary optic neuropathy, Best's disease;
adult vitelliform macular dystrophy; X-linked retinoschisis; a
color vision disorder; age-related macular degeneration; wet
age-related macular degeneration; geographic atrophy; diabetic
retinopathy; a retinal vein occlusion; retinal ischemia; Familial
Exudative Vitreoretinopathy (FEVR); COATs disease; and Sorsby's
fundus dystrophy.
55. The method according to claim 47, wherein the method further
comprises identifying the subject as having a cone-associated
disorder.
56. The method according to claim 47, wherein the method further
comprises detecting an improvement in vision following the
administering step.
57. The method according to claim 47, wherein the subject is a
primate.
58. The method according to claim 47, wherein the retinal disorder
is a color vision disorder.
59. The method according to claim 58, wherein the color vision
disorder is blue cone monochromacy.
60. The method according to claim 58, wherein the color vision
disorder is color vision deficiency.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 15/554,664, filed Aug. 30, 2017, which
is a U.S. National Phase Application of International Patent
Application No. PCT/US2016/020482, filed Mar. 2, 2016; which claims
the benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application No. 62/127,194, filed on Mar. 2, 2015, and U.S.
Provisional Application No. 62/134,466, filed on Mar. 17, 2015;
each of which is incorporated by reference herein in its
entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is AVBI_006_04
US_ST25.txt. The text file is 74 KB, was created on May 5, 2021,
and is being submitted electronically via EFS-Web.
FIELD OF THE INVENTION
[0003] This invention pertains to viral-based gene therapy of
retinal disorders.
BACKGROUND OF THE INVENTION
[0004] Photoreceptors are a specialized type of neuron found in the
retina that are capable detecting light and converting that light
signal into electrical signals. There are two types of
photoreceptors in the retina: rod photoreceptors, which are more
sensitive to light and hence support vision in dim lighting; and
cone photoreceptors, which are sensitive to specific wavelengths of
light and hence support the perception of color, and which respond
faster to stimuli than rods so perceive finer detail and more rapid
changes in images than rods and hence support high acuity
vision.
[0005] A number of vision disorders are associated with a loss of
viability or function of the cone photoreceptors, including, for
example, those associated with defects within cones, i.e.
cone-intrinsic defects, such as Stargardt's macular dystrophy, cone
dystrophy, cone-rod dystrophy, Spinocerebellar ataxia type 7, and
Bardet-Biedl syndrome-1, as well as color vision disorders,
including achromotopsia, blue cone monochromacy, and protan,
deutan, and tritan defects; and those that are associated with
retinal disorders that affect the central macula, such as
age-related macular degeneration, macular telangiectasia, retinitis
pigmentosa, diabetic retinopathy, retinal vein occlusions,
glaucoma, Sorsby's fundus dystrophy, adult vitelliform macular
dystrophy, Best's disease, and X-linked retinoschisis. It is
expected that these cone cell disorders may be treated by
delivering to cone photoreceptors a therapeutic gene that, when
expressed by the cone photoreceptors, complements the deficiency
and "rescues" the cone cell viability and/or function.
[0006] The highest density of cone photoreceptors exist at the 1.5
mm depression located in the center of the macula of the retina.
This region, called the "fovea centralis" or "foveal pit", is
responsible for sharp central vision (also called foveal vision),
which is necessary in humans for activities where visual detail is
of primary importance, such as reading and driving. The fovea
centralis consists of two sub-regions: the foveola, a 0.35 mm
diameter rod-free region of retina at the center of the pit; and
the fovea, a 1.5 mm-diameter cone-enriched region of retina that
surrounds the foveola and forms the slopes of the pit. Surrounding
the fovea centralis is the parafovea, which forms the lip of the
depression and is comprised of all cells of the retina, cone
photoreceptors being represented in reduced numbers relative to in
the fovea centralis. Beyond the parafovea is the perifovea, a
region of retina which contains an even more diminished density of
cones. Because cone cells of the fovea constitute the vast majority
of cone photoreceptors in the retina, these cells are ideal target
recipients of therapeutic genes delivered for the treatment of
cone-associated disorders (Oster 1935).
[0007] Some success at delivering genes to cells of the retina has
been achieved by employing viral vectors such as adeno-associated
virus (AAV) or lentivirus. However, these vectors must be
administered by subretinal injection, a procedure that disrupts the
structure of the retina and carries with it a risk of creating
additional damage to retinal tissue that is often already damaged
by the disorder being treated. One alternative is to deliver the
viral vector to the retina intravitreally, i.e., by injecting the
vector into the vitreous of the eye and hoping that the vector
permeates the retina and transduces the retinal cells. However, as
demonstrated by the art, foveal cone cells are notoriously
resistant to transduction by viral vectors delivered intravitreally
to the retina.
[0008] Thus, there is a need in the art for viral vectors that
transduce cone cells with high efficiency when delivered from the
vitreous of the eye. The present invention addresses these
issues.
SUMMARY OF THE INVENTION
[0009] Methods and compositions are provided for intravitreally
delivering a polynucleotide to cone photoreceptors. Aspects of the
methods include injecting a recombinant adeno-associated virus
comprising a polynucleotide of interest into the vitreous of the
eye. These methods and compositions find particular use in treating
ocular disorders associated with cone dysfunction and/or death.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures.
[0011] FIG. 1 illustrates how intravitreally-delivered AAV2 variant
AAV2-7m8 transduces retinal cells in the fovea centralis and
parafovea of primates more efficiently than
intravitreally-delivered AAV2. 5.times.10.sup.11 vector genomes of
AAV2.CMV.GFP (upper left); AAV-2.5T.CMV.GFP (upper right) (Excoffon
K. J., et al. 2009. Proc. Natl. Acad. Sci. U.S.A 106:3865-3870);
(lower left) AAV2-7.8.CMV.GFP (Dalkara D, et al. Sci Transl Med.
2013 Jun. 12; 5(189):189ra76); or AAV-ShH10.CMV.GFP (lower right)
(Klimczak R R et al. PLoS One. 2009 Oct. 14; 4(10):e7467) was
injected into the vitreous of an African green monkey in a volume
of 50 uL, and GFP expression was observed 8 weeks later by OCT
fluorescence imaging in vivo.
[0012] FIG. 2 illustrates how the robustly the AAV2-7m8 capsid
transduces foveal cones of primates. (a-b) AAV2-7m8.MNTC.GFP was
injected into the central vitreous of a baboon and expression was
observed (a) 5 weeks and (b) 8 weeks later by fundus fluorescence.
(c and d) Natural GFP fluorescence within a 15 micron section of
the fovea at approximately 6 months after injection with
AAV2-7m8.MNTC.GFP at low magnification (c) and high magnification
(d).
[0013] FIG. 3 illustrates robust and cone-specific gene expression
in the cones of a mouse retina following intravitreal injection of
AAV-7m8 delivered MNTC.GFP. (a-b) Examples of GFP fluorescence 11
weeks after mice received intravitreal injections of
5.04.times.10.sup.10 vector genomes via intravitreal injection.
(c-e) retinas were harvested for histology 14 weeks after injection
and cone outer segments were labeled with an antibody to L/M opsin
(red). In (c) the red channel is turned off so only the native GFP
is visible, (d) is the same image with the red channel on to allow
visualization of cone outer segments. Comparison of (c) and (d)
shows that most if not all cones were transduced by the virus. (e)
Image from the same retina as in c and d from different angle
showing profiles of cone photoreceptors.
[0014] FIG. 4A-4B illustrates gene expression directed by the pMNTC
regulatory cassette in the cones of the Mongolian gerbil retina.
1.times.10.sup.10-2.times.10.sup.10 vector genomes of virus
carrying GFP under the control of the CMV, pR2.1, or MNTC promoter
were injected in a volume of 5 uL into the vitreous of a Mongolian
gerbil, and GFP expression visualized at the designated time points
by fundus fluorescence imaging. (a) Expression of GFP directed by
AAV2-7m8.CMV.GFP and AAV2-7m8.MNTC.GFP, visualized 4 weeks after
intravitreal administration. Gerbils 12-10, 12-11, and 12-12 were
injected with AAV2-7m8.CMV.GFP, while gerbils 12-13, 12-14, and
12-15 were injected with AAV2-7m8.MNTC.GFP. OD, oculus dexter
(right eye). OS, oculus sinister (left eye). (b) Expression of GFP
directed by AAV2-7m8.pR2.1.GFP and AAV2-7m8.MNTC.GFP, 4 and 8 weeks
later as detected by fundus fluorescence imaging.
[0015] FIG. 5A-5D demonstrate that the pMNTC regulatory cassette
provides for more robust gene expression in foveal cones of
primates than the cone promoter pR2.1. 5.times.10.sup.11 vector
genomes of AAV2-7m8.MNTC.GFP or AAV2-7m8.pR2.1.GFP were injected in
a volume of 50 uL into the vitreous of African Green Monkeys as
indicated (AAV2-7m8.MNTC.GFP into animals 271 and 472;
AAV2-7m8.pR2.1.GFP into animals 500 and 509). Retinas were
visualized in vivo at (a) 2 weeks, (b) 4 weeks, (c) 8 weeks, and
(d) 12 weeks for GFP using a fundus fluorescence camera (a, b, c,
d) or autofluorescence on Heidelberg Spectralis OCT (a, b; data not
shown for weeks 8 and 12). OD, oculus dexter (right eye). OS,
oculus sinister (left eye).
[0016] FIG. 6A-6D demonstrate the contribution of each of the
optimized pMNTC elements to the more robust expression observed.
(a) The pMNTC and pR2.1 expression cassettes. (b) The experimental
expression cassettes, in which each element in pMNTC is replaced
one-by-one by the corresponding element in pR2.1. (c,d) Expression
of the luciferase transgene in the retinas of gerbils
intravitreally injected with each of the test articles (n=6-8 eyes
per construct) as detected (c) 4 weeks and (d) 8 weeks after
injection by IVIS imaging. "7m8.CMV" served as the positive
control.
[0017] FIG. 7 illustrates cone-specific gene expression directed by
the pR2.1 regulatory cassette in a non-human primate (NHP).
5.times.10.sup.11 vector genomes of AAV2-7m8.pR2.1.GFP were
injected in a volume of 50 uL into the vitreous of an African Green
Monkey. GFP transgene expression was observed by stereo
fluorescence microscopy of an 8 .mu.m cross-section of the retina.
GFP was stained with an anti-GFP antibody (green; chicken
polyclonal; Abcam Cat #13970); opsin cone cells were stained with
an anti-L/M Opsin antibody specific for opsin cones (red; rabbit
polyclonal; Abcam Cat #5405); rod cells were stained with an
anti-rhodopsin antibody (1D4 pink; mouse monoclonal; Abcam Cat
#5417); and nuclei were stained with Dapi (blue/all nuclei;
Invitrogen REF #D21490. GFP staining co-localized with L/M opsin
staining but not rhodopsin staining. GFP transgene expression was
present in L/M-opsin cones across the photoreceptor layer, but GFP
transgene expression was not observed in rods. Arrows indicate
illustrative cone cells double-stained for both GFP and opsin.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Methods and compositions are provided for intravitreally
delivering a polynucleotide to cone photoreceptors. Aspects of the
methods include injecting a recombinant adeno-associated virus
comprising the polynucleotide of interest into the vitreous of the
eye. These methods and compositions find particular use in treating
ocular disorders associated with cone dysfunction and/or death.
These and other objects, advantages, and features of the invention
will become apparent to those persons skilled in the art upon
reading the details of the compositions and methods as more fully
described below.
[0019] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0020] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0021] 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 materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described.
[0022] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. It is understood
that the present disclosure supersedes any disclosure of an
incorporated publication to the extent there is a
contradiction.
[0023] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0024] It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely", "only" and the like in connection with the recitation
of claim elements, or the use of a "negative" limitation.
[0025] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the polynucleotide" includes reference to one or
more polynucleotides and equivalents thereof, e.g. nucleic acid
sequences, known to those skilled in the art, and so forth.
[0026] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Definitions
[0027] A "vector" as used herein refers to a macromolecule or
association of macromolecules that comprises or associates with a
polynucleotide and which can be used to mediate delivery of the
polynucleotide to a cell. Illustrative vectors include, for
example, plasmids, viral vectors (virus or the viral genome
thereof), liposomes, and other gene delivery vehicles.
[0028] By a "virus" it is meant a viral particle comprising a viral
capsid and a viral genome. For example, an adeno-associated virus
refers to a viral particle comprising at least one adeno-associated
virus capsid protein or variant thereof and an encapsidated
adeno-associated virus vector genome or variant thereof.
[0029] By a viral "capsid" it is meant the protein shell of a
virus. Viral capsids typically comprise several oligomeric
structural subunits made of protein called protomers. The capsid
encloses, or "encapsidates", the genetic material, or "genome", of
the virus. In some viruses, the capsid is enveloped, meaning that
the capsid is coated with a lipid membrane known as a viral
envelope.
[0030] By a viral "genome" (referred to interchangeably herein as
"viral genome", "viral vector DNA" and "viral DNA"), it is meant a
polynucleotide sequence comprising at least one, and generally two,
viral terminal repeats (e.g. inverted terminal repeats (ITRs), long
terminal repeats (LTR)) at its ends.
[0031] By a "recombinant viral genome" it is meant a viral genome
comprising a heterologous nucleic acid sequence and at least one,
and generally two, viral terminal repeats at its ends. By a
"recombinant virus" it is meant a viral particle comprising a
recombinant viral genome.
[0032] As used herein, the term "heterologous" means derived from a
genotypically distinct entity from that of the rest of the entity
to which it is being compared. For example, a polynucleotide
introduced by genetic engineering techniques into a plasmid or
vector derived from a different species, e.g. a viral genome, is a
heterologous polynucleotide. As another example, a promoter removed
from its native coding sequence and operatively linked to a coding
sequence with which it is not naturally found linked is a
heterologous promoter. As a third example, a heterologous gene
product, e.g. RNA, protein, is a gene product not normally encoded
by a cell in which it is being expressed.
[0033] The term "replication defective" as used herein relative to
the viruses of the disclosure refers to a virus that cannot
independently replicate and package its genome. For example, when a
cell of a subject is infected with recombinant virions, the
heterologous gene is expressed in the infected cells; however, due
to the fact that the infected cells lack AAV rep and cap genes and
accessory function genes, the recombinant virus is not able to
replicate further.
[0034] The term "AAV" is an abbreviation for adeno-associated
virus. When used herein, the term AAV may be used to refer to the
virus itself or derivatives thereof, e.g. the viral capsid, the
viral genome, and the like. The term "AAV" encompasses all
subtypes, both naturally occurring and recombinant forms, and
variants thereof except where required otherwise.
[0035] By "naturally occurring" or "wild-type" AAV it is meant any
adeno-associated virus or derivative thereof comprising a viral
capsid that consists of viral capsid proteins that occur in nature.
Non-limiting examples of naturally occurring AAV include AAV type 1
(AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4
(AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7
(AAV-7), AAV type 8 (AAV-8), AAV9, AAV10, AAV11, AAV12, rh10, avian
AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate
AAV, and ovine AAV. "Primate AAV" refers to AAV that infect
primates, "non-primate AAV" refers to AAV that infect non-primate
mammals, "bovine AAV" refers to AAV that infect bovine mammals,
etc.
[0036] By an "AAV variant" or a "variant AAV" it is meant to
include an AAV viral particle comprising a variant, or mutant, AAV
capsid protein. Examples of variant AAV capsid proteins include AAV
capsid proteins comprising at least one amino acid difference
(e.g., amino acid substitution, amino acid insertion, amino acid
deletion) relative to a corresponding parental AAV capsid protein,
i.e. an AAV capsid protein from which it was derived, a wild type
AAV capsid protein, etc., where the variant AAV capsid protein does
not consist of an amino acid sequence present in a naturally
occurring AAV capsid protein. In addition to differing
structurally, i.e. at the sequence level, from the corresponding
parental AAV, the AAV variant may differ functionally from the
corresponding parental AAV. Put another way, the variant capsid
protein comprising the at least one amino acid difference relative
to a corresponding parental AAV capsid protein may confer
functional characteristics on the AAV variant that are not
possessed by the corresponding parental AAV. For example, the AAV
variant may have a different cellular tropism, i.e. a different
affinity for and/or ability to infect a particular type of cell,
e.g. the AAV variant may bind to a cell, e.g. a retinal cell, with
an increased (or decreased) affinity than the parental AAV, and/or
infect/transduce a cell, e.g. a retinal cell, with an increased (or
decreased) efficiency than the parental AAV such that more (or
less) cells of a cell population is transduced/infected with the
same titer of viral particles. As a second example, the AAV variant
may have a greater (or lesser) affinity for antibodies produced by
the host animal, e.g. the AAV variant may bind with greater (or
lesser) affinity to neutralizing antibodies and be cleared from the
host tissue to a greater (or lesser) extent.
[0037] By "recombinant AAV", or "rAAV" it is meant to include any
AAV that comprises a heterologous polynucleotide sequence in its
viral genome. In general, the heterologous polynucleotide is
flanked by at least one, and generally by two naturally occurring
or variant AAV inverted terminal repeat sequences (ITRs). The term
rAAV vector encompasses both rAAV vector particles and rAAV vector
plasmids. Thus, for example, an rAAV that comprises a heterologous
polynucleotide sequence would be an rAAV that includes a nucleic
acid sequence not normally included in a naturally-occurring,
wild-type AAV, for example, a transgene (e.g. a non-AAV RNA-coding
polynucleotide sequence, non-AAV protein-coding polynucleotide
sequence), a non-AAV promoter sequence, a non-AAV poly-adenylation
sequence, etc.
[0038] As used herein, the term "expression vector" refers to a
vector comprising a region which encodes a gene product of
interest, and is used for effecting the expression of a gene
product in an intended target cell. An expression vector also
comprises control elements operatively linked to the encoding
region to facilitate expression of the protein in the target. The
combination of control elements and a gene or genes to which they
are operably linked for expression is sometimes referred to as an
"expression cassette," a large number of which are known and
available in the art or can be readily constructed from components
that are available in the art.
[0039] As used herein, the term "expression" refers to the
transcription and/or translation of a coding sequence, e.g. an
endogenous gene, a heterologous gene, in a cell.
[0040] As used herein, the terms "gene" or "coding sequence" refer
to a polynucleotide sequence that encodes a gene product, and
encompasses both naturally occurring polynucleotide sequences and
cDNA. A gene may or may not include regions preceding and following
the coding region, e.g. 5' untranslated (5' UTR) or "leader"
sequences and 3' UTR or "trailer" sequences, or intervening
sequences (introns) between individual coding segments (exons).
[0041] As used herein, the term "gene product" refers the desired
expression product of a polynucleotide sequence such as a
polypeptide, peptide, protein or RNA including, for example, a
ribozyme, short interfering RNA (siRNA), miRNA or small hairpin RNA
(shRNA). The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The terms also encompass an amino acid polymer that has
been modified; for example, disulfide bond formation,
glycosylation, lipidation, phosphorylation, or conjugation with a
labeling component.
[0042] As used herein, the terms "operatively linked" or "operably
linked" refers to a juxtaposition of genetic elements on a single
polynucleotide, wherein the elements are in a relationship
permitting them to operate in the expected manner. For instance, a
promoter is operatively linked to a coding region if the promoter
helps initiate transcription of the coding sequence. There may be
intervening residues between the promoter and coding region so long
as this functional relationship is maintained. The combination of
control elements, e.g. promoter, enhancer(s), etc. and a gene or
genes to which they are operably linked for expression is sometimes
referred to as an "expression cassette," a large number of which
are known and available in the art or can be readily constructed
from components that are available in the art.
[0043] By a "promoter" it is generally meant a DNA sequence that
directs the binding of RNA polymerase and thereby promotes RNA
synthesis, i.e., a minimal sequence sufficient to direct
transcription. Promoters and corresponding protein or polypeptide
expression may be ubiquitous, meaning strongly active in a wide
range of cells, tissues and species or cell-type specific,
tissue-specific, or species-specific. Promoters may be
"constitutive," meaning continually active, or "inducible," meaning
the promoter can be activated or deactivated by the presence or
absence of biotic or abiotic factors.
[0044] By an "enhancer" it is generally meant a cis-acting
regulatory element that stimulates, i.e. promotes or enhances,
transcription of an adjacent genes. By a "silencer" it is meant a
cis-acting regulatory element that inhibits, i.e. reduces or
suppresses, transcription of an adjacent gene, e.g. by actively
interfering with general transcription factor assembly or by
inhibiting other regulatory elements, e.g. enhancers, associated
with the gene. Enhancers can function (i.e., can be associated with
a coding sequence) in either orientation, over distances of up to
several kilobase pairs (kb) from the coding sequence and from a
position downstream of a transcribed region. Enhancer sequences
influence promoter-dependent gene expression and may be located in
the 5' or 3' regions of the native gene. Enhancer sequences may or
may not be contiguous with the promoter sequence. Likewise,
enhancer sequences may or may not be immediately adjacent to the
gene sequence. For example, an enhancer sequence may be several
thousand basepairs from the promoter and/or gene sequence.
[0045] A "termination signal sequence" within the meaning of the
invention may be any genetic element that causes RNA polymerase to
terminate transcription, such as for example a polyadenylation
signal sequence. A polyadenylation signal sequence is a recognition
region necessary for endonuclease cleavage of an RNA transcript
that is followed by the polyadenylation consensus sequence AATAAA.
A polyadenylation signal sequence provides a "polyA site", i.e. a
site on a RNA transcript to which adenine residues will be added by
post-transcriptional polyadenylation.
[0046] The terms "identical" or percent "identity" in the context
of two or more nucleotide sequences 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
herein, e.g. the Smith-Waterman algorithm, or by visual
inspection.
[0047] As used herein, the term "sequence identity" refers to the
degree of identify between nucleotides in two or more aligned
sequences, when aligned using a sequence alignment program. The
term "% homology" is used interchangeably herein with the term "%
identity" herein and refers to the level of nucleic acid or amino
acid sequence identity between two or more aligned sequences, when
aligned using a sequence alignment program. For example, as used
herein, 80% homology means the same thing as 80% sequence identity
determined by a defined algorithm, and accordingly a homologue of a
given sequence has greater than 80% sequence identity over a length
of the given sequence. Sequence identity may be determined by
aligning sequences using any of a number of publicly available
alignment algorithm tools, e.g., the local homology algorithm of
Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), the homology
alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48: 443
(1970), the search for similarity method of Pearson & Lipman,
Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), by the BLAST
algorithm, Altschul et al., J Mol. Biol. 215: 403-410 (1990), with
software that is publicly available through the National Center for
Biotechnology Information (www.ncbi.nlm.nih.gov/), or by visual
inspection (see generally, Ausubel et al., infra).
[0048] The terms "complement" and "complementary" refer to two
antiparallel nucleotide sequences capable of pairing with one
another upon formation of hydrogen bonds between the complementary
base residues in the antiparallel nucleotide sequences.
[0049] The term "native", when used in the context of a
polynucleotide or polypeptide herein, refers to a polynucleotide or
polypeptide sequence that is found in nature; i.e., that is present
in the genome of a wild-type virus or cell.
[0050] The term "variant`, when used in the context of a
polynucleotide or polypeptide herein, refers to a mutants of a
native polynucleotide or polypeptide having less than 100% sequence
identity with the native sequence or any other native sequence.
Such variants may comprise one or more substitutions, deletions, or
insertions in the corresponding native gene or gene product
sequence. The term "variant" also includes fragments of the native
gene or gene product, and mutants thereof, e.g. fragments
comprising one or more substitutions, deletions, or insertions in
the corresponding native gene or gene product fragment. In some
embodiments, the variant retains a functional activity of the
native gene product, e.g. ligand binding, receptor binding, protein
signaling, etc., as known in the art.
[0051] The term "fragment," when referring to a recombinant protein
or polypeptide of the invention means a polypeptide having an amino
acid sequence which is the same as part of, but not all of, the
amino acid sequence of the corresponding full length protein or
polypeptide, which retains at least one of the functions or
activities of the corresponding full length protein or polypeptide.
The fragment preferably includes at least 20-100 contiguous amino
acid residues of the full length protein or polypeptide.
[0052] As used herein, the terms "biological activity" and
"biologically active" refer to the activity attributed to a
particular gene product, e.g. RNA or protein, in a cell line in
culture or in vivo. For example, the "biological activity" of an
RNAi molecule refers to the ability of the molecule to inhibit the
production of a polypeptide from a target polynucleotide
sequence.
[0053] As used herein, the term "antagonist" refers a molecule that
acts to inhibit the activity of a target molecule. Antagonists
include both structural antagonists that inhibit the activity of
the target molecule by, for example, binding directly to the target
or inactivating its receptor and functional antagonists, which, for
example, decrease production of the target in a biological system
or increase production of inhibitors of the target in a biological
system.
[0054] The terms "administering" or "introducing", as used herein
refer to contacting a cell, tissue, or subject with a vector for
the purposes of delivering a polynucleotide to the cell or to cells
and or organs of the subject. Such administering or introducing may
take place in vivo, in vitro or ex vivo. A vector for expression of
a gene product may be introduced into a cell by transfection, which
typically means insertion of heterologous DNA into a cell by
physical means (e.g., calcium phosphate transfection,
electroporation, microinjection or lipofection); infection, which
typically refers to introduction by way of an infectious agent,
i.e. a virus; or transduction, which typically means stable
infection of a cell with a virus or the transfer of genetic
material from one microorganism to another by way of a viral agent
(e.g., a bacteriophage).
[0055] "Transformation" or "transfection" as used herein refers to
the delivery of a heterologous DNA to the interior of a cell, e.g.
a mammalian cell, an insect cell, a bacterial cell, etc. by a
vector. A vector used to "transform" a cell may be a plasmid,
minicircle DNA, or other vehicle. Typically, a cell is referred to
as "transduced", "infected"; "transfected" or "transformed"
dependent on the means used for administration, introduction or
insertion of heterologous DNA (i.e., the vector) into the cell. The
terms "transfected" and "transformed" are used interchangeably
herein to refer to the introduction of heterologous DNA by
non-viral methods, e.g. electroporation, calcium chloride
transfection, lipofection, etc., e.g. as when preparing the subject
viral vectors for use in the subject methods. The terms
"transduced" and "infected" are used interchangeably herein to
refer to introduction of the heterologous DNA to the cell in the
context of a viral particle.
[0056] The term "host cell", as used herein refers to a cell which
has been transduced, infected, transfected or transformed with a
vector. The vector may be a plasmid, a viral particle, a phage,
etc. The culture conditions, such as temperature, pH and the like,
are those previously used with the host cell selected for
expression, and will be apparent to those skilled in the art. It
will be appreciated that the term "host cell" refers to the
original transduced, infected, transfected or transformed cell and
progeny thereof.
[0057] As used herein, a "therapeutic" gene refers to a gene that,
when expressed, confers a beneficial effect on the cell or tissue
in which it is present, or on a mammal in which the gene is
expressed. Examples of beneficial effects include amelioration of a
sign or symptom of a condition or disease, prevention or inhibition
of a condition or disease, or conferral of a desired
characteristic. Therapeutic genes include genes that correct a
genetic deficiency in a cell or mammal.
[0058] The terms "treatment", "treating" and the like are used
herein to generally mean obtaining a desired pharmacologic and/or
physiologic effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or symptom thereof,
e.g. reducing the likelihood that the disease or symptom thereof
occurs in the subject, and/or may be therapeutic in terms of a
partial or complete cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, and includes: (a) preventing
the disease from occurring in a subject which may be predisposed to
the disease but has not yet been diagnosed as having it; (b)
inhibiting the disease, i.e., arresting its development; or (c)
relieving the disease, i.e., causing regression of the disease. The
therapeutic agent may be administered before, during or after the
onset of disease or injury. The treatment of ongoing disease, where
the treatment stabilizes or reduces the undesirable clinical
symptoms of the patient, is of particular interest. Such treatment
is desirably performed prior to complete loss of function in the
affected tissues. The subject therapy will desirably be
administered during the symptomatic stage of the disease, and in
some cases after the symptomatic stage of the disease.
[0059] The terms "individual," "subject," "host," and "patient,"
are used interchangeably herein and refer to any mammalian subject
for whom diagnosis, treatment, or therapy is desired, including,
but not limited to, human and non-human primates, including simians
and humans; mammalian sport animals (e.g., horses); mammalian farm
animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats,
etc.); and rodents (e.g., mice, rats, etc.); particularly
humans.
[0060] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms "a", "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise. Furthermore, to the extent
that the terms "including", "includes", "having", "has", "with", or
variants thereof are used in either the detailed description and/or
the claims, such terms are intended to be inclusive in a manner
similar to the term "comprising".
[0061] By "comprising" it is meant that the recited elements are
required in, for example, the composition, method, kit, etc., but
other elements may be included to form the, for example,
composition, method, kit etc. within the scope of the claim. For
example, an expression cassette "comprising" a gene encoding a
therapeutic polypeptide operably linked to a promoter is an
expression cassette that may include other elements in addition to
the gene and promoter, e.g. poly-adenylation sequence, enhancer
elements, other genes, linker domains, etc.
[0062] By "consisting essentially of", it is meant a limitation of
the scope of the, for example, composition, method, kit, etc.,
described to the specified materials or steps that do not
materially affect the basic and novel characteristic(s) of the, for
example, composition, method, kit, etc. For example, an expression
cassette "consisting essentially of" a gene encoding a therapeutic
polypeptide operably linked to a promoter and a polyadenylation
sequence may include additional sequences, e.g. linker sequences,
so long as they do not materially affect the transcription or
translation of the gene. As another example, a variant polypeptide
fragment "consisting essentially of" a recited sequence has the
amino acid sequence of the recited sequence plus or minus about 10
amino acid residues at the boundaries of the sequence based upon
the full length naive polypeptide from which it was derived, e.g.
10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the recited
bounding amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
residues more than the recited bounding amino acid residue.
[0063] By "consisting of", it is meant the exclusion from the
composition, method, or kit of any element, step, or ingredient not
specified in the claim. For example, an expression cassette
"consisting of" a gene encoding a therapeutic polypeptide operably
linked to a promoter and a polyadenylation sequence consists only
of the promoter, polynucleotide sequence encoding the therapeutic
polypeptide, and polyadenlyation sequence. As another example, a
polypeptide "consisting of" a recited sequence contains only the
recited amino acid sequence.
[0064] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, preferably up
to 10%, more preferably up to 5%, and more preferably still up to
1% of a given value. Alternatively, particularly with respect to
biological systems or processes, the term can mean within an order
of magnitude, preferably within 5-fold, and more preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated the term "about"
meaning within an acceptable error range for the particular value
should be assumed.
Methods and Compositions
[0065] In some aspects of the invention, methods and compositions
are provided for delivering a polynucleotide to cone
photoreceptors. As discussed above, cone photoreceptors, referred
to interchangeably herein as "cone cells", "retinal cones", and
most simply, "cones," are one of two subtypes of photoreceptor
cells in the retina of the eye, the other being rod photoreceptors.
Cone photoreceptors may be readily distinguished from rod
photoreceptors by a number of physical, biochemical, and functional
characteristics. For example, cone photoreceptors comprise an outer
segment region that is shaped like a cone, whereas rod
photoreceptors comprise an outer segment that is shaped like a rod.
Cone photoreceptors express a number of proteins that are not
expressed by rod photoreceptors, including, e.g., L-opsin (OPN1LW,
the nucleic acid and amino acid sequences for which may be found at
GenBank Accession No:
[0066] NM 020061.5), M-opsin (OPN1MW, the nucleic acid and amino
acid sequences for which may be found at GenBank Accession No:
NM_000513.2), or S-opsin (OPN1SW, the nucleic acid and amino acid
sequences for which may be found at GenBank Accession No:
NM_001708.2); whereas rod photoreceptors express a number of
proteins that are not expressed by cone photoreceptors, e.g.
rhodopsin (RHO, the nucleic acid and amino acid sequences for which
may be found at GenBank Accession No: NM_000539.3) and rod-derived
cone viability factor (RDCVF, also known as NXNL1, the nucleic acid
and amino acid sequences for which may be found at GenBank
Accession No: NM_138454.1). Functionally, cone photoreceptors
differ from rod photoreceptors in that cone photoreceptors are
responsible for color vision and function best in relatively bright
light, whereas rod photoreceptors support vision at low light
levels and function best in dim light; cones and rods can be
distinguished based on this difference using an electroretinogram
(ERG) or color ERG (cERG). Finally, cone photoreceptors may be
distinguished from rod photoreceptors by their location in the
retina. As discussed above, the vast majority of cone
photoreceptors--all of them L- and M-cone photoreceptors--are
densely packed in a 1.5 mm depression located in the center of the
macula of the retina, called the fovea centralis, with the
remaining L- and M-cone photoreceptors and the S-cone
photoreceptors scattered in the parafovea, the perifovea, and the
peripheral retina. In contrast, rod photoreceptors are excluded
from the foveola and are poorly represented in the fovea, instead
being primarily found in the parafovea, the perifovea, and the
peripheral retina.
[0067] As discussed above, prior to the present disclosure, it was
common understanding in the art that cone photoreceptors--and more
particularly, the L- and M-cone photoreceptors in the fovea--were
resistant to transduction by AAV delivered from the vitreous.
However, as demonstrated by the working examples herein, foveal
cones can, in fact, be transduced by intravitreally delivery using
the methods and compositions of the present disclosure. In some
embodiments, the cone photoreceptors that are transduced by the
subject methods and compositions reside anywhere in the retina,
i.e. the macula (the foveal centralis, the parafovea, the
perifovea), or the periphery. In some embodiments, the cone
photoreceptors reside in the fovea centralis. In certain
embodiments, the cone photoreceptors are foveal cones, that is,
they are L- or M-cones that reside within the fovea, this being the
region of the fovea centralis spanning from about 0.175 mm from the
center of the fovea centralis to about 0.75 mm from the center of
the fovea centralis.
rAAV Virions
[0068] In practicing the subject methods, the polynucleotide of
interest is delivered to cone photoreceptors by injecting into the
vitreous of the eye a recombinant viral particle comprising the
polynucleotide of interest as a heterologous sequence within its
genome. In some instances, the recombinant viral particles are
recombinant adeno-associated virus (rAAV) particles. In some
embodiments, the rAAV are of a wild-type serotype; that is, they
comprise a viral capsid that consists of viral capsid proteins that
occur in nature. In other embodiments, the rAAV are an AAV serotype
variant, i.e., they comprise a variant AAV capsid protein, that is,
an AAV capsid protein that comprises at least one amino acid
difference relative to a corresponding parental AAV capsid protein,
e.g. a wild type AAV capsid protein, and does not consist of an
amino acid sequence present in a naturally occurring AAV capsid
protein.
[0069] As demonstrated in the working examples of the present
application, rAAV virions comprising a variant AAV capsid protein
comprising at least one amino acid difference in the GH loop, or
more particularly, in subloop IV of the GH loop, demonstrate an
increased infectivity of cone photoreceptors relative to rAAV
virions comprising wild type AAV capsid protein when delivered
intravitreally. By "increased infectivity," it is meant that the
variant rAAV virion is better able to transduce the target cell
than the wild type AAV capsid protein. Improvements in the ability
of an AAV to transduce a cell can be observed by observing more
polynucleotide being delivered to each cell and more cells being
transduced in a tissue, resulting in an increase in the amount of
polynucleotide delivered to each cell and to the tissue.
Accordingly, in some aspects of the invention, methods are provided
for the improved delivery of a polynucleotide of interest to cone
photoreceptors, the improvement comprising delivering to the
vitreous of the eye an effective amount of a rAAV variant, the rAAV
variant comprising i) a variant AAV capsid protein that comprises
at least one amino acid difference relative to a corresponding
parental AAV capsid protein, e.g. a wild type AAV capsid protein,
and does not consist of an amino acid sequence present in a
naturally occurring AAV capsid protein, and ii) the polynucleotide
of interest as a heterologous sequence within the viral genome.
[0070] Of particular interest in the subject disclosure are rAAV
variants that comprise at least one amino acid difference in the GH
loop, or "loop IV", of an AAV capsid protein relative to a
corresponding parental AAV capsid protein. By the GH loop, or loop
IV, it is meant the loop created between the G and H strands of the
jelly-roll .beta.-barrel of the AAV capsid protein VP1, as
described in, e.g., Xie et al. (2002) PNAS 99(16):10405-10410, van
Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J.
Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. In some
instances, the at least one amino acid difference is within subloop
4 of the GH loop, i.e., the solvent-accessible portion of the GH
loop, consisting essentially of about amino acids 571-612 of AAV1
VP1 (SEQ ID NO:1), about amino acids 570-611 of AAV2 VP1 (SEQ ID
NO:2), about amino acids 571-612 of AAV3 VP1 (SEQ ID NO:3), about
amino acids 569-610 of AAV4 VP1 (SEQ ID NO:4), about amino acids
560-601 of AAV5 VP1 (SEQ ID NO:5), about amino acids 571 to 612 of
AAV6 VP1 (SEQ ID NO:6), about amino acids 572 to 613 of AAV7 VP1
(SEQ ID NO:7), about amino acids 573 to 614 of AAV8 VP1 (SEQ ID
NO:8), about amino acids 571 to 612 of AAV9 VP1 (SEQ ID NO:9),
about amino acids 573 to 614 of AAV10 VP1 (SEQ ID NO:10); or about
the corresponding amino acid range of a variant thereof. In certain
instances, the at least one amino acid difference is within the
range of amino acids consisting essentially of amino acids 581-596
of AAV1 VP1, 580-595 of AAV2 VP1, 581-596 of AAV3 VP1, 579-594 of
AAV4, 570-585 of AAV5 VP1, 581-596 of AAV6 VP1, 582-597 of AAV7
VP1, 583-598 of AAV8 VP1, 581-596 of AAV9 VP1, 583-598 of AAV10
VP1, or within the corresponding amino acid range of a variant
thereof. Those skilled in the art would know, based on a comparison
of the amino acid sequences of capsid proteins of various AAV
serotypes, where the amino acids "corresponding to amino acids
570-611 of VP1 from AAV2", for example, would be in a capsid
protein of any given AAV serotype.
[0071] In some embodiments, the at least one amino acid difference
is an insertion of a peptide between two amino acids in the GH loop
of the AAV capsid protein, e.g. between about amino acids 571-612
of AAV1 VP1 (SEQ ID NO:1), about amino acids 570-611 of AAV2 VP1
(SEQ ID NO:2), about amino acids 571-612 of AAV3 VP1 (SEQ ID NO:3),
about amino acids 569-610 of AAV4 VP1 (SEQ ID NO:4), about amino
acids 560-601 of AAV5 VP1 (SEQ ID NO:5), about amino acids 571 to
612 of AAV6 VP1 (SEQ ID NO:6), about amino acids 572 to 613 of AAV7
VP1 (SEQ ID NO:7), about amino acids 573 to 614 of AAV8 VP1 (SEQ ID
NO:8), about amino acids 571 to 612 of AAV9 VP1 (SEQ ID NO:9),
about amino acids 573 to 614 of AAV10 VP1 (SEQ ID NO:10); or about
the corresponding amino acid range of a variant thereof; for
example, between two amino acids within amino acids 581-596 of AAV1
VP1, 580-595 of AAV2 VP1, 581-596 of AAV3 VP1, 579-594 of AAV4,
570-585 of AAV5 VP1, 581-596 of AAV6 VP1, 582-597 of AAV7 VP1,
583-598 of AAV8 VP1, 581-596 of AAV9 VP1, 583-598 of AAV10 VP1, or
within the corresponding amino acid range of a variant thereof. For
example, the insertion site can be between amino acids 580 and 581,
amino acids 581 and 582, amino acids 582 and 583, amino acids 583
and 584, amino acids 584 and 585, amino acids 585 and 586, amino
acids 586 and 587, amino acids 587 and 588, amino acids 588 and
589, amino acids 589 and 590, amino acids 590 and 591, amino acids
591 and 592, amino acids 592 and 593, amino acids 593 and 594, or
amino acids 594 and 595 of AAV2 VP1, or the corresponding amino
acids in another AAV VP1 or variant thereof.
[0072] Of particular interest in some embodiments of the present
disclosure are the rAAV variants comprising a peptide insertion as
disclosed in PCT Publication No. WO 2012/145601, the full
disclosure of which is incorporated herein by reference. These rAAV
variants comprise a peptide insert having 5 to 11 amino acids in
length, that is, the inserted peptide comprises 5 amino acids, 6
amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino
acids, or 11 amino acids.
[0073] One exemplary peptide of particular interest is a peptide of
Formula I:
TABLE-US-00001 (SEQ ID NO: 20)
Y.sub.1Y.sub.2X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7Y.sub.3Y.s-
ub.4
where: each of Y1-Y4, if present, is independently selected from
Ala, Leu, Gly, Ser, and Thr; X1, if present, is selected from Leu,
Asn, and Lys; X2 is selected from Gly, Glu, Ala, and Asp; X3 is
selected from Glu, Thr, Gly, and Pro; X4 is selected from Thr, Ile,
Gln, and Lys; X5 is selected from Thr and Ala; X6 is selected from
Arg, Asn, and Thr; X7, if present, is selected from Pro and Asn. In
certain embodiments, X1 and/or X7 is absent.
[0074] A second exemplary peptide of particular interest is a
peptide of Formula II:
TABLE-US-00002 (SEQ DI NO: 21)
Y.sub.1Y.sub.2X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7Y.sub.3Y.s-
ub.4
where: each of Y1-Y4, if present, is independently selected from
Ala, Leu, Gly, Ser, and Thr; each of X1-X4 is any amino acid;
X5 is Thr
X6 is Arg; and
X7 is Pro.
[0075] In certain embodiments, any one or more of Y1-Y4 are
absent.
[0076] A third exemplary peptide of particular interest is a
peptide of Formula III:
TABLE-US-00003 (SEQ ID NO: 22)
Y.sub.1Y.sub.2X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7Y.sub.3Y.s-
ub.4
where: each of Y1-Y4, if present, is independently selected from
Ala, Leu, Gly, Ser, and Thr; X1, if present, is selected from Leu
and Asn; X2, if present, is selected from Gly and Glu; X3 is
selected from Glu and Thr; X4 is selected from Thr and Ile;
X5 is Thr;
X6 is Arg; and
X7 is Pro.
[0077] In certain embodiments, any one or more of Y1-Y4, X1 and X2
are absent.
[0078] A fourth exemplary peptide of particular interest is a
peptide of Formula IV:
TABLE-US-00004 (SEQ ID NO: 23) Y1Y2X1X2X3X4X5X6X7Y3Y4
where: each of Y1-Y4, if present, is independently selected from
Ala, Leu, Gly, Ser, and Thr; X1, if present, is selected from Leu,
Asn, Arg, Ala, Ser, and Lys; X2 is selected from Gly, Glu, Ala,
Val, Thr, and Asp; X3 is selected from Glu, Thr, Gly, Asp, or Pro;
X4 is selected from Thr, Ile, Gly, Lys, Asp, and Gln; X5 is
selected from Thr, Ser, Val, and Ala; X6 is selected from Arg, Val,
Lys, Pro, Thr, and Asn; and X7 is selected from Pro, Gly, Phe, Asn,
and Arg. In certain embodiments, any one or more of Y1-Y4 and X1
are absent.
[0079] Exemplary insertion peptides of particular interest having
these formulas include peptides comprising the sequence LGETTRP
(SEQ ID NO:11) and NETITRP (SEQ ID NO:12), or variants thereof. In
some cases, the insertion peptide has from 1 to 4 spacer amino
acids (Y1-Y4) at the amino terminus and/or at the carboxyl
terminus. Suitable spacer amino acids include, but are not limited
to, leucine, alanine, glycine, and serine. For example, in some
cases, an insertion peptide has the amino acid sequence: LALGETTRPA
(SEQ ID NO:13); LANETITRPA (SEQ ID NO:14), As another example, in
some cases, the insertion peptide has the amino acid sequence
AALGETTRPA (SEQ ID NO:15) or AANETITRPA (SEQ ID NO:16), As yet
another example, in some cases, an insertion peptide has the amino
acid sequence GLGETTRPA (SEQ ID NO:17) or GNETITRPA (SEQ ID
NO:18).
[0080] In some embodiments, a subject rAAV virion capsid does not
include any amino acid substitutions, insertions, or deletions,
other than an insertion of from about 5 to 11 amino acids in the GH
loop or subregion thereof relative to a corresponding parental AAV
capsid protein. In other embodiments, a subject rAAV virion capsid
may include from 1 to about 25 amino acid insertions, deletions, or
substitutions, compared to the parental AAV capsid protein, in
addition to an insertion of from about 5 to 11 amino acids in the
GH loop or subregion thereof as described above. For example, a
number of amino acid sequence alterations have been disclosed in
the art, any of which may be included in the subject rAAV. In some
embodiments, a subject rAAV virion capsid is a chimeric capsid,
e.g., the capsid comprises a portion of an AAV capsid of a first
AAV serotype and a portion of an AAV capsid of a second serotype;
and comprises an insertion of from about 5 amino acids to about 11
amino acids in the GH loop or subregion thereof relative to a
corresponding parental AAV capsid protein.
[0081] In some embodiments, a subject rAAV virion comprises a
capsid protein comprising an amino acid sequence having a sequence
identity of 80% or more to the VP1 capsid protein of the
corresponding parental capsid protein, e.g. 85% or more, 90% or
more, 95% or more or 97 C % identity or more to the corresponding
parental capsid protein and an insertion of from about 5 to 11
amino acids in the GH loop or subregion thereof relative to a
corresponding parental AAV capsid protein. For example a sequence
identity of 80% or more to the 7m8 VP1 sequence described in SEQ ID
NO:19, e.g. 85% identity or more, 90% identity or more, or 95%
identity or more to the 7m8 VP1 sequence, in some instances 97%
identity or more, 98% identity or more, or at least about 99%
sequence identity to the amino acid sequence provided in SEQ ID
NO:19.
[0082] rAAV variants that are encompassed by the subject
compositions and that find use in the subject methods may be
readily validated as such by determining the efficacy by which they
transduce cone photoreceptors, e.g. foveal cone photoreceptors. For
example, viral particles may be created comprising an AAV viral
genome comprising an expression cassette comprising GFP operably
linked to a cone promoter as known in the art, packaged into the
subject rAAV, and the viral particles injected into the vitreous of
a mammalian eye, e.g. the eye of a mouse, rat, rabbit, gerbil,
hamster, squirrel, or primate, e.g. non-human primate. rAAV virions
encompassed by the present disclosure will typically exhibit at
least a 2-fold, at least a 5-fold, at least a 10-fold, at least a
15-fold, at least a 20-fold, at least a 25-fold, at least a
50-fold, in some instances, more than 50-fold, e.g. at least a
60-fold, at least a 70-fold, at least an 80-fold, at least a
90-fold, for example, a 100-fold increased infectivity of cone
photoreceptors or more when administered via intravitreal injection
as compared to the infectivity of cone photoreceptors by an AAV
virion comprising the corresponding parental AAV capsid protein.
Put another way, rAAV virions suitable for use in the subject
methods will infect at least 10-fold more, at least 15-fold more,
at least 20-fold more, at least 50-fold more, in some instances
more than 50-fold more cone photoreceptors, e.g. at least 60-fold,
at least 70-fold, at least 80-fold, at least 90-fold, for example,
a 100-fold more cone photoreceptors than AAV virions comprising the
corresponding parental AAV capsid protein.
[0083] In some embodiments, the method may further comprise the
step of detecting the presence of the delivered polynucleotide in
the cone photoreceptor. Any convenient method may be employed for
detecting the presence of the polynucleotide. For example, the
polynucleotide may be detecting using, e.g., PCR, Next Gen
sequencing, and the like, or the expression of a gene product
encoded by the polynucleotide may be detected by, e.g., RT-PCR,
Northern blot, RNAse protection, Western blot, ELISA,
immunohistochemistry, and the like. These methods are particularly
suited to preclinical studies. In clinical studies, in may be
preferably to detect the presence of the polynucleotide by
detecting the presence of a functional gene product, that is, by
detecting the impact of the gene product on the viability or
function of the cone photoreceptor in the subject. For example, if
the gene product encoded by the polynucleotide improves the
viability of the cone photoreceptor, an improvement in viability of
the cone photoreceptor may be detected by, e.g., fundus
photography, Optical coherence tomography (OCT), Adaptive Optics
(AO), and the like, as a way of detecting the presence of the
polynucleotide. If the gene product encoded by the polynucleotide
alters the activity of the cone photoreceptor, the modified
activity of the cone photoreceptor may be detected by, e.g.,
electroretinogram (ERG) and color ERG (cERG); color vision tests
such as pseudoisochromatic plates (Ishihara plates,
Hardy-Rand-Ritter polychromatic plates), the Farnsworth-Munsell 100
hue test, the Farnsworth's panel D-15, the City university test,
Kollner's rule, and the like; and visual acuity tests such as the
ETDRS letters test, Snellen visual acuity test, and the like, as a
way of detecting the presence of the delivered polynucleotide.
[0084] As discussed above, in some embodiments, the polynucleotide
that is delivered by the subject compositions and methods is
expressed by the cone photoreceptor to which it is delivered. In
other words, in some aspects of the invention, methods are provided
for expressing a gene product in a cone photoreceptor, the methods
comprising delivering to the cone photoreceptor a polynucleotide
that encodes the gene product of interest. As will be well
understood by the ordinarily skilled artisan, expression by a cone
cell of a polynucleotide of interest typically requires that the
polynucleotide of interest be operably linked to a promoter. As
will also be appreciated by the ordinarily skilled artisan, there
are a number of ways in which this can be achieved. For example,
the polynucleotide may be delivered to the host cell, i.e. the cone
photoreceptor, operatively linked to a promoter. In other words,
the viral genome comprising the polynucleotide of interest also
comprises a promoter, wherein the promoter is operably linked to
the polynucleotide to form an expression cassette. As another
example, the polynucleotide may be delivered to the host cell i.e.
the cone photoreceptor, flanked by sequences that promoter the
integration of the polynucleotide into the host genome. In other
words, the viral genome comprising the polynucleotide of interest
comprises sequences flanking the polynucleotide of interest that
are homologous to sequences flanking the 3' end of a host cell
promoter and promote the recombination of the polynucleotide of
interest into the host genome such that it is operably linked to
the host cell promoter. Other arrangements of the recombinant viral
genome that may be employed to ensure the expression of the
polynucleotide of interest will be readily envisioned by the
ordinarily skilled artisan; see, for example, US Application
Publication No. 2013/0280222, the full disclosure of which is
incorporated herein by reference.
[0085] Accordingly, in some instances, the viral genome comprised
by the rAAV comprises a promoter operably linked to the
polynucleotide of interest. In some instances, the promoter is a
ubiquitous promoter, i.e., it is a promoter that is active in a
wide range of cells, tissues and species. In other instances, the
promoter is a cone promoter. By a cone promoter it is meant a
promoter that is active in cone photoreceptors, i.e., that promotes
the expression in cone photoreceptors of a polynucleotide to which
it is operably linked. Non-limiting examples of cone promoters that
find use in the subject compositions include the pMNTC promoter as
disclosed in U.S. Provisional Application Nos. 61/954,330 and
62/127,185; the pR2.1 promoter or variants thereof (e.g. pR1.7,
pR1.5, pR1.1, etc.) as disclosed in, e.g., US Application No.
2013/0317091; or the synthetic IRBP/GNAT2 promoter as disclosed in
US Application No. 2014/0275231; the full disclosures of which are
incorporated herein by reference. In other instances, the viral
genome comprised by the rAAV comprises two sequences having
homology to a target integration site in the host genome, a first
sequence that is homologous to the region 5' of the integration
site and located 5' to the polynucleotide on the viral genome, and
a second sequence that is homologous to the region 3' of the
integration site and located 3' to the polynucleotide on the viral
genome, wherein the target integration site is 3' to and operably
linked to a host promoter, e.g. a cone promoter, e.g. an L-opsin
promoter, an M-opsin promoter.
[0086] In some embodiments, transduction is enhanced relative to
expression as observed when a wild type or other parental capsid is
employed. By enhanced, it is meant transduction that is elevated,
increased, or augmented for example, at least 2-fold, at least
5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at
least 25-fold, at least 50-fold, in some instances, more than
50-fold, e.g. at least 60-fold, at least 70-fold, at least 80-fold,
at least 90-fold, for example, 100-fold in a subject's cone
photoreceptors over levels that would be observed using a wild type
or other parental capsid protein, and usually to an amount to have
an impact on cone viability and/or function, e.g. to provide a
therapeutic benefit to the subject.
[0087] Enhanced transduction of cone cells by the subject variant
rAAVs is expected to result in enhanced expression of
polynucleotides, e.g., expression cassettes, being delivered to
those cells by the variant rAAV. Enhanced expression of a
polynucleotide by the rAAVs of the subject disclosure may be
observed in a number of ways. For example, enhanced expression may
be observed by detecting the expression of the polynucleotide
following contact of the variant rAAV to the cone cells sooner,
e.g. 7 days sooner, 2 weeks sooner, 3 weeks sooner, 4 weeks sooner,
8 weeks sooner, 12 weeks sooner, or more, than expression would be
detected if the polynucleotide were delivered by the parental rAAV.
Enhanced expression may also be observed as an increase in the
amount of gene product per cell. For example, there may be a 2-fold
increase or more, e.g. a 3-fold increase or more, a 4-fold increase
or more, a 5-fold increase or more, or a 10-fold increase or more
in the amount of gene product per cone cell. Enhanced expression
may also be observed as an increase in the number of cone cells
that express detectable levels of the polynucleotide carried by the
variant rAAV. For example, there may be a 2-fold increase or more,
e.g. a 3-fold increase or more, a 4-fold increase or more, a 5-fold
increase or more, or a 10-fold increase or more in the number of
cone cells that express detectable levels of the polynucleotide. As
another example, the polynucleotide of the present invention may
promote detectable levels of the polynucleotide in a greater
percentage of cells as compared to a parental rAAV; for example,
where a parental rAAV may promote detectable levels of
polynucleotide expression in, for example, less than 5% of the cone
cells in a certain region, the rAAV of the present invention
promotes detectable levels of expression in 5% or more of the cone
cells in that region; e.g. 10% or more, 15% or more, 20% or more,
25% or more, 30% or more, 35% or more, 40% or more, or 45% or more,
in some instances 50% or more, 55% or more; 60% or more, 65% or
more, 70% or more, or 75% or more, for example 80% or more, 85% or
more, 90% or more, or 95% or more of the cone cells that are
contacted, will express detectable levels of gene product. Enhanced
expression may also be observed as an alteration in the viability
and/or function of the cone cells, e.g. as measured using
assessment tools such as fundus photography, OCT, adaptive optics,
cERG, color vision tests, visual acuity tests, and the like, as
known in the art and as described herein.
[0088] In some embodiments, the method may further comprise the
step of detecting the expression of the polynucleotide in the cone
photoreceptor. In such embodiments, any convenient method as known
in the art or described herein may be employed for detecting the
expression of the polynucleotide, including, for example, detecting
the gene product, i.e., the encoded RNA or protein, e.g., by
RT-PCR, Northern blot, RNAse protection, Western blot, ELISA,
immunohistochemistry, and the like; detecting the impact of the
gene product on the viability of the cone photoreceptor, e.g., by
fundus photography, Optical coherence tomography (OCT), Adaptive
Optics (AO); or detecting the impact of the gene product on cone
function, e.g. electroretinography (ERG), color vision tests,
visual acuity tests, etc., any of which may be employed in the
subject methods.
[0089] rAAV virions comprising the polynucleotide of interest of
the present disclosure may be produced using any convenient
methodologies, AAV packaging cells, and packaging technology as
known to those of skill in the art. For example, an AAV expression
vector (that is, a plasmid comprising the rAAV genome as well as
elements useful for the cloning of the genomic elements in, e.g.
bacteria, e.g. origin of replication, selectable marker, etc.) may
be transfected into mammalian producer cells. Also transfected into
the mammalian producer cells is an AAV helper construct, i.e. a
plasmid comprising AAV REP and CAP coding regions that can be
expressed in the producer cell, which complement AAV helper
functions absent from the AAV expression vector. The
dually-transfected producer cells are then infected by a helper
virus, e.g. adenovirus, or transfected with a plasmid comprising
helper virus accessory genes that promote AAV vector replication,
e.g., regions VA, E2A, E4, so as to promote efficient rAAV virus
production. The producer cells are then cultured to produce rAAV,
and AAV vectors are purified and formulated using standard
techniques known in the art.
[0090] As another example, an AAV expression vector may be packaged
as a baculovirus and introduced into insect producer cells, e.g.
Sf9 cells. Also introduced into the insect cells by another
baculovirus are the AAV REP and CAP genes. Baculovirus-being a
virus--comprises the genes encoding the accessory functions
necessary for efficient rAAV virus production. Accordingly, upon
infection of the insect cells by the two baculoviruses, the
producer cells can be cultured to produce rAAV, and AAV vectors
purified and formulated using standard techniques known in the
art.
[0091] Examples of these and other methods may be found in, for
example, U.S. Pat. Nos. 5,436,146; 5,753,500, 6,040,183, 6,093,570
and 6,548,286, expressly incorporated by reference herein in their
entirety. Further compositions and methods for packaging are
described in Wang et al. (US 2002/0168342), also incorporated by
reference herein in its entirety.
[0092] Any convenient host cells used in the art for producing rAAV
virions may be employed in the production of the subject vectors,
including, for example, mammalian cells, insect cells,
microorganisms and yeast, e.g. SF-9, 293, A549, HeLa cells, etc. In
some instances, the host cells are packaging cells in which the AAV
rep and cap genes are stably maintained in the host cell. In some
instances, the host cells are producer cells in which the AAV
vector genome is stably maintained and packaged.
Pharmaceutical Compositions and Unit Dosages
[0093] In some embodiments, e.g. gene therapy uses, it will be
desirable to formulate the subject rAAV as a pharmaceutical
composition. In certain embodiments, a pharmaceutical composition
comprises a vector or virion (e.g., rAAV) described herein and one
or more pharmaceutically acceptable carriers, diluents or
excipients. Pharmaceutical compositions suitable for use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersion. For intravenous administration, suitable carriers
include physiological saline, bacteriostatic water, or phosphate
buffered saline (PBS). In all cases, the composition must be
sterile and should 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 (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can 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. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
manitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the internal compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
[0094] Sterile solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a 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, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0095] In one embodiment, active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0096] The pharmaceutical compositions of the subject disclosure
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other compound which, upon administration to
an animal comprising a human, is capable of providing (directly or
indirectly) the biologically active metabolite or residue thereof.
Accordingly, for example, the disclosure is also drawn to prodrugs
and pharmaceutically acceptable salts of the compounds of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bio-equivalents.
[0097] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions.
[0098] The term "pharmaceutically acceptable salt" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto.
[0099] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metals or
organic amines. Metals used as cations comprise sodium, potassium,
magnesium, calcium, and the like. Amines comprise
N--N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base
addition salts of said acidic compounds are prepared by contacting
the free acid form with a sufficient amount of the desired base to
produce the salt in the conventional manner. The free acid form may
be regenerated by contacting the salt form with an acid and
isolating the free acid in the conventional manner. The free acid
forms differ from their respective salt forms somewhat in certain
physical properties such as solubility in polar solvents, but
otherwise the salts are equivalent to their respective free acid
for purposes of the present invention.
[0100] As used herein, a "pharmaceutical addition salt" comprises a
pharmaceutically acceptable salt of an acid form of one of the
components of the compositions of the invention. These comprise
organic or inorganic acid salts of the amines. Preferred acid salts
are the hydrochlorides, acetates, salicylates, nitrates and
phosphates. Other suitable pharmaceutically acceptable salts are
well known to those skilled in the art and comprise basic salts of
a variety of inorganic and organic acids, such as, for example,
with inorganic acids, such as for example hydrochloric acid,
hydrobromic acid, sulfuric acid or phosphoric acid; with organic
carboxylic, sulfonic, sulfo or phospho acids or N-substituted
sulfamic acids, for example acetic acid, propionic acid, glycolic
acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic
acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic
acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,
benzoic acid, cinnamic acid, mandelic acid, salicylic acid,
4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic
acid, embonic acid, nicotinic acid or isonicotinic acid; and with
amino acids, such as the 20 alpha-amino acids involved in the
synthesis of proteins in Nature, for example glutamic acid or
aspartic acid, and also with phenylacetic acid, methanesulfonic
acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,
ethane-1,2-disulfonic acid, benzenesulfonic acid,
4-methylbenzenesulfoic acid, naphthalene-2-sulfonic acid,
naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,
glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation
of cyclamates), or with other acid organic compounds, such as
ascorbic acid. Pharmaceutically acceptable salts of compounds may
also be prepared with a pharmaceutically acceptable cation.
Suitable pharmaceutically acceptable cations are well known to
those skilled in the art and comprise alkaline, alkaline earth,
ammonium and quaternary ammonium cations. Carbonates or hydrogen
carbonates are also possible. For oligonucleotides, preferred
examples of pharmaceutically acceptable salts comprise but are not
limited to: (I) salts formed with cations such as sodium,
potassium, ammonium, magnesium, calcium, polyamides such as
spermine and spermidine, and the like; (II) acid addition salts
formed with inorganic acids, for example hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and
the like; (III) salts formed with organic acids such as, for
example, acetic acid, oxalic acid, tartaric acid, succinic acid,
maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,
ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic
acid, polyglutamic acid, napthalenesulfonic acid, methanesulfonic
acid, p-toluenesulfonic acid, naphthalenedisulfonic acid,
polygalacturonic acid, and the like; and (IV) salts formed from
elemental anions such as chlorine, bromine, and iodine.
[0101] Pharmaceutical compositions of the present invention
comprise, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that comprise, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0102] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
co-administration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extra circulatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate oligonucleotide in hepatic tissue can be
reduced when it is co-administered with polyinosinic acid, dextran
sulphate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
[0103] The subject recombinant AAV can be incorporated into
pharmaceutical compositions for administration to mammalian
patients, particularly humans. The virions can be formulated in
nontoxic, inert, pharmaceutically acceptable aqueous carriers,
preferably at a pH ranging from 3 to 8, more preferably ranging
from 6 to 8. Such sterile compositions will comprise the vector or
virion containing the nucleic acid encoding the therapeutic
molecule dissolved in an aqueous buffer having an acceptable pH
upon reconstitution.
[0104] In some embodiments, the pharmaceutical composition provided
herein comprise a therapeutically effective amount of a vector or
virion in admixture with a pharmaceutically acceptable carrier
and/or excipient, for example saline, phosphate buffered saline,
phosphate and amino acids, polymers, polyols, sugar, buffers,
preservatives and other proteins. Exemplary amino acids, polymers
and sugars and the like are octylphenoxy polyethoxy ethanol
compounds, polyethylene glycol monostearate compounds,
polyoxyethylene sorbitan fatty acid esters, sucrose, fructose,
dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol,
galactitol, xylitol, lactose, trehalose, bovine or human serum
albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine,
arginine, carnitine, alanine, glycine, lysine, valine, leucine,
polyvinylpyrrolidone, polyethylene and glycol. Preferably, this
formulation is stable for at least six months at 4.degree. C.
[0105] In some embodiments, the pharmaceutical composition provided
herein comprises a buffer, such as phosphate buffered saline (PBS)
or sodium phosphate/sodium sulfate, tris buffer, glycine buffer,
sterile water and other buffers known to the ordinarily skilled
artisan such as those described by Good et al. (1966) Biochemistry
5:467. The pH of the buffer in which the pharmaceutical composition
comprising the tumor suppressor gene contained in the adenoviral
vector delivery system, may be in the range of 6.5 to 7.75,
preferably 7 to 7.5, and most preferably 7.2 to 7.4.
[0106] In some embodiments, the pharmaceutical composition provided
herein comprises substances which increase the viscosity of the
suspension, such as sodium carboxymethyl cellulose, sorbitol, or
dextran, in the amount about 1-10 percent, such as 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 percent.
[0107] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0108] In some instances, e.g. for administration intraocularly,
orally, or parentally, it may be especially advantageous to
formulate the pharmaceutical composition in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form
as used herein refers to physically discrete units suited as
unitary dosages for the subject to be treated; each unit containing
a predetermined quantity of active compound calculated to produce
the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0109] In some cases, the unit dose of the pharmaceutical
composition of the disclosure may be measured as pfu (plaque
forming units). In some cases, the pfu of the unit dose of the
pharmaceutical composition of the disclosure may be about
1.times.10.sup.8 to about 5.times.10.sup.10 pfu. In some cases, the
pfu of the unit dose of the pharmaceutical composition of the
disclosure is at least about 1.times.10.sup.8, 2.times.10.sup.8,
3.times.10.sup.8, 4.times.10.sup.8, 5.times.10.sup.8,
6.times.10.sup.8, 7.times.10.sup.8, 8.times.10.sup.8,
9.times.10.sup.8, 1.times.10.sup.9, 2.times.10.sup.9,
3.times.10.sup.9, 4.times.10.sup.9, 5.times.10.sup.9,
6.times.10.sup.9, 7.times.10.sup.9, 8.times.10.sup.9,
9.times.10.sup.9, 1.times.10.sup.10, 2.times.10.sup.10,
3.times.10.sup.10, 4.times.10.sup.10, and 5.times.10.sup.10 pfu. In
some cases, the pfu of the unit dose of the pharmaceutical
composition of the disclosure is at most about 1.times.10.sup.8,
2.times.10.sup.8, 3.times.10.sup.8, 4.times.10.sup.8,
5.times.10.sup.8, 6.times.10.sup.8, 7.times.10.sup.8,
8.times.10.sup.8, 9.times.10.sup.8, 1.times.10.sup.9,
2.times.10.sup.9, 3.times.10.sup.9, 4.times.10.sup.9,
5.times.10.sup.9, 6.times.10.sup.9, 7.times.10.sup.9,
8.times.10.sup.9, 9.times.10.sup.9, 1.times.10.sup.10,
2.times.10.sup.10, 3.times.10.sup.10, 4.times.10.sup.10, and
5.times.10.sup.10 pfu.
[0110] In some cases, the viral vector of the disclosure may be
measured as vector genomes. In some cases, the unit dose of the
pharmaceutical composition of the disclosure is 1.times.10.sup.8
vector genomes or more, e.g. 1.times.10.sup.9, 1.times.10.sup.10,
1.times.10.sup.11, 1.times.10.sup.12, or 1.times.10.sup.13 vector
genomes or more, in certain instances, 1.times.10.sup.14 vector
genomes or more, and usually no more than 1.times.10.sup.15 vector
genomes. In some embodiments, the unit dose of the pharmaceutical
composition of the disclosure is at most about 1.times.10.sup.15
vector genomes, e.g. 1.times.10.sup.14 vector genomes or less, for
example 1.times.10.sup.13, 1.times.10.sup.12, 1.times.10.sup.11,
1.times.10.sup.10, or 1.times.10.sup.9 vector genomes or less, in
certain instances 1.times.10.sup.8 vector genomes, and typically no
less than 1.times.10.sup.8 vector genomes. In some cases, the unit
dose of the pharmaceutical composition of the disclosure is
1.times.10.sup.10 to 1.times.10.sup.11 vector genomes. In some
cases, the unit dose of the pharmaceutical composition of the
disclosure is 1.times.10.sup.10 to 3.times.10.sup.12 vector
genomes. In some cases, the unit dose of the pharmaceutical
composition of the disclosure is 1.times.10.sup.9 to
3.times.10.sup.13 vector genomes. In some cases, the unit dose of
the pharmaceutical composition of the disclosure is
1.times.10.sup.8 to 3.times.10.sup.14 vector genomes.
[0111] In some cases, the unit dose of the pharmaceutical
composition of the disclosure may be measured using multiplicity of
infection (MOI). In some cases, MOI may refer to the ratio, or
multiple of vector or viral genomes to the cells to which the
nucleic may be delivered. In some cases, the MOI may be
1.times.10.sup.6. In some cases, the MOI may be
1.times.10.sup.5-1.times.10.sup.7. In some cases, the MOI may be
1.times.10.sup.4-1.times.10.sup.8. In some cases, recombinant
viruses of the disclosure are at least about 1.times.10.sup.1,
1.times.10.sup.2, 1.times.10.sup.3, 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10,
1.times.10.sup.11, 1.times.10.sup.12, 1.times.10.sup.13,
1.times.10.sup.14, 1.times.10.sup.15, 1.times.10.sup.16,
1.times.10.sup.17, and 1.times.10.sup.18 MOI. In some cases,
recombinant viruses of this disclosure are 1.times.10.sup.8 to
3.times.10.sup.14 MOI. In some cases, recombinant viruses of the
disclosure are at most about 1.times.10.sup.1, 1.times.10.sup.2,
1.times.10.sup.3, 1.times.10.sup.4, 1.times.10.sup.5,
1.times.10.sup.6, 1.times.10.sup.7, 1.times.10.sup.8,
1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
1.times.10.sup.12, 1.times.10.sup.13, 1.times.10.sup.14,
1.times.10.sup.15, 1.times.10.sup.16, 1.times.10.sup.17, and
1.times.10.sup.18 MOI.
[0112] In some aspects, the pharmaceutical composition comprises
about 1.times.10.sup.8 to about 1.times.10.sup.15 recombinant
viruses, about 1.times.10.sup.9 to about 1.times.10.sup.14
recombinant viruses, about 1.times.10.sup.10 to about
1.times.10.sup.13 recombinant viruses, about 1.times.10.sup.109 to
about 3.times.10.sup.12 recombinant viruses, or about
1.times.10.sup.11 to about 3.times.10.sup.12 recombinant
viruses.
Methods of Administration
[0113] The pharmaceutical composition of the present invention may
be administered to the eye of the subject by any convenient method,
e.g. intraocularly, intravenously, intraperitoneally, etc. In some
instances, the administration is intraocular, e.g. by intravitreal
injection or subretinal injection. The general methods for
delivering a vector via intravitreal injection or via subretinal
injection may be illustrated by the following brief outlines. These
examples are merely meant to illustrate certain features of the
methods, and are in no way meant to be limiting.
[0114] In preferred embodiments, the subject rAAV is delivered
intravitreally. For intravitreal administration, the vector can be
delivered in the form of a suspension. Initially, topical
anesthetic is applied to the surface of the eye followed by a
topical antiseptic solution. The eye is held open, with or without
instrumentation, and the vector is injected through the sclera with
a short, narrow, for example a 30 gauge needle, into the vitreous
cavity of the eye of a subject under direct observation.
Intravitreal administration is generally well tolerated. At the
conclusion of the procedure, there is sometimes mild redness at the
injection site. There is occasional tenderness, but most patients
do not report any pain. No eye patch or eye shield is necessary
after this procedure, and activities are not restricted. Sometimes,
an antibiotic eye drop is prescribed for several days to help
prevent infection.
[0115] In some embodiments, the subject rAAV is delivered
subretinally. For subretinal administration, the vector can be
delivered in the form of a suspension injected subretinally under
direct observation using an operating microscope. This procedure
may involve vitrectomy followed by injection of vector suspension
using a fine cannula through one or more small retinotomies into
the subretinal space.
[0116] Briefly, an infusion cannula can be sutured in place to
maintain a normal globe volume by infusion (of e.g. saline)
throughout the operation. A vitrectomy is performed using a cannula
of appropriate bore size (for example 20 to 27 gauge), wherein the
volume of vitreous gel that is removed is replaced by infusion of
saline or other isotonic solution from the infusion cannula. The
vitrectomy is advantageously performed because (1) the removal of
its cortex (the posterior hyaloid membrane) facilitates penetration
of the retina by the cannula; (2) its removal and replacement with
fluid (e.g. saline) creates space to accommodate the intraocular
injection of vector, and (3) its controlled removal reduces the
possibility of retinal tears and unplanned retinal detachment.
[0117] In practicing the subject methods, the subject rAAV virion
is delivered to the eye in an amount effective to deliver the
polynucleotide of interest to 5% or more of the subject's cone
photoreceptors, for example, 10% or more, 20% or more, 30% or more,
40% or more, or 50% or more of the subject's cone photoreceptors,
e.g. 60% or more, 70% or more, 80% or more, or 90% or more of the
subject's cone photoreceptors, in some instance, 95% or more, 98%
or more, or 100% of the subject's cone photoreceptors to provide
therapeutic benefit to the subject individual. Put another way,
following the administering, 5% or more of the subject's cone
photoreceptors, e.g. 10% or more, 20% or more, 30% or more, 40% or
more, or 50% or more, in some instance 60% or more, 70% or more,
80% or more, or 90% or more, e.g. 95%, 98%, or 100% of the cones,
will comprise a sufficient amount of the polynucleotide of interest
to have an impact on cone viability and/or function, e.g. to treat
or prevent a disorder. In some embodiments, the transduced cones
photoreceptors will be located throughout the retina. In some
embodiments, the transduced cone photoreceptors will be cones in
the fovea and foveola. In some embodiments, the transduced cone
photoreceptors will be foveal cones, i.e. L- or M-cones located in
the fovea.
[0118] Typically, an effective amount will be about
1.times.10.sup.8 vector genomes or more of the subject rAAV, e.g.
1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
1.times.10.sup.12, or 1.times.10.sup.13 vector genomes or more, in
certain instances, 1.times.10.sup.14 vector genomes or more, and
usually no more than 1.times.10.sup.15 vector genomes. In some
cases, the amount of vector genomes that is delivered is at most
about 1.times.10.sup.15 vector genomes, e.g. 1.times.10.sup.14
vector genomes or less, for example 1.times.10.sup.13,
1.times.10.sup.12, 1.times.10.sup.11, 1.times.10.sup.10, or
1.times.10.sup.9 vector genomes or less, in certain instances
1.times.10.sup.8 vector genomes, and typically no less than
1.times.10.sup.8 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.10 to
1.times.10.sup.11 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.10 to
3.times.10.sup.12 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.9 to
3.times.10.sup.13 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.8 to
3.times.10.sup.14 vector genomes.
[0119] In some cases, the amount of pharmaceutical composition to
be administered may be measured using multiplicity of infection
(MOI). In some cases, MOI may refer to the ratio, or multiple of
vector or viral genomes to the cells to which the nucleic may be
delivered. In some cases, the MOI may be 1.times.10.sup.6. In some
cases, the MOI may be 1.times.10.sup.5-1.times.10.sup.7. In some
cases, the MOI may be 1.times.10.sup.4-1.times.10.sup.8. In some
cases, recombinant viruses of the disclosure are at least about
1.times.10.sup.1, 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, 1.times.10.sup.14, 1.times.10.sup.15,
1.times.10.sup.16, 1.times.10.sup.17, and 1.times.10.sup.18 MOI. In
some cases, recombinant viruses of this disclosure are
1.times.10.sup.8 to 3.times.10.sup.14 MOI. In some cases,
recombinant viruses of the disclosure are at most about
1.times.10.sup.1, 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, 1.times.10.sup.14, 1.times.10.sup.15,
1.times.10.sup.16, 1.times.10.sup.17, and 1.times.10.sup.18 MOI. In
some aspects, the amount of pharmaceutical composition comprises
about 1.times.10.sup.8 to about 1.times.10.sup.15 recombinant
viruses, about 1.times.10.sup.9 to about 1.times.10.sup.14
recombinant viruses, about 1.times.10.sup.10 to about
1.times.10.sup.13 recombinant viruses, about 1.times.10.sup.10 to
about 3.times.10.sup.12 recombinant viruses, or about
1.times.10.sup.11 to about 3.times.10.sup.12 recombinant
viruses.
Utility
[0120] Methods and compositions for the intravitreal delivery of
polynucleotides to cone photoreceptors, and more particular foveal
cones, find many uses in research and in medicine.
[0121] For example, such methods and compositions may be used in
research to test the function of the gene product encode by the
polynucleotide in vivo, e.g. to better understand the function of
the cone photoreceptor and/or whether the gene product will impact
the viability and/or function of the cone photoreceptor.
[0122] As alluded to above, the subject rAAVs, referred to
collectively herein as "subject compositions", find use in
expressing a transgene in cone cells of an animal, for example, in
foveal cones of an animal. For example, the subject compositions
may be used in research, e.g. to determine the effect that the gene
has on cone cell viability and/or function. As another example, the
subject compositions may be used in medicine, e.g. to treat a cone
cell disorder. Thus, in some aspects of the invention, methods are
provided for the expression of a gene in cone cells, the method
comprising contacting cone cells with a composition of the present
disclosure. In some embodiments, contacting occurs in vitro. In
some embodiments, contacting occurs in vivo, i.e., the subject
composition is administered to a subject.
[0123] For instances in which cone cells are to be contacted in
vitro with a subject rAAV, the cells may be from any mammalian
species, e.g. rodent (e.g. mice, rats, gerbils, squirrels), rabbit,
feline, canine, goat, ovine, pig, equine, bovine, primate, human.
Cells may be from established cell lines, e.g. WERI cells, 661W
cells, or they may be primary cells, where "primary cells",
"primary cell lines", and "primary cultures" are used
interchangeably herein to refer to cells and cells cultures that
have been derived from a subject and allowed to grow in vitro for a
limited number of passages, i.e. splittings, of the culture. For
example, primary cultures are cultures that may have been passaged
0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times,
but not enough times go through the crisis stage. Typically, the
primary cell lines of the present invention are maintained for
fewer than 10 passages in vitro.
[0124] If the cells are primary cells, they may be harvested from a
mammal by any convenient method, e.g. whole explant, biopsy, etc.
An appropriate solution may be used for dispersion or suspension of
the harvested cells. Such solution will generally be a balanced
salt solution, e.g. normal saline, PBS, Hank's balanced salt
solution, etc., conveniently supplemented with fetal calf serum or
other naturally occurring factors, in conjunction with an
acceptable buffer at low concentration, generally from 5-25 mM.
Convenient buffers include HEPES, phosphate buffers, lactate
buffers, etc. The cells may be used immediately, or they may be
stored, frozen, for long periods of time, being thawed and capable
of being reused. In such cases, the cells will usually be frozen in
10% DMSO, 50% serum, 40% buffered medium, or some other such
solution as is commonly used in the art to preserve cells at such
freezing temperatures, and thawed in a manner as commonly known in
the art for thawing frozen cultured cells.
[0125] To promote expression of the transgene, the subject rAAV
will be contacted with the cells for about 30 minutes to 24 hours
or more, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5
hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16
hours, 18 hours, 20 hours, 24 hours, etc. The subject rAAV may be
provided to the subject cells one or more times, e.g. one time,
twice, three times, or more than three times, and the cells allowed
to incubate with the agent(s) for some amount of time following
each contacting event e.g. 16-24 hours, after which time the media
is replaced with fresh media and the cells are cultured further.
Contacting the cells may occur in any culture media and under any
culture conditions that promote the survival of the cells. For
example, cells may be suspended in any appropriate nutrient medium
that is convenient, such as Iscove's modified DMEM or RPMI 1640,
supplemented with fetal calf serum or heat inactivated goat serum
(about 5-10%), L-glutamine, a thiol, particularly
2-mercaptoethanol, and antibiotics, e.g. penicillin and
streptomycin. The culture may contain growth factors to which the
cells are responsive. Growth factors, as defined herein, are
molecules capable of promoting survival, growth and/or
differentiation of cells, either in culture or in the intact
tissue, through specific effects on a transmembrane receptor.
Growth factors include polypeptides and non-polypeptide
factors.
[0126] Typically, an effective amount of subject rAAV is provided
to produce the expression of the transgene in cells. As discussed
elsewhere herein, the effective amount may be readily determined
empirically, e.g. by detecting the presence or levels of transgene
gene product, by detecting an effect on the viability or function
of the cone cells, etc. Typically, an effect amount of subject rAAV
will promote greater expression of the transgene in cone cells than
the same amount of parental rAAV from which its capsid was derived.
Typically, expression will be enhanced 2-fold or more relative to
the expression from parental rAAV, for example 3-fold, 4-fold, or
5-fold or more, in some instances 10-fold, 20-fold or 50-fold or
more, e.g. 100-fold.
[0127] In some embodiments, as when the transgene is a selectable
marker, the population of cells may be enriched for those
comprising the transgene by separating the modified cells from the
remaining population. Separation may be by any convenient
separation technique appropriate for the selectable marker used.
For example, if the transgene is a fluorescent marker, cells may be
separated by fluorescence activated cell sorting, whereas if the
transgene is a cell surface marker, cells may be separated from the
heterogeneous population by affinity separation techniques, e.g.
magnetic separation, affinity chromatography, "panning" with an
affinity reagent attached to a solid matrix, or other convenient
technique. Techniques providing accurate separation include
fluorescence activated cell sorters, which can have varying degrees
of sophistication, such as multiple color channels, low angle and
obtuse light scattering detecting channels, impedance channels,
etc. The cells may be selected against dead cells by employing dyes
associated with dead cells (e.g. propidium iodide). Any technique
may be employed which is not unduly detrimental to the viability of
the cells. Cell compositions that are highly enriched for cells
comprising the transgene are achieved in this manner. By "highly
enriched", it is meant that the genetically modified cells will be
70% or more, 75% or more, 80% or more, 85% or more, 90% or more of
the cell composition, for example, about 95% or more, or 98% or
more of the cell composition. In other words, the composition may
be a substantially pure composition of genetically modified
cells.
[0128] For instances in which cone cells are to be contacted in
vivo with the subject rAAV, the subject may be any mammal, e.g.
rodent (e.g. mice, rats, gerbils), rabbit, feline, canine, goat,
ovine, pig, equine, bovine, or primate. In certain embodiments, the
subject is a primate of the Parvorder Catarrhini. As is known in
the art, Catarrhini is one of the two subdivisions of the higher
primates (the other being the New World monkeys), and includes Old
World monkeys and the apes, which in turn are further divided into
the lesser apes or gibbons and the great apes, consisting of the
orangutans, gorillas, chimpanzees, bonobos, and humans. In a
further preferred embodiment, the primate is a human.
[0129] The subject rAAV may be administered to the retina of the
subject by any suitable method. For example, the subject
composition may be administered intraocularly via intravitreal
injection or subretinal injection. The general methods for
delivering a vector via intravitreal injection or via subretinal
injection may be illustrated by the following brief outlines. These
examples are merely meant to illustrate certain features of the
methods, and are in no way meant to be limiting.
[0130] For subretinal administration, the subject rAAV can be
delivered in the form of a suspension injected subretinally under
direct observation using an operating microscope. Typically, a
volume of 1 to 200 uL, e.g. 50 uL, 100 uL, 150 ul, or 200 uL, but
usually no more than 200 uL, of the subject composition will be
administered by such methods. This procedure may involve vitrectomy
followed by injection of vector suspension using a fine cannula
through one or more small retinotomies into the subretinal space.
Briefly, an infusion cannula can be sutured in place to maintain a
normal globe volume by infusion (of e.g. saline) throughout the
operation. A vitrectomy is performed using a cannula of appropriate
bore size (for example 20 to 27 gauge), wherein the volume of
vitreous gel that is removed is replaced by infusion of saline or
other isotonic solution from the infusion cannula. The vitrectomy
is advantageously performed because (1) the removal of its cortex
(the posterior hyaloid membrane) facilitates penetration of the
retina by the cannula; (2) its removal and replacement with fluid
(e.g. saline) creates space to accommodate the intraocular
injection of vector, and (3) its controlled removal reduces the
possibility of retinal tears and unplanned retinal detachment.
[0131] For intravitreal administration, the subject rAAV can be
delivered in the form of a suspension. Initially, topical
anesthetic is applied to the surface of the eye followed by a
topical antiseptic solution. The eye is held open, with or without
instrumentation, and the rAAV is injected through the sclera with a
short, narrow, for example a 30 gauge needle, into the vitreous
cavity of the eye of a subject under direct observation. Typically,
a volume of 1 to 100 uL, e.g. 25 uL, 50 uL, or 100 uL, and usually
no more than 100 uL, of the subject composition may be delivered to
the eye by intravitreal injection without removing the vitreous.
Alternatively, a vitrectomy may be performed, and the entire volume
of vitreous gel is replaced by an infusion of the subject
composition. In such cases, up to about 4 mL of the subject
composition may be delivered, e.g. to a human eye. Intravitreal
administration is generally well tolerated. At the conclusion of
the procedure, there is sometimes mild redness at the injection
site. There is occasional tenderness, but most patients do not
report any pain. No eye patch or eye shield is necessary after this
procedure, and activities are not restricted. Sometimes, an
antibiotic eye drop is prescribed for several days to help prevent
infection.
[0132] The subject methods and/or compositions may be used in
medicine to express a therapeutic polynucleotide in cone
photoreceptors as a therapy to treat or prevent a retinal disorder.
The terms "treatment", "treating" and the like are used herein to
generally mean obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof, e.g. reducing
the likelihood that the disease or symptom thereof occurs in the
subject, and/or may be therapeutic in terms of a partial or
complete cure for a disease and/or adverse effect attributable to
the disease. "Treatment" as used herein covers any treatment of a
disease in a mammal, and includes: (a) preventing the disease from
occurring in a subject which may be predisposed to the disease but
has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; or (c) relieving the
disease, i.e., causing regression of the disease. The therapeutic
agent may be administered before, during or after the onset of
disease or injury. The treatment of ongoing disease, where the
treatment stabilizes or reduces the undesirable clinical symptoms
of the patient, is of particular interest. Such treatment is
desirably performed prior to complete loss of function in the
affected tissues. The subject therapy will desirably be
administered during the symptomatic stage of the disease, and in
some cases after the symptomatic stage of the disease.
[0133] There are a number of retinal disorders that may be treated
or prevented using the subject methods and/or compositions. Of
particular interest are cone-associated disorders; that is,
disorders that are associated with a loss of cone viability and/or
a reduction in cone function. As discussed above, cone
photoreceptors are responsible for color vision and high acuity
foveal vision, and are densely packed in a 1.5 mm depression
located in the center of the macula of the retina, called the fovea
centralis. Consistent with this, disorders associated with cone
dysfunction and viability typically manifest in the macula and
impact color vision and high acuity vision. Non-limiting examples
of cone-associated disorders include rod-cone dystrophy; cone-rod
dystrophy; progressive cone dystrophy; retinitis pigmentosa (RP);
Stargardt Disease; macular telangiectasia, Leber hereditary optic
neuropathy, Best's disease; adult vitelliform macular dystrophy;
X-linked retinoschisis; color vision disorders such as blue cone
monochromacy, achromatopsia, incomplete achromatopsia, protan
defects, deutan defects, and tritan defects; and retinal disorders
that affect the central macula, such as, for example, age-related
macular degeneration, wet age-related macular degeneration,
geographic atrophy, macular telangiectasia, retinitis pigmentosa,
diabetic retinopathy, retinal vein occlusions, glaucoma, Sorsby's
fundus dystrophy, adult vitelliform macular dystrophy, Best's
disease, and X-linked retinoschisis.
[0134] Stargardt's macular dystrophy. Stargardt's macular
dystrophy, also known as Stargardt Disease and fundus
flavimaculatus, is an inherited form of juvenile macular
degeneration that causes progressive vision loss usually to the
point of legal blindness. The onset of symptoms usually appears
between the ages of six and thirty years old (average of about
16-18 years). Mutations in several genes, including ABCA4, CNGB3,
ELOVL4, PROM1, are associated with the disorder. Symptoms typically
develop by twenty years of age, and include wavy vision, blind
spots, blurriness, impaired color vision, and difficulty adapting
to dim lighting. The main symptom of Stargardt disease is loss of
visual acuity, which ranges from 20/50 to 20/200. In addition,
those with Stargardt disease are sensitive to glare; overcast days
offer some relief. Vision is most noticeably impaired when the
macula is damaged, which can be observed by fundus exam.
[0135] Cone dystrophy. Cone dystrophy (COD) is an inherited ocular
disorder characterized by the loss of cone cells. The most common
symptoms of cone dystrophy are vision loss (age of onset ranging
from the late teens to the sixties), sensitivity to bright lights,
and poor color vision. Visual acuity usually deteriorates
gradually, but it can deteriorate rapidly to 20/200; later, in more
severe cases, it drops to "counting fingers" vision. Color vision
testing using color test plates (HRR series) reveals many errors on
both red-green and blue-yellow plates. It is believed that the
dystrophy is primary, since subjective and objective abnormalities
of cone function are found before ophthalmoscopic changes can be
seen. However, the retinal pigment epithelium (RPE) rapidly becomes
involved, leading to a retinal dystrophy primarily involving the
macula. The fundus exam via ophthalmoscope is essentially normal
early on in cone dystrophy, and definite macular changes usually
occur well after visual loss. The most common type of macular
lesion seen during ophthalmoscopic examination has a bull's-eye
appearance and consists of a doughnut-like zone of atrophic pigment
epithelium surrounding a central darker area. In another, less
frequent form of cone dystrophy there is rather diffuse atrophy of
the posterior pole with spotty pigment clumping in the macular
area. Rarely, atrophy of the choriocapillaris and larger choroidal
vessels is seen in patients at an early stage. Fluorescein
angiography (FA) is a useful adjunct in the workup of someone
suspected to have cone dystrophy, as it may detect early changes in
the retina that are too subtle to be seen by ophthalmoscope.
Because of the wide spectrum of fundus changes and the difficulty
in making the diagnosis in the early stages, electroretinography
(ERG) remains the best test for making the diagnosis. Abnormal cone
function on the ERG is indicated by a reduced single-flash and
flicker response when the test is carried out in a well-lit room
(photopic ERG). Mutations in several genes, including GUCA1A,
PDE6C, PDE6H, and RPGR, are associated with the disorder.
[0136] Cone-rod dystrophy. Cone-rod dystrophy (CRD, or CORD) is an
inherited retinal dystrophy that belongs to the group of pigmentary
retinopathies. CRD is characterized by retinal pigment deposits
visible on fundus examination, predominantly localized to the
macular region and the loss of both cone and rod cells. In contrast
to rod-cone dystrophy (RCD) resulting from the primary loss in rod
photoreceptors and later followed by the secondary loss in cone
photoreceptors, CRD reflects the opposite sequence of events:
primary cone involvement, or, sometimes, by concomitant loss of
both cones and rods. Symptoms include decreased visual acuity,
color vision defects, photoaversion and decreased sensitivity in
the central visual field, later followed by progressive loss in
peripheral vision and night blindness. Mutations in several genes,
including ADAM9, PCDH21, CRX, GUCY2D, PITPNM3, PROM1, PRPH2, RAX2,
RIMS1, RPGR, and RPGRIP1, are associated with the disorder.
[0137] Spinocerebellar ataxia type 7. Spinocerebellar ataxia is a
progressive, degenerative, inherited disease characterized by
slowly progressive incoordination of gait and is often associated
with poor coordination of hands, speech, and eye movements. There
are multiple types of SCA, with Spinocerebellar ataxia type 7
(SCA-7) differing from most other SCAs in that visual problems can
occur in addition to poor coordination. SCA-7 is associated with
automosmal dominant mutations in the ATXN7/SCA7 gene. When the
disease manifests itself before age 40, visual problems rather than
poor coordination are typically the earliest signs of disease.
Early symptoms include difficulty distinguishing colors and
decreased central vison. In addition, symptoms of ataxia
(incoordination, slow eye movements, and mild changes in sensation
or reflexes) may be detectable. Loss of motor control, unclear
speech, and difficulty swallowing become prominent as the disease
progresses.
[0138] Bardet-Biedl syndrome-1. Bardet-Biedl syndrome-1 (BBS-1) is
a pleiotropic disorder with variable expressivity and a wide range
of clinical variability observed both within and between families.
The main clinical features are rod-cone dystrophy, with
childhood-onset visual loss preceded by night blindness; postaxial
polydactyly; truncal obesity that manifests during infancy and
remains problematic throughout adulthood; specific learning
difficulties in some but not all individuals; male hypogenitalism
and complex female genitourinary malformations; and renal
dysfunction, a major cause of morbidity and mortality. Vision loss
is one of the major features of Bardet-Biedl syndrome. Problems
with night vision become apparent by mid-childhood, followed by
blind spots that develop in the peripheral vision. Over time, these
blind spots enlarge and merge to produce tunnel vision. Most people
with Bardet-Biedl syndrome also develop blurred central vision
(poor visual acuity) and become legally blind by adolescence or
early adulthood. Bardet-Biedl syndrome can result from mutations in
at least 14 different genes (often called BBS genes) known or
suspected to play critical roles in cilia function, with mutations
in BBS1 and BBS10 being the most common.
[0139] Achromatopsia. Achromatopsia, or Rod monochromatism, is a
disorder in which subjects experience a complete lack of the
perception of color, such that the subject sees only in black,
white, and shades of grey. Other symptoms include reduced visual
acuity, photophobia, nystagmus, small central scotoma, and
eccentric fixation. The disorder is frequently noticed first in
children around six months of age by their photophobic activity
and/or their nystagmus. Visual acuity and stability of the eye
motions generally improve during the first 6-7 years of life (but
remain near 20/200). Mutations in CNGB3, CNGA3, GNAT2, PDE6C, and
PDE6HI have been associated with the disorder.
[0140] Incomplete achromatopsia. Incomplete achromatopsia is
similar to Achromatopsia but with less penetrance. In incomplete
achromatopsia, the symptoms are similar to those of complete
achromatopsia except in a diminished form. Individuals with
incomplete achromatopsia have reduced visual acuity with or without
nystagmus or photophobia. Furthermore, these individuals show only
partial impairment of cone cell function but again have retained
rod cell function.
[0141] Blue cone monochromacy. Blue cone (S cone) monochromatism
(BCM) is a rare X-linked congenital stationary cone dysfunction
syndrome, affecting approximately 1 in 100,000 individuals.
Affected males with BCM have no functional long wavelength
sensitive (L) or medium wavelength sensitive (M) cones in the
retina, due to mutations at the genetic locus for the L and M-opsin
genes. Color discrimination is severely impaired from birth, and
vision is derived from the remaining preserved S cones and rod
photoreceptors. BCM typically presents with reduced visual acuity
(6/24 to 6/60), pendular nystagmus, photophobia, and patients often
have myopia. The rod-specific and maximal electroretinogram (ERG)
usually show no definite abnormality, whereas the 30 Hz cone ERG
cannot be detected. Single flash photopic ERG is often recordable,
albeit small and late, and the S cone ERG is well preserved.
[0142] Color vision deficiency. Color vision deficiency (CVD), or
color blindness, is the inability or decreased ability to see
color, or perceive color differences, under normal lighting
conditions. Individuals suffering from color blindness may be
identified as such using any of a number of color vision tests,
e.g., color ERG (cERG), pseudoisochromatic plates (Ishihara plates,
Hardy-Rand-Ritter polychromatic plates), the Farnsworth-Munsell 100
hue test, the Farnsworth's panel D-15, the City University test,
Kollner's rule, etc. Examples of color vision deficiencies include
protan defects, deutan defects, and tritan defects. Protan defects
include protanopia (an insensitivity to red light) and protanomaly
(a reduced sensitivity to red light), and are associated with
mutations in the L-Opsin gene (OPN1LW). Deutan defects include
deuteranopia (an insensitivity to green light) and deutanomaly (a
reduced sensitivity to green light), and are associated with
mutations in the M-Opsin gene (OPN1MW). Tritan defects include
tritanopia (an insensitivity to blue light) and tritanomaly (a
reduced sensitivity to blue light), and are associated with
mutations in the S-Opsin gene (OPN1SW).
[0143] Age-related macular degeneration. Age-related macular
degeneration (AMD) is one of the leading causes of vision loss in
people over the age of 50 years. AMD mainly affects central vision,
which is needed for detailed tasks such as reading, driving, and
recognizing faces. The vision loss in this condition results from a
gradual deterioration of photoreceptors in the macula. Side
(peripheral) vision and night vision are generally not
affected.
[0144] Researchers have described two major types of age-related
macular degeneration, known as the dry, or "nonexudative" form, and
the wet, or "exudative" or "neovascular", form, both of which may
be treated by delivering transgenes packaged in the subject
rAAV.
[0145] Dry AMD is characterized by a buildup of yellow deposits
called drusen between the retinal pigment epithelium and the
underlying choroid of the macula, which may be observed by Fundus
photography. This results in a slowly progressive loss of vision.
The condition typically affects vision in both eyes, although
vision loss often occurs in one eye before the other. Other changes
may include pigment changes and RPE atrophy. For example, in
certain cases called central geographic atrophy, or "GA", atrophy
of the retinal pigment epithelial and subsequent loss of
photoreceptors in the central part of the eye is observed. Dry AMD
has been associated with mutations in CD59 and genes in the
complement cascade.
[0146] Wet AMD is a progressed state of dry AMD, and occurs in abut
10% of dry AMD patients. Pathological changes include retinal
pigment epithelial cells (RPE) dysfunction, fluid collecting under
the RPE, and choroidal neovascularization (CNV) in the macular
area. Fluid leakage, RPE or neural retinal detachment and bleeding
from ruptured blood vessels can occur in severe cases. Symptoms of
wet AMD may include visual distortions, such as straight lines
appearing wavy or crooked, a doorway or street sign looking
lopsided, or objects appearing smaller or farther away than they
really are; decreased central vision; decreased intensity or
brightness of colors; and well-defined blurry spot or blind spot in
the field of vision. Onset may be abrupt and worsen rapidly.
Diagnosis may include the use of an Amsler grid to test for defects
in the subject's central vision (macular degeneration may cause the
straight lines in the grid to appear faded, broken or distorted),
fluorescein angiogram to observe blood vessel or retinal
abnormalities, and optical coherence tomography to detect retina
swelling or leaking blood vessels. A number of cellular factors
have been implicated in the generation of CNV, among which are
vascular endothelial growth factor (VEGF), platelet-derived growth
factor (PDGF), pigment epithelium-derived factor (PEDF), hypoxia
inducible factor (HIF), angiopoietin (Ang), and other cytokines,
mitogen-activated protein kinases (MAPK) and others.
[0147] Macular telangiectasia. Macular telangiectasia (MacTel) is a
form of pathologically dilated blood vessels (telangiectasia) in
the parafoveal region of the macula. The tissue deteriorates and
the retinal structure becomes scarred due to the development of
liquid-filled cysts, which impairs nutrition of the photoreceptor
cells and destroys vision permanently. There are two types of
MacTel, type 1 and type 2. Macular telangiectasia type 2 is a
bilateral disease, whose prevalence has recently been shown to be
as high as 0.1% in persons 40 years and older. Biomicroscopy may
show reduced retinal transparency, crystalline deposits, mildly
ectatic capillaries, blunted venules, retinal pigment plaques,
foveal atrophy, and neovascular complexes. Fluorescein angiography
shows telangiectatic capillaries predominantly temporal to the
foveola in the early phase and a diffuse hyperfluorescence in the
late phase. High-resolution optical coherence tomography (OCT) may
reveal disruption of the photoreceptor inner segment-outer segment
border, hyporeflective cavities at the level of the inner or outer
retina, and atrophy of the retina in later stages. In Type 1
macular telangiectasia, the disease almost always occurs in one
eye, which differentiates it from Type 2. While MacTel does not
usually cause total blindness, it commonly causes loss of the
central vision, which is required for reading and driving vision,
over a period of 10-20 years.
[0148] Retinitis pigmentosa. Retinitis Pigmentosa (RP) is a group
of inherited disorders characterized by progressive peripheral
vision loss and night vision difficulties (nyctalopia) that can
lead to central vision loss. Presenting signs and symptoms of RP
vary, but the classic ones include nyctalopia (night blindness,
most commonly the earliest symptom in RP); visual loss (usually
peripheral, but in advanced cases, central visual loss); and
photopsia (seeing flashes of light). Because RP is a collection of
many inherited diseases, significant variability exists in the
physical findings. Ocular examination involves assessment of visual
acuity and pupillary reaction, as well as anterior segment,
retinal, and funduscopic evaluation. In some instances, the RP is
one aspect of a syndrome, e.g. syndromes that are also associated
with hearing loss (Usher syndrome, Waardenburg syndrome, Alport
syndrome, Refsum disease); Kearns-Sayre syndrome (external
ophthalmoplegia, lid ptosis, heart block, and pigmentary
retinopathy); Abetalipoproteinemia (Fat malabsorption, fat-soluble
vitamin deficiencies, spinocerebellar degeneration, and pigmentary
retinal degeneration); mucopolysaccharidoses (eg, Hurler syndrome,
Scheie syndrome, Sanfilippo syndrome); Bardet-Biedl syndrome
(Polydactyly, truncal obesity, kidney dysfunction, short stature,
and pigmentary retinopathy); and neuronal ceroid lipofuscinosis
(Dementia, seizures, and pigmentary retinopathy; infantile form is
known as Jansky-Bielschowsky disease, juvenile form is
Vogt-Spielmeyer-Batten disease, and adult form is Kufs syndrome).
Retinitis pigmentosa is most commonly associated with mutations in
the RHO, RP2, RPGR, RPGRIP1, PDE6A, PDE6B, MERTK, PRPH2, CNGB1,
USH2A, ABCA4, BBS genes.
[0149] Diabetic retinopathy. Diabetic retinopathy (DR) is damage to
the retina caused by complications of diabetes, which can
eventually lead to blindness. Without wishing to be bound by
theory, it is believed that hyperglycemia-induced intramural
pericyte death and thickening of the basement membrane lead to
incompetence of the vascular walls. These damages change the
formation of the blood-retinal barrier and also make the retinal
blood vessels become more permeable.
[0150] There are two stages of diabetic retinopathy:
non-proliferative diabetic retinopathy (NPDR), and proliferative
diabetic retinopathy (PDR). Nonproliferative diabetic retinopathy
is the first stage of diabetic retinopathy, and is diagnosed by
fundoscopic exam and coexistent diabetes. In cases of reduced
vision, fluorescein angiography may be done to visualize the
vessles in the back of the eye to and any retinal ischemia that may
be present. All people with diabetes are at risk for developing
NPDR, and as such, would be candidates for prophylactic treatment
with the subject vectors. Proliferative diabetic retinopathy is the
second stage of diabetic retinopathy, characterized by
neovascularization of the retina, vitreous hemorrhage, and blurred
vision. In some instances, fibrovascular proliferation causes
tractional retinal detachment. In some instances, the vessels can
also grow into the angle of the anterior chamber of the eye and
cause neovascular glaucoma. Individuals with NPDR are at increased
risk for developing PDR, and as such, would be candidates for
prophylactic treatment with the subject vectors.
[0151] Diabetic macular edema. Diabetic macular edema (DME) is an
advanced, vision-limiting complication of diabetic retinopathy that
affects nearly 30% of patients who have had diabetes for at least
20 years, and is responsible for much of the vision loss due to DR.
It results from retinal microvascular changes that compromise the
blood-retinal barrier, causing leakage of plasma constituents into
the surrounding retina and, consequently, retinal edema. Without
wishing to be bound by theory, it is believed that hyperglycemia,
sustained alterations in cell signaling pathways, and chronic
microvascular inflammation with leukocyte-mediated injury leads to
chronic retinal microvascular damage, which triggers an increase in
intraocular levels of VEGF, which in turn increases the
permeability of the vasculature.
[0152] Patients at risk for developing DME include those who have
had diabetes for an extended amount of time and who experience one
or more of severe hypertension (high blood pressure), fluid
retention, hypoalbuminemia, or hyperlipidemia. Common symptoms of
DME are blurry vision, floaters, double vision, and eventually
blindness if the condition is allowed to progress untreated. DME is
diagnosed by funduscopic examination as retinal thickening within 2
disc diameters of the center of the macula. Other methods that may
be employed include Optical coherence tomography (OCT) to detect
retinal swelling, cystoid edema, and serous retinal detachment;
fluorescein angiography, which distinguishes and localizes areas of
focal versus diffuse leakage, thereby guiding the placement of
laser photocoagulation if laser photocoagulation is to be used to
treat the edema; and color stereo fundus photographs, which can be
used to evaluate long-term changes in the retina. Visual acuity may
also be measured, especially to follow the progression of macular
edema and observe its treatment following administration of the
subject pharmaceutical compositions.
[0153] Retinal vein occlusions. A retinal vein occlusion (RVO) is a
blockage of the portion of the circulation that drains the retina
of blood. The blockage can cause back-up pressure in the
capillaries, which can lead to hemorrhages and also to leakage of
fluid and other constituents of blood.
[0154] Glaucoma. Glaucoma is a term describing a group of ocular
(eye) disorders that result in optic nerve damage, often associated
with increased fluid pressure in the eye (intraocular
pressure)(IOP). The disorders can be roughly divided into two main
categories, "open-angle" and "closed-angle" (or "angle closure")
glaucoma. Open-angle glaucoma accounts for 90% of glaucoma cases in
the United States. It is painless and does not have acute attacks.
The only signs are gradually progressive visual field loss, and
optic nerve changes (increased cup-to-disc ratio on fundoscopic
examination). Closed-angle glaucoma accounts for less than 10% of
glaucoma cases in the United States, but as many as half of
glaucoma cases in other nations (particularly Asian countries).
About 10% of patients with closed angles present with acute angle
closure crises characterized by sudden ocular pain, seeing halos
around lights, red eye, very high intraocular pressure (>30
mmHg), nausea and vomiting, suddenly decreased vision, and a fixed,
mid-dilated pupil. It is also associated with an oval pupil in some
cases. Modulating the activity of proteins encoded by DLK, NMDA,
INOS, CASP-3, Bcl-2, or Bcl-xl may treat the condition.
[0155] Sorsby's fundus dystrophy. Sorsby's fundus dystrophy is an
autosomal dominant, retinal disease associated with mutations in
the TIMP3 gene. Clinically, early, mid-peripheral, drusen and
colour vision deficits are found. Some patients complain of night
blindness. Most commonly, the presenting symptom is sudden acuity
loss, manifest in the third to fourth decades of life, due to
untreatable submacular neovascularisation. Histologically, there is
accumulation of a confluent lipid containing material 30 .mu.m
thick at the level of Bruch's membrane.
[0156] Vitelliform macular dystrophy. Vitelliform macular dystrophy
is a genetic eye disorder that can cause progressive vision loss.
Vitelliform macular dystrophy is associated with the buildup of
fatty yellow pigment (lipofuscin) in cells underlying the macula.
Over time, the abnormal accumulation of this substance can damage
cells that are critical for clear central vision. As a result,
people with this disorder often lose their central vision, and
their eyesight may become blurry or distorted. Vitelliform macular
dystrophy typically does not affect side (peripheral) vision or the
ability to see at night.
[0157] Researchers have described two forms of vitelliform macular
dystrophy with similar features. The early-onset form (known as
Best disease) usually appears in childhood; the onset of symptoms
and the severity of vision loss vary widely. It is associated with
mutations in the VMD2/BEST1 gene. The adult-onset form (Adult
vitelliform macular dystrophy) begins later, usually in
mid-adulthood, and tends to cause vision loss that worsens slowly
over time. It has been associated with mutations in the PRPH2 gene.
The two forms of vitelliform macular dystrophy each have
characteristic changes in the macula that can be detected during an
eye examination.
[0158] Rod-cone dystrophy. Rod-cone dystrophies are a family of
progressive diseases in which rod dysfunction, which leads to night
blindness and loss of peripheral visual field expanses, is either
the prevailing problem or occurring at least as severely as cone
dysfunction. A scallop-bordered lacunar atrophy may be seen in the
midperiphery of the retina. The macula is only mildly involved by
clinical examination although central retinal thinning is seen in
all cases. Dyschromatopsia is mild early and usually becomes more
severe. The visual fields are moderately to severely constricted
although in younger individuals a typical ring scotoma is present.
The peripheral retina contains `white dots` and often resembles the
retinal changes seen in retinitis punctate albescens. Retinitis
pigmentosa is the main group of diseases included under this
definition and, as a whole, is estimated to affect approximately
one in every 3,500 people. Depending on the classification criteria
used, about 60-80% of all retinitis pigmentosa patients have a
clear-cut rod-cone dystrophy pattern of retinal disease and once
other syndromic forms are taken into account, about 50-60% of all
retinitis pigmentosas fall in the rod-cone dystrophy nonsyndromic
category.
[0159] Leber's congenital amaurosis. Leber's congenital amaurosis
(LCA) is a severe dystrophy of the retina that typically becomes
evident in the first year of life. Visual function is usually poor
and often accompanied by nystagmus, sluggish or near-absent
pupillary responses, photophobia, high hyperopia, and keratoconus.
Visual acuity is rarely better than 20/400. A characteristic
finding is Franceschetti's oculo-digital sign, comprising eye
poking, pressing, and rubbing. The appearance of the fundus is
extremely variable. While the retina may initially appear normal, a
pigmentary retinopathy reminiscent of retinitis pigmentosa is
frequently observed later in childhood. The electroretinogram (ERG)
is characteristically "nondetectable" or severely subnormal.
Mutations in 17 genes are known to cause LCA: GUCY2D (locus name:
LCA1), RPE65 (LCA2), SPATA7 (LCA3), AIPL1 (LCA4), LCAS (LCAS),
RPGRIP1 (LCA6), CRX (LCA7), CRB1 (LCAS), NMNAT1 (LCA9), CEP290
(LCA10), IMPDH1 (LCA11), RD3 (LCA12), RDH12 (LCA13), LRAT (LCA14),
TULP1 (LCA15), KCNJ13 (LCA16), and IQCB1. Together, mutations in
these genes are estimated to account for over half of all LCA
diagnoses. At least one other disease locus for LCA has been
reported, but the gene is not known.
[0160] X-linked retinoschisis. X-linked retinoschisis (XLRS) is
characterized by symmetric bilateral macular involvement with onset
in the first decade of life, in some cases as early as age three
months. Fundus examination shows areas of schisis (splitting of the
nerve fiber layer of the retina) in the macula, sometimes giving
the impression of a spoke wheel pattern. Schisis of the peripheral
retina, predominantly inferotemporally, occurs in approximately 50%
of individuals. Affected males typically have vision of 20/60 to
20/120. Visual acuity often deteriorates during the first and
second decades of life but then remains relatively stable until the
fifth or sixth decade. The diagnosis of X-linked juvenile
retinoschisis is based on fundus findings, results of
electrophysiologic testing, and molecular genetic testing. RS1 is
the only gene known to be associated with X-linked juvenile
retinoschisis.
[0161] An individual affected by a cone cell disorder or at risk
for developing a cone cell disorder can be readily identified using
techniques to detect the symptoms of the disorder as known in the
art, including, without limitation, fundus photography; Optical
coherence tomography (OCT); adaptive optics (AO);
electroretinography, e.g. ERG, color ERG (cERG); color vision tests
such as pseudoisochromatic plates (Ishihara plates,
Hardy-Rand-Ritter polychromatic plates), the Farnsworth-Munsell 100
hue test, the Farnsworth's panel D-15, the City university test,
Kollner's rule, and the like; and visual acuity tests such as the
ETDRS letters test, Snellen visual acuity test, visual field test,
contrast sensitivity test, and the like; as will be known by the
ordinarily skilled artisan. Additionally or alternatively, the
individual affected by a cone cell disorder or at risk for
developing a cone cell disorder can be readily identified using
techniques to detect gene mutations that are associated with the
cone cell disorder as known in the art, including, without
limitation, PCR, DNA sequence analysis, restriction digestion,
Southern blot hybridization, mass spectrometry, etc. In some
embodiments, the method comprises the step of identifying the
individual in need of a cone cell therapy. In such instances, any
convenient method for determining if the individual has the
symptom(s) of a cone cell disorder or is at risk for developing a
cone cell disorder, for example by detecting the symptoms described
herein or known in the art, by detecting a mutation in a gene as
herein or as known in the art, etc. may be utilized to identify the
individual in need of a cone cell therapy.
[0162] In practicing the subject methods, the subject composition
is typically delivered to the retina of the subject in an amount
that is effective to result in the expression of the transgene in
the cone cells. In some embodiments, the method comprises the step
of detecting the expression of the transgene in the cone cells.
[0163] There are a number of ways to detect the expression of a
transgene, any of which may be used in the subject embodiments. For
example, expression may be detected directly, i.e. by measuring the
amount of gene product, for example, at the RNA level, e.g. by
RT-PCR, Northern blot, RNAse protection; or at the protein level,
e.g. by Western blot, ELISA, immunohistochemistry, and the like. As
another example, expression may be detected indirectly, i.e. by
detecting the impact of the gene product on the viability or
function of the cone photoreceptor in the subject. For example, if
the gene product encoded by the transgene improves the viability of
the cone cell, the expression of the transgene may be detected by
detecting an improvement in viability of the cone cell, e.g. by
fundus photography, Optical coherence tomography (OCT), Adaptive
Optics (AO), and the like. If the gene product encoded by the
transgene alters the activity of the cone cell, the expression of
the transgene may be detected by detecting a change in the activity
of the cone cell, e.g. by electroretinogram (ERG) and color ERG
(cERG); functional adaptive optics; color vision tests such as
pseudoisochromatic plates (Ishihara plates, Hardy-Rand-Ritter
polychromatic plates), the Farnsworth-Munsell 100 hue test, the
Farnsworth's panel D-15, the City university test, Kollner's rule,
and the like; and visual acuity tests such as the ETDRS letters
test, Snellen visual acuity test, visual field test, contrast
sensitivity test, and the like, as a way of detecting the presence
of the delivered polynucleotide. In some instances, both an
improvement in viability and a modification in cone cell function
may be detected.
[0164] In some embodiments, the subject method results in a
therapeutic benefit, e.g. preventing the development of a disorder,
halting the progression of a disorder, reversing the progression of
a disorder, etc. In some embodiments, the subject method comprises
the step of detecting that a therapeutic benefit has been achieved.
The ordinarily skilled artisan will appreciate that such measures
of therapeutic efficacy will be applicable to the particular
disease being modified, and will recognize the appropriate
detection methods to use to measure therapeutic efficacy. For
example, therapeutic efficacy in treating macular degeneration may
be observed as a reduction in the rate of macular degeneration or a
cessation of the progression of macular degeneration, effects which
may be observed by, e.g., fundus photography, OCT, or AO, by
comparing test results after administration of the subject
composition to test results before administration of the subject
composition. As another example, therapeutic efficacy in treating a
progressive cone dysfunction may be observed as a reduction in the
rate of progression of cone dysfunction, as a cessation in the
progression of cone dysfunction, or as an improvement in cone
function, effects which may be observed by, e.g., ERG and/or cERG;
color vision tests; functional adaptive optics; and/or visual
acuity tests, for example, by comparing test results after
administration of the subject composition to test results before
administration of the subject composition and detecting a change in
cone viability and/or function. As a third example, therapeutic
efficacy in treating a color vision deficiency may be observed as
an alteration in the individual's perception of color, e.g. in the
perception of red wavelengths, in the perception of green
wavelengths, in the perception of blue wavelengths, effects which
may be observed by, e.g., cERG and color vision tests, for example,
by comparing test results after administration of the subject
composition to test results before administration of the subject
composition and detecting a change in cone viability and/or
function.
[0165] Expression of a transgene delivered by the subject rAAV is
expected to be robust. Accordingly, in some instances, the
expression of the transgene, e.g. as detected by measuring levels
of gene product, by measuring therapeutic efficacy, etc, may be
observed two months or less after administration, e.g. 4, 3 or 2
weeks or less after administration, for example, 1 week after
administration of the subject composition. Expression of the
transgene is also expected to persist over time. Accordingly, in
some instances, the expression of the transgene, e.g. as detected
by measuring levels of gene product, by measuring therapeutic
efficacy, etc., may be observed 2 months or more after
administration of the subject composition, e.g., 4, 6, 8, or 10
months or more, in some instances 1 year or more, for example 2, 3,
4, or 5 years, in certain instances, more than 5 years.
[0166] In certain embodiments, the method comprises the step of
detecting expression of the polynucleotide delivered by the subject
rAAV in the cone cells, wherein expression is enhanced relative to
expression from an AAV not comprising a 7-10 amino acid insert in
the GH loop. i.e. a reference control, e.g. a parental rAAV into
which the peptide has been inserted. Typically, expression will be
enhanced 2-fold or more relative to the expression from a
reference, e.g. a parental rAAV, for example 3-fold, 4-fold, or
5-fold or more, in some instances 10-fold, 20-fold or 50-fold or
more, e.g. 100-fold, as evidenced by, e.g. earlier detection,
higher levels of gene product, a stronger functional impact on the
cells, etc.
[0167] Typically, an effective amount to achieve a change in will
be about 1.times.10.sup.8 vector genomes or more, in some cases
1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
1.times.10.sup.12, or 1.times.10.sup.13 vector genomes or more, in
certain instances, 1.times.10.sup.14 vector genomes or more, and
usually no more than 1.times.10.sup.15 vector genomes. In some
cases, the amount of vector genomes that is delivered is at most
about 1.times.10.sup.15 vector genomes, e.g. 1.times.10.sup.14
vector genomes or less, for example 1.times.10.sup.13,
1.times.10.sup.12, 1.times.10.sup.11, 1.times.10.sup.10, or
1.times.10.sup.9 vector genomes or less, in certain instances
1.times.10.sup.8 vector genomes, and typically no less than
1.times.10.sup.8 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.10 to
1.times.10.sup.11 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.10 to
3.times.10.sup.12 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.9 to
3.times.10.sup.13 vector genomes. In some cases, the amount of
vector genomes that is delivered is 1.times.10.sup.8 to
3.times.10.sup.14 vector genomes.
[0168] In some cases, the amount of pharmaceutical composition to
be administered may be measured using multiplicity of infection
(MOI). In some cases, MOI may refer to the ratio, or multiple of
vector or viral genomes to the cells to which the nucleic may be
delivered. In some cases, the MOI may be 1.times.10.sup.6. In some
cases, the MOI may be 1.times.10.sup.5-1.times.10.sup.7. In some
cases, the MOI may be 1.times.10.sup.4-1.times.10.sup.8. In some
cases, recombinant viruses of the disclosure are at least about
1.times.10.sup.1, 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, 1.times.10.sup.14, 1.times.10.sup.15,
1.times.10.sup.16, 1.times.10.sup.17, and 1.times.10.sup.18 MOI. In
some cases, recombinant viruses of this disclosure are
1.times.10.sup.8 to 3.times.10.sup.14 MOI. In some cases,
recombinant viruses of the disclosure are at most about
1.times.10.sup.1, 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, 1.times.10.sup.14, 1.times.10.sup.15,
1.times.10.sup.16, 1.times.10.sup.17, and 1.times.10.sup.18 MOI. In
some aspects, the amount of pharmaceutical composition comprises
about 1.times.10.sup.8 to about 1.times.10.sup.15 particles of
recombinant viruses, about 1.times.10.sup.9 to about
1.times.10.sup.14 particles of recombinant viruses, about
1.times.10.sup.10 to about 1.times.10.sup.13 particles of
recombinant viruses, or about 1.times.10.sup.11 to about
3.times.10.sup.12 particles of recombinant viruses.
[0169] Individual doses are typically not less than an amount
required to produce a measurable effect on the subject, and may be
determined based on the pharmacokinetics and pharmacology for
absorption, distribution, metabolism, and excretion ("ADME") of the
subject composition or its by-products, and thus based on the
disposition of the composition within the subject. This includes
consideration of the route of administration as well as dosage
amount, which can be adjusted for subretinal (applied directly to
where action is desired for mainly a local effect), intravitreal
(applied to the vitreaous for a pan-retinal effect), or parenteral
(applied by systemic routes, e.g. intravenous, intramuscular, etc.)
applications. Effective amounts of dose and/or dose regimen can
readily be determined empirically from preclinical assays, from
safety and escalation and dose range trials, individual
clinician-patient relationships, as well as in vitro and in vivo
assays such as those described herein and illustrated in the
Experimental section, below.
[0170] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0171] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
EXAMPLES
[0172] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
[0173] General methods in molecular and cellular biochemistry can
be found in such standard textbooks as Molecular Cloning: A
Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory
Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel
et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag
et al., John Wiley & Sons 1996); Nonviral Vectors for Gene
Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors
(Kaplift & Loewy eds., Academic Press 1995); Immunology Methods
Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue
Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998), the disclosures of which
are incorporated herein by reference. Reagents, cloning vectors,
and kits for genetic manipulation referred to in this disclosure
are available from commercial vendors such as BioRad, Stratagene,
Invitrogen, Sigma-Aldrich, and ClonTech.
Example 1
Background
[0174] New therapies are needed for the treatment of many cone
photoreceptor associated disorders, including macular dystrophies
such as cone-rod dystrophy, cone dystrophy, Stargardt macular
dystrophy, and achromatopsia; color vision disorders such as
protan, deutan, and tritan defects; and vision disorders of the
central macula such as age-related macular degeneration, macular
telangiectasia, retinitis pigmentosa, diabetic retinopathy, retinal
vein occlusions, glaucoma, Sorsby's fundus dystrophy, adult
vitelliform macular dystrophy, Best's disease, and X-linked
retinoschisis. As these vision disorders are associated with a loss
of function and/or viability of the cone photoreceptors, it is
hypothesized that these disorders may be treatable by delivering a
therapeutic gene to cone photoreceptors to rescue cone viability
and function.
[0175] To that end, the polynucleotide cassette "pMNTC" was
designed in which enhancer, promoter, 5'UTR, intron, Kozak, and
polyadenylation sequences were designed for cone-specific
expression (FIG. 6a). The cassette included an LCR enhancer
sequence from the L- and M-opsin genomic locus and a truncated
promoter sequence from the M-Opsin gene, comprising about 140
nucleotides upstream of the transcriptional start site. In
addition, the cassette included a 5' untranslated region (5' UTR)
based on the M-opsin 5'UTR but modified to have minimal secondary
structure and to include additional sequence at its 3' end into
which an intron was inserted. The intronic sequence used was a pSI
chimeric intron having the 5'-donor site from the first intron of
the human .beta.-globin gene and the branch and 3'-acceptor site
from the intron that lies between the leader and the body of an
immunoglobulin gene heavy chain variable region (Bothwell, A. L. et
al. (1981) Heavy chain variable region contribution to the NPb
family of antibodies: Somatic mutation evident in a gamma 2a
variable region. Cell 24, 625-37). The sequences of the donor and
acceptor sites, along with the branchpoint site, were changed to
match the consensus sequences for splicing (Senapathy, P., Shapiro,
M. B. and Harris, N. L. (1990) Meth. Enzymol. 183, 252-78). Also
included in the pMNTC polynucleotide cassette was a strong Kozak
sequence and an SV40 polyadenylation sequence.
[0176] Experiments were also performed to identify the best AAV
with which to deliver transgenes to cone cells. Successful delivery
of polynucleotides to cells of the retina for the purposes of gene
therapy has been achieved using viral vectors such as AAV and
lentivirus. However, these viruses must be injected subretinally to
reach the cells of the non-human primate (NHP) retina, a procedure
that carries with it the risk of retinal damage. A less disruptive
approach is administration by intravitreal injection. However,
efficient transduction of cone photoreceptors following
intravitreal delivery of AAV or lentivirus has never been
demonstrated: while reports exist of AAVs with the ability to
transduce retinal cone cells with high efficiency (Merigan et al.
IOVS 2008, 49 E-abstract 4514), later reports have questioned the
efficacy of these vectors (Yin et al. IOVS 2011,
52(5):2775-2783).
Results
[0177] Directed evolution of AAV2 has led to the identification of
the viral variant "7m8" that is able to transduce photoreceptors
better than wild type AAV2 (Dalkara et al. Sci Transl Med 2013).
However, the retina contains two types of photoreceptors--rods and
cones--and no reports exist demonstrated whether AAV2-7m8 can
transduce cone photoreceptors, per se, and more particularly, cone
photoreceptors in the highly cone-enriched area of the fovea. To
test this possibility, we delivered AAV2-7m8 carrying an expression
cassette of the ubiquitous promoter CMV operably linked to GFP to
the retina of African Green monkey by intravitreal injection.
Intravitreally delivered AAV2-7m8.CMV.GFP appeared to transduce
retinal cells in the fovea centralis (the 0.35 mm diameter rod-free
region of retina at the center of the foveal pit) and parafovea
(the lip of the depression) of primates more efficiently than
intravitreally-delivered AAV2 or other AAV variants previously
shown in the art to transduce retinal cells. Neither AAV2-7m8 nor
the other AAVs tested appeared to be able to transduce the cones of
the primate fovea, the 1.5 mm-diameter cone-enriched region of
retina that surrounds the foveola and forms the slopes of the pit
(FIG. 1).
[0178] We next packaged a genome comprising pMNTC operably linked
to GFP within the AAV2-7m8 capsid, and assessed the ability of this
vector composition to express the GFP transgene in cone cells in
vivo when injected intravitreally. Expression was evaluated in a
number of species with varying numbers of retinal cones cells among
total photoreceptors, including mouse (3% cones), rat (1% cones),
gerbil (13% cones), and nonhuman primate (5% cones). Contrary to
our results in FIG. 1, strong gene expression could be detected
throughout the nonhuman primate fovea (FIG. 2). These data indicate
that intravitreally delivered AAV2-7m8 can, in fact, transduce
retinal cones, and that pMNTC acts as a robust expression cassette
in cone cells. Robust reporter gene expression was also seen in the
intravireally injected retina of the rat (data not shown) and
gerbil (FIG. 4A), with expression levels and anatomic location
correlating with cone abundance and location in all species.
[0179] To determine the cell-specificity of pMNTC-directed
expression, whole mounts of transduced mouse retina were analyzed
by immunohistochemistry using an antibody that is specific for cone
L and M opsins. The expression of L/M opsin, which labels the outer
segments of cone photoreceptors only, was observed in virtually all
of the cones of the mouse retina that expressed GFP from the
AAV2-7m8.MNTC.GFP vector (FIG. 3), indicating that MNTC-directed
expression of transgenes is highly cone-specific. Moreover 80% or
more of the cone outer segments that were labelled by the L/M
opsin-specific antibody also expressed the GFP transgene,
indicating that AAV2-7m8 transduces cones highly efficiently (FIG.
3).
[0180] We also determined the cell-specificity of pR2.1-directed
expression by packaging a genome comprising pR2.1 operably linked
to GFP within the AAV2-7m8 capsid (AAV2-7m8pR2.1.GFP vector). pR2.1
comprises the human L/M opsin enhancer ("LCR") and the promoter
region from the human L-Opsin gene. In addition, pR2.1 comprises
the L-Opsin 5'UTR fused to additional 5'UTR sequence at its 3' end,
into which modified SV40 late 16s intronic sequence has been
inserted. This is followed by the L-Opsin Kozak sequence, which is
then typically linked in-frame to a transgene. At the end of the
cassette is an SV40 polyA tail. The ability of this vector
composition to express the GFP transgene in cone cells in vivo was
assessed 12 weeks after intravitreal injection in an African green
monkey (non-human primate; NHP). Briefly, the NHP received
bilateral intravitreal administrations of 50 uL of
1.0.times.10.sup.13 vg/mL AAV2-7m8pR2.1.GFP to yield a final dose
of 5.times.10.sup.11 vg per eye. Retinal examination, including
fundus color and fluorescence photography, was performed by using a
Topcon TRC-50EX retinal camera with Canon 6D digital imaging
hardware and a Spectralis OCT Plus at baseline and at weeks 4, 8,
and 12 post-intravitreal vector injection. The animal was
terminated at 12 weeks and eyes processed. A cross-section of a
treated retina from the NHP was stained with a chicken polyclonal
anti-GFP antibody (Abcam Cat #13970; Cambridge, UK); a rabbit
polyclonal anti-L/M Opsin antibody specific for opsin cones (Abcam
Cat #5405); a 1D4 mouse monoclonal anti-rhodopsin antibody (Abcam
Cat #5417); and Dapi to stain all nuclei (Invitrogen Ref #D21490).
GFP-tagged transgene containing cells were imaged by multispectral
analysis along with the antibody probes and DIC (differential
interference contract for topology). As flattened stacks of optical
planes through the entire section. Cell analysis for transgene was
optimized using morphology and colocalization with probes. GFP
(transgene) staining co-localized with L/M opsin staining and not
with rhodopsin staining, indicating that pR2.1 promotes expression
in cone cells specifically (FIG. 7). GFP transgene signal was
observed at fovea, mid and far periphery; GFP transgene signal
colocalized with L/M-opsin, calbindin and PNA probe; clear
exclusion of 1D4-containing cells in fovea was observed; and there
was no GFP transgene positive cell association with rods or other
probe-containing cells. (FIG. 7). In summary, cells double-stained
for GFP (transgene expression) and L/M opsin were observed, but
there was a lack of cells double-staining for both GFP and
rhodopsin, indicating that the AAV2-7m8pR2.1.GFP vector
specifically directed expression in cone cells and not rod
cells.
[0181] We next compared the ability of pMNTC to promote expression
in cone cells to that of pR2.1. Viral preparations of
AAV2-7m8.MNTC.GFP and AAV2-7m8.pR2.1.GFP were delivered
intravitreally to the retinas of gerbils and nonhuman primates in
vivo, and the retinas imaged in vivo 2 weeks, 4 weeks, 8 weeks, and
12 weeks later by fundus autofluorescence and OCT. GFP reporter
expression was detected sooner, more strongly, and in more cones in
gerbil retina transduced with rAAV carrying the pMNTC.GFP
expression cassette than in gerbil retinas carrying the pR2.1.GFP
expression cassette (FIG. 4B). Likewise, GFP reporter expression
was detected sooner and in more cones in nonhuman primate retinas
transduced with rAAV carrying the pMNTC.GFP expression cassette as
compared to NHP retinas transduced with the pR2.1 expression
cassette (FIG. 5, n=4 eyes). In both gerbils and NHP, GFP was
consistently observed to be stronger from pMNTC than from pR2.1
throughout the duration of the study.
[0182] To determine the contribution of each of the elements in the
pMNTC expression cassette to the overall improvement in expression,
a series of expression constructs were cloned in which each of the
elements in pMNTC was substituted one-by-one with the corresponding
element from the pR2.1 expression cassette. These constructs were
then packaged into AAV2-7m8 and delivered by intravitreal injection
to the gerbil retina. Gerbil retinas were assessed 4 and 8 weeks
later in vivo by in vivo bioluminescence (IVIS imaging system,
PerkinElmer), which provides a quantitative readout of reporter
expression across the entire eye.
[0183] As expected, expression of the luciferase reporter under the
control of pMNTC was higher than expression of the luciferase
reporter under the control of pR2.1 (FIG. 6). Replacement of the
pMNTC promoter sequence with the pR2.1 promoter sequence having the
most sequence homology to it reduced expression (construct
pMNTC_pR2.1 L3'P), as did the inclusion of pR2.1 promoter sequence
that lies more distal to the 5'UTR of pR2.1 (construct
pMNTC_pR2.1-L5'P). Expression was also reduced by the introduction
into the pMNTC 5'UTR of two false start sequences ("AUG1" and
"AUG2") that were observed in the pR2.1 5'UTR (construct
pMNTC_2.1-AUG1/2). Interestingly, expression was not reduced when
the pMNTC 5'UTR was replaced with a modified pR2.1 5'UTR sequence
in which these false starts had been removed (nucleotide 17 changed
to C, nt 61 and 62 changed to CA) (pMNTC_pR2.1-5'UTR), suggesting
that the pR2.1 5'UTR would promote strong expression in cone cells
but for the false AUGs in the pR2.1 5'UTR element. Also
interestingly, the pR2.1 intron appeared to provide more robust
expression than the pSI chimeric intron of pMNTC, suggesting that
inclusion of the pR2.1 intron in the polynucleotide cassettes of
the present disclosure may be used to further improve expression in
cone cells. Lastly, removal of the L/M enhancer (found in both
pR2.1 and pMNTC) reduced expression as well. While the polyA tailed
seemed at first to also have a significant impact on expression,
re-sequencing of the pMNTC construct comprising this pR2.1 element
revealed that the polyA tail was not operably linked to the
transgene, thereby explaining why only background levels of
expression were observed from this construct. Thus, the L/M opsin
LCR, the inclusion of the M opsin core promoter rather than the L
opsin promoter, and the exclusion of false starts in the 5'UTR all
contribute to the enhancement in gene expression achieved using the
pMNTC promoter.
[0184] In conclusion, we have identified an AAV variant, the AAV
variant comprising a 7m8 peptide in the GH loop, which may be used
for the intravitreal delivery of polynucleotides to retinal cones.
Likewise, we have identified a number of polynucleotide cassette
elements that may be used to promote strong expression in cone
photoreceptors. Together, these discoveries represent improvements
that may facilitate the development of therapeutic agents for
cone-associated disorders.
Materials and Methods
[0185] Transgene expression in vitro in WERI-RB-1 cells. WERI-Rb-1
retinoblastoma cells expressing cone photoreceptor pigments cells
are transfected with a polynucleotide cassette of the present
disclosure according to the method described by Shaaban and Deeb,
1998; IOVS 39(6)885-896. The polynucleotide cassettes are
transfected as plasmid DNA using well established techniques of
molecular biology, such as cloning (Maniatis et al.) or via de novo
DNA synthesis. All regulatory elements are placed in the cassette
and used to drive the enhanced GFP protein. Plasmid DNA is then
introduced into cells using established techniques for non-viral
transfection, for example using a lipid-based transfection reagent
(Altogen Biosystems, NV) or Lipofectamine LTX (Life Technologies).
Cells are then cultured for 72 hours and eGFP expression is
measured using flow cytometry and fluorescence microscopy.
Transgene expression in cells transfected with the polynucleotide
cassette of the present invention (i.e., constructs designed for
cone photoreceptor expression) is compared to the un-optimized
counterparts (i.e., those based on pR2.1) and is found to be
stronger from cassettes carrying improved elements
[0186] In vitro expression is also evaluated using other mammalian
cell lines that express cone opsins, such as 661W cells (Tan et
al., IOVS 2004; 45(3) 764-768).
[0187] Similarly, in vitro expression is evaluated using
non-photoreceptor cell lines that have been engineered to express
cone photoreceptor-specific proteins. Such a system has been
described with HEK293 cells that have been genetically engineered
to express CRX/Sp1 (Khani et al., IOVS 2007; 48: 3954). Marker
genes are also used (eGFP, dsRed, mCherry, luciferase) as well as
physiologic genes (opsin, ACHR genes). Physiologic genes are tested
by examining mRNA levels (e.g., by RT-PCR) or protein levels (e.g.,
by ELISA or Western blot).
[0188] Animal care. All experiments conformed to the principles
regarding the care and use of animals adopted by the American
Physiological Society and the Society for Neuroscience, and were
approved by the Institutional Animal Care and Use Committee
(IACUC).
[0189] Small animal studies. The expression of the gene products
encoded by the coding sequence of the expression cassettes was
evaluated in vivo in mice, rats, and gerbils. This was accomplished
by intravitreal injection in vivo of an rAAV preparation comprising
the expression cassette (Li et al., 2008; Mol Vis 48: 332-338).
Note that electroporation of plasmid DNA may be performed instead
(Matsuda/Cepko).
[0190] Mouse studies. Mice used in this study were C57BL/6. Animals
were anesthetized with ketamine/xylazine (110 mg/kg
intraperitoneal). A beveled 34 gauge disposable needle loaded with
test article was inserted into the vitreous of the eye, and
5.04.times.1010 vector genomes of rAAV in a volume of 1.5.mu.l was
injected into the vitreous.
[0191] Gerbil and rat studies. Mongolian gerbils (Meriones
unguiculatus) and brown Norway rats were used in this study. Pupils
were dilated with 10% phenylephrine and 0.5% tropicamide. Animals
were anesthetized with an intraperitoneal or intramuscular
injection of 0.1-0.2 mL of a ketamine/xylazine solution (70 mg/mL
ketamine and 10 mg/mL xylazine for rats; 25 mg/mL ketamine and 0.3
mg/mL xylazine for gerbils). A beveled 34 gauge disposable needle
loaded with test article in a 100 .mu.L Hamilton syringe was
inserted into the vitreous of the eye through the sclera at an
optimized superior-temporal point about 1 mm from Limbus.
1.times.1010-2.times.1010 vector genomes of test article
(2.times.1010 vg of rAAV.GFP, or 1.15.times.1010 vg of
rAAV.luciferase) in a 5 uL volume was injected slowly with a
micro-injection pump into the vitreous, after which the needle tip
was held in the injected eye at the injected position for 10
seconds so as to ensure adequate test article dispensing. The
needle was then withdrawn.
[0192] Non-human primate (NHP) studies. The polynucleotide
cassettes and expression vectors were also tested in large animals.
This was done by using AAV, for example using the techniques of
Mancuso et al. Briefly, an AAV cassette was made, the AAV
encapsidating the expression cassette was manufactured, and the
viral prep was injected intravitreally (up to 170 uL in the
vitreous) or subretinally (up to 3, 100 uL injections at different
locations; vitrectomy may be performed prior to injection) in
nonhuman primates. Expression was evaluated by reporter (GFP),
color ERG, and/or behavioral testing using the Cambridge Color Test
or on animals trained to make a saccade (eye movement) when a
target enters the field of view. The saccades are monitored using
an eye tracker. Prior to treatment animals are trained to perform a
color vision test or to make a saccade when it sees a colored
target. An ERG is performed to estimate the spectral sensitivity of
the cones present. Data from the color vision test performance and
the ERG provide evidence that the animal is dichromatic
(colorblind). For animals that receive a vector carrying the GFP
gene, expression is monitored using fundus imaging with RetCam II
or similar device under light that produces excitation of the GFP.
For animals receiving a photopigment gene that differs in spectral
sensitivity compared to the animal's endogenous pigments,
expression is monitored using the multifocal color ERG to measure
spectral sensitivity at up to 106 different retinal locations, and
by behavioral testing.
[0193] Baboons were sedated with 10-15 mg/kg ketamine following by
sevofluorane. African Green monkeys were sedated with an
intramuscular injection of 5:1 ketamine:xylazine mix (0.2 ml/kg of
100 mg/ml ketamine and 20 mg/ml xylazine). Mydriasis was achieved
with topical 10% phenylephrine. An eye speculum was placed in the
eye to facilitate injections. A drop of proparacaine hydrochloride
0.5% and then 5% betadine solution was applied, followed by a rinse
with sterile saline. Baboons (FIG. 2) received 60 .mu.l of a
3.4.times.10.sup.13 vg preparation of rAAV by intravitreal (ITV)
injection to yield a final dose of 2.02.times.10.sup.12 vg per eye.
African Green monkeys received 50 uL of a 1.times.10.sup.13
preparation of rAAV vector by ITV injection to yield a final dose
of 5.times.10.sup.11 vg per eye. ITV injections to the central
vitreous were administered using a 31-gauge 0.375 inch needle
(Terumo) inserted inferotemporally at the level of the or a serrata
.about.2.5 mm poster to the limbus under a surgical magnification
to allow full visualization of extraocular and intraocular needle
placement. Central vitreous placement was confirmed by direct
observation of the needle tip at the time of the injection.
Following ITV injections a topical triple antibiotic ointment was
administered.
[0194] Slit-lamp biomicroscopy. The anterior segment of each monkey
eye was examined by slit-lamp biomicroscopy during baseline
screening and at week 4 (day 28), week 8 (day 56) and week 12 (day
84) post-injection to monitor inflammation. No abnormalities were
observed.
[0195] NHP Necropsy and Eye Processing. Animals were euthanized
with pentobarbital 12 weeks post intravitreal injection. Eyes were
tagged with a suture at the 12 o'clock position before enucleating
and trimming of extraocular tissues. Posterior cups were isolated
by removing tissues anterior to the limbus and fixed by immersion
in 4% paraformaldehyde and stored in 70% ethanol.
[0196] Immunolabeling. Eyes were rehydrated into water then PBS
buffer before flattening and delaminating retina as whole mounts.
Preparations were imaged by stereo fluorescence microscopy
(Discovery FI V20, Carl Zeiss Microscopy, LLC, Thornwood, N.Y.) for
GFP. Quadrant with fovea was detached from flat-mount, mounted
under coverslip and imaged as full montage (5.times. tiling and
stitching, Axio Observer Z1, Zeiss). Strip of retina was isolated
central at fovea out to periphery, cryoprotected in sucrose and
frozen in OCT. 8 .mu.m sections were immunostained with antibodies
to proteins enriched in specific retinal cell populations,
including, L/M- and S-opsins, glutamine synthetase (GS), calbindin,
rhodopsin (1D4), .beta.-III Tubulin, Laminin, peanut agglutinin
(PNA) and/or others. GFP-tagged transgene containing cells were
imaged by multispectral analysis along with antibody probes and DIC
(differential interference contract for topology) as flattened
stacks of optical plains through entire section (Axio Observer Z1,
with Apotome, Zeiss). Cell analysis for transgene was optimized
using morphology and colocalization with probes.
[0197] Fundus examination and photography. Eye examination and
fundus photography of rat and gerbil retinas was performed using a
Phoenix Micron IV fundus microscope. All animals received a
baseline screening/photographing to confirm ocular health, and then
photographed at the designated timepoints to monitor the expression
of the GFP transgene. Any change to the optic nerves and retina or
appearance of gross lesions were recorded by a color fundus
photography and expression of GFP was visualized using fluorescence
fundus imaging with a fluorescein filter.
[0198] Retinal examination, fundus color and fluorescence
photography, and autofluorescence OCT of NHP were performed by
using a Topcon TRC-50EX retinal camera with Canon 6D digital
imaging hardware and New Vision Fundus Image Analysis System
software and Spectralis OCT Plus. All animals received a baseline
imaging. GFP expression was also documented at week 2, 4, 8, and 12
post-intravitreal vector injection.
[0199] IVIS Imaging System. Expression of luciferase in the retina
following delivery of rAAV.luciferase was quantified in vivo 2, 4
and 8 weeks post-intravitreal injection using an IVIS Imaging
System. Gerbils were injected subcutaneously with 150 mg/kg
luciferin (PerkinElmer) (15 mg/ml luciferin at a dose of 15 ml/kg).
Approximately 22 minutes later, animals were sedated by inhalation
of 4% isoflurane for 3-5 minutes. Immediately thereafter, animals
were placed on the imaging platform in pairs, and the luminescence
of the one eye of each animal quantified followed immediately by
imaging of the contralateral eye. A naive gerbil was used as a
negative standard, with background levels of luminescence typically
registering a luminescence of 1.times.10.sup.4 photons/second.
Bioluminescence verification using a phantom mouse (XPM-2 Perkin
Elmer phantom mouse for bioluminescence imaging) was performed
prior to imaging to ensure calibration of the imaging system.
[0200] Immunohistochemistry. Mice were euthanized with a lethal
dose of sodium pentobarbital and tissues fixed via cardiac
perfusion first with 0.13M phosphate buffered saline (PBS) pH
7.2-7.4 containing 2 units of heparin per mL, followed by 4%
paraformaldehyde (PFA) in PBS, followed by 4% paraformaldehyde plus
1% glutaraldehyde in PBS. Glutaraldehyde served to keep the neural
retina attached to the RPE so that the cone outer segments would
remain intact. Each solution was warmed to .about.37.degree. C.
just prior to administration and .about.35-40 mL of perfusate was
delivered at each stage. Once the perfusion was stopped, the mouse
was wrapped in a moist paper towel and left to further fix for 2-3
hours before enucleation and dissection.
[0201] Permanent ink was used to mark the orientation of the eye,
the anterior segment was removed, and the eye-cup was fixed in 4%
PFA overnight at 4.degree. C. and then stored in PBS at 4.degree.
C. Retinal whole-mounts were made by flattening the dissected
retina between tissues soaked in 4% PFA for two hours and then
transferring them to a culture plate for 6 more hours of fixation.
Afterward, the PFA was replaced with PBS containing 0.03% sodium
azide (Sigma).
[0202] Antibody labeling was carried out on a rotating table
shaker. To block non-specific labeling, whole mounts were incubated
overnight at 4.degree. C. with a solution containing 5% donkey
serum (Jackson ImmunoResearch, Cat #004-000-120), 1 mg/ml BSA
(Jackson ImmunoResearch, Cat #001-000-161), and 0.03% Triton X-100
in PBS (pH 7.4). The primary antibody used in this study was rabbit
anti red-green (L/M) opsin diluted 1:200 (Millipore, Cat #AB5405.
Specimens were washed in PBS 3 times for 30 minutes each, then
incubated at 4.degree. C. overnight with DAPI
(4',6-diamidino-2-phenylindole, dihydrochloride 1:10,000;
Invitrogen, Cat #D-21490) plus secondary antibodies. The secondary
antibody for the L/M-opsin antibody was Alexa Fluor 488 labeled
donkey anti-rabbit IgG(H+L) diluted 1:200 in antibody dilution
buffer (Invitrogen, Cat #A21206). The incubation with secondary
antibody was followed by three 30 minute PBS washes, 30 minutes of
post-fixation with 4% paraformaldehyde, and three more 30 minute
PBS washes. Finally, the retinal slices were placed on slides with
2% DABCO in glycerol and covered with cover slips.
[0203] Microscopy. Widefield images of mouse retina whole mounts
were acquired using a Nikon Eclipse E1000 with a 20.times.
(open-air) objective and camera set with a 1.5.times. optical zoom.
For each specimen, 50 optical sections were taken 0.5 .mu.m apart
and the M-opsin Z-stack was reconstructed in ImageJ. The Z-stack
was oriented so that the lengths of the outer segments were in
plane, and the distance between where antibody staining began and
ended was measured as an estimate of the length of the outer
segments. Further, a 3D projection of the Z-stack was generated and
the number of cones with visible M-opsin in the outer segment could
be quantified.
[0204] Confocal image slices were acquired using an Olympus
FluoView.TM. FV1000. Sections were imaged using a 20.times. oil
immersion lens (40 images taken 0.5 .mu.m apart) and the Z-stacks
were reconstructed in ImageJ. Channel exposure levels were balanced
within and across images using Adobe Photoshop. For the retinal
whole mounts, images were taken using a 10.times. open-air lens and
mosaics were constructed with Adobe Photoshop's native mosaic
construction software.
[0205] Experiments testing the tissue specificity of the
polynucleotide cassettes. In this instance, a construct encoding
GFP is injected via one or more routes of administration, such as
intravitreal, subretinal, or intravenously. The animal is then
sacrificed and tissues are analyzed by qPCR--to detect DNA
sequences indicating presence of the construct--and GFP
expression--to detect areas where the construct is actively
expressed. Whereas absence of DNA sequence indicates lack of
biodistribution to a given tissue, the presence of DNA sequence
together with the lack of transgene expression (mRNA or protein
level) indicates presence of vector but lack of expression in that
tissue. In this way, the level of specificity for cone
photoreceptors can be established, and used to determine the
utility of this invention in terms of restricting expression to
target cone photoreceptor cells without expression in non-targeted
tissues such as optic nerve, liver, spleen, or brain tissue.
Intravitreal AAV is known to biodistribute to the brain (Provost et
al) so highly expressed, improved constructs for targeting cone
photoreceptors would be useful to limit expression to target cells
of the retina and limit potential adverse events associated with
off-target transgene expression.
[0206] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of the present invention is embodied by the
appended claims.
[0207] All publications and patent applications described herein
are hereby incorporated by reference in their entireties.
Sequence CWU 1
1
231736PRTAdeno-associated virus - 1 1Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp
Ser Ser Ser Gly Ile Gly145 150 155 160Lys Thr Gly Gln Gln Pro Ala
Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser
Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro
Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala
Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215
220Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr
Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly
Ala Ser Asn Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285His Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val
Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330
335Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
Pro Ala 355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr
Leu Asn Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly
Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Glu Val Pro Phe
His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met
Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr
Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455
460Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu
Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr
Lys Thr Asp Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala
Ser Lys Tyr Asn Leu Asn 500 505 510Gly Arg Glu Ser Ile Ile Asn Pro
Gly Thr Ala Met Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe
Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540Lys Glu Ser Ala
Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr
Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570
575Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala
580 585 590Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val
Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala
Lys Ile Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu
Met Gly Gly Phe Gly Leu625 630 635 640Lys Asn Pro Pro Pro Gln Ile
Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu
Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser
Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys
Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695
700Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly
Leu705 710 715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu
Thr Arg Pro Leu 725 730 7352735PRTAdeno-associated virus - 2 2Met
Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser1 5 10
15Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu
Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly
Glu Pro 50 55 60Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys
Ala Tyr Asp65 70 75 80Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu
Lys Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu
Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln
Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Glu
Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu His
Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly145 150 155 160Lys
Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170
175Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly
Ser Gly 195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly
Val Gly Asn Ser 210 215 220Ser Gly Asn Trp His Cys Asp Ser Thr Trp
Met Gly Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp
Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser
Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270Phe Gly Tyr
Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285Cys
His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295
300Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln
Val305 310 315 320Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile
Ala Asn Asn Leu 325 330 335Thr Ser Thr Val Gln Val Phe Thr Asp Ser
Glu Tyr Gln Leu Pro Tyr 340 345 350Val Leu Gly Ser Ala His Gln Gly
Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365Val Phe Met Val Pro Gln
Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380Gln Ala Val Gly
Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser385 390 395 400Gln
Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410
415Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg
420 425 430Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser
Arg Thr 435 440 445Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu
Gln Phe Ser Gln 450 455 460Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser
Arg Asn Trp Leu Pro Gly465 470 475 480Pro Cys Tyr Arg Gln Gln Arg
Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495Asn Ser Glu Tyr Ser
Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510Arg Asp Ser
Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525Asp
Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535
540Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile
Thr545 550 555 560Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala
Thr Glu Gln Tyr 565 570 575Gly Ser Val Ser Thr Asn Leu Gln Arg Gly
Asn Arg Gln Ala Ala Thr 580 585 590Ala Asp Val Asn Thr Gln Gly Val
Leu Pro Gly Met Val Trp Gln Asp 595 600 605Arg Asp Val Tyr Leu Gln
Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620Asp Gly His Phe
His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys625 630 635 640His
Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650
655Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
660 665 670Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu
Gln Lys 675 680 685Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr
Thr Ser Asn Tyr 690 695 700Asn Lys Ser Val Asn Val Asp Phe Thr Val
Asp Thr Asn Gly Val Tyr705 710 715 720Ser Glu Pro Arg Pro Ile Gly
Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 7353736PRTAdeno-associated
virus - 3 3Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn
Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val
Pro Gln Pro 20 25 30Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly
Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu
Asp Lys Gly Glu Pro 50 55 60Val Asn Glu Ala Asp Ala Ala Ala Leu Glu
His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn
Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg
Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro 115 120 125Leu Gly Leu
Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Gly 130 135 140Ala
Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly145 150
155 160Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln
Thr 165 170 175Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly
Glu Pro Pro 180 185 190Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met
Ala Ser Gly Gly Gly 195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Asn Ser 210 215 220Ser Gly Asn Trp His Cys Asp
Ser Gln Trp Leu Gly Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys
Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265
270Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
Asn Trp 290 295 300Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe
Asn Ile Gln Val305 310 315 320Arg Gly Val Thr Gln Asn Asp Gly Thr
Thr Thr Ile Ala Asn Asn Leu 325 330 335Thr Ser Thr Val Gln Val Phe
Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350Val Leu Gly Ser Ala
His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365Val Phe Met
Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380Gln
Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser385 390
395 400Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe
Glu 405 410 415Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp Arg 420 425 430Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Asn Arg Thr 435 440 445Gln Gly Thr Thr Ser Gly Thr Thr Asn
Gln Ser Arg Leu Leu Phe Ser 450 455 460Gln Ala Gly Pro Gln Ser Met
Ser Leu Gln Ala Arg Asn Trp Leu Pro465 470 475 480Gly Pro Cys Tyr
Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn 485 490 495Asn Asn
Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn 500 505
510Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys
515 520 525Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile
Phe Gly 530 535 540Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp
Asn Val Met Ile545 550 555 560Thr Asp Glu Glu Glu Ile Arg Thr Thr
Asn Pro Val Ala Thr Glu Gln 565 570 575Tyr Gly Thr Val Ala Asn Asn
Leu Gln Ser Ser Asn Thr Ala Pro Thr 580 585 590Thr Gly Thr Val Asn
His Gln Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605Asp Arg Asp
Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620Thr
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu625 630
635 640Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro
Ala 645 650 655Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser
Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile
Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn Ser Lys Arg Trp Asn Pro
Glu Ile Gln Tyr Thr Ser Asn 690 695 700Tyr Asn Lys Ser Val Asn Val
Asp Phe Thr Val Asp Thr Asn Gly Val705 710 715 720Tyr Ser Glu Pro
Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730
7354734PRTAdeno-associated virus - 4 4Met Thr Asp Gly Tyr Leu Pro
Asp Trp Leu Glu Asp Asn Leu Ser Glu1 5 10 15Gly Val Arg Glu Trp Trp
Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys 20 25 30Ala Asn Gln Gln His
Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly 35 40 45Tyr Lys Tyr Leu
Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val 50 55 60Asn Ala Ala
Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln65 70 75 80Gln
Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp 85 90
95Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly Asn
100 105 110Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu
Pro Leu 115 120 125Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly
Lys Lys Arg Pro 130 135 140Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser
Ser Thr Gly Ile Gly Lys145 150 155 160Lys Gly Lys Gln Pro Ala Lys
Lys Lys Leu Val Phe Glu Asp Glu Thr 165 170 175Gly Ala Gly Asp Gly
Pro Pro Glu Gly Ser Thr Ser Gly Ala Met Ser 180 185 190Asp Asp Ser
Glu Met Arg Ala Ala Ala Gly Gly Ala Ala Val Glu Gly 195 200 205Gly
Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys 210 215
220Asp Ser Thr Trp Ser Glu Gly His Val Thr Thr Thr Ser Thr Arg
Thr225 230 235 240Trp Val Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys
Arg Leu Gly
Glu 245 250 255Ser Leu Gln Ser Asn Thr Tyr Asn Gly Phe Ser Thr Pro
Trp Gly Tyr 260 265 270Phe Asp Phe Asn Arg Phe His Cys His Phe Ser
Pro Arg Asp Trp Gln 275 280 285Arg Leu Ile Asn Asn Asn Trp Gly Met
Arg Pro Lys Ala Met Arg Val 290 295 300Lys Ile Phe Asn Ile Gln Val
Lys Glu Val Thr Thr Ser Asn Gly Glu305 310 315 320Thr Thr Val Ala
Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp 325 330 335Ser Ser
Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu Gly Ser 340 345
350Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr
355 360 365Cys Gly Leu Val Thr Gly Asn Thr Ser Gln Gln Gln Thr Asp
Arg Asn 370 375 380Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met
Leu Arg Thr Gly385 390 395 400Asn Asn Phe Glu Ile Thr Tyr Ser Phe
Glu Lys Val Pro Phe His Ser 405 410 415Met Tyr Ala His Ser Gln Ser
Leu Asp Arg Leu Met Asn Pro Leu Ile 420 425 430Asp Gln Tyr Leu Trp
Gly Leu Gln Ser Thr Thr Thr Gly Thr Thr Leu 435 440 445Asn Ala Gly
Thr Ala Thr Thr Asn Phe Thr Lys Leu Arg Pro Thr Asn 450 455 460Phe
Ser Asn Phe Lys Lys Asn Trp Leu Pro Gly Pro Ser Ile Lys Gln465 470
475 480Gln Gly Phe Ser Lys Thr Ala Asn Gln Asn Tyr Lys Ile Pro Ala
Thr 485 490 495Gly Ser Asp Ser Leu Ile Lys Tyr Glu Thr His Ser Thr
Leu Asp Gly 500 505 510Arg Trp Ser Ala Leu Thr Pro Gly Pro Pro Met
Ala Thr Ala Gly Pro 515 520 525Ala Asp Ser Lys Phe Ser Asn Ser Gln
Leu Ile Phe Ala Gly Pro Lys 530 535 540Gln Asn Gly Asn Thr Ala Thr
Val Pro Gly Thr Leu Ile Phe Thr Ser545 550 555 560Glu Glu Glu Leu
Ala Ala Thr Asn Ala Thr Asp Thr Asp Met Trp Gly 565 570 575Asn Leu
Pro Gly Gly Asp Gln Ser Asn Ser Asn Leu Pro Thr Val Asp 580 585
590Arg Leu Thr Ala Leu Gly Ala Val Pro Gly Met Val Trp Gln Asn Arg
595 600 605Asp Ile Tyr Tyr Gln Gly Pro Ile Trp Ala Lys Ile Pro His
Thr Asp 610 615 620Gly His Phe His Pro Ser Pro Leu Ile Gly Gly Phe
Gly Leu Lys His625 630 635 640Pro Pro Pro Gln Ile Phe Ile Lys Asn
Thr Pro Val Pro Ala Asn Pro 645 650 655Ala Thr Thr Phe Ser Ser Thr
Pro Val Asn Ser Phe Ile Thr Gln Tyr 660 665 670Ser Thr Gly Gln Val
Ser Val Gln Ile Asp Trp Glu Ile Gln Lys Glu 675 680 685Arg Ser Lys
Arg Trp Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly 690 695 700Gln
Gln Asn Ser Leu Leu Trp Ala Pro Asp Ala Ala Gly Lys Tyr Thr705 710
715 720Glu Pro Arg Ala Ile Gly Thr Arg Tyr Leu Thr His His Leu 725
7305724PRTAdeno-associated virus - 5 5Met Ser Phe Val Asp His Pro
Pro Asp Trp Leu Glu Glu Val Gly Glu1 5 10 15Gly Leu Arg Glu Phe Leu
Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys 20 25 30Pro Asn Gln Gln His
Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly 35 40 45Tyr Asn Tyr Leu
Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val 50 55 60Asn Arg Ala
Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu65 70 75 80Gln
Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp 85 90
95Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn
100 105 110Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu
Pro Phe 115 120 125Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr
Gly Lys Arg Ile 130 135 140Asp Asp His Phe Pro Lys Arg Lys Lys Ala
Arg Thr Glu Glu Asp Ser145 150 155 160Lys Pro Ser Thr Ser Ser Asp
Ala Glu Ala Gly Pro Ser Gly Ser Gln 165 170 175Gln Leu Gln Ile Pro
Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr 180 185 190Met Ser Ala
Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala 195 200 205Asp
Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp 210 215
220Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu
Pro225 230 235 240Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser
Gly Ser Val Asp 245 250 255Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr
Ser Thr Pro Trp Gly Tyr 260 265 270Phe Asp Phe Asn Arg Phe His Ser
His Trp Ser Pro Arg Asp Trp Gln 275 280 285Arg Leu Ile Asn Asn Tyr
Trp Gly Phe Arg Pro Arg Ser Leu Arg Val 290 295 300Lys Ile Phe Asn
Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr305 310 315 320Thr
Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp 325 330
335Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys
340 345 350Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr
Gly Tyr 355 360 365Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr
Glu Arg Ser Ser 370 375 380Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys
Met Leu Arg Thr Gly Asn385 390 395 400Asn Phe Glu Phe Thr Tyr Asn
Phe Glu Glu Val Pro Phe His Ser Ser 405 410 415Phe Ala Pro Ser Gln
Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp 420 425 430Gln Tyr Leu
Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln 435 440 445Phe
Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp 450 455
460Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser
Gly465 470 475 480Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr
Asn Arg Met Glu 485 490 495Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro
Gln Pro Asn Gly Met Thr 500 505 510Asn Asn Leu Gln Gly Ser Asn Thr
Tyr Ala Leu Glu Asn Thr Met Ile 515 520 525Phe Asn Ser Gln Pro Ala
Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu 530 535 540Gly Asn Met Leu
Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg545 550 555 560Val
Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser 565 570
575Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro
580 585 590Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro
Ile Trp 595 600 605Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro
Ser Pro Ala Met 610 615 620Gly Gly Phe Gly Leu Lys His Pro Pro Pro
Met Met Leu Ile Lys Asn625 630 635 640Thr Pro Val Pro Gly Asn Ile
Thr Ser Phe Ser Asp Val Pro Val Ser 645 650 655Ser Phe Ile Thr Gln
Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu 660 665 670Trp Glu Leu
Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln 675 680 685Tyr
Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp 690 695
700Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr
Leu705 710 715 720Thr Arg Pro Leu6736PRTAdeno-associated virus - 6
6Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5
10 15Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys
Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val
Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys
Gly Glu Pro 50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp
Lys Ala Tyr Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr
Leu Arg Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln
Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe
Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Phe Gly Leu Val Glu
Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu
Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly145 150 155
160Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu
Pro Pro 180 185 190Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala
Ser Gly Gly Gly 195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly Ala
Asp Gly Val Gly Asn Ala 210 215 220Ser Gly Asn Trp His Cys Asp Ser
Thr Trp Leu Gly Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg
Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln
Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270Tyr
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280
285His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn
290 295 300Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn
Ile Gln305 310 315 320Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr
Thr Ile Ala Asn Asn 325 330 335Leu Thr Ser Thr Val Gln Val Phe Ser
Asp Ser Glu Tyr Gln Leu Pro 340 345 350Tyr Val Leu Gly Ser Ala His
Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365Asp Val Phe Met Ile
Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380Ser Gln Ala
Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro385 390 395
400Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
405 410 415Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp 420 425 430Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Asn Arg 435 440 445Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn
Lys Asp Leu Leu Phe Ser 450 455 460Arg Gly Ser Pro Ala Gly Met Ser
Val Gln Pro Lys Asn Trp Leu Pro465 470 475 480Gly Pro Cys Tyr Arg
Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495Asn Asn Ser
Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510Gly
Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520
525Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly
530 535 540Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val
Met Ile545 550 555 560Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro
Val Ala Thr Glu Arg 565 570 575Phe Gly Thr Val Ala Val Asn Leu Gln
Ser Ser Ser Thr Asp Pro Ala 580 585 590Thr Gly Asp Val His Val Met
Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605Asp Arg Asp Val Tyr
Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620Thr Asp Gly
His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu625 630 635
640Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala
645 650 655Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe
Ile Thr 660 665 670Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu
Trp Glu Leu Gln 675 680 685Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu
Val Gln Tyr Thr Ser Asn 690 695 700Tyr Ala Lys Ser Ala Asn Val Asp
Phe Thr Val Asp Asn Asn Gly Leu705 710 715 720Tyr Thr Glu Pro Arg
Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730
7357737PRTAdeno-associated virus - 7 7Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Ala Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro Gln Arg Ser Pro
Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys Gly Gln Gln Pro
Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr Gly Asp Ser Glu
Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185 190Pro Ala Ala
Pro Ser Ser Val Gly Ser Gly Thr Val Ala Ala Gly Gly 195 200 205Gly
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn 210 215
220Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg
Val225 230 235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr
Tyr Asn Asn His 245 250 255Leu Tyr Lys Gln Ile Ser Ser Glu Thr Ala
Gly Ser Thr Asn Asp Asn 260 265 270Thr Tyr Phe Gly Tyr Ser Thr Pro
Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285Phe His Cys His Phe Ser
Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300Asn Trp Gly Phe
Arg Pro Lys Lys Leu Arg Phe Lys Leu Phe Asn Ile305 310 315 320Gln
Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn 325 330
335Asn Leu Thr Ser Thr Ile Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu
340 345 350Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro
Phe Pro 355 360 365Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu
Thr Leu Asn Asn 370 375 380Gly Ser Gln Ser Val Gly Arg Ser Ser Phe
Tyr Cys Leu Glu Tyr Phe385 390 395 400Pro Ser Gln Met Leu Arg Thr
Gly Asn Asn Phe Glu Phe Ser Tyr Ser 405 410 415Phe Glu Asp Val Pro
Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430Asp Arg Leu
Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ala 435 440 445Arg
Thr Gln Ser Asn Pro Gly Gly Thr Ala Gly Asn Arg Glu Leu Gln 450 455
460Phe Tyr Gln Gly Gly Pro Ser Thr Met Ala Glu Gln Ala Lys Asn
Trp465 470 475 480Leu Pro Gly Pro Cys Phe Arg Gln Gln Arg Val Ser
Lys Thr Leu Asp 485 490 495Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
Gly Ala Thr Lys Tyr His 500 505 510Leu Asn Gly Arg Asn Ser Leu Val
Asn Pro
Gly Val Ala Met Ala Thr 515 520 525His Lys Asp Asp Glu Asp Arg Phe
Phe Pro Ser Ser Gly Val Leu Ile 530 535 540Phe Gly Lys Thr Gly Ala
Thr Asn Lys Thr Thr Leu Glu Asn Val Leu545 550 555 560Met Thr Asn
Glu Glu Glu Ile Arg Pro Thr Asn Pro Val Ala Thr Glu 565 570 575Glu
Tyr Gly Ile Val Ser Ser Asn Leu Gln Ala Ala Asn Thr Ala Ala 580 585
590Gln Thr Gln Val Val Asn Asn Gln Gly Ala Leu Pro Gly Met Val Trp
595 600 605Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys
Ile Pro 610 615 620His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met
Gly Gly Phe Gly625 630 635 640Leu Lys His Pro Pro Pro Gln Ile Leu
Ile Lys Asn Thr Pro Val Pro 645 650 655Ala Asn Pro Pro Glu Val Phe
Thr Pro Ala Lys Phe Ala Ser Phe Ile 660 665 670Thr Gln Tyr Ser Thr
Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu 675 680 685Gln Lys Glu
Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser 690 695 700Asn
Phe Glu Lys Gln Thr Gly Val Asp Phe Ala Val Asp Ser Gln Gly705 710
715 720Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
Asn 725 730 735Leu8738PRTAdeno-associated virus - 8 8Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly
Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys
Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40
45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr
Asp65 70 75 80Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr
Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr
Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys
Lys Arg Val Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Gly Ala
Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro
Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys
Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185
190Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly
195 200 205Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val
Gly Ser 210 215 220Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu
Gly Asp Arg Val225 230 235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala
Leu Pro Thr Tyr Asn Asn His 245 250 255Leu Tyr Lys Gln Ile Ser Asn
Gly Thr Ser Gly Gly Ala Thr Asn Asp 260 265 270Asn Thr Tyr Phe Gly
Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285Arg Phe His
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300Asn
Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn305 310
315 320Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile
Ala 325 330 335Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser
Glu Tyr Gln 340 345 350Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly
Cys Leu Pro Pro Phe 355 360 365Pro Ala Asp Val Phe Met Ile Pro Gln
Tyr Gly Tyr Leu Thr Leu Asn 370 375 380Asn Gly Ser Gln Ala Val Gly
Arg Ser Ser Phe Tyr Cys Leu Glu Tyr385 390 395 400Phe Pro Ser Gln
Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr 405 410 415Thr Phe
Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425
430Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu
435 440 445Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr
Leu Gly 450 455 460Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln
Ala Lys Asn Trp465 470 475 480Leu Pro Gly Pro Cys Tyr Arg Gln Gln
Arg Val Ser Thr Thr Thr Gly 485 490 495Gln Asn Asn Asn Ser Asn Phe
Ala Trp Thr Ala Gly Thr Lys Tyr His 500 505 510Leu Asn Gly Arg Asn
Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr 515 520 525His Lys Asp
Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile 530 535 540Phe
Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val545 550
555 560Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala
Thr 565 570 575Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln
Asn Thr Ala 580 585 590Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala
Leu Pro Gly Met Val 595 600 605Trp Gln Asn Arg Asp Val Tyr Leu Gln
Gly Pro Ile Trp Ala Lys Ile 610 615 620Pro His Thr Asp Gly Asn Phe
His Pro Ser Pro Leu Met Gly Gly Phe625 630 635 640Gly Leu Lys His
Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655Pro Ala
Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe 660 665
670Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu
675 680 685Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln
Tyr Thr 690 695 700Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala
Val Asn Thr Glu705 710 715 720Gly Val Tyr Ser Glu Pro Arg Pro Ile
Gly Thr Arg Tyr Leu Thr Arg 725 730 735Asn
Leu9736PRTAdeno-associated virus 9 9Met Ala Ala Asp Gly Tyr Leu Pro
Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp
Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30Lys Ala Asn Gln Gln His
Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu
Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala
Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln
Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp
Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro
115 120 125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ala Gly Ile Gly145 150 155 160Lys Ser Gly Ala Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Thr Glu Ser Val Pro
Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190Ala Ala Pro Ser Gly
Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Val
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser
Asn Asp Asn 260 265 270Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
Phe Asp Phe Asn Arg 275 280 285Phe His Cys His Phe Ser Pro Arg Asp
Trp Gln Arg Leu Ile Asn Asn 290 295 300Asn Trp Gly Phe Arg Pro Lys
Arg Leu Asn Phe Lys Leu Phe Asn Ile305 310 315 320Gln Val Lys Glu
Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335Asn Leu
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345
350Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro
355 360 365Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu
Asn Asp 370 375 380Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys
Leu Glu Tyr Phe385 390 395 400Pro Ser Gln Met Leu Arg Thr Gly Asn
Asn Phe Gln Phe Ser Tyr Glu 405 410 415Phe Glu Asn Val Pro Phe His
Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430Asp Arg Leu Met Asn
Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445Lys Thr Ile
Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460Val
Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro465 470
475 480Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln
Asn 485 490 495Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp
Ala Leu Asn 500 505 510Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala
Met Ala Ser His Lys 515 520 525Glu Gly Glu Asp Arg Phe Phe Pro Leu
Ser Gly Ser Leu Ile Phe Gly 530 535 540Lys Gln Gly Thr Gly Arg Asp
Asn Val Asp Ala Asp Lys Val Met Ile545 550 555 560Thr Asn Glu Glu
Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575Tyr Gly
Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585
590Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln
595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
Pro His 610 615 620Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly
Gly Phe Gly Met625 630 635 640Lys His Pro Pro Pro Gln Ile Leu Ile
Lys Asn Thr Pro Val Pro Ala 645 650 655Asp Pro Pro Thr Ala Phe Asn
Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700Tyr
Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val705 710
715 720Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn
Leu 725 730 73510738PRTAdeno-associated virus 10 10Met Ala Ala Asp
Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile
Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala
Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly
Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55
60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65
70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His
Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe
Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg
Val Leu Glu Pro 115 120 125Leu Gly Leu Val Glu Glu Ala Ala Lys Thr
Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Pro Ser Pro Gln Arg
Ser Pro Asp Ser Ser Thr Gly Ile145 150 155 160Gly Lys Lys Gly Gln
Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175Thr Gly Glu
Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185 190Pro
Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly 195 200
205Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser
210 215 220Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp
Arg Val225 230 235 240Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro
Thr Tyr Asn Asn His 245 250 255Leu Tyr Lys Gln Ile Ser Asn Gly Thr
Ser Gly Gly Ser Thr Asn Asp 260 265 270Asn Thr Tyr Phe Gly Tyr Ser
Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285Arg Phe His Cys His
Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300Asn Asn Trp
Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn305 310 315
320Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala
325 330 335Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu
Tyr Gln 340 345 350Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys
Leu Pro Pro Phe 355 360 365Pro Ala Asp Val Phe Met Ile Pro Gln Tyr
Gly Tyr Leu Thr Leu Asn 370 375 380Asn Gly Ser Gln Ala Val Gly Arg
Ser Ser Phe Tyr Cys Leu Glu Tyr385 390 395 400Phe Pro Ser Gln Met
Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr 405 410 415Thr Phe Glu
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430Leu
Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440
445Ser Arg Thr Gln Ser Thr Gly Gly Thr Gln Gly Thr Gln Gln Leu Leu
450 455 460Phe Ser Gln Ala Gly Pro Ala Asn Met Ser Ala Gln Ala Lys
Asn Trp465 470 475 480Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val
Ser Thr Thr Leu Ser 485 490 495Gln Asn Asn Asn Ser Asn Phe Ala Trp
Thr Gly Ala Thr Lys Tyr His 500 505 510Leu Asn Gly Arg Asp Ser Leu
Val Asn Pro Gly Val Ala Met Ala Thr 515 520 525His Lys Asp Asp Glu
Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met 530 535 540Phe Gly Lys
Gln Gly Ala Gly Arg Asp Asn Val Asp Tyr Ser Ser Val545 550 555
560Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
565 570 575Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Ala Asn
Thr Gly 580 585 590Pro Ile Val Gly Asn Val Asn Ser Gln Gly Ala Leu
Pro Gly Met Val 595 600 605Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly
Pro Ile Trp Ala Lys Ile 610 615 620Pro His Thr Asp Gly Asn Phe His
Pro Ser Pro Leu Met Gly Gly Phe625 630 635 640Gly Leu Lys His Pro
Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655Pro Ala Asp
Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe 660 665 670Ile
Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 675 680
685Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr
690 695 700Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn
Thr Glu705 710 715 720Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr
Arg Tyr Leu Thr Arg 725 730 735Asn Leu117PRTArtificial SequencerAAV
peptide insert 11Leu Gly Glu Thr Thr Arg Pro1 5127PRTArtificial
SequencerAAV peptide insert 12Asn Glu Thr Ile Thr Arg Pro1
51310PRTArtificial
SequencerAAV peptide insert 13Leu Ala Leu Gly Glu Thr Thr Arg Pro
Ala1 5 101410PRTArtificial SequenceLANETITRPA 14Leu Ala Asn Glu Thr
Ile Thr Arg Pro Ala1 5 101510PRTArtificial SequencerAAV peptide
insert 15Ala Ala Leu Gly Glu Thr Thr Arg Pro Ala1 5
101610PRTArtificial SequencerAAV peptide insert 16Ala Ala Asn Glu
Thr Ile Thr Arg Pro Ala1 5 10179PRTArtificial SequencerAAV peptide
insert 17Gly Leu Gly Glu Thr Thr Arg Pro Ala1 5189PRTArtificial
SequencerAAV peptide insert 18Gly Asn Glu Thr Ile Thr Arg Pro Ala1
519745PRTArtificial SequenceVariant VP1 capsid protein with peptide
insertion 19Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr
Leu Ser1 5 10 15Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro
Pro Pro Pro 20 25 30Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly
Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu
Asp Lys Gly Glu Pro 50 55 60Val Asn Glu Ala Asp Ala Ala Ala Leu Glu
His Asp Lys Ala Tyr Asp65 70 75 80Arg Gln Leu Asp Ser Gly Asp Asn
Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg
Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Leu Gly Leu
Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro
Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly145 150
155 160Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln
Thr 165 170 175Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly
Gln Pro Pro 180 185 190Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met
Ala Thr Gly Ser Gly 195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Asn Ser 210 215 220Ser Gly Asn Trp His Cys Asp
Ser Thr Trp Met Gly Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys
Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265
270Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
Asn Trp 290 295 300Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe
Asn Ile Gln Val305 310 315 320Lys Glu Val Thr Gln Asn Asp Gly Thr
Thr Thr Ile Ala Asn Asn Leu 325 330 335Thr Ser Thr Val Gln Val Phe
Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350Val Leu Gly Ser Ala
His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365Val Phe Met
Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380Gln
Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser385 390
395 400Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
Glu 405 410 415Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp Arg 420 425 430Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Ser Arg Thr 435 440 445Asn Thr Pro Ser Gly Thr Thr Thr Gln
Ser Arg Leu Gln Phe Ser Gln 450 455 460Ala Gly Ala Ser Asp Ile Arg
Asp Gln Ser Arg Asn Trp Leu Pro Gly465 470 475 480Pro Cys Tyr Arg
Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495Asn Ser
Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505
510Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp
515 520 525Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe
Gly Lys 530 535 540Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys
Val Met Ile Thr545 550 555 560Asp Glu Glu Glu Ile Arg Thr Thr Asn
Pro Val Ala Thr Glu Gln Tyr 565 570 575Gly Ser Val Ser Thr Asn Leu
Gln Arg Gly Asn Leu Ala Leu Gly Glu 580 585 590Thr Thr Arg Pro Ala
Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln 595 600 605Gly Val Leu
Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln 610 615 620Gly
Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro625 630
635 640Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln
Ile 645 650 655Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr
Thr Phe Ser 660 665 670Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr
Ser Thr Gly Gln Val 675 680 685Ser Val Glu Ile Glu Trp Glu Leu Gln
Lys Glu Asn Ser Lys Arg Trp 690 695 700Asn Pro Glu Ile Gln Tyr Thr
Ser Asn Tyr Asn Lys Ser Val Asn Val705 710 715 720Asp Phe Thr Val
Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile 725 730 735Gly Thr
Arg Tyr Leu Thr Arg Asn Leu 740 7452011PRTArtificial
SequencePeptide Insertion Formula IMISC_FEATURE(1)..(2)Xaa = Ala,
Leu, Gly, Ser, Thr or is absentMISC_FEATURE(3)..(3)Xaa = Leu, Asn
and LysMISC_FEATURE(4)..(4)Xaa = Gly, Glu, Ala and
AspMISC_FEATURE(5)..(5)Xaa = Glu, Thr, Gly and
ProMISC_FEATURE(6)..(6)Xaa = Thr, Ile, Gln, and
LysMISC_FEATURE(7)..(7)Xaa = Thr and AlaMISC_FEATURE(8)..(8)Xaa =
Arg, Asn and ThrMISC_FEATURE(9)..(9)Xaa = Pro and
AsnMISC_FEATURE(10)..(11)Xaa = Ala, Leu, Gly, Ser, Thr or is absent
20Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
102111PRTArtificial SequencePeptide Insertion Formula
IIMISC_FEATURE(1)..(2)Xaa = Ala, Leu, Gly, Ser, Thr or is
absentMISC_FEATURE(3)..(6)Xaa = any amino
acidmisc_feature(10)..(11)Xaa can be any naturally occurring amino
acid 21Xaa Xaa Xaa Xaa Xaa Xaa Thr Arg Pro Xaa Xaa1 5
102211PRTArtificial SequencePeptide Insertion Formula
IIIMISC_FEATURE(1)..(2)Xaa = Ala, Leu, Gly, Ser, Thr or is
absentMISC_FEATURE(3)..(3)Xaa = Leu and AsnMISC_FEATURE(4)..(4)Xaa
= Gly and GluMISC_FEATURE(5)..(5)Xaa = Glu and
ThrMISC_FEATURE(6)..(6)Xaa = Thr and IleMISC_FEATURE(10)..(11)Xaa =
Ala, Leu, Gly, Ser, Thr or is absent 22Xaa Xaa Xaa Xaa Xaa Xaa Thr
Arg Pro Xaa Xaa1 5 102311PRTArtificial SequencePeptide Insertion
Formula IVMISC_FEATURE(1)..(2)Xaa = Ala, Leu, Gly, Ser, Thr or is
absentMISC_FEATURE(3)..(3)Xaa = Leu, Asn, Arg, Ala, Ser and
LysMISC_FEATURE(4)..(4)Xaa =Gly, Glu, Ala, Val, Thr and
AspMISC_FEATURE(5)..(5)Xaa = Glu, Thr, Gly, Asp and
ProMISC_FEATURE(6)..(6)Xaa = Thr, Ile, Gly, Lys, Asp and
GlnMISC_FEATURE(7)..(7)Xaa = Thr, Ser, Val and
AlaMISC_FEATURE(8)..(8)Xaa = Arg, Val, Lys, Pro, Thr and
AsnMISC_FEATURE(9)..(9)Xaa = Pro, Gly, Phe, Asn and
ArgMISC_FEATURE(10)..(11)Xaa = Ala, Leu, Gly, Ser, Thr or is absent
23Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
* * * * *